New gene therapy strategies for hepatic fibrosis

The liver is the largest internal organ of the body, which may suffer acute or chronic injury induced by many factors, leading to cirrhosis and hepatocarcinoma. Cirrhosis is the irreversible end result of fibrous scarring and hepatocellular regeneration, characterized by diffuse disorganization of the normal hepatic structure, regenerative nodules and fibrotic tissue. Cirrhosis is associated with a high co-morbidity and mortality without effective treatment, and much research has been aimed at developing new therapeutic strategies to guarantee recovery. Liver-based gene therapy has been used to downregulate specific genes, to block the expression of deleterious genes, to delivery therapeutic genes, to prevent allograft rejection and to augment liver regeneration. Viral and non-viral vectors have been used, with viral vectors proving to be more efficient. This review provides an overview of the main strategies used in liver-gene therapy represented by non-viral vectors, viral vectors, novel administration methods like hydrodynamic injection, hybrids of two viral vectors and blocking molecules, with the hope of translating findings from the laboratory to the patient′s bed-side.

Molecular therapy for hepatic injury and fibrosis:Where are we?

Hepatic fibrosis is a wound healing response, involving pathways of inflammation and fibrogenesis. In response to various insults, such as alcohol, ischemia, viral agents, and medications or hepatotoxins, hepatocyte damage will cause the release of cytokines and other soluble factors by Kupffer cells and other cell types in the liver. These factors lead to activation of hepatic stellate cells, which synthesize large amounts of extracellular matrix components. With chronic injury and fibrosis, liver architecture and metabolism are disrupted, eventually manifesting as cirrhosis and its complications. In addition to eliminating etiology, such as antiviral therapy and pharmacological intervention, it is encouraging that novel strategies are being developed to directly address hepatic injury and fibrosis at the subcellular and molecular levels. With improvement in understanding these mechanisms and pathways, key steps in injury, signaling, activation, and gene expression are being targeted by molecular modalities and other molecular or gene therapy approaches. This article intends to provide an update in terms of the current status of molecular therapy for hepatic injury and fibrosis and how far we are from clinical utilization of these new therapeutic modalities.

Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

AIM： To investigate the potential mechanism of Arg- Gly-Asp （RGD） peptide-labeled liposome loading oxy- matrine （OM） therapy in CCI4-induced hepatic fibrosis in rats. METHODS： We constructed a rat model of CCh- induced hepatic fibrosis and treated the rats with dif- ferent formulations of OM. To evaluate the antifibrotic effect of OM, we detected levels of alkaline phospha- tase, hepatic histopathology （hematoxylin and eosin stain and Masson staining） and fibrosis-related gene expression of matrix metallopeptidase （MMP）-2, tis- sue inhibitor of metalloproteinase （TIMP）-I as well as type I procollagen via quantitative real-time poly- merase chain reaction. To detect cell viability and apop- tosis of hepatic stellate cells （HSCs）, we performed 3-（4,5）-dimethylthiahiazo（-z-yl）-3,5-diphenytetrazoli- umromide assay and flow cytometry. To reinforce the combination of oxymatrine with HSCs, we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM, and its targeting of HSCs was examined by fluorescent microscopy. RESULTS： OM attenuated CCh-induced hepatic fibro- sis, as defined by reducing serum alkaline phosphatase （344.47± 27.52 U/L vs 550.69 ± 43.78 U/L, P 〈 0.05）, attenuating liver injury and improving collagen deposits （2.36% ± 0.09% vs 7.70% ±0.60%, P 〈 0.05） and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen （P 〈 0.05）. OM inhibited cell viability and induced apoptosis of HSCs in vitro. RGD promoted OM targeting of HSCs and en- hanced the therapeutic effect of OM in terms of serum alkaline phosphatase （272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L, P 〈 0.05）, liver injury, collagen deposits （0.26%± 0.09% vs 2.36% ± 0.09%, P 〈 0.05） and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen （P 〈 0.05）. Moreover, in vitro assay demonstrated that RGD en- hanced the effect of OM on HSC viability and apoptosis. CONCLUSION： OM attenuated hepatic fibrosis by in- hibiting viability and inducing apoptosis of HSCs. The RGD-labeled formulation enhanced the targeting effi- ciency for HSCs and the therapeutic effect.

