CN112076309A - Application of cyclic erythropoietin-derived peptide in kidney injury and cyclosporin A injury protection - Google Patents

Abstract

The invention discloses an application of cyclic erythropoietin-derived peptide in kidney injury and cyclosporin A injury protection, wherein a control group comprises the following components: exposing the abdominal cavity and renal pedicles; IR: two renal pedicles were separated, clamped closed with vascular clamps for 30 minutes to allow the kidneys to change color, and then perfused for 2 or 8 weeks; IR + CsA: dissolving CsA in olive oil, and intragastrically administering to IR mice every day; IR + CHBP: dissolving CHBP in saline, and injecting the IR mice into the abdominal cavity once every 3 days; IR + CsA + CHBP: simultaneous treatment of IR mice with CsA and CHBP; urine albumin/creatinine, serum creatinine and histology, apoptosis, caspase-3 and HMGB1 were evaluated, and intracellular signaling pathways were screened through a protein chip; a renal epithelial cell model is established, renal injury is simulated, and the influence of CsA, CHBP and/or caspase-3 siRNA on TCMK-1 is researched.

Description

The role of a cyclic erythropoietin-derived peptide in the protection of renal injury and cyclosporine A injury application

technical field

The invention belongs to the field of medicine, and in particular relates to the application of a cyclic erythropoietin-derived peptide in the protection of kidney injury and cyclosporine A injury.

Background technique

Kidney transplantation is the main treatment for patients with end-stage renal disease, but transplantation dysfunction and donor shortage are still the major concerns of today. Ischemia-reperfusion (IR) injury is closely related to delayed graft function, acute rejection and subsequent chronic graft dysfunction. Cyclosporine A (CsA) is the most commonly used immunosuppressant after renal transplantation, but its nephrotoxicity cannot be ignored.

IR injury is an inevitable injury in kidney transplantation, which can trigger immune response, oxidative damage, inflammatory response and cell death, manifested as tubular epithelial cell (TEC) death, inflammatory cell infiltration, cytokine and chemokine production, caspase -3 activation. TECs are most vulnerable to IR injury, but are also involved in regeneration, which may be involved in alleviating damage and promoting repair. Surviving TECs dedifferentiate and enter the cell cycle within hours of injury, initiating proliferation and maintaining homeostasis.

The 32kD caspase-3 precursor is cleaved into a 17kD active subunit and plays apoptotic and inflammatory roles in renal IR-related injury. HMGB1 is an injury-associated molecule that is rapidly released from the nucleus to the extracellular domain, mediating apoptosis and inflammation in acute kidney injury and subsequent fibrosis. Our previous study showed that caspase-3 and HMGB1 levels correlated with renal IR injury and degree of fibrosis. Different treatments, including caspase-3 siRNA, ameliorated kidney damage and also decreased the expression of caspase-3 and HMGB1.

CsA is one of the most important immunosuppressants in various organ transplantation including kidney. CsA belongs to the group of calcineurin inhibitors and has a large effect in increasing organ and patient survival. The use of CsA reduces the incidence of acute rejection and early graft loss after renal transplantation. However, due to the nephrotoxicity of CsA, it did not improve the long-term survival of transplanted kidneys. CsA nephrotoxicity manifests as interstitial fibrosis and hyaline degeneration of renal arteriole wall, which can lead to chronic graft injury and progressive renal impairment.

Erythropoietin (EPO) protects different organs including the brain, heart and kidneys from IR damage. This protection is achieved through the heterodimeric EPO receptor and beta-co-receptor (EPOR/βcR) (also known as the innate repair receptor), which is pharmacologically distinct from the homodimer of erythropoietin (EPOR). Our previous study showed that in IR injury, EPO reduced tubular apoptosis but promoted inflammatory cell apoptosis. However, high doses of EPO often cause side effects such as high blood pressure and thrombosis. Therefore, some scholars have developed a helical B surface peptide (HBSP) that only interacts with EPOR/βcR. HBSP consists of 11 amino acids (QEQLERALNSS) derived from the water surface of helix B in the EPO 3D structure, but has a short half-life of only a few minutes. To improve its plasma stability, a novel metabolically stable cyclic HBSP (CHBP)) was further produced with extended half-life and potent tissue protection.

This study further investigated the role and mechanism of CHBP in acute and chronic injury-related models. We hypothesized that CHBP protects kidneys and TECs from IR and/or CsA-induced damage. The renoprotection of CHBP may involve signaling pathways of several mechanisms, which warrants further investigation of how the underlying protective mechanisms differ between 2 and 8 weeks after IR and/or CsA-induced injury, or whether CHBP has comparable immune responses to CsA regulating effect.

SUMMARY OF THE INVENTION

Technical problems solved: In view of the above-mentioned technical problems such as large doses of EPO usually cause side effects, and the short half-life of linear erythropoietin-derived peptides in vivo, we chose to use cyclic erythropoietin-derived peptides as the research object. Since the dosage and efficiency of the cyclic erythropoietin-derived peptide in vivo are not yet clear, the present application preliminarily sets the dosage of 24 nmol/kg body weight as the experimental dosage in the preliminary experimental work. It is not known how the cyclic erythropoietin-derived peptide enters the body to target and act on the kidney. The present application is designed to inject from the abdominal cavity, enter the systemic circulation, and finally act on the kidney tissue. One of the objectives of the present invention is to provide the application of a cyclic erythropoietin-derived peptide in the protection of acute and chronic kidney injury and cyclosporine A injury.

Technical solutions:

Application of a cyclic erythropoietin-derived peptide in the protection of renal injury and cyclosporine A injury.

