法尼酯X受体对肝脂酶表达的影响

目的:探讨法尼酯X受体(farnesoid X receptor,FXR)对肝脂酶(hepatic lipase,HL)表达的影响。方法:用FXR激动剂CDCA作用人肝癌细胞株(HepG2),用RT-PCR和Western blotting检测HL的表达情况。结果:用不同浓度的CDCA(25μmol/L、50μmol/L、75μmol/L)分别作用HepG2细胞6h、12h、24h、48h后,HL的mRNA和蛋白质水平呈时间和剂量依赖性下调。结论:FXR激动剂可抑制肝脂酶的表达。     

肝癌细胞粘附和收缩调节对其Rho蛋白表达和迁移影响的实验研究

运用定量显微形态分析技术、免疫荧光实验技术和MTT分析法,分别对裱衬纤维连接蛋白(fibronectin,FN)和应用肌球蛋白轻链激酶(myosin light chain kinase,MLCK)抑制剂ML-7作用下,肝细胞L02与肝癌细胞HepG2细胞骨架装配和Rho蛋白表达变化,以及细胞迁移能力进行检测和定量表征,了解肝癌细胞侵袭转移的胞内分子基础。结果显示：1)L02 Rho蛋白表达水平明显低于HepG2,裱衬FN低浓度（1～5 μg/mL）使 L02 Rho蛋白表达进一步下调,HepG2 Rho蛋白表达水平却明显增高,而裱衬 FN高浓度（10～40 μg/mL）L02Rho蛋白升高超过对照组,HepG2 Rho蛋白表达下降,且远低于对照组水平;2）随着裱衬FN浓度的增加,HepG2增殖抑制明显;3）裱衬低浓度FN使HepG2迁移运动能力增强,而高浓度FN使细胞净位移和迁移轨迹离散度减少;4）ML-7低浓度（6 μmol/L）使L02 Rho蛋白表达下调,而对HepG2 Rho蛋白表达无明显影响,L02细胞骨架解聚较HepG2明显;ML-7高浓度（10 μmol/L）HepG2Rho蛋白表达减少,且迁移速率降低;L02 Rho蛋白表达无进一步下调。说明：1)细胞粘附状态对调节HepG2 和L02Rho蛋白表达呈相反趋势;2)HepG2对骨架收缩抑制的响应较L02迟缓;3)HepG2Rho蛋白表达水平与其迁移能力呈正相关。     

烟酸对HepG2细胞载脂蛋白M表达的影响及其机制

目的:探讨烟酸对HepG2细胞载脂蛋白M表达的影响及其机制。方法:分别以不同浓度的烟酸(0、0.25、0.5、1.0、2.0mmol/L)干预HepG2细胞24h。提取各组细胞总RNA和蛋白质,分别采用实时R T-PCR和Western Blot检测载脂蛋白M的mRNA和蛋白的表达。采用实时R T-PCR检测肝细胞核因子-1αmRNA的表达。结果:烟酸呈剂量依赖性上调载脂蛋白M基因和蛋白、肝细胞核因子-1α基因的表达(P<0.05)。结论:烟酸可上调载脂蛋白M表达,其机制可能是通过上调肝细胞核因子-1α来实现的。     

