The Study Of The Protection Effect Of Ginsenoside Rg1 Combined With Human Amniotic Mesenchymal Stem Cells On The Cisplatin Induced Acute Kidney Injury In Mice

Objective: To investigate the protective effect of ginsenoside Rg1(Rg1)combined with human amniotic mesenchymal stem cells(h AMSCs)on cisplatin induced acute kidney injury in mice and its related mechanisms.Methods: h AMSCs were isolated by trypsin and collagenase digestion.Flow cytometry was used to identify the phenotype of the subcultured h AMSCs.Acute kidney injury(AKI)model was established by a single intraperitoneal injection of cisplatin(10 mg/ kg).h AMSCs were transplanted by tail vein,and Rg1(0.25 mg/kg/d)was continuously intraperitoneal injected for 7 days.The general state of the animal was observed.On the 3th and 7th day after transplantation,the renal function was assessed by blood biochemistry the histopathological changes were observed by HE staining.The renal injury related proteins were detected by immunohistochemistry,And the ultrastructural changes of renal tubular epithelial cells were obversed by transmission electron microscopy.The renal colonization and distribution of h AMSCs were detected by immunofluorescence and small animal in vivo imaging on the 2th and 8th day after h AMSCs transplantation.Results:(1)The fusiform h AMSCs was subcultured by differential adherent method to passage 3-5.Flow cytometry analysis showed that at least 90% the h AMSCs were expression of CD90,CD105,CD44,CD73,and no expression of CD34,CD45,CD11 b,CD19 and HLA-DR,while high expression of vimentin in cytoplasm of h AMSCs.(2)AKI mice showed different degrees of body weight loss,arch it's back,thin sloppy stool,and so on.(3)On the 3th day after transplantation,the concentration of serum cystatin c(Cyc)was significantly higher in AKI group than the control group(P<0.05).Compared with AKI group,the Cyc levels of Rg1 and Rg1+h AMSCs treated mice were dereased,but was no change was found in the h AMSCs treated mice.On the 7th day after transplantation,the Cyc levels of Rg1,h AMSCs and Rg1+h AMSCs group were obviously reduced.The Cyc level was especially lower in Rg1+h AMSCs group than in the h AMSCs and Rg1 groups(P<0.05).The decrease degree of Cyc in all the four experimental groups on the 7th day were higher than that on the 3th day.(4)There were no significant difference on the levels of urea and Scr between each group(P<0.05).On the 7th day after transplantation,the urea levels of the four experimental groups were significantly lower than that in the AKI group(P<0.05).(5)On 3th day after transplantation,the HE staining showed that the typical histopathologic changes of acute tubular necrosis,exfoliation and a lot of urinary cylinder were found in the AKI group.The above changes were significantly alleviated in the Rg1 group and Rg1+h AMSCs group.On the 7th day after transplation,the pathological injury of Rg1,h AMSCs and Rg1+ h AMSCs groups were significantly improved.The organization structure of Rg1+ h AMSCs group was closest to the control group.(6)Immunohistochemistry test showed that HIF-1?,NAG,Kim-1 proteins all significantly increased in AKI group than that in the control group(P<0.05).On the 3th day,the expression level of HIF-1?,in Rg1 and Rg1+h AMSCs groupwere higher than that in the AKI group(P<0.05),while the expression levels of NAG and Kim-1 were lower than that in the AKI group(P<0.05).The expression of HIF-1? in the four experimental groups on the 7th day were higher than that on the 3th day,while the expression of NAG and Kim-1 on the 7th day were lower than that on the 3th day.The expressions of HIF-1? were higher in Rg1+h AMSCs than that in the Rg1 and h AMSCs groups,but the expression of NAG and Kim-1 were lower in the Rg1+h AMSCs group.(7)On the 7th day after transplantation,the transmission electron microscopy examination revealed severe mitochondrial damage in the AKI group.The damage degree in the Rg1,h AMSCs,Rg1+h AMSCs groups were significantly less than that in the AKI group,and the Rg1+h AMSCs group had the least damage,while there was no significant difference between Rg1 and h AMSCs groups.(8)The MAB281 positive green fluorescence of renal tissue were not seen in the control,AKI and Rg1 groups.on the 3th and 7th day after transplantation,the number of green fluorescent cells in Rg1+h AMSCs group was significantly higher than that in the h AMSCs group.(9)In vivo imaging of small animals showedthat the spleen,liver and lungs of the h AMSCs group and the Rg1+h AMSCs group had the strongest fluorescence,followed by the kidney,and the fluorescence intensity of the Rg1+h AMSCs group was higher than that in the h AMSCs group.Conclusion: Rg1 combined with h AMSCs transplantation has obvious protective effect on cisplatin induced AKI in mice.The efficacy of Rg1+h AMSCs is better than Rg1 or h AMSCs alone.The mechanism may be related to the promotion of h AMSCs homing,colonization,survival,and involves the protection of mitochondria in renal tubular epithelial cells.It may also be related to HIF-1? protein expression up-regulation.