Further, the steps are:

Step 1: Establish a mouse kidney injury model: 25-30g male BALB/c mice were randomly divided into 5 groups with 6 mice in each group, ie n=6. Anesthesia with 1% pentobarbital ml/g; IR group: separate two renal pedicles, clamp them with vascular forceps for 30 minutes to discolor the kidneys, then release the vascular forceps to reperfuse blood for 2 or 8 weeks; IR+CsA group: 2mg CsA was dissolved in 1ml olive oil, and the mice in the IR group were gavaged every day according to the standard of 35mg/kg body weight; IR+CHBP group: 0.11mg CHBP was dissolved in 32ml 0.9% normal saline, According to the standard of 24nmol/kg body weight, the mice in the IR group were injected intraperitoneally, once every 3 days; the IR+CsA+CHBP group: the mice in the IR group were treated with CsA by gavage every day and CHBP once every 3 days;

Step 2: Urine samples were collected at weeks 2, 4, 6 and 8 using metabolic cages. At 2 or 8 weeks, animals were sacrificed and blood samples and kidney samples were collected. Kidney samples were treated with 10% (w/v) Fixed in formalin buffer, quick-frozen in liquid nitrogen and stored at -80°C for later use, urine samples were measured using an automatic biochemical analyzer for urinary albumin/creatinine, and blood samples were measured using an automatic biochemical analyzer for serum creatinine (SCr). ;

Step 3: The kidney sample was paraffin-embedded into sections, the sections were stained with hematoxylin and eosin (H&E), and the semi-quantitative assessment of the degree of tubulointerstitial damage (TID) was graded on a scale of 0-4: the percentage of damaged areas was less than 5 % scored 0; 5%-25% scored 1; 25%-50% scored 2; 50%-75% scored 3; more than 75% scored 4; parameters assessed include tubular lumen Dilation and vacuolation, interstitial expansion, inflammatory cell infiltration, protein casts, intraluminal cells or cell debris; 12 cortical fields were randomly selected for each section at 200x magnification for scoring;

Step 4: Masson's trichrome staining was used to detect tubulointerstitial fibrosis. 20 cortical fields were randomly selected for each section at 400x magnification, and the collagen deposition was assessed by scanning with Image-Pro Plus software;

Step 5: In situ labeling of fragmented DNA (ISEL) with terminal deoxynucleotidyl transferase (TdT) using the ApopTag peroxidase kit: Digest the sections with 40 μg/mL proteinase K at 37°C for 15 minutes, The sections were then incubated with TdT and digoxigenin-dUTP for 60 minutes at 37°C, and after the addition of digoxigenin-peroxidase complex for 30 minutes, sections were incubated with 3′-amino-9-ethyl 20 visual fields were selected in 200x field to detect apoptotic cells in tubules, lumen and interstitial regions;

The sixth step, Western blot analysis: prepare 25 micrograms of kidney protein, separate it on a 12-15% polyacrylamide denaturing gel, and transfer it to PVDF membrane with caspase-3 antibody, HMGB1 antibody, internal reference Before β-actin incubation, block with 5% milk, after incubation with peroxidase-conjugated secondary antibody, use molecular imager Chemi Doc XRS+ system to scan and develop, and perform semi-quantitative analysis by Image Lab software;

The seventh step, real-time quantitative PCR: detection of Caspase-3 mRNA, Caspase-3 and housekeeping gene GAPDH in kidney and TCMK-1 cells by reverse transcription (RT) real-time quantitative polymerase chain reaction (qPCR) in StepOnePlus real-time PCR system The probe was labeled with 6-carboxyfluorescein (FAM), 2 μg total RNA extracted with Trizol reagent was used for reverse transcription into cDNA, and 2 μl cDNA was amplified with Taq polymerase in qPCR reaction buffer containing 900 nM forward, reverse The reaction system of primers and 250nM probe was carried out at 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The normal group was used as the control, GAPDH was used as the correction, and the caspase was calculated using the 2- ^ΔΔCt method. -3 mRNA expression;

The eighth step, protein chip analysis: The protein chip in the protein chip kit uses 50 μg of protein to simultaneously detect 18 kinds of phosphorylated or cleaved signal molecules. According to the instructions of the chip, the image is obtained by briefly exposing the slide to the molecular imaging system. And use Alpha View software 3.3 to conduct semi-quantitative analysis by scanning bulk density;

The ninth step, TCMK-1 cell culture: TCMK-1 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100 units/ml penicillin G and 100 μg/ml streptomycin, with/without CHBP Condition, when the cells were confluent to 60-70%, they were treated with CsA for 24 hours, and Caspase-3 siRNA was transfected into TCMK-1 cells using Lipofectamine@RNAiMAX. The sequence of double-stranded CASP-3 siRNA was: sense strand 5′ - GCUUCUUCAGAGGCGACUAtt-3' and antisense strand 5'-UAGUCGCCUCUGAAGAAGCta-3', where the negative control siRNA did not target any known mammalian gene, siRNA-transfected cells were cultured for 4-6 hours with/without CHBP treatment CsA was added to the cells;

The tenth step, cell apoptosis was measured by flow cytometry: the adherent TCMK-1 cells were collected, the cell pellet was resuspended in buffer, and incubated with annexin-V and PI in the dark for 15 minutes. V or PI and no dye group were used as controls. The samples were analyzed by BD FACS Calibur flow cytometer using Cell Quest research software, and 10,000 cells were counted. The result quadrant plot showed: viable cells (Annexin V-/PI- ), early apoptotic cells (Annexin V+/PI-), late apoptotic cells (Annexin V+/PI+) or necrotic cells (Annexin V-/PI+), the number of cells of each type is expressed as a percentage of the total gated cells ;

Step 11, Statistical analysis: Statistical analysis was performed using GraphPad Prism 6.0 software, data were expressed as mean ± standard error of the mean (SEM), and analysis of variance (ANOVA) was used to compare the results among three or more groups , a two-sided t-test was used to compare results between the two groups, P ≤ 0.05 was considered statistically significant, and all data from cell culture studies were representative of at least three independent experiments.

