| Diabetic nephropathy(DN)is one of the most common and serious chronic complications of diabetes mellitus(DM).At present,with the rapid increasing of diabetes mellitus incidence year by year,the incidence of DN is also rising,and becomes the leading factor of end-stage renal disease.Studies have found that inflammation can affect the occurrence and development of DN.Therefore,it is of great significance to investigate the changes of inflammation pathway and inflammatory factors in kidney during DM to prevent and treat DN.Sericin is a water-soluble protein composed of a variety of amino acids,which is biocompatible and biodegradable.According to our previous study,sericin could reduce blood glucose(BG)of diabetic rats and protect kidney damage caused by diabetes,but the specific mechanism is still unclear.In this study,the type 2 diabetes mellitus rat’s model was established by intraperitoneal injection of streptozotocin(STZ)combined with high-fat and high-sugar diet.The effects of sericin on p38 MAPK signal transduction pathway and NLRP3 inflammatory body activation in kidney of type 2 diabetic rats were investigated,in order to provided a new idea for prevention and treatment of DN.Objective:To investigate the effects of sericin on p38 MAPK signal transduction pathway and NLRP3 inflammatory body activation in kidney of type 2 diabetic rats by observing the expression of p38 mitogen-activated protein kinase(p38 MAPK),phosphorylated p38MAPK(p-p38MAPK),MAPK kinase 6(MKK6),nuclear factor-kappa B(NF-κB),nucleotide-binding oligomerized domain-like receptor protein 3(Nod-like receptor protein 3,NLRP3),caspase-1,interleukin-1β(IL-1β)and interleukin-6(IL-6)using immunohistochemical staining,Western blotting,ELISA and Real time PCR.Methods:1.60 male SPF-class SD rats were divided at random into normal control group(NC group),diabetic model group(DM group),low dose sericin treatment group(LS group),high dose sericin treatment group(HS group),positive control group(PC group),12 rats in each group.Rats in DM group,LS group,HS group and PC group were injected STZ(35mg/kg,twice)into peritoneal cavity combined with high-fat and high-sugar diet to establish the model of type 2 diabetes.Rats with fasting BG≥11.1 mmol/L was regarded as the successful model.After the model was successfully established,the rats in HS group and LS group were given different dose sericin(2.4g/kg/d,1.8g/kg/d)for 35 days,the rats in PC group were given metformin(55.33mg/kg/d)for 35 days,and the rats in NC group and DM group were given the same dose of saline for 35 days.2.Glucose oxidase method was used to detect BG.3.HE staining was used to observe the renal morphology and structure changes.4.Immunohistochemical staining was used to detect the expression of MKK6,p-p38 MAPK,NF-κB,NLRP3,caspase-1,IL-1β and IL-6 protein in kidney.5.Western blotting was used to detect the expression of MKK6,p38 MAPK,p-p38 MAPK,NF-κB,NLRP3 and caspase-1 protein in kidneys.6.ELISA was used to detect the content of IL-1β and IL-6 in kidney.7.Real time PCR was used to detect the expression of MKK6,p38 MAPK,NF-κB,NLRP3,caspase-1,IL-1β and IL-6 mRNA in kidney.Results:1.BG of rats in each group:Compared with the BG [(10.83±2.03)mmol/L] of rats in NC group,the BG [(29.45±4.82)mmol/L] of rats in DM group increased significantly(P<0.05).Compared with DM group rats,the BG of LS group rats [(13.18±2.30)mmol/L],HS group rats [(13.20±4.09)mmol/L],PC group rats [(10.04±2.29)mmol/L] significantly decreased(P<0.05);and there had no significant difference in BG level among LS group,HS group and PC group(P>0.05).2.The changes of renal morphology and structure in rats of each groupHE staining showed glomerular hypertrophy,mesangial hyperplasia,basement membrane thickening and extracellular matrix accumulation in kidney of rats DM group.Compared with DM group,the renal pathological changes of rats in LS group,HS group and PC group were significantly improved.3.The expression of MKK6 in kidney of rats in each groupMKK6 immunopositive products were brown or sepia,which were mainly found in the cytoplasm of renal tubular epithelial cells and mesangial cells.The MKK6 protein and mRNA expression in the