Effect Of Yishen Traditional Chinese Medicine Compound Mixture On NRK-52E Cells And Biosafety Evaluation

ObjectiveChronic renal failure?CRF?usually accompanies by renal fibrosis,there is a close correlation between renal fibrosis and epithelial mesenchymal transition?EMT?,and related studies on the use of NRK-52E cells for EMT have been mature.In order to investigate the effects of the Yishen mixture on normal rat kidney cells?NRK-52E?and abnormal NRK-52E cells with EMT,and evaluate the biosafety of the Yishen mixture on zebrafish embryo hatching and larvae feeding.The purpose is providing further evidence of the Yishen mixture for CRF treatment.MethodsThe appropriate concentration and time of the Yishen mixture were selected through cell proliferation assay,cell reactive oxygen species?ROS?level,and Na⁺-K⁺-ATPase test with NRK-52E cells.TGF-?1 was used to induce EMT of NRK-52E,which was determined through cell proliferation,cell morphology,and cell migration.Further,cell apoptosis and the activity of Na⁺-K⁺-ATPase were observed to check the effects of intervention by the Yishen mixture.Finally,the embryo and larval of zebrafish were used to evaluate the biosafety of the Yishen mixture.Results?1?Different concentrations of the Yishen mixture could promote the proliferation of NRK-52E cell for 6 h?p<0.05?.However,each group failed to promote cell proliferation for 12 h.In the meantime,the ROS level of almost all groups significantly increased compared to the control except for treating 6 h with the 0.75 mg/mL Yishen mixture?p<0.05?.?2?NRK-52E cells had arisen successfully epithelial-mesenchymal transition?EMT?induced by TGF-?1.When treated with the 0.75 mg/mL Yishen mixture for 6 h,the apoptosis rate and activity of Na⁺-K⁺-ATPase of the abnormal NRK-52E cells with EMT both decreased?p<0.05?.?3?When zebrafish embryos were exposed to the Yishen mixture,just the 800?g/mL group delayed the incubation,and the zebrafish in each group had normal physiological morphology with yolk in 120 hpf.When zebrafish larvae were exposed to the Yishen mixture for 96 h,the mortality rate increased as the concentration increased,but the96h-LC₅₀ was greater than 2500?g/mL.In addition,there was no significant concentration dependence for mortality at both embryonic and larval levels.According to the acute toxicity rating scale of Fish and Wildlife Authority?FSW?,when the median lethal concentration exceeds 100?g/mL,it is almost non-toxic.The results showed that the Yishen mixture is safe and non-toxic to fish.ConclusionThe Yishen mixture can promote the normal NRK-52E cells proliferation and reduce the activity of Na⁺-K⁺-ATPase of the abnormal NRK-52E cells with EMT,and preliminarily reveal the mechanism of the Yishen mixture in the treatment of CRF.