CN108420811B - Application of the lipid-lowering medicine in terms of hyperhomocysteinemiainjury treatment - Google Patents

Abstract

The invention discloses the application of a lipid-lowering drug in the treatment of hyperhomocysteinemia. The study found that HHcy can significantly promote the expression levels of autophagy and plasmacytization-related genes and proteins in B lymphocytes, while autophagy and plasmacytization inhibitory factors are down-regulated, indicating that HHcy can stimulate autophagy and plasmacytization of B lymphocytes. Pretreatment of B lymphocytes with lipid-lowering drugs can reverse the up-regulation of autophagy and plasmacytization-related genes and proteins and the down-regulation of related inhibitors caused by HHcy. The study also found that HHcy can significantly promote the secretion of anti-β2GPI by B lymphocytes, and pretreatment of B lymphocytes with lipid-lowering drugs can reverse the promoting effect of HHcy on the secretion of anti-β2GPI by B lymphocytes. anti-β2GPI is an antiphospholipid antibody, and its elevation will lead to an increase in the level of antiphospholipid antibody in the blood, which has the risk of inducing antiphospholipid syndrome, and the intervention of lipid-lowering drugs can prevent this risk. Lipid-lowering drugs can also reduce the inflammatory activation of macrophages by anti-β2GPI by inhibiting the secretion of anti-β2GPI by B lymphocytes caused by hyperhomocysteinemia.

Description

Application of lipid-lowering drugs in the treatment of hyperhomocysteinemia

technical field

The invention belongs to the field of medicine and relates to the application of lipid-lowering drugs in the treatment of hyperhomocysteinemia.

Background technique

Homocysteine (Hcy) is an intermediate product of methionine metabolism in the body, and it is a non-essential amino acid containing a sulfhydryl group. Abnormal methionine metabolism will lead to the accumulation of Hcy, an intermediate metabolite in the blood. If the level of Hcy in the blood rises above 10 μM, it is defined as hyperhomocysteinemia (HHcy).

Studies have shown that HHcy is related to many diseases including cardiovascular diseases.

Contents of the invention

The purpose of the present invention is to provide the application of the lipid-lowering drug in the treatment of hyperhomocysteinemia.

Above-mentioned purpose of the present invention is achieved by following technical scheme:

Application of the lipid-lowering drug in the preparation of a drug for inhibiting autophagy of B lymphocytes caused by hyperhomocysteinemia. In vivo and in vitro studies have found that HHcy can significantly promote the expression levels of autophagy-related genes and proteins in B lymphocytes, and autophagy inhibitory factors are significantly down-regulated, which indicates that HHcy can stimulate and promote autophagy in B lymphocytes in vivo and in vitro. The lipid-lowering drug fenofibrate pretreatment of B lymphocytes can reverse the up-regulation of autophagy-related genes and proteins and the down-regulation of autophagy inhibitors caused by HHcy.

Application of the lipid-lowering drug in the preparation of a drug for inhibiting plasmacytization of B lymphocytes caused by hyperhomocysteinemia. Those skilled in the art know that when activated, B lymphocytes undergo immunoglobulin type transformation and recombination, and finally differentiate into plasma cells. BLIMP1 and IRF4 have the effect of promoting plasmacytosis, and the molecules that inhibit B lymphocyte plasma cells include BCL6 and PAX5; the plasmacytization of B lymphocytes can be reflected by measuring the expression levels of BLIMP1, IRF4, BCL6 and PAX5 in B lymphocytes degree. In vivo and in vitro studies have found that HHcy can significantly promote the expression levels of B lymphocyte plasmacytization-related genes and proteins, while plasmacytization inhibitors are significantly down-regulated, which indicates that HHcy can stimulate and promote B lymphocyte plasmacytization in vivo and in vitro. Pretreatment of B lymphocytes with the lipid-lowering drug fenofibrate can reverse the up-regulation of plasmacytization-related genes and proteins and the down-regulation of plasmacytization inhibitory factors induced by HHcy.

