WO2022121110A1 - Mechanism based on gastrointestinal symptoms caused by sars-cov-2 and application therefor - Google Patents

Abstract

The present invention relates to the field of medical technology. Mechanistic studies have found that the SARS-CoV-2 Spike protein activates an Ras-Raf-MEK-ERK pathway by means of binding to ACE2 receptors of intestinal epithelial cells, thereby promoting the secretion of VEGF by intestinal epithelial cells. However, the pathway is not altered in endothelial cells. An ERK or VEGF blocker can remedy Spike protein-enhanced vascular permeability and alleviate gastrointestinal symptoms in mice. The results indicate that in gastrointestinal tracts, especially in duodenum in which ACE2 is highly expressed, the SARS-CoV-2 Spike protein can directly bind to ACE2 in the intestinal epithelial cells, activate a downstream ERK/VEGF signal pathway, and induce an increase in vascular permeability, thereby leading to inflammation-related gastrointestinal symptoms. The present study provides an explanatory mechanism and a treatment strategy for gastrointestinal symptoms in patients having COVID-19.

Description

Mechanisms and applications of gastrointestinal symptoms based on SARS-CoV-2 technical field

The present invention relates to the field of medical technology, and more particularly, to the mechanism and application of gastrointestinal symptoms caused by SARS-CoV-2.

Background technique

COVID-19 is a severe acute respiratory illness caused by the SARS-CoV-2 virus that has caused a worldwide pandemic since its outbreak in late 2019. After SARS-CoV-2 infection, the common feature is typical respiratory symptoms, and the airway is also identified as a target organ. However, as SARS-CoV-2 infection has been further studied, clinical reports of cases with gastrointestinal symptoms, which typically manifest as diarrhea, anorexia, nausea, and vomiting, continue to increase. In addition, some patients infected with SARS-CoV-2 exhibited only digestive symptoms at the time of onset or even during the course of the disease, indicating that the gastrointestinal tract is highly susceptible to SARS-CoV-2.

In these patients with gastrointestinal symptoms, pathological upper abdominal CT showed thickening of the bowel wall. Notably, thickening of the small bowel wall was especially observed in ICU patients. However, the systemic characteristics of susceptible sites in the gastrointestinal tract of patients with COVID-19 and the mechanisms underlying the development of gastrointestinal symptoms remain unclear. Gastrointestinal symptoms in COVID-19 patients often manifest as inflammatory cell infiltration and interstitial edema, suggesting impaired intestinal vascular permeability. While the respiratory system is the primary target of SARS-CoV-2, the vascular system is also compromised, with clinically occurring vascular-related complications such as myocardial infarction, mesenteric ischemia, and intestinal endotheliitis, suggesting that endothelial cells may be the source of the virus direct target. In fact, evidence for direct viral infection of endothelial cells has recently emerged. However, whether gastrointestinal inflammation and altered vascular permeability are the result of endothelial infection or secondary effects arising from the response of surrounding cells to the virus is unclear.

technical problem

The technical problem to be solved by the present invention is to overcome the defect that the mechanism of gastrointestinal symptoms caused by SARS-CoV-2 in the prior art is not clear. Gastrointestinal symptoms in patients with -19 provide an explanatory mechanism and a therapeutic strategy.

Angiotensin-converting enzyme (ACE2) maintains circulatory homeostasis by regulating angiogenesis, thrombosis, and vascular remodeling, and it has been reported that ACE2 is the major binding receptor for SARS-CoV-2. ACE2 is expressed in many organs, including the digestive and vascular systems. Notably, human ACE2 is most highly expressed in the small intestine. Two studies have demonstrated that SARS-CoV-2 replicates in intestinal epithelial cells using ACE2 as an entry receptor using human intestinal organoids and a colon epithelial cancer cell line (Caco-2), respectively. SARS-CoV-2 can also bind to ACE2 in other vertebrates, such as mice and rats, without being infected, raising the question of whether SARS-CoV-2 binding to ACE2 is possible in these organisms cause any signaling.

Here, we investigated SARS-CoV-2 Spike/ACE2-mediated signaling pathways in the gastrointestinal tract and its associated pathology and clinical symptoms, which may provide insights into the repair of intestinal vascular barrier damage and the gastrointestinal tract in patients infected with COVID-19 Relief of symptoms provides a therapeutic target.

technical solutions

The object of the present invention is achieved through the following technical solutions:

The present invention first provides a method of inhibiting SARS-CoV-2-induced damage to the vascular barrier in gastrointestinal tissue, comprising administering to the gastrointestinal tissue a vascular rejection targeting signal.

Here, we investigate SARS-CoV-2 Spike/ACE2-mediated signaling pathways in the gastrointestinal tract and its associated pathology and clinical outcomes, and we have identified SARS-CoV-2 Possible pathways and mechanisms of Spike protein-induced intestinal vascular permeability and gastrointestinal inflammation. Our data suggest that by binding to ACE2, Spike protein induces VEGF production by activating the Ras-Raf-MEK-ERK pathway in intestinal epithelial cells, resulting in VE-cad-mediated vascular hyperpermeability, ultimately leading to Tissue inflammation and edema. Blockade of ERK or VEGF was able to alleviate gastrointestinal symptoms induced by Spike stimulation.

We found edema and inflammatory cell infiltration in the staining of intestinal tissue sections of patients with COVID-19, and speculated that vascular permeability had changed, and VEGF was an important factor affecting vascular permeability, so we verified SARS-CoV- 2 Spike The protein can regulate the phosphorylation of downstream ERK by binding to intestinal epithelial ACE2, thereby promoting the secretion of VEGF. Secreted VEGF stimulated lower VE-cad expression or higher VE-cad phosphorylation in vascular endothelial cells. Increase vascular permeability. For this process, the use of ERK inhibitors can reduce the phosphorylation of pERK and the production of VEGF, thereby restoring the expression of VE-cad in vascular endothelial cells, indicating that ERK inhibitors can be used as reference drugs. VEGF antagonists inhibit the binding of VEGF to VEGF receptors, thereby inhibiting the increase of vascular endothelial permeability.

Therefore, preferably, the repulsive targeting signal includes an ERK inhibitor and/or a VEGF antagonist, the ERK inhibitor refers to a substance that can bind to ERK and block the biological activity of ERK, and the VEGF antagonist refers to Substances that bind to VEGF and block the biological activity of VEGF.

Specifically, the ERK inhibitors include clinical stage inhibitors SCH772984, GDC-0994, Ulixertinib, KO-947, LY3214996, MK-8353, CC-90003, LTT462, etc., and those in preclinical stage or biological activity evaluation stage The VEGF antagonists include bevacizumab, ramucirumab, ranibizumab, aflibercept, conbercept and the like.

More specifically, the above-mentioned ERK inhibitor is SCH772984, and the VEGF antagonist is bevacizumab.

In the research process of the present invention, it is found that in the gastrointestinal tract, the duodenum is the most sensitive to the SARS-CoV-2 Spike protein, so preferably, the above-mentioned gastrointestinal tract tissue is the duodenum. The above study of the present invention also highlights the sensitivity of the duodenum to SARS-CoV-2 Spike-induced increased vascular permeability and its potential pathways, providing a potential therapeutic target for gastrointestinal symptoms in COVID-19 patients.

The present invention also provides a method for screening or evaluating an agent that inhibits SARS-CoV-2-induced damage to the vascular barrier in the gastrointestinal tract, specifically: determining the effect of the agent on the VEGF-mediated increase in the vascular permeability of the gastrointestinal tract. ability.