Effects of augmentation of liver regeneration recombinant plasmid on rat hepatic fibrosis

AIM: To investigate the effects of eukaryotic expression of plasmid on augmentation of liver regeneration (ALR) in rat hepatic fibrosis and to explore their mechanisms. METHODS: Ten rats were randomly selected from 50 Wistar rats as normal control group. The rest were administered intraperitoneally with porcine serum twice weekly. After 8 wk, they were randomly divided into: model control group, colchicine group (Col), first ALR group (ALR1), second ALR group (ALR2). Then colchicine ALR recombinant plasmid were used to treat them respectively. At the end of the 4th wk, rats were killed. Serum indicators were detected and histopathological changes were graded. Expression of type Ⅰ, Ⅲ, collagen and TIMP-1 were detected by immunohisto-chemistry and expression of TIMP-1 mRNA was detected by semi-quantified RT-PCR. RESULTS: The histologic examination showed that the degree of the rat hepatic fibrosis in two ALR groups was lower than those in model control group. Compared with model group, ALR significantly reduced the serum levels of ALT, AST, HA, LN, PCIII and IV (P<0.05). Immunohistochemical staining showed that expression of type Ⅰ, Ⅲ, collagen and TIMP-1 in two ALR groups was ameliorated dramatically compared with model group (I collagen: 6.94±1.42,5.80±1.66 and 10.83±3.58 in ALR1, ALR2 and model groups, respectively; Ⅲ collagen: 7.18±1.95, 4.50±1.67 and 10.25±2.61, respectively; TIMP-1: 0.39±0.05,0.20±0.06 and 0.53±0.12, respectively,P<0.05 or P<0.01). The expression level of TIMP-1 mRNA in the liver tissues was markedly decreased in two ALR groups compared with model group (TIMP-1 mRNA/β-actin: 0.89±0.08, 0.65±0.11 and 1.36±0.11 in ALR1, ALR2 and model groups respectively, P<0.01). CONCLUSION: ALR recombinant plasmid has beneficial effects on rat hepatic fibrosis by enhancing regeneration of injured liver cells and inhibiting TIMP-1 expressions.

Assessing significant fibrosis using imaging-based elastography in chronic hepatitis B patients: Pilot study

BACKGROUND Accurate detection of significant fibrosis(fibrosis stage 2 or higher on the METAVIR scale)is important especially for chronic hepatitis B(CHB)patients with high viral loads but with normal or mildly elevated alanine aminotransferase(ALT)levels because the presence of significant fibrosis is accepted as the indication for antiviral treatment.Liver biopsy is the reference standard for diagnosing significant fibrosis,but it is an invasive procedure.Consequently,noninvasive imaging-based measurements,such as magnetic resonance elastography(MRE)or two-dimensional shear-wave elastography(2DSWE),have been proposed for the quantitative assessment of liver fibrosis.AIM To explore MRE and 2D-SWE to identify fibrosis stage,and to compare their performance with that of serum-based indices.METHODS The study enrolled 63 treatment-na?ve CHB patients with high viral loads but with normal or mildly elevated ALT levels who underwent liver biopsy before a decision was made to initiate antiviral therapy.MRE and 2D-SWE were performed,and serum-based indices,such as FIB-4 and aspartate transaminase to platelet ratio index(APRI),were calculated.The diagnostic performances of MRE,2D-SWE,FIB-4,and APRI for assessing significant fibrosis(≥F2)and cirrhosis(F4)were evaluated with liver histology as the reference standard,using receiver operating characteristic analyses.RESULTS The liver fibrosis stage was F0/F1 in 19,F2 in 14,F3 in 14,and F4 in 16 patients,respectively.MRE significantly discriminated F2 from F0/1(P=0.022),whereas 2D-SWE showed a broad overlap in distinguishing those stages.MRE showed a higher correlation coefficient value with fibrosis stage than 2D-SWE with fibrosis stage(0.869 vs 0.649,Spearman test;P<0.001).Multivariate linear regression analyses showed that fibrosis stage was the only factor affecting the values of MRE(P<0.001),whereas body mass index(P=0.042)and fibrosis stage(P<0.001)were independent factors affecting 2D-SWE values.MRE performance for diagnosing significant fibrosis was better[area under the curve(AUC)=0.906,positive predictive value(PPV)97.3%,negative predictive value(NPV)69.2%]than that of FIB-4(AUC=0.697,P=0.002)and APRI(AUC=0.717,P=0.010),whereas the performance of 2D-SWE(AUC=0.843,PPV 86%,NPV 65%)was not significantly different from that of FIB-4 or APRI.CONCLUSION Compared to SWE,MRE might be more precise non-invasive assessment for depicting significant fibrosis and for making-decision to initiate antiviral-therapy in treatment-na?ve CHB patients with normal or mildly-elevated ALT levels.

