CN112076309A - Application of cyclic erythropoietin-derived peptide in kidney injury and cyclosporin A injury protection - Google Patents
Application of cyclic erythropoietin-derived peptide in kidney injury and cyclosporin A injury protection Download PDFInfo
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- CN112076309A CN112076309A CN202010740606.4A CN202010740606A CN112076309A CN 112076309 A CN112076309 A CN 112076309A CN 202010740606 A CN202010740606 A CN 202010740606A CN 112076309 A CN112076309 A CN 112076309A
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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
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of cyclic erythropoietin-derived peptide in kidney injury and cyclosporin A injury protection.
Background
Kidney transplantation is the primary treatment for patients with end-stage renal disease, but graft dysfunction and donor shortages remain major concerns of widespread concern today. Ischemia-reperfusion (IR) injury is closely related to delayed function of transplanted organs, acute rejection and subsequent chronic graft dysfunction. Cyclosporin a (Cyclosporine a, CsA) is the most commonly used immunosuppressant after renal transplantation, but its nephrotoxicity is not negligible.
IR injury is an inevitable injury in kidney transplants that can trigger immune responses, oxidative damage, inflammatory responses and cell death, manifested by Tubular Epithelial Cell (TEC) death, inflammatory cell infiltration, cytokine and chemokine production, caspase-3 activation. TECs are most susceptible to IR damage, but are also involved in regeneration, which may be relevant to reduce damage and promote repair. Surviving TECs dedifferentiate and enter the cell cycle within hours after injury, initiating proliferation and maintaining homeostasis.
The 32kD caspase-3 precursor cleaves to the 17kD active subunit, playing apoptotic and inflammatory roles in renal IR-related injury. HMGB1 is an injury-associated molecule that is rapidly released from the nucleus into the extracellular domain, mediating apoptosis and inflammation in acute kidney injury and subsequent fibrosis. Our previous studies showed that caspase-3 and HMGB1 levels correlated with renal IR injury and the degree of fibrosis. Various treatments, including caspase-3 siRNA, improved renal injury and reduced caspase-3 and HMGB1 expression.
CsA is one of the most important immunosuppressive agents in many organ transplants, including the kidney. CsA belongs to the group of calcineurin inhibitors and has a great effect in increasing organ and patient survival rates. The use of CsA reduces the incidence of acute rejection and early graft loss after renal transplantation. However, CsA does not improve the long-term survival of transplanted kidneys due to its nephrotoxicity. CsA nephrotoxicity is manifested by interstitial fibrosis and hyaline degenerative degeneration of the arteriolar duct wall, which can lead to chronic graft injury and progressive renal function impairment.
Erythropoietin (EPO) is capable of protecting different organs including the brain, heart and kidneys from IR damage. This protection is achieved by a heterodimeric EPO receptor and a β -co-receptor (EPOR/β cR), also known as the innate repair receptor, which is pharmacologically distinct from the homodimer of Erythropoietin (EPOR). Our previous studies have shown that EPO can reduce tubular apoptosis but promote inflammatory apoptosis in IR injury. However, high doses of EPO often cause side effects such as hypertension and thrombosis. Therefore, researchers developed a Helical B Surface Peptide (HBSP) that interacts only with EPOR/β cR. HBSP consists of 11 amino acids derived from the surface of helix B in the EPO 3D structure (QEQLERALNSS), but has a short half-life of only a few minutes. To improve its plasma stability, a novel metabolically stable Cyclic HBSP (CHBP)) is further produced, with a prolonged half-life and potent tissue protection.