Application of the lipid-lowering drug in the preparation of a drug for inhibiting the secretion of anti-β2GPI by B lymphocytes caused by hyperhomocysteinemia. In vivo and in vitro studies have found that HHcy can significantly promote the secretion of anti-β2GPI by B lymphocytes, and pretreatment of B lymphocytes with the lipid-lowering drug fenofibrate can reverse the promoting effect of HHcy on the secretion of anti-β2GPI by B lymphocytes.

Application of the lipid-lowering drug in the preparation of a drug that inhibits the secretion of anti-β2GPI by B lymphocytes caused by hyperhomocysteinemia, thereby increasing the level of antiphospholipid antibodies in blood. Anti-β2GPI is a typical antiphospholipid antibody (APL), and the increase of anti-β2GPI will lead to a significant increase in the level of antiphospholipid antibody in the blood. Those skilled in the art know that excessive APL can cause a kind of autoimmune disease antiphospholipid syndrome (APS). That is to say, for HHcy patients, the increased anti-β2GPI secretion level of B lymphocytes leads to excessive APL in the blood, which has the risk of causing APS autoimmune diseases, and the intervention of lipid-lowering drug fenofibrate can prevent this risk.

Application of the lipid-lowering drug in the preparation of a drug that inhibits the secretion of anti-β2GPI by B lymphocytes caused by hyperhomocysteinemia and promotes the inflammatory activation of macrophages. The results of the study found that anti-β2GPI IgG can directly promote the polarization of macrophage M1 and inhibit the polarization of anti-inflammatory M2, promote the secretion of inflammatory factors by macrophages, and trigger or aggravate inflammation in the body. According to the above results, the lipid-lowering drug fenofibrate can inhibit the secretion of anti-β2GPI by B lymphocytes, so those skilled in the art can know that lipid-lowering drugs can inhibit the secretion of anti-β2GPI by B lymphocytes caused by hyperhomocysteinemia. β2GPI reduces the inflammatory activation effect of anti-β2GPI on macrophages.

In a specific embodiment, the lipid-lowering drug is fenofibrate.

Description of drawings

Figure 1: HHcy stimulates the body to produce APL IgG and anti-β2GPI IgG. Homocysteine drinking water feeding (1.8g/l) caused HHcy model in C57BL/6J mice. ELISA detection of mouse plasma APL (A), anti-β2GPI (B) and ACL (C) IgG levels. n=3-5. *Compared with the control group; P<0.05 means significant difference.

Figure 2: Hcy stimulates B lymphocytes to produce APL IgG and anti-β2GPI IgG. Hcy stimulated spleen B lymphocytes of C57BL/6J mice for 72 hours. ELISA was used to detect the levels of APL (A), anti-β2GPI (B) and ACL (C) IgG in the culture supernatant of B lymphocytes after Hcy stimulation. n=6. *Compared with the control group; P<0.05 means significant difference.

Figure 3: HHcy promotes plasmacytization of mouse B lymphocytes. After C57BL/6J mice were given normal drinking water or Hcy (1.8g/l) drinking water for two weeks, spleen B lymphocytes were isolated for detection. (A) Western blot was used to detect the expression levels of BLIMP1 and IRF4 proteins in B lymphocytes; eIF5 was used as an internal reference. n=3. (B) Quantitative PCR detection of the expression levels of Pax5, Irf4 and Blimp1 genes in B lymphocytes. n=3-5. *Compared with the control group; P<0.05 significantly different.

Figure 4: Hcy promotes plasmacytization of mouse spleen B lymphocytes. Spleen B lymphocytes were isolated from C57BL/6J mice and stimulated with Hcy for 48 hours. (A) The expression levels of BLIMP1 and IRF4 proteins in B lymphocytes were detected by Western blot, and eIF5 was used as an internal reference. n=3. (B) Quantitative PCR detection of the expression levels of Pax5, Irf4 and Blimp1 genes in B lymphocytes. n=3-5. *Compared with the control group; P<0.05 means significant difference.

