亚砷酸钠对SD大鼠肝组织纤维化及上皮-间质转化相关蛋白表达的影响

Effects of sodium arsenite on liver fibrosis and expression of epithelial-mesenchymal transformation-related proteins in SD rats

[背景]长期砷暴露易造成其在肝脏内蓄积,从而导致肝脏病变。相关研究显示间充质细胞在肝纤维化中起着重要作用,且上皮-间质转化(EMT)是间充质细胞的主要来源之一。[目的]探讨不同剂量亚砷酸钠染毒对SD大鼠肝组织纤维化及EMT相关蛋白表达的影响。24只健康初断乳SD大鼠随机分为4组,每组6只,雌雄各半,分别为对照组(10 mL·kg^(-1)生理盐水)、2.5 mg·kg^(-1)亚砷酸钠组、5.0 mg·kg^(-1)亚砷酸钠组和10.0 mg·kg^(-1)亚砷酸钠组,灌胃染毒,每天一次,每周六次。每周称重大鼠一次,持续36周后收集大鼠血清及肝组织,称重并计算脏器系数。苏木精-伊红(HE)染色和马松(Masson)三色染色观察大鼠肝纤维化损伤病理改变程度;酶联免疫吸附法(ELISA)检测大鼠血清中肝纤维化相关蛋白透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原氨基端肽(PⅢNP)和Ⅳ型胶原(COL-Ⅳ)分泌水平;Western blotting法检测大鼠肝组织中肝星状细胞(HSCs)活化相关蛋白α-平滑肌肌动蛋白(α-SMA)和转化生长因子β1(TGF-β1),以及EMT相关标志蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(Ncadherin)、波形蛋白(Vimentin)、锌指转录因子(Snail)表达水平。[结果]与对照组相比,染毒第36周时,10.0 mg·kg^(-1)亚砷酸钠组雌、雄大鼠体重均降低(P<0.05),肝脏系数增加(P<0.05)。病理染色结果显示:各亚砷酸钠染毒组大鼠肝组织可见大量炎性细胞浸润,肝实质细胞液化坏死、变性,同时,大鼠肝组织胶原纤维阳性着色面积均且随染砷剂量增加而逐渐上升(P<0.05)。ELISA和Western blotting结果显示:5.0、10.0 mg·kg^(-1)亚砷酸钠组HA、LN、PⅢNP、COL-Ⅳ分泌水平较对照组和2.5 mg·kg^(-1)亚砷酸钠组增加(P<0.05);与对照组相比,各染砷剂量组大鼠肝组织中α-SMA、TGF-β1蛋白表达均上升(P<0.05),E-cadherin蛋白表达水平下降(P<0.05),而N-cadherin、Vimentin、Snail表达水平升高(P<0.05)。[结论]亚砷酸钠暴露可诱导SD大鼠HSCs活化及肝纤维化损伤,导致细胞外基质分泌水平增加,同时伴有肝组织EMT,提示EMT与砷致肝纤维化损伤过程密切相关。

[Background]Long-term exposure to sodium arsenite leads to its accumulation in the liver and liver injury as a result.Previous studies showed that mesenchymal cells play an important role in hepatic fibrosis,and epithelial-mesenchymal transformation(EMT)is considered to be a main source of mesenchymal cells.[Objective]To investigate the effects of sodium arsenite at different doses on liver fibrosis and EMT-related protein expressions in SD rats.[Methods]Twenty-four healthy weaned SD rats,half male and half female,were randomly divided into four groups according to body weight,with 6 rats in each group.The four groups were control group(gavage with 10.0 mL·kg^(-1)physiological saline),2.5 mg·kg^(-1)sodium arsenite group,5.0 mg·kg^(-1)sodium arsenite group,and 10.0 mg·kg^(-1)sodium arsenite group.All rats were gavaged 6 d per week for 36 weeks and weighed once a week,the serum and liver tissues of rats were collected and weighed,then the organ coefficient was calculated.Hematoxylin-eosin staining and Masson’s trichrome staining were used to determine the pathological changes of hepatic fibrosis in rats.The serum secretion levels of hyaluronic acid(HA),laminin(LN),procollagenⅢN-terminal propeptide(PⅢNP),and collagenⅣ(COL-Ⅳ)in rats were detected by enzyme-linked immunosorbent assay(ELISA).The protein expressions of HSCs activation-related proteins,such asα-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1),as well as EMT-related markers,such as E-cadherin,N-cadherin,Vimentin,and Snail,were detected by Western blotting.[Results]Compared with the control group,the 10.0 mg·kg^(-1)sodium arsenite group showed decreased body weight(P<0.05)and increased liver coefficient(P<0.05)of female and male rats.The pathological staining showed that,compared with the control group,a large number of inflammato ry cells were observed in liver tissue of rats exposed to sodium arsenite,liver parenchymal cells we re also liquefied,necrotic,and denatured,and the collagen positive staining area of liver tissue showed an upward trend along with the increase of arsenic exposure dose(P<0.05).The results of ELISA and Western blotting showed that the serum secretion levels of HA,LN,PⅢNP,and COL-Ⅳin the 5.0and 10.0 mg·kg^(-1)sodium arsenite groups were higher than those in the control group and the 2.5 mg·kg^(-1)sodium arsenite group(P<0.05).Compared with the control group,the expressions ofα-SMA and TGF-β1 proteins in liver tissue were increased in each sodium arsenite exposure group(P<0.05),the expression levels of E-cadherin protein were decreased(P<0.05),and the expression levels of Ncadherin,Vimentin,and Snail were increased(P<0.05).[Conclusion]Sodium arsenite exposure can induce HSCs activation and liver fibrosis injury in SD rats,resulting in increased extracellular matrix secretion levels,accompanied by EMT in liver tissue,suggesting that EMT is closely related to the process of liver fibrosis caused by arsenic.