Metformin attenuates angiotensin II induced cardiac fibrosis and transforming growth factor-β1 production through the inhibition of hepatocyte nuclear factor4

Aim In diabetic patients, metformin appears to provide cardiovascular protection that cannot be attribu- ted only to its antihyperglycemic effects. Metformin is also known as the AMP-activated protein kinase （AMPK） ac- tivator. Our previous study suggested that metformin inhibits transforming growth factor-β1 （TGF-β1） production in a mouse heart failure model of pressure overload. TGF-β1 is a key factor in cardiac fibrosis and is usually induced by Angiotensin Ⅱ （Ang Ⅱ ） in the pressure overload mouse models. This study investigated the effect of metformin on cardiac fibrosis and TGF-β production induced by AngII and the underlying mechanisms. Methods C57/BL6 wild-type and AMPKα2 knockout mice were used. AngII （3 mg · kg-1 · d-1） was infused subcutaneously into mice for 7 days. Adult mouse cardiac fibroblasts were isolated and treated with AngII （ 1 μmol · L-1） and/or met- formin （1 mmol · L-l）. Results In C57/BL6 mice, metformin inhibits AngII-induced cardiac fibrosis. In cardi-ac fibroblasts, metformin inhibits TGF-β1 expression and production induced by AngII. AMPK inhibitor, com- pound C, reversed the effects of metformin. In vivo, AMPKα2 deficiency further increases AngII-induced TGF-β1 production. In cardiac fibroblasts, metformin inhibited AngII induced hepatocyte nuclear factor4 （HNF4ot protein level increase and HNF4α binding with TGF-β1 promoter using chromatin immunoprecipitation assay. In vivo, AMPKα2 deficiency further increased AngII-induced HNF4α protein level. Using HNF4α adenovirus, overexpress- ing HNF4α led to a 1.5-fold increase in TGF-β1 mRNA expression. HNF4a siRNA blocked AngII induced TGF- β1 production. Luciferase reporter with deleted HNF4a binding sites showed decreased TGFbl transcriptional activ- ity induced by AngII. In AMPK or2-/- heart, the inhibition of metformin on HNF4a protein was attenuated. Con- clusion Metformin inhibits AngII induced cardiac fibrosis and TGF-β1 production through AMPK activation. The underlying mechanism is that AMPK activation inhibits AngII induced HNF4α and then decreases TGF-β1 expres- sion.

Hepatocyte nuclear factor 4-alpha involvement in liver and intestinal inflammatory networks

Hepatocyte nuclear factor 4-alpha(HNF4-α)is a nuclear receptor regulating metabolism,cell junctions,differentiation and proliferation in liver and intestinal epithelial cells.Mutations within the HNF4A gene are associated with human diseases such as maturityonset diabetes of the young.Recently,HNF4A has also been described as a susceptibility gene for ulcerative colitis in genome-wide association studies.In addition,specific HNF4A genetic variants have been identified in pediatric cohorts of Crohn’s disease.Results obtained from knockout mice supported that HNF4-αcan protect the intestinal mucosae against inflammation.However,the exact molecular links behind HNF4-αand inflammatory bowel diseases remains elusive.In this review,we will summarize the current knowledge about the role of HNF4-αand its isoforms in inflammation.Specific nature of HNF4-αP1 and P2 classes of isoforms will be summarized.HNF4-αrole as a hepatocyte mediator for cytokines relays during liver inflammation will be integrated based on documented examples of the literature.Conclusions that can be made from these earlier liver studies will serve as a basis to extrapolate correlations and divergences applicable to intestinal inflammation.Finally,potential functional roles for HNF4-αisoforms in protecting the intestinal mucosae from chronic and pathological inflammation will be presented.

Role of hepatocyte nuclear factor 4-alpha in gastrointestinal and liver diseases

Hepatocyte nuclear factor 4-alpha(HNF4α)is a highly conserved member of nuclear receptor superfamily of ligand-dependent transcription factors that is expressed in liver and gastrointestinal organs(pancreas,stomach,and intestine).In liver,HNF4αis best known for its role as a master regulator of liver-specific gene expression and essential for adult and fetal liver function.Dysregulation of HNF4αexpression has been associated with many human diseases such as ulcerative colitis,colon cancer,maturity-onset diabetes of the young,liver cirrhosis,and hepatocellular carcinoma.However,the precise role of HNF4αin the etiology of these human pathogenesis is not well understood.Limited information is known about the role of HNF4αisoforms in liver and gastrointestinal disease progression.There is,therefore,a critical need to know how disruption of the expression of these isoforms may impact on disease progression and phenotypes.In this review,we will update our current understanding on the role of HNF4αin human liver and gastrointestinal diseases.We further provide additional information on possible use of HNF4αas a target for potential therapeutic approaches.

