Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

Bcl‐2 overexpression in hepatic stellate cell line CFSC‐2G,induces a pro‐fibrotic state

Background and Aim: Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro‐ and anti‐apoptotic molecules. Since Bcl‐2 overexpression preserves viability against OS, our objective was to address the effect of Bcl‐2 overexpression in the hepatic stellate cells (HSC) cell‐line CFSC‐2G under acetaldehyde and H₂O₂ challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. Methods: To induce Bcl‐2 overexpression, HSC cell line CFSC‐2G was transfected by lipofection technique. Green fluorescent protein‐only CFSC‐2G cells were used as a control. Cell survival after H₂O₂ treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation‐rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue‐inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a‐actin (α‐SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor‐β (TGF‐β) mRNA. Results: Cells overexpressing Bcl‐2 survived ≈ 20% more than control cells when exposed to H₂O₂ and ≈ 35% proteins were protected from oxidation, but Bcl‐2 did not slow proliferation or induced senescence. Bcl‐2 overexpression did not change α‐SMA levels, but it increased TIMP‐1 (55%), tissue transglutaminases (tTG) (25%) and TGF‐β mRNA (49%), when exposed to acetaldehyde, while MMP‐13 content decreased (47%). Conclusions: Bcl‐2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP‐1, tTG and TGF‐β mRNA levels and decreased MMP‐13 content, suggesting that Bcl‐2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.     

Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis.

BACKGROUND/AIMS: Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP-2 and MMP-14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis. METHODS: MMP and tissue inhibitor of metalloproteinase (TIMP)-2 expression in liver extracts was examined by ribonuclease protection assay, Western blotting and gelatin zymography. MMP activity was examined by (14)C gelatin degradation. RESULTS: In human cirrhotic liver, MMP-14 mRNA was increased to 230-330% of normal liver expression. Both 63 kDa proenzyme and 60 kDa activated form were present. Cirrhotic livers had 270-320% of normal liver expression of MMP-2 protein with 20-25% being the 62 Da activated form. Protein and mRNA for MMP-2 and MMP-14 progressively increased during 8 weeks of CCl(4) treatment in rats. Between 3 and 7 days of resolution from CCl(4) liver fibrosis, MMP-2 and MMP-14 persisted at elevated levels. Gelatinolytic activity in liver homogenates peaked at 7 days of recovery, being 140% above that in livers at peak fibrosis. CONCLUSIONS: Increased expression and activation of MMP-2 and -14 occurs even under conditions of elevated TIMPs during liver fibrogenesis. During liver fibrosis resolution, as TIMP expression decays, the persistence of MMP-2 and MMP-14 may permit collagen degradation.     

Antifibrotic and fibrolytic properties of celecoxib in liver damage induced by carbon tetrachloride in the rat

Background: Transforming growth factor‐β (TGF‐β) plays a pivotal role in liver fibrosis, because it activates hepatic stellate cells, stimulating extracellular matrix deposition. Cyclooxygenase‐2 (COX‐2) has been associated with TGF‐β because its inhibition decreases TGF‐β expression and collagen production in some cultured cell types. Aim: The aim of this work was to evaluate the ability of celecoxib (a selective COX‐2 inhibitor) to prevent and to reverse the liver fibrosis induced by CCl₄. Methods: We established experimental groups of rats including vehicle and drug controls, damage induced by chronic CCl₄ administration and CCl₄ plus pharmacological treatment in both prevention and reversion models. We determined: alanine aminotransferase, alkaline phosphatase, γ‐glutamyl transpeptidase, COX and metalloproteinase‐2 and ‐9 activities, lipid peroxidation, glutathione levels, glycogen and collagen content and TGF‐β expression. Results: Celecoxib prevented and aided to the recovery of livers with necrotic and cholestatic damage. Celecoxib exhibited anti‐oxidant properties by restoring the redox equilibrium (lipid peroxidation and glutathione levels). Glycogen was decreased by CCl₄, while celecoxib partially prevented and reversed this effect. Celecoxib inhibited COX‐2 activity, decreased TGF‐β expression, induced metalloproteinase‐2 activity and, consequently, prevented and reversed collagen accumulation. Conclusion: Our findings indicate that celecoxib exerts strong antifibrogenic and fibrolytic effects in the CCl₄ model of cirrhosis.     