The present invention studies the mechanism of gastrointestinal symptoms caused by SARS-CoV-2, namely SARS-CoV-2 Spike protein directly binds to ACE2 on intestinal epithelial cells, activates the downstream ERK/VEGF signaling pathway, and induces increased vascular permeability. Specifically, SARS-CoV-2 Spike protein directly binds to ACE2 on intestinal epithelial cells, activates the upstream molecules of ERK Ras, Raf and MEK in intestinal epithelial cells, further activates ERK, promotes the secretion of VEGF from intestinal epithelial cells, and makes intestinal endothelial cells VE-cad Decreased expression or increased phosphorylation results in increased intestinal endothelial vascular permeability.

It can be seen from the above mechanism that inhibiting ERK, MEK, Ras, Raf, and VEGF can alleviate the degree of damage to the gastrointestinal vascular barrier caused by SARS-CoV-2. Therefore, it is preferable to determine that the agent affects the VEGF-mediated gastrointestinal tract The ability to increase vascular permeability refers to determining the ability of the agent to inhibit ERK, MEK, Ras, Raf, VEGF.

As a preferred embodiment, in an in vitro experiment, the effect of the agent on the increase in vascular permeability of the gastrointestinal tract mediated by VEGF is determined by the following steps:

(1) Prepare a candidate agent group consisting of exposure to SARS-CoV-2 Intestinal epithelial cell secretions, endothelial cells and candidate agents of Spike protein;

(2) Prepare a placebo group consisting of exposure to SARS-CoV-2 Intestinal epithelial secretions of Spike protein, endothelial cells and placebo composition;

(3) Among them, the endothelial cells in the candidate drug group can detect higher VE-cad expression or lower VE-cad phosphorylation level than the endothelial cells in the placebo group, indicating that the candidate drug can Inhibits VEGF-mediated increase in gastrointestinal vascular permeability.

Preferably, the placebo described in step (2) is selected from PBS or DMEM.

As another embodiment, when in vivo, said agent affects VEGF-mediated increase in gastrointestinal vascular permeability as determined by the following steps:

(1) Construct an animal model of SARS-CoV-2 gastrointestinal inflammation;

(2) Intraperitoneal injection or no placebo injection to the model animals of (1);

(3) Inject the candidate drug into the model animal of (1) by intraperitoneal injection;

(4) wherein, a lower expression level of VEGF can be detected in the gastrointestinal tract tissue of the animals after (3) treatment than in the gastrointestinal tract tissue of the animals after treatment (2), indicating that the agent can Inhibits VEGF-mediated increase in gastrointestinal vascular permeability.

Preferably, the placebo described in step (2) is selected from PBS or DMEM.

Preferably, the gastrointestinal tract tissue in step (4) is the duodenum.

Preferably, the construction method of the SARS-CoV-2 gastrointestinal inflammation animal model in step (1) includes the following steps:

S1. Mice were anesthetized after fasting for 16-24 hours;

S2. Inject acetic acid from the anus of the mouse, turn the mouse upside down, and lavage the gastrointestinal tract of the mouse;

S3. After a period of time, mice were intraperitoneally injected with SARS-CoV-2 Spike protein;

S4. The animal model of SARS-CoV-2 gastrointestinal inflammation was successfully obtained after a few hours.

Before intraperitoneal injection of SARS-CoV-2 Spike protein in the present invention, acid enema treatment is performed on the epithelium of the gastrointestinal tract, and then SARS-CoV-2 Spike protein stimulation is more conducive to the binding of SARS-CoV-2 Spike protein; the animal model obtained in this way is consistent with the clinical symptoms of COVID-19 patients reported in the present, so the animal model constructed by the method of the present invention can replicate clinical symptoms Gastrointestinal manifestations in patients with COVID-19.

Preferably, in the construction method of the above-mentioned animal model, the SARS-CoV-2 described in step S3 The dosage of Spike protein was 5.5 nM/kg mouse body weight.

Similarly, the action time of intraperitoneal injection of SARS-CoV-2 Spike protein was optimized, and it was found that the clinical manifestations of gastrointestinal damage in patients with COVID-19 could be achieved after 6 hours. There was no significant difference in the performance of intestinal injury. Therefore, preferably, in the above-mentioned method for constructing an animal model, it is characterized in that the several hours in step S4 refers to 6-24 hours.

More specifically, in vivo experiments, the effect of the agent on the VEGF-mediated increase in gastrointestinal vascular permeability was determined by the following steps:

(1) Mice were anesthetized after fasting for 16-24 hours;

(2) Inject acetic acid from the anus of the mouse, turn the mouse upside down, and lavage the gastrointestinal tract of the mouse;

(3) The mice were fed normally. After 16-24 hours, the candidate drugs were injected into the mice through the abdominal cavity respectively to obtain the mice in the candidate drug group; the placebo was injected into the mice through the abdominal cavity respectively to obtain the mice in the placebo group. ;

(4) After 1-2 hours, the mice in the candidate drug group and the placebo group were injected with SARS-CoV-2 by intraperitoneal injection. Spike protein, mice in the control group were intraperitoneally injected with the same amount of IgG-Fc protein;

(5) After 1-2 hours, the candidate drug was injected intraperitoneally to the mice in the candidate drug group again, and the placebo was intraperitoneally injected to the mice in the placebo group;

(6) The gastrointestinal tissues of the mice in the control group, the mice in the candidate drug group and the mice in the placebo group were taken respectively. Compared with the mice in the control group, the gastrointestinal tissues of the mice in the placebo group were able to detect higher Compared with the mice in the placebo group, the gastrointestinal tissue of the candidate drug group could detect a lower VEGF expression level, indicating that the candidate drug can inhibit the VEGF-mediated gastrointestinal vascular permeability Increase.

As a preferred embodiment, in the in vivo experiment, the concentration of acetic acid in the above step (2) is 1-2% v/v, and the volume is 500 μL to 1000 μL.

As a preferred embodiment, in the in vivo experiment, the SARS-CoV-2 described in the above step (4) The dosage of Spike protein was 5.5nM/kg mouse body weight.

The present invention also provides a method for treating or preventing gastrointestinal symptoms caused by SARS-CoV-2, characterized by comprising the following steps:

(1) identify subjects with gastrointestinal symptoms caused by said SARS-CoV-2, and,

(2) administering to the gastrointestinal tract of a subject a repulsive targeting signal that binds to ACE2 and/or ERK and/or VEGF.

Specifically, the repulsive guidance signal includes an ERK inhibitor and/or a VEGF antagonist, the ERK inhibitor refers to a substance that can bind to ERK and block the biological activity of ERK, and the VEGF antagonist refers to a substance that can interact with VEGF Substances that bind to and block the biological activity of VEGF.

Specifically, the ERK inhibitors include clinical stage inhibitors SCH772984, GDC-0994, Ulixertinib, KO-947, LY3214996, MK-8353, CC-90003, LTT462, etc., which are in the preclinical stage or the biological activity evaluation stage. Inhibitors FR180204, VTX-11e, BL-EI-001, etc.; the VEGF antagonists include bevacizumab, ramucirumab, ranibizumab, aflibercept, conbercept and the like.

More specifically, the above-mentioned ERK inhibitor is SCH772984, and the VEGF antagonist is the anti-VEGF drug bevacizumab.

The present invention also provides a medicament for treating or preventing or alleviating gastrointestinal symptoms caused by SARS-CoV-2, the medicament having at least one of the following functions:

(1) Inhibit the expression of VEGF in intestinal epithelial cells;

(2) Inhibit the expression of ERK in intestinal epithelial cells;

(3) Lower VE-cad expression or higher phosphorylation levels were detected in the vascular endothelium caused by rescue of the SARS-CoV-2 spike protein.