Hepatocyte nuclear factor 4α induces a tendency of differentiation and activation of rat hepatic stellate cells

AIM: To investigate the effect of hepatocyte nuclear factor 4α(HNF4α) on the differentiation and transformation of hepatic stellate cells(HSCs).METHODS: By constructing the recombinant adenovirus vector expressing HNF4α and HNF4αshRNA vector, and manipulating HNF4α expression in HSC-T6 cells, we explored the influence of HNF4α and its induction capacity in the differentiation of rat HSCs into hepatocytes.RESULTS: With increased expression of HNF4αmediated by AdHNF4α, the relative expression of Nanog was downregulated in HSC-T6 cells(98.33 ±12.33 vs 41.33 ± 5.67, P < 0.001). Consequently, the expression of G-P-6 and PEPCK was upregulated(G-P-6:14.34 ± 3.33 vs 42.53 ± 5.87, P < 0.01; PEPCK: 10.10± 4.67 vs 56.56 ± 5.25, P < 0.001), the expression of AFP and ALB was positive, and the expression of Nanog, Type Ⅰ collagen, α-SMA, and TIMP-1 was significantly decreased. HNF4α also downregulated vimentin expression and enhanced E-cadherin expression. The ultrastructure of HNF4α-induced cells had more mitochondria and ribosomes compared with the parental cells. After silencing HNF4α expression,EPCK, E-cadherin, AFP, and ALB were downregulated and α-SMA and vimentin were upregulated.CONCLUSION: HNF4α can induce a tendency of differentiation of HSCs into hepatocyte-like cells. These findings may provide an effective way for the treatmentof liver diseases.

Berberine retarded the growth of gastric cancer xenograft tumors by targeting hepatocyte nuclear factor 4α

BACKGROUND Gastric cancer is the third deadliest cancer in the world and ranks second in incidence and mortality of cancers in China.Despite advances in prevention,diagnosis,and therapy,the absolute number of cases is increasing every year due to aging and the growth of high-risk populations,and gastric cancer is still a leading cause of cancer-related death.Gastric cancer is a consequence of the complex interaction of microbial agents,with environmental and host factors,resulting in the dysregulation of multiple oncogenic and tumor-suppressing signaling pathways.Global efforts have been made to investigate in detail the genomic and epigenomic heterogeneity of this disease,resulting in the identification of new specific and sensitive predictive and prognostic biomarkers.Trastuzumab,a monoclonal antibody against the HER2 receptor,is approved in the first-line treatment of patients with HER2+tumors,which accounts for 13%-23%of the gastric cancer population.Ramucirumab,a monoclonal antibody against VEGFR2,is currently recommended in patients progressing after first-line treatment.Several clinical trials have also tested novel agents for advanced gastric cancer but mostly with dis-appointing results,such as anti-EGFR and anti-MET monoclonal antibodies.Therefore,it is still of great significance to screen specific molecular targets for gastric cancer and drugs directed against the molecular targets.AIM To investigate the effect and mechanism of berberine against tumor growth in gastric cancer xenograft models and to explore the role of hepatocyte nuclear factor 4α(HNF4α)-WNT5a/β-catenin pathways played in the antitumor effects of berberine.METHODS MGC803 and SGC7901 subcutaneous xenograft models were established.The control group was intragastrically administrated with normal saline,and the berberine group was administrated intragastrically with 100 mg/kg/d berberine.The body weight of nude mice during the experiment was measured to assess whether berberine has any adverse reaction.The volume of subcutaneous tumors during this experiment was recorded to evaluate the inhibitory effect of berberine on the growth of MGC803 and SGC7901 subcutaneous transplantation tumors.Polymerase chain reaction assays were conducted to evaluate the alteration of transcriptional expression of HNF4α,WNT5a andβ-catenin in tumor tissues and liver tissues from the MGC803 and SGC7901 xenograft models.Western blotting and IHC were performed to assess the protein expression of HNF4α,WNT5a andβ-catenin in tumor tissues and liver tissues from the MGC803 and SGC7901 xenograft models.RESULTS In the both MGC803 and SGC7901 xenograft tumor models,berberine significantly reduced tumor volume and weight and thus retarded the growth rate of tumors.In the SGC7901 and MGC803 subcutaneously transplanted tumor models,berberine down-regulated the expression of HNF4α,WNT5a andβ-catenin in tumor tissues from both transcription and protein levels.Besides,berberine also suppressed the protein expression of HNF4α,WNT5a andβ-catenin in liver tissues.CONCLUSION Berberine retarded the growth of MGC803 and SGC7901 xenograft model tumors,and the mechanism behind these anti-growth effects might be the downregulation of the expression of HNF4α-WNT5a/β-catenin signaling pathways both in tumor tissues and liver tissues of the xenograft models.