The anti‐inflammatory effect of celecoxib does not prevent liver fibrosis in bile duct‐ligated rats

Background/Aims: Celecoxib was used in the treatment of inflammation in patients with cirrhosis. However, data on the progression of liver fibrosis after treatment by celecoxib are not available. This study aims to elucidate the effects of celecoxib on cholestatic liver fibrosis in rats. Methods: Rats underwent bile duct ligation (BDL) for 1 or 2 weeks to induce hepatic fibrosis. Celecoxib was introduced on day 1 after operation. The effects of celecoxib were assessed by comparison of the severity of hepatic fibrosis. Results: Infiltration of inflammatory cells and proliferation of bile ducts was seen after 1 week of BDL and fibrosis was induced after 2 weeks. Reduced alanine aminotransferase (ALT) levels and blunted expression of inflammatory factors [tumour necrosis factor‐α, interleukin (IL)‐1β and IL‐6] were seen in the liver of BDL‐treated rats that received celecoxib at week 1. Although celecoxib was sufficient in suppressing the cyclo‐oxygenase (COX)‐2 expression in the control organ (kidney), it failed to suppress the enhanced hepatic COX‐2 expression. At week 2, celecoxib did not alter the ALT level, the severity of fibrosis and hepatic collagen contents. This was associated with unchanged α‐smooth muscle actin protein expression and tissue inhibitor of metalloproteinase‐2 (TIMP‐2), matrix metalloproteinase (MMP)‐2 and MMP‐9 mRNA expressions in the liver. Celecoxib had no effect on the BDL‐dependent increase in bilirubin levels at any time point. Conclusions: The present study provides morphological and molecular biological evidences for the role of celecoxib in cholestatic liver fibrosis. Celecoxib protects against hepatic inflammation in the early stage of BDL rats, but does not have an effect on liver fibrosis.     

Active matrix metalloproteinase‐2 promotes apoptosis of hepatic stellate cells via the cleavage of cellular N‐cadherin

Background and Aims: Hepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix‐degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP‐1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N‐cadherin at the cell surface. Results: N‐cadherin expression was upregulated in human HSC during activation in culture. Addition of function‐blocking antibodies or a peptide targeting the extracellular domain of N‐cadherin, to cultured HSC, promoted apoptosis. During apoptosis, there was cleavage of N‐cadherin into 20–100 kDa fragments. MMP‐2 became activated early during HSC apoptosis and directly cleaved N‐cadherin in vitro. Addition of activated MMP‐2 to HSCs in culture resulted in enhanced apoptosis and loss of N‐cadherin. Conclusions: Together, these studies identify a role for both N‐cadherin and MMP‐2 in mediating HSC apoptosis, where N‐cadherin works to provide a cell survival stimulus and MMP‐2 promotes HSC apoptosis concomitant with N‐cadherin degradation.     

Transforming growth factor‐α attenuates hepatic fibrosis: possible involvement of matrix metalloproteinase‐1

