金银花提取物对哮喘模型小鼠气道平滑肌细胞增殖和凋亡的影响

Effect of honeysuckle extract on proliferation and apoptosis of airway smooth muscle cells in asthmatic model mice

目的:探讨金银花提取物对哮喘小鼠气道平滑肌细胞(ASMCs)增殖和凋亡的影响,阐明其相关的作用机制。方法:构建小鼠哮喘模型,分离原代ASMCs并进行鉴定。采用白细胞介素4(IL-4)诱导巨噬细胞RAW264.7 M2极化并进行鉴定。RAW264.7细胞分为对照组及低、中和高剂量金银花提取物组,对照组诱导RAW264.7细胞M2极化后与ASMCs共培养,低、中和高剂量金银花提取物组分别用不同浓度(50、100和200 mg·L^(-1))金银花提取物处理RAW264.7细胞1 h,再用60μg·L^(-1)IL-4处理6 h,随后将ASMCs与RAW264.7细胞共培养24 h。流式细胞术检测各组RAW264.7细胞中巨噬细胞表面标记蛋白CD86和CD206阳性细胞百分率,酶联免疫吸附试验(ELISA)检测各组ASMCs培养上清液中白细胞介素10(IL-10)水平,MTT法检测各组ASMCs增殖活性,流式细胞术检测各组ASMCs凋亡率。结果:细胞形态表现和免疫荧光染色结果证明所提取的细胞为ASMCs。RAW264.7细胞M2极化鉴定结果显示已诱导成为M2巨噬细胞。与对照组比较,低、中和高剂量金银花提取物组RAW264.7细胞中CD86阳性细胞百分率明显升高(P<0.05),CD206阳性细胞百分率明显降低(P<0.05),ASMCs培养上清液中IL-10水平和ASMCs增殖活性明显降低(P<0.05),ASMCs凋亡率明显升高(P<0.05);与低剂量金银花提取物组比较,中和高剂量金银花提取物组RAW264.7细胞中CD86阳性细胞百分率明显升高(P<0.05),CD206阳性细胞百分率明显降低(P<0.05),ASMCs培养上清液中IL-10水平和AMSC增殖活性明显降低(P<0.05),ASMCs凋亡率明显升高(P<0.05);与中剂量金银花提取物组比较,高剂量金银花提取物组RAW264.7细胞中CD86阳性细胞百分率明显升高(P<0.05),CD206阳性细胞百分率明显降低(P<0.05),ASMCs培养上清液中IL-10水平和ASMCs增殖活性明显降低(P<0.05),ASMCs凋亡率明显升高(P<0.05)。结论:金银花提取物可通过抑制巨噬细胞M2极化进而抑制哮喘小鼠ASMCs细胞增殖并促进其凋亡。

Objective:To discuss the effect of honeysuckle extract on the proliferation and apoptosis of airway smooth muscle cells(ASMCs)in the asthmatic mice,and to clarify the related mechanism.Methods:The asthma models of mice were constructed,and the primary ASMCs were isolated and identified.The M2 polarization of macrophages RAW264.7 was induced and identified.The RAW264.7 cells were divided into control group,low,medium and high doses of honeysuckle extract groups.The RAW264.7 cells in control group were induced M2 polarization and co-cultured with ASMCs.The RAW264.7 cells in low,medium,and high doses of honeysuckle extract groups were treated with different concentrations(50,100,and 200 mg·L^(-1) )of honeysuckle extract for 1 h and then treated with interleukin-4(IL-4)(60μg·L^(-1) )for 6 h.Then the RAW264.7 cells and ASMCs were co-cultured for 24 h.The percentages of CD86 and CD206 positive cells in various groups were detected by flow cytometry;the levels of interleukin-10(IL-10)in culture supernatant of the ASMCs in various groups were detected by enzyme-linked immunosorbent assay(ELISA)method;the proliferation activities of cells in various groups were detected by MTT assay;the apoptotic rates of ASMCs in various groups were detected by flow cytometry.Results:The morphology of the cells and immunofluorescence staining results proved that the extracted cells were the ASMCs.The M2 polarization identification results showed that the RAW264.7 cells were induced into the M2 macrophages.Compared with control group,the percentages of CD86 positive cells in the ASMCs in low,medium and high doses of honeysuckle extract groups were significantly increased(P<0.05),while the percentages of CD206 positive cells were decreased(P<0.05);the IL-10 levels in culture supernatant of the ASMCs and the proliferation activities of ASMCs were significantly decreased(P<0.05),and the apoptotic rates of the ASMCs were increased(P<0.05).Compared with low dose of honeysuckle extract group,the percentages of CD86 positive cells in medium and high doses of honeysuckle extract groups were significantly increased(P<0.05),while the percentages of CD206 positive cells were decreased(P<0.05);the IL-10 levels in the culture supernatant and proliferation activities of the cells were significantly decreased(P<0.05),and the apoptotic rates of the ASMCs were increased(P<0.05).Compared with medium dose of honeysuckle extract group,the percentages of CD86 positive cells in high dose of honeysuckle extract group was significantly increased(P<0.05),while the percentage of CD206 positive cells was decreased(P<0.05);the IL-10 levels in the culture supernatant and proliferation activities of the cells were significantly decreased(P<0.05),and the apoptotic rates of the ASMCs were increased(P<0.05).Conlusion:Honeysuckle extract can inhibit the proliferation and promote the apoptosis of the ASMCs in the asthmatic mice by inhibiting the M2 polarization of the macrophages.