IL-8受体拮抗剂G31P对糖尿病小鼠糖尿病肾病纤维化的影响及机制

Effect and its mechanism of IL-8 receptor antagonist G31P on fibrosis of diabetic nephropathy in diabetic mice

目的探讨IL-8受体拮抗剂CXCL8_((3-72)) K11R/G31P(简称G31P)对糖尿病小鼠糖尿病肾病(DN)纤维化的影响及机制。方法将40只雄性C57BL/6J小鼠随机均分为四组:C、D组给予小鼠高脂饮食喂养8周联合链脲佐菌素50 mg/kg腹腔注射构建糖尿病模型,而A、B组给予普通饲料喂养8周联合等量柠檬酸盐缓冲液腹腔注射。糖尿病模型成功建立后,B、D组隔天1次皮下注射G31P 0.5 mg/kg,连续8周;而A、C组给予注射等量生理盐水。8周后测定血糖、尿量、血尿素氮及尿微量白蛋白/尿肌酐比值。采用Masson染色及天狼猩红染色观察各组肾脏纤维化及胶原沉积情况。采用免疫组化染色观察各组肾脏胶原Ⅳ、MMP-2和MMP-9表达。采用qRT-PCR检测各组MMP-2、MMP-9、纤维黏连蛋白(FN)、C-X-C基序趋化因子受体1(CXCR1)及CXCR2水平。采用Western blot检测各组肾脏细胞外调节蛋白激酶(ERK1/2)通路蛋白表达。结果与A组相比,C组尿量、血尿素氮、尿微量白蛋白/尿肌酐比值、肾脏胶原Ⅳ、FN、CXCR1、CXCR2、磷酸化ERK1/2均增高(P<0.01或P<0.05),但D组上述指标较C组均降低(P<0.01或P<0.05)。与A组相比,C组小鼠肾间质及肾小球区域有大量胶原纤维聚集,而D组较C组小鼠肾脏胶原纤维沉积减少(P<0.01)。C组MMP-2和MMP-9表达低于A组(P<0.01),而D组其表达高于C组(P<0.05)。结论 G31P通过抑制ERK1/2通路延缓DN纤维化进展,为防治DN提供新的抗炎靶点。

Objective To explore the effect and underlying mechanism of IL-8 receptor antagonist CXCL8_((3-72)) K11R/G31P(G31P) on diabetic nephropathy(DN) fibrosis in diabetic mice.Methods Forty male C57 BL/6 J mice were randomly divided into four groups with 10 rats each.The mice in groups of C and D were fed with high-fat diet for 8 weeks and injected intraperitoneally with STZ 50 mg/kg to establish diabetes models, while the mice in groups of A and B were given normal feed feeding for 8 weeks and injected intraperitoneally with an equal amount of citrate buffer solution.After the establishment of diabetes models successfully, the mice in groups of B and D were injected subcutaneously with G31 P 0.5 mg/kg, evey other day for 8 weeks, while the mice in groups of A and C were given the same amount of normal saline for 8 weeks.Eight weeks later, the blood glucose, urine output, blood urea nitrogen and the ratio of urine microalbumin to urine creatinine were measured.Masson staining and sirius scarlet staining were used to observe the kidney fibrosis and collagen deposition.Immunohistochemical staining was used to observe kidney collagen Ⅳ,MMP-2,MMP-9 in each group.qRT-PCR was used to detect MMP-2,MMP-9,fibronectin(FN),CXC motif chemokine receptor 1(CXCR1) and CXCR2 levels.Western blot was used to detect the protein expression of extracellular regulatory protein kinase(ERK1/2) pathway.Results Compared with group A,the urine output, blood urea nitrogen, urine microalbumin/urine creatinine ratio, kidney collagen Ⅳ,FN,CXCR1,CXCR2,phosphorylated ERK1/2 levels were increased in group C(P<0.01 or P<0.05),while the above indicators in group D were lower than those in group C(P<0.01 or P<0.05).Compared with group A,the renal interstitium and glomerular area of the mice in group C had a large amount of collagen fiber accumulation, which in group D was significantly less than that of group C(P<0.01).The expressions of MMP-2 and MMP-9 in group C were lower than those in group A(P<0.01),which in group D were higher than those in group C(P<0.05).Conclusion G31 P could attenuate the progression of renal fibrosis in DN mice via ERK1/2 pathways, which may provide a new anti-inflammatory target for the prevention and treatment of DN.