Toll样受体4介导的炎症信号通路在无机砷致大鼠肝纤维化损伤中的作用

The role of Toll-like receptor 4-mediated immune inflammation in inorganic arsenic-induced liver fibrosis in rats

目的观察无机砷中毒大鼠肝组织中Toll样受体4(Toll-like receptor 4,TLR4)信号通路相关蛋白及其磷酸化蛋白表达水平,探讨TLR4介导的炎症信号通路在砷致肝纤维化损伤中的作用。方法将健康初断乳SD大鼠18只,按体重(80~100 g)采用随机数字表法分为3组,每组6只,雌雄各半。对照组给予10 ml/kg生理盐水灌胃;亚砷酸钠(NaAsO_(2))染毒组给予10 mg/kg NaAsO_(2)灌胃;TAK-242干预组给予10 mg/kg NaAsO_(2)灌胃,12周后同时给予0.5 mg/kg TLR4抑制剂TAK-242腹腔注射。每周给药6 d,持续36周。实验结束后采集各组大鼠肝组织和血清,HE、Masson染色法光镜下观察肝组织病理学及胶原纤维沉积情况;全自动生化分析仪检测血清肝功能指标丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)含量;蛋白质印迹法检测大鼠肝组织纤维化蛋白α-平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)、波形蛋白(Vimentin)表达水平及TLR4信号通路相关蛋白TLR4,核转录因子κB(NF-κB)-p65亚基(p65)、NF-κB-p50亚基(p50)及其磷酸化蛋白p-p65、p-p50表达水平;酶联免疫吸附试验(ELISA)检测肝组织炎症因子白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)、IL-10分泌水平。结果HE、Masson染色结果显示,与对照组比较,NaAsO_(2)染毒组出现明显的炎性细胞浸润、肝细胞坏死及胶原纤维沉积,而TAK-242干预组炎性细胞浸润情况较NaAsO_(2)染毒组有所改善,并且胶原纤维沉积减少。血清肝功能指标ALT、AST、ALP含量检测结果显示,NaAsO_(2)染毒组均高于对照组,而TAK-242干预组均低于NaAsO_(2)染毒组(均P<0.05)。蛋白质印迹法检测结果显示,NaAsO_(2)染毒组大鼠肝组织纤维化蛋白α-SMA、TGF-β1、Vimentin(1.04±0.19、0.92±0.14、1.20±0.21),TLR4信号通路相关蛋白及其磷酸化蛋白TLR4、p50、p-p50、p-p65表达水平(1.16±0.21、0.95±0.16、1.24±0.23、1.56±0.25)均高于对照组(0.44±0.08、0.42±0.08、0.72±0.07,0.69±0.15、0.71±0.11、0.46±0.07、0.54±0.11,均P<0.05),TAK-242干预组(0.60±0.13、0.59±0.16、0.49±0.11,0.47±0.08、0.86±0.09、0.79±0.14、1.02±0.17)表达水平均低于NaAsO_(2)染毒组(均P<0.05);p65表达水平3组间比较,差异无统计学意义(F=14.29,P=0.053)。ELISA检测结果显示,NaAsO_(2)染毒组大鼠肝组织IL-6、TNF-α分泌水平[(98.89±4.58)、(83.25±4.57)ng/g]均高于对照组[(27.30±3.92)、(27.77±1.83)ng/g,均P<0.05],而IL-10分泌水平[(36.88±3.86)ng/g]低于对照组[(77.96±7.87)ng/g,P<0.05];TAK-242干预组与NaAsO_(2)染毒组比较,IL-6、TNF-α分泌水平[(44.32±3.60)、(36.51±2.93)ng/g]均较低,IL-10分泌水平[(60.40±4.94)ng/g]较高(均P<0.05)。结论TLR4介导的炎症信号通路相关蛋白及其磷酸化蛋白在无机砷暴露大鼠肝组织中均高表达,抑制TLR4信号通路可明显减轻无机砷致大鼠肝纤维化损伤程度。