The present study further investigated the role and mechanism of CHBP in acute and chronic injury-related models. We hypothesized that CHBP protects kidney and TEC from IR and/or CsA induced damage. Renal protection of CHBP may involve several mechanisms of signaling, which necessitates 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 a comparable modulating effect on the immune response of CsA.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the technical problems that the high-dose EPO generally causes side effects, the half-life of the linear erythropoietin-derived peptide in vivo is short, and the like, the cyclic erythropoietin-derived peptide is selected to be used as a research object. Since the dose, efficiency, etc. of the cyclic ghrelin-derived peptide used in vivo are not clear, the present application preliminarily set the dose of 24nmol/kg body weight as the experimental dose in the preliminary experimental work. How to enter the body to target and act on the kidney by the cyclic erythropoietin-derived peptide is not known at present, and the cyclic erythropoietin-derived peptide is injected from the abdominal cavity and enters the systemic circulation to finally act on the kidney tissue. The invention aims to provide application of cyclic erythropoietin-derived peptide in acute and chronic kidney injury and cyclosporin A injury protection.
The technical scheme is as follows:
an application of cyclic erythropoietin-derived peptide in protecting kidney injury and cyclosporin A injury.
Further, the method comprises the following steps:
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 gastric lavage 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 μ g/mL proteinase K at 37 ℃ for 15 minutes, then incubating with TdT and digoxigenin-dUTP at 37 ℃ for 60 minutes, adding digoxigenin-peroxidase complex for 30 minutes, developing the section with 3' -amino-9-ethylcarbazole (AEC) substrate, and selecting 20 fields of view to detect apoptotic cells of tubules, lumen and interstitial region in 200x field of view;
sixth step, western blot analysis: preparing 25 microgram of kidney protein for separation on 12-15% polyacrylamide denaturing gel, 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 the 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, Taq polymerase was used for qPCR reaction bufferMu.l of cDNA were amplified in solution, wherein the reaction system containing 900nM forward and reverse primers and 250nM probe was subjected to 40 cycles at 95 ℃ for 10 min, followed by 95 ℃ for 15 sec and 60 ℃ for 1 min, with the normal group as control and GAPDH as correction, using 2-ΔΔCtThe method calculates the expression of caspase-3 mRNA;
eighth step, protein chip analysis: the protein chip in the protein chip kit utilizes 50 mu g of protein to simultaneously detect 18 types of phosphorylated or cracked signal molecules, obtains images by exposing a glass slide to a molecular imaging system for a short time according to the instructions of the chip, and uses Alpha View software 3.3 to perform semi-quantitative analysis by scanning the volume density;
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, and the cells were transfected with Caspase-3 siRNA into TCMK-1 cells using Lipofectamine @ RNAiMAX for 24 hours with/without CHBP fusion to 60-70% cell length, the sequence of the double stranded CASP-3siRNA being: sense strand 5'-GCUUCUUCAGAGGCGACUAtt-3' and antisense strand 5'-UAGUCGCCUCUGAAGAAGCta-3', wherein the negative control siRNA does not target any known mammalian gene, siRNA transfected cells were cultured for 4-6 hours, and CsA was added to cells with/without CHBP treatment;
step ten, determining apoptosis by a flow cytometer: collecting TCMK-1 cells attached to the wall, resuspending the Cell pellet in buffer, and incubating with annexin-V and PI away from light for 15 minutes, taking the incubation of annexin-V or PI only and the dye-free set as controls, analyzing the sample by BD FACS Calibur flow cytometer using Cell Quest research software, counting 10000 cells, and the result quadrant dot plot shows: 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.
Further, in the first step, 2mg of CsA was dissolved in 1ml of olive oil, and the mice in the group IR were gavaged daily at 35mg/kg body weight, while 0.11mg of CHBP was dissolved in 32ml of 0.9% physiological saline every 3 days, and the mice in the group IR were intraperitoneally injected at 24nmol/kg body weight.
Further, the third step is interstitial expansion as edema or fibrosis.
Further, the sixth step produces 25 micrograms of kidney protein: taking out tissue from-80 deg.C, thawing, adding 1ml protein lysate (prepared by mixing protease inhibitor PMSF and protein lysate RIPA at a ratio of 1: 100) per 100mg tissue, homogenizing on ice homogenizer, mixing in ice bath for 30min, transferring into EP tube, centrifuging at 4 deg.C and 12000g for 15min, and collecting supernatant. And (3) determining the protein concentration by using a BCA method of a protein quantification kit, taking a certain amount of protein liquid, adding 6 Xprotein loading and diluting to 1X, boiling for 5 minutes in boiling water to denature the protein, and storing at 4 ℃.