USP9X低表达通过下调Mcl-1促进肝癌细胞凋亡

研究抑制泛素特异性蛋白酶9X（ubiquitin-specific protease 9X,USP9X）对人肝癌（primary hepatocellular carcinoma,HCC）细胞SMMC7721和HepG2中髓细胞白血病-1（myeloid cell leukemia-1,Mcl-1）蛋白的表达调控及对细胞凋亡和生长活力的影响。实验分为USP9X-siRNA组和阴性对照NC组两组进行分析。通过Western blot技术分别检测USP9X在肝癌细胞SMMC7721、HepG2和正常人肝细胞株L02中的蛋白表达情况;应用化学合成USP9X-siRNA转染肝癌细胞SMMC7721和HepG2,通过Western blot、流式细胞仪和MTT检测转染前后Mcl-1的蛋白表达差异以及细胞凋亡和生长活力变化。结果表明,USP9X在肝癌细胞SMMC7721和HepG2中的蛋白表达水平均高于正常肝细胞L02（t=15.155,P=0.000;t=9.171,P=0.001）;SMMC7721和HepG2细胞中抑制USP9X能明显下调Mcl-1的蛋白表达,并导致细胞凋亡增加和生长活力降低。提示,肝癌细胞SMMC7721和HepG2中USP9X表达上调;USP9X表达降低可能通过下调Mcl-1的蛋白表达进而诱导人肝癌细胞SMMC7721和HepG2的凋亡。     

辛伐他汀和非诺贝特对HepG2细胞载脂蛋白M表达的影响

目的:观察辛伐他汀和非诺贝特及两者联合对HepG2细胞载脂蛋白M表达的影响。方法:分别以不同浓度的辛伐他汀(0、1、5、10、25μmol/L)和非诺贝特(0、50、100mmol/L)及辛伐他汀(5.0μmol/L)+非诺贝特(50mmol/L)干预HepG2细胞24h。提取各组细胞总RNA和蛋白质,分别采用实时RT-PCR和WesternBlot检测apoM的mRNA和蛋白的表达。结果:辛伐他汀和非诺贝特均呈剂量依赖性上调载脂蛋白M基因和蛋白的表达(P<0.05)。联合用药比单药更能显著上调载脂蛋白M的表达(P<0.05)。结论:他汀类和贝特类药物均可上调载脂蛋白M表达,两药联合的作用更为显著。     

磷脂爬行酶1对干扰素抑制乙型肝炎病毒(HBV)作用的影响

研究磷脂爬行酶1(Phospholipid scramblase 1,PLSCR1)对干扰素抑制HBV作用的影响。设计合成PLSCR1特异性小干扰RNA(siRNA),以完全随机序列的阴性小干扰(NCsiRNA)作为对照,转染HepG2细胞,于转染48h后分别检测PLSCR1mRNA和蛋白水平表达量的变化,筛选出对PLSCR1具有沉默作用的siRNA;将HepG2细胞分为正常对照组和干扰素处理组,将1.3倍乙型肝炎病毒(HBV)全基因真核细胞表达载体HBV1.3质粒分别与PLSCR1siRNA或NCsiRNA共同转染HepG2细胞或干扰素处理的HepG2细胞,转染48h后检测各组细胞中PLSCR1mRNA表达量及培养液上清中HBsAg表达水平。PLSCR1特异性小干扰RNA siRNA911转染后能够显著抑制HepG2细胞中PLSCR1基因在mRNA和蛋白水平的表达;与HepG2细胞对照组比较,干扰素处理组细胞转染HBV1.3质粒、NCsiRNA+HBV1.3质粒后,细胞培养液中HBsAg表达水平均显著降低(P0.05);而PLSCR1siRNA与HBV1.3共转染IFN处理的HepG2细胞组与共转染HepG2细胞组相比较,细胞培养液中HBsAg的表达水平没有显著差异。提示抑制PLSCR1的siRNA可抑制干扰素的抗HBV活性,提示PLSCR1在干扰素抑制HBV复制中具有重要作用。     

