从少火生气理论探讨肾气丸促巨噬细胞M2型极化影响成骨的作用

目的 基于少火生气理论探讨肾气丸原组方（SQW）和肾气丸去除桂枝、附子（SQQ）诱导巨噬细胞M2型极化对小鼠骨髓间充质干细胞（BMSC）成骨分化作用的效果差异。方法 采用CCK8法分别检测SQW、SQQ对RAW264.7细胞存活率的影响,以确定药物干预的安全作用浓度。将RAW264.7细胞分为CON组、IL-4组、IL-4+SQW组、IL-4+SQQ组。将不同药物组诱导巨噬细胞M2型极化后分泌的细胞上清液作为条件培养基培养BMSC,评估BMSC成骨分化的能力。用碱性磷酸酶染色法（ALP）及茜素红染色法（ARS）检测各组BMSC成骨分化能力;使用免疫荧光技术（IF）检测BMSC成骨标志蛋白骨形态发生蛋白4（BMP4）、骨保护素（OPG）的分布和表达;使用蛋白免疫印迹法（Western Blot）检测M2型巨噬细胞极化相关的精氨酸酶1（ARG1）、白细胞介素10（IL-10）的表达以及BMSC成骨相关的BMP4、RUNT相关转录因子2（RUNX2）的表达。结果 与IL-4+SQQ组相比,IL-4+SQW组更加显著促进M2型巨噬细胞极化相关因子ARG1、IL-10上调（P＜0.01）,且其条件培养基培养的BMSC的ALP及ARS染色增强,成骨蛋白BMP4、OPG、RUNX2表达升高（P＜0.05）。结论 肾气丸原组方与肾气丸去除桂枝、附子两味药相比,具备更强的促进巨噬细胞M2型极化与增强BMSC成骨分化能力,为少火生气理论提供了实验依据。

The effect of the kidney Qi pill on osteogenesis by promoting M2-type polarization of macrophages based on the theory of mild fire producing Qi

Objective To investigate the different effects of the original ingredient recipe of kidney Qi pill (SWQ) and kidney Qi pill after removal of casmanthus twigs and monkseed (SQQ), on the osteogenic differentiation of mouse bone marrow mesenchyml stem cells (BMSC) by inducing the M2-type polarization of macrophages, based on the theory of mild fire producing Qi. Methods CCK8 method was used to detect the effects of SQW and SQQ on the survival rate of RAW264.7 cells, so as to determine the safe concentration of drug intervention. RAW264.7 cells were divided into CON group, IL-4 group, IL-4+SQW group, and IL-4+SQQ group in vitro. The cell supernatant secreted by M2-type polarization of macrophages after polarization induced by different drug groups was used as conditioned medium to culture BMSC. The osteogenic differentiation ability of BMSC was evaluated. The osteogenic differentiation ability of BMSC in each group was detected with alkaline phosphatase staining (ALP) and Alizarin red staining (ARS). The distribution and expression of bone morphogenetic protein 4 (BMP4) and osteoprotein (OPG) were detected with immunofluorescence (IF). The expressions of polarity-related arginase 1 (ARG1) and interleukin 10 (IL-10) in M2-type macrophages and the expressions of BMSC osteogenic BMP4 and Runt-related transcription factor 2 (RUNX2) were detected using Western blotting. Results Compared with IL-4+SQQ group, IL-4+SQW group significantly promoted the up-regulation of M2-type macrophage polariation-related factors ARG1 and IL-10 (P< 0.01), and the conditioned medium culture enhanced the ALP and ARS staining of BMSC, and improved the expressions of BMP4, OPG and RUNX2 in osteoblasts (P< 0.05). Conclusion Compared with SQQ, SQW has a stronger ability to promote M2-type polarization of macrophages and to enhance osteogenic differentiation of BMSC. This provides experimental basis for the theory of mild fire producing Qi.