温肾通络止痛方通过SENP1-SIRT3通路调控巨噬细胞极化改善骨髓间充质干细胞成骨分化和去睾丸小鼠的骨丢失

Wenshen Tongluo Zhitong Prescription regulating the polarization of macrophages through SENP1-SIRT3 pathway to improve the osteogenic differentiation of bone marrow mesenchymal stem cells and bone loss in orchiectomy mice

目的:探讨温肾通络止痛方是否通过巨噬细胞极化促进骨髓间充质干细胞(BMSCs)成骨分化从而发挥抗骨质疏松效应。方法:Micro-CT分析小鼠股骨的骨微结构,HE染色检测小鼠股骨病理变化,免疫组化检测小鼠股骨中OCN、CD86、CD206、SIRT3蛋白表达量,免疫荧光检测小鼠股骨中CD206、F4/80蛋白的表达量。免疫荧光检测RAW264.7细胞iNOS、CD206的荧光强度。qPCR检测RAW264.7细胞CD206、IL-6、iNOS mRNA表达水平。Western Blot检测SENP1、SIRT3蛋白表达量。ALP染色和茜素红染色评估含药血清处理的巨噬细胞条件培养基对BMSCs成骨分化的影响。免疫荧光和Real-time qPCR检测巨噬细胞极化水平。结果:与模型组比较,复方和阿仑膦酸钠处理后BMD、BV/TV增加(P<0.01),OCN阳性细胞数量增加(P<0.05),而脂多糖(LPS)激活M1巨噬细胞后可逆转以上作用(P<0.05,P<0.01)。与对照组比较,模型组CD86阳性细胞数目增加,CD206阳性细胞数目减少(P<0.01);复方和阿仑膦酸钠干预之后,CD86表达减少,CD206表达增加(P<0.01),而LPS干预逆转了复方对巨噬细胞极化的影响(P<0.01)。与模型组比较,含药血清组iNOS、IL-6表达下调(P<0.05,P<0.01),CD206表达上调(P<0.05,P<0.01)。含药血清处理的巨噬细胞条件培养基可增加ALP阳性细胞数、钙化结节数量。复方及其含药血清可促进巨噬细胞SENP1、SIRT3蛋白表达(P<0.05,P<0.01)、小鼠股骨SIRT3的荧光强度(P<0.05)。SENP1抑制剂Momordin Ic可阻断复方对M2标志分子CD206的影响(P<0.05)。结论:温肾通络止痛方可能通过SENP1-SIRT3通路调控巨噬细胞极化改善BMSCs成骨分化从而发挥抗骨质疏松效应。

Objective:To investigate whether Wenshen Tongluo Zhitong Prescription promotes osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)through macrophage polarization and thus plays an anti-osteoporosis effect.Methods:The bone microstructure of mouse femur was analyzed by Micro-CT,the pathological changes of mouse femur were detected by HE staining,the expression levels of OCN,CD86,CD206 and SIRT3 proteins in mouse femur were detected by immunohistochemistry,and the expressions of CD206 and F4/80 proteins in mouse femur were detected by immunofluorescence.The fluorescence intensity of iNOS and CD206 in RAW264.7 cells was detected by immunofluorescence.The mRNA expression levels of CD206,IL-6 and iNOS in RAW264.7 cells were detected by Real-time qPCR.The protein expression levels of SENP1 and SIRT3 were detected by Western Blot.ALP staining and alizarin red staining were used to evaluate the effect of osteogenic differentiation of macrophage conditioned medium BMSCs treated with medicated serum.The polarization level of macrophages was detected by immunofluorescence and qPCR.Results:Compared with the model group,BMD and BV/TV were increased(P<0.01)after treatment with compound and alendronate sodium,and the number of OCN-positive cells was increased(P<0.05).The above effects could be reversed after LPS activated M1 macrophages(P<0.05,P<0.01).Compared with the control group,the number of CD86 positive cells increased and the number of CD206 positive cells decreased in the model group(P<0.01).The expression of CD86 was decreased and CD206 was increased(P<0.01)after treatment with sodium alenophosphate and compound compound(P<0.01),while LPS intervention reversed the effect of compound compound on macrophage polarization(P<0.01).Compared with model group,the expression of iNOS and IL-6 in drug-containing serum group was down-regulated(P<0.05,P<0.01),and the expression of CD206 was up-regulated(P<0.05,P<0.01).The number of ALP positive cells and the number of calcified nodules were increased by the macrophage conditioned medium treated with medicated serum.The compound and its drug-containing serum could promote the protein expression of SENP1 and SIRT3 in macrophages(P<0.05,P<0.01),and the fluorescence intensity of SIRT3 in mouse femur(P<0.05).Momordin Ic,a SENP1 inhibitor,blocked the effect of the compound on M2 marker CD206(P<0.05).Conclusion:Wenshen Tongluo Zhitong Prescription may improve the osteogenic differentiation of BMSCs by regulating the polarization of macrophages through SENP1-SIRT3 pathway,thus playing an anti-osteoporosis effect.