Regulation of hepatic micro RNA expression by hepatocyte nuclear factor 4 alpha

AIM To uncover the role of hepatocyte nuclear factor 4 alpha(HNF4α) in regulating hepatic expression of micro RNAs.METHODS Microarray and real-time PCR were used to determine hepatic expression of micro RNAs in young-adult mice lacking Hnf4α expression in liver(Hnf4α-Liv KO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4α, RNA polymerase-Ⅱ, and histone modifications to loci of micro RNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4α on the promoters of mouse and human micro RNAs as well as effects of micro RNAs on the untranslated regions(3'UTR) of two genes in human hepatoma cells. RESULTS Microarray data indicated that most micro RNAs remained unaltered by Hnf4α deficiency in Hnf4α-Liv KO mice. However, certain liver-predominant micro RNAs were down-regulated similarly in young-adult male and female Hnf4α-Liv KO mice. The down-regulation of mi R-101, mi R-192, mi R-193 a, mi R-194, mi R-215, mi R-802, and mi R-122 as well as induction of mi R-34 and mi R-29 in male Hnf4α-Liv KO mice were confirmed by real-timePCR. Analysis of public chromatin immunoprecipitationsequencing data indicates that HNF4α directly binds to the promoters of mi R-101, mi R-122, mi R-194-2/mi R-192 and mi R-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4α markedly activated the promoters of mouse and human mi R-101b/mi R-101-2 and the mi R-194/mi R-192 cluster. Additionally, mi R-192 and mi R-194 significantly decreased activities of luciferase reporters for the 3'UTR of histone H3F3 and chromodomain helicase DNA binding protein 1(CHD1), respectively, suggesting that mi R-192 and mi R-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1.CONCLUSION HNF4α is essential for hepatic basal expression of a group of liver-enriched micro RNAs, including mi R-101, mi R-192, mi R-193 a, mi R-194 and mi R-802, through which HNF4α may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.

Inhibiting the expression of hepatocyte nuclear factor 4 alpha attenuates lipopolysaccharide/ D-galactosamine-induced fulminant hepatic failure in mice

BACKGROUND: Hepatocyte nuclear factor 4 alpha (HNF4α) plays an important role in regulating cytokine-induced inflammatory responses. This study aimed to investigate the role of HNF4α in the development of fulminant hepatic failure (FHF) induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN). METHODS: The FHF model was induced by simultaneous intraperitoneal injection of LPS/D-GalN in mice. Three days prior to LPS/D-GalN administration, HNF4α short-hairpin interfering RNA expression plasmid or physiological saline was injected via the tail vein with a hydrodynamics-based procedure. The degree of hepatic damage and cumulative survival rate were subsequently assessed. RESULTS: The expression of HNF4α was increased in the early stage after LPS/D-GalN administration. Inhibiting the expression of HNF4α reduced serum levels of alanine aminotransferase and aspartate aminotransferase, alleviated histological injury, and improved the survival of mice with FHF. In addition, both serum and hepatic tumor necrosis factor alpha expression were suppressed when HNF4α expression was inhibited in mice with FHF. CONCLUSION: Inhibiting HNF4α expression protects mice from FHF induced by LPS/D-GalN, but the exact mechanism behind this needs further investigation.

