Berberine Alleviates Cisplatin-induced Acute Kidney Injury Via Regulating Autophagy And Apoptosis

Background and ObjectiveCisplatin,as a drug for tumor treatment is widely used in clinic.However,the toxicity and side effects are great.Acute kidney disease(AKI)is a common adverse reaction of cisplatin.The characteristic pathological changes of cisplatin induced AKI are tubule-stromal acute injury,proximal tubular epithelial cell injury and inflammatory response.Berberine can inhibit inflammation and regulate immunity in a variety of diseases,but its role in cisplatin-induced AKI needs further study.Method(1)Male C57BL/6 mice aged 6-8 weeks were randomly divided into control group(saline),cisplatin group(cisplatin)and cisplatin+BBR(5,10mg/Kg)group(BBR).In cisplatin group and BBR group,mice were injected intraperitoneally with 15 mg/kg of cisplatin.In BBR group,mice were given BBR 24h,48h,72h and 30 minutes before and 24h,48 h after cisplatin injection,respectively.All the mice were sacrificed 72 h after cisplatin injection,and serum were collected for detecting serum creatinine(Cre)and BUN levels.Kidney samples were collected for detecting protein expression levels of KIM-1,caspase3,cleaved-caspase3,Bcl-2,LC3Ⅱ/LC3Ⅰ,P62,ULK1,p-ULK1,mTOR,p-mTOR in the renal tissue by Western blotting.The pathological changes in the renal tissues were observed using hematoxylin-eosin staining(HE)and Periodic Acid-Schiff staining(PAS).(2)NRK-52E cells and HKC cells were incubated with various doses(12.5,25,50,100,200 μM)of cisplatin at different time(6 h,12 h,24 h).Cell viability were evaluated by CCK8.Then we evaluated the apoptosis proteins(caspase 3)and autophagy proteins(LC3Ⅱ/LC3Ⅰ,p62)at 12h.Then,cells were pretreated with different concentrations(1μM,2μM,4μM)of berberine.Changes in cell apoptosis were detected and mitochondrial membrane potential(MMP)were detected by flow cytometry,and changes in autophagy and apoptosis-related proteins(LC3μ/LC3Ⅰ,p62,ULK1,pULK1,mTOR,p-mTOR,caspase 3,cleaved-caspase 3,Bcl-2)were detected by Western blot.Changes of apoptosis-related proteins caspase 9,Bax and Cytc in mitochondrial pathway were detected by Western blot.We evaluate the changes of autophagy and apoptosis-related proteins induced by autophagy inhibitors chloroquine(CQ)and caspase inhibitor(Z-VAD-FMK).Results(1)Compared with the control group,the cisplatin-treated mice showed higher serum CRE(P<0.0001)and BUN(P<0.0001).Treatment with BBR at doses of 5 and 10 mg/kg for six days significantly reduced serum CRE(P<0.0001)and BUN(P<0.0001).PAS further revealed that BBR ameliorated cisplatin-induced nephrotoxicity and reduced cisplatin-induced increase in protein expression levels of caspase 3,KIM-1,and p-mTOR.Compared with cisplatin-treated mice,the mice treated with BBR showed increased Bcl-2,LC3Ⅱ/LC3Ⅰ,p-ULKl and decreased p62.(2)When cells were incubated with cisplatin(50μM)for 12 h,cell viability were decreased significantly,the expression of autophagy protein LC3II/LC3I were decreased,and the expression of caspase 3 were up-regulated.Then cells were incubated with BBR for 24 h,LC3Ⅱ/LC3Ⅰ,p-ULK1/ULK1(P<0.05)expression in cells treated with BBR were up-regulated,p62,p-mTOR/mTOR(p<0.05)level were reduced.BBR reversed cell apoptosis and changes in MMP induced by cisplatin.After inhibiting autophagy with CQ,the expression of caspase 3 was up-regulated.Hower,when inhibiting caspase with Z-VAD-FMK,the expression of LC3Ⅱ/LC3Ⅰ were upregulated and the expression of p62 were decreased.Compared with cells incubated with cisplatin,BBR significantly decreased the expression of caspase 9,Bax and Cytc(P<0.05).Conclusions(1)Cisplatin decreased renal function and increased tubular apoptosis in C57BL/6 mice.The intervention of BBR could ameliorate cisplatin-induced renal dysfunction and the apoptosis of renal tubules.In the BBR intervention group,the level of autophagy in renal tissues increased and the expression of apoptotic protein decreased.(2)BBR reduced cell apoptosis induced by cisplatin,improving the decline of MMP,and reduced apoptosis by up-regulating of autophagy of renal tubular epithelial cells.BBR up-regulated autophagy through mTOR/ULK1 pathway and alleviated apoptosis through the mitochondrial pathway.