Further, the IR+CsA+CHBP group in the first step is to dissolve 2mg CsA in 1ml olive oil, and gavage the mice in the IR group every day according to the standard of 35mg/kg body weight, while adding 0.11mg CHBP every 3 days. It was dissolved in 32 ml of 0.9% normal saline, and the mice in the IR group were intraperitoneally injected once according to the standard of 24 nmol/kg body weight.

Further, in the third step, the expansion of the interstitium is edema or fibrosis.

Further, the sixth step is to prepare 25 micrograms of kidney protein: take out the tissue from -80°C, add 1 ml of protein lysis solution (protease inhibitor PMSF and protein lysis solution RIPA according to the ratio of 1:100) for every 100 mg of tissue after thawing. Mix and prepare), homogenize with a homogenizer on ice, mix in an ice bath for 30 min, transfer to an EP tube, centrifuge at 12000g for 15 min at 4°C, and take the supernatant. Use the protein quantification kit BCA method to determine the protein concentration, take a certain amount of protein solution, add 6× protein loading and dilute to 1×, boil in boiling water for 5 minutes to denature the protein, and store at 4°C.

Further, in the ninth step, the TCMK-1 cells are mouse kidney epithelial cell line CCL139 ^™ .

Further, in the ninth step, the CsA treatment was continued for 24 hours, wherein the CsA concentrations were 2.5, 5, 10, 20 and 40 μg/ml, respectively.

Further, in the ninth step, Caspase-3 siRNA was transfected into TCMK-1 cells, wherein the CASP-3 siRNA was 10, 20, 30 and 40 nM.

Further, the sequences of the double-stranded CASP-3 siRNA in the ninth step are: sense strand 5'-GCUUCUUCAGAGGCGACUAtt-3' and antisense strand 5'-UAGUCGCCUCUGAAGAAGCta-3'.

Further, the negative control siRNA in the ninth step is NCsiRNA, #4390843, Thermo Fisher Scientific.

Beneficial effects:

1. The use of a novel cyclic erythropoietin-derived peptide (CHBP) for the protection of acute and chronic renal injury, and the protection of cyclosporine A (CsA) nephrotoxic injury during renal transplantation.

2. To establish a stable mouse model of IR and CsA toxic kidney injury, to study the mechanism of acute and chronic kidney injury, and to observe the effect and mechanism of CHBP with or without CsA therapeutic intervention; to explore the signaling pathways that may involve several mechanisms in the renal protection of CHBP , it is necessary to further investigate how the underlying protective mechanisms differ between 2 and 8 weeks after IR and/or CsA-induced injury to screen biological markers for effective and low-invasive diagnostic and therapeutic monitoring; design new specific genes treatment (caspase-3 siRNA) to improve the survival of transplanted kidneys.

3. In the 2-8 week model, urinary albumin/creatinine increased after IR injury, and further increased after CsA intervention, but CHBP could reverse this injury. Similar to active caspase-3 expression changes, but no corresponding changes in SCr.

4. At the 2nd and/or 8th week, IR led to an increase in apoptotic cells, and CsA and CHBP intervention could reduce cell apoptosis.

5. The expression of p70 S6 kinase, mTOR, S6 ribosomal protein, GSK-3β and caspase-3 was reduced by CsA treatment at two time points with or without CHBP intervention, while the addition of CHBP in the IR+CsA group could reduce the expression of p70 S6 kinase, mTOR, S6 ribosomal protein, GSK-3β and caspase-3 at both time points. Increased expression of p53 and S6RP.

6. The protective effect of CHBP is reflected in the combined effect of CASP-3siRNA in the acute phase of injury, which can reduce the apoptosis of TCMK-1 cells induced by CsA.

7. In the acute stage of renal injury, the protective effect of CHBP is reflected in the repair of IR injury, while in the process of chronic injury, the protective effect of CHBP is reflected in the protection of CsA nephrotoxic injury, and the underlying mechanisms are different. Urinary albumin/creatinine is a better biomarker than Scr. The synergistic effect of CHBP and CASP-3siRNA can effectively reduce CsA-induced apoptosis of tubular epithelial cells.

8. CHBP reduced urinary albumin and increased urinary albumin/creatinine ratio (mg/μmol) in the CsA-treated group compared with the IR group at 2 and 4 weeks, 6 and 8 weeks The ratio of urinary albumin/creatinine was also increased in the IR group compared with the control group (Fig. 1C,D). CHBP treatment in the IR group significantly decreased this ratio at all time points with/without CsA treatment (Figure 1A-D).

In addition, the dynamic distribution of urinary albumin/creatinine was higher in the IR group than in the control and CHBP-treated groups at all time points during the following 2-8 weeks, while CsA further increased the urinary albumin/creatinine ratio (Fig. 1E). However, the differences between the control and IR groups appeared to widen over time, and CHBP combined with CsA/no CsA treatment narrowed their differences. The trends in the CsA group with/without CHBP were unexpectedly similar, with both peaking at week 4, declining at week 6, and then trending towards week 8, despite the large distance between the two groups. Stablize.