Preferably, the drugs include, but are not limited to: ERK inhibitors and/or VEGF antagonists.

Specifically, the ERK inhibitors include clinical stage inhibitors SCH772984, GDC-0994, Ulixertinib, KO-947, LY3214996, MK-8353, CC-90003, LTT462, etc., which are in the preclinical stage or the biological activity evaluation stage. Inhibitors FR180204, VTX-11e, BL-EI-001, etc.; the VEGF antagonists include bevacizumab, ramucirumab, ranibizumab, aflibercept, conbercept and the like.

Growing evidence that SARS-CoV-2 can infect endothelial cells in the gut and lung raises the question of whether endothelial manifestations upon infection are due to direct targeting of endothelial cells or secondary cells targeting other cells. effect. In this study, we elucidated that Spike-triggered signaling does not impair vascular integrity by affecting endothelial permeability, but rather by modulating the VEGF pathway in perivascular intestinal epithelial cells. VEGF, a key factor in acute respiratory distress syndrome (ARDS), surged in the serum of COVID-19 patients. Consistent in part with this study, we found elevated VEGF in local tissue infected with Spike, which may be responsible for damage to the vascular barrier in the gastrointestinal tract.

Our study provides the first evidence that the ACE2/ERK/VEGF axis in intestinal epithelial cells plays a critical role in SARS-CoV-2-induced intestinal inflammation.

beneficial effect

Compared with the prior art, the present invention has the following beneficial effects:

The present study found that the SARS-CoV-2 Spike protein can combine with human and mouse ACE2 to induce increased vascular permeability. Through co-culture of endothelial and epithelial cells, it was found that Spike protein activates the Ras-Raf-MEK-ERK pathway by binding to ACE2 of intestinal epithelial cells, and promotes the secretion of VEGF from epithelial cells, which does not exist in endothelial cells. ERK or VEGF blockade rescued Spike protein-enhanced vascular permeability in vivo and alleviated gastrointestinal symptoms. These results suggest that in the gastrointestinal tract, especially the duodenum, the SARS-CoV-2 Spike protein can directly bind to ACE2 in intestinal epithelial cells, activate the downstream ERK/VEGF signaling pathway, and induce increased vascular permeability. This study provides a mechanistic explanation and a rational treatment strategy for gastrointestinal symptoms in patients with COVID-19.

Description of drawings

Figure 1a shows the co-localization of ACE2 and SARS-CoV-2 Spike protein observed in the gastrointestinal tissue of COVID-19 patients; Figure 1b shows the local inflammation in H&E stained gastrointestinal tissue;

Figure 2a shows the construction process of the animal model; Figure 2b shows the HE staining images of different parts of the experimental group (Spike-Fc) mice and the control group (Control-Fc) mice; Figure 2c and Figure 2d show the experimental group (Spike-Fc) mice. Fc) Immunofluorescence staining of different parts of the gastrointestinal tract of mice and control (Control-Fc) mice;

Figure 3a shows the evaluation of vascular barrier function (using TRITC-dextran tracer) in experimental (Spike-Fc) and control (Control-Fc) mice (n=9); data are expressed as mean ± SD , P value is by t test, * represents p<0.05; Figure 3b shows the effect of experimental group (Spike-Fc) and control group (Control-Fc) on the permeability of HUVECs cells (using TRITC-dextran tracer) ), n=9, data are expressed as mean ± SD, P value is expressed by t test, ns means no significance; Figure 3c shows that the HUVECs of experimental group (Spike-Fc) and control group (Control-Fc) were determined by WB The expression of adhesion proteins VE-cad and pVE-cad (Y731) in cells, the histogram represents the ratio of protein expression relative to β-actin quantification, the experimental data were averaged three times, the data are expressed as mean ± SD, and the p value is paired with t-test, ns represents no significance; Figure 3d shows the process of in vitro permeation experiments; Figure 3e shows the permeability of HUVEC barriers evaluated by the permeability of dextran, data are expressed as mean ± SD, n=8, p-values are expressed by t Test, *** represents p<0.0001;

Figure 4a shows the pull-down results of Spike-Fc protein and human ACE2 in Caco-2 cells and 239T cells, respectively, where Input represents total cell lysate, and IP:Fc represents the protein pulled down by Fc peptide; Figure 4b shows Spike-Fc Fluorescent staining results of Fc protein and human ACE2 in 239T cells;

Figure 5a shows the detection of cohesin ZO-1, VE-cad, pVE-cad (Y658) and pVE-cad (Y731) in the supernatant of the co-culture system of Caco-2-HUVECs incubated with Spike-Fc or Control-Fc by WB. ) expression; the histogram shows that the protein expression relative to the amount of β-actin was quantified by densitometric scanning, the experimental data were averaged three times, the data were expressed as mean ± SD, the p value was tested by paired t test, * represents p < 0.05, ns Representatives are not significant; Figure 5b and Figure 5c show VE-cad and IHC plot of pVE-cad(Y731), the scale bar is 100 μm, and the scatter plot shows the expression levels of VE-cad and pVE-cad(Y731) in the duodenum, jejunum, colon and rectum (data are presented as mean ± SD represents, p value is by t test, ns represents no significance, * represents p<0.05, ** represents p<0.01); Figure 5d and Figure 5e show VE-cad in duodenum of healthy and COVID-19 patients and the detection of pVE-cad (Y731) (data are represented by mean ± SD, p value is represented by t test, ns represents no significance, ** represents p<0.01, *** represents p<0.0001);

Figure 6a shows the RT-PCR analysis of VEGF transcript levels in the duodenum, jejunum, colon and rectum of experimental (Spike-Fc) and control (Control-Fc) mice (data are expressed as mean ± SD, The p value was tested by t-test, * represents p<0.05); Figure 6b shows the ELISA analysis of VEGF in the duodenum, jejunum, colon and rectum of experimental (Spike-Fc) and control (Control-Fc) mice The scatter plot shows the protein concentration of VEGF in intestinal tissue (data are represented by mean ± SD, p value is by t test, ns represents no significance); The amount of VEGF secreted by Cntrol-Fc-treated Caco-2 cells; Figure 6d shows the amount of VEGF in plasma of mice treated with Spike-Fc or Control-Fc was analyzed by ELISA; scatter plot shows the protein of VEGF in plasma Concentration (data are represented by mean ± SD, p value by t test, ** represents p<0.01);

Figure 7 shows immunoblot analysis of ERK or pERK in Caco-2 cells treated with Spike-Fc or Control-Fc; histograms show quantification of protein expression relative to the amount of β-actin by densitometric scanning. Data are represented by mean ± SD, p value is by paired t test, * represents p < 0.05;

Figure 8a shows the detection of Caco-2 siRNA (with three siRNA knockdown sequences 01, 02 and 03) by western blot. Compared with the control sequence (NC), all three sequences showed obvious knockdown effect, and the endogenous ACE2 After knockdown, the expressions of Ras, C-Raf, pMEK, pERK and p-P90RSK were significantly increased; Figure 8b shows that after stimulating HUVCE cells with Control-Fc or Spike-Fc protein for 1 h, the expression of ERK and pERK proteins was detected by western blotting Happening;

