The Study On The Mechanism Of The Crosstalk Between The Anti-oxidative And Anti-inflammatory Effects Of Quercetin In Acute Alcoholic Liver Injury

Background and Objective:Alcohol abuse is a worldwide social problem,especially in China.Untill now we still lack new and powerful governance model and medicines,although the morbidity of liver diseases caused by this reason still stays in a high position without going down.Due to the lack of effective preventive and therapeutic measurements,severe alcoholic liver disease(ALD)still maintain a high rate of disability and poor clinic outcomes despite of the rapid development of modern medicine.Unlike the rapid development of antiviral treatment for viral hepatitis,there are still only two major effective choices for patients with severe alcoholic hepatitis,which are the glucocorticoids and liver transplantation.Therefore,there is an urgent need for exploration of new drugs based on the gap between the upgoing morbidity and the stagnant treatment methods.Since the immune system network is gradually taking shape in different human organs and systems,people’s recognition of its functions have entered a new phase.The role of the innate immune system in various diseases and its complicated regulatory network have become a brandnew hotspot.There are a great deal of innate immune cells in the liver.It has been confirmed that innate immune system activation plays an important role in various liver diseases such as nonalcoholic fatty liver disease,ischemia-reperfusion injury,acute liver failure and liver malignancy.As a multiprotein complex in the cytoplasm,the inflammasome is accumulated and spliced by a variety of proteins(including various receptors such as the NOD-like receptor family).After sensing the intrinsicly or externally pathophysiological stimulation signal(such as oxidative stress stimulation),the inflammasome will perform polymerization,remodeling and activation and act as the core of the inflammatory response.It will promote the secretion of pro-inflammatory cytokines and evoke local feedback related cascade response.The inflammasome plays a key role in self-renewal and protective maintenance.Normally,it acts as a protector for appropriate elimination of abnormal stimuli in physiological conditions.However,excessive and uncontrolled activation of this process may lead to recognitive disorders and control imbalance,which induces and aggravates tissue destruction pathologically.Many studies have proven the delicated function and the corresponding network regulatory mode of NLRP3 inflammasome.Unfortunately,few of them shed light on the pharmaceutical development of alcoholic liver disease that can modulate the impaired innate immune balance.As a countury with a long history of alcohol,we possess a large amount of patients troubled by ALD.As to now,we still face the dilemma of the limited traditional measurements and the inadequent new choices.Deepened exploration of the native defentive system has opened up a new era for ALD.Oxidative stress and inflammatory response used to be considered as two major footstones in ALD.Now the innate immune system may provide a link between these two and the NLRP3 inflammasome may become a potential choice.Drugs the can modulate inflammasome will provide the possibility of cutting the interaction of these two footstones,which may act as a more competitive clinic choice.Therefore,here we want to explore: 1.As a reducible plant component,whether quercetin can save the liver destruction occurred in AALI? 2.Whether quercetin can remold the inflammatory balance in AALI? And what is its influence on NLRP3 inflammasome? 3.Search for the underlying cross-talk between the two protective phenotypes of quercetin and try to find the keypoints and modulatory mode in this process.All in all,we intend to take steps for new directions and clues for further prevention and treatment of ALD.Methods: Part I: To detect the differences in expression of antioxidant heme oxygenase-1(HO-1)through analysis of data of patients with alcoholic hepatitis in the GEO(Gene Expression Omnibus datasets)database and establishment of animal models of acute alcoholic liver injury.Five groups of rats were included in our experiment: normal control group,quercetin control group,AALI model group,quercetin treatment group and quercetin + Znpp IX(HO-1 inhibitor)group.Rats were scrified and the corresponding liver tissues were well prepared,after which double-blind pathological scoring were performed to evaluate the effectiveness of the animal model and the differences in histopathological changes in each experimental group.For all the rats,we compared the transcriptional and protein expressional