kidney tissue of DM group were markedly higher than those of NC group(P<0.05);the MKK6 protein and mRNA expression in kidney of LS group,HS group and PC group were evidently lower than those of DM group(P<0.05).4.The expression of p38 MAPK in kidney of rats in each groupThere had no striking difference in p38 MAPK protein and mRNA expression among each group(P>0.05).5.The expression of p-p38 MAPK in kidney of rats in each groupp-p38 MAPK immunopositive products were brown or sepia,which were mainly found in the cytoplasm of renal tubular epithelial cells and mesangial cells.The p-p38 MAPK protein expression in the kidney tissue of DM group were markedly higher than those of NC group(P<0.05);the p-p38 MAPK protein expression in kidney of LS group,HS group and PC group were evidently lower than those of DM group(P<0.05);and the p-p38 MAPK protein expression in kidney of HS group were greatly lower than those of LS group(P<0.05).6.The expression of NLRP3 in kidney of rats in each groupNLRP3 immunopositive products were brown or sepia,which were mainly found in the cytoplasm of renal tubular epithelial cells.The NLRP3 protein and mRNA expression in the kidney tissue of DM group were markedly higher than those of NC group(P<0.05);the NLRP3 protein and mRNA expression in kidney of LS group,HS group and PC group were evidently lower than those of DM group(P<0.05).7.The expression of caspase-1 in kidneys of rats in each groupCaspase-1 immunopositive products were brown or sepia,which were mainly found in renal tubular epithelial cells and mesangial cells.The caspase-1 protein and mRNA expression in the kidney tissue of DM group were markedly higher than those of NC group(P<0.05);the caspase-1 protein and mRNA expression in kidney of LS group,HS group and PC group were evidently lower than those of DM group(P<0.05);and the caspase-1 mRNA expression in kidney of HS group were greatly lower than those of LS group(P<0.05).8.The expression of NF-κB in kidney of rats in each groupNF-κB immunopositive products were brown or sepia,which were mainly found in the renal tubular epithelial cells and mesangial cells.The NF-κB protein and mRNA expression in the kidney tissue of DM group were markedly higher than those of NC group(P<0.05);the NF-κB protein and mRNA expression in kidney of LS group,HS group and PC group were evidently lower than those of DM group(P<0.05);and the NF-κB mRNA expression in kidney of HS group were greatly lower than those of LS group(P<0.05).9.The expression of IL-1β in kidneys of rats in each groupIL-1β immunopositive products were brown or sepia,which were mainly found in the renal tubular epithelial cells and mesangial cells.The IL-1β protein and mRNA expression in the kidney tissue of DM group were markedly higher than those of NC group(P<0.05);the IL-1β protein and mRNA expression in kidney of LS group,HS group and PC group were evidently lower than those of DM group(P<0.05);and the IL-1β mRNA expression in kidney of HS group were greatly lower than those of LS group(P<0.05).10.The expression of IL-6 in kidney of rats in each groupIL-6 immunopositive products were brown or sepia,which were mainly found in the renal tubular epithelial cells and mesangial cells.The IL-6 protein and mRNA expression in the kidney tissue of DM group were markedly higher than those of NC group(P<0.05);the IL-6 protein and mRNA expression in kidney of LS group,HS group and PC group were evidently lower than those of DM group(P<0.05);and the IL-6 protein expression in kidney of HS group were greatly lower than those of LS group(P<0.05).Conclusions:1.The up-regulation of MKK6,p-p38 MAPK,NF-κB,NLRP3,caspase-1,IL-1β and IL-6 in kidney of type 2 diabetic rats suggests that the activation of p38 MAPK signal transduction pathway and NLRP3 inflammatory body may be involved in the development of diabetic kidney damage.2.Sericin can inhibit activation of p38 MAPK signal transduction pathway and NLRP3 inflammatory body by down-regulating the expression of MKK6,p-p38 MAPK,NF-κB,NLRP3,caspase-1,IL-1β and IL-6 in kidney of diabetic rats,so has protective effects on kidney injury duiring DM. |
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