Figure 5: HHcy promotes autophagy of mouse spleen B lymphocytes. C57BL/6J mice were given normal drinking water or Hcy (1.8g/l) drinking water for two weeks, and the spleen B lymphocytes were isolated for detection. (A) The expression levels of BLIMP1, IRF4, p62 and LC3II proteins in B lymphocytes were detected by Western blot, and eIF5 was used as an internal reference. n=3. (B) Quantitative PCR detection of the expression levels of Blimp1, Irf4, Lc3ii, Atg5, Atg7, Atg9 and Atg12 genes in B lymphocytes. n=3-5. * Compared with the control group; P<0.05 means significant difference.

Figure 6: Hcy promotes autophagy in mouse spleen B lymphocytes. Spleen B lymphocytes of C57BL/6J mice were isolated and stimulated with Hcy for 48 hours. (A) The expression levels of BLIMP1, IRF4, p62 and LC3II proteins in B lymphocytes were detected by Western blot, and eIF5 was used as an internal reference. n=3. (B) Quantitative PCR detection of Blimp1, Irf4, Lc3ii, Atg5, Atg7, Atg9 and Atg12 gene expression levels in B lymphocytes. n=3-5. *Compared with the control group; P<0.05 means significant difference.

Figure 7: The lipid-lowering drug fenofibrate inhibits plasmacytization, autophagy and anti-β2GPI IgG secretion of B lymphocytes. Spleen B lymphocytes of C57BL/6J mice were isolated, and after B lymphocytes were pretreated with fenofibrate (10 μM) for 30 minutes, they were stimulated with Hcy (100 μM) for 48 hours or 72 hours. (A) After Hcy (100 μM) stimulation for 48 hours, the expression levels of BLIMP1, IRF4, p62 and LC3II proteins in B lymphocytes were detected by Western blot, and eIF5 was used as an internal reference. n=3. (B) After Hcy (100 μM) stimulated B lymphocytes for 72 hours, the anti-β2GPIIgG level in the cell culture supernatant was detected by ELISA. n=6. *compared with the control group; #compared with the Hcy group; P<0.05 has a significant difference.

Figure 8: Anti-β2GPI activates macrophages. Anti-β2GPI (1 ng/μl) stimulated macrophages for 24 hours. (A) Immunofluorescent staining to detect the distribution of β2-GPI (red) and Hochest33324 (blue) on macrophages. n=6. (B) Quantitative PCR detection of genes associated with M1 class transformation of macrophages. (C) Quantitative PCR detection of genes associated with M2 class transformation of macrophage cells. n=6. *Compared with the control group; P<0.05 means significant difference.

Figure 9: Anti-β2GPI activates macrophages to secrete inflammatory factors. Anti-?2GPI (1 ng/µl) stimulated macrophages for 24 hours. CBA detection of mouse inflammation test to detect the secretion of inflammatory factors such as TNF-α, MCP1, IL-6 and IL-10 in macrophages. n=6. *Compared with the control group. P<0.05 means significant difference.

Detailed ways

The following describes the substantive content of the present invention in detail in conjunction with the drawings and embodiments, but does not limit the protection scope of the present invention.

1. Experimental materials

Anti-mouse LC3II, BLIMP1, IRF4, Phospho-DRP1 (Ser637): American CST Company; anti-mouse eIF5 antibody: American Santa Cruz Biotechnology Company; anti-mouse p62 antibody: Japan MBL International Company; Anti-: American Rockland Company; anti-mouse APOH antibody: American Biossantibodies Company; horseradish enzyme-labeled goat anti-rabbit IgG (H+L): Zhongshan Jinqiao Technology Co., Ltd.

Mouse anticardiolipin antibody IgG (ACA-IgG) detection kit: Shanghai Guduo Biotechnology Co., Ltd.; mouse β2 glycoprotein I antibody IgG (anti-β2GPIIgG): Shanghai Guduo Biotechnology Co., Ltd.; mouse antiphospholipid Antibody (AplAPA): Shanghai Guduo Biotechnology Co., Ltd.; beta2-GlycoproteinI IgG/IgM ELISAKit: Abnova Company of the United States; BD CBA Mouse Inflammation Detection Kit: BD Company of the United States; BCA Protein Quantification Kit: Pierce Company of the United States; Recording kit: American Promega Company.