Further, the TCMK-1 cells in the ninth step are mouse kidney epithelial cell line CCL139TM。
Further, the CsA treatment in said ninth step was continued for 24 hours with CsA concentrations of 2.5, 5, 10, 20 and 40 μ g/ml, respectively.
Further, Caspase-3 siRNAs were transfected into TCMK-1 cells in the ninth step, where the CASP-3 siRNAs were 10, 20, 30 and 40 nM.
Further, the double-stranded CASP-3siRNA in the ninth step has the sequence: 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.
Has the advantages that:
1. the novel cyclic erythropoietin-derived peptides (CHBPs) are used for protection against acute and chronic kidney injury, as well as against nephrotoxic injury from cyclosporin a (csa) during renal transplantation.
2. Establishing a stable mouse model of the toxic kidney injury of IR and CsA, researching the mechanism of acute and chronic kidney injury and observing the effect and mechanism of CHBP whether CsA treatment intervention exists; exploring the signaling pathways that the renal protection of CHBP may involve several mechanisms, it is necessary to further investigate how the potential protective mechanisms are different 2 and 8 weeks after IR and/or CsA-induced injury, in order to screen biological indicators for effective and low-invasive diagnostic and therapeutic monitoring; a novel specific gene therapy (caspase-3 siRNA) was designed to improve the survival of transplanted kidneys.
3. In the 2-8 week model, urinary albumin/creatinine was elevated following IR injury and further elevated following CsA intervention, but CHBP reversed the injury with a trend similar to tubulointerstitial injury, fibrosis, HMGB1 and active caspase-3 expression changes, but no corresponding change in SCr.
4. At weeks 2 and/or 8, IR leads to an increase in apoptotic cells and CsA and CHBP intervention can reduce apoptosis.
5. At both time points CsA treatment reduced the expression of p 70S 6 kinase, mTOR, S6ribosomal protein, GSK-3 β and caspase-3, with or without CHBP intervention, while the addition of CHBP to the IR + CsA group increased the expression of p53 and S6 RP.
6. The protection effect of CHBP is reflected in that the combined effect of the injury acute phase and CASP-3siRNA can reduce the CSA-induced TCMK-1 cell apoptosis.
7. The protection of CHBP during acute phase of renal injury is manifested by repair of IR injury, while during chronic injury, the protection of CHBP is manifested by protection of CsA nephrotoxic injury, with different underlying mechanisms. Urinary albumin/creatinine is a better biomarker compared to Scr. The CHBP and CASP-3siRNA synergistic effect can effectively reduce the CsA-induced tubular epithelial cell apoptosis.
8. CHBP decreased urinary albumin, and at weeks 2 and 4, the CsA treated group increased the urinary albumin/creatinine ratio (mg/. mu.mol) compared to the IR group (FIGS. 1A, B), and at weeks 6 and 8, the IR group increased the urinary albumin/creatinine ratio compared to the control group (FIGS. 1C, D). CHBP treatment in the IR group significantly reduced this ratio at all time points with/without CsA treatment (FIGS. 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 of the subsequent 2-8 weeks, and the urinary albumin/creatinine ratio was further increased by CsA (fig. 1E). However, the difference between the control and IR groups appeared to expand over time, while CHBP treatment with/without CsA reduced the difference between them. The trends for the two CsA groups with/without CHBP were unexpectedly similar, both peaking at week 4, declining at week 6, and then settling at week 8, despite the remote distance between the two.
However, at weeks 2 and 8, there was no significant difference in SCR levels in all groups (FIG. 1F, G).
9. CHBP improved tissue damage and semi-quantitative analysis of TID was performed in H & E stained sections. TID scores were significantly higher in the IR group at weeks 2 and 8 than in the control group (fig. 2A-D), and CsA was further higher at week 8 than in the IR group. Most interestingly, the IR group improved TID by CHBP treatment only at 2 weeks, and the CsA group improved TID in both time points.
