| Objective: To treat the rats of acute liver failure induced by D-galactosamine with lipopolysaccharide,using the Chinese traditional medicine of“Clean-heat,cool the blood,disintoxication and Removing blood stasis ” in combination with stem cell transplantation;and to compare the hepatocytes apoptosis of one treatment alone,analyse from the point of view of apoptosis,to explore further the therapeutic mechanism and the synergistic effect.Methods :(1)We isolated rat bone marrow mesenchymal stem cells by using whole bone marrow adherence method,subcultured them in vitro and observed microscopically,and did phenotype identification by flow cytometry.(2)We took 72 clean SD rats(180g-220g),and allocated randomly 6 rats into a blank group and the remaining 66 rats into a model group with acute liver failure induced by D-galactosamine(10%,1.2g/kg)and LPS(0.005%,5 μ g/kg)through intraperitoneal injection,12 hours each in turn,and then observed the model rats’ activities and physiological conditions;12 hours later we took the dead rats to do liver pathological observation,and the preliminary model was established.(3)Another 12 hours later,we randomly allocated the living rats into a model group(a control group),a positive controlgroup,an intervention group(Chinese traditional medicine group),a stem cell transplantation group(a stem cell group)and a combined treatment group(involving stem cell transplantation and Chinese traditional medicine).(4)We had the rats in the combined treatment group injected through portal vein of BMSCs(1.2ml),and the rats in other groups injected of saline(1.2ml).The combined treatment group was given by gavage twice a day with Tradition Chinese drugs pharmaceutics “Yin Chen Si Ling”(1.5g/kg),the Chinese traditional medicine group twice a day withTradition Chinese drugs pharmaceutics “Yin Chen Si Ling”(1.5g/kg),the positive control group twice a day with compound glycyrrhizin tablet(1.5mg/kg)and the rats in other groups with equal volume of saline until the rats were killed.(5)We counted the survival rate of rats within 48 hours and 120 hours of treatment;we took the rats’ blood sample and liver tissue in 48 hours and 120 hours,and determined ALT,AST and TBIL to see if liver function in each group improved and to what extent;we observed the liver tissue pathological changes by HE staining,calculated the apoptosis index by Tunel staining and determined the protein expressions of Caspase-3 by Western-blot,and of Bcl-2 and Bax mRNA by RT-PCR.Results:(1)The primary cell in rats cell was isolated,and after adherence purification and subculture,there were some cells,being fusiform and polygonal,with pseudopodia,in spiring shape and blaze shape.That the positive rate of cell in CD29 was up to 97.9%,CD44-98.2%,CD34-18.7%,CD45-12.9% met BMSCs,which was identified by flow cytometer.(2)The rats were dispirited,somnolent,anorexic 6 hours after the model establishment,and seizures and twitching occurred in a few rats.12 hours later,6 rats were dead in whose liver tissue badly inflammatory cell infiltration was observed by HE staining,necrotizing,bleeding and spreading;hepatic cords were in disorder;hepatic lobules had distinctive damages;vacuolar degeneration was seen in some cells;the acute liver injury was induced so a hepatic failure model came into being,with a suggestion of liver function test results;(3)After hepatic failure model builded 12 hours later,the remaining 58 living rats and 8 rats in the blank group entered into the next phase.There were survival rates of rats in each group(a control group,a positive control group,Chinese traditional medicine group,a stem cell groupand a combined treatment group);48 hours after the treatment,the survival rates is 45.5%,66.7%,72.7%,66.7%,72.7%;120 hours after the treatment,the survival rates is 33.3%,50%,63.6%,58.3%,72.7%.(4)48hours later and 120 hours later,the liver function test told that the rats in each group except the blank one appeared significantly abnormal,in which the combined treatment group was statistically better than other groups(p<0.05),concerning AST,ALT,and TBIL.(5)48 hours later,there were lots of apoptotic hepatocytes in liver tissue was observed by Tunel staining.The apoptosis index of combined treatment group and Chinese traditional medicine group were statistically more decreased than all the control groups(p<0.05).120 hours later,The apoptosis index of each group but for the blank one was significantly idecreased,in which the combinedtreatment group was statistically best(p<0.05).(6)48 hours and 120 hours later,Caspase-3 in each group but for the blank one was significantly increasingly expressed,in which the combined treatment group was statistically better than all the control groups(p<0.05).(7)48 hours and 120 hours later,the expression of Bax mRNA in each group is gradually decreased,while Bcl-2 mRNA increased,where Bax mRNA expression of the combined group was statistically lower all the other groups(p<0.05),and Bcl-2 mRNA higher than the other groups.Conclusion:(1)Purified marrow mesenchymal stem cells are isolated from SD rats bone marrow,by adherence(many times)method,and subculture.(2)We have SD rats injected intraperitoneally of D-galactosamine(10%,1.2g/kg)and LPS(0.005%,5μg/kg)every 12 hours in sequence,so we establish a model of acute liver failure with a low death rate,while the livers have distinctive damages.(3)The combined treatment(the Chinese traditional medicine and the stem cell transplantation)is against the liver dysfunction,protects the liver tissue and inhibits the hepatic apoptosis,whose effect is better than using either of them alone.(4)Tradition Chinese drugs pharmaceutics “Yin Chen Si Ling” betters homeotasis in the rats of acute liver failure,protects from liver damages,decreases the protein expression concerning hepatic apoptosis,and regulates apoptotic gene expression,which,with stem cell transplantation,inhibits apoptosis through the internal environment in cells. |
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