Mechanism of action and targeted therapy of stellate cells in liver fibrosis

The incidence of liver fibrosis is increasing worldwide,and if left untreated,it will later develop into cirrhosis with a high mortality rate.In this paper,the activation pathway and related mechanism of stellate cells in liver fibrosis are introduced,and some current therapeutic methods are summarized.These results suggest that stellate cells play an important role in liver fibrosis,and targeted therapy for the purpose of inhibiting the activation of stellate cells and inducing their apoptosis is expected to be an effective regimen to reverse liver fibrosis.However,there are some problems such as insufficient in-depth study of related mechanisms and imperfect experiments.In future animal experiments and clinical trials,more studies can be carried out to provide high-quality protocols for the treatment of liver fibrosis.

Effects and mechanism of adenovirus-mediated phosphatase and tension homologue deleted on chromosome ten gene on collagen deposition in rat liver fibrosis

AIM To evaluate the effects of phosphatase and tension homologue deleted on chromosome ten(PTEN) gene on collagen metabolism in hepatic fibrosis and the underlying mechanisms.METHODS rat primary hepatic stellate cells(HSCs) and human LX-2 cells were transfected with adenovirus containing c DNA constructs encoding wild-type PTEN(Ad-PTEN), PTEN mutant G129 E gene(Ad-G129E), and r NA interference constructs targeting the PTEN sequence PTEN short hairpin r NA to up-regulate and downregulate the expression of PTEN. HSCs were assayed using fluorescent microscopy, real-time polymerase chain reaction, and western blotting. Moreover, a CCl_4-induced rat hepatic fibrosis model was established to investigate the in vivo effects. Hematoxylin and eosin, and Masson's trichrome were used to assess the histological changes. The expression of collagen Ⅰ and Ⅲ was assessed using immunohistochemistry and western blot analysis.RESULTS Elevated expression of PTEN gene reduced serum levels of alanine transaminase and aspartate transaminase, decreased collagen deposition in the liver, and reduced hepatocyte necrosis. In contrast, knockdown of PTEN expression had an opposite effect, such as increased collagen deposition in the liver, and was molecularly characterized by the increased expression of matrix metalloproteinase(MMP)-13(P < 0.01) and MMP-2(P < 0.01), as well as decreased expression of the tissue inhibitor of metalloproteinase(TIMP)-1(P < 0.01) and TIMP-2(P < 0.01).CONCLUSION These data indicated that gene therapy using recombinant adenovirus encoding PTEN might be a novel way of treating hepatic fibrosis.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

Evaluation of the prognostic value of liver stiffness in patients with hepatitis C virus treated with triple or dual antiviral therapy:A prospective pilot study