However, there were no significant differences in SCr levels among all groups at weeks 2 and 8 (Fig. 1F,G).

9. CHBP can improve tissue damage, and semi-quantitative analysis of TID was performed in H&E-stained sections. The TID score in the IR group was significantly higher than that in the control group at the 2nd and 8th week (Fig. 2A-D), and at the 8th week, the CsA was further increased than that in the IR group. Most interestingly, the IR group improved TID with CHBP treatment only at 2 weeks, and the CsA group improved TID at both time points.

Masson's trichrome staining showed that interstitial fibrosis was significantly increased in the IR group compared to the control group, but was ameliorated by CHBP at both time points (Fig. 3A-D). At week 8, CHBP in the CsA-treated group significantly reduced the interstitial fibrosis score.

10. CHBP and CsA can reduce the apoptosis of renal cells. The apoptotic cells detected by ISEL are mainly located in the renal tubule and interstitial area, and some cells have polymorphic nuclei (Fig. 4A, C). Apoptosis is rarely seen in the glomerular area. cell. From the total number of apoptotic cells in the tubule, interstitial and lumen, IR can significantly increase the total number of apoptotic cells, CsA can reduce the total number of apoptotic cells at 2 weeks and 8 weeks, CHBP only at 8 weeks decreased (Fig. 4B).

11. CHBP changed the mRNA and protein of Caspase-3. The expression of Caspase-3 mRNA and protein were detected by qPCR and Western blot respectively. Caspase-3 mRNA levels increased in the IR group at both time points, further increased in the CsA group at 2 weeks, and decreased under CHBP treatment at 8 weeks (Fig. 5A,E).

At 8 weeks, the expression of the 32kD caspase-3 precursor was reduced by CsA (Fig. 5B,C, and F,G). The level of 17kD active caspase-3 increased in the IR group at 2 weeks and continued to increase in the CsA group at 8 weeks, but both were reversed by CHBP treatment (Fig. 5D,H).

12. CHBP reduced HMGB1 protein, and the expression of HMGB1 was detected by Western blotting (Fig. 6A). Compared with the IR group, CsA was only elevated at 8 weeks, and CHBP treatment decreased the expression of HMGB1 in the 2-week IR group and the 8-week CsA-treated group (Fig. 6B,C).

13. Different proteins were different at week 2 and week 8, and the protein chip was used to detect 18 proteins in the kidney at the same time (Fig. 7A, B). At 2 weeks, the expressions of p70 S6 kinase, mTOR and caspase-3 were all decreased in the CsA group with or without CHBP treatment compared with the IR group (Fig. 7C-E), whereas in the CsA treated group, CHBP The expression of p53 could be increased (Fig. 7F).

At 8 weeks, CsA with/without CHBP could down-regulate the expression of S6 ribosomal protein, GSK-3β and caspase-3 in IR group, and caspase-3 in IR group was also decreased by CHBP. However, in the CsA-treated group, CHBP increased the expression of S6ribosomal proteins (Fig. 7G-I).

14. CHBP reversed the increase in caspase-3 expression and apoptosis in TCMK-1 cells caused by CsA, and the expression level of caspase-3 mRNA increased with increasing CsA concentration (2.5-20 μg/ml) (Figure 8A). . At 20 and 40 μg/ml, the percentage of early apoptotic cells was significantly increased (Fig. 8B, C), but the percentage of early apoptotic cells was significantly decreased after CHBP treatment, and the lowest was at 20 ng/ml (Fig. 8D, E).

15. In the TCMK-1 cell model, CASP-3siRNA reduced caspase-3 mRNA and apoptosis, compared with the control using 20 μg/ml CsA with or without NCsiRNA, the caspase-3 mRNA in the CASP-3siRNA treated group. Expression decreased from 10-30 nM, with the greatest inhibition at 30 nm/ml, which decreased to 64.85% (Fig. 9A). Caspase-3 mRNA expression was significantly increased by CsA, but was reversed by CHBP with/without CASP-3 siRNA (Fig. 9B). Likewise, CsA significantly increased early apoptotic cells, but was reversed by CHBP in the presence or absence of CASP-3 siRNA or NCsiRNA (Fig. 9C,D). More interestingly, the combined treatment of CHBP and CASP-3siRNA further reduced early apoptotic cells compared with the CsA+CHBP+NCsiRNA group.

16. The main protective effect of CHBP against IR injury was 2 weeks, and the protective effect against CsA nephrotoxicity was 8 weeks, and urinary albumin/creatinine was a better biomarker than SCr. Signaling pathways underlying CHBP renoprotection were associated with different intercellular proteins, such as mTOR at 2 weeks and GSK-3β at 8 weeks, but were associated with caspase-3 at both time points. CHBP combined with CASP-3 siRNA has a profound protective effect on CsA-induced damage in TECs.

Description of drawings

Fig. 1 is the analysis diagram of urine albumin/creatinine and SCr in Example 1 of the application;

Fig. 2 is a semi-quantitative TID score chart of H&E stained sections in Example 2 of the application.

Fig. 3 is a score chart of Masson's trichrome staining section in Example 2 of the application.

FIG. 4 is a staining diagram of apoptotic cells in Example 3 of the present application.

Figure 5 is a graph of caspase-3 mRNA expression detected by qPCR and caspase-3 protein expression detected by western blot in Example 5 of the application.

FIG. 6 is a graph showing the expression of HMGB1 protein detected by western blot in Example 4 of the application.