Trichrome staining by Masson showed a significant increase in interstitial fibrosis in the IR group compared to the control group, but was improved by CHBP at both time points (fig. 3A-D). At week 8, CHBP in the CsA treated group significantly reduced the score of interstitial fibrosis.
10. CHBP and CsA can reduce renal cell apoptosis, ISEL detected apoptotic cells mainly located in renal tubule and interstitial region, and some cells have polymorphonuclear (FIG. 4A, C), and the glomerular region has few apoptotic cells. From the total number of apoptotic cells in the small tract, interstitial and luminal areas, IR significantly increased the total apoptotic cell number, CsA decreased the total apoptotic cell number at 2 and 8 weeks, and CHBP decreased only at 8 weeks (fig. 4B).
11. CHBP changes Caspase-3 mRNA and protein, and qPCR and Western blot are respectively used for detecting the expression of Caspase-3 mRNA and protein. Caspase-3 mRNA levels at both time points increased with the IR group, further increased in the CsA group at 2 weeks, and decreased under CHBP treatment at 8 weeks (FIGS. 5A, E).
At 8 weeks, expression of the 32kD caspase-3 precursor was reduced by CsA (FIGS. 5B, C, and F, G). Levels of 17kD active caspase-3 were elevated in the IR group at 2 weeks and continued to be elevated in the CsA group at 8 weeks, but were all reversed by CHBP treatment (FIG. 5D, H).
12. The CHBP reduces HMGB1 protein, and expression of HMGB1 is detected by Western blotting (FIG. 6A). CsA was elevated only at 8 weeks compared to the IR group, and expression of HMGB1 in the 2-week IR group and the 8-week CsA-treated group was reduced after CHBP treatment (fig. 6B, C).
13. The different proteins differed between week 2 and week 8, and the protein chip was used to simultaneously detect 18 proteins in the kidney (fig. 7A, B). At week 2, p 70S 6 kinase, mTOR, and caspase-3 were reduced in expression compared to the IR group in CsA group with or without CHBP treatment (FIG. 7C-E), whereas in CsA treated group CHBP increased p53 expression (FIG. 7F).
At 8 weeks, CsA expression of IR group S6ribosomal protein, GSK-3 β and caspase-3 was downregulated by the presence/absence of CHBP, and the IR group caspase-3 was also decreased by CHBP. However, CHBP increased the expression of S6ribosomal protein in the CsA-treated group (FIGS. 7G-I).
14. CHBP reversed the increase in caspase-3 expression and apoptosis in TCMK-1 cells caused by CsA, and caspase-3 mRNA expression levels increased with CsA concentration (2.5-20. mu.g/ml) (FIG. 8A). At 20, 40 μ g/ml, the percentage of early apoptotic cells increased significantly (fig. 8B, C), but the proportion of early apoptosis decreased significantly after treatment with CHBP, with a minimum at 20ng/ml (fig. 8D, E).
15. In the TCMK-1 cell model, caspase-3 siRNA reduced caspase-3 mRNA and apoptosis, and caspase-3 mRNA expression was reduced from 10-30nM in the CASP-3siRNA treated group compared to the control with or without NCsiRNA using 20. mu.g/ml CsA, with maximal inhibition at 30nM/ml, reduced to 64.85% (FIG. 9A). Caspase-3 mRNA expression was significantly increased under CsA but was reversed by CHBP with/without CASP-3siRNA (FIG. 9B). Similarly, CsA significantly increased early apoptotic cells, but was reversible by CHBP with or without CASP-3siRNA or NCsiRNA (FIGS. 9C, D). More interestingly, combination treatment with CHBP and CASP-3siRNA could further reduce early apoptotic cells compared to the CsA + CHBP + NCsiRNA group.
