目的 观察回阳生肌方的拆方(益气温阳方、活血通络方)对巨噬细胞表型转化的影响.方法 人单核细胞株(THP-1)细胞用佛波醇酯类多克隆刺激剂(PMA)刺激成巨噬细胞后,加入脂多糖(LPS)、γ-干扰素(INF-γ)刺激48 h,转化为M1型巨噬细胞.洛伐他汀(40.50 mg/L)作为阳性对照药物;采用CCK-8方法观察益气温阳方、活血通络方对巨噬细胞活性的影响;以中性红法检测其对巨噬细胞吞噬功能的影响.M1型巨噬细胞加入洛伐他汀、益气温阳方、活血通络方24 h后, PCR法检测诱导型一氧化氮合酶(iNOS)mRNA、精氨酸酶1(Arg-1)mRNA的表达;CBA法检测上清液中肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-12(IL-12)、白细胞介素-10(IL-10)和生长因子(VEGF)的含量;ELISA法检测上清液中转化生长因子β(TGF-β)的含量.结果 益气温阳方在浓度4、0.8、0.16 mg/L时、活血通络方在浓度32、6.4、1.28 mg/L时对巨噬细胞增殖无影响,但可促进巨噬细胞对中性红的吞噬,分别作为益气温阳方、活血通络方的高、中、低浓度.加入LPS、INF-γ后,M1型巨噬细胞标志物iNOS mRNA显著升高,而加入洛伐他汀、益气温阳方及活血通络方后显著降低(P<0.01);M2型巨噬细胞标志物Arg-1 mRNA表达相对于正常组显著降低,而洛伐他汀、益气温阳方和活血通络方都明显促进Arg-1 mRNA表达(P < 0.01),但益气温阳方、活血通络方与洛伐他汀相比,Arg-1 mRNA表达显著降低.以上药物作用M1型巨噬细胞细胞24 h后,TNF-α、IL-6、IL-1含量显著降低,VEGF、TGF-β含量显著升高,益气温阳方高浓度使IL-10含量显著升高(P<0.05).与洛伐他汀相比,活血通络方和益气温阳方高浓度组VEGF、TGF-β差异有统计学意义(P< 0.05).结论 益气温阳方和活血通络方均在一定程度上抑制M1型巨噬细胞标志物iNOS mRNA表达和诱导M2型巨噬细胞标志物Arg-1 mRNA的表达,并且可抑制M1型巨噬细胞因子的分泌,促进M2型巨噬细胞因子的分泌.%Objective To observe the influence of Huiyang Shengji Fang(Restoring Yang and Promoting Tissue Regeneration Decoction, HYSJF) and its decomposed formulas-Yiqi Wenyang Fang (Qi-supplementing and Yang-warming Decoction,YQWYF) and Huoxue Tongluo Fang(Blood-activating and collateral-freeing Decoction,HXTLF) on macrophage phenotype inversion,and discuss the mechanism of YQWYF and HXTLF in regulating macrophage phenotype inversion during the period of chronic skin ulcer. Methods THP-1 cells were stimulated and transformed into macrophages with PMA. After stimulated with LPS and INF-γ for 48 h, the macrophages were transformed into M1 macrophages. Lovastatin (10 μmol /L) was taken as control drug. The influence of YQWYF and HXTLF on activities of macrophages was observed by using CCK-8 method, and their influence on the phagocytosis of macrophages was detected by using neutral red uptake assay (NRU). The expressions of iNOS mRNA and Arg-1 mRNA were detected by using polymerase chain reaction (PCR) after M1 macrophages stimulated with lovastatin,YQWYF and HXTLF for 24 h. The levels of supernate TNF-α,IL-6,IL-12, IL-10 and VEGF were detected by using CBA method. The level of supernate TGF-β was detected by using ELISA. Results YQWYF,in dose of 4 mg/L (low-dose YQWYF group),0.8 mg/L (mid-dose YQWYF group) and 0.16 mg/L (high-dose YQWYF group), and HXTLF, in dose of 32 mg/L (low-dose HXTLF group),6.4 mg/L (mid-dose HXTLF group) and 1.28 mg/L (high-dose HXTLF group) had no influence on proliferation of macrophages but improved phagocytosis of NRU of macrophages. The expression of iNOS mRNA of M1 macrophages increased significantly after stimulated by LPS and INF-γ, and decreased significantly in lovastatin group, YQWYF group and HXTLF group compared with model group (P <0.01). The expression of Arg-1 mRNA of M2 macrophages decreased significantly after stimulated by LPS and INF-γ compared with normal group, was improved significantly in lovastatin group, YQWYF group and HXTLF group compared with model group (P <0. 01), and decreased significantly in YQWYF group and HXTLF group compared with lovastatin group. After M1 macrophages stimulated by above drugs for 24 h, the levels of TNF-α, IL-6 and IL-1 decreased significantly, and levels of VEGF and TGF-β increased significantly. The level of IL-10 increased significantly in high-dose YQWYF group (P<0.05),The levels of IL-10,VEGF and TGF-β had statistical difference in HXTLF group and high-dose YQWYF group compared with lovastatin group. Conclusion YQWYF and HXTLF can inhibit expression of iNOS mRNA of M1 macrophages and induce expression of Arg-1 mRNA of M2 macrophages, and can inhibit the secretion of M1 macrophage cytokine and improve secretion of M2 macrophage cytokine.
展开▼