FIG. 7 is a graph of 18 proteins detected by the protein chip in Example 6 of the present application.

Figure 8 is a graph showing the effects of CsA and CHBP on the expression of caspase-3 mRNA and apoptosis in TCMK-1 cells in Example 7 of the present application.

Fig. 9 is a graph showing the effect of CASP-3 siRNA in Example 8 of the present application on caspase-3 mRNA expression and apoptosis in TCMK-1 cells.

Detailed ways

The specific steps of the present invention are illustrated by the following examples, but are not limited by the examples.

Terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

The present invention will be described in further detail below with reference to specific examples and data. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

25-30 g male BALB/c mice were purchased from the Experimental Animal Center of Nantong University. CHBP was purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and protein chip kit was purchased from Chemiescence Readout, Danfoss, USA. All animal manipulations were performed in accordance with the requirements of the Animal Care and Use Committee of Nantong University and the Animal Care Ethics Committee of Jiangsu Province.

Example 1

Application of a cyclic erythropoietin-derived peptide in the protection of kidney injury and cyclosporine A injury to establish a mouse model of kidney injury: 25-30g male BALB/c mice were randomly divided into 5 groups, each group of 6 Only, i.e. n=6, in which control group: exposed abdominal cavity and renal pedicle, and anesthetized with 0.01ml/g of 1% pentobarbital; IR group: separated two renal pedicles and clamped them with vascular forceps for 30 minutes The kidneys were discolored, then the vascular clamp was released, and the blood was reperfused for 2 or 8 weeks; IR+CsA group: 2 mg of CsA was dissolved in 1 ml of olive oil, and IR mice were given 35 mg/kg body weight by gavage every day; IR +CHBP group: Dissolve 0.11mg CHBP in 32ml 0.9% saline, inject IR mice intraperitoneally according to the standard of 24nmol/kg body weight, once every 3 days; IR+CsA +CHBP group: dissolve 2mg CsA in 1ml olive oil In the IR group, 0.11 mg CHBP was dissolved in 32 ml of 0.9% normal saline every 3 days, and the mice in the IR group were intraperitoneally injected according to the standard of 24 nmol/kg body weight. Once, IR mice were treated with CsA and CHBP simultaneously.

Urine samples were collected at weeks 2, 4, 6 and 8 using metabolic cages, animals were sacrificed at weeks 2 or 8, blood samples and kidney samples were collected, kidney samples were treated with 10% (w/v) neutral formaldehyde The samples were fixed in Lin buffer, snap-frozen in liquid nitrogen and stored at -80°C for later use. Urine albumin/creatinine was measured using an automatic biochemical analyzer, and serum creatinine (SCr) was measured using an automatic biochemical analyzer for blood samples.

As shown in Figure 1A,B, the CsA treatment group increased the ratio of urinary albumin/creatinine (mg/μmol) compared with the IR group at 2 and 4 weeks, and at 6 and 8 weeks, as shown in Figure 1C,D The ratio of urinary albumin/creatinine was also increased in the IR group compared to the control group, and CHBP treatment in the IR group significantly decreased this ratio at all time points with/without CsA treatment, as shown in Figure 1A-D. ratio.

As shown in Figure 1E, the dynamic distribution of urinary albumin/creatinine was higher in the IR group than in the control and CHBP-treated groups at all time points during the subsequent 2-8 weeks, while CsA increased the urinary albumin/creatinine ratio in both To further increase, however, the differences between the control and IR groups appeared to widen over time, while CHBP combined with CsA/no CsA treatment narrowed their differences. The trends in the CsA group with/without CHBP were unexpectedly similar, with both peaking at week 4, declining at week 6, and then trending towards week 8, despite the large distance between the two groups. Stablize.

As shown in Figures 1F and G, SCr levels were not significantly different among all groups at weeks 2 and 8.

Example 2

Histological evaluation:

Paraffin-embedded sections of kidney samples, sections were stained with hematoxylin and eosin (H&E), and a semi-quantitative assessment of the degree of tubulointerstitial damage (TID) was scored on a scale of 0-4: the percentage of damaged area was less than 5%, scored 0 5%-25% score 1; 25%-50% score 2; 50%-75% score 3; more than 75% score 4; parameters assessed include tubular dilation and vacuolation within the tubular lumen , interstitial expansion i.e. edema or fibrosis, inflammatory cell infiltration, protein casts, intraluminal cells or cellular debris; 12 cortical fields were randomly selected for each section at 200x magnification for scoring.

As shown in Figure 2A-D, the semi-quantitative analysis of TID in H&E-stained sections showed that the TID score of the IR group was significantly higher than that of the control group at 2 and 8 weeks, and the CsA was further increased than that of the IR group at 8 weeks. , the IR group improved TID by CHBP treatment only at 2 weeks, and the CsA group improved TID at both time points.

Masson's trichrome staining was used to detect tubulointerstitial fibrosis, and 20 cortical fields were randomly selected from each section at 400x magnification and scanned using Image-Pro Plus software to assess collagen deposition.

As shown in Figure 3A-D, Masson's trichrome staining showed that interstitial fibrosis was significantly increased in the IR group compared to the control group, but was ameliorated by CHBP at both time points, and at week 8, CsA treatment CHBP in the group significantly reduced the score of interstitial fibrosis.