Expression of hepatocyte nuclear factor 4 alpha,wingless-related integration site,andβ-catenin in clinical gastric cancer

BACKGROUND Gastric cancer(GC)is the second most common cause of cancer-related deaths worldwide.Hepatocyte nuclear factor 4 alpha(HNF4α)that belongs to the nuclear hormone receptor superfamily,is overexpressed in GC tissues,and might be involved in the development of GC by regulating its downstream winglessrelated integration site(WNT)/β-catenin signaling.AIM To clarify the expression of HNF4α/WNT5a/β-catenin signaling proteins in clinical GC tissues.METHODS We immunohistochemically stained pathological blocks of GC and matched paracancerous tissues.The intensity of HNF4α,WNT5a andβ-catenin staining in the tumor cells was determined according to cell rates and staining intensity.The correlations between GC and HNF4α,WNT5a,andβ-catenin expression using chisquare and paired chi-square tests.Relationships between double-positive HNF4αand WNT5a expression and types of gastric tumor tissues were assessed using regression analysis.Correlations between HNF4αand WNT5a expression at the RNA level in GC tissues found in the TCGA database were analyzed using Pearson correlation coefficients.RESULTS We found more abundant HNF4αand WNT5a proteins in GC,especially in mucinous adenocarcinoma and mixed GC than in adjacent tissues(P<0.001).Low and high levels of cytoplasmicβ-catenin respectively expressed in GC and adjacent tissues(P<0.001)were not significantly associated with pathological parameters.CONCLUSION The expressions of HNF4αand WNT5a could serve as early diagnostic biomarkers for GC.

Corticosteroid-induced bradycardia in multiple sclerosis and maturity-onset diabetes of the young due to hepatocyte nuclear factor 4-alpha mutation:A case report

BACKGROUND Intravenous steroid pulse therapy is the treatment of choice for acute exacerbation of multiple sclerosis(MS).Although steroid administration is generally welltolerated,cases of cardiac arrhythmia have been reported.Herein,we describe a young woman who developed marked sinus bradycardia and T-wave abnormalities after corticosteroid administration.We also present plausible explanations for the abnormalities observed in this patient.CASE SUMMARY An 18-year-old woman experienced vertiginous dizziness and binocular diplopia 1 wk prior to admission.Neurological examination revealed left internuclear ophthalmoplegia with left peripheral-type facial palsy.The initial laboratory results were consistent with those of type 2 diabetes.Brain magnetic resonance imaging revealed multifocal,non-enhancing,symptomatic lesions and multiple enhancing lesions.She was diagnosed with MS and maturity-onset diabetes of the young.Intravenous methylprednisolone was administered.On day 5 after methylprednisolone infusion,marked bradycardia with T-wave abnormalities were observed.Genetic evaluation to elucidate the underlying conditions revealed a hepatocyte nuclear factor 4-alpha(HNF4A)gene mutation.Steroid treatment was discontinued under suspicion of corticosteroid-induced bradycardia.Her electrocardiogram changes returned to normal without complications two days after steroid discontinuation.CONCLUSION Corticosteroid-induced bradycardia may have a significant clinical impact,especially in patients with comorbidities,such as HNF4A mutations.

Role of Hepatocyte Nuclear Factor 4α in Regulating Hepatic Differentiation and the Inflammatory Response in HCC

Limited treatment options are available for hepatocellular carcinoma(HCC),especially in the advanced stage,which is associated with a poor prognosis.Many studies have demonstrated that hepatocyte nuclear factor 4α(HNF 4α)plays an important role in hepatic differentiation and the carcinogenesis of HCC.HNF 4αcritically regulates hepatic differentiation by controlling a large number of genes involved in hepatic functions including metabolism,xenobiotic detoxification,bile acid synthesis,and serum protein production.It has also been confirmed to play an important role in the inflammatory environment in HCC.Thus,HNF 4αis considered to be a promising target for the treatment of HCC.Some studies have demonstrated that regulating HNF 4αexpression in HCC had beneficial effects in in vivo and in vitro experiments.We herein review the role of HNF 4αin regulating hepatic metabolism and the inflammatory response,aiming to provide some ideas on induced hepatic differentiation therapy and regulating the inflammatory microenvironment for the treatment of advanced HCC.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Association of A Common Haplotype of Hepatocyte Nuclear Factor 1α With Type 2 Diabetes in Chinese Population