2. Experimental method

1. Establishment of experimental animals and disease models

1) Experimental animals: We selected female mice aged 6-8 weeks for the experiment, among which C57BL/6J mice were provided by the Experimental Animal Center of Peking University Health Science Center, and all mice were raised in a specific pathogen-free (SPF) environment.

2) Hyperhomocysteinemia mouse model: To induce hyperhomocysteinemia in mice, we administered 6-week-old female C57BL/6J mice containing 1.8g/lDL-homocysteine The mice were fed with drinking water for four weeks to confirm that homocysteinemia was successfully induced.

3) All animal experiments were carried out in accordance with the guidelines of the Experimental Animal Ethics Branch of the Biomedical Ethics Committee of Peking University Health Science Center.

2. Spleen B lymphocyte extraction and culture

1) Preparation of spleen cell suspension: After the mice were killed by cervical dislocation, they were disinfected with 75% alcohol, and the abdominal cavity was quickly opened to separate the spleen and placed in PBS (4°C) for cells. The fascia, fat, and connective tissue around the spleen were peeled off, placed on a 300-mesh nylon mesh, PBS was added and the spleen was ground to prepare a single-cell suspension of the spleen.

2) Isolation of mononuclear cells: Transfer the single cell suspension of the spleen to a 15ml centrifuge tube, centrifuge (1000rpm×10min), discard the supernatant, and add 4°C pre-cooled red blood cells to lyse the red blood cells (add to each mouse spleen 3-5ml), centrifuged (1000rpm×10min), then resuspended and washed with PBS, and centrifuged (1000rpm×10min) to collect single cells.

3) Anti-CD19 magnetic beads positively sort B lymphocytes (take the isolation of 1×10 ⁸ spleen cells as an example): resuspend the isolated mononuclear cells in PBS to 1×10 ⁸ cells/900 μl, then add anti-CD19 magnetic beads After 100 μl, incubate in a refrigerator at 4°C for 18 minutes, then add 10 times the volume of PBS to stop the reaction, centrifuge (1000rpm×10min) and obtain CD19 ⁺ B lymphocytes by LS-positive magnetic column separation, and centrifuge again (1000rpm×10min) to obtain B lymphocytes.

4) Cultivation of B lymphocytes: resuspend the cells with RPMI-1640 medium containing 10% serum, count the cells, inoculate them in a cell culture plate at 3×10 ⁶ cells/ml, add LPS (final concentration is 0.1 μg/ml) ml) and cultured in a cell incubator with 5% CO ₂ at 37°C.

5) Hcy stimulation experiment: add Hcy (final concentration 100 μM) to the culture medium containing B cells, and stimulate for a specific time.

6) Addition of inhibitors or blocking agents: Add autophagy inhibitor 3-MA (final concentration: 10mM) to B cells half an hour before Hcy stimulation, and lipid-lowering drug fenofibrate (final concentration: 10μM) Incubate for half an hour.

3. Isolation of primary mouse peritoneal macrophages

1) Female 8-week-old C57BL/6J mice were intraperitoneally injected with 2ml of 4% thioglycolate broth.

2) After 3 days, the mice were killed by cervical dislocation, sterilized with 75% alcohol, injected with 8ml of peritoneal lavage solution containing 10% FBS in the lower abdomen, and massaged the abdomen for 3 minutes to fully lavage the peritoneal cavity.

3) Cut the skin to expose the abdominal cavity, and use a 10ml syringe to extract the peritoneal fluid.

4) Centrifuge the peritoneal fluid (1000rpm×5min), discard the supernatant; add 5ml of RPMI-1640 containing 10% FBS to wash the cells, centrifuge (1000rpm×5min), discard the supernatant.