Example 3

In situ end-labeling of apoptotic cells:

Sections were in situ labeled with terminal deoxynucleotidyl transferase (TdT) fragmented DNA (ISEL) using the ApopTag peroxidase kit, and sections were digested with 40 μg/mL proteinase K at 37 °C for 15 min, and then incubated at 37 °C After incubation with TdT and digoxigenin-dUTP for 60 minutes, and after adding digoxigenin-peroxidase complex for 30 minutes, the sections were treated with 3′-amino-9-ethylcarbazole ( AEC) substrates were developed, and 20 fields were selected in a 200x field to detect apoptotic cells in tubules, luminal and interstitial regions.

As shown in Figure 4A-C, the apoptotic cells detected by ISEL were mainly located in the renal tubule and interstitial areas, some cells had polymorphic nuclei, and few apoptotic cells were found in the glomerular area. In terms of the total number of apoptotic cells in the cavity, IR can significantly increase the total number of apoptotic cells, and CsA can reduce the total number of apoptotic cells at 2 weeks and 8 weeks. As shown in Figure 4C, CHBP only decreased at 8 weeks. .

Example 4

Western blot analysis:

Prepare 25 micrograms of kidney protein to separate on 12-15% polyacrylamide denaturing gel and transfer it to PVDF membrane with 5 % milk was blocked, incubated with peroxidase-conjugated secondary antibodies, scanned and developed using a molecular imager Chemi Doc XRS+ system, and semi-quantitative analysis was performed by Image Lab software.

As shown in Figure 6A, the expression of HMGB1 was detected by Western blotting. As shown in Figure 6B and C, compared with the IR group, CsA was only elevated at 8 weeks, and CHBP treatment could reduce the expression of HMGB1 in the 2-week IR group and the 8-week CsA-treated group.

Example 5

Real-time quantitative PCR:

Detection of Caspase-3 mRNA in kidney and TCMK-1 cells by reverse transcription (RT) real-time quantitative polymerase chain reaction (qPCR) in the StepOne Plus real-time PCR system, the probes for Caspase-3 and the housekeeping gene GAPDH were fluoresced by 6-carboxy 2 μg total RNA extracted with Trizol reagent was used for reverse transcription into cDNA, and 2 μl cDNA was amplified with Taq polymerase in qPCR reaction buffer containing 900 nM forward and reverse primers and 250 nM probe. The reaction system was performed at 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The normal group was used as a control and GAPDH was used as a correction, and the expression of caspase-3 mRNA was calculated using the 2- ^ΔΔCt method.

As shown in Figure 5A and E, the expression of Caspase-3 mRNA and protein was detected by qPCR and Western blot. The caspase-3 mRNA level at both time points increased with the IR group, and further increased in the CsA group at 2 weeks , decreased under CHBP treatment at 8 weeks.

As shown in Figure 5B,C,F,G, at 8 weeks, the expression of 32kD precursor caspase-3 was decreased by CsA, and the level of 17kD active caspase-3 was increased in the IR group at 2 weeks and in the CsA group at 8 weeks. The elevations persisted, as shown in Figure 5D, H, but were all reversible by CHBP treatment.

Example 6

Protein chip analysis:

Protein chip kit (Chemiescence Readout, Danfoss, USA), the protein chip can simultaneously detect 18 kinds of phosphorylated or cleaved signal molecules with 50μg protein, according to the instructions of the chip, by briefly exposing the slide to the molecular imaging system to obtain images , and semi-quantitative analysis by scanning bulk density using Alpha View software 3.3.

As shown in Figure 7A,B, the protein chip was used to simultaneously detect 18 proteins in the kidney, and at 2 weeks, the CsA group had p70 S6 kinase with or without CHBP treatment compared with the IR group, as shown in Figure 7C-E. showed that the expressions of mTOR and caspase-3 were both lower than those of the IR group, while in the CsA-treated group, as shown in Figure 7F, CHBP could increase the expression of p53.

As shown in Figure 7G-I, at 8 weeks, CsA with/without CHBP down-regulated the expression of S6 ribosomal protein, GSK-3β and caspase-3 in the IR group, and caspase-3 in the IR group was also decreased by CHBP. However, in the CsA-treated group, CHBP increased the expression of S6ribosomal proteins.

Example 7

TCMK-1 cell culture:

TCMK-1 cells (mouse kidney epithelial cell line, CCL139 ^™ ) were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100 units/ml penicillin G and 100 μg/ml streptomycin in the presence/absence of CHBP Under the condition of 60-70% confluence, the cells were treated with CsA (2.5, 5, 10, 20 and 40 μg/ml) for 24 hours, and Caspase-3 siRNA (CASP-3 siRNA, 10, 20 μg/ml) was treated with Lipofectamine@RNAiMAX. , 30 and 40 nM) were transfected into TCMK-1 cells, and the sequences of double-stranded CASP-3 siRNA were: sense strand 5′-GCUUCUUCAGAGGCGACUAtt-3′ and antisense strand 5′-UAGUCGCCUCUGAAGAAGCta-3′, in which the negative control siRNA (NCsiRNA , #4390843, Thermo Fisher Scientific) does not target any known mammalian gene, siRNA-transfected cells were cultured for 4-6 hr and CsA was added to cells with/without CHBP treatment.

As shown in Fig. 8A, the expression level of aspase-3 mRNA increased with the increase of CsA concentration (2.5-20 μg/ml), and at 20 and 40 μg/ml, as shown in Fig. 8B, C, the percentage of early apoptotic cells significantly increased, but the proportion of early apoptosis decreased significantly after CHBP treatment, as shown in Figure 8D, E, the lowest at 20ng/ml.