Figure 9a shows the expression levels of each protein in the Ras-Raf-MEK-ERK signaling pathway after Spike-Fc, Control-Fc and SCH772984 treatment of Caco-2 cells, the histogram shows the protein expression relative to β-actin was quantified by protein grayscale The experimental data were repeated three times, the data were represented by the mean ± SD, the p value was tested by paired t test, * represents p<0.05, ns represents no significance; Figure 9b shows that Spike-Fc, Control-Fc and SCH772984 were compared with Caco After the -2 cells were co-cultured, the amount of VEGF was determined by ELISA, the data were expressed as mean ± SD, the p value was expressed by t test, * represents p < 0.05; Figure 9c shows the experimental group (Spike-Fc) and the control group (Control-Fc) Fc) IHC map of ERK and pERK in intestinal tissues (duodenum, jejunum, ileum, colon and rectum) of model mice injected with SCH772984 intraperitoneally, the scale bar is 100 μm, and the scatter plot shows the duodenum, jejunum and ileum , expression levels of ERK and pERK in colon and rectum (data are represented by mean ± SD, p values are represented by t-test, ns represents no significance, * represents p<0.05, ** represents p<0.01); Figure 9d and Figure 9 9e shows the detection of ERK and pERK in the duodenum of healthy people and patients with COVID-19, (data are expressed as mean ± SD, p-values are expressed by t-test, ns means no significance, ** means p<0.01;

Figure 10a shows the expression and localization of VE-cad in Caco-2 cells treated with Spike-Fc or Control-Fc or SCH772984 or Bevacizumab, and Figure 10b shows the vascular barrier of the co-culture system (using TRITC-dextran tracer ), the co-culture system was co-cultured with HUVEC cells and the supernatant of Caco-2 cells treated with Spike-Fc or Control-Fc or SCH772984 or Bevacizumab; data are expressed as mean ± SD, n=8, p-values were tested by t test , ns means no significance, *** means p<0.0001; Figure 10c shows the experimental group (Spike-Fc) and the control group (Control-Fc) intraperitoneal injection of SCH772984 or Bevacizumab model mice intestinal tissue (12 IHC plots of VE-cad and pVE-cad (Y731) in the denum, jejunum, ileum, colon and rectum), the scale bar is 100 μm, and the scatter plot shows VE-cad and pVE-cad in the duodenum, jejunum and ileum, colon and rectum. The expression level of pVE-cad (Y731) (data are represented by mean ± SD, n=4, p value is by t test, ns means no significance, * means p<0.05, ** means p<0.01, *** for p<0.0001);

Figure 11a shows the use of ELISA to analyze the expression of VEGF in the intestinal tissues (duodenum, jejunum, colon and rectum) of model mice injected with SCH772984 or Bevacizumab in the experimental group (Spike-Fc) and the control group (Control-Fc) by intraperitoneal injection. Amount, data are represented by mean ± SD, n=4, p value is by t test, ns represents no significance, * represents p<0.005; Figure 11b shows the experimental group (Spike-Fc) and the control group (Control-Fc) ) or immunoblot analysis of ERK or pERK in intestinal tissues (duodenum, jejunum, colon, and rectum) of model mice injected with SCH772984 or Bevacizumab intraperitoneally; The amount of β-actin, the experimental data was repeated three times, the data were expressed as mean ± SD, and the p value was expressed by paired t test, *represents p<0.05, **represents p<0.01, and ***represents p<0.0001;

Figure 12a shows that after acid stimulation, the intestinal tissue ( Evans of duodenum, jejunum, colon and rectum) Blue dye exudation experiment, data are expressed as mean ± SD, n=6, p value is by t test, ns means no significance, * means p<0.05, ** means p<0.01, *** means p<0.0001 ; Figure 12b shows the evens permeated per unit weight (g) of intestinal tissue (duodenum, jejunum, colon and rectum) The OD value of blue dye; Figure 12c shows the experimental group (Spike-Fc) and the control group (Control-Fc) or the intestinal tissue (duodenum, jejunum, colon and rectum) of model mice injected with SCH772984 or Bevacizumab intraperitoneally ) of H&E staining; yellow arrows represent inflammatory infiltrates; red stars represent edema, and no inflammation, moderate inflammation, and severe inflammation were assessed in the intestinal tissue; histograms show the proportion of negative, mild, and moderate inflammation assessed in the digestive tract, and the data are shown in Mean ± SD, 4 mice in each group;

Figure 13 shows the model mechanism of SARS-CoV-2 Spike protein-mediated vascular hyperpermeability and intestinal tissue inflammation.

BEST MODE FOR CARRYING OUT THE INVENTION

The specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help the understanding of the present invention, but do not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. The test methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

Patient information and data collection: Clinical specimens, including duodenum and rectum, were collected from patients with COVID-19 (n=17) using endoscopy according to the guidelines of the Chinese Center for Disease Control and Prevention (CDC). The clinical data of 17 patients are shown in Supplementary Table 1. Ethically approved by the Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-Sen University (No. 2020L029-1), all patients signed informed consent.

Cell lines and cell culture: Human umbilical vein endothelial cells (HUVEC) were purchased from ScienCell (Cat. No. 8000, Cat. No. 5000) in ECM medium (ScienCell, Cat. No. 5000) supplemented with 10% fetal bovine serum. 1001) in culture. Human colorectal adenocarcinoma cells (Caco-2) were purchased from (Guangzhou IGE Biotechnology Company, Guangzhou) and cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum, 50 U /mL penicillin and 50 mg/mL streptomycin (Gibco, catalog number 15140-122). A murine endothelial cell line (C166) was obtained from the American Biological Resource Center (ATCC, Manassas, USA), supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg of Dulbecco's modified Eagle's medium ( Gibco). Cells were cultured in a humidified 37°C incubator with 5% _CO . The following studies were approved by the Fifth Affiliated Hospital of Sun Yat-Sen University. For all animal experiments, the permission of the Experimental Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University was obtained. Statistical analysis: Statistical analysis was performed using SPSS v13.0 software. Mean values and errors were calculated for statistical analysis, and all data were analyzed using GraphPad Prism software (version 5.0). Differences between groups were assessed using t-tests when only two groups were analyzed; differences between groups were assessed by paired t-tests when comparing quantitative WB. Pearson correlation analysis was performed to analyze the correlation between protein expressions, and P<0.05 was considered to be statistically significant.

one, SARS-CoV-2 Spike Protein preferentially induces duodenal interstitial edema

1. H&E staining and immunofluorescence staining

(1) Tissue sections: Clinical specimens from COVID-19 patients were formalin-fixed, paraffin-embedded and sectioned (4 µm).

(2) Immunofluorescence staining

Sections need to be placed in 10% goat serum prepared in PBST, incubated at room temperature for 1 hour, and then added with primary antibodies (anti-ACE2, Santa Cruz, sc390851, 1:100; anti-SARS-Cov-2 Spike, Sino Biological, 40150-R007, 1:500), the sections were incubated at 4°C overnight. The next day, wash 3 times with PBST and add fluorescent secondary antibodies (AlexaFluor® 647-conjugated goat anti-rabbit IgG, bs-0296G-AF647, Bioss, 1:100; Dylight-550 goat anti-rabbit IgG secondary antibody BA1135, 1:200 ) at room temperature for 1 h. Nuclei were then counterstained with 4',6-diamidino-2-phenylindole (DAPI) after 3 washes with PBST. Slides were imaged using a laser scanning confocal microscope (LSM880, Carl Zeiss Microscopy).

RESULTS: Using double immunofluorescence staining, co-localization of ACE2 and SARS-CoV-2 Spike protein was observed in the duodenum in gastrointestinal tissues obtained by endoscopy from patients with COVID-19, whereas in the duodenum Spike proteins were detectable in the denum (Fig. 1a). H&E staining further showed marked edema of the mucosal lamina propria interstitium, local inflammation with plasma cell and lymphocyte infiltration (Fig. 1b). Importantly, interstitial edema was significantly associated with disease type, acid reflux, total bilirubin, glutamate-pyruvate aminotransferase (ALT) and aspartate aminotransferase (AST) (Table 2), suggesting progression of the disease prediction features.

two, SARS-CoV-2 Construction of an animal model of gastrointestinal inflammation

All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised in 1996) and the "Ethical Guidelines for Animal Experiment Institutions" formulated by the Fifth Affiliated Hospital of Sun Yat-sen University. Fifty-four C57BL/6J female and male mice (7-8 weeks old) were placed in a light/dark cycle (14/10 h), constant temperature (26°C) environment with ad libitum access to food and water.

