The Studies Of The Molecular Mechanism Of Down-regulation Of Hepatic Glutathione S-transferase A1/A4 Expression In Human Obstructive Cholestasis

Background & ObjectiveCholestasis is a common clinical syndrome.The hallmark of cholestasis is an impairment of bile secretion and flow followed by a lack of bile in the intestine and accumulation of potentially toxic bile salts in the liver and the systemic circulation.In general,the pathogeny of cholestasis can be divided into two categories;hereditary transporter defects and acquired cholestatic diseases.Hereditary defects of hepatobiliary ATP-binding cassette(ABC)transporters have been linked to a broad spectrum of hepatobiliary disorders ranging from progressive familial intrahepatic cholestasis(PFIC),benign recurrent intrahepatic cholestasis(BRIC)to intrahepatic cholestasis of pregnancy(ICP),and so on.The acquired cholestatic diseases include obstructive cholestasis,biliary obstruction,viral hepatitis,drug-induced cholestasis,and inflammation-induced cholestasis.Bile acids accumulating in the hepatocytes are a common clinical manifestation of these diseases.Under physiological condition,BAs exert their multiple physiological functions including stimulation of bile flow,intestinal absorption of lipophilic nutrients,solubilization and excretion of cholesterol,maintainting intestinal flora balance,as well as antimicrobial and metabolic effects.In cholestasis,however,the consequent accumulation of bile salts in the hepatocytes leads to mitochondria damage,oxidative stress,pro-inflammatory cytokine production,apoptosis and severe hepatocellular injury.In addition,BAs may disrupt cell membranes through their detergent action on lipid components and can promote the generation of reactive oxygen species that,in turn,oxidatively modify lipids,proteins,and nucleic acids,and eventually cause hepatocyte necrosis and apoptosis.Unfortunately,there are few treatment methods for anti-cholestasis.So far,the only approved drug for treatment of cholestatic disorders is ursodeoxycholic acid(UDCA),which is a hydrophilic bile acid and normally accounts for only small parts of the human bile acid pool.For dealing with the accumulation of bile salts,the hepatocytes can clear those bile acids through the following 4 phase: phase 0,hepatic uptake;phase I,metabolism(e.g.,hydroxylation);phase II,detoxification(e.g.,conjugation);and phase III,excretion.However,some studies have confirmed that the activities of the detoxification enzymes,including superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx),glutathione reductase,and glutathione S-transferase(GSTs),were decreased in the livers of bile duct-ligated(BDL)rats.It is well established that GSTs are crucial for the detoxification of a variety of endogenous or exogenous toxic compounds by conjugating lipophilic electrophiles to glutathione(GSH).The GSTs can bind non-substrate ligands,including bile acids and bilirubin,which makes them less toxic and better substrates for alternative elimination pathways.As phase II detoxifying enzymes,the GST functions include the repair of macromolecules that are oxidized via reactive oxygen species,anti-apoptosis,biosynthesis of physiologically important metabolites,and regeneration of S-thiolated proteins.Among the GSTs,the Alpha-class GSTs contribute the majority of phospholipid hydroperoxide activity and the ensuing toxicity.Moreover,GSTA1 and GSTA4 play key roles in the oxidative stress protection mechanisms that catalyze the conjugation of glutathione with peroxide.GSTA1 is crucial for the binding of hydrophobic ligands and steroid hormones,and it plays an important protective role in JNK-associated apoptosis by suppressing JNK signaling activation.Some recent studies confirmed that Gsta4-null mice have a reduced ability to conjugate 4-HNE,increased susceptibility to CCl4,mitochondrial dysfunction,increased protein carbonylation,reduced antioxidant capacity and increased apoptosis.However,the activity and expression of GSTs were reportedly reduced under obstructive cholestasis conditions.The molecular mechanism of this reduction remains unclear.A recent study indicated that tumor necrosis factor alpha(TNF?)decreases Gsta4 expression in 3T3-L1 adipocytes.In addition,curcumin,the main polyphenolic active compound,was found to significantly decrease hepatic TNF? levels and increase GST enzyme activities.We,and others,have found that the levels of TNF? are significantly increased in both cholestatic patients and BDL rats.Therefore,we hypothesized that the down-regulation of hepatic GSTA1 and GSTA4 expression in cholestatic patients may be mediated by TNF?.Here,we investigate the molecular mechanism of GSTA1/GSTA4 reduction in BDL rats and human hepatoma