ASPP2过表达可增强DRAM介导的自噬对HepG2细胞的促调亡作用

p53凋亡刺激蛋白2（apoptosis stimulating protein 2 of p53, ASPP2）能特异性地与p53蛋白结合并增强其促凋亡功能,进而发挥抗肿瘤作用.最近文献提示,自噬对肿瘤发生、发展及肿瘤细胞对抗肿瘤药物的反应都具有重要作用.在本研究中,甲基磺酸(MMS)处理HepG2细胞24 h后,用calcein AM/PI和M30染色检测细胞凋亡,可引起早期（M30免疫组化阳性）和晚期细胞凋亡（PI染色阳性）. 给HepG2细胞转染GFP-LC3质粒后,发现MMS处理24 h可引起自噬的发生. ASPP2腺病毒（rAd-ASPP2）感染HepG2细胞引起ASPP2过表达后,再用MMS处理24 h,能引起更明显的早期、晚期细胞凋亡和自噬. 荧光定量PCR检测发现,rAd-ASPP2诱导了更高的BCL-2相关X蛋白基因（BAX）和p53蛋白的目的基因p53诱导的自噬调节蛋白（p53-induced modulator of autophagy,DRAM）的表达. 但仅用rAd-ASPP2处理HepG2细胞不能引起自噬和凋亡.利用2条DRAM特异性的siRNA下调DRAM的表达,发现rAd-ASPP2引起的自噬被完全抑制, 早期和晚期凋亡均部分被抑制,同时BAX 的mRNA水平也明显下降. 以上结果说明,ASPP2可通过上调BAX和DRAM基因的转录而促进MMS引起的HepG2细胞凋亡; 另外,DRAM介导的自噬是ASPP2促进MMS引起的肿瘤细胞凋亡的机制之一. 该研究可为肝癌的基因治疗提供新的思路.     

乙型肝炎病毒X蛋白上调环加氧酶2表达促进肝癌细胞增殖的实验研究

乙肝病毒X蛋白（Hepatitis B virus X protein,HBx）高表达与乙肝相关肝癌的发病密切相关,但HBx发挥促癌作用的机制并不清楚。环加氧酶2（Cyclooxygenase,COX2）具有促进肝癌细胞增殖的功能,乙肝相关肝癌中COX2的表达增加且与HBx呈正相关,提示上调COX2可能是HBx促进肝癌细胞增殖的分子机制。为了阐明HBx是否通过上调COX2促进肝癌细胞增殖,本实验培养了肝癌HepG2细胞并分为对照组、转染pcDNA3.1-HBx质粒的HBx组、转染NC siRNA的si-NC组、转染NC siRNA及pcDNA3.1-HBx质粒的si-NC+HBx组、转染COX2 siRNA及pcDNA3.1-HBx质粒的si-COX2+HBx组。检测细胞增殖活力OD_490nm的水平,COX2、B淋巴细胞瘤2基因（BCL2）、生存素（Survivin）的表达量,前列腺素E2（英文名,PGE2）的含量。实验结果显示,HBx组的OD_490nm水平,细胞中COX2、BCL2、Survivin的表达量,培养基中PGE2的含量均高于对照...     

研究: PCSK9/LDLR通路介导姜黄素烟酸酯促进HepG2摄取脂质

为研究PCSK9/LDLR通路介导姜黄素烟酸酯(CurTn)降低血浆低密度脂蛋白胆固醇(LDL-C),减少动脉内膜下脂质沉积的分子机制,用5、10、15 μmo/L姜黄素烟酸酯与25 mg/L LDL共孵育HepG2细胞24 h,分别采用油红O染色、胆固醇荧光定量试剂盒、DiI-LDL摄取检测细胞内胆固醇含量及LDL摄取情况,用逆转录定量聚合酶链反应(RT-Q-PCR)检测LDLR及SREBP2的mRNA表达,蛋白质印迹检测LDLR、SREBP2及PCSK9蛋白表达．随姜黄素烟酸酯作用浓度的增高细胞内脂滴显著增多,细胞内游离胆固醇(FC)、总胆固醇(TC)含量增高,细胞内胆固醇摄取增多;RT-Q-PCR和蛋白质印迹检测发现,与对照组(Control)比较,5、10、15 μmo/L 姜黄素烟酸酯处理组LDLR 蛋白表达增高,SREBP2 mRNA表达水平升高,PCSK9蛋白表达降低,但对LDLR mRNA及SREBP2 蛋白表达无影响．结果表明：姜黄素烟酸酯通过降低PCSK9、减少LDLR降解、升高LDLR蛋白表达,促进HepG2细胞胆摄取胆固醇．初步说明CurTn可能通过抑制PCSK9介导LDLR溶酶体降解,促进肝脏清除血浆LDL-C水平．     