5) Resuspend the cells with RPMI-1640 medium containing 10% serum, count the cells, inoculate 3×10 ⁶ cells/well in a cell culture 6-well plate (for protein level detection), and inoculate 1.5×10 ⁶ cells Each well was seeded in a cell culture 12-well plate (for gene level detection). Place them in a cell incubator with 5% CO ₂ at 37°C for culture, and replace them with fresh RPMI-1640 medium containing 10% serum after 2 hours of adherence, and give them to the treatment experiment after resting for 12 hours.

4. Extraction and quantification of cellular proteins

1) Extraction of total cell protein: collect the suspended B cells into a 1.5ml EP tube, centrifuge (4°C, 350g×10min), discard the supernatant, add PBS to wash the cells, and centrifuge again (4°C, 350g×10min) Finally, add whole cell lysate (40 μl for every 1×10 ⁷ B cells), and place on ice for 30 min until the cells are fully lysed. After centrifugation (4°C, 10000rpm×10min), transfer the supernatant to a new 0.6ml EP tube to obtain the total cell protein lysate.

2) Protein quantification: Use the BCA protein quantification kit from Pierce to configure albumin standard solutions (0, 50, 100, 200, 300, 400, 500, 1000 mg/ml) and add them to 96-well microtiter plates (each Well 25μl), the protein sample to be tested was diluted to 25μl with water (2.5μl sample + 22.5μl water) and then added to the microtiter plate, and finally 200μl of BCA working solution was added to each well (reagent A and reagent B were mixed at a concentration of 50:1) , and incubated at 37° C. for 30 min, with 570 nm as the measuring wavelength and 630 nm as the calibration wavelength, and using a microplate reader to measure and record the absorbance value. Use Prism6 software to make a standard curve according to the absorbance value of the standard protein, and then calculate the protein concentration of the sample according to the absorbance value of the sample.

5. Western blotting

1) Protein electrophoresis: configure a discontinuous SDS polyacrylamide gel (the upper layer is a 6% stacking gel, and the lower layer is a 10% separating gel), and use the Bio-Rad Mini-PROTEAN Tetra cell system to add protein samples (protein and 5× loading Buffer (mixed at a ratio of 1:4) and protein molecular weight marker for electrophoresis, 55V constant voltage electrophoresis until bromophenol blue reaches the separation gel interface, after the protein marker is initially separated, change the voltage to 110V, constant voltage electrophoresis until bromophenol blue reaches the separation gel Stop electrophoresis after the gel subsides the border.

2) Protein transfer: cut out a 7×5cm gel, use the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell system to electroporate the gel at a constant current (250mA) for 2 hours, and transfer it to a nitrocellulose membrane.

3) Immunoblotting: After transferring to the membrane, take out the blotted membrane and block it with 5% milk or BSA for 1 hour at room temperature, then add the primary antibody (1:1000 concentration dilution), and incubate overnight at 4°C in a shaker. Take out the blotted membrane the next day, wash the membrane with TBST (3 times × 10min), add the blotted membrane to IRDye700 or 800-linked fluorescent secondary antibody (1:10000-20000 dilution) and incubate at room temperature for 1 hour in the dark, then wash with TBST film (3 times x 10 min).

4) Protein imaging: using the Odyssey infrared imaging system, select the corresponding fluorescent channel to detect the fluorescent bands of the blotted membrane.

5) Image analysis: use Odyssey software, after removing the background, quantify the protein expression according to the gray value of the band, and export the corresponding image.

6. Total RNA extraction and quantification

1) Liquid phase separation of total RNA: collect suspended B cells into 1.5ml EP tube, centrifuge (4°C, 350g×10min), discard the supernatant, add PBS to wash the cells, and centrifuge again (4°C, 350g×10min ), add RNATRIzol (1ml per ¹ ×107 B cells), place on ice for 10min until the cells are fully lysed; add 200μl chloroform to the EP tube, mix vigorously by hand for 15s, place on ice to separate layers, Centrifuge (4°C, 13000rpm×15min); carefully transfer the upper liquid phase (about 600μl) to a new 1.5ml EP tube.