Hep G2 cells.Research methods1.HepG2 Cells Culture and Treatment1.1 We transfected HepG2 cells with Hep G2-NTCP-OE or Hep G2-CTR to obtain stably NTCP-transfected HepG2 cell lines allowing NTCP over-expression.1.2 We treated cultured HepG2 cells with CA and CDCA,NTCP-transfected HepG2 cells allowing NTCP over-expression with TCA,GCA,TCDCA and GCDCA at different doses for designated times,respectively.Then,the expressions of GSTA1 and GSTA4 were measured by western blotting.1.3 We treated Hep G2 cells with TNF? and detected the expression of GSTA1 and GSTA4 by Real time qPCR and western blotting.1.4 We treated HepG2 cells with TNF? 100ng/ml for 12 h,and then extracted both total and nuclear protein.Both protein levels of total p65,the main form of nuclear factor kappa B(NF-?B),in the nuclear fraction and phosphorylated p65 nuclear factor E2 related factor 2(Nrf2)in total protein were analyzed by western blotting.1.5 Hep G2 cells were treated with 50?M BAY 11-7082(NF-?B signaling inhibitor)for 1h,and then 100ng/ml TNF? was added to the culture for 24 h.The expressions of GSTA1 and GSTA4 were measured by Real time qPCR and western blotting.2.Animals and experimental proceduresForty-two healthy male Sprague-Daw-ley(SD)rats,aged seven to eight weeks and weighing between 200 and 250 g,were used in this study.The rats were acclimated in plastic cages individually in temperature–controlled(20-23°C)rooms with humidity ranging between 40% and 60% and light cycles set to 12-hour light and 12-hour dark,and fed a standard diet of rodent pellets and purified water.All 42 rats were randomly divided into two groups: the BDL group and the sham operation group.The animals were anesthetized using sodium pentobarbital 50 mg/kg intraperitoneally(21 rats per group).After the midline abdominal incision,the common bile duct(CBD)was isolated,and doubly ligated with 4– 0 silk sutures in the BDL group.In the sham operation group,rats received sham-operation without ligating the CBD.Seven rats in each group were sacrificed according to standard protocol on days 3,7,and 14 post-surgery,respectively.The rat livers were cut into small pieces immediately and then kept in liquid nitrogen until used.Results1.The bile acids did not correlated with the expressions of GSTA1 and GSTA4 in HepG2 cells.2.The expressions of GSTA1/GSTA4 were repressed by TNF? at both the m RNA and protein levels in a dose-and time-dependent manner.3.TNF? activated NF-?B signaling in HepG2 cells,and did not reduced Nrf2 protein expression.4.Inhibition of NF-?B activation by BAY 11-7082 attenuated TNF?-mediated decrease in GSTA1 and GSTA4 expressions.5.Gsta2,Gsta3,and Gsta4 m RNA levels in liver samples of BDL rats were significantly decreased starting from 3 days post-surgery compared to the Sham group;whereas Gstm1,Gstm2 and Gstm4 m RNA levels were significantly decreased starting from 7 days post-surgery.6.Compared with the Sham group rats,increased association of NF-?B and CBP of BDL group rats was observed.At the same time,the binding of Nrf2 and CBP was decreased.7.Compared with the Sham group,the binding of Nrf2 and ARE located in Gsta1 promoter area was decreased in BDL group rats.DiscussionThe induction of detoxification enzymes is one of essential adaptive protective responses in cholestatic hepatocytes.In this study,however,we demonstrated that TNF? could repress GSTA1 and GSTA4 expressions at both the m RNA and protein levels in HepG2 cells in a dose-and time-dependent maner.Furthermore,we found that inhibiting NF-?B signaling pathway could attenuate the reduction of GSTA1 and GSTA4 expressions by TNF?.These results indicated that TNF? repressed GSTA1 and GSTA4 expressions via activating NF-?B signaling pathway in HepG2 cells.We then observed that the expressions of Gsta2-4,Gstm1,Gstm2 and Gstm4 were decreased in liver samples of BDL rats.Meanwhile,we demonstrated that NF-?B can competitively bind to CBP with Nrf2 in liver tissue of BDL rats.As a result,the binding of Nrf2 and ARE located in Gsta1 promoter area was decreased in BDL group.It was verified that Hep G2 cells possess a spectrum of normal hepatocyte proteins,including GSTs.So,the data from this study might reflect the situation in normal human hepatocytes.The accumulation of toxic bile acids in hepatocytes likely plays a trigger role in cholestatic liver injury.However,the results obtained in this study indicated that bile acids were not involved in GSTA1 and GSTA4 down-regulation.Obstructive cholestasis due to bile duct obstruction is always accompanied by various inflammatory cytokines such as TNF?,interleukin(IL)-1?,and IL-6.Among these inflammatory cytokines,TNF? is involved in regulating some transporters expression.Actually,others and we have found that the level of TNF? is significantly increased in both cholestatic patients and BDL rats.In present investigation,we demonstrated that GSTA1 and GSTA4 expressions were repressed by TNF? in Hep G2 cells,similar to the results from 3T3-L1 adipocytes.TNF-? is a proinflammatory cytokine that has many important pathological roles,including cell necrosis,apoptosis and tumorigenesis.Considering GSTA play a crucial role in against oxidant stress and apoptosis,TNF?