Example 8

Determination of apoptosis by flow cytometry:

The adherent TCMK-1 cells were collected, the cell pellet was resuspended in buffer, and incubated with annexin-V and PI for 15 minutes in the dark. The annexin-V or PI and no dye groups were used as controls by BD FACS Calibur flow cytometer, using Cell Quest research software, the sample was analyzed, 10,000 cells were counted, and the quadrant plot of the results showed: viable cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-) , late apoptotic cells (Annexin V+/PI+) or necrotic cells (Annexin V-/PI+), and the number of cells of each type was expressed as a percentage of the total gated cells.

As shown in Figure 9A, the expression of caspase-3 mRNA in the CASP-3siRNA-treated group decreased from 10-30nM compared to the control using 20μg/ml CsA with or without NCsiRNA, and the inhibition was the greatest at 30nM, which decreased to 64.85% , caspase-3 mRNA expression was significantly increased under the action of CsA, as shown in Figure 9B, but could be reversed by CHBP with/without CASP-3 siRNA, similarly, CsA significantly increased early apoptotic cells, but in the presence or absence of CASP In the case of -3siRNA or NCsiRNA, as shown in Figures 9C and D, both can be reversed by CHBP. Compared with the CsA+CHBP+NCsiRNA group, the combined treatment of CHBP and CASP-3siRNA can further reduce the early apoptotic cells.

Example 9

Statistical Analysis:

Statistical analysis was performed using GraphPad Prism 6.0 software, data were expressed as mean ± standard error of the mean (SEM), analysis of variance (ANOVA) was used to compare results between three or more groups, and two-sided t-test was used for comparison Results between the two groups, P ≤ 0.05 were considered statistically significant, and all data from cell culture studies were representative of at least three independent experiments.

sequence listing

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Affiliated Hospital of Nantong University

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<213> Artificial Sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

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Claims (10)

1. An application of cyclic erythropoietin-derived peptide in protecting kidney injury and cyclosporin A injury.

2. The use of a cyclic erythropoietin-derived peptide according to claim 1 for kidney injury and cyclosporin a injury protection, characterized by the steps of:

the first step is as follows: establishing a mouse kidney injury model: male BALB/c mice, 25-30g, were randomly divided into 5 groups of 6 mice each, i.e. n =6, with control groups: the abdominal cavity and renal pedicles were exposed and anesthetized with 0.01ml/g of 1% pentobarbital; IR group: separating two renal pedicles, clamping them for 30min with vascular clamps to discolor the kidneys, and then releasing the vascular clamps to allow reperfusion of the blood for 2 or 8 weeks; IR + CsA group: 2mg CsA was dissolved in 1ml olive oil and the IR group mice were gavaged daily at 35mg/kg body weight; IR + CHBP group: dissolving 0.11mg CHBP in 32ml 0.9% physiological saline, and injecting into IR group mice abdominal cavity according to 24nmol/kg body weight standard once every 3 days; IR + CsA + CHBP group: the IR group mice were treated simultaneously with CsA gavage and CHBP once every 3 days;

the second step is that: collecting urine samples at weeks 2, 4, 6 and 8 using a metabolism cage, at week 2 or 8, sacrificing the animals, collecting blood samples and kidney samples, fixing the kidney samples with 10% (w/v) neutral formalin buffer solution, quick freezing in liquid nitrogen and storing at-80 ℃ for later use, measuring urine albumin/creatinine using an automated biochemical analyzer for urine samples, and measuring serum creatinine (SCr) using an automated biochemical analyzer for blood samples;

the third step: kidney specimens were paraffin-embedded sections, stained with hematoxylin and eosin (H & E), and semi-quantitative assessment of the degree of tubulointerstitial damage (TID) was scored from 0 to 4 points: the percentage of the damaged area is less than 5 percent, and 0 is obtained; 5% -25% to obtain 1 point; 25% -50% to obtain 2 points; 50% -75% to obtain 3 points; 4 points are obtained when the content exceeds 75 percent; the parameters evaluated include tubular dilation and vacuoles within the tubular lumen, interstitial dilation, inflammatory cell infiltration, protein casts, intraluminal cells or cell debris; each section was scored by randomly selecting 12 cortical fields at 200x magnification;

the fourth step: masson trichrome staining, detecting tubulointerstitial fibrosis, randomly selecting 20 cortical fields for each section under 400x magnification, and scanning using Image-Pro Plus software to assess collagen deposition;

fifth, the sections were labeled in situ with terminal deoxynucleotidyl transferase (TdT) fragmented dna (isel) using the ApopTag peroxidase kit: digesting the section with 40 mug/mL proteinase K for 15 minutes at 37 ℃, then incubating the section with TdT and digoxigenin-dUTP for 60 minutes at 37 ℃, adding digoxigenin-peroxidase complex for 30 minutes, developing the section with 3' -amino-9-ethylcarbazole (AEC) substrate, and selecting 20 fields in 200x fields to detect apoptotic cells of tubules, lumen and interstitial region;

sixth step, western blot analysis: preparing 25 microgram of kidney protein to separate on 12-15% polyacrylamide denaturing gel and transferring it to PVDF membrane, blocking with 5% milk before incubation with caspase-3 antibody, HMGB1 antibody, internal reference beta-actin, after incubation with peroxidase-coupled secondary antibody, scanning and developing using molecular imager Chemi Doc XRS + system, and semi-quantitative analysis by Image Lab software;

