| [Background and objective]Cirrhosis is the proliferation of hepatic fibrous connective tissue associated with diffuse nodular regeneration of liver cells. Reduction of the normal lobular architecture, disappearance of all forms of cirrhosis of the most important feature. Also shows significant liver necrosis, Diiffusivity deposition of fibrous connective tissue, the proliferation of different levels of regeneration nodules, normal lobular reduction, structural disorders (necrosis, portal area connecting the portal vein and central vein bridging necrosis), blood hemodynamic changes (due to liver necrosis, intrahepatic portosystemic shunt, the formation of fibrous septa). Specific mechanisms include cirrhosis:hepatic lobule, the portal area around the portal vein prefabricated fiber proliferation; liver cell damage caused by fibrosis; lobule and necrosis caused by the bridge network hard fibrous scaffold collapse (collagen fibers did not increase); Most new fiber, degradation reduced; caused by the proliferation of intrahepatic bile duct fibrosis. Liver fibrosis, collagen fibers by building skeleton for other extracellular matrix implantation. A large number of extracellular matrix proliferation and deposition in the Disse space and interstitial cells of the liver sinusoidal around central veins, formed under the layer of sinusoidal endothelial basement membrane, impeding blood flow and hepatic sinusoidal cells material exchange. Ultimately, the lobule structure changes, stenosis and obstruction of small hepatic and portal vein pressure increased. Hepatic stellate cells in liver cirrhosis and portal hypertension in the development of an important role. Activated hepatic stellate cell migration and adhesion and chemotactic response of inflammatory cells increased rough endoplasmic reticulum and morphological changes, transformed into myofibroblasts-like cells, secreting large amounts of collagen type I, represented by extracellular matrix in liver fibrosis and proliferation, particularly within the hepatic lobule play an important role in fibrosis. At the molecular level, this process received a variety of cytokines, including transforming growth factorβ1 as a strong concern the role of fibroblasts, transforming growth factorβ1 signaling through a variety of activated hepatic stellate cells via activation of type I collagen promoter, increased expression of collagen I mRNA.ET was isolated from porcine aortic endothelial cells by Japanese scholars in 1988, It's a strong vasoactive peptide involved in the maintenance of vascular tone and systemic hemodynamics regulation, and its role in the treatment of cardiovascular diseases has been widely recognized. It is a group composed of 21 amino acid peptide small molecule family, including three kinds of isomers, ET A 1, ET A 2, ET-3, in which the vascular effect of ET-1 is the most intense one. Meanwhile, endothelin also has a strong role in promoting the growth and induced fibrosis. The role of endothelin mainly by two kinds of receptor subtypes, namely endothelin A and endothelin B receptor. In the liver, endothelin A receptor is mainly distributed in hepatic stellate cells, endothelin B receptor evenly distributed in the hepatic stellate cells and sinus endothelial cells. In cirrhotic patients, plasma endothelin concentrations were significantly increased, and increased the level of endothelin and the severity of liver cirrhosis, whether there is ascites, hepatorenal syndrome are related. Plasma endothelin concentrations in patients with cirrhosis, and the expression of its receptor mRNA increased in parallel, hepatic stellate cell activation, endothelin induced contraction of activated hepatic stellate cells enhanced, resulting in intrahepatic vascular resistance and increased portal pressure may be portal hypertension in one of the important pathological mechanism; the other hand, endothelin led to hepatic stellate cells into fibroblasts, the secretion of type I collagen as the representative of the extracellular matrix, resulting in the development of liver cirrhosis, that play an important role. Therefore, endothelin-induced contraction of hepatic stellate cells to suppress may be liver cirrhosis and portal hypertension in new ways. With endothelin-induced portal hypertension in liver an important role in the occurrence and development of in-depth understanding, the role of inhibition of endothelin methods developed rapidly. At present, there are, endothelin receptor antagonistic agents and endothelin-converting enzyme inhibitors. As most of the endothelin-1 could transformed without the mediation of endothelin-converting enzyme, so the effectiveness of the ECE inhibitor limited its use. More current focus is endothelin receptor antagonist. Endothelin receptor antagonist-mediated receptor according to two types of selective endothelin receptor antagonists and non-selective endothelin receptor antagonist, this experiment was to investigate the effects of ET receptor antagonists on the expression of hepatic collagen typeland TGFβ1 mRNA in carbon tetrachloride (CCl4)-induced cirrhosis rats.[Materials and methods]1 ReagentsCarbon tetrachloride, analytical grade, molecular weight 153.82, Beijing Chemical Plant. Drug BQ-123, BQ-788 was purchased from CALBIOCHEM company. RT-PCR-related reagents were purchased from TaKaRa Biotechnology (Dalian) Co, Ltd.2 Preparation of animal model and experimental groups40 clean male Sprague-Dawley rats were purchased from Experimental Animal Center of Zhengzhou University, weight 370-400g. Random divided into normal control group:(NL, n=8), normal feeding, no treatment; the remaining 32 Preparation of liver cirrhosis model (50% mass fraction of carbon tetrachloride, irrigation by 3ml/kg weight stomach, the interval 6d/times,10 times). Model group, 32 rats were further divided into:model control group (CC14, n=8), endothelin A receptor antagonist group (ETRAa, n=8), endothelin B receptor antagonist group (ETRBa, n=8), the combined treatment group (ETRA+B, n=8),3 