Objective To analyze the association of variants of hepatocyte nuclear factor-1α （HNF-1α） gene with type 2 diabetes in Chinese population. Methods In 152 unrelated type 2 diabetes patients and 93 unrelated controls, eleven single nucleotide polymorphisms （SNPs） were identified and genotyped. Statistical analyses were performed to investigate whether these SNPs were associated with diabetes status in our samples. Results In the individual SNP study, no SNP differed significantly in frequency between type 2 diabetes patients and controls. In the haplotype analysis, two haplotype blocks were identified. In haplotype block 1, no evidence was found between common HNF-1α haplotypes and type 2 diabetes. However, in haplotype block 2, a common haplotype GCGC formed by four tagging SNPs （tSNPs） was found to be associated with decreased risk of type 2 diabetes （odds ratio [OR] 0.6011, 95% confidence interval [CI] 0.4138-0.8732, P=0.0073, empirical P=0.0511, permutation test）. A similar trend was also observed in the diplotype analysis, indicating that the increasing copy number of the haplotype GCGC was associated with the decreased frequency of diabetes （P=0.0193）. Conclusion The results of this study provide evidence that the haplotype of HNF-1α decreases the risk of type 2 diabetes in Chinese individuals.

转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化

Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

糖尿病小鼠肾小管上皮细胞HNF4A和MUCDHL下调促进肾纤维化

目的:探索肝细胞核因子4α(HNF4A)和μ-原钙黏蛋白(MUCDHL)在糖尿病小鼠肾脏中的作用和联系。方法:(1)选取12周龄的db/m小鼠和db/db小鼠各6只,正常饮食喂养至16周,Western blot检测肾组织纤维连接蛋白(FN)、Ⅲ型胶原(Col-Ⅲ)、上皮钙黏素(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、HNF4A、Snail和MUCDHL的蛋白水平,免疫组织化学染色观察FN、HNF4A和MUCDHL蛋白的表达及分布情况。(2)体外培养小鼠肾小管上皮细胞(mRTEC),分为正常糖(NG)组、高糖(HG)组、过表达对照组(NG+vector组和HG+vector组)、过表达组(NG+OE-MUCDHL组、HG+OE-MUCDHL组、NG+OE-HNF4A组和HG+OE-HNF4A组)、敲减对照组(NG+control组和HG+control组)和敲减组(NG+si-MUCDHL组、HG+si-MUCDHL组、NG+si-HNF4A组和HG+si-HNF4A组),Western blot检测相关指标的蛋白水平。结果:(1)与db/m组相比,db/db组体重、血糖和尿白蛋白/肌酐比值均显著升高(P<0.05),提示db/db小鼠有明显肾损伤;与db/m组相比,db/db组FN、Col-Ⅲ、α-SMA和Snail蛋白表达升高,E-cadherin、HNF4A和MUCDHL蛋白表达降低(P<0.05);MUCDHL主要表达在肾小管上皮细胞顶端膜,FN主要表达在肾小管间质,HNF4A主要表达在肾小管细胞浆和细胞核。(2)与NG组相比,HG组FN、Col-Ⅲ、α-SMA和Snail蛋白表达升高,E-cadherin、HNF4A和MUCDHL蛋白表达降低(P<0.05);过表达MUCDHL后FN、Col-Ⅲ、α-SMA和Snail蛋白表达降低,E-cadherin和MUCDHL蛋白表达升高(P<0.05),HNF4A表达不变;敲减MUCDHL后相关指标表达与上述效应相反(P<0.05),HNF4A表达不变;过表达HNF4A可使MUCDHL表达增加,其余指标的表达变化与过表达MUCDHL一致;敲减HNF4A表达可使上述效应反转(P<0.05);MUCDHL可能是HNF4A的下游靶基因。结论:HNF4A和MUCDHL在糖尿病小鼠肾小管中表达降低。HNF4A可能通过上调MUCDHL的表达延缓糖尿病肾病肾纤维化进程。

Facilitating effects of berberine on rat pancreatic islets through modulating hepatic nuclear factor 4 alpha expression and glucokinase activity