AIM:To evaluate the association between liver stiffness(LS) prior to the initiation of dual/triple therapy and viral response.METHODS:LS was measured in all patients before treatment was administered.The therapeutic approach was based on hepatic,virological,and immunological evaluations and considered the fact that patients with severe fibrosis(F3)or compensated cirrhosis(F4)in Child-Pugh class A are the primary candidates for triple therapy.In total,65 hepatitis C virus(HCV)patients were treated with Peg-interferon/ribavirin(Peg-IFN/RBV);24patients were classified as genotypes 1/4(36.92%),and41 patients were classified as genotypes 2/3(63.08%)(dual therapy).In addition,20 HCV treatment-experienced genotype 1 patients were treated with Peg IFN-RBV and boceprevir(triple therapy).Wilcoxon rank-sum tests were used to compare the groups.RESULTS:LS significantly differed between dual therapy and triple therapy(P=0.002).The mean LS value before dual therapy treatment was 8.61±5.79k Pa and was significantly different between patients achieving a sustained virologic response(SVR)24weeks after therapy and those who did not(7.23±5.18 k Pa vs 11.72±5.99 k Pa,respectively,P=0.0003).The relative risk of non-response to therapy was 4.45(95%CI:2.32-8.55).The attributable risk of non-response to therapy was 49%.The mean LSvalue before triple therapy treatment was 13.29±8.57k Pa and was significantly different between patients achieving and not achieving SVR24(9.41±5.05 vs19.11±9.74,respectively;P=0.008).The relative risk of non-response to therapy was 5.57%(95%CI:1.50-20.65).The attributable risk of non-response to therapy(70%)was increased compared with dual therapy patients.Pre-treatment stiffness>12 k Pa was significantly associated with non-SVR(P<0.025)in both groups.CONCLUSION:Pre-treatment liver stiffness may be useful for predicting the response to treatment in patients treated with either dual or triple anti-HCV therapy.

Interplays of liver fibrosis-associated microRNAs:Molecular mechanisms and implications in diagnosis and therapy

microRNAs(miRNAs)are a class of non-coding functional small RNA composed of 21e23 nucleotides,having multiple associations with liver fibrosis.Fibrosis-associated miRNAs are roughly classified into pro-fibrosis or anti-fibrosis types.The former is capable of activating hepatic stellate cells(HSCs)by modulating pro-fibrotic signaling pathways,mainly including TGF-b/SMAD,WNT/b-catenin,and Hedgehog;the latter is responsible for maintenance of the quiescent phenotype of normal HSCs,phenotypic reversion of activated HSCs(aHSCs),inhibition of HSCs proliferation and suppression of the extracellular matrix-associated gene expression.Moreover,several miRNAs are involved in regulation of liver fibrosis via alternative mechanisms,such as interacting between hepatocytes and other liver cells via exosomes and increasing autophagy of aHSCs.Thus,understanding the role of these miRNAs may provide new avenues for the development of novel interventions against hepatic fibrosis.

水飞蓟素联合复方蛋氨酸胆碱治疗酒精性肝炎患者疗效及其对血清肝纤维化指标的影响

目的观察应用水飞蓟素联合复方蛋氨酸胆碱治疗酒精性肝炎(AH)患者的临床疗效及其对血清肝纤维化指标的影响。方法2021年1月~2023年1月我院诊治的AH患者101例,采用随机数字表法将患者分为对照组50例和观察组51例,分别给予复方蛋氨酸胆碱或复方蛋氨酸胆碱联合水飞蓟素治疗6个月。采用放射免疫分析法检测血清层粘连蛋白(LN)、透明质酸(HA)、Ⅲ型前胶原N端肽(PCⅢ)和Ⅳ型胶原(Ⅳ-C),采用ELISA法检测血清过氧化物酶体增殖物激活受体γ(PPARγ)、瘦素(LEP)、脂联素(APN)和白细胞介素-18(IL-18)水平。结果经治疗,观察组血清ALT和AST水平分别为(41.8±4.9)IU/L和(42.5±4.9)IU/L,均显著低于对照组【分别为(75.3±6.8)IU/L和(62.7±5.8)IU/L,P<0.05】;观察组血清HA、PCⅢ和Ⅳ-C水平分别为(103.6±13.5)μg/L、(94.2±9.8)μg/L和(75.2±8.6)μg/L,均显著低于对照组【分别为(146.2±15.3)μg/L、(125.8±13.3)μg/L和(112.3±14.5)μg/L,P<0.05】;观察组血清PPARγ、LEP和IL-18水平分别为(231.7±26.8)pg/mL、(5.4±0.7)μg/L和(92.5±11.7)ng/mL,均显著低于对照组【分别为(285.3±29.4)pg/mL、(8.3±0.9)μg/L和(118.5±12.4)ng/mL,P<0.05】,而血清APN水平为(15.4±1.7)ng/L,显著高于对照组【(11.3±1.9)ng/L,P<0.05】。结论应用水飞蓟素联合复方蛋氨酸胆碱治疗AH患者效果较好,有利于肝功能指标恢复,可能对延缓肝纤维化进展、降低机体炎症反应和调节脂代谢有帮助。