-induced reduction of GSTA1/GSTA4 may be one of the mechanisms of biliary fibrosis,cirrhosis and finally end-stage liver disease during chronic obstructive cholestasis.The expressions of GSTA,as one of the phase II enzymes,are cooperatively con trolled by the Nrf2.To test whether the TNF?-induced reduction of GSTA1 and GSTA4 was related to the decrease of Nrf2 expression,we detected total Nrf2 protein levels in Hep G2 cells after TNF? treatment.Interestingly,the data showed that the reduction was not accompanied by the change of Nrf2 protein,consistent with our previously findings from clinical liver samples of obstructive cholestasis.These results also suggest that the transcriptional activity of Nrf2 is impaired during obstructive cholestas is.It was recently reported that some proteins,such as NF-?B,Bach 1and estrogen receptor,could antagonize Nrf2 activity.Since TNF? has long been known to induce NF-?B signaling,which is implicated in diverse pathological processes including infenction,inflammation and cancer,we tested whether NF-?B signaling was activated by TNF? and involved in regulation of GSTA1 and GSTA4 in HepG2 cells.We observed that total p65 protein in the nuclear fraction and phosphorylated p65 in total protein were significantly increased after only TNF? treatment.Then,we used BAY11-7082(inhibitor of NF-?B)to demonstrate that NF-?B signaling pathway was involved in the down-regulation of GSTA1 and GSTA4 expressions by TNF?.These findings indicate that NF-?B may derease Nrf2 transcriptional activity under obstructive cholestasis condition.Some studies have confirmed that p65 could recruit either coactivators,such as CREB binding protein(CBP),or corepressors,such as histone deacetylases(HDACs)in nucleus.Meanwhile,CBP and small Maf protein(MafK,MafG or MafF)are well-established coactivators of Nrf2,which required for the transcriptional activity of Nrf2.To further confirm the above conclusions,we construc ted the animal models of cholestasis by ligating rat common bile duct.Firstly,we confirmed the down-regulation of multiple GST in liver tissue of BDL rats.Secondly,we demonstrated that NF-?B can competitively bind to CBP with Nrf2 in liver tissue of BDL rats.At the same time,the binding of Nrf2 and ARE located in Gsta1 promoter area was decreased in BDL group.As some pathological stimuli,such as lipopolysaccharide(LPS),cigarette smoke,flow shear stresses,and reactive oxygen species activate both NF-?B signaling and Nrf2-ARE(the antioxidant-response element)pathway,there may be an implicated crosstalk between these two pathways in different pathological processes.For example,a recently study reported that NF-?B subunits p50 and p65 induce transcription of Nrf2 in AML cells,human monocytes and THP-1 cells at a specific promoter ?B-site.Conversely,another study reported that NF-?B also antagonizes Nrf2-ARE signaling.Our finds are similar to the latter.Taken together,in obstructive cholestasis,the possible reason for the TNF?-induced reduction of GSTA1/GSTA4 is NF-?B inhibis Nrf2 interaction with its coactivators,resulting in impairment the ability of Nrf2 binding to ARE.In summary,our data demonstrate that TNF? can repress the expressions of GSTA1 and GSTA4 in transcriptional level through the NF-?B signaling pathway and inhibiting activation of NF-?B signaling pathway could reverse this repression.Targeting NF-?B signaling pathway by inhibitor may be a possible therapy to improve liver detoxification ability and attenuate liver injury in human obstructive cholestasis.ConclusionIn the current studies,we observed that bile acids were not involved in GSTA1 and GSTA4 down-regulation in Hep G2 cells.Furthermore,we demonstrated that expressions of GSTA1/GSTA4 were repressed by TNF? at both the mRNA and protein levels in a dose-and time-dependent manner.Meanwhile,inhibiting NF-?B signaling pathway could attenuate the reduction of GSTA1/GSTA4 in HepG2 cells that had been caused by TNF? treatment.Then,we found that the expressions of Gsta2-4,Gstm1,Gstm2 and Gstm4 were decreased in liver samples of BDL rats.Furthermor,we demonstrated that NF-?B can competitively bind to CBP with Nrf2 in liver tissue of BDL rats.At the same time,the binding of Nrf2 and ARE located in Gsta1 promoter area was decreased in BDL group.