Background: The effect of transforming growth factor (TGF)‐α on fibrosis varies between cell types and the role of TGF‐α in hepatic fibrosis has not been fully elucidated. Methods: We examined the effect of TGF‐α on hepatic fibrosis using TGF‐α‐expressing transgenic mice fed a methionine‐ and choline‐deficient (MCD) diet and human hepatic stellate cells (HSCs) line LX‐2, rat and human primary HSCs. Results: Although the expression levels of the tissue inhibitor of metalloproteinases‐1 and α1(I) collagen mRNA were unchanged, feeding the TGF‐α transgenic mice the MCD diet resulted in greater expression of the murine functional analogue of matrix metalloproteinase‐1 (MMP‐1), MMP‐13 mRNA and protein and attenuated hepatic fibrosis compared with wild‐type mice. TGF‐α overexpression did not affect the extent of the steatosis, oxidative stress and hepatic inflammation in the MCD diet‐fed mice. The effect of TGF‐α on the fibrogenic and anti‐fibrogenic gene expressions varied between cell types in vitro. TGF‐α increased MMP‐1 mRNA expressions that were completely blocked by gefitinib in LX‐2 cells. The extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase and p38 pathways were involved in MMP‐1 mRNA expression in LX‐2 cells. Although TGF‐α increased the phosphorylation of p38, the p38 inhibitor activated the RAS‐ERK pathway and increased TGF‐α‐induced MMP‐1 mRNA expression, which suggested that there may be a crosstalk between the RAS‐ERK and the p38 pathways in LX‐2 cells. Conclusions: The TGF‐α may attenuate hepatic fibrosis in part because of upregulation of the expression of MMP‐1. The balance between fibrogenic and anti‐fibrogenic gene expression and between the activity of the RAS‐ERK and the p38 pathways may be crucial for the fibrotic process.     

MMP‐9 and TIMP‐1 in the cord blood of premature infants developing BPD

We investigated matrix metalloproteinase‐9 (MMP‐9) and tissue inhibitor of metalloproteinase 1 (TIMP‐1) levels in the cord blood of 29 premature infants who were <30 weeks gestation. One, 8, and 14 infants developed severe, moderate and mild bronchopulmonary dysplasia (BPD), respectively, and 6 did not. MMP‐9 and TIMP‐1 levels in the cord blood were determined by ELISA. MMP‐9/TIMP‐1 ratios in the cord blood of infants who developed severe or moderate BPD (n = 9) were significantly higher than those who developed mild BPD or did not develop BPD (n = 20; P = 0.015). Multivariate linear regressions demonstrated that MMP‐9 levels and MMP‐9/TIMP‐1 ratios in the cord blood of the premature infants correlated with the oxygen supplementation period (r = 0.58, P = 0.003 and r = 0.41, P = 0.030, respectively). The MMP‐9 levels and MMP‐9/TIMP‐1 ratios correlated with the severity of maternal chorioamnionitis (both trend P = 0.006). The MMP‐9 levels and MMP‐9/TIMP‐1 ratios in the cord blood may be related to the pathogenesis and severity of BPD and maternal chorioamnionitis. Pediatr Pulmonol. 2009; 44:267–272. © 2009 Wiley‐Liss, Inc.     

软肝化坚颗粒对肝纤维化C57小鼠肝组织中MMP-13和TIMP-1 mRNA表达的影响

目的观察软肝化坚颗粒对肝纤维化模型C57小鼠血清中基质金属蛋白酶13(MMP-13)和基质金属蛋白酶抑制剂1(TIMP-1)的含量和肝组织中MMP-13和TIMP-1 mRNA表达的影响。方法 40只C57小鼠被随机分为4组,每组10只,即正常对照组、模型对照组、模型软肝化坚颗粒组和模型秋水仙碱组。采用四氯化碳腹腔注射制造小鼠肝纤维化模型。分别采用ELISA法和实时荧光定量PCR检测小鼠血清MMPs、TIMP-1含量及肝组织中MMP-13和TIMP-1 mRNA的表达。同一指标的多组间计量资料的比较采用方差分析。结果与正常对照组相比较,模型对照组血清中MMPs的含量明显降低,TIMP-1的含量明显升高,P=0.003、0.027,模型软肝化坚颗粒组与正常对照组相比差异均无统计学意义,P=0.364、0.217。与正常对照组相比,模型软肝化坚颗粒组小鼠肝组织中MMP-13 mRNA的表达量显著升高,P=0.005,TIMP-1 mRNA的表达量差异无统计学意义,P=0.997;模型对照组小鼠肝组织中TIMP-1 mRNA的表达量明显升高,差异具有统计学意义,P=0.009。结论软肝化坚颗粒不但可促进MMPs的产生,而且可抑制TIMP-1产生,进而促进ECM的降解,这可能也是软肝化坚颗粒抗肝纤维化的重要机制之一。     