基于索磷布韦的直接抗病毒药物治疗丙型肝炎肝硬化患者疗效评价

目的探讨基于索磷布韦(SOF)的直接抗病毒药物(DAAs)治疗代偿期丙型肝炎肝硬化(CHC-CLC)和失代偿期丙型肝炎肝硬化(CHC-DLC)患者的临床疗效。方法2019年7月~2022年12月我科诊治的CHC-CLC患者39例和CHC-DLC患者23例,分别接受SOF联合维帕他韦(VEL)或在此联合方案的基础上加用利巴韦林治疗12 w。采用实时荧光定量RT-PCR法检测血清HCV RNA载量,采用基因分型芯片检测HCV基因型,常规检测血常规和血生化指标,计算天冬氨酸氨基转移酶(AST)和血小板(PLT)比值指数(APRI)和肝纤维化4因子指数(FIB-4),使用Fibroscan行肝硬度检测(LSM)。结果到治疗结束时,CHC-DLC患者死亡2例(8.7%);在生存患者中,CHC-CLC组治疗早期病毒学应答率、治疗结束应答率、持续病毒学应答率(SVR24)和SVR48分别为92.3%、100.0%、100.0%和100.0%,显著优于CHC-DLC组(分别为80.9%、100.0%、76.2%和66.7%,P<0.05);治疗后,两组均获得病毒学应答,但CHC-CLC组血小板计数和白蛋白水平分别为(140.6±26.3)×10^(9)/L和(36.4±1.8)g/L,均显著高于CHC-DLC组【分别为(70.5±27.0)×10^(9)/L和(33.4±2.7)g/L,P<0.05】;CHC-CLC组APRI、FIB-4和LSM分别为(1.1±0.4)、(3.0±1.0)和(13.8±2.0)kPa,均显著低于CHC-DLC组【分别为(1.7±0.7)、(5.1±1.7)和(26.2±2.5)kPa,P<0.05】。结论基于SOF的DAAs治疗方案治疗CHC-CLC或CHC-DLC患者具有较为理想的病毒学应答率,肝功能指标得到改善,值得临床应用。

恩替卡韦联合复方鳖甲软肝片治疗慢性乙型肝炎的临床疗效和远期结局

目的探讨应用恩替卡韦联合复方鳖甲软肝片治疗慢性乙型肝炎(CHB)的临床疗效。方法选取2013年5月至2015年4月陆军军医大学第一附属医院收治的120例CHB病人为研究对象,其中联合组应用恩替卡韦(ETV)联合复方鳖甲软肝片治疗60例,对照组应用ETV治疗60例,均治疗72周。随后两组继续服用ETV,随访至少8年。观察两组病人治疗72周的疗效、两次肝活检组织病理学的变化以及分析逆转肝组织纤维化的相关因素,统计两组随访8年的肝癌发生率和病死率。结果治疗72周,联合组肝脏硬度测量值(LSM)下降率58.333%与对照组28.333%相比,差异有统计学意义(P<0.05)。接受两次肝穿的CHB病人,联合组肝纤维化逆转率78.000%与对照组48.485%相比,差异有统计学意义(P<0.05)。单因素分析结果显示,年龄和纤维化分期(F)与肝组织纤维化逆转有关(P<0.05)。多因素分析结果显示,年龄和纤维化分期(F)是肝组织纤维化逆转的独立保护因素(P<0.05)。经过长达8年以上的随访,联合组肝癌发生率和肝脏相关病死率与对照组比较,差异无统计学意义(P>0.05)。结论ETV联合复方鳖甲软肝片治疗72周可显著降低CHB病人的LSM值,逆转进展期肝纤维化。早期抗病毒联合抗纤维化治疗对逆转肝纤维化有重要意义,或可影响长期抗病毒治疗的远期结局。