1. The construction process of SARS-CoV-2 gastrointestinal inflammation animal model, including the following steps:

(1) 8-9 weeks old C57BL/6J mice were randomly divided into two groups (experimental group and control group); (2) all mice were fasted for 24 hours; (3) after fasting for 24 hours, the abdominal cavity of each mouse was Inject 100 μL of 1% pentobarbital for anesthesia; (4) After 5 minutes of anesthesia, apply petroleum jelly on the surface of the hose, and then gently insert it from the anus to a depth of 4 cm; (5) Connect a syringe to one end of the hose and inject 500 μL of 1% acetic acid, and the control group was injected with an equal volume of PBS; (6) the mice were inverted for 1 min, and then lavaged with 500 μL of PBS twice to wash off the injected acetic acid; (7) The mice were put back into the cage and supplemented with food; (8) 16-24 hours later, the S protein (SARS-CoV-2 Spike protein) 5μg/mice (or 5.5 nmol/kg) was intraperitoneally injected, and the control group was injected with the isotype control Mouse IgG1-Fc protein; (9) After 6 hours, the mice were sacrificed, and the duodenum, colon, jejunum and rectum tissues were taken, embedded and dehydrated, and sliced for HE staining; and immunofluorescence staining was performed to investigate the co-localization of ACE2 and SARS-CoV-2. .

2. Co-immunoprecipitation

Spike was co-immunoprecipitated with interacting proteins using the Dynabeads Protein G Immunoprecipitation Kit (ThermoFisher).

Results: As can be seen from Figure 2b and Table 3, the inflammation caused by Spike-Fc was mainly in the duodenum, and there was no significant change in the jejunum, colon or rectum. It can be seen from Figure 2c and Figure 2d: Spike-Fc Fc and ACE2 achieved co-localization in the duodenum, and no significant changes were found in the jejunum, colon or rectum. These phenomena are consistent with existing reported clinical symptoms of COVID-19 patients, indicating that the SARS-CoV-2 gastrointestinal inflammation animal model constructed by the above method can be used to replicate the gastrointestinal manifestations of clinical COVID-19 patients.

Table 3 Changes of tissue inflammation and interstitial edema after SARS-CoV-2 Spike protein acts on mouse intestinal tissue

three, SARS-CoV-2 Spike disrupt the intestinal vascular barrier

1. In vivo permeability measurement

Experimental procedure: SARS-CoV-2 gastrointestinal inflammation model mice and control groups (Mouse IgG1-Fc protein) was injected into the tail vein of TRITC-dextran, and after half an hour, the peritoneal cavity was washed with 2.5 mL of PBS; after taking the ascites, the ascites was centrifuged at 1500 rpm for 10 min, and the excitation and emission wavelengths were 540 nm and 590 using a microplate reader, respectively. Fluorescence was measured at nm; duodenal, jejunum, colon and rectal tissues were taken after the mice were sacrificed, embedded and dehydrated, and then sliced and stained with H&E.

Results: Following Spike protein delivery, mice were intravenously injected with TRITC-dextran, followed by quantitative detection of dextran leaking into the abdominal cavity. Greater leakage was observed in mice treated with Spike-Fc compared to controls (Control-Fc) (Fig. 3a), suggesting that Spike proteins can cause impairment of the intestinal vascular barrier.

2. In vitro permeability assay

ACE2 has been reported to be expressed in various cell types including endothelial cells, so here we first investigated whether Spike proteins could mediate permeability by directly affecting the endothelium. We incubated human umbilical vein endothelial cells (HUVEC) with Spike-Fc and assessed permeability by dextran permeability assay as follows:

(1) The endothelial cells were seeded in the upper chamber of a transwell chamber (0.4 μm, Corning) at a density of 2.5×10 ⁵ per well, so that the cells were distributed in a monolayer; (2) 700ul ECM containing 5% serum was added to the lower chamber Culture medium, 37°C, 5% CO ₂ for 24 hours; (3) Then add 200ul of 70-KDa TRITC-dextran with a final concentration of 2 mg/ml to the upper chamber of the transwell chamber; (4) Remove the upper chamber after 5 hours , respectively, take 100ul of culture medium from the lower chamber and seed it into a 96-well plate; (5) detect with a multi-function microplate detector (excitation wavelength 540nm, absorption wavelength 590nm).

Results: Spike protein did not significantly affect the permeability of HUVEC cells (Fig. 3b). According to previous reports, ACE2 is the main binding receptor of SARS-CoV-2. ACE2 is expressed in a variety of organs, including the digestive and vascular systems. There are also two studies using human intestinal-like organoids and a colon epithelial cancer cell line (Caco-2) to demonstrate that SARS-CoV-2 replicates in intestinal epithelial cells using ACE2 as an entry receptor, while the in vivo permeability experiments described above give Therefore, it is reasonable to speculate that Spike protein induces endothelial permeability by affecting intestinal epithelial cells.

Next, HUVEC cells were incubated with conditioned medium of Spike-Fc-treated intestinal epithelial cells Caco-2, as determined by permeability, using Spike-Fc (0.25 mg/mL) for Caco-2 (5× 10 ⁵ ), the control group was treated with IgG-Fc (0.25mg/mL) for 24h, and then the cultured Caco-2 cell supernatant was filtered through a 0.22 μm filter. HUVEC cells (1 x ¹⁰⁵ ) were plated in a monolayer in the upper chamber of a 24-well plate overnight. After the incubation time required for cell adhesion, the wells were pipetted into the same 24-well plate, medium with 1% FBS was added until the HUVECs reached confluency for 6 h, and then the filtered Caco-2 supernatant was replaced to the bottom of the Transwell chamber and co-cultured with HUVEC for 12 h. To test the permeability of HUVECs in vitro, the upper chamber medium was removed, and tetramethylrhodamine isothiocyanate-Dextran (T1162, Sigma) (2 mg/mL) was added to the upper wells. After 3 hours, the Dextran-added cells were collected. culture medium, and fluorescence was measured at excitation and emission wavelengths of 540 nm and 590 nm, respectively, using a microplate reader.

RESULTS: Significant infiltration of endothelial cells exposed to Spike protein-stimulated Caco-2 supernatants was detected (Fig. 3e).

3. SARS-CoV-2 Spike protein binding assay

Recombinant SARS-CoV-2 Spike protein (RBD, Fc tag) was purchased from Sino Bioloical. For in vitro binding assays, Caco-2 cells and cells were treated with Spike-Fc (0.25 mg/mL) or control, respectively IgG-Fc (0.25 mg/mL) was incubated at 37 °C for 1 h, and then the protein in the lysate was pulled down with Protein G Sepharose for immunoblotting. in Caco-2 Neutralized with Spike-Fc (0.25mg /mL) or control IgG-Fc (0.25mg /mL) at 37°C for 1 h or 24 h for downstream molecular analysis. Cell lysates were analyzed by western blot and quantitative PCR, and cell culture supernatants (24 h) were filtered through 0.22 μm filters (MILLEX GP) and stored at -80°C for Elisa and co-culture experiments.

Results: The binding of Spike-Fc protein to human ACE2 was confirmed by pull-down assay (Fig. 4a) and immunofluorescence staining (Fig. 4b). The above experimental data indicate that Spike protein can bind to ACE2 on intestinal epithelial cells and may disrupt the intestinal vascular barrier system through the paracrine pathway.