Polaprezinc attenuates liver fibrosis in a mouse model of non-alcoholic steatohepatitis

Background and Aim: The effect of polaprezinc, a zinc‐carnosine chelate compound, on the development of non‐alcoholic steatohepatitis (NASH) was investigated in dietary methionine and choline deficient (MCD) mice. Methods: Mice were fed the MCD diet with or without polaprezinc (2.2 g/kg diet) for 10 weeks. Liver histopathology, triglyceride and lipid peroxide levels, and the expression of genes linked to fibrosis were then assessed. Results: MCD mice developed steatohepatitis accompanied by mild fibrosis with an increase in lipid peroxidation, hepatic stellate cell (HSC) activation, and the augmented mRNA expression of tumor necrosis factor‐α, transforming growth factor‐β1 and procollagen α1(I). The mRNA expression levels of matrix metalloproteinase (MMP)‐2 and tissue inhibitors of metalloproteinase (TIMP)‐1 and TIMP‐2 were also enhanced. Histopathologically, polaprezinc supplementation did not influence the development of steatosis but it apparently attenuated fibrosis. Polaprezinc slightly reduced lipid peroxidation and suppressed HSC activation as well as the mRNA expression of pro‐inflammatory cytokines. Polaprezinc affected the MCD diet‐enhanced expression of TIMP‐1 even when administered relatively late. Conclusion: These results suggest that polaprezinc attenuates fibrosis in NASH by reducing inflammation and lipid peroxidation and, during a later phase, promoting fibrolysis via the inhibition of TIMP expression in the liver. Further investigation is required to clarify the clinical efficacy of polaprezinc in patients with NASH.     

血清学诊断肝纤维化新指标--金属蛋白酶组织抑制因子检测方法学建立及应用评价

目的：自行建立一种改良固相致敏红细胞吸附技术（SPASE）用于快速检测肝硬化病人血清中金属蛋白酶组织抑制因子－1，2（TIMP－1和TIMP－2），同时研究TIMP－1和TIMP－2在肝化病人肝组织中的定位及表达状态，以及肝组织TIMPs与血清TIMPs的相关性。探讨血清中TIMP－1和TIMP－2可否作为肝纤维化的血清学诊断新指标。方法：应用自行建立的SPASE检测1082例肝病病人（其中肝硬化310例）血清标本中的TIMP－1和TIMP－2。用原位杂交及免疫组织化学技术分别检测40例肝硬化病人活检肝组织中TIMP－1和TIMP－2 mRNA及相关抗原的表达，并与病人自身血清检测结果对比。另检测20例正常肝组织作为对照。结果：SPASE法特异性中和抑制试验示中和抑制率均在70％以上，检测310例肝硬化病人血清中TIMP－1和TIMP－2的阳性率分别为79．68％和65．16％。40例肝硬化病人肝组织中TIMP－1和TIMP－2 mRNA及相关抗原表达阳性率为100％，TIMP－1表达强度高于TIMP－2（P＜0．01）；阳性信号主要位于肝细胞胞质内，未见细胞核表达。40例肝硬化肝活检病人血清检测结果，TIMP－1阳性率为100％，TIMP－1阳性率为77．5％（31／40）。正常肝组织无一例有TIMPs相关抗原表达。结论：TIMPs定位于肝硬化病人的肝细胞胞质内，在肝硬化组织内呈程度不等的阳性表达；血清中TIMPs与肝组织中TIMPs表达有明显相关性，因而血清中TIMP－1和TIMP－2可作为肝纤维化较为有用的血清学诊断新指标，尤其是TIMP－1的诊断意义更大。SPASE法检测血清中TIMPs具有较好的特异性。     