富马酸丙酚替诺福韦联合复方鳖甲软肝片治疗乙型肝炎肝硬化患者疗效和安全性评估

目的观察富马酸丙酚替诺福韦(TAF)联合复方鳖甲软肝片治疗乙型肝炎肝硬化患者的效果。方法2021年6月~2023年6月我院收治的乙型肝炎肝硬化患者116例,被随机分为两组,每组58例。给予对照组患者口服TAF治疗,给予观察组TAF联合复方鳖甲软肝片治疗,两组均治疗观察6个月。常规检测血清HBV标记物和HBV DNA载量,采用放射免疫分析法检测血清透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PIIIP)和Ⅳ型胶原(CIV),使用超声检测肝脏剪切波弹性成像(SWE)和门脉指标。结果在治疗6个月末,观察组血清ALT水平为(43.1±6.2)U/L,显著低于对照组【(62.6±7.8)U/L,P<0.05】,而两组血清TBIL、ALB和INR比较,无显著性差异(P>0.05);两组血清HBV DNA均转阴,但血清HBsAg和HBeAg水平均无变化;观察组血清透明质酸、Ⅲ型前胶原和Ⅳ型胶原水平分别为(82.2±10.3)ng/L、(94.8±11.5)ng/mL和(61.3±7.5)ng/mL,均显著低于对照组【分别为(123.1±11.9)ng/L、(132.1±13.8)ng/mL和(89.7±9.1)ng/mL,P<0.05】;观察组SWE为(11.2±2.0)kPa,显著低于对照组【(14.1±2.5)kPa,P<0.05】,而两组门静脉内径和脾静脉内径比较,无显著性差异(P>0.05)。结论应用TAF联合复方鳖甲软肝片治疗乙型肝炎肝硬化患者可加速肝功能指标的恢复,抗肝纤维化作用明显,其长期疗效还有待于进一步观察。

Guidelines for diagnosis and treatment of hepatic fibrosis with integrated traditional Chinese and Western medicine(2019 edition)

In 2006, the Hepatology Committee of Chinese Association of Integrative Medicine issued the "Guidelines for the Prevention and Treatment of Liver Fibrosis with Integrated Traditional Chinese and Western Medicine." In recent years, the fields of Chinese medicine, Western medicine, and integrative medicine have made rapid advances in basic and clinical research into chronic liver disease, and accumulated new evidence for the prevention and treatment of hepatic fibrosis. Therefore, in order to meet clinical needs, liver disease experts of integrated traditional Chinese and Western medicine were united to revise the previous guidelines in order to help physicians make correct and reasonable decisions in the diagnosis and treatment of hepatic fibrosis.

Sustained virological response: A milestone in the treatment of chronic hepatitis C

AIM: To evaluate the long-term eradication of hepatitis C virus (HCV) infection and liver-related complications in chronically infected patients that have achieved sustained virological response. METHODS: One hundred and fifty subjects with chronic hepatitis C (CHC) or cirrhosis and sustained virological response (SVR) between the years of 1989 and 2008 were enrolled in a long-term clinical follow-up study at the Gastrointestinal and Liver Unit of the University Hospital of Naples "Federico Ⅱ". At the beginning of the study, the diagnosis of HCV infection was made on the basis of serum positivity for antibodies to HCV and detection of HCV RNA transcripts, while a diagnosis of chronic hepatitis was formulated using imaging techniques and/or a liver biopsy. SVR was achieved by interferon-based therapy, both conventional and pegylated, with and without ribavirin treatment. The patients were evaluated for follow-up at a median length of 8.6 years, but ranged from 2-19.9 years. Among them, 137 patients had pre-treatment CHC and 13 had cirrhosis. The patients were followed with clinical, biochemical, virological, and ultrasound assessments on a given schedule. Finally, a group of 27 patients underwent a liver biopsy at the beginning of the study and transient elastography at their final visit to evaluate changes in liver fibrosis. RESULTS: The median follow-up was 8.6 years (range 2-19.9 years). HCV RNA remained undetectable in all patients, even in patients who eventually developed liver-related complications, indicating no risk of HCV recurrence. Three liver-related complications were observed: two cases of hepatocellular carcinoma and one case of bleeding from esophageal varices resulting in an incidence rate of 0.23%/person per year. Further, all three complications took place in patients diagnosed with cirrhosis before treatment began. Only one death due to liver-related causes occurred, resulting in a mortality rate of 0.077% person per year. This amounts to a 99.33% survival rate in our cohort of patients after therapy for HCV infection. Finally, of the 27 patients who underwent a liver biopsy at the beginning of the study, a reduction in liver fibrosis was observed in 70.3% of the cases; only three cases registering values of liver stiffness indicative of significant fibrosis. CONCLUSION: Patients with CHC and SVR show an excellent prognosis with no risk of recurrence and a very low rate of mortality. Our data indicate that viruseradication following interferon treatment can last up to 20 years.

