脂多糖对肺成纤维细胞胸腺细胞分化抗原-1及整合素β3表达的调控并抑制自噬的研究

Lipopolysaccharide regulates the expression of thymocyte differentiation antigen-1 and integrin β3 of lung fibroblasts and inhibit autophagy

目的 明确脂多糖（lipopolysaccharide, LPS）对小鼠肺成纤维细胞胸腺细胞分化抗原-1（thymocyte differentiation antigen-1, Thy-1）和整合素β3蛋白表达的调控作用，并观察其与肺成纤维细胞自噬抑制的关系。 方法 将原代培养的小鼠肺成纤维细胞采用随机数字法分为4组（每组3孔）：PBS对照组（Con组）、0.25 mg/L LPS组（LPS0.25组）、0.50 mg/L LPS组（LPS0.50组）、1.00 mg/L LPS组（LPS1.00组），随后以不同浓度LPS（0.25、0.50、1.00 mg/L）刺激，于刺激后6 h通过Western blot检测不同浓度LPS刺激小鼠肺成纤维细胞后Thy-1和整合素β3蛋白的表达情况。于刺激后30 h比较肺成纤维细胞自噬相关蛋白LC3-Ⅱ/Ⅰ和Beclin-1的表达情况，同时通过透射电子显微镜观察细胞内自噬小体形成情况。 结果 随着LPS刺激浓度的增加，小鼠肺成纤维细胞的Thy-1蛋白表达量下降，其中LPS1.00组较Con组有明显下降（P〈0.05）；整合素β3蛋白表达量升高，其中LPS1.00组较Con组有明显升高（P〈0.05）。LPS1.00组刺激小鼠肺成纤维细胞30 h后明显抑制其自噬相关蛋白LC3-Ⅱ/Ⅰ和Beclin-1的表达（P〈0.05），并抑制自噬小体形成（P〈0.05）。 结论 LPS可下调Thy？蛳1蛋白表达、上调整合素β3蛋白表达，同时抑制自噬相关蛋白LC3-Ⅱ/Ⅰ和Beclin-1的表达及自噬小体的生成，该过程可能与LPS诱导肺成纤维细胞自噬抑制以及肺纤维化进展有关。

Objective To clarify the effect of lipopolysaccharide （LPS） on the expression of thymocyte differentiation antigen-1（Thy-1） and integrin β3 in mouse lung fibroblasts and its relationship with autophagy inhibition. Methods Primary cultured mouse lung fibroblasts were divided into four groups （n=3） based on different concentration of LPS stimulation： PBS control group （Con group）,0.25 mg/L LPS group （LPS0.25 group）, 0.50 mg/L LPS group （LPS0.50 group）, 1.00 mg/L LPS group （LPS1.00 group）. Then the total cellular proteins were extracted. The expression of Thy-1 and integrin β3 were detected by Western blot. The expression of autophagy-related proteins LC3-Ⅱ/Ⅰ and Beclin-1 in cells was compared at 30 h after LPS stimulatin. The formation of autophagic bodies was observed by using transmission electron microscopy. Results Thy-1 protein expression was decreased in lung fibroblasts with increasing concentration of LPS stimulation. Compared with the control group（P〈0.05）, the level of Thy-1 protein in lung fibroblasts was significantly decreased in 1.00 mg/L LPS group. Integrin β3 protein expression was increased with increasing concentration of LPS stimulation. As a result, the expression leve of integrin in 1.00 mg/L LPS group was significantly higher than the level in control group （P〈0.05）. At the same time, LPS significantly inhibited the expression of autophagy-related proteins LC3-Ⅱ/Ⅰ（P〈0.05） and Beclin-1 （P〈0.05） in mouse lung fibroblasts and the formation of autophagic bodies（P〈0.05）. Conclusions LPS can down-regulate the expression of Thy-1 but up-regulate the expression of integrin β3. It also inhibits the expression of autophagy-related proteins LC3-Ⅱ/Ⅰ, Beclin-1 and the formation of autophagic bodies. This process may be associated with LPS-induced autophagy inhibition of lung fibroblasts and the progress of lung fibrosis.