Hypoxia stimulates hepatocyte epithelial to mesenchymal transition by hypoxia‐inducible factor and transforming growth factor‐β‐dependent mechanisms

Background/Aims: During development of liver fibrosis, an important source of myofibroblasts is hepatocytes, which differentiate into myofibroblasts by epithelial to mesenchymal transition (EMT). In epithelial tumours and kidney fibrosis, hypoxia, through activation of hypoxia‐inducible factors (HIFs), is an important stimulus of EMT. Our recent studies demonstrated that HIF‐1α is important for the development of liver fibrosis. Accordingly, the hypothesis was tested that hypoxia stimulates hepatocyte EMT by a HIF‐dependent mechanism. Methods: Primary mouse hepatocytes were exposed to room air or 1% oxygen and EMT evaluated. In addition, bile duct ligations (BDLs) were performed in control and HIF‐1α‐deficient mice and EMT quantified. Results: Exposure of hepatocytes to 1% oxygen increased expression of α‐smooth muscle actin, vimentin, Snail and fibroblast‐specific protein‐1 (FSP‐1). Levels of E‐cadherin and zona occludens‐1 were decreased. Upregulation of FSP‐1 and Snail by hypoxia was completely prevented in HIF‐1β‐deficient hepatocytes and by pretreatment with SB431542, a transforming growth factor‐β (TGF‐β) receptor inhibitor. HIFs promoted TGF‐β‐dependent EMT by stimulating activation of latent TGF‐β1. To determine whether HIF‐1α contributes to EMT in the liver during the development of fibrosis, control and HIF‐1α‐deficient mice were subjected to BDL. FSP‐1 was increased to a greater extent in the livers of control mice when compared with HIF‐1α‐deficient mice. Conclusions: Results from these studies demonstrate that hypoxia stimulates hepatocyte EMT by a HIF and TGF‐β‐dependent mechanism. Furthermore, these studies suggest that HIF‐1α is important for EMT in the liver during the development of fibrosis.     

基质金属蛋白酶及组织抑制剂在肝癌中的表达及与预后的关系

目的：探讨基质金属蛋白酶（MMPs)及其组织抑制剂（TIMPs）在肝癌中的表达及其意义。方法：应用免疫组化、Northern印迹杂交及图像分析技术对人肝细胞癌（HCC）、肝癌细胞株7721、全反式维甲酸处理的7721细胞（RA－7721）和正常人肝细胞株L－02作MMP－1、2、9和TIMP－2的表达分析。结果;肝癌细胞浆内可表达MMP－1、2和9,但癌内阴性组5年生存率明显高于相应的阳性组（P＜0．05）。体外MMP－9mRNA在7721细胞表达明显高于RA－7721以及L－02细胞,而TIMP－2mRNA的表达与MMP－9相反,MMP－2mRNA在7721细胞中的表达仅略高于L－02细胞。结论：HCC组织内MMP－1和9的表达与患者的预后密切相关;癌细胞高表达MMP－9、低表达TIMP－2,可能是肝癌细胞浸润、转移的主要基础,而MMP-2并无重要作用。     

All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

Serum levels of matrix metalloproteinase‐9, tissue inhibitors of metalloproteinase‐1 and their ratio are associated with impaired lung function in the elderly: A population‐based study

Background and objective: Matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), regulate homeostasis and turnover of the extra cellular matrix. The aim of this study was to investigate the associations of serum MMP‐9 and TIMP‐1 with lung function. Methods: Spirometry was performed in a population‐based sample of 888 subjects aged 70 years. Serum MMP‐9 and TIMP‐1 concentrations were measured by ELISA. Results: Lower FEV₁ values were associated with higher serum levels of MMP‐9 (P = 0.001) and TIMP‐1 (P < 0.001), and a higher ratio of MMP‐9 to TIMP‐1 (P = 0.02). These associations were significant after adjustment for gender, weight, height, BMI, current smoking, pack years of smoking and the time for which samples were frozen. After stratification for gender, the associations between FEV₁ and MMP‐9, TIMP‐1, and their ratio, were significant in men but not in women. Conclusions: Lower FEV₁ was significantly but weakly associated with higher serum levels of MMP‐9, TIMP‐1 and a higher MMP‐9/TIMP‐1 ratio. This association was stronger in men than in women, suggesting a possible role for extracellular matrix remodelling in the development of impaired lung function. These associations may also partly explain the association between low FEV₁ and cardiovascular disease.     