A small molecule fibrokinase inhibitor in a model of fibropolycystic hepatorenal disease

AIM To evaluate the novel platelet-derived growth factor receptor and vascular endothelial growth factor receptor dual kinase inhibitor ANG3070 in a polycystic kidney disease-congenital hepatic fibrosis model.METHODS At 6 wk of age, PCK rats were randomized to vehicle or ANG3070 for 4 wk. At 10 wk, 24 h urine and left kidneys were collected and rats were continued on treatment for 4 wk. At 14 wk, 24 h urine was collected, rats were sacrificed, and liver and right kidneys were collected for histological evaluation. For Western blot studies, PCK rats were treated with vehicle or ANG3070 for 7 d and sacrificed approximately 30 min after the last treatments.RESULTS Compared to the wild-type cohort, the PCK kidney(Vehicle cohort) exhibited a marked increase in kidney and liver mass, hepato-renal cystic volume, hepato-renal fibrosis and hepato-renal injury biomarkers. Intervention with ANG3070 in PCK rats decreased kidney weight, reduced renal cystic volume and reduced total kidney hydroxyproline, indicating significantly reduced rental interstitial fibrosis compared to the PCK-Vehicle cohort. ANG3070 treatment also mitigated several markers of kidney injury, including urinary neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, cystatin C and interleukin-18 levels. In addition, this treatment attenuated key indices of renal dysfunction, including proteinuria, albuminuria and serum blood urea nitrogen and creatinine, and significantly improved renal function compared to the PCK-Vehicle cohort. ANG3070 treatment also significantly decreased liver enlargement, hepatic lesions, and liver fibrosis, and mitigated liver dysfunction compared to the PCK-Vehicle cohort.CONCLUSION These results suggest that ANG3070 has the potential to slow disease, and may serve as a bridge toward hepato-renal transplantation in patients with fibropolycystic disease.

Current treatment for hepatitis C virus/human immunodeficiency virus coinfection in adults

Hepatitis C virus(HCV)/human immunodeficiency virus(HIV)coinfection is a major problem among HIV-infected patients,resulting in increased morbidity and mortality rates due to the acceleration of liver fibrosis progression by HIV,leading to liver cirrhosis and hepatocellular carcinoma.Although the efficacy of directacting antiviral therapy in patients with HIV/HCV coinfection and HCV monoinfection are similar in terms of sustained virologic response rate,there are some additional complications that arise in the treatment of patients with HIV/HCV coinfection,including drug-drug interactions and HCV reinfection due to the high risk behavior of these patients.This review will summarize the current management of HIV/HCV coinfection.

Nationwide retrospective study of hepatitis B virological response and liver stiffness improvement in 465 patients on nucleos(t)ide analogue

BACKGROUND Hepatitis B virus(HBV)nucleos(t)ide analog(NA)therapy reduces liver disease but requires prolonged therapy to achieve hepatitis B surface antigen(HBsAg)loss.There is limited North American real-world data using non-invasive tools for fibrosis assessment and few have compared 1st generation NA or lamivudine(LAM)to tenofovir disoproxil fumarate(TDF).AIM To assess impact of NA on virological response and fibrosis regression using liver stiffness measurement(LSM)(i.e.,FibroScan®).METHODS Retrospective,observational cohort study from the Canadian HBV Network.Data collected included demographics,NA,HBV DNA,alanine aminotransferase(ALT),and LSM.Patients were HBV monoinfected patients,treatment naïve,and received 1 NA with minimum 1 year follow-up.RESULTS In 465(median 49 years,37%female,35%hepatitis B e antigen+at baseline,84%Asian,6%White,and 9%Black).Percentage of 64(n=299)received TDF and 166 were LAM-treated with similar median duration of 3.9 and 3.7 years,respectively.The mean baseline LSM was 11.2 kPa(TDF)vs 8.3 kPa(LAM)(P=0.003).At 5-year follow-up,the mean LSM was 7.0 kPa in TDF vs 6.7 kPa in LAM(P=0.83).There was a significant difference in fibrosis regression between groups(i.e.,mean-4.2 kPa change in TDF and-1.6 kPa in LAM,P<0.05).The last available data on treatment showed that all had normal ALT,but more TDF patients were virologically suppressed(<10 IU/mL)(n=170/190,89%)vs LAM-treated(n=35/58,60%)(P<0.05).None cleared HBsAg.CONCLUSION In this real-world North American study,approximately 5 years of NA achieves liver fibrosis regression rarely leads to HBsAg loss.

