白桦酯酸介导STAT3信号通路调控肝纤维化进程

Betulinic acid mediates STAT3 signaling pathway in regulating hepatic fibrosis

目的探讨白桦酯酸(betulinic acid,BA)介导STAT3信号通路改善肝纤维化的进程。方法Raw264.7细胞给予LPS(1 mg·L-1)孵育12 h,收集上清液。BA(25μmol·L-1)预处理HSCs 2 h后,给予上述上清液孵育0-6 h;不同浓度BA(0-25μmol·L-1)预处理HSCs 2 h后,给予上述上清液孵育1 h,分析BA对细胞外基质、炎性因子以及相关信号通路的影响。BA(50 mg·kg-1)预处理C57BL/6♂小鼠4 d后,腹腔注射10%CCl 4,24 h给予小鼠安乐死,收集小鼠血清和肝脏。结果Raw264.7细胞在LPS刺激后,测得细胞上清液IL-6含量因时间延长而增加,12 h后达最高值。取LPS刺激Raw264.7细胞12 h上清液活化HSCs,BA可明显降低活化HSCs中α-SMA、collagen-Ⅰ蛋白表达,降低TIMP-1及MMP-13比值,呈现时间和剂量依赖关系。BA可明显增加活化HSCs中STAT3磷酸化蛋白表达,明显降低TLR4和CD14蛋白表达,调控AMPK-LKB1磷酸化,呈现时间和剂量依赖性。结论BA可能介导炎性上清液活化HSCs中STAT3磷酸化蛋白表达,调控TLR4及AMPK/LKB1磷酸化,进而逆转肝纤维化。

Aim To survey the effect of betulinic acid against hepatic fibrosis via STAT3 signaling pathway.Methods Raw264.7 cells were incubated with LPS(1 mg·L-1)for 12 h,and the supernatant was collected.HSCs were pretreated with betulinic acid(25μmol·L-1)for 2 h,and then incubated with the supernatant for 0-6 h.HSCs were pretreated with betulinic acid(0-25μmol·L-1)for 2 h,followed with the above supernatants for next 1 h.The effects of betulinic acid on extracellular matrix,inflammatory factors and related signaling pathways were analyzed.C57BL/6 male mice were pretreated with BA for 4 d,and then injected with 10%CCl 4 intraperitoneally.The mice were euthanized for 24 h.The serum and liver were collected.Results When Raw264.7 cells were stimulated by LPS,the content of IL-6 in the supernatant of Raw264.7 cells was improved over time and arrived at the highest level at 12 h.HSCs were activated by the supernatant from Raw264.7 stimulated by LPS for 12 h.Betulinic acid significantly decreased the protein expression ofα-SMA and collagen-Ⅰprotein,and decreased the ratio of TIMP-1 and MMP-13 in a time-and dose-dependent manner.Betulinic acid significantly improved the expression of STAT3 phosphorylation,decreased the expressions of TLR4 and CD14,and regulated the AMPK and LKB1 phosphorylation in a time-and dose-dependent manner.Conclusions Betulinic acid may mediate STAT3 phosphorylation to regulate TLR4 and AMPK/LKB1 signaling in activated HSCs,which further ameliorates hepatic fibrosis.