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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
作者:刘春芳  陈超  毛豪丽 
单位:上海交通大学附属第九人民医院 麻醉科, 上海 200011
关键词:盐酸戊乙奎醚 脂多糖 气道平滑肌细胞 炎性损伤 P38MAPK/JNK通路 
分类号:R363.1;R562.25
出版年·卷·期(页码):2021·49·第一期(52-58)
摘要:

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-1 LPS;1、10、100 μmol·L-1 PHC+LPS组分别在LPS组基础上加入终质量浓度为1、10、100 μmo·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、100 μmol·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 P38MAPK/JNK pathway.Methods: HumanASMCwere cultured in vitro. ASMCwere 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 P38MAPK/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), ASMCwere 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 P38MAPK/JNK pathway activation.

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