半边旗二萜类成分PAG通过ROS诱导肝癌细胞凋亡的作用研究

探讨半边旗二萜类成分Pteisolic acid G(PAG)对人肝癌细胞HepG2增殖和凋亡的影响及作用机制。用不同浓度的PAG处理HepG2细胞后,采用MTT法检测细胞存活率;采用PI单染法检测细胞周期分布;采用Annexin V-FITC/PI双染法检测细胞凋亡率;采用RT-PCR和Western Blotting检测细胞内mRNA和蛋白表达情况;采用DCFH-DA法检测细胞内ROS水平,采用ROS抑制剂乙酰半胱氨酸(NAC)评价PAG细胞增殖抑制作用对ROS的依赖性。结果表明,在24 h、48 h和72 h时,PAG可剂量依赖性地抑制HepG2细胞的增殖(p0.05),IC_(50)分别为64.8μmol/L,38.5μmol/L和24.8μmol/L;用药24 h时PAG可剂量依赖性地使HepG2细胞阻滞在G_2/M期,同时增加HepG2细胞凋亡率(p0.05);PAG可剂量依赖性地降低HepG2细胞内Bcl-2 mRNA和caspase 3、PARP、Bcl-2蛋白的表达(p0.05),增加Bax mRNA和actived-caspase 3、cleaved-PARP、Bax蛋白的表达(p0.05)。当使用1 mmol/L的ROS抑制剂NAC预处理HepG2细胞时,PAG对HepG2细胞增殖抑制作用被显著阻断。上述结果表明,半边旗二萜类成分PAG可提高Bax/Bcl-2的基因和蛋白表达比值,从而诱导肝癌细胞HepG2凋亡,该作用可能是通过升高细胞内ROS水平来实现的。     

Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.     

Bile acids induce the expression of the human peroxisome proliferator-activated receptor alpha gene via activation of the farnesoid X receptor

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. PPARalpha is also reported to influence bile acid formation and bile composition. Farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that mediates the effects of bile acids on gene expression and plays a major role in bile acid and possibly also in lipid metabolism. Thus, both PPARalpha and FXR appear to act on common metabolic pathways. To determine the existence of a molecular cross-talk between these two nuclear receptors, the regulation of PPARalpha expression by bile acids was investigated. Incubation of human hepatoma HepG2 cells with the natural FXR ligand chenodeoxycholic acid (CDCA) as well as with the nonsteroidal FXR agonist GW4064 resulted in a significant induction of PPARalpha mRNA levels. In addition, hPPARalpha gene expression was up-regulated by taurocholic acid in human primary hepatocytes. Cotransfection of FXR/retinoid X receptor in the presence of CDCA led to up to a 3-fold induction of human PPARalpha promoter activity in HepG2 cells. Mutation analysis identified a FXR response element in the human PPARalpha promoter (alpha-FXR response element (alphaFXRE)] that mediates bile acid regulation of this promoter. FXR bound the alphaFXRE site as demonstrated by gel shift analysis, and CDCA specifically increased the activity of a heterologous promoter driven by four copies of the alphaFXRE. In contrast, neither the murine PPARalpha promoter, in which the alphaFXRE is not conserved, nor a mouse alphaFXRE-driven heterologous reporter, were responsive to CDCA treatment. Moreover, PPARalpha expression was not regulated in taurocholic acid-fed mice. Finally, induction of hPPARalpha mRNA levels by CDCA resulted in an enhanced induction of the expression of the PPARalpha target gene carnitine palmitoyltransferase I by PPARalpha ligands. In concert, these results demonstrate that bile acids stimulate PPARalpha expression in a species-specific manner via a FXRE located within the human PPARalpha promoter. These results provide molecular evidence for a cross-talk between the FXR and PPARalpha pathways in humans.     

