Mechanism Study Of Kaempferol's Protective Effects Against Alcoholic Liver Injury

Alcoholic liver disease?ALD?is the most widespread cause of severe liver disease with a global health concern;it can be induced by chronic and binge alcohol consumption.Dehydrogenase?ADH?and Cytochrome P450?CYP450?are in charge of the metabolism of alcohol after administrated.The metabolites of alcohol are acetaldehyde and reactive oxidative species?ROS?,which are the main harmful factors of liver oxidative stress.Human beings are the main infected target,so the suitable and reasonable animal ALD models should be established according to the ethics.Nowadays the treatment for ALD is based on the abstinence or liver transplant,because there are no specific and effective medicines for the ALD treatment.Our study is aimed to select the effective natural products from flavonoids by an improved in vitro drug selection system and explorer its effect on ALD and the potential mechanism behind.The flavonoids have the activities of anti-oxidative,anti-inflammatory and anticancer as well.We improved the traditional drug selection method targeting to CYP2E1,the microsomal CYP2E1 was instead by active liver homogenate-mitochondrial CYP2E1.This improved method was much more sensitive and effective than the microsomal system.The selected kaempferol has a wide range of biological activities and is widely presented in various vegetables,fruits and tea.The ALD model in mice was successfully constructed and kaempferol's effects were evaluated on this model.Based on the results of pharmacodynamics study on kaempferol,the further study was performed to explore the potential mechanism.The main investigations and results are as follows:1.Construction of ALD mode in mice.The ethanol at analytically pure level?Ethanol group?and edible alcohol?Alcohol group?were given to the normal mice byintragastric administration for 4 weeks separately.The ethanol groups were divided into 2 groups fed with high fat liquid diet and normal diet individually,as well as the alcohol group.The following indices were tested:the clinical symptom,body weight,relative liver ratio,activities of glutamic oxalacetic transaminase?GOT/AST?and glutamate pyruvate transaminase?GPT/ALT?and histopathological observation.Take all the results into account,the alcohol group with high fat liquid diet was the most suitable method to set up the ALD model in mice.2.Development of improved in vitro drug selection system targeting to CYP2E1.The traditional method was performed to select the inhibitors targeting to CYP2E1 in liver microsome.The liver microsome was mainly composed with endoplasmic reticulum?ER?.Protease was abounding in ER and CYP2E1 was degradation easily.In this study,the improved in vitro drug selection system includes liver homogenate with bioactivities and mitochondria.Comparing to the original method,the improved system is much more stable,effective,sensitive and efficient.The best natural product was kaempferol with IC₅₀ 16.28?M which was much lower than other flavones.3.Effects of kaempferol against ALD.To evaluate kaempferol effects on liver function,serum AST and ALT activities,anti-oxidative defense activities and ROS contents were detected and histopathology.According to the serum biochemistry test and histopathology assay,kaempferol at the dose of 40 mg/g.b.w and 80 mg/g.b.w was proved to be a potential preventive and therapeutic drug for ALD.4.The in vivo mechanism study on ALD intervention by kaempferol.Alcohol dehydrogenase,cytochrome 2E1?CYP2E1?and specificity protein 1?SP1?were determined and the down-regulation of CYP2E1 and SP1 was confirming that kaempferol decrease the generation of reactive oxidative species?ROS?in ALD.The antioxidative level was measured and the results showed that glutamate-cysteine ligase,glutathione and nuclear factor-E2-related factor 2 were increased,which suggested that kaempferol could accelerate the ROS clearance.The protective mechanism for kaempferol was through CYP2E1down-regulation and antioxidant defense up-regulation.5.Kaempferol blocked the CYP2E1 translocation targeting to mitochondria by inhibiting its molecular chaperone in vitro.The mitochondrial CYP2E1 was incubated with kaempferol and its activity was reduced.It is demonstrated that kaempferol could interact with CYP2E1.The primary hepatocyte incubated with alcohol or kaempferol or both were collected after 72h.Extracted the total protein,microsomal protein and mitochondrial protein and the expressions of CYP2E1 were detected by Western blot.The results showed that kaempferol inhibit the CYP2E1 protein levels in different sources,especially the mitochondrial CYP2E1.Same results were shown in kaempferol inhibiting CYP2E1 activities.The mitochondrial CYP2E1was transported from the ribosome with the company of molecular chaperone,such as HSP70and HSP90.It was demonstrated that kaempferol reduced the cytoplasm HSP70 expression significantly,while the cytoplasm HSP90 was affected by kaempferol.The hepatocyte morphology also showed that kaempferol protected the hepatocyte from alcohol injury.Hence,the mitochondrial could be a potential targeting organelle for kaempferol protecteingliver against alcoholic injury.In this study,the in vivo and in vitro experiments illustrated that kaempferol protected against the alcoholic liver injury by multipath.The potential mechanism is through different aspects:the interaction with CYP2E1,inhibiting the CYP2E1 mRNA level by SP1,increasing GSH by enhancing Nrf2-GCLC,inhibiting the CYP2E1 targeting to mitochondria by HSP70 inhibition.