levels of nuclear factor-erythroid 2 related factor 2(Nrf2)and HMOX1.In addition,we detected levels of oxidative stress products such as malonaldehyde(MDA)and reactive oxygen species(ROS)in each group of experimental animals by ELISA.Part II: We verified the detailed effect of quercetin on the two primary parts of innate immune,which are the pro- and anti-inflammatory equilibrium and NLRP3 inflammasome.The concrete method was comparision of levels of pro-inflammatory cytokines(IL-1β,IL-18 and TNF-α)and anti-inflammatory factors(IL-10)in the rats of the five groups.Meanwhile we tested the transcriptional and protein expressional levels of inflammasome components in the livers of rats in the corresponding five groups.Prt III: To explore the possibility of the cross-talk between the two above major phenotypes in AALI.GO analysis and pathway analysis of the HMOX1 gene were performed on the NCBI,KEGG and Reactome websites.Furtherly,the activity of NF-κB transcription factor were investigated by ELISA in the five experimental groups.Results: 1.The results of GEO database analysis proved the differential levels of HMOX1 gene expression in patients with alcoholic hepatitis and the controls.Down-regulated HMOX1 gene level was seen in AH patients(p<0.05).2.The classic changes of ALD were seen in AALI animal models in the field of liver histopathology.Significantly elevated pathological scores and serum liver enzyme levels were found in AALI models(p<0.05).3.Rats in the medicine treatment group possessed much higher m RNA transcriptional expression levels of Nrf2 & HO-1 and HO-1 protein levels related to controls and AALI models(p<0.05).Combination of Znpp IX leads to lower m RNA and protein level of HO-1 than single medicine pretreatment(p<0.05).4.Dramatic elevations of oxidative products(MDA and ROS)had been seen in the serum and liver of AALI models,as well as the decreased reducible GSH-Px activity(p < 0.05).Single pre-treatment of quercetin partially blocked the burst of the two above oxidative indexes and significantly improved the reducible activity of GSH-Px.Elevated MDA and ROS levels and depressed GSH-Px activity had been seen again when using quercetin combined with Znpp IX(p<0.05).5.Acute ethanol stimuli evoked the burst of pro-inflammatory cytokines(TNF-α,IL-1β and IL-18)in the AALI model group(p<0.05).However,no remarkable difference of IL-10 levels had been recorded in the controls and AALI models(p>0.05).Rats pretreated with quercetin alone menifested much lower levels of pro-inflammatory cytokines(p<0.05)and higher results of anti-inflammatory factors than AALI models.When combined with Znpp IX,quercetin’s protective effect on inhibiting inflammatory factors were significantly weakened than using single medicine(p<0.05).No statistical difference of IL-10 was recorded in the groups of quercetin with or without Znpp IX(p>0.05).6.As to the three basic components of NLRP3 inflammasome,there were significant elevations of their m RNA and protein expression levels in AALI group(p<0.05).Compared with the AALI model group,quercetin alleviated the overexpression of NLRP3,ASC and caspase-1 in both m RNA transcription and protein synthesis(p<0.05).The m RNA and protein expression levels of NLRP3,ASC and caspase-1 increased again when quercetin were combined with Znpp IX.7.GO analysis and pathway analysis of HMOX1 hinted that HO-1 may take part in the intervention of the interleukin family and nuclear transcription factor NF-κB.8.Acute alcohol stimuli promoted the NF-κB p65 activity in AALI models(p<0.05).Its activity was much lower when pre-treated with quercetin(p<0.05).Combined use of Znpp IX upregulated the NF-κB p65 activity in liver again.Conclusion:1.HMOX1(the encoding gene of HO-1)expression was down-regulated in AH patients.2.The AALI models showed characteristic liver and enzyme features.The upregulation of HO-1 protein by quercetin in AALI can be attributed to the activation of Nrf2/HO-1 axis,which can be inhibited by Znpp IX.3.Quercetin exerted anti-oxidative action in AALI,which was HO-1 dependent.4.In AALI,quercetin can inhibite the secretion of pro-inflammatory cytokines,which was HO-1 dependent.Meanwhile,it can promote secretion of anti-inflammatory IL-10 in a HO-1 independent way.5.Quercetin can reduce the overexpression of the three basic components of NLRP3 inflammasome in AALI which was related to HO-1.6.We found a underlying cross-talk between the two major hepatoprotective phenotypes of quercetin in AALI.That is,quercetin upregulated reducible HO-1 and scavenged ROS,which furtherly downregulated NF-κB and inhibited overexpression of NLRP3 inflammasome.Finally it decreased release of pro-inflammatory cytokines.