回阳生肌膏不同拆方对M1型巨噬细胞调控成纤维细胞分泌功能的影响

Effects of Different Split Recipes of Huiyang Shengji Ointment on the Regulation of Fibroblast Secretory Function by M1 Macrophages

目的观察回阳生肌膏不同拆方益气温阳方和活血通络方对M1型巨噬细胞调控成纤维细胞表型的影响。方法用丙二醇甲醚醋酸酯(2-Acetoxy-1-methoxypropane,PMA)、脂多糖(Lipopolysaccharide,LPS)和干扰素γ(Interferon-γ,IFN-γ)刺激人单核细胞株(THP-1)转化为M1型巨噬细胞,分别加入不完全培养液、益气温阳方(4、0.8、0.16 mg/L)和活血通络方(32、6.4、1.28 mg/L)药物,24 h后收集培养上清液。依次将培养上清液加入到人成纤维细胞株(HSF),模型组加入M1型巨噬细胞上清液,益气温阳方、活血通络方分别加入高、中、低药物组M1型巨噬细胞上清液,空白组或对照组加入HSF细胞培养液。采用细胞计数试剂盒(Cell Counting Kit-8,CCK-8)方法观察细胞增殖情况;luminex法检测上清液中趋化因子2(C-C motif chemokine ligand 2,CCL2)、趋化因子7(C-C motif chemokine ligand 7,CCL7)、白细胞介素-6(interleukin-6,IL-6)、基质金属蛋白酶(matrix metalloproteinase,MMPs)和基质金属蛋白酶抑制剂-1(tissue inhibitor of matrix metalloproteinase 1,TIMP-1)的含量;realtime-PCR法检测细胞内CCL2、CCL7、IL-6、MMPs、TIMP-1、Ⅰ型胶原蛋白基因α1(collagen typeⅠalpha gene,COL1A1)和Ⅲ型胶原蛋白基因α1(collagen typeⅢalpha gene,COL3A1)mRNA的表达。结果与模型组相比,益气温阳组和活血通络组各剂量组均可促进HSF细胞增殖;抑制HSF细胞炎症因子CCL2、CCL7、IL-6的分泌及其mRNA的表达(P<0.01);抑制HSF细胞基质金属蛋白酶1(matrix metalloproteinase 1,MMP-1)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)、基质金属蛋白酶3(matrix metalloproteinase 3,MMP-3)的分泌及其mRNA的表达(P<0.01),促进TIMP-1的分泌及mRNA的表达(P<0.01),抑制MMP-3 mRNA的表达(P<0.01);促进HSF细胞COL1A1和COL3A1 mRNA的表达(P<0.01)。结论益气温阳方主要促进成纤维细胞增殖,抑制HSF细胞炎症因子和MMPs的分泌蛋白及mRNA的表达,促进基质金属蛋白酶抑制剂(tissue inhibitor of matrix metalloproteinases,TIMPs)的分泌和mRNA表达,以促使创面成纤维细胞活化,而活血通络方主要促进成纤维细胞胶原生成,促进其向肌成纤维细胞转化,两方相辅相成,共同促进创面愈合。

Objective To observe the effects of different split recipes of Huiyang Shengji Ointment(HYSJ)on the regulation of fibroblast phenotype by M1 macrophages. Methods Human mononuclear cell lines(THP-1)were transformed into M1 macrophages stimulated by PMA,LPS and IFN-γ. All cells were cultured by incomplete culture medium,Yiqi Wenyang Decoction(YQWY)(4,0.8,0.16 mg/L),and Huoxue Tongluo Decoction(HXTL)(32,6.4,1.28 mg/L)respectively,and cell culture supernatants were collected after 24 hours. Then cell culture supernatants were sequentially added to the human fibroblast cell line(HSF),model group(M1 macrophage supernatant,)and YQWY and HXTL were added to the different drug groups of M1 macrophage supernatant respectively. The cell proliferation was observed by CCK-8 methods;The content of CCL2,CCL7,IL-6,MMPs and TIMP-1 in the supernatants were detected by luminex. The expressions of CCL2,CCL7,IL-6,MMPs,TIMP-1,COL1 A1 and COL3 A1 mRNA were detected by realtime-PCR. Results Compared with the model group,YQWY and HXTL could promote the proliferation of HSF cells,inhibit the secretion of HSF cell inflammatory factors CCL2,CCL7,IL-6 and their m RNA expressions(P<0.01),also inhibit the secretion of MMP-2 and MMP-14 and their mRNA expression(P<0.01),promote the secretion of TIMP-1 and mRNA expressions(P<0.01),and inhibit the expressions of MMP-3 mRNA(P<0.01),promote the expression of COL1 A1 and COL3 A1 mRNA in HSF cells(P<0.01). Conclusion YQWY mainly promote fibroblast proliferation,inhibit the the secretion of inflammatory factors,MMPs and mRNA expression of HSF cells,promote the secretion of TIMPs and mRNA expression to promote fibroblasts activation. Meanwhile,HXTL mainly promote fibroblast collagen formation,promote its transformation into myofibroblasts,and the two complement each other to promote wound healing.