16. The primary protection of CHBP for IR injury was at 2 weeks and protection for CsA nephrotoxicity was at 8 weeks, urinary albumin/creatinine as a better biomarker than SCr. The potential signaling pathway for renal protection by CHBP is associated with different intercellular proteins, such as mTOR at 2 weeks and GSK-3 β at 8 weeks, but caspase-3 is associated at both time points. The CHBP combined with CASP-3siRNA has a profound protective effect on CsA-induced injury in TECs.
Drawings
FIG. 1 is a graph of urine albumin/creatinine and SCR analysis according to example 1 of the present application;
FIG. 2 is a TID semi-quantitative score chart of H & E stained sections in example 2 of the present application.
FIG. 3 is a graph showing the scores of Masson trichrome stained sections in example 2 of the present application.
FIG. 4 is a graph of apoptotic cell staining in example 3 of the present application.
FIG. 5 is a graph showing the expression of caspase-3 mRNA detected by qPCR and the expression of caspase-3 protein detected by western blot in example 5 of the present application.
FIG. 6 is an expression diagram of HMGB1 protein detected by western blot in example 4 of the present application.
FIG. 7 is a diagram of detection of 18 proteins on the protein chip in example 6 of the present application.
FIG. 8 is a graph showing the effect of CsA and CHBP on TCMK-1 cell caspase-3 mRNA expression and apoptosis in example 7 of the present application.
FIG. 9 is a graph showing the effect of CASP-3siRNA on TCMK-1 cell caspase-3 mRNA expression and apoptosis in example 8 of the present application.
Detailed Description
The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.
The terms used in the present invention have meanings generally understood by those of ordinary skill in the art unless otherwise specified.
The invention is described in further detail below with reference to specific examples and with reference to data. It should be understood that these examples are 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.
25-30g of male BALB/c mice were purchased from the university of Nantong laboratory animal center. CHBP was purchased from Shanghai institute of medicine, Chinese academy of sciences, and protein chip kit was purchased from Chemiesce Readout, Denfoss, USA. All animal handling was performed according to the requirements of the university of southwest university committee on animal care and use and the ethical committee on animal care in Jiangsu province.
Example 1
The application of cyclic erythropoietin-derived peptide in kidney injury and cyclosporin A injury protection is to establish 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: dissolving 2mg CsA in 1ml olive oil, and intragastrically administering to IR mice per day according to 35mg/kg body weight standard; IR + CHBP group: dissolving 0.11mg CHBP in 32ml 0.9% saline, and injecting into IR mouse abdominal cavity once every 3 days according to 24nmol/kg body weight standard; IR + CsA + CHBP group: IR mice were treated with CsA and CHBP simultaneously by intraabdominal injection of 2mg CsA in 1ml olive oil, intragastric administration of 35mg/kg body weight standard to IR mice per day, intraabdominal injection of 0.11mg CHBP in 32ml 0.9% saline every 3 days, and intraabdominal injection of 24nmol/kg body weight standard to IR mice.
Urine samples were collected at weeks 2, 4, 6 and 8 using a metabolism cage, animals were sacrificed at weeks 2 or 8, blood samples and kidney samples were collected, kidney samples were fixed with 10% (w/v) neutral buffered formalin, stored at-80 ℃ for later use after snap freezing in liquid nitrogen, urine samples were measured for urinary albumin/creatinine using an automated biochemical analyzer, and blood samples were measured for serum creatinine (SCr) using an automated biochemical analyzer.
As shown in FIG. 1A, B, at weeks 2 and 4, the CsA-treated group increased the urinary albumin/creatinine ratio (mg/. mu.mol) compared to the IR group, and at weeks 6 and 8, as shown in FIG. 1C, D, the IR group increased the urinary albumin/creatinine ratio compared to the control group, and at all time points with/without CsA treatment, as shown in FIGS. 1A-D, the CHBP treatment in the IR group significantly decreased the ratio.