Effects of farnesoid X receptor on the expression of the fatty acid synthetase and hepatic lipase

The farnesoid X receptor (FXR) is a nuclear receptor that regulates gene expression in response to bile acids (BAs). FXR plays an important role in the homeostasis of bile acid, cholesterol, lipoprotein and triglyceride. In this report, we identified fatty acid synthase (FAS) and hepatic lipase (HL) genes as novel target genes of FXR. Human hepatoma HepG2 cells were treated with chenodeoxycholic acid, the natural FXR ligand, and the messenger RNA and protein levels of FAS and HL were determined by RT-PCR and Western blot analysis, respectively. Chenodeoxycholic acid (CDCA) down-regulated the expression of FAS and HL genes in a dose and time-dependent manner in human hepatoma HepG2 cells. In addition, treatment of mice with CDCA significantly decreased the expression of FAS and HL in mouse liver and the activity of HL. These results demonstrated that FAS and HL might be FXR-regulated genes in liver cells. In view of the role of FAS and HL in lipogenesis and plasma lipoprotein metabolism, our results further support the central role of FXR in the homeostasis of fatty acid and lipid.     

Farnesoid X Receptor, through the Binding with Steroidogenic Factor 1-responsive Element, Inhibits Aromatase Expression in Tumor Leydig Cells

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. It is expressed in the liver and the gastrointestinal tract, but also in several non-enterohepatic tissues including testis. Recently, FXR was identified as a negative modulator of the androgen-estrogen-converting aromatase enzyme in human breast cancer cells. In the present study we detected the expression of FXR in Leydig normal and tumor cell lines and in rat testes tissue. We found, in rat Leydig tumor cells, R2C, that FXR activation by the primary bile acid chenodeoxycholic acid (CDCA) or a synthetic agonist GW4064, through a SHP-independent mechanism, down-regulates aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. Transient transfection experiments, using vector containing rat aromatase promoter PII, evidenced that CDCA reduces basal aromatase promoter activity. Mutagenesis studies, electrophoretic mobility shift, and chromatin immunoprecipitation analysis reveal that FXR is able to compete with steroidogenic factor 1 in binding to a common sequence present in the aromatase promoter region interfering negatively with its activity. Finally, the FXR-mediated anti-proliferative effects exerted by CDCA on tumor Leydig cells are at least in part due to an inhibition of estrogen-dependent cell growth. In conclusion our findings identify for the first time the activators of FXR as negative modulators of the aromatase enzyme in Leydig tumor cell lines.     

Farnesoid X receptor protects liver cells from apoptosis induced by serum deprivation in vitro and fasting in vivo

The farnesoid X receptor (FXR) is a key metabolic regulator in the liver by maintaining the homeostasis of liver metabolites. Recent findings suggest that FXR may have a much broader function in liver physiology and pathology. In the present work, we identify a novel role of FXR in protecting liver cell from apoptosis induced by nutritional withdrawal including serum deprivation in vitro or starvation in vivo. Two FXR ligands, chenodeoxycholic acid (CDCA) and GW4064, rescued HepG2 cells from serum deprivation-induced apoptosis in a dose-dependent manner. This effect of FXR on apoptotic suppression was compromised when FXR was knocked down by short interfering RNA. Similarly, the effects of both CDCA and GW4064 were abolished after inhibition of the MAPK pathway by a specific inhibitor of MAPK kinase 1/2. Immunoblotting results indicated that FXR activation by CDCA and GW4064 induced ERK1/2 phosphorylation, which was attenuated by serum deprivation. In vivo, FXR(-/-) mice exhibited an exacerbated liver apoptosis and lower levels of phosphorylated-ERK1/2 compared to wild-type mice after starvation. In conclusion, our results suggest a novel role of FXR in modulating liver cell apoptosis.     

