| Cholestasis is a common clinical symptom of many diseases, which are caused by thebile formation disorder or flow obstacle.Cholestasis is a clinical manifestations of manyhepatobiliary diseases, such as gallstones, bile duct inflammation, cholangiocarcinoma. Inrecent years, there is an upward trend in the intrahepatic cholestasis due to iatrogenic bileduct injury with the development of laparoscopic technique popularity. Cholestasis ischaracterized by the continued retention of high concentrations of toxic bile acids in theblood and liver, which may lead to hepatocellular damage, bile duct proliferation, liverfibrosis and even liver failure if not correctly treated. A big progress has been made in thestudy of the molecular mechanisms of cholestasis leading to hepatocellular damage, however,we are still short of effective treatment, especially for the patients who are unable to carry outsurgical therapy. In summary, our study explore the molecular mechanism of liver cell injurycaused by cholestasis, which may provide a new idea for drug treatment of cholestasis.Autophagy is a cellular process in which cells degrade some impaired organelles andmacromolecules with lysosomes, providing energy, amino acids for cells to maintain thebalance of cellular homoeostasis and protein metabolism. As a protective mechanism,autophagy plays an important role in protecting from apoptosis in many disadvantagedenvironment and keeping cells alive. It plays a positive role when cells are lack of growthfactor and nutriment deprivation, especially in cells with injury. The study has confirmed thatautophagy can protect liver cells from injury induced by glycochenodeoxycholate(GCDC),but the role of autophagy in the process remains unclear. To solve the problem, this studyproceeded with the following three aspects:1.To examine the effect of cholestasis onhepatocytes in vitro experiments and in vivo animal bile duct ligation model;2. Investigatethe role of autophagy in cholestatic liver injury;3. Study the specific molecular mechanismswhether autophagy protect liver cells from injury induced by cholestasis through decreaseingROS, and explore the role of ROS in cholestasis-induced autophagy.Part1Validation of hepatocellular damage model induced by cholestasis in vitroand in vivo.First of all, we established the GCDC-induced hepatocellular damage model in vitro. Normal liver cell line L02was treated with different doses of GCDC and was detected atdifferent times. The results suggested that GCDC can lead to half of liver cells death atconcentration of200μM when treated with24hours. GCDC has positive dose and timedependent effect on liver cells death. We carried out bile duct ligation (BDL) experiment atthe same time to simulate the pathophysiological process of cholestasis in vivo. Mice serumwas detected for bilirubin, ALT, AST to affirm whether the cholestasis model wassuccessfully constructed. The results suggested that the bilirubin, ALT and AST of mouseserum in BDL group had significantly increased, indicating that cholestasis model wasestablished successfully.Part2The role of autophagy in hepatocellular damage induced by cholestasisOn one hand, L02cells were transfected with plasmid expressing GFP-LC3which can beused as a marker of autophagy, and then treated with GCDC(200μM) for24hours. ThenGFP-LC3was observed to examine autophagy indirectly under fluorescence microscope andLC3expression at the protein level was confirmed by western blotting. Meanwhile,autophagy inhibitor chloroquine (CQ) or autophagy inducer rapamycin were applicated toL02, and then examine the change of apoptosis and necrosis by flow cytometry and TUNELassay in order to further clarify whether autophagy was involved in liver cell injury caused bycholestasis in vitro. On the other hand, CQ or rapamycin was used in BDL model to observesurvival time and pathologic changes of the mice under a fluorescence microscope andwestern blotting. The result showed that boht hepatocyte apoptosis and necrosis weresignificantly induced by inhibiting autophagy. In BDL model, survival time of CQ group wassignificantly shorten and liver necrosis was increased significantly while rapamycin groupgave the opposite effect. We concluded that GCDC activated autophagy can protecthepatocytes from cholestasis by inhibiting apoptosis and necrosis.Part3Autophagy protect liver cells from cholestasis induced injury throughdecreasing accumulated ROS.Further experiment were carried out to explore the molecular mechanisms of ROS inGCDC induced hepatocellular damage. First, we examined ROS activity treated with GCDC. We found that GCDC was able to induce the generation of ROS which can be inhibited byantioxidant NAC. Then, in order to explore the relationship between ROS and autophagy,western blotting was done to detect the change of LC3expression in different group. Theresults showed thae NAC partly decreased expression of LC3, indicating that expression ofROS may be partly involved in the regulation of autophagy induced by GCDC. In vivoexperiment,8-OHdG which is a indicator of ROS was detected to validate the result. Wefound that the level of8-OHdG in the BDL group was higher than that in CQ+BDL group,indicating that ROS activity was enhanced after inhibition of autophagy. It suggested thatROS may be involved in the BDL-induced autophagy. At last, in order to study therelationship between protective role of ROS and hepatocellular damage induced by GCDC,we detected the the level of ROS in different group, and found that ROS increased in CQ+GCDC group; MTT assay aslo confirmed that cell survival rate was significantly increasedwhen NAC was added. All these results suggested that autophagy can protect liver cells fromcholestasis induced damage by diminishing the level of ROS.To sum up, we conclude as follows:1. Cholestasis can induce autophagy in hepatocytes; autophagy protects hepatocytes fromdamage by reducing apoptosis, which will be reversed while autophagy is inhibited;2. ROS is involved in GCDC induced autophagy which can protect liver cells fromcholestasis induced damage by diminishing the level of ROS. |
