Regulation Of Yinchen Siling Granule Combined With Bone Marrow Mesenchymal Stem Cells On Mitochondrial Respiratory Chain In Rats With Acute Liver Failure

Object: To investigate the regulatory effects of Yinchen Siling Granule combined with bone marrow mesenchymal stem cells(BMSCs)transplantation on acute liver failure(ALF)rats’ mitochondrial respiratory chain complexes and Adenosine Triphosphate(ATP).Methods: Adopted Yinchen Siling granule combined with BMSCs transplantation were used to treat ALF,detection of liver function,associated mitochondrial apoptosis proteins in ALF rats,and observation of liver pathology and hepatocyte mitochondrial ultrastructure,also determination of respiratory chain complexes I,II,III and mitochondrial ATP activity.The specific experimental steps were as followed:1.Extraction and identification of Rats Bone Marrow Mesenchymal Stem Cells: BMSCs were isolated in vitro from SD rats’ bones,cells were purified,cultured,and passed.Cell surface markers were identified by flow cytometry after passing to the 3rd generation.2.Grouping and Molding :60 SD healthy male rats were randomly and equally divided into normal group,combined group,Chinese medicine group,stem cell group,control group and model group(ALF group).The normal group was injected with 0.9% Nacl solution intraperitoneally,and the remaining 5 groups were injected intraperitoneally with thioacetamide(TAA,300 mg/kg)to construct ALF model,repeated once after 24 h.3.Interventions: After the 2nd intraperitoneal injection of TAA,Normal and model groups: gavage of equal amounts of 0.9% Nacl solution,Chinese medicine group:1.5g/kg of the solution of Yinchen Siling granule by gavage,Control group: gavage of compound glycyrrhizin tablet solution 7.5 mg/kg,Stem cell group: Tail vein injection of BMSCs suspension(cell count approximately 1.0 × 106 cells/m L)in the cell group,Combined group: gavage of Yinchen Siling solution 1.5g/kg + tail vein injection of BMSCs suspension(cell count about 1.0×106 cells/m L),total intervention 48 h,q12h intervention 1time.4.Result detection:72 hours after the rats were intervened,detection liver function(ALT,AST,TBIL),observed pathological structure of the liver tissues,and by transmission electron microscopy(Transmission Electron Microscope,TEM)observed the ultrastructural changes of hepatocyte mitochondria.Changes in the activity of respiratory chain complexes(I,II,III)were detected by UV spectrophotometer.By phosphomolybdic acid colorimetry,determination of mitochondrial ATP in liver and hepatocytes,and the expressions of apoptosis proteins(Bcl-2 and Caspase-3)were detected by Western blot.Results:1.Successful identification of rat BMSCs: Flow cytometric identification of 3rd generation cell surface markers: strong positive for CD29/90,negative for CD45/34.2.Rat liver function results: After 72 h of intervention,liver function(AST,ALT,TBIL)was least impaired in the combined group compared with each intervention group(P<0.05).3.Rats’ histopathological results of liver tissues: Yinchen Siling granule combined with BMSCs transplantation could reduce mitochondrial damage in ALF rats hepatocytes.4.Rats’ liver c mitochondrial complexes activity and ATP concentration results: Compared with the model group,the activities of Complex I and Complex III and the concentration of ATP in the liver mitochondrial extract of the rats in the combined group were increased,and the difference was significant(P<0.05).5.Results of apoptosis protein Bcl-2,Caspase-3: Compared to each intervention group,The highest mean level of Bcl-2 protein expression(P<0.05)and the lowest mean level of Caspase-3protein expression(P<0.05)were found in the combined group.Conclusion:1.The liver function injury was the least in the Yinchen Siling granule combined with BMSCs transplantation group.2.Yinchen Siling granule combined with BMSCs could increase the survival rate of ALF rats,reduced the degree of hepatocyte and hepatocyte mitochondrial degeneration and necrosis,and inhibited hepatocyte mitochondrial damage.3.The mitochondrial respiratory chain of ALF rats was regulated by combining Yinchen Siling granule with BMSCs.The mechanism might be related to increased hepatocyte mitochondrial respiratory chain complexes(I,II,III)and ATPase activity,enhanced mitochondrial energy metabolism,upregulated apoptotic protein Bcl-2,and inhibited of Caspase-3 levels.