Farnesoid X receptor suppresses constitutive androstane receptor activity at the multidrug resistance protein-4 promoter

Multidrug resistance protein-4 (MRP4) is a member of the multidrug resistance associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive up-regulation in response to cholestatic injury or bile acid feeding. In this study we demonstrate that farnesoid X receptor (FXR) regulates MRP4 in vivo and in vitro. In vivo deletion of FXR induces MRP4 gene expression. In vitro treatment of HepG2 cells with FXR ligands, chenodeoxycholic acid (CDCA), cholic acid (CA) and the synthetic ligand GW-4064 suppresses basal mRNA level of the MRP4 gene as well as the co-treatment with CDCA and 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), an activator of constitutive androstane receptor (CAR). We found in the human MRP4 promoter a CAR responsive element (CARE) embedded within an FXR responsive element (FXRE). We cloned this region and found that FXR suppresses CAR activity in luciferase assay. Finally, we demonstrated that FXR competes with CAR for binding to this overlapping binding site. Our results support the view that FXR activation in obstructive cholestasis might worsen liver injury by hijacking a protective mechanism regulated by CAR and provides a new molecular explanation to the pathophysiology of cholestasis.     

Dehydrodiconiferyl alcohol,a lignan from Herpetospermum pedunculosum,alleviates cholestasis by activating pathways associated with the farnesoid X receptor

BackgroundIn our previous study, we demonstrated the hepatoprotective effect of Herpetospermum pedunculosum in cholestatic rats. A bioassay-guided study also led to the identification and isolation of a lignan, dihydrodiconiferyl alcohol (DA) from the seeds of H. pedunculosum.PurposeTo investigate whether DA could alleviate cholestasis and determine the mechanisms underlying such action.MethodsMale Sprague-Dawley (SD) rats were administered with DA (10, 20 or 40 mg/kg) intragastrically once daily for 7 days prior to treatment with α-naphthylisothiocyanate (ANIT) (60 mg/kg). We then evaluated the levels of a range of serum indicators, determined bile flow, and carried out histopathological analyses. Western blotting was then used to investigate the levels of inflammatory mediators and the Farnesoid X Receptor (FXR), proteins involved in the downstream biosynthesis of bile acids, and a range of transport proteins. Molecular docking was used to simulate the interaction between DA and FXR. Cell viability of human hepatocytes (L-02) cells was determined by MTT. Then, we treated guggulsterone-inhibited L-02 cells, Si-FXR L-02 cells, and FXR-overexpression cells with the FXR agonist GW4064 (6 μM) or DA (25, 50 and 100 μM) for 24 h before detecting gene and protein expression by RT-PCR and western blotting, respectively.ResultsDA significantly attenuated ANIT-induced cholestasis in SD rats by reducing liver function indicators in the serum, increasing bile flow, improving the recovery of histopathological injuries in the liver, and by alleviating pro-inflammatory cytokines in the liver. DA also increased the expression levels of FXR and altered the levels of downstream proteins in the liver tissues, thus indicating that DA might alleviate cholestasis by regulating the FXR. Molecular docking simulations predicted that DA was as an agonist of FXR. In vitro mechanical studies further showed that DA increased the mRNA and protein expression levels of FXR, Small Heterodimer Partner 1/2, Bile Salt Export Pump, Multidrug Resistance-associated Protein 2, and Na+/taurocholate Co-transporting Polypeptide, in both guggulsterone-inhibited and Si-FXR L-02 cells. Moreover, DA enhanced the mRNA and protein expression of FXR, and its downstream genes and proteins, in L-02 cells containing an FXR-overexpression plasmid.ConclusionDA may represent an effective agonist for FXR has significant therapeutic potential for the treatment of cholestatic liver injury.