Protective Effect Of Chinese Herbal Medicine On Hepatocytes And Its Mitochondrial Mechanism

Hepatocytes are the most abundant cells of the liver (about80%). Although hepatocytes are not the main sources of ECM, hepatocyte damage plays a critical role in the development of hepatic fibrosis, particularly hepatocyte apoptosis. Mitochondria are the main sources of ROS and are the key action site of ROS. Therefore, finding new natural antioxidants inhibit liver cell apoptosis by protecting mitochondria against ROS, which is beneficial to hepatic fibrosis therapy.Cordyceps sinensis is a tonic traditional Chinese medicine, which have many useful biological activities including antitumor, antiinfluenza virus, immunopotentiation, hypoglycemic activity, hypocholesterolemic. Many evidences appear that cordyceps plays antioxidant activities in oxidative stresses induced cell injury. Cordceps contains many effective gradients such as cordyceps polysaccharide, cordycepin, cordycepic acid etc. The aim of our study is to investigate the mitochondrial protection of CPS, cordycepin, cordycepic acid on hydrogen peroxide induced heptocyte apoptosis. MTT screen the optimum drug concentration, Hoechst33258measures cell apoptosis, and detecting ROS, MMP, ATP and Cyto C, western blotting determines the expression of Bax and Bcl-2. Our results show that CPS increases cell viability and MMP, elevates ATP level, eliminates ROS, inhibits the release of Cyto C, descends the ratio of Bax/Bcl-2. These data indicate that CPS protects mitochondria from oxidative stress induced cell apoptosis by eliminating free radicals. However, cordycepin and cordycepic acid are not significant protection of mitochondria.Salvianolic radix, one of the most commonly used traditional Chinese herbs, was widely used for treating many types of diseases, including cerebrovscular, hepatic cirrhosis, hepatic fibrosis, cardiovascular etc. Many researches find that Salvianolic radix acts key roles in hepatic fibrosis therapy; the mechanism of salvianolic radix is its antioxidant activities. Salvianolic radix’s effective gradients include lipophilic compounds and hydrophilic compounds, the hydrophilic compounds take main roles especially phenolic acids compounds which have strong antioxidant activities.The aim of our study is to investigate the mitochondrial protection of SalB, Salvianic acid A sodium, protocatechualdehyde on hydrogen peroxide induced heptocyte apoptosis. The experiments are same as the part of cordyceps. The results show that SalB increases cell viability and MMP, elevates ATP level, eliminates ROS, inhibits the release of Cyto C, descends the ratio of Bax/Bcl-2and suppresses the pro-caspase3 activation. These data indicate that Sa1B protects mitochondria from oxidative stress induced cell apoptosis by eliminating free radicals. However, Salvianic acid A sodium and protocatechualdehyde don’t have significant mitochondrial protection.Mitochondria are dynamic organelles that constantly fusion and fission, the dynamic balance of fusion and fission maintain mitochondrial morphology and function. Mitochondrial morphology is regulated by mitochondrial fusion proteins (Mfn1, Mfn2and Opal) and mitochondrial fission proteins (Drpl, Hfis1and MTP18). In response to an oxidative stress, cells undergo mitochondrial fission generated fragmented mitochondria. Mortalin is a molecular chaperone70family member that is located primarily in mitochondria, where it functions to maintain quality control, stress reaction, cell signal transduction. Our previous findings appear that mortalin over expression can eliminate cellular ROS, keep MMP stability, inhibit the release of Cyto C, suppress Bax conformational change, prevent glucose deprivation induced cell apoptosis. The second part results show that Sa1B prevent mitochondrial dysfunction from oxidative stress. Therefore, our aims are to study the protective effects of Sa1B and mortalin on hydrogen peroxide induced mitochondrial fragmentation.MTG mark mitochondrial morphology, CLSM observes mitochondrial dynamic, construct mortalin over-expression and knockdown plasmid, real time PCR detects mRNA expression of mitochondrial fusion and fission relative factors, western blotting detects protein expression level of fusion and fission relative factors. These results show that SalB and mortalin over-expression suppress hydrogen peroxide induced mitochondrial fragmentation. Mortalin knockdown have no significant alterations of mitochondrial morphology, but mitochondrial shape obvious truncated and fragmented in mortalin knockdown group under hydrogen peroxide induced stress conditions. Cells treatment with Sa1B can elevates mortalin protein expression level. Mortalin over-expression ascends the mRNA expression level of Mfn1, Drp1and Opal, descends mRNA expression level of Hfis1and MTP18, has no effects on Mfh2. SalB decreases the ratio of Bax/Bcl-2and has no effects on the protein expression level of Mfn2and increases Hfisl. Mortalin over-expression decreases Bax/Bcl-2and Hfis1, no effects on the protein expression level of Drpl. In conclusion, Sa1B up-regulate mortalin and mortalin protects mitochondrial morphology by modulating fusion and fission relative factors.