Four, SARS-CoV-2 Spike Mechanisms of disruption of the intestinal vascular barrier

1. Immunohistochemistry

For tissue samples, immunohistochemistry was performed to determine the expression levels of ERK, pERK, VE-cad and pVE-cad(Y731) proteins in the duodenum, jejunum, colon and rectum. Subsequently, HE staining was also performed on human/mouse tissue sections to assess tissue morphological characteristics and distribution of target proteins.

Results: Tight junctions and adherent junctions are the basic components of the intestinal vascular barrier. Changes in tight junction proteins such as ZO-1 were first analyzed, but no significant changes were found. However, the expression of a key adhesion protein, VE-cadherin (VE-cad), was reduced, accompanied by an increase in its phosphorylation at Tyr731 but not at 658 (pVE-cad) (Fig. 5a). Then, we measured the expression and phosphorylation levels of VE-cad in vivo. Different parts of the gastrointestinal tract of mice treated with Spike protein were analyzed by western blot and immunohistochemistry (IHC). The results showed a persistent decrease in VE-cad in the duodenum (Fig. 5b) and a persistent increase in pVE-cad (Fig. 5c). In addition, we also confirmed this change in the tissues of COVID-19 patients through experiments (Fig. 5d, Fig. 5e). The above results suggest that Spike promotes endothelial permeability by inhibiting VE-cad expression while increasing its phosphorylation.

five, SARS-CoV-2 Spike protein through ERK/VEGF pathway mediates vascular permeability

1. RNA isolation and qRT-PCR

Tissues were immediately excised from SARS-CoV-2 gastrointestinal inflammation model mice (including control and experimental groups) fixed in liquid nitrogen. Total RNA from tissues was isolated using TRIzol by conventional RNA extraction protocols. Total RNA from cells was isolated with Total RNA Kit I. The isolated RNA was reverse transcribed into cDNA (Vazyme, Nanjing, China). qRT-PCR was performed using a real-time PCR system (Bio-Rad, America) and ChamQ Universal SYBR qPCR master mix (Vazyme, Nanjing, China). Primers were designed and synthesized by Guangzhou IGE Biotechnology Company. GAPDH was used as an internal control and all reactions were repeated three times. Relative RNA expression ^was calculated using the 2 ^{- ΔΔCt} method.

2. ELISA

The concentration of VEGF produced from cells, tissues and serum was detected by ELISA. For cell culture supernatants, Quantikine ELISA human VEGF immunoassay (cat. No. DVE00, R&D) Determination of VEGF concentration. For mouse samples, use mouse VEGF according to the manufacturer's instructions Simplestep ELISA kit (cat. No. ab209882, Abcam) to measure VEGF levels in tissue homogenates (prepared in cold PBS using an electric homogenizer) and serum.

Results: In the duodenal tissue of Spike-treated mice, the expression of VEGF was significantly increased at both mRNA and protein levels (Fig. 6a, 6b). Notably, VEGF levels in the serum of these mice were not altered (Fig. 6d). The conclusion here is therefore consistent with the in vitro co-culture results that it is epithelial cells, but not endothelial cells, that produce more VEGF upon Spike stimulation (Fig. 6c).

VEGF, known as a potent vascular permeability factor, was upregulated in blood samples from COVID-19 patients. Studies using cellular models have shown that SARS-CoV-2 infection significantly increases VEGF expression in human lung epithelial cells. Based on these findings, we speculate that VEGF can be induced by viruses in enterocytes, which may be responsible for gastrointestinal symptoms in COVID-19 patients. The above experimental data verified our expectation.

3. Construction of Caco-2 cells knocking down ACE2, including the following steps:

(1) Caco-2 cells were attached to a six-well plate; (2) Lipofectamine was used LTX (Invitrogen, ThermoFisher Scientific Corporation) transfection reagent, and the negative control siRNA and ACE2 siRNA were transfected into Caco-2 cells; (3) After 48h, use RIPA buffer containing protease/phosphatase inhibitor mixture to lyse Caco-2, and extract the protein; (4) Western blot was used to detect the knockdown efficiency and the expression of related proteins.

Caco-2 was transfected with negative control siRNA and ACE2 siRNA for 48 hours using Lipofectamine LTX (Invitrogen, ThermoFisher Scientific), then Caco-2 was lysed using RIPA buffer containing a protease/phosphatase inhibitor cocktail in preparation for a western blot.

So far, we have shown that the intestinal epithelium is a producer of VEGF, which plays a key role in Spike-induced permeability. Activation of the ERK signaling pathway has been reported to lead to overexpression of VEGF in human colon cells. We validated this pathway in Caco-2 cells treated with Spike protein. Reliable ERK activation was detected by immunoblotting, including increases in total ERK and phosphorylated ERK (pERK) (Figure 7).

We detected ERK upstream molecules Ras, Raf and MEK in ACE2 knockdown Caco-2 cells. to ensure that it functions downstream of ACE2, thus clarifying the pathway (Figure 8a). Next, we examined each molecule in this pathway in Spike-treated Caco-2 cells and confirmed alterations in each (Fig. 9a). Furthermore, this change could be suppressed by using the ERK inhibitor SCH772984 (Fig. 9a). demonstrated that SCH772984 completely reversed overexpressed VEGF in Spike-treated Caco-2 cells (Figure 9b). Meanwhile, the ERK pathway in Spike-treated HUVEC cells remained unchanged (Fig. 8b). Importantly, alterations in ERK and pERK were also demonstrated in animal tissues (Fig. 9c) and biopsy specimens from COVID-19 patients (Figs. 9d, 9e). Taken together, these findings define a critical role for the ERK/VEGF pathway in Spike-mediated permeability.

6. Blocking ERK/VEGF Pathways can be rescued SARS-CoV-2 Spike Induced vascular hyperpermeability

1. In vitro cell permeability assay

Caco-2 (5×10 ⁵ ) was treated with Spike-Fc (0.25mg/mL), control group was treated with IgG-Fc (0.25mg/mL), Spike-Fc with SCH772984 (1uM), bevacizumab Bevacizumab (25ug/ml) cells were treated for 24h, and then the cultured Caco-2 cell supernatant was filtered through a 0.22 μm filter. A monolayer of HUVEC ( ¹ x 105) was plated in the upper chamber of a 24-well plate overnight. After the incubation time required for cell adhesion, the wells were pipetted into the same 24-well plate, medium with 1% FBS was added until the HUVEC cells reached confluency for 6 h, and then the filtered Caco-2 cell supernatant was added. The solution was changed to the bottom of the Transwell chamber and co-cultured with HUVEC cells for 12 h. To test the permeability of HUVEC cells in vitro, the upper chamber medium was removed, and tetramethylrhodamine isothiocyanate-Dextran (T1162, Sigma) (2 mg/mL) was added to the upper well. After 3 hours, the collection was added with Dextran of medium, and measured fluorescence using a microplate reader at excitation and emission wavelengths of 540 nm and 590 nm, respectively.