AIM: To observe the effect of berberine on insulin secretion in rat pancreatic islets and to explore its possible molecular mechanism. METHODS: Primary rat islets were isolated from male Sprague-Dawley rats by collagenase digestion and treated with different concentrations (1,3,10 and 30 μmol/L) of berberine or 1 μmol/L Glibenclamide (GB) for 24 h. Glucose-stimulated insulin secretion (GSIS) assay was conducted and insulin was determined by radioimmunoassay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cytotoxicity. The mRNA level of hepatic nuclear factor 4 alpha (HNF4α) was determined by reverse transcription polymerase chain reaction (RT-PCR). Indirect immunofluorescence staining and Western blot analysis were employed to detect protein expression of HNF4α in the islets. Glucokinase (GK) activity was measured by spectrophotometric method. RESULTS: Berberine enhanced GSIS rather than basal insulin secretion dose-dependently in rat islets and showed no significant cytotoxicity on islet cells at the concentration of 10 μmol/L. Both mRNA and protein expressions of HNF4α were up-regulated by berberine in a dose-dependent manner,and GK activity was also increased accordingly. However,GB demonstrated no regulatory effects on HNF4α expression or GK activity. CONCLUSION: Berberine can enhance GSIS in rat islets,and probably exerts the insulinotropic effect via a pathway involving HNF4α and GK,which is distinct from sulphonylureas (SUs).

4-HNE通过抑制TNF-α介导的NF-κB活化诱导酒精性肝损伤

目的:通过体外细胞及体内动物实验,研究肿瘤坏死因子α(TNF-α)诱导的4-羟基壬烯酸(4-HNE)致敏肝细胞发生死亡的作用及机制。方法:以人肝细胞株HepG2及小鼠原代肝细胞为细胞模型,通过乳酸脱氢酶(LDH)释放及MTT比色法检测4-HNE对TNF-α诱导的肝细胞死亡的作用,利用Western blotting技术检测细胞内4-HNE与蛋白质形成的加合物水平,通过Western blotting和ELISA技术检测细胞核内NF-κB(p65)的表达及其与DNA结合活性。以C57BL/6小鼠为动物模型,利用HE染色、ELISA、Western blotting及TUNEL等技术,检测长期摄入酒精前后动物肝组织形态、甘油三酯(TG)水平、4-HNE水平、TNF-α水平及血浆丙氨酸氨基转移酶(ALT)活性的变化。结果:(1)4-HNE可以显著增加HepG2细胞及小鼠原代肝细胞对TNF-α杀伤作用的敏感性,从而使TNF-α诱导4-HNE致敏的肝细胞死亡。(2)4-HNE可显著提高HepG2细胞内4-HNE-蛋白质加合物的水平。(3)4-HNE抑制HepG2细胞内TNF-α介导的NF-κB活化。(4)长期摄入酒精导致小鼠肝细胞内4-HNE和TNF-α水平升高,引起肝细胞内TG水平升高,血浆ALT活性升高,肝细胞死亡增多。结论:长期摄入酒精使肝细胞发生氧化应激,其产物4-HNE可作为一种肝细胞致敏因子,通过抑制肝细胞内TNF-α介导的NF-κB抗细胞凋亡信号通路,诱导酒精性肝损伤。这可能是一种新的酒精性肝病发病机制。

白藜芦醇在缺氧/复氧诱导的肝细胞损伤中的保护作用及与TLR4/NF-κB通路的关系

目的探讨白藜芦醇(resveratrol,RES)在缺氧/复氧(hypoxia reoxygenation,H/R)诱导的大鼠肝细胞损伤中的保护作用及其相关分子机制。方法建立BRL-3A细胞(大鼠肝细胞株)H/R模型。建模前分别用RES和TLR4抑制剂(HTA125)预处理细胞。建模完成后,检测细胞存活率及细胞凋亡,观察细胞形态变化,检测细胞培养液中谷丙转氨酶(alanine transaminase,ALT)及炎症因子含量,检测细胞TLR4、NF-κB p65基因mRNA及蛋白水平表达及NF-κB p65入核情况。结果 H/R条件使细胞存活率明显降低,细胞凋亡率增加(P<0.01),受损细胞明显增多,ALT、IL-1β含量增多(P<0.01),TLR4和NF-κB p65表达水平显著提高(P<0.01),p65入核细胞比例提高(P<0.01)。经RES及HTA125预处理后,细胞存活率显著提高,细胞凋亡比例显著缩小(P<0.01),受损细胞减少,ALT、IL-1β含量降低(P<0.01),TLR4和NF-κB p65表达水平明显降低(P<0.01),p65入核细胞比例下降(P<0.01)。结论 RES可以减轻H/R诱导的BRL-3A肝细胞损伤,该作用可能与抑制TLR4/NF-κB信号通路有关。