groups after the endothelin receptor antagonist treatment 2 times a day (every 10 h), were injected subcutaneously endothelin receptor antagonist BQ-123 (12.5μg/Kg,10 nmol/min) and BQ-788 (15μg/Kg,10 nmol/min), and BQ-123+BQ-788 (12.5μg/kg+15μg/kg, 10 nmol/min). Based on pre-trial [4], the BQ-123 and BQ-788 dose allows the application of the peak plasma concentration of 10-7mol/L, this concentration will be able to have the greatest blocking effect.3d last CCl4 orally after fasting 12h,1% 50mg/Kg intraperitoneal injection of pentobarbital sodium anesthesia. A portion of liver tissue fixed in 10% formaldehyde, underwent routine pathology testing, set the remaining liver tissue stored in liquid nitrogen, ready to extract total RNA.3 Liver CollagenⅠmRNA and TGFβ1-mRNA determination①Liver total RNA extraction:Extraction of liver tissue by TRIZOL reagent total RNA. Spectrophotometer extracted total RNA content and purity requiring OD260/OD280 values between 1.8-2.0.②Reverse transcription polymerase chain reaction (RT-PCR):The total liver tissue RNA 4μg, ThermoScriptTM RT total reaction 20μl, at 65℃for 1h.③CollagenⅠ, TGFβ1, GAPDH andβ-actin primers are as follows:④Polymerase chain reaction:Take RT product cDNA 1μl, the total volume of 25μl, the PCR amplification. Reaction conditions:94℃for denaturing 2min,94℃denaturation 30s,55℃annealing 30 s,72℃extension 50s,30 cycles,72℃extension after 7min.⑤Quantitative analysis of PCR products:RT-PCR results can be seen in the DNA amplification of 443bp and 298bp specific band, respectively, rat liver tissue Collagen I-cDNA and TGFβ1-cDNA fragments to Kodak 120 gel imaging system for DNA amplification with image analysis to Collagen I/GAPDH ratio and TGFβ1/β-actin that Collagen I mRNA and TGFβ1-mRNA relative expression.4 StatisticalSoftware used SPSS18.0 statistical analysis of the data processing, measurement data using mean±standard deviation (x±s) that among groups of the comparative group design with random data unit of analysis of variance, p<0.05 for the the difference was statistically significant.[Results]1 PathologyLobular architecture, normal control group, no degeneration and necrosis of liver cells and other diseases, individual portal area in a few scattered lymphoid cells and monocytes. CC14 cirrhosis model group, the most important inflammatory response, the normal liver lobule structure disappeared, liver parenchyma surrounded by fibrous septa, separated into different size fake leaflets, showing a larger range of diffuse liver damage. Application of endothelin receptor antagonist group than in model control group of liver inflammation and fibrosis was significantly reduced, can be seen scattered across the fibers and inflammatory cell infiltration in portal area.2 The effect of Endothelin receptor antagonist on TGFβ1 mRNA expression in cirrhosis ratsLiver TGFβ1-mRNA expression studies suggest that:the model group increased expression of TGFβ1 mRNA in liver tissue 142%, significantly higher than the control group (0.75±0.15 vs.0.31±0.05, P<0.05); endothelin A receptor antagonist, endothelin B receptor antagonist group and the combined treatment group, the hepatic expression of TGFβ1 mRNA levels were lower than the control group, which TGFβ1 mRNA combined treatment group was lowest, about 28% lower but there is no statistically significant difference (P> 0.05).3 The effect of Endothelin receptor antagonist on type I 6wcollagen mRNA expression in cirrhosis rats: Liver CollagenⅠmRNA expression studies suggest that:the model group CollagenⅠmRNA expression in liver tissue compared with normal control group was significantly higher (0.87±0.17 vs 0.43±0.08, P<0.05); application of endothelin receptor antagonists, regardless of endothelin A receptor antagonist, endothelin B receptor antagonist or the combined treatment group, the liver CollagenⅠmRNA expression were lower than model control group, P<0.05. And the combined treatment group CollagenⅠmRNA expression level of the lowest 37% reduction[Conclusion]Endothelin receptor antagonists can inhibit the expression of hepatic collagen typeⅠin cirrhotic rats effectively,and reduce inflammation reaction and liver fibrosis. Objective:This study investigated the hepatic vascular diseases induced by heat stress in rats and its clinical significance. Methods:The acute heat stress model: Male SPF Wistar rats were exposed under temperature of 42±0.5℃and relative humidity of 40±5% in a temperature-control box for 100 minutes. Core temperature (Tc) was measured with a rectal probe. When the Tc rose to 40±1.45℃, the portal vein pressure, blood coagulation indicators, endotoxin concentration was measured and the liver was sampled for pathological examination. The chronic heat stress model:The temperature was controlled with the aforementioned method; the heat exposure was given once a week and maintained 4 weeks; and then kept on feeding for 4 weeks. The portal vein pressure and pathology of liver specimens were examined. Results:①The acute heat stress model:The heat stress induced a increase in portal vein pressure in experimental group(7.69±0.843 cmH2O) compared with the control group (6.75±0.92 cm H2O) (p<0.05); The coagulation function was compromised in experimental group with a significant difference (P<0.05). The plasma endotoxin concentration were not significantly different.②The model of chronical heat stress:The heat stress induced a significant increase in portal vein pressure in experimental group (10.46±1.635 cmH2O) compared with that in the control group (6.95±0.497 cmH20) (p<0.01). The pathological and immunohistochemical examination indicated that there were inflammatory cell infiltration, vascular endothelial cells damage, hepatic veins stenosis/thrombosis and regenerated vascular could be found. Conclusions:The heat stress compromises the integrity of the coagulation conditions, causes hepatic vascular lesions and leads to hepatic vascular diseases coupled with portal hypertension finally. |
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