The Study On Syk/JNK/NLRP3 Signal Transduction Pathway In Diabetic Nephropathy

Objective Diabetes mellitus(DM)is a group of chronic metabolic disease that is common,persistent hyperglycemia can lead to metabolism disorder of glucose,protein,fat,water and electrolyte,resulting in damage of target organs and great vessels and causing diabetic foot,retinopathy,renal injury and other complications.Diabetes nephrophathy(DN)is the one of the common microvascular complication secondary to diabetes and the final end stage renal disease(ESRD)caused by DN is the main causation of death in diabetic patients.It has been reported that the NLRP3 inflammasome was involved in the pathogenesis of DN,however,its specific role in the pathogenesis of DN has not been fully illuminated.Studies showed that the spleen tyrosine kinase(Syk)or c-Jun N-terminal kinase(JNK)may participate in the regulation of NLRP3 activation,but it is unclear that whether Syk plays a great role in the mechanism of DN with inflammatory injury and what's the concrete relationship among Syk,JNK and NLRP3 inflammasome in DN.Therefore,we detected the expression of Syk,JNK and NLRP3 inflammasome in type 1 diabetic rats and renal cells induced by high glucose,and explored the role of Syk/JNK/NLRP3 signal transduction pathway in the pathogenesis of diabetic nephropathy.Methods1.Intraperitoneal injection of 65 mg/kg STZ solution(1 % STZ)to establish animal model of type 1 diabetes in SD rats2.Twenty four hours urinary protein and the ratio of KW/BW(kidney weight/body weight)were measured to identify a rat model of diabetic nephropathy in SD rats.3.HE(Hematoxylin-eosin)and PAS(Periodic Acid-Schiff)staining were used to observe the pathological changes of renal tissue structure in SD rats under light microscope.4.Western Blot,RT-PCR and immunohistochemical staining were used to detect the expression of NLRP3 inflammasome and IL-1? in kidney tissue of DN rats.5.Western Blot was used to detect the expression of p-Syk and p-JNK in kidney tissue of DN rats.6.Expression of p-Syk,p-JNK,NLRP3,ASC,caspase-1 and IL-1? in high glucose-induced human renal tubular epithelial cell line HK2 were detected by Western Blot.7.HK2 cell line was pretreated with SP(JNK inhibitor)for 2 h and cultured for 24 h with high glucose and Western blot was used to detect the expression of NLRP3 inflammasome and IL-1?.8.HK2 cell line and rat glomerular mesangial cells were pretreated with BAY61-3606(Syk inhibitor)or Syk-si RNA,cultured in high glucose for 10 min or 24 h.Western Blot was used to detect the expression level of p-JNK or NLRP3,ASC,and caspase-1,IL-1?.9.Western Blot and flow cytometry was used to detect high glucose-induced apoptosis of HK2 cells.Results1.We successfully constructed animal models of type 1 diabetes in SD rats: After a single intraperitoneal injection of large doses of STZ for 72 hours,the random blood glucose in the DN group rats was higher than 16.7 mmol/L,showing:Polyuria,drink more,eat more and lose weight symptoms.The blood glucose concentration in DN group rats ware significantly higher than that in Con group rats(P < 0.001),and the body weight was significantly lower(P < 0.05).2.Successful establishment of an SD rat animal model of diabetic nephropathy: the24-hours proteinuria in DN rats was significantly elevated(P < 0.01),and the KW/BW value was significantly higher in DN group rats than that in Con group(P < 0.05).3.The results of HE staining showed that compared with the Con group,the renal tissue structure of the DN group was disordered,the fibrosis was obvious,and the number of renal tubular epithelial cells showed swelling and vacuolar degeneration.The results of PAS staining showed that the basement membrane thickened and the mesangial matrix deposition was seen in the kidney tissue of DN rats compared with the Con group.4.Western Blot results of rat kidney tissue showed that the expression of NLRP3 inflammasome and IL-1? protein in kidney tissue of DN group was significantly higher than that of Con group(P < 0.01).RT-PCR results showed that the m RNA levels of NLRP3 inflammasomes and IL-1? in kidney tissue of DN rats were significantly higher than those in Con group(P < 0.05).Immunohistochemical staining showed that the expressions of NLRP3,caspase-1,and mature IL-1? in DN rat kidney were significantly higher than that in Con group(P < 0.01).5.The Western Blot results of rat kidney tissues showed that compared with Con group,the expressions of p-Syk and p-JNK protein in kidney tissues of DN group was significantly increased(P < 0.001).6.The results of Western Blot showed that the expression of p-Syk,p-JNK,NLRP3,ASC,caspase-1 and IL-1? in HK2 cells induced by high glucose(HG)was significantly higher than that in Con group(normal glucose)(P < 0.05).7.The results of Western Blot showed that the expression of NLRP3 inflammasomes and IL-1? in HK2 cells was significantly lower in the inhibitor group(SP pretreatment for 2 h,then 25 mmol/L glucose culture)than in the high glucose group(25 mmol/L)(P < 0.01).8.Western Blot results showed that HK2 cell line and rat glomerular mesangial cells were pretreated with BAY61-3606 or Syk-si RNA and then incubated with 25mmol/L high glucose,the expression levels of p-JNK or NLRP3 inflammasomes and IL-1? in the inhibitor group and Syk-si RNA interference group were significantly lower than those in the high glucose group(P < 0.05).9.Western Blot results showed that the apoptotic level of HK2 cells in high glucose group was significantly higher than that in normal control group(P < 0.001).The apoptosis of HK2 cells was significantly lower in the inhibitor group(BAY61-3606 pretreatment for 2 hours,then 25 mmol/L glucose culture)than in the high glucose group(P < 0.05);the result of flow cytometry was consistent with that of Western Blot.Conclusions1.We constructed a rat model of type 1 diabetes in SD rats;2.We established a rat model of diabetic nephropathy in SD rats successfully;3.The p-Syk,p-JNK,NLRP3 inflammasomes and IL-1? participate in the pathogenesis of Diabetic Nephropathy;4.Syk/JNK/NLRP3 signal transduction pathway is involved in the pathogenesis of diabetic nephropathy;5.Syk-mediated apoptosis of HK2 cells is closely associated with the pathogenesis of DN.