高糖诱导下肝星形细胞活化与p38MAPK信号通路的关系

Relationship between high glucose-induced hepatic stellate cell activation and the p38MAPK signaling pathway

目的探讨高糖诱导下肝星形细胞（r／sc）活化与p38丝裂原活化蛋白激酶（p38MAPK）信号通路的相互关系。方法体外培养人HSC并进行形态学鉴定，选取60份样本，分为对照组、高糖组和阻断组各20份；对照组常规培养，高糖组在常规培养基础上加入葡萄糖（终浓度25mol／L），阻断组在高糖组基础上加入p38MAPK阻断剂SB203580（终浓度40mol／L）进行培养，培养120h后用链霉亲和素一生物素复合物法（SABC）及Western印迹法检测各样本的α-肌动蛋白（α-SMA）和p-p38MAPK蛋白表达情况。结果经鉴定，培养后的HSC呈扁平状，胞体大，具有发育良好的应力纤维，胞浆内缺乏脂肪滴。SABC法原位检验结果显示：高糖组的α-SMA阳性信号强于对照组，而阻断组的α-SMA阳性信号则近似于对照组。3组HSC的仅．SMA和p-p38MAPK蛋白的相对表达量差异均有统计学意义（F=31．36，78．49，P均〈0．01）。其中，高糖组的α-SMA表达相对量为（34．61±4．07）％，高于对照组和阻断组的（23．90±6．02）％和（26．48±4．41）％，差异均有统计学意义（t=7．20和-5．37，P均〈0．01）；高糖组表达相对量高糖组为（22．79±3.80）％，对照组和阻断组分别为（8．13±4．95）％和（10．66±4．15）％，差异均有统计学意义（t=-11．01和．10．41，p均〈0．01）。结论活化程度较高的HSC具有较高的p38MAPK信号强度，而阻断p38MAPK信号通路能够降低HSC的活化程度。

Objective To explore the relationship between p38-mitogen activated protein kinase （p38MAPK） signaling pathway and high glucose-induced hepatic stellate cell （HSC） activation. Methods HSC was cultivated, and morphological identification was conducted. Twenty human HSC samples were randomly collected to represent each experimental group including the control group, high glucose group and blocking group. Cells from the control group were cultured under normal conditions. Cells from the high glucose group were cuhured under high glucose conditions of which the ultimate concentration was 25mol/L. The blocking group was cultured under high glucose conditions in the presence of the p38MAPK inhibitor SB203580 of which the uhimate concentration was 40mol/L. After incubation for 120 h, SABC and Western blot analysis were used to detect α-SMA expression in each sample to determine the degree of hepatic stellate cell activation. Western blot analysis was also used to detect the expression of phospho- p38MAPK protein in each group. Results According to identification, the cultured HSC was flat with big cell body, and had well-developed stress fiber. There was less fat droplet in the cytoplasm. The result of SABC situ test showed that, compared with the high glucose group, positive signal of α-SMA proteins was stronger than that of control group which had similar positive signal as the blocking group. The differences of both HSCα-SMA and p-p38MAPK protein expression levels in three groups had statistical significance （F=31.36, 78.49, P all〈0.01 ）. Among them, the expression level of α-SMA in the high glucose group was （34.61±4.07）% and higher than （23.90±6.02）% of control group and （26.48±4.41 ）% of blocking group, of which the differences were statistically significant （t=-7.20 and -5.37, P both 〈0.01 ）. The expression level of p-p38MAPK was （22.79±3.80）% in the high glucose group, （8.13±4.95）% in the control group and （10.66±4.15）% in the blocking group, of which the differences had statistical significance （t=- 11.01 and - 10.41, P both 〈0.01 ）. Conclusion The higher level of HSC represents the higher signal strength of p38MAPK. However, blocking the p38MAPK signaling pathway can reduce the activation of hepatic stellate cells.