Study Of Transcription Factor Nrf2Inhibited LPS Induced Rat Liver Kupffer Cells NF-κB Activation

Non-alcoholic fatty liver disease（NAFLD） is described by the exclusion ofexcessive alcohol intake(intake of more than210g in week, more than five consecutiveyears), or there are other specific causes (such as malnutrition, drugs, viruses,ect.)liver fatdeposition in the case,is similar with a liver histological changes with alcoholic liverdisease (ALD), but no history of excessive alcohol consumption clinicopathologicalsyndrome[1], including simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosisand liver carcinoma, is a common clinical chronic liver disease[2-3]. In developed countries,the ordinary adult NAFLD prevalence rate of20%to33%, in Europe and the UnitedStates and other Western countries, its prevalence in children and adolescents in the UnitedStates is eatimated at5%to10%, up to70%of in metamorphosis obese population[1]. Theincidence of NAFLD in China in recent years, also increased significantly, and showedrapidly growth and younger age trend, has become China′s important liver disease and one of the major cause of cryptogenic cirrhosis. Nevertheless, NAFLD pathogenesis iscomplex and has not yet been fully elucidated, is still a lack of really effective treatment, itis the study of the pathogenesis and interventions research focus.Insulin resistance(IR) and inflammation are the two main features of NAFLD. Theaccepted explanation NAFLD incidence of “two-hit” theory that the liver is suffered thefirst blow for the surrounding IR induced liver fat accumulation[2], and the second timeagainst oxidative stress, ROS and RNS increased activation of NF-κB, the expression ofinflammatory cytokines are raised. In recent years, the role of the gut microflora in thepathogenesis of NAFLD has been confirmed[5-10], the normal intestinal flora interactedwith the high-fat diet, a metabolic endotoxemia, Lipopolysaccharide(LPS) via the portalsystem into the liver induced liver Kupffer cells (KCs) infiltration, activation, synthesis ofa variety of inflammatory factors trigger liver IR, These findings clearly different from the“two-hit” thory. And study confirmed that LPS-activated KCs originating link, Theshort-term high-fat diet can cause KC activation, leading to liver IR selectively cleared KCcan eliminate this effect[12,14]. Inflammatory cytokines IL-1α、IL-1β、IL-6and TNF-α causeliver IR involved in IR and type two diabetes[11]. That the activatived KCs induced byLPS of a high fat diet, regulating the expression of inflammatory cytokines via NF-κB,which is caused liver IR[15,16]. Therefore, the inhibition of NF-κB activation simulated byLPS induced KCs can help to prevent NAFLD.Nuclear transcription factor Nrf2is the central link of the cells against endogenousand exogenous oxidative stress，which is via upregulating the expression of the phase-Ⅱdetoxifying enzymes to reduce the level of intracellular ROS, which can play the role ofpreventing this progress and protecting the NAFLD[16,17]. We turned out that curcumin caninduce nuclear transcription Nrf2translocating from cytoplasm to nuclear, thereforecuricumin decreased the activity of NF-κB indirectly and reduced the expression ofinflammatory cytokines TNF-α, IL-1β and IL-6, which suggests promoting thetranslocation of Nrf2can suppress the activity of NF-κB. However, the mechanism of thatNrf2regulate the activity of NF-κB of kupffer cells is rare in protecting NAFLD. ObjectiveIn vitro, we observed the level of oxidative stress, the expression of NF-κB andinflammatory cytokines, which produced by LPS-induced Kupffer cells.After LPSinduced Kupffer cells,then curcumin stimulated, we observed the level of oxidative stress,the translocation of Nrf2and expression of phase Ⅱenzymes(HO-1), the expression ofNF-κB and inflammatory cytokines. Which these changes plays a great role in thepathogenesis of NAFLD.Methods1．We isolated, cultured and identified Kupffer cells of Sprague Dawley rat liver.2．We divided Kupffer cells into control group and LPS group, then we observed thetranslocation of NF-κB by immunofluorescence staining, the level of malondialdehyde(MDA)and glutathione(GSH) by spectrophotometric method, the expression of NF-κBand inflammatory cytokines TNF-α, IL-1β and IL-6.3．We divided Kupffer cells into control group, LPS group and curcumin group, henwe observed the translocation of Nrf2by immunofluorescence staining, the expression ofphase Ⅱenzymes(HO-1) by PCR, the level of malondialdehyde and glutathione byspectrophotometric method, the expression of NF-κB and inflammatory cytokinesTNF-α, IL-1β and IL-6.Results1．We isolated kupffer cell and identified in success.2．We found out that the level of MDA of LPS group was significantly higher thancontrol group（P＜0.05）, but the GSH in the opposite result（P＜0.05）, compared withcontrol group, that immunofluorescence results showed LPS can induced NF-κBtranslocating from cytoplasm into nucleus, western blot displayed the activation of NF-κBby LPS（P＜0.05）, the level of TNF-α, IL-1β and IL-6was obviously more than controlgroup（P＜0.05）. 3．That immunofluorescence results showed LPS can induced Nrf2translocatingfrom cytoplasm into nucleus and curcumin can boost this process. PCR method resultsrevealed that the expression of HO-1mRNA of LPS group was significantly highercompared with control group(P <0.05), the expression of HO-1mRNA of curcumin groupwas obviously more than that in LPS group (P <0.05). Spectrophotometry resulted thatlevel of MDA in LPS group was markly higher (P <0.05), however, curcumin group waslower than that in LPS group (P <0.05), but was still higher than that control group (P<0.05). But the level of GSH in LPS group was lower than that control group (P <0.05),the GSH of curcumin group was higher than that in LPS group (P <0.05), but lower thanthat in control group(P <0.05). Western blot results displayed that expression of NF-κBp65of LPS group was significantly higher than that control group (P <0.05), but lowerthan curcumin group (P <0.05). ELISA results appeared that the expression of TNF-α,IL-1β and IL-6in LPS group was significantly higher than control group,(P <0.05), butdistinctly lower than curcumin group (P <0.05).ConclusionIn experiments, we successfully isolated, cultured and identified the kupffer cells, wealso turned out that LPS can increase the level of oxidative stress, and promotetranslocation of NF-κB, decrease the expression of inflammatory cytokines. At the sametime, we confirmed that curcumin can effectively promote nuclear translocation of Nrf2toreduce level of oxidative stress, inhibit activation of NF-κB to lessen expression ofinflammatory cytokines. Now, we take the Nrf2and kupffer cells as targets to treat andprevent NAFLD, which provide new theory and experimental evidence for us.