黄芩苷通过抑制TLR4/MyD88/NF-κB信号通路减轻大鼠肝纤维化的作用

Baicalin alleviates hepatic fibrosis in rats by inhibiting TLR4/MyD88/NF-κB signaling pathway

目的观察黄芩苷对肝纤维化(HF)大鼠Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核转录因子-κB(NF-κB)信号通路的相关因子表达,探讨其干预HF的分子机制。方法将清洁级SD雄性大鼠随机分为正常组和造模组,造模组每周2次腹腔注射40%CCl4溶液,连续8周。造模成功后,将造模大鼠随机分为模型组、黄芩苷组和TAK-242组,每组10只。黄芩苷(25 mL·kg^(-1))及TAK-242组(5 mg·kg^(-1))每天给药一次,连续5 d。苏木素-伊红(HE)及Masson染色观察各组大鼠肝组织病理变化;生化仪检测大鼠血清中天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、白蛋白(ALB)、碱性磷酸酶(ALP)含量;ELISA检测大鼠血清中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、透明质酸(HA)、层粘蛋白(LN)、III型前胶原(PCⅢ)和IV型胶原(CⅣ)及肝组织中羟脯氨酸(HYP)、超氧化物歧化酶(SOD)和丙二醛(MDA)的水平;Real-time RT-PCR检测大鼠肝脏中MyD88、TLR4、NF-κB mRNA表达;Western blot检测大鼠肝组织MyD88、TLR4和NF-κB蛋白表达。结果HE及Masson染色结果显示模型组的肝组织有炎性细胞浸润及纤维组织增生,黄芩苷组和TAK-242组的肝组织部分细胞肿胀,有纤维间隔,黄芩苷组改善效果优于TAK-242。生化仪检测结果显示,同正常组比,模型组血清中AST、ALT和ALP含量显著增加(P<0.01),ALB的含量显著降低(P<0.01);同模型组相比,黄芩苷可显著降低血清中AST、ALT、ALP含量(P<0.01),升高ALB含量(P<0.01);ELISA检测结果显示,同正常组比,模型组血清中IL-6、IL-1β、TNF-α、HA、LN、PCⅢ和CⅣ及肝组织中HYP、MDA含量显著增加(P<0.01),SOD的含量显著降低(P<0.01)。同模型组比,黄芩苷可明显降低IL-6、IL-1β、TNF-α、HA、LN、PCⅢ和CⅣ及肝组织中HYP、MDA含量(P<0.01),增加SOD的含量(P<0.01)。与正常组比,模型组TLR4、MyD88和NF-κB表达显著升高(P<0.01)。与模型组比,黄芩苷可显著降低TLR4、MyD88和NF-κB表达(P<0.01)。结论黄芩苷组可通过抑制TLR4/MyD88/NF-κB通路的活化,对HF发挥潜在治疗效果。

Objective To observe the expression of related factors of baicalin on Toll-like receptor 4(TLR4)/myeloid differen-tiation factor 88(MyD88)/nuclear transcription factor-κB(NF-κB)signaling pathway in hepatic fibrosis(HF)rats,and to explore the molecular mechanism of its intervention in HF.Methods The cleaning stage SD male rats were randomly divided into normal groups and model groups,and the model group was injected a 40%CCl4 solution twice a week for 8 consecutive weeks.After successful modeling,the model rats were randomly divided into model groups,baicalin groups and TAK-242 groups,10 per group.Baicalin(10 ml·kg^(-1))and TAK-242 group(5 mg·kg^(-1))were administered once a day,and 5 days were con-tinuous.Hematoxylate-eosin(HE)and Masson were observed to change the pathological changes of liver tissue of each group;biochemical instrument detected Aspartate aminotransferase(AST),Alanine aminotransferase(ALT),Albumin(ALB),alkaline phosphatase(ALP)content in rat serum;ELISA detected Interleukin-6(IL-6),Interleukin-1β(IL-1β),Tumor necrosis factor-α(TNF-α),Haluronic acid(HA),laminin(LN),procollagen III(PCII)and collagen type IV(CIV)in rat serum and levels of Hydroxyproline(HYP),Superoxide dismutase(SOD)and Malondialdehyde(MDA);Real-time RT-PCR de-tects MyD88,TLR4,NF-κB mRNA expression in rat liver;Western blot detects rat liver Tissue MYD88,TLR4 and NF-κB protein expression.Results HE and Masson staining results showed that there were inflammatory cell infiltration and fibrous tissue hyperplasia in the liver tissue of model group,while some cells were swollen and fibrous septum in the liver tissue of baicalin group and TAK-242 group,and the improvement effect of baicalin group was better than TAK-242 group.Compared with nor-mal group,the serum AST,ALT and ALP contents in model group were significantly increased(P<0.01),and the serum ALB content was significantly decreased(P<0.01).Compared with model group,baicalin significantly decreased the contents of AST,ALT and ALP in serum(P<0.01),and increased the content of ALB(P<0.01).ELISA results showed that com-pared with normal group,the contents of IL-6,IL-1β,TNF-α,HA,LN,PCⅢand CⅣin serum and the contents of HYP and MDA in liver tissue in model group were significantly increased(P<0.01),while the content of SOD was significantly de-creased(P<0.01).Compared with the model group,baicalin significantly decreased the contents of IL-6,IL-1β,TNF-α,HA,LN,PCⅢand CⅣand the contents of HYP and MDA in liver tissue(P<0.01),and increased the content of SOD(P<0.01).Compared with normal group,the expression of TLR4,MyD88 and NF-κB in model group was significantly increased(P<0.01).Compared with model group,baicalin significantly decreased the expression of TLR4,MyD88 and NF-κB(P<0.01).Baicalin group has potential therapeutic effect on HF by inhibiting the activation of TLR4/MyD88/NF-κB pathway.Con-clusion Baicalin group can exert a potential therapeutic effect on HF by inhibiting the activation of TLR4/MyD88/NF-κB path-way.