多西环素对乳腺癌移植瘤小鼠MMP-2及TIMP-2 mRNA表达的影响

目的 研究多西环素在小鼠乳腺癌肺转移模型中对基质金属蛋白酶-2(MMP-2)及金属蛋白酶组织抑制因子-2(TIMP-2)mRNA表达的影响.方法 30只TAⅡ近交系小鼠建立肺转移性乳腺癌BCML-TAⅡ99模型,随机分为对照组及给药组,每组15只,于可触及肿物后次日,给药组腹腔注射盐酸多西环素0.1 ml(10 mg/ml),对照组给予等量生理盐水.7 w后处死动物,剥离肿瘤称重,部分新鲜组织采用逆转录多聚酶链反应(RT-PCR)方法 检测肿瘤组织MMP-2及TIMP-2 mRNA表达水平.结果 多西环素给药组小鼠肿瘤生长大小及转移率分别为(2.718±0.337)g和13.3%均低于对照组[(4.313±0.468)g,53.3%,P＜0.01,P＜0.05].与对照组比较,多西环素治疗组MMP-2 mRNA为0.637±0.088,明显低于对照组 (1.035±0.097,P＜0.05);TIMP-2 mRNA为0.778±0.148,明显高于对照组 (0.484±0.125,P＜0.05).结论 多西环素通过调节MMP-2及TIMP-2 mRNA的表达水平,对小鼠乳腺癌肺转移模型中肿瘤生长及转移发挥抑制作用.     

Combined use of aspartate aminotransferase,platelet count and matrix metalloproteinase 2 measurements to predict liver fibrosis in HIV/hepatitis C virus‐coinfected patients

Objective Noninvasive tests that can be used in place of liver biopsy to diagnose fibrosis have major limitations. They either leave a significant proportion of patients without a definitive diagnosis or produce inaccurate results. Moreover, the performance of these tests is lower in HIV/hepatitis C virus (HCV) coinfection. Against this background, we examined the utility of serum matrix metalloproteinase 2 (MMP‐2) and tissue inhibitor of metalloproteinase 1 (TIMP‐1) measurements in combination with routine clinical data to predict fibrosis in HIV/HCV‐coinfected patients. Methods Patients with a liver biopsy who had not received anti‐HCV therapy were included in the study. A model including variables independently associated with fibrosis was constructed. Diagnostic accuracy was determined by measuring the area under the receiver operating characteristic curve (AUROC). Positive (PPV) and negative (NPV) predictive values were calculated. Results Ninety patients were included in the study. Aspartate aminotransferase (AST), platelet count and MMP‐2 were predictors of significant fibrosis (F≥2) and cirrhosis (F4). A score constructed using these variables yielded an AUROC of 0.76 for F≥2 and 0.88 for F4. Score cut‐offs detected (value ≥3.5) and excluded (value ≤1.5) F≥2 with a PPV of 87% and an NPV of 88%. Thirty‐one patients (34%) were correctly diagnosed using these cut‐offs, with four (13%) incorrect classifications. Cirrhosis was excluded with a certainty of 98% and diagnosed with a probability of 83%. Two (17%) of 12 patients were misclassified as having cirrhosis. The AST to platelet count index and MMP‐2 levels were sequentially applied to detect F≥2. Forty‐one patients (46%) were identified with this approach, with six (15%) misclassifications. Conclusion MMP‐2 levels can be used in combination with AST and platelet count to aid the diagnosis of liver fibrosis in HIV/HCV‐coinfected patients.