2) RNA precipitation: add an equal volume of isopropanol to the upper liquid phase, invert and mix well, place on ice for 15 min, and centrifuge (4° C., 13000 rpm×15 min).

3) RNA washing: discard the supernatant after centrifugation, add 1ml of 75% ethanol (diluted with DEPC water), invert and mix well and then centrifuge (4°C, 13000rpm×10min), discard the supernatant and let stand at room temperature to dry the RNA.

4) RNA dissolution and resuspension: add nuclease-free water to dissolve RNA (10-15 μl per 1×10 ⁷ B cells) to dissolve RNA precipitate.

5) Quantification of RNA: Aspirate DEPC water for measurement as a blank control, then aspirate 1 μl of each sample for concentration determination, and record RNA concentration and absorbance OD260nm/280nm.

7. Real-time quantitative PCR analysis

1) RNA reverse transcription: pipette 1.5 μg of total RNA into EP tubes, denature at 70°C for 10 minutes, place on ice, use the Promega reverse transcription system, add the following reagents to the EP tubes:

25mM _MgCl2 4μl

10× reverse transcription buffer 2 μl

10mM dNTP mix 2μl

Oligo(dT)15 primer 1μl

Recombinant reverse transcriptase inhibitor 0.5 μl

AMV reverse transcriptase 0.6 μl

Finally make up to a total volume of 20 μl with nuclease-free water.

Put the EP tube in a water bath at 42°C for 1 hour, denature at 100°C for 5 minutes, place it on ice, and store it at -20°C.

2) Design of PCR primers: PCR primers were designed according to Primer on the NCBI website, primers were screened using Oligo software, and finally specific primers were obtained by using Blast on the NCBI website to perform comparative analysis of primer specificity. The specific primer sequences are shown in the table below:

3) Quantitative PCR and data analysis:

The cDNA obtained by reverse transcription was tested according to the qPCR system. The system is as follows:

10× PCR buffer 2.5 μl

2.5mM dNTP mix 2μl

0.5 μl of 10 μM upstream primer

0.5 μl of 10 μM downstream primer

Reverse transcription reaction mix 5 μl

TaKaRaTaq 0.25 μl

SYRB Green 1.25μl

Make up to 25 μl with deionized water

Pre-denaturation at 94°C for 5min; (denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s) x 40 cycles; extension at 72°C for 5min. Data were collected and analyzed statistically using Stratagene Mx Pro software.

8. Determination of APL, ACA and anti-β2GPI IgG by ELISA

1) Sample collection: For cell supernatant samples, the isolated B lymphocytes (1×10 ⁶ cells/500 μl) were implanted into a 48-well cell culture plate, and the cell supernatant was collected after stimulation for a corresponding time; for plasma samples, collected Mouse blood, centrifuge (3000rpm×20min), transfer the plasma to a new EP tube, and store the sample at -80°C.

2) Experimental determination: using the mouse-specific APL antibody, ACL antibody and aβ2GPI IgG from Shanghai Guduo Biotechnology Co., Ltd.

3) ELISA kit for determination.

9. Statistical analysis

All experimental data were calculated using GraphPad Prism 6 software, and expressed as mean ± standard deviation (Mean ± SD). When comparing two groups of data, the Student's t test method was used; when comparing multiple groups of data, one-way analysis of variance was used, and Tukey's test was further used. P value <0.05 was taken as the criterion of significant difference.

3. Experimental results

1. High homocysteine promotes the secretion of anti-β2GPI by B lymphocytes

The HHcy model was induced by feeding 6-week-old C57BL/6J female mice with Hcy (1.8g/l) drinking water for 4 weeks. Then we detected the total antiphospholipid antibody (APL) produced by activated B lymphocytes and two typical antiphospholipid antibodies, anti-β2GPI and anticardiolipin antibody (ACL) IgG secretion levels. As shown in Figure 1, compared with the control group, the total APL IgG in the peripheral blood of HHcy mice increased significantly (3609.67±585.70mg/l rose to 8402.67±1014.18mg/l), and the anti-β2GPI IgG level also increased significantly (2.45 ±0.47mg/l rose to 5.82±1.63mg/l). However, ACL IgG levels did not change significantly. The results showed that HHcy promoted the production and secretion of APL and anti-β2GPIIgG in mouse B lymphocytes.

