Effects Of CXCR1/2 Antagonist By G31P For Renal Injury On Diabetic Nephropathy Mice

Objective: Diabetic nephropathy(DN)is a microvascular disease caused by hyperglycemia in diabetic patients,and it is one of the most serious complications of diabetes mellitus(DM).With the further research on DN,it has been found that inflammation plays an important role in the occurrence and development of DN.CXCL8(3-72)K11R/G31P(briefly G31P),as a CXCR1/2 receptor antagonist,competes with CXCL8 to bind CXCR1/2 receptors,thereby inhibiting the biological activity of CXCL8 and exerting antiinflammatory effects.In this study,we established DM mouse model to explore the inhibitory effect of G31 P on DN renal injury.Method:1.In vivo experiments1.1 Generating a DM mouse model and grouping: 6 weeks old C57BL/6J male mice were selected and DM mouse model was generated by high fat diet(HFD)combined with streptozotocin(STZ).According to the research content,mice were divided into the following four groups: Normal control group(the following abbreviated as Cntl group),Cntl+G31P group,DM group,DM+G31P group.Cntl+G31P group and DM+G31P group were subcutaneously injected with G31 P once every two days for one month,at a dose of 0.5 mg/kg,and Cntl and DM groups were injected with the same volume of normal saline.1.2 Animal tissue samples collection: During treatment,the mice were monitored weekly for changes in body weight,food intake,water intake and blood glucose.After one month of treatment,all mice were sacrificed to collect samples: took the mice eye blood,after standing for 1h at room temperature,centrifuged the blood,took serum and stored at-80?.Took out the kidneys of mice,weighed and took pictures.1.3 Experimental methods: Part of the renal tissues were fixed in 4% paraformaldehyde solution,for the test of H&E,Periodic acid schiff(PAS),Sirius red,and immunohistochemistry(IHC):The pathological changes of renal tissue were observed by H&E staining,the degree of glomerular sclerosis was observed by PAS staining,Sirius red staining was used to observe the collagen deposition,the expression of Collagen IV,MPO,F4/80,p-NF-kB,CXCR1,CXCR2,Podocin and Nephrin were detected by IHC;Part of the kidney tissues were fixed with 3% glutaraldehyde and 1% osmium tetraoxide to evaluate the thickness of glomerular basement membrane(GBM)and the podocyte fusion.The remaining renal tissues were used to extract RNA and protein,RT-PCR was used to detect the expression of inflammation and fibrosis related factors and Western blotting was used to detect the expression of ERK1/2,JAK2 and STAT3 phosphorylated proteins in the signaling pathway.2.In vitro experiments:2.1 Cell culture and grouping: 6-15 passage human glomerular mesangial cells(HRMCs)were cultured and divided into four groups as follows:(a)normal blood glucose group(NG group),cells were cultured in DMEM medium;(b)mannitol control group(MC group),cells were cultured in normal medium with additional 24.4 mM mannitol supplement which was served as osmolarity control;(c)high glucose group(HG group),cells were cultured in medium supplemented additional 24.5 mM glucose;(d)G31P treated group(HG+G31P group),cells were treated with 100 ng/mL G31 P 1 h before high glucose stimulation.2.2 Experimental methods: The cytotoxicity of G31 P to HRMCs was detected by CCK-8 assay;The expression of CXCL8,TNF-a,TGF-? and CTGF in each group were detected by RT-PCR;Western blotting was used to detect the expression levels of ERK1/2,JAK2 and STAT3 phosphorylated proteins in HRMCs signaling pathway.Results:1.In vivo experiments:1.1 G31 P conferred renoprotection in DN:by observing the kidney size and calculating the ratio of kidney weight to body weight,the result showed that G31 P treatment could decrease the renal hypertrophy induced by DM and the ratio of kidney weight/body weight decreased(P<0.01);DM mice were significantly increased in blood glucose,food intake,water intake and urine volume,while G31 P significantly reduced the water intake and urine volume of DM mice(P<0.01);G31P treatment significantly reduced the levels of BUN,Ccr and UACR in the DM group(P<0.01);H&E staining showed that DM group mice had glomerular hypertrophy,mesangial matrix increased,part of the renal tubular epithelial cells had hypertrophy,vacuolar degeneration,atrophy and necrosis.After G31 P treatment,these phenomena had improved.TEM showed that the basement membrane of DM group was diffuse thickening,foot width widening,fusion or even disappear,the number of podocytes decreased,and after G31 P treatment,the pathological damage was significantly relieved;RT-PCR and IHC showed that the expression of Nephrin and Podocin in DM group were significantly lower than that in Cntl group,G31 P partly improved their expression levels(P<0.05).1.2 G31 P inhibited the inflammatory reaction in DM mice renal tissues: RT-PCR showed that the expression levels of IL-1?,IL-6 and TNF-? in DM group were significantly increased,and G31 P could reduce their expression(P<0.01);IHC staining showed that the expression of NF-?B,MPO and F4/80 in the kidney tissues of DM mice were significantly increased,and after G31 P treatment the expression of those three were significantly decreased(P<0.01);The ELISA,RT-PCR and IHC results showed that the levels of CXCL8,CXCR1 and CXCR2 in serum or kidney tissues were significantly increased in DM mice and decreased after G31 P treatment(P<0.05,P<0.01);Western blotting showed that the phosphorylated protein levels of ERK1/2,JAK2 and STAT3 were significantly increased in DM group,and the expression levels of those three were significantly decreased after G31 P treatment(P <0.05,P <0.01).1.3 G31 P could improve the fibrosis of renal tissues in DM mice: PAS staining showed that the PAS positive staining was significantly increased in the DM group,and the glomerular sclerosis index(GSI)was increased,after G31 P treatment,the GSI level was decreased(P<0.01);According to the Sirius red,the G31 P treatment could significantly improve the collagen deposition in DM mice(P<0.01);IHC showed that the expression of Collagen IV in DM mice was significantly higher than that in Cntl group,after G31 P treatment,the Collagen IV level was decreased(P<0.05);RT-PCR and Western blotting showed that TGF-? and CTGF were highly expressed in renal tissues of DM mice,and their expression were significantly decreased after G31 P treatment(P<0.05,P<0.01).2.In vitro experiments:The CCK-8 assay result showed that appropriate dose of G31 P had no effect on the proliferation of HRMCs,indicating that it had no cytotoxicity;RT-PCR showed that in HRMCs,the expression levels of TNF-?,TGF-? and CTGF in HG group were significantly higher than those in NG group,G31 P treatment could significantly reduce those three mRNA expression levels(P<0.05).In addition,under the stimulation of 30 mmol/L high glucose,the expression of CXCL8 mRNA was significantly increased,and the peak appeared at 6 h after stimulation;High glucose stimulation could induce the expression of ERK1/2,JAK2 and STAT3 phosphorylated protein in HRMCs,and those three levels were significantly decreased after G31 P treatment(P<0.05,P<0.01).Conclusion:(1)In the kidney of DN mice,a large number of inflammatory cells were accumulated and inflammation-related factors were significantly increased,it was speculated that inflammation played an important role in the development and progression of DN.(2)By combining with CXCR1/2 recepters,G31 P could reduce renal damage in DN mice and exert its protective effect on the kidney by reducing the expression of proinflammatory and fibrotic factors,providing a new idea and theoretical basis for the clinical treatment of DN.