Objective To observe the expression levels of Toll-like receptor 4(TLR4)signaling pathway-related proteins and their phosphorylation in the liver tissues of rats with inorganic arsenic poisoning,and to explore the role of TLR4-mediated inflammatory signaling pathway in arsenic-induced liver fibrosis injury.Methods Eighteen healthy weanling SD rats were divided into 3 groups according to their body weight(80-100 g)using a random number table(6 rats in each group,half males and half females).The control group was given 10 ml/kg of normal saline by gavage.The sodium arsenite(NaAsO_(2))exposure group was given 10 mg/kg of NaAsO_(2) by gavage.The TAK-242 intervention group was given 10 mg/kg of NaAsO_(2) by gavage,and 0.5 mg/kg of TAK-242 was also administered intraperitoneally to inhibit TLR4 after 12 weeks.All rats were administered 6 days a week for 36 weeks.At the end of the treatment,the liver tissues and serum of the rats in each group were collected.HE and Masson staining were used to observe the pathological and fibrotic changes of the liver tissues.Automatic biochemical analyzer was used to detect serum liver function indexes of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and alkaline phosphatase(ALP).Western blot was used to detect the expression changes of rat liver fibrosis proteinα-smooth muscle actin(α-SMA),transforming growth factor-β1(TGF-β1),Vimentin and TLR4 signaling pathway-related proteins TLR4,nuclear factorκB(NF-κB)-p65 subunit(p65),NF-κB-p50 subunit(p50)and their phosphorylation p-p65 and p-p50 expression levels.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion levels of inflammatory related factors interleukin(IL)-6,tumor necrosis factor-α(TNF-α)and IL-10.Results HE and Masson staining results showed that compared with the control group,the NaAsO_(2) exposure group showed significant inflammatory cell infiltration,hepatocyte necrosis and collagen fibrous deposition,while the TAK-242 intervention group showed improvement of the inflammatory cell infiltration and reduction of collagen fibrous deposition compared with the NaAsO_(2) exposure group.The results of serum liver function indexes showed that ALT,AST and ALP in NaAsO_(2) exposure group were increased compared with the control group,but the TAK-242 intervention group was significantly decreased compared with the NaAsO_(2) exposure group(P<0.05).Western bolt results showed that in NaAsO_(2) exposure group,the expression levels of fibrosis proteinα-SMA,TGF-β1 and Vimentin(1.04±0.19,0.92±0.14,1.20±0.21)and TLR4 signaling pathway-related proteins and their phosphorylation TLR4,p50,p-p50 and p-p65(1.16±0.21,0.95±0.16,1.24±0.23,1.56±0.25)were higher than the control group(0.44±0.08,0.42±0.08,0.72±0.07,0.69±0.15,0.71±0.11,0.46±0.07,0.54±0.11,P<0.05),and the TAK-242 intervention group(0.60±0.13,0.59±0.16,0.49±0.11,0.47±0.08,0.86±0.09,0.79±0.14,1.02±0.17)were lower than the NaAsO_(2) exposure group(P<0.05).There was no significant difference in the expression level of TLR4 signal pathway-related protein p65 among the three groups(F=14.29,P=0.053).ELISA results showed that the secretion levels of IL-6 and TNF-α[(98.89±4.58),(83.25±4.57)ng/g]in rats liver tissues of the NaAsO_(2) exposure group were higher than the control group[(27.30±3.92),(27.77±1.83)ng/g,P<0.05],while the secretion level of IL-10[(36.88±3.86)ng/g]was lower than the control group[(77.96±7.87)ng/g,P<0.05].In TAK-242 intervention group,IL-6 and TNF-αsecretion levels[(44.32±3.60),(36.51±2.93)ng/g]were lower and IL-10 secretion level[(60.40±4.94)ng/g]was higher compared with the NaAsO_(2) exposure group(P<0.05).Conclusion TLR4-mediated inflammatory signaling pathway-related proteins and their phosphorylation are highly expressed in the liver tissues of rats with inorganic arsenic poisoning,and inhibition of TLR4 signaling pathway could significantly reduce the degree of liver fibrosis injury caused by inorganic arsenic in rats.