The Study Of Mesenchymal Stem Cells Enhanced By Icatritin On Acute Liver Failure

Acute liver failure（ALF）is a kind of syndrome caused by a variety of reasons.It can cause liver cells extensively necrosis,liver function damaged,and cause hepatic encephalopathy in the short term.Although ALF mainly harm liver,but it can further cause multiple organ dysfunction.So its clinical mortality is extremely high（80%to 90%）.Currently,clinical treatment methods such as internal medicine,artificial liver,liver transplantation and hepatocyte transplantation are not effective.In recent years,mesenchymal stem cell（MSC）is started to be used in the treatment of liver failure.Experiments have proved that MSC can improve the survival rate of animal models with liver failure,and improve liver failure patients’symptoms,liver function,survival rate.However,after MSC transplantation,the cells’survival rate and conversion rate are low.Therefore,traditional Chinese medicine can tonify deficiency.So,this study suggested that ICT can strengthen the scientific hypothesis and verify the efficacy of MSC on the treatment of ALF,and provided more ideas for the treatment of acute liver failure.ObjectiveThis study provided evidence-based basis for using MSC to treat acute liver failure and provides experimental evidence for using MSC combined with ICT to treat acute liver failure through literature research and meta-analysis.The study screened the optimal concentration of ICT that can enhance MSC’s anti-apoptotic ability and discussed whether it can enhance the efficacy of MSC in the treatment of acute hepatic failure,and preliminarily explored its possible mechanism.MethodsSuch databases as Pubmed,Embase,CBM,CNKI,VIP database and WanFang Data were searched to collect randomized clinical trials on the effectiveness and safety of Mesenchymal Stromal Cells contrast using medicine for Liver Failure.According to the inclusion and exclusion criteria,literature was screened,data were extracted,and the methodological quality of included studies was also assessed.Then,Meta-analysis was performed using RevMan 5.3 software.In vitro experiment,to establish the apoptosis model of UC-MSC,UC-MSC was cultured in vitro,and the cell apoptosis model was established by H₂O₂ with different concentrations in UC-MSC.The optimal concentration of H₂O₂ was selected by MTS method.Then used different concentrations of ICT to pretreat UC-MSC,and tested UC-MSC’s activity by MTS method.Used QPCR and western blot methods to detect the Cleaved Caspase-3,Bcl-2,Bax mRNA and protein expression level of UC-MSC,and screening the optimal concentration of ICT pre protecting UC-MSC apoptosis state caused by H₂O₂.In vivo experiment,established the rat acute liver failure model by intraperitoneal injected of D-GalN/LPS,and all the rats were randomLy divided into ALF group,UC-MSC group and ICT+UC-MSC group.In addition,normal group rats were not disposed of any treatment.After building the model for 1h,ALF group rats were injected with 1 mL saline into the abdomen,UC-MSC group rats were injected with 1 mL UC-MSC suspension into the abdomen,and ICT+UC-MSC group rats were injected with 1 mL UC-MSC within 0.1μM ICT suspension.The survival rate,serum AST and ALT levels,liver HE staining,the protein expressions of Cleaved caspase-3,bcl-2 and Bax in liver tissues were observed.Results26 RCTs were included eventually among a total of 367 cases on Mesenchymal Stromal Cells in the treatment of Liver Failure.The analysis shows that the effectiveness and safety of MSC in the treatment of Liver Failure is superior than using medicine,which is drawn from total effective rate of treatment group[RR=1.45,95%CI（1.15,1.83）,P<0.005],The TBIL[MD=-66.19,95%CI（-108.11,-24.27）,P<0.005],ALT[MD=-36.91,95%CI（-39.42,-34.39）,P<0.0001]and MELD score[MD=-2.09,95%CI（-2.91,-1.27）,P<0.00001]are both superior to control group after treatment.In vitro experiment,MTS results showed that compared with control group,200μM to 900μM groups’OD values were lower simulated by H₂O₂ after cultivated 6h to 24h,and the difference had statistical significance（P<0.01）,but it did not show obvious concentration and time dependence.We chose using 400μM H₂O₂ to stimulate UC-MSC 6h for further studies.Then using different concentrations of ICT to pre-protect UC-MSC for 2h,and 400μM H₂O₂ stimulated UC-MSC for 6h.MTS results showed that each element of ICT concentration groups all can heighten the OD value of UC-MSC cells,of which 0.1μM group set of OD value most,and differences were statistically significant（P<0.01）;compared with the 0.1μM group,the OD value of0.2μM group and 0.5μM group were decreased,and the differences were statistically significant（P<0.05）.QPCR results showed that,compared with control group,each group of UC-MSC’s Cleaved Caspase-3 and Bax mRNA relative expression were reduced,and the difference had statistical significance（P<0.01）,and 0.1μM group was decreased significantly.Comparison with 0.1μM group,the relative expression levels of Cleaved caspase-3 and Bax in other ICT concentration groups were higher,and the differences were statistically significant（P<0.05）.Compared with control group,the mRNA relative expression level of Bcl-2 in each group was increased,and the increasing was more significant in 0.1μM group,and the difference was statistically significant（P<0.01）.Compared with 0.1μM group,the mRNA expression level of Bcl-2 of other groups were lower,and the differences were statistically significant（P<0.01）.Western blot results showed that,compared with control group,each group’s Cleaved Caspase-3 and Bax protein relative expression were reduced,and the difference had statistical significance（P<0.01）,of which 0.1μM group decreased significantly.Compared with 0.1μM group,the relative expression levels of Cleaved caspase-3 and Bax in the other groups were higher,and the differences were statistically significant（P<0.01）.Compared with the control group,the protein relative expression of bcl-2in each group was increased,and the difference was statistically significant（P<0.01）.Comparison with 0.1μM group,the relative expression of bcl-2 in the other groups was lower.In vivo experiment,the 48h survival rate of ICT+UC-MSC group was 80%,the 48h survival rate of UC-MSC group was 60%,and ALT group was 20%.Compared with ALF group,the serum AST,ALT levels of UC-MSC group and ICT+UC-MSC group were decreased（P<0.01）,and both of the two groups’protein expressions of Cleaved Caspase-3,Bax were reduced,and Bcl-2 was increased（P<0.05,P<0.01）.Compared with UC-MSC group,the serum AST,ALT levels of ICT+UC-MSC group were reduced（P<0.05）,the protein expressions of Cleaved Caspase-3,Bax were reduced（P<0.05,P<0.05）,and Bcl-2 was increased after establishing the models for 24h（P<0.05）.ConclusionThe clinical effects of MSC in the Treatment of Liver Failure is superior to the medicine group.ICT can enhance UC-MSC’s anti-apoptosis ability in vitro.ICT pre-cultivated UC-MSC could improve the acute liver failure rats’survival rate,liver function and pathological changes.The mechanism might be related to regulating the expressions of Cleaved Caspase3,Bax and Bcl-2 genes and proteins.