We further verified the role of HHcy in stimulating B lymphocytes to produce antiphospholipid antibodies in vitro. After isolating splenic B lymphocytes from C57BL/6J mice, they were stimulated with Hcy (100 μM) for 72 hours, and the anti-β2GPI IgG increased significantly (from 0.023±0.0039mg/l to 0.10±0.020mg/l) (B in Figure 2) . However, APL IgG and ACL IgG levels did not change compared with controls (A and C in Figure 2). The results further confirmed that HHcy promotes the production of anti-phospholipid antibodies in mouse B lymphocytes, especially the production and secretion of anti-β2GPI IgG.

2. High homocysteine promotes plasmacytization of B lymphocytes

Those skilled in the art know that when activated, B lymphocytes undergo immunoglobulin type transformation and recombination, and finally differentiate into plasma cells. BLIMP1 and IRF4 have the effect of promoting plasmacytosis, and the molecules that inhibit B lymphocyte plasma cells include BCL6 and PAX5; the plasmacytization of B lymphocytes can be reflected by measuring the expression levels of BLIMP1, IRF4, BCL6 and PAX5 in B lymphocytes degree.

As shown in A in Figure 3, HHcy significantly up-regulated the protein expression levels of BLIMP1 and IRF4 in splenic B lymphocytes in vivo. At the same time, the results of quantitative PCR in B in Figure 3 showed that the gene levels of Blimp1 and transcription factor interferon regulatory factor 4 (Irf4) in the spleen of HHcy mice were also significantly up-regulated. In addition, the gene Pax5, a transcription factor that inhibits plasmacytosis of B lymphocytes, showed a downtrend. The above results indicated that Hcy stimulation in vivo promoted the differentiation of B lymphocytes into plasma cells.

In vitro experiments, we further verified the plasmacytization effect of Hcy on B lymphocytes. As shown in A in Figure 4, after splenic B lymphocytes were isolated from C57BL/6J mice, and given Hcy (100 μM) stimulation for 48 hours, the protein expression levels of plasmacytization markers BLIMP1 and IRF4 expressed by B lymphocytes were significantly up-regulated. At the same time, the quantitative PCR results of B in Figure 4 showed that Hcy stimulated mouse spleen B lymphocytes in vitro, and the gene expression levels of plasmacytization markers Blimp1 and Irf4 were significantly up-regulated. In addition, the transcription factor Pax5 gene that inhibits B lymphocyte plasmacytization is consistent with the results of HHcy stimulation of B lymphocytes in vivo, showing a downward trend.

The above results suggest that HHcy significantly up-regulates the protein and gene expression levels of BLIMP1 and IRF4 in B lymphocytes in vitro and in vitro, and at the same time down-regulates the gene expression level of transcription factor Pax5, thereby promoting the plasmacytization of B lymphocytes.

3. High homocysteine promotes autophagy of B lymphocytes

Those skilled in the art know that the plasmacytization of B lymphocytes and the pathogenesis of antiphospholipid syndromes such as systemic lupus erythematosus depend on the autophagy of B lymphocytes.

As shown in Figure 5A, HHcy promotes plasmacytosis of B lymphocytes in vivo, that is, when the protein expression levels of BLIMP1 and IRF4 are significantly upregulated, the level of autophagy marker protein (LC3II) expressed by B lymphocytes is significantly upregulated; the protein level of p62 is significantly upregulated decline. At the same time, the quantitative PCR results in B in Figure 5 showed that the expression levels of Blimp1 and Irf4 genes related to plasmatization of splenic B lymphocytes stimulated by HHcy in vivo were significantly up-regulated; Pax5 gene was significantly down-regulated. The mRNA expressions of autophagy-related genes Lc3ii, Atg5, Atg7, Atg9, and Atg12 were significantly upregulated. These results indicate that HHcy stimulates plasmacytization of B lymphocytes and promotes autophagy of B lymphocytes.