结直肠癌组织中HNF4α的细胞内定位及临床意义

目的检测肝细胞核因子4α(HNF4α)在结直肠癌组织中的细胞内定位及其表达情况,探讨HNF4α定位及其表达与结直肠癌临床各病理因素的关系。方法应用免疫组化方法检测132例结直肠癌组织标本及其癌旁正常组织中HNF4α的细胞内定位及其表达情况。结果结直肠癌组织中HNF4α蛋白分子的表达定位于细胞浆或细胞核;结直肠癌组织细胞浆HNF4α的阳性率(76.5%)显著高于癌旁正常组织(3.8%,P<0.05);结直肠癌组织细胞核HNF4α的阳性率(12.9%)显著低于癌旁正常组织(64.4%,P<0.05);HNF4α在结直肠癌组织细胞浆中的表达与组织分化程度及Dukes分期有关(P=0.02、P=0.03),与性别、年龄、肿瘤位置、有无淋巴结转移等临床病理特征无明显相关性(P均>0.05)。结论结直肠癌组织中HNF4α在细胞浆中高表达或在细胞核中低表达,HNF4α的细胞内定位异常可为结直肠癌的诊断及预后判断提供客观依据。

过表达肝细胞核因子4alpha对人骨髓间充质干细胞向肝样细胞分化的作用

目的应用转基因技术将肝细胞核因子4alpha(HNF-4α)转导入人骨髓间充质干细胞(MSCs)内,使其连续过表达HNF-4α并促进MSCs向肝样细胞分化。方法 HNF-4α基因通过慢病毒表达载体pLV/Final-puro-hHNF4α-hrGFP转入人MSCs(UE7T-13细胞)内,用流式细胞分析法检测及分选后,收集hrGFP阳性的UE7T-13细胞进行扩增。体外成肝诱导一周后,免疫荧光染色检测过表达HNF-4α基因的UE7T-13细胞(E7-hHNF-4α细胞)的白蛋白(ALB)和细胞角蛋白(CK18)的表达情况,糖原染色检测UE7T-13细胞及E7-hHNF-4α细胞的糖原储存功能。结果建立稳定、过表达hHNF-4α基因的E7-hHNF-4α细胞,持续过表达HNF-4α促进MSCs向肝样细胞分化,经过7天体外成肝诱导,E7-hHNF-4α细胞具有成熟肝样细胞表达ALB和CK18蛋白及糖原储存功能。结论利用Gateway技术可将HNF-4α高效转导入人MSCs细胞中,并有效促进MSCs向肝样细胞分化。

丝胶对2型糖尿病大鼠肝脏肝细胞核因子-4α表达的影响

目的研究丝胶对2型糖尿病大鼠肝脏肝细胞核因子(HNF)-4α表达的影响。方法雄性SD大鼠48只随机分为:正常对照组、糖尿病模型组、丝胶治疗组和丝胶预防组,每组12只大鼠。链脲佐菌素连续腹腔注射制作2型糖尿病大鼠模型,待模型成功建立后,模型组大鼠不做任何处理,丝胶治疗组大鼠给予2.4 g·kg-1·d-1的丝胶连续灌胃35 d;丝胶预防组大鼠在链脲佐菌素连续腹腔注射前,给予丝胶(2.4 g·kg-1·d-1)灌胃,时间与丝胶治疗组相同。分别采用Western印迹法和RT-PCR法检测大鼠肝脏HNF-4α蛋白和mRNA的表达。结果与正常对照组大鼠比较,糖尿病模型组大鼠肝脏HNF-4α蛋白和mRNA的表达明显升高(P<0.01);与糖尿病模型组大鼠比较,丝胶治疗组、丝胶预防组大鼠肝脏HNF-4α蛋白和mRNA的表达明显降低(P<0.01)。结论丝胶改善糖代谢的作用可能与其调节肝脏HNF-4α的表达有关。