Study On The Mechanism Of Shenshuai Ⅱ Recipe On Aerobic Glycolysis In Improving Renal Interstitial Fibrosis Based On IL-1β/IRAK4/c-Myc Pathway

Objective: The effect of Shenshuai Ⅱ recipe(SSR)on Interleukin(IL)-1β/IRAK4/c-Myc pathway and aerobic glycolysis was observed in the 5/6(ablation/infarction,A/I)rat model of chronic renal failure in vivo and the activation model of rat renal fibroblasts(NRK-49F)induced by IL-1β in vitro.The purpose of this study is to reveal the mechanism of SSR in improving renal interstitial fibrosis(RIF).Methods: 1.In vivo experiment: 55 SD male rats were randomly selected to establish the rat model of chronic renal failure by 5/6 renal ablation and infarction(5/6(A/I)),and another 12 rats were selected as sham operation group.The rats were randomly divided into sham operation group,model group(5/6(A/I)),Chinese medicine group(5/6(A/I)+SSR)and western medicine group(5/6(A/I)+LOS).8 weeks after treatment,the general condition of rats was observed.The renal function(urinary creatinine,serum creatinine(Scr),blood urea nitrogen(BUN),uric acid(UA)),liver function(alanine aminotransfease(ALT)aspartate transaminase(AST))were detected by automatic biochemical analyzer.HE staining,PAS staining and Masson staining were used to observe the changes of renal histomorphology and interstitial collagen deposition.The concentration of lactic acid in serum and renal tissue and pyruvate acid in renal tissue were measured by colorimetry.The activity of hexokinase(HK)was measured by ultraviolet method.The expression of α-smooth muscle actin(α-SMA),HK2 was detected by immunohistochemistry.The expression of HK2,M2-pyruvate kinase(PKM2),lactate dehydrogenase A(LDHA),IL-1 receptor Ⅰ(IL-1RⅠ),IRAK4 and c-Myc m RNA in rat kidney was measured by Real time PCR(RT-PCR).The protein expressions of fibronectin(FN),collagen Ⅰ(Col-Ⅰ),α-SMA,proliferating cell nuclear antigen(PCNA),HK2,phosphofructokinase FB3(PFKFB3),PKM2,phosphorylated-PKM2(P-PKM2),IL-1β,IL-1RⅠ,IRAK4 and c-Myc were detected by Western Blot(WB).2.In vitro experiment: NRK-49 F cells were stimulated by IL-1β(10ng/ml)for 24 hours to establish NRK-49 F activation model.First of all,the effects of different concentrations of SSR on NRK-49 F cell viability were detected by CCK-8 method,and the optimum concentration of SSR was determined.Secondly,the experiment was divided into control group,model group,low dose group of SSR(SSRL,200mg/L),high dose group of SSR(SSRH,400mg/L),and control group of 2-deoxy-D-glucose(2-DG,10 m M).The concentration of lactic acid in medium was detected by colorimetry,the concentration of glucose in medium was detected by glucose oxidase method.The expression of α-SMA and HK2 protein was detected by immunofluorescence.The protein expressions of FN,α-SMA,PCNA and HK2 were measured by WB.Finally,NRK-49 F cells were silenced by IRAK4 si RNA and c-Myc si RNA genes,IRAK4 m RNA and protein expression were detected by RT-PCR and WB in IRAK4 si RNA group and IRAK4 si RNA+SSRH group.The concentration of lactic acid and glucose in the culture medium,the m RNA expression of c-Myc and the protein expression of c-Myc,FN,Col-Ⅲ,PCNA and HK2 in c-Myc si RNA group and c-Myc si RNA+SSRH group were detected.Results: 1.In vivo experiment: Compared with the sham operation group,the general condition of rats in the model group was worse,Scr,BUN,UA increased,Ccr decreased;renal morphology and structure were abnormal: glomerulosclerosis,thickening of glomerular basement membrane,inflammatory cell infiltration,interstitial collagen deposition significantly increased.Lactic acid concentration in serum and renal tissue increased;lactic acid/pyruvate ratio increased,renal tissue HK activity increased;the m RNA expression of HK2,PKM2,LDHA,IL-1RⅠ,IRAK4,c-Myc and the protein expression of FN,Col-I,α-SMA,PCNA,HK2,PFKFB3,PKM2,P-PKM2,IL-1β,IL-1RⅠ,IRAK4 and c-Myc were increased.Compared with the model group,SSR could improve the general state and pathological changes of kidney tissue,decrease the levels of Scr,BUN and UA,and increase the level of Ccr.Decreased serum and renal tissue lactic acid,renal tissue lactic acid/pyruvate ratio,and renal tissue HK activity,down-regulated the m RNA expression of HK2,PKM2,LDHA,IL-1RⅠ,IRAK4,c-Myc,and decreased the protein expression of FN,Col-Ⅰ,α-SMA,PCNA,HK2,PFKFB3,PKM2,P-PKM2,IL-1β,IL-1RⅠ,IRAK4 and c-Myc.2.In vitro experiment:(1)Compared with the con group,SSR with the concentration below 400mg/L had no significant effect on the viability of NRK-49 F cells.(2)Compared with the con group,the p H value and the glucose concentration of culture medium decreased,the concentration of lactic acid increased,and the expression of FN,α-SMA,PCNA and HK2 in NRK-49 F cells increased in the IL-1β group.Compared with the IL-1β group,the p H value and glucose concentration of cell culture medium in each intervention group increased,and the concentration of lactic acid significantly decreased.The protein expression of FN,α-SMA,PCNA and HK2 in NRK-49 F cells decreased,but only SSRH group and 2-DG group decreased significantly.(3)Compared with the con group,the expression of IRAK4,c-Myc m RNA and protein in the IL-1βgroup increased.Compared with the IL-1β group,the expression of IRAK4,c-Myc m RNA and protein in SSRH group,IRAK4/c-Myc si RNA group and IRAK4/c-Myc si RNA+SSRH group decreased significantly,especially in IRAK4/c-Myc si RNA+SSRH group.In addition,the p H value of cell culture medium and glucose concentration in c-Myc si RNA group increased,lactic acid concentration decreased,and the expression of FN,Col-Ⅲ,PCNA and HK2 protein decreased.(4)Compared with c-Myc si RNA group,the p H value of culture medium,glucose concentration,lactic acid concentration,FN,Col-Ⅲ,PCNA and HK2 protein expression in c-Myc si RNA+SSRH group had no significant difference.Conclusion: 1.SSR can effectively improve the renal function and renal histopathology of rats with chronic renal failure,reduce the deposition of extracellular matrix and the proliferation and activation of fibroblasts,and has definite anti-fibrosis effect.2.SSR can reduce the expression of the transcription factor c-Myc in the renal tissue of rats with chronic renal failure and inhibit c-Myc-mediated aerobic glycolysis,thereby reducing the renal interstitial fibrosis.3.SSR can inhibit renal interstitial fibrosis in rats with chronic renal failure and proliferation and activation of IL-1β-induced NRK-49 F cells.The mechanism may be related to the inhibition of SSR on IL-1β-induced activation of IRAK4/c-Myc pathway,which in turn inhibits renal aerobic glycolysis.