2. In vivo animal experiments, including the following steps:

(1) 8-9 weeks old C57BL/6J mice were randomly divided into six groups: control group, placebo group I, placebo group II, Spike protein group, SCH772984 group and bevacizumab group; (2) All mice were fasted for 24 hours; (3) After fasting for 24 hours, each mouse was anesthetized by intraperitoneal injection of 100 μL of 1% pentobarbital; (4) After 5 minutes of anesthesia, the surface of the hose was smeared with Vaseline, and then injected from the anus. Gently insert, the insertion depth is 4cm; (5) Connect a syringe to one end of the hose, inject 500 μL of 1% acetic acid, and the control group is injected with an equal volume of PBS; (6) Invert the mouse for 1 min, and then use 500 μL of PBS for Lavage, lavage twice, in order to wash off the injected acetic acid; (7) Put the mice back in the cage and supplement with food; (8) After 16-24 hours, the mice in the SCH772984 group were injected intraperitoneally at a dose of 50 mg/kg, The bevacizumab group was injected intraperitoneally at a dose of 5 mg/kg, the placebo group I mice were intraperitoneally injected with the solvent of SCH772984, the placebo group II mice were intraperitoneally injected with the bevacizumab solvent, and the control group did not. (9) After 1-2 hours, the SCH772984 group, the bevacizumab group, the placebo group I, and the placebo group II were given intraperitoneal injection of S protein (SARS-CoV-2 Spike protein) 5 μg / animal (or 5.5 μg) respectively. nmol/kg), the control group was injected with the same type control Mouse IgG1-Fc protein; (10) 1~2h, the mice in the SCH772984 group were again injected intraperitoneally at a dose of 50 mg/kg, the bevacizumab group was injected at a dose of 5 mg/kg, and the placebo group I was injected with SCH772984 (11) TRITC-dextran was injected into the tail vein after 4-6 hours, and 2.5ml of PBS was washed in the abdominal cavity after half an hour; (12) after taking the ascites, the ascites was rotated at 1500rpm. Centrifuge for 10 min, and use a microplate reader to measure the fluorescence at excitation and emission wavelengths of 540 nm and 590 nm, respectively; (13) The mice were sacrificed, and the duodenum, colon, jejunum, ileum, and rectum were taken, and some tissues were embedded, dehydrated, and sliced. After HE staining; and immunofluorescence staining experiments were performed to investigate the co-localization of ACE2 and SARS-CoV-2. The content of VEGF in some fresh tissues was detected by Elisa kit.

3. Immunofluorescence analysis

To detect endothelial adherent junctions, HUVECs were seeded into 15 mm glass bottom cell culture dishes (2.5×10 ⁵ ) and co-cultured with the supernatant of Caco-2 (treated with Spike-Fc and IgG-Fc, respectively) for 24 h. It was then fixed with 4% paraformaldehyde. Samples were stained sequentially with the following antibodies or fluorescent dyes: VE-Cadherin (Cat.No.44-1145G, ThermoFisher, 1:1,000), Dylight-488 Goat Anti-mouse IgG secondary antibody (Cat.No. BA1126, BOSTER, 1:200), Antifade Mounting Medium with DAPI (Cat. No. Ab104139, Abcam). The results were acquired with a confocal microscope, and the sample pictures were taken by a Zeiss 880 with a 60x objective lens, and image processing was performed using ImageJ software.

Results: To assess the efficacy of blocking the ERK/VEGF pathway in vascular barrier function, we introduced the ERK inhibitor SCH772984 and the anti-VEGF drug Bevacizumab, which is commonly used to treat cancer vascular permeability For high first-line drugs, enteroepithelial-endothelial cell co-cultures were performed separately. Both SCH772984 and bevacizumab significantly inhibited the internalization of VE-cad (Fig. 10a) and rescued the SARS-CoV-2 Spike Protein-induced osmosis (Fig. 10b). To confirm these findings in vivo, Spike-treated animals were administered SCH772984 or bevacizumab. The results showed that VE-Cad was restored (Fig. 10c), accompanied by a decrease in VEGF production with either drug treatment (Fig. 11a), most likely through ERK/ Downregulation of pERK (Fig. 11b). These data suggest that blocking the ERK/VEGF pathway restores the vascular barrier function disrupted by the Spike protein.

seven, SCH772984 or bevacizumab SARS-CoV-2 Spike protein-induced inflammation

1. Evans Blue test, including the following steps:

(1) The mice were fasted for 24 hours in advance; (2) 100ul of 1% pentobarbital was injected intraperitoneally into the mice; (3) After the mice were anesthetized for about 5 minutes, apply Vaseline to the surface of the tube, and the tube was removed from the anus. Gently insert it with a depth of 4cm; (4) The hose is connected to a syringe at one end, and 500ul of 1% acetic acid is injected; (5) After injection, the mouse is inverted for 1 min, and then lavaged with 1ml of PBS, and lavaged twice; ( 6) Put the mice back into the cage and supplement with food; (7) After 14 hours, the mice in the SCH772984 group were injected intraperitoneally according to the dosage of SCH772984: 50 mg/kg, and the mice in the bevacizumab group were intraperitoneally injected with the dosage of 5 mg/kg , the mice in the placebo group I were intraperitoneally injected with the solvent of SCH772984, and the mice in the placebo group II were injected with the solvent of bevacizumab, and the control group was not treated; (8) SCH772984 group, bevacizumab group and Placebo group I and placebo group II were intraperitoneally injected with S protein (SARS-CoV-2 Spike protein) 5 μg/mouse (or 5.5 nmol/kg), respectively, and the control group was injected with the isotype control Mouse IgG1-Fc protein; (9) 2 hours later The mice in the SCH772984 group were again injected intraperitoneally at a dose of 50 mg/kg, the bevacizumab group was injected at a dose of 5 mg/kg, the placebo group I was injected with the solvent of SCH772984, and the placebo group II was injected with bevacizumab The solvent of monoclonal antibody; (10) 4 hours later, evens blue dye was injected into the tail vein, and the injection amount was 25 mg/kg, and the mice were sacrificed after half an hour; (11) duodenum, jejunum, ileum, colon and rectum were collected after the mice were sacrificed. , take pictures; (12) Weigh the mouse tissue after taking pictures, add formamide in an amount of 1ml/100mg, 50°C, shake in an incubator for 48h; (13) 8000g, centrifuge for 10min, take 100ul of supernatant The OD value of the solution was measured at 620 nm.

Results: To investigate the potential of targeting the ERK/VEGF pathway in a preclinical setting to improve SARS-CoV-2-induced permeability and inflammation, the gastrointestinal tract following SCH772984 or bevacizumab treatment was first identified by Evans Blue assay. Permeability changes. Evans infiltrated into duodenal tissue from SCH772984 or bevacizumab treatment groups The amount of Blue dye was almost the same as that of the control group (Fig. 12a left). As expected, no significant effects were detected in either the jejunum, colon or rectum (Fig. 12b). Pathologically, duodenal inflammation was reduced with SCH772984 or bevacizumab treatment, reflecting a stronger barrier (Fig. 12c). The above results suggest that ERK/VEGF inhibition rescues the damaged vascular barrier and secondary inflammation in the gastrointestinal tract induced by the SARS-CoV-2 Spike protein, thereby highlighting a novel approach for the treatment of gastrointestinal symptoms in patients with COVID-19. Strategy.