化滞柔肝颗粒减轻非酒精性脂肪性肝炎相关肝纤维化的机制研究

目的结合网络药理学研究方法,探讨化滞柔肝颗粒(HZRG)减轻非酒精性脂肪性肝炎(NASH)相关肝纤维化的作用机制。方法借助网络药理学分析聚焦HZRG治疗NASH的潜在靶点。将雄性C57BL/6小鼠随机分为对照组、模型组和HZRG低、高剂量组,每组8只。除对照组外,其余各组小鼠给予蛋氨酸胆碱缺乏饮食诱导NASH相关纤维化模型,造模的同时给予相应药物干预,连续4周。HE染色观察肝组织病理变化,并行非酒精性脂肪性肝病活动度评分(NAS);天狼猩红和Masson染色法观察肝脏胶原沉积情况;免疫组化法观察肝组织中α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原蛋白α1链(Col1a1)的表达情况,并用ImageJ软件分析阳性表达面积。试剂盒检测肝组织总胆固醇(TC)、三酰甘油(TG)水平。采用生化分析仪检测各组小鼠血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)、碱性磷酸酶(ALP)水平,ELISA试剂盒检测血清肿瘤坏死因子-α(TNF-α)水平。Western blot法和免疫荧光法检测α-SMA、Col1a1、p53蛋白表达水平;RT-qPCR法检测p53 mRNA相对表达量。不同浓度转化生长因子-β1(TGF-β1)或Tenovin-6处理人源性肝星状细胞系LX2细胞24 h,检测相关指标。结果①网络药理学研究结果提示,p53是HZRG发挥效应的潜在靶点。②HE染色、Masson染色和天狼猩红染色结果显示,模型组小鼠肝脏脂质累积、炎细胞浸润、纤维化形成,与对照组比较,模型组小鼠肝组织病理α-SMA、Col1a1阳性表达面积增加(P<0.05),NAS评分升高(P<0.05);HZRG低、高剂量组小鼠肝细胞内脂滴减少,肝脏脂肪变和炎细胞浸润程度明显减轻,与模型组比较,HZRG低、高剂量组肝组织病理α-SMA、Col1a1阳性表达面积减少(P<0.05),NAS评分降低(P<0.05)。③与对照组比较,模型组小鼠血清ALT、AST、LDH、ALP水平升高(P<0.05);与模型组比较,HZRG低、高剂量组小鼠血清ALT、AST、LDH、TNF-α水平降低(P<0.05),HZRG低剂量组小鼠血清ALP水平降低(P<0.05)。④与对照组比较,模型组小鼠肝组织TG、α-SMA蛋白水平升高(P<0.05);与模型组比较,HZRG高剂量组小鼠肝组织α-SMA蛋白水平降低(P<0.05),HZRG低、高剂量组小鼠肝组织TG、Col1a1蛋白水平降低(P<0.05)。⑤各组小鼠肝组织p53 mRNA表达水平比较,差异无统计学意义(P>0.05)。⑥p53蛋白在NASH小鼠肝组织及活化的LX2细胞中表达降低,HZRG及Tenovin-6可上调其表达;p53蛋白表达升高,可缓解NASH小鼠肝组织纤维化进展、抑制LX2细胞活化。结论HZRG减轻NASH相关肝纤维化的机制可能与上调p53蛋白表达有关。