In order to further verify the regulatory effect of HHcy on autophagy of B lymphocytes in vivo. After we isolated splenic B lymphocytes from C57BL/6J mice, they were stimulated with Hcy (100 μM) for 48 hours. As shown in Figure 6A, the protein expression levels of BLIMP1 and IRF4 in Hcy stimulated B lymphocytes were significantly up-regulated; spleen B lymphocytes The expressed autophagy marker protein (LC3II) protein level was significantly up-regulated; the autophagy substrate p62 protein level was significantly decreased. The quantitative PCR results of B in Figure 6 showed that Hcy-stimulated spleen B lymphocytes Blimp1 and Irf4 gene levels were significantly up-regulated, the inhibitory factor Pax5 gene expression was significantly down-regulated, and the mRNA expressions of autophagy-related genes Lc3ii, Atg5, Atg7, Atg9, and Atg12 level increased significantly. The above results further indicated that Hcy stimulated autophagy in B lymphocytes in vivo and in vitro.

4. The lipid-lowering drug fenofibrate blocks Hcy to up-regulate the plasmacytization, autophagy and anti-β2GPI secretion of B lymphocytes

As shown in Figure 7A, Western blotting results showed that pretreatment of B lymphocytes with fenofibrate effectively reversed the protein expression levels of plasmacytosis markers such as BLIMP1 and IRF4 in B lymphocytes that were up-regulated by Hcy stimulation. At the same time, both the up-regulation of autophagy marker protein (LC3II) protein expression level and the down-regulation of p62 protein level caused by Hcy stimulation were reversed. The ELISA results of B in Figure 7 showed that fenofibrate significantly reduced the production of anti-β2GPI IgG after Hcy stimulation of B lymphocytes for 72 hours (from 0.12±0.02mg/l to 0.05±0.01mg/l). These results indicate that lipid metabolism is involved in the process of Hcy promoting B lymphocyte plasmacytization and anti-β2GPI IgG secretion, and the lipid-lowering drug fenofibrate can block Hcy up-regulation of B lymphocyte plasmacytization, autophagy and anti-β2GPI secretion.

5. Anti-β2GPI IgG promotes the inflammatory polarization of macrophage M1 and inhibits its M2 anti-inflammatory polarization

Macrophages were stimulated with commercially available anti-β2GPI IgG, and the effect of the antibody on macrophage polarization was observed. As shown in Figure 8A, compared with the control group, laser confocal imaging observed the recruitment of β2GPI to the macrophage membrane on the surface of macrophages treated with anti-β2GPI IgG. The results in B in Figure 8 show that after antibody treatment of macrophages, the gene expression of various inflammatory cytokines such as Il-1β, Tnf-α, Mcp1 and Il-6 increased. The results of C in Figure 8 show that after antibody treatment of macrophages, the gene expressions of anti-inflammatory M2 macrophage marker molecules Arg-1, Mrc1, Mmgl2, Clec10a and Chi3l3 were significantly reduced. These results suggest that anti-β2GPI directly promotes macrophage M1 polarization and inhibits anti-inflammatory M2 polarization.

We also detected the effect of anti-β2GPI IgG on the secretion level of the first-related inflammatory factors after acting on macrophages. The results of flow CBA in Figure 9 show that anti-β2GPI IgG can effectively induce macrophage tumor necrosis factor (TNF-α), monocyte chemoattractant protein-1 (MCP1), interleukin-6 (IL- 6) and the secretion of inflammatory factors such as interleukin-10 (IL-10).

The purpose of the above-mentioned embodiments is to specifically introduce the substantive content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims (1)

1. fenofibrate is suffered from due to anti-phospholipid antibody level increases in blood in preparation prevention and treatment Patients with Hyperhomocysteine Suffer from the application in the drug of autoimmune disease antiphospholipid syndrome.