Claims (19)

1. A method of inhibiting SARS-CoV-2-induced damage to the vascular barrier in gastrointestinal tissue, comprising administering to the gastrointestinal tissue a vascular rejection targeting signal.
2. The method for inhibiting vascular barrier damage in gastrointestinal tract tissue caused by SARS-CoV-2 according to claim 1, wherein the repulsive guidance signal comprises an ERK inhibitor and/or a VEGF antagonist, and the An ERK inhibitor refers to a substance that can bind to ERK and block the biological activity of ERK, and the VEGF antagonist refers to a substance that can bind to VEGF and block the biological activity of VEGF.
3. The method for inhibiting vascular barrier damage in gastrointestinal tract tissue caused by SARS-CoV-2 according to claim 2, wherein the ERK inhibitors include but are not limited to SCH772984, GDC-0994, Ulixertinib, KO- 947, LY3214996, MK-8353, CC-90003, LTT462, FR180204, VTX-11e, BL-EI-001.
4. The method for inhibiting vascular barrier damage in gastrointestinal tissue caused by SARS-CoV-2 according to claim 2, wherein the VEGF antagonists include but are not limited to bevacizumab, ramucirumab Antibiotic, ranibizumab, aflibercept, conbercept.
5. The method for inhibiting damage to the vascular barrier in gastrointestinal tract tissue caused by SARS-CoV-2 according to any one of claims 1 to 4, wherein the gastrointestinal tract tissue is duodenum.
6. A method of screening or evaluating an agent that inhibits SARS-CoV-2-induced impairment of the vascular barrier in gastrointestinal tissue, characterized by determining the ability of the agent to affect VEGF-mediated increase in vascular permeability of the gastrointestinal tract.
7. The method for screening or evaluating an agent that inhibits vascular barrier damage in gastrointestinal tract tissue caused by SARS-CoV-2 according to claim 6, wherein the agent is determined to affect VEGF-mediated vascular access in the gastrointestinal tract The ability to increase permeability refers to determining the ability of the agent to inhibit ERK, MEK, Ras, Raf, VEGF.
8. The method for screening or evaluating an agent that inhibits SARS-CoV-2-induced damage to the vascular barrier in gastrointestinal tissue according to claim 7, wherein the agent affects VEGF-mediated vascular permeability of the gastrointestinal tract Increased sexuality is determined by the following steps:

   (1) Prepare a candidate agent group, which consists of secretions of intestinal epithelial cells exposed to SARS-CoV-2 Spike protein, endothelial cells and candidate agents;

   (2) Prepare a placebo group, which consists of secretions of intestinal epithelial cells exposed to SARS-CoV-2 Spike protein, endothelial cells and placebo;

   (3) Among them, the endothelial cells in the candidate drug group can detect higher VE-cad expression or lower VE-cad phosphorylation level than the endothelial cells in the placebo group, indicating that the candidate drug can Inhibits VEGF-mediated increase in gastrointestinal vascular permeability.
9. The method for screening or evaluating an agent that inhibits SARS-CoV-2-induced damage to the vascular barrier in gastrointestinal tissue according to claim 7, wherein the agent affects VEGF-mediated vascular permeability of the gastrointestinal tract Increased sexuality is determined by the following steps:

   (1) Construct an animal model of SARS-CoV-2 gastrointestinal inflammation;

   (2) Intraperitoneal injection or no placebo injection to the model animals of (1);

   (3) Inject the candidate drug into the model animal of (1) by intraperitoneal injection;

   (4) wherein, a lower expression level of VEGF can be detected in the gastrointestinal tract tissue of the animals after (3) treatment than in the gastrointestinal tract tissue of the animals after treatment (2), indicating that the agent can Inhibits VEGF-mediated increase in gastrointestinal vascular permeability.
10. The method for screening or evaluating agents for inhibiting vascular barrier damage in gastrointestinal tract tissue caused by SARS-CoV-2 according to claim 9, wherein the SARS-CoV-2 gastrointestinal inflammation in step (1) The construction method of the animal model includes the following steps:

    S1. Mice were anesthetized after fasting for 16-24 hours;

    S2. Inject acetic acid from the anus of the mouse, turn the mouse upside down, and lavage the gastrointestinal tract of the mouse;

    S3. After a period of time, mice were intraperitoneally injected with SARS-CoV-2 Spike protein;

    S4. The animal model of SARS-CoV-2 gastrointestinal inflammation was successfully obtained after a few hours.
11. The method for screening or evaluating an agent that inhibits SARS-CoV-2-induced vascular barrier damage in gastrointestinal tissue according to claim 10, wherein the agent affects VEGF-mediated gastrointestinal vascular permeability Increased sexuality is determined by the following steps:

    (1) Mice were anesthetized after fasting for 16-24 hours;

    (2) Inject acetic acid from the anus of the mouse, turn the mouse upside down, and lavage the gastrointestinal tract of the mouse;

    (3) The mice were fed normally. After 16-24 hours, the candidate drugs were injected into the mice through the abdominal cavity respectively to obtain the mice in the candidate drug group; the placebo was injected into the mice through the abdominal cavity respectively to obtain the mice in the placebo group. ;

    (4) After 1-2 hours, the mice in the candidate agent group and the placebo group were intraperitoneally injected with SARS-CoV-2 Spike protein respectively, and the mice in the control group were intraperitoneally injected with the same amount of IgG-Fc protein;

    (5) After 1-2 hours, the candidate drug was injected intraperitoneally to the mice in the candidate drug group again, and the placebo was intraperitoneally injected to the mice in the placebo group;

    (6) The gastrointestinal tissues of the mice in the control group, the mice in the candidate drug group and the mice in the placebo group were taken respectively. Compared with the mice in the control group, the gastrointestinal tissues of the mice in the placebo group were able to detect higher Compared with the mice in the placebo group, the gastrointestinal tissue of the candidate drug group could detect a lower VEGF expression level, indicating that the candidate drug can inhibit the VEGF-mediated gastrointestinal vascular permeability Increase.
12. The method for screening or evaluating agents for inhibiting vascular barrier damage in gastrointestinal tract tissue caused by SARS-CoV-2 according to claim 11, wherein the concentration of acetic acid in step (2) is 1-2% v/ v, the volume is 500 μL to 1000 μL.
13. The method for screening or evaluating a drug that inhibits vascular barrier damage in gastrointestinal tract tissue caused by SARS-CoV-2 according to claim 12, wherein the step (4) of the SARS-CoV-2 Spike protein The dosage was 5.5nM/kg mouse body weight.
14. The method for screening or evaluating an agent that inhibits damage to the vascular barrier in gastrointestinal tract tissue caused by SARS-CoV-2 according to any one of claims 6 to 13, wherein the gastrointestinal tract tissue is twelve the colon.
15. A method for treating or preventing gastrointestinal symptoms caused by SARS-CoV-2, characterized by comprising the following steps:

    (1) Identifying subjects with gastrointestinal symptoms caused by said SARS-CoV-2;

    (2) administering to the gastrointestinal tract of a subject a repulsive targeting signal that binds to ACE2 and/or ERK and/or VEGF.
16. The method for treating or preventing gastrointestinal symptoms caused by SARS-CoV-2 according to claim 15, wherein the rejection-directed signal comprises an ERK inhibitor and/or a VEGF antagonist, and the ERK inhibitor It refers to a substance that can bind to ERK and block the biological activity of ERK, and the VEGF antagonist refers to a substance that can bind to VEGF and block the biological activity of VEGF.
17. The method for treating or preventing gastrointestinal symptoms caused by SARS-CoV-2 according to claim 16, wherein the ERK inhibitors include but are not limited to SCH772984, GDC-0994, Ulixertinib, KO-947, LY3214996 , MK-8353, CC-90003, LTT462, FR180204, VTX-11e, BL-EI-001.
18. The method for treating or preventing gastrointestinal symptoms caused by SARS-CoV-2 according to claim 16, wherein the VEGF antagonists include but are not limited to bevacizumab, ramucirumab, Zizumab, Aflibercept, Conbercept.
19. A drug for treating or preventing or alleviating gastrointestinal symptoms caused by SARS-CoV-2, characterized in that the drug has at least one of the following functions:

    (1) Inhibit the expression of VEGF in intestinal epithelial cells;

    (2) Inhibit the expression of ERK in intestinal epithelial cells;

    (3) Rescue SARS-CoV-2 Spike protein-induced inhibition of endothelial VE-cad expression or up-regulation of VE-cad phosphorylation.