基于PPARγ研究补肺益肾方对肺泡巨噬细胞胞葬功能障碍的改善机制

Bufei Yishen Prescription Improves Impaired Efferocytosis of Alveolar Macrophages through PPARγ

目的:在前期确立香烟烟雾提取物(Cigarette smoke extract, CSE)诱导的肺泡巨噬细胞胞葬功能障碍体外模型后,观察补肺益肾方对胞葬功能、胞葬辅助因子及过氧化物酶体增殖物激活受体γ(Peroxisome proliferator-activated receptor gamma, PPARγ)信号通路相关蛋白的影响,探讨其对慢阻肺的作用机制。方法:将NR8383细胞分为正常对照组(20%大鼠空白血清)、模型对照组(20%大鼠空白血清、5%CSE)、18.5 g/kg补肺益肾组(20%大鼠含药血清、5%CSE)、GW9662组(20%大鼠空白血清、10μmol/L GW9662)、补肺益肾方合GW9662组(20%大鼠含药血清、10μmol/L GW9662),采用流式细胞术检测NR8383细胞胞葬功能;酶联免疫吸附实验检测胞葬辅助因子GAS6、MFG-E8的含量;免疫印迹法检测PPARγ信号通路活性及通路下游膜识别受体CD36的表达。:结果:与正常对照组相比,模型对照组的胞葬功能、GAS6、MFG-E8含量均明显降低(P<0.05或P<0.01)。PPARγ核蛋白、CD36蛋白表达均明显下调(P<0.05或P<0.01);与模型对照组相比,18.5 g/kg补肺益肾20%含药血清组胞葬功能、GAS6、MFG-E8含量明显升高;PPARγ核蛋白、CD36蛋白表达均明显上调(P<0.05或P<0.01),与GW9662组相比,补肺益肾方合GW9662组胞葬功能、GAS6、MFG-E8含量、PPARγ核蛋白、CD36蛋白表达均明显升高(P<0.05或P<0.01)。结论:补肺益肾方可改善香烟烟雾诱导的肺泡巨噬细胞胞葬功能障碍,其机理与调控PPARγ通路、上调胞葬辅助因子(MFG-E8、GAS6)及膜识别蛋白CD36有关。

Objective:To observe the effects of Bufei Yishen Prescription on efferocytosis, accessory factors for efferocytosis and related proteins in peroxisome proliferator-activated receptor gamma(PPARγ) signaling pathway in a previously confirmed in vitro model of alveolar macrophages with cigarette smoke extract(CSE)-induced impairment in efferocytosis, and explore its mechanism against chronic obstructive pulmonary disease(COPD). Methods: NR8383 cells were divided into the normal control group(20% rat blank serum), model group(20% rat blank serum and 5% CSE), 18.5 g/kg Bufei Yishen Prescription group(20% rat drug-containing serum and 5% CSE), GW9662 group(20% rat blank serum and 10 μmol/L GW9662) and Bufei Yishen Prescription combined with GW9662 group(20% rat drug-containing serum and 10 μmol/L GW9662). The efferocytosis of NR8383 cells was determined by flow cytometry. Enzyme-linked immunosorbent assay was used to detect the levels of GAS6 and MFG-E8, accessory factors for efferocytosis. Western blotting was carried out to measure the activity of PPARγ signaling pathway and the expression of the downstream membrane recognition receptor CD36. Results:Compared with the normal control group, the model group displayed significantly reduced efferocytosis and GAS6 and MFG-E8(P<0.05 or P<0.01) and down-regulated PPARγ nuclear protein and CD36 protein expression(P<0.05 or P<0.01). Compared with the model group, the 18.5 g/kg Bufei Yishen Prescription group resulted in a significant increase in GAS6 and MFG-E8 levels and up-regulation of PPARγ nuclear protein and CD36 protein expression(P<0.05 or P<0.01). Compared with the GW9662 group, Bufei Yishen Prescription combined with GW9662 obviously elevated the GAS6 and MFG-E8 levels and up-regulated the PPARγ nuclear protein and CD36 protein expression(P<0.05 or P<0.01). Conclusion: Bufei Yishen Prescription improves the impaired efferocytosis of alveolar macrophages induced by CSE, which may be related to the up-regulation of MFG-E8 and GAS6 and membrane recognition protein CD36 in the PPARγ signaling pathway.