As shown in fig. 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 of the subsequent 2-8 weeks, while CsA further increased the urinary albumin/creatinine ratio, but the difference between the control and IR groups appeared to increase with time, while CHBP combined CsA/CsA-free treatment diminished the difference between them. The trends for the two CsA groups with/without CHBP were unexpectedly similar, both peaking at week 4, declining at week 6, and then settling at week 8, despite the remote distance between the two.
As shown in FIG. 1F, G, there was no significant difference in the SCr levels between all groups at week 2 and week 8.
Example 2
Histological evaluation:
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 renal tubule dilation and vacuole in the renal tubular lumen, interstitial dilation i.e. edema or fibrosis, inflammatory cell infiltration, protein casts, intraluminal cells or cell debris; each section was scored by randomly selecting 12 cortical fields at 200x magnification.
As shown in fig. 2A-D, semi-quantitative analysis of TID in H & E stained sections, TID scores were significantly higher for the IR group at weeks 2 and 8 than for the control group, CsA was further higher at week 8 than for the IR group, TID was improved by CHBP treatment only at week 2 for the IR group, and TID was improved by the CsA group at both time points.
Masson trichrome staining, with tubulointerstitial fibrosis detected, 20 cortical fields were randomly selected at 400x magnification per section and scanned using Image-Pro Plus software to assess collagen deposition.
As shown in FIGS. 3A-D, trichrome staining by Masson showed a significant increase in interstitial fibrosis in the IR group compared to the control group, but was improved by CHBP at both time points, with the CsA treated group having CHBP that significantly reduced the score of interstitial fibrosis at week 8.
Example 3
In situ end-labeling of apoptotic cells:
sections the fragmented dna (isel) was labeled in situ with terminal deoxynucleotidyl transferase (TdT) using the ApopTag peroxidase kit, sections were digested with 40 μ g/mL proteinase K for 15 minutes at 37 ℃, then incubated with TdT and digoxigenin-dUTP for 60 minutes at 37 ℃, after adding digoxigenin-peroxidase complex for 30 minutes, sections were visualized with 3' -amino-9-ethylcarbazole (AEC) substrate and 20 fields of view were selected for detection of apoptotic cells in tubules, luminal and interstitial regions in 200x fields.
As shown in fig. 4A-C, the apoptotic cells detected by ISEL were mainly localized in the tubular and interstitial regions, some cells had polymorphonuclear nuclei, and apoptotic cells were rarely found in the glomerular region, and the total apoptotic cell count was greatly increased by IR, decreased by CsA at 2 and 8 weeks, and decreased by CHBP only at 8 weeks, as seen in fig. 4C.
Example 4
Western blot analysis:
preparation of 25 microgram of kidney protein was isolated on 12-15% polyacrylamide denaturing gel and transferred to PVDF membrane, blocked with 5% milk prior to incubation with caspase-3 antibody, HMGB1 antibody, internal reference beta-actin, followed by incubation with peroxidase-coupled secondary antibody, scanning for visualization using molecular imager Chemi Doc XRS + system, and semi-quantitative analysis by Image Lab software.
As shown in fig. 6A, expression of HMGB1 was detected by Western blotting. As shown in fig. 6B, C, CsA was elevated only at 8 weeks compared to the IR group, and expression of HMGB1 in the 2-week IR group and the 8-week CsA-treated group was reduced after CHBP treatment.
Example 5
Real-time quantitative PCR:
detection of Caspase-3 mRNA, Caspase-3 and housekeeping gene GAPDH probes in kidney and TCMK-1 cells in the 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 to cDNA, 2. mu.l of cDNA was amplified in qPCR reaction buffer with Taq polymerase, where the reaction system containing 900nM forward, reverse primers and 250nM probe was performed at 95 ℃ for 10 min followed by 40 cycles at 95 ℃ for 15 sec and 60 ℃ for 1 min, with normal as control and GAPDH as correction, using 2-ΔΔCtThe method calculates caspase-3 mRNA expression.
As shown in FIG. 5A, E, Caspase-3 mRNA and protein expression was measured by qPCR and Western blot, and Caspase-3 mRNA levels at two time points were increased in the IR group, further increased in the CsA group at 2 weeks, and decreased under CHBP treatment at 8 weeks.
