Regulatory Effects Of Sericin On Rho/ROCK Signal Transduction Pathway In Kidney Of Type 2 Diabetic Rats

Diabetic nephropathy is the primary cause of endstage renal disease and renal replacement therapy in developed countries,and it is also becoming the first cause of endstage renal disease in developing countries including China.The typical pathological changes of diabetic nephropathy are extracellular matrix accumulation and basement membrane thickeningchanges.Studies have shown that Rho/ROCK signaling pathway is closely related to the accumulation of extracellular matrix in kidney,and inhibition of Rho/ROCK signaling pathway may provide a new therapeutic strategy for diabetic nephropathy.Therefore,further study of the role of Rho/ROCK signaling pathway in diabetic nephropathy has important significance.Sericin is a highmolecular watersoluble protein coated on the periphery of silk fibroin.The molecular weight of sericin is 10-300 KD,which is mainly composed of 18 kinds of amino acids,and has the characteristics of biocompatibility and biodegradability.In our country,there had prescriptions for soaking silkworm cocoons in boiling water to control blood sugar.Our previous study also showed that sericin could effectively reduce the blood glucose of type 2 diabetic rats,and had certain preventive effects on blood glucose increasing,but the specific mechanism is still unclear.In this study,the rat model of type 2 diabetes was established by highfat and highsugar diet combined with intraperitoneal injection of streptozotocin,to investigate the regulatory effects of sericin on Rho/ROCK signal transduction pathway in kidney of type 2 diabetic rats,an provide new idea for diabetes mellitus and its complications.Objective:To investigate the regulatory effects of sericin on Rho/ROCK signal transduction pathway in kidney of type 2 diabetic rats by observingthe changes of protein kinase C(PKC),Rho-associated coiledcoilcontainingprotein kinase1(ROCK1),and extracellular matrix related IV collagen(Collagen IV,COL-IV),laminin(LN),biglycan(BGN)and elastin(ELN)expression in kidney of rats model.Methods:1.60 healthy male SD rats were randomly divided into normal(NC)group,type 2 diabetes model(DM)group,low dosesericin(LS)group,high dose sericin(HS)group and metformin positive control(PC)group,with 12 rats in each group.The rats model of type 2 diabetes mellituswas prepared by high fat and high sugar diet combined with intraperitoneal injection of streptozotocin(35mg/kg,2 times).Fasting blood glucose ?11.1 mmol/L was takens as the model standard.After the model was successfully established,the rats in LS group were given 1.8g/kg/d sericin for 35 days,the rats in HS group were given 2.4g/kg/d sericin for 35 days,the rats in PC group were given metformin 55.33mg/kg/d also for 35 days;and rats in NC group and DM group were given the same dose of normal saline for 35 days.2.Glucose oxidase method was used to detect the fasting blood glucose of rats in each group.3.Masson staining was used to observe the degree of renal tissue fibrosis.4.Immunohistochemical staining and Western Blotting were used to detect the expression of PKC,ROCK1,COL-IV,LN,ELN and BGN protein in kidney.5.Realtime PCR was used to detect the PKC,ROCK1,COL-IV,LN,ELN and BGNmRNA expression inkidney.Results:1.Blood glucose of rats in each group:The blood glucose of rats in NC group was(10.831±2.034)mmol/L,and the blood glucose of rats in DM group was(29.455±4.822)mmol/L,which was significantly higher than that in NC group(P<0.05).the blood glucose of rats in LS group,HS group,and PC groupwere respectively(13.202±2.300)mmol/L,(13.200±4.091)mmol/Land(10.040±2.292)mmol/L,which were all significantly lower than DM group(P<0.05).There was no significant differencesabout blood glucose among LS group,HS group and PC group(P>0.05).2.Results of Masson staining of kidney:Compared with NC group,large amount of blue fibers deposited in glomerulus and tubulointerstitium of rats in DM group,the mesangial area enlarged and mesangial matrix was proliferated.Which indicated that the degree of renal fibrosis of rats in DM group was more serious.Compared with DM group,the degree of renal fibrosis of rats in LS group,HS group and PC group alleviated.3.The expression of PKC in kidney of rats in each group:The PKC protein immunopositive products were mainly located in renaltubular epithelial cells,which were brownish yellow and/or tan particles.Compared with NC group,the expression of PKC protein and mRNA in kidney of rats in DM group were significantly increased(P<0.05).Compared with DM group,the expression of PKC protein and mRNA in kidney of rats in LS group,HS group and PC group were significantly decreased(P<0.05);There was no significant differencesabout PKC protein and mRNA expression among LS group,HS group and PC group(P>0.05).4.The expression of ROCK1 in kidney of rats in each group:The ROCK1 protein immunopositive productsmainly located in renal tubular epithelial cells,whichwerebrownish yellow and/or tan particles.Compared with NC group,the expression of ROCK1 protein and mRNA in kidney of rats in DM group were significantly increased(P<0.05).Compared with DM group,the expression of ROCK1 protein and mRNA in kidney of rats in LS group,HS group and PC group were significantly decreased(P<0.05);There was no significant differencesabout ROCK1 protein and mRNA expression among LS group,HS group and PC group(P>0.05).5.The expression of COL-IV in kidney of rats in each group:The COL-IV protein immunopositive products mainly located in the tubulointerstitial and renal tubular epithelial cells,whichwere brownish yellow and/or tan particles.Compared with NC group,the expression of COL-IV protein and mRNA in kidney of rats in DM group were significantly increased(P<0.05).Compared with DM group,the expression of COL-IV protein and mRNA in kidney of rats in LS group,HS group and PC group were significantly decreased(P<0.05);the expression of COL-IV protein and mRNA in the kidney of rats in HS group were significantly lower than LS group(P<0.05).6.The expression of LN in kidney of rats in each group:The LN immunopositive productsmainly located in renal tubular epithelial cells,whichwerebrownish yellow and/or tan particles.Compared with NC group,the expression of LN protein and mRNA in kidney of rats in DM group were significantly increased(P<0.05).Compared with DM group,the expression of LN protein and mRNA in kidney of rats in LS group,HS group and PC group were significantly decreased(P<0.05);There was no significant differencesabout LN protein and mRNA expression among LS group,HS group and PC group(P>0.05).7.The expression of ELN in kidney of rats in each group:The ELN immunopositive productsmainly located in renal tubular epithelial cells and which werebrownish yellow and/or tan particles.Compared with NC group,the expression of ELN protein and mRNA in kidney of rats in DM group were significantly increased(P<0.05).Compared with DM group,the expression of ELN protein and mRNA in kidney of rats in LS group,HS group and PC group were significantly decreased(P<0.05);the expression of ELN protein and mRNA in the kidney of rats in HS group were significantly lower than LS group(P<0.05).8.The expression of BGN in kidney of rats in each group:The BGN immunopositive products mainly located in the distal convoluted tubules and collecting ducts,whichwere brownish yellow and/or tan particles.Compared with NC group,the expression of BGN protein and mRNA in kidney of rats in DM group were significantly increased(P<0.05).Compared with DM group,the expression of BGN protein and mRNA in kidney of rats in LS group,HS group and PC group were significantly decreased(P<0.05);There was no significant differencesabout BGN protein and mRNA expression among LS group,HS group and PC group(P>0.05).Conclusions:Sericin can improve the abnormality of Rho/ROCK signal transduction pathway in kidney during diabetes mellitus by downregulating the expression of PKC and ROCK1,so to inhibit the accumulation of extracellular matrix and delay development of diabetic nephropathy.