seventh step, real-time quantitative PCR: detection of Caspase-3 mRNA, Caspase-3 and housekeeping gene GAPDH probes in kidney and TCMK-1 cells in StepOne Plus real-time PCR System by Reverse Transcription (RT) real-time quantitative polymerase chain reaction (qPCR) were labeled with 6-carboxyfluorescein (FAM), 2. mu.g of total RNA extracted with Trizol reagent was used for reverse transcription into cDNA, 2. mu.l of cDNA was amplified in qPCR reaction buffer with Taq polymerase, wherein the reaction system containing 900nM forward, reverse primer and 250nM probe was in a StepOne Plus real-time PCR system10 min at 95 ℃ followed by 40 cycles of 15 sec at 95 ℃ and 1 min at 60 ℃ with normal group as control and GAPDH as correction, 2^-ΔΔCtThe method calculates the expression of caspase-3 mRNA;

eighth step, protein chip analysis: the protein chip in the protein chip kit simultaneously detects 18 phosphorylated or cracked signal molecules by using 50 mug protein, obtains an image by briefly exposing a glass slide to a molecular imaging system according to a chip use instruction, and performs semi-quantitative analysis by scanning volume density by using Alpha View software 3.3;

ninth step, TCMK-1 cell culture: TCMK-1 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100 units/ml penicillin G and 100. mu.g/ml streptomycin, the cells were treated with CsA for 24 hours with/without CHBP at 60-70% cell length fusion, Caspase-3 siRNA was transfected into TCMK-1 cells using Lipofectamine @ RNAImax, the sequence of double stranded CASP-3siRNA was: 5'-GCUUCUUCAGAGGCGACUAtt-3' and 5'-UAGUCGCCUCUGAAGAAGCta-3', wherein the negative control siRNA does not target any known mammalian genes, the siRNA transfected cells are cultured for 4-6 hours and CsA is added to the cells with/without CHBP treatment;

step ten, determining apoptosis by a flow cytometer: TCMK-1 cells attached to the wall were collected, the Cell pellet resuspended in buffer and incubated with annexin-V and PI away from light for 15 minutes, and the samples were analyzed by BD FACS Calibur flow cytometer using Cell Quest research software, with annexin-V or PI incubated alone and no dye set as controls, and 10000 cells counted, with the results shown in a quadrant dot plot: viable cells (Annexin V-/PI-), early apoptotic cells (Annexin V +/PI-), late apoptotic cells (Annexin V +/PI +) or necrotic cells (Annexin V-/PI +), the number of each type of cell being expressed as a percentage of total gated cells;

eleventh, statistical analysis: statistical analysis was performed using GraphPad Prism 6.0 software, data expressed as mean ± Standard Error of Mean (SEM), analysis of variance (ANOVA) was used to compare results between three or more groups, two-sided t-test was used to compare results between two groups, P ≦ 0.05 was considered statistically significant, and all data from cell culture studies represent at least three independent experiments.

3. The use of a cyclic erythropoietin-derived peptide according to claim 2 for kidney injury and cyclosporin a injury protection, wherein: in the first step, 2mg of CsA is dissolved in 1ml of olive oil, the mice in the IR group are subjected to intragastric administration according to the standard of 35mg/kg of body weight every day, and 0.11mg of CHBP is dissolved in 32ml of 0.9% physiological saline every 3 days and is subjected to intraperitoneal injection once according to the standard of 24nmol/kg of body weight.

4. The use of a cyclic erythropoietin-derived peptide according to claim 3 for kidney injury and cyclosporin A injury protection, wherein: the third step is interstitial expansion as edema or fibrosis.

5. The use of a cyclic erythropoietin-derived peptide according to claim 1 for kidney injury and cyclosporin a injury protection, wherein: the sixth step prepares 25 micrograms of kidney protein: taking out the tissue from-80 ℃, adding 1ml of protein lysate (prepared by mixing protease inhibitor PMSF and protein lysate RIPA according to the ratio of 1: 100) into every 100mg of tissue after thawing, homogenizing on an ice homogenizer, mixing in an ice bath for 30min, then transferring into an EP tube, centrifuging for 15min at 4 ℃ and 12000g, taking the supernatant, measuring the protein concentration by using a protein quantification kit BCA method, taking a certain amount of protein solution, adding 6 times of protein loading and diluting to 1 x, boiling for 5min with boiling water to denature the protein, and storing at 4 ℃.

6. The use of a cyclic erythropoietin-derived peptide according to claim 1 for kidney injury and cyclosporin a injury protection, wherein: in the ninth step, TCMK-1 cells are mouse renal epithelial cell line CCL139 cells.

7. The use of a cyclic erythropoietin-derived peptide according to claim 1 for kidney injury and cyclosporin a injury protection, wherein: the CsA treatment in the ninth step was continued for 24 hours with CsA concentrations of 2.5, 5, 10, 20 and 40. mu.g/ml, respectively.

8. The use of a cyclic erythropoietin-derived peptide according to claim 1 for kidney injury and cyclosporin a injury protection, wherein: caspase-3 siRNA was transfected into TCMK-1 cells in the ninth step, where CASP-3siRNA was 10, 20, 30 and 40 nM.

9. The use of a cyclic erythropoietin-derived peptide according to claim 1 for kidney injury and cyclosporin a injury protection, wherein: the sequence of the double-stranded CASP-3siRNA in the ninth step is as follows: sense strand 5'-GCUUCUUCAGAGGCGACUAtt-3' and antisense strand 5'-UAGUCGCCUCUGAAGAAGCta-3'.

10. The use of a cyclic erythropoietin-derived peptide according to claim 1 for kidney injury and cyclosporin a injury protection, wherein: the negative control siRNA in the ninth step is NCsiRNA, #4390843, Thermo Fisher Scientific.

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