As shown in FIG. 5B, C, F, G, at 8 weeks, expression of the 32kD caspase-3 precursor was reduced by CsA, 17kD active caspase-3 levels were elevated in the IR group at 2 weeks and continued to be elevated in the CsA group at 8 weeks, as shown in FIG. 5D, H, but all were reversible by CHBP treatment.
Example 6
Protein chip analysis:
protein chip kit (Chemiescence Readout, denver, usa) which can simultaneously detect 18 phosphorylated or cleaved signal molecules using 50 μ g protein, acquire images by briefly exposing slides to a molecular imaging system according to the chip instructions, and perform semi-quantitative analysis by scanning the bulk density using Alpha View software 3.3.
As shown in FIG. 7A, B, the protein chip was used to detect 18 proteins in kidney simultaneously, and at 2 weeks, p 70S 6 kinase was present in CsA group with or without CHBP treatment, as shown in FIG. 7C-E, and both mTOR and caspase-3 expression were reduced compared to IR group, while in CsA treated group, CHBP increased p53 expression as shown in FIG. 7F.
As shown in FIGS. 7G-I, at 8 weeks, expression of IR group S6ribosomal protein, GSK-3 β and caspase-3 was downregulated by CsA with/without CHBP, and the IR group caspase-3 was also decreased by CHBP. However, in the CsA-treated group, CHBP increased the expression of S6ribosomal protein.
Example 7
TCMK-1 cell culture:
TCMK-1 cells (mouse renal epithelial cell line, CCL 139) were cultured in DMEM/F12 medium containing 10% fetal bovine serum, 100 units/ml penicillin G and 100. mu.g/ml streptomycinTM) Caspase-3 siRNAs (CASP-3 siRNAs, 10, 20, 30, and 40nM) were transfected into TCMK-1 cells using Lipofectamine @ RNAiMAX for 24 hours at cell length fusions of 60-70% with/without CHBP (2.5, 5, 10, 20, and 40 μ g/ml), the sequence of the double-stranded CASP-3siRNA being: sense strand 5'-GCUUCUUCAGAGGCGACUAtt-3' and antisense strand 5'-UAGUCGCCUCUGAAGAAGCta-3', in which the negative control siRNA (NCsiRNA, #4390843, Thermo Fisher Scientific) did not target any known mammalian genes, siRNA transfected cells were cultured for 4-6 hours and CsA was added to cells with/without CHBP treatment.
As shown in FIG. 8A, the expression level of caspase-3 mRNA increased with increasing CsA concentration (2.5-20. mu.g/ml), and at 20, 40. mu.g/ml, the percentage of early apoptotic cells was significantly increased as shown in FIG. 8B, C, but the proportion of early apoptosis after treatment with CHBP was significantly decreased, as shown in FIG. 8D, E, which was lowest at 20 ng/ml.
Example 8
Flow cytometry for apoptosis:
TCMK-1 cells were harvested, Cell pellets resuspended in buffer and incubated with annexin-V and PI away from light for 15 minutes, and samples were analyzed by BD FACS Calibur flow cytometer using Cell Quest research software, with annexin-V or PI alone and no dye set as controls, counting 10,000 cells, with quadrant dot plots showing: 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.
As shown in FIG. 9A, the caspase-3 mRNA expression in the CASP-3siRNA treated group was reduced from 10-30nM, with the maximum inhibition at 30nM being 64.85% compared to the control with or without NCsiRNA using 20. mu.g/ml CsA, and the caspase-3 mRNA expression was significantly increased by CsA, as shown in FIG. 9B, but was reversed by the presence/absence of CASP-3siRNA, and similarly, CsA significantly increased early apoptotic cells, but with or without CASP-3siRNA or NCsiRNA, as shown in FIG. 9C, D, the combined treatment with CHBP and CASP-3siRNA further reduced early apoptotic cells compared to the CsA + CHBP + NCsiRNA group.
Example 9
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.
Sequence listing
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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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