盐酸戊乙奎醚对脂多糖诱导气道平滑肌细胞炎性损伤及P38MAPK/JNK通路的影响

Effects of penehyclidine hydrochloride on lipopolysaccharide-induced inflammatory injury of airway smooth muscle cells and P38MAPK/JNK pathway

目的:探讨盐酸戊乙奎醚(PHC)对脂多糖(LPS)诱导气道平滑肌细胞(ASMC)炎性损伤及P38MAPK/JNK通路的影响。方法:体外培养人ASMC,将ASMC随机分成5组。对照组只含ASMC;LPS组在ASMC中加入终质量浓度为500 ng·ml-1LPS;1、10、100μmol·L-1PHC+LPS组分别在LPS组基础上加入终质量浓度为1、10、100μmo·L-1PHC。采用CCK-8法检测ASMC增殖情况;显微镜下观察细胞形态学变化;采用酶联免疫吸附法(ELISA)检测ASMC细胞因子乳酸脱氢酶(LDH)、丙二醛(MDA)水平;采用Annexin V/PI双染法流式细胞术检测细胞凋亡;采用Western blotting检测凋亡及P38MAPK/JNK通路相关蛋白表达情况。结果:与对照组相比,LPS组ASMC 48 h时的吸光度(A)值显著降低(P <0.05),细胞体积扁小,形态偏圆,胞质空亮,胞质内出现大小不等的颗粒,细胞因子LDH、MDA水平均显著升高(P <0.05),细胞凋亡率显著升高(P <0.05),抑凋亡基因Bcl-2蛋白表达水平显著降低(P <0.05),促凋亡基因Bax蛋白表达水平显著升高(P <0.05),p-JNK、p-P38蛋白表达水平显著升高(P <0.05);给予PHC处理后,与LPS组相比,1、10、100μmol·L-1PHC+LPS组细胞48 h时的A值均显著升高(P <0.05),细胞无明显形态学变化,且贴壁细胞数量多,细胞因子LDH、MDA水平均显著降低(P <0.05),细胞凋亡率显著降低(P <0.05),抑凋亡基因Bcl-2蛋白表达水平显著升高(P <0.05),促凋亡基因Bax蛋白表达水平显著降低(P <0.05),P-JNK、P-p38蛋白表达水平显著降低(P <0.05),且呈剂量依赖性。结论:PHC可抑制LPS诱导的ASMC炎性损伤,其机制可能与抑制P38MAPK/JNK通路的活化有关。

Objective: To investigate the effects of penehyclidine hydrochloride( PHC) on lipopolysaccharide( LPS)-induced inflammatory injury of airway smooth muscle cells( AMSC) and P38 MAPK/JNK pathway.Methods: Human ASMC were cultured in vitro. ASMC were randomly divided into 5 groups: control group( only ASMC cells were included),LPS group( the final concentration of the above cells was 500 ng·ml-1 LPS),1,10,100 μmol·L-1 PHC + LPS group( the final concentration of 1,10,100 μmol·L-1 PHC was added on the basis of LPS group). The proliferation of ASMC was detected using CCK-8 method;the changes of cell morphology were observed under microscope;LDH and MDA levels of ASMC were detected by enzyme-linked immunosorbent assay( ELISA);Annexin V/PI double staining was used to detect apoptosis;Western blotting was used to detect apoptosis and P38 MAPK/JNK pathway related protein expression. Results: Compared with the control group,the absorbance( A) value at 48 h of LPS group decreased significantly( P < 0. 05),ASMC were small and flat,round in shape,empty and bright in cytoplasm,the levels of LDH and MDA were significantly increased( P < 0. 05),the apoptosis rate increased significantly( P < 0. 05),the expression level of Bcl-2 decreased significantly( P <0. 05),the expression level of Bax was significantly increased( P < 0. 05),and the expression levels of p-JNK and p-P38 were significantly increased( P < 0. 05);After treatment of penehyclidine hydrochloride,compared with LPS group,the A value at 48 h of cells in 1,10,100 μmol·L-1 ICA + LPS group increased significantly( P <0. 05),there was no obviously morphological change,and the number of adherent cells was more than that in LPS group,the levels of LDH and MDA decreased significantly( P < 0. 05),the apoptotic rate decreased significantly( P < 0. 05),the expression level of Bcl-2 was significantly increased( P < 0. 05),the expression level of Bax protein decreased significantly( P < 0. 05),the expression levels of p-JNK and p-P38 decreased significantly( P <0. 05),which showed a dose-dependent manner. Conclusion: PHC can inhibit the inflammatory injury of ASMC induced by LPS,and the mechanism may be related to the inhibition of P38 MAPK/JNK pathway activation.