摘要
目的:观察防己黄芪消肿方(FHDP)对Hulth法膝骨关节炎(KOA)大鼠关节滑膜巨噬细胞极化情况及滑膜炎情况的干预作用。方法:36只大鼠随机分为6组,分别为假手术组、模型组、FHDP高、中、低剂量组(29.16、14.58、7.29 g·kg^(-1))及洛索洛芬钠组(16.2 mg·kg^(-1))。采用改良Hulth法造KOA大鼠模型。术后6周根据分组给予高、中、低3个剂量的FHDP药液、生理盐水(NS)及洛索洛芬钠药液进行干预,给药2周后处死动物。苏木素-伊红(HE)染色后观察滑膜及软骨的组织病理学改变,流式细胞术(FCM)及免疫荧光(IF)评估M1/M2巨噬细胞极化情况,分别采用免疫组织化学(IHC)及酶联免疫吸附测定法(ELISA)检测关节组织和血清中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)、金属基质蛋白酶-13(MMP-13)等巨噬细胞极化相关蛋白的表达水平。结果:与假手术组比较,模型组Krenn及Mankin评分显著增高(P<0.01);与模型组比较,各给药组均能降低Krenn评分(P<0.05,P<0.01),Mankin评分差异无统计学意义。与假手术组比较,模型组M1/mø(CD38+)显著升高(P<0.01),M2/mø(CD206+)降低(P<0.05);与模型组比较,FHDP高、中、低剂量组可降低M1/mø(P<0.05,P<0.01),各给药组均提高M2/mø,但差异无统计学意义。与假手术组比较,模型组M1/M2显著升高(P<0.01);与模型组比较,各FHDP剂量组M1/M2均显著降低(P<0.01),FHDP高、中剂量组较洛索洛芬钠组M1/M2更低(P<0.05)。与假手术组比较,模型组滑膜及软骨中TNF-α、IL-1β、MMP-13的水平显著增高(P<0.01),IL-10的水平显著降低(P<0.01);与模型组比较,各给药组均能降低滑膜中TNF-α、IL-1β水平(P<0.05),对滑膜中MMP-13及IL-10的水平影响差异无统计学意义,各给药组均未能影响软骨中各炎症介质的水平。与假手术组比较,模型组血清TNF-α、IL-β水平显著升高(P<0.01),IL-10水平降低(P<0.05);与模型组比较,FHDP高剂量组可降低TNF-α水平(P<0.05),各给药组均能升高IL-10水平(P<0.05,P<0.01),各给药组IL-β的水平差异无统计学意义。结论:防己黄芪消肿方可改善KOA大鼠关节滑膜炎,可通过抑制M1巨噬细胞极化影响促炎细胞因子及金属基质蛋白酶的分泌,对M2巨噬细胞极化无明显影响作用,调控巨噬细胞极化失衡是该方缓解滑膜炎症治疗KOA的可能机制。
Objective:To assess the curative effects of Fangji Huangqi detumescence prescription(FHDP)on synovitis and polarization of synovial macrophages of knee osteoarthritis(KOA)model in rats induced by Hulth method.Method:Thirty-six rats were randomly divided into sham operation group,model group,high-dose,medium-dose,and low-dose(29.16,14.58,and 7.29 g·kg^(-1))FHDP groups,and loxoprofen sodium(16.2 mg·kg^(-1))group.KOA model in rats was induced by modified Hulth method.Six weeks after the operation,rats were given high,medium,and low concentrations of FHDP,normal saline(NS),and loxoprofen sodium according to the group to intervene,and sacrificed after 2-week administration.Synovium and cartilage histopathological changes were observed after hematoxylin-eosin(HE)staining.Flow cytometry(FCM)and immunofluorescence(IF)test were used to evaluate the polarization of M1/M2 macrophages.Immunohistochemistry(IMC)and enzyme-linked immunosorbent assay(ELISA)were used to detect the related protein expression levels of macrophage polarization,such as interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),interleukin-10(IL-10),and matrix metalloproteinase-13(MMP-13)in joint tissues and serum.Result:Compared with the sham operation group,Krenn and Mankin scores in the model group were significantly increased(P<0.01).Compared with the model group,Krenn score was decreased in all administration groups(P<0.05,P<0.01),but there was no significant difference in Mankin score in any administration groups.Compared with the sham operation group,M1/mø(CD38+)ratio in the model group was significantly increased(P<0.01),and M2/mø(CD206+)ratio in the model group was decreased(P<0.05).Compared with the model group,M1/møratio in the high,medium,and low-dose FHDP groups was decreased(P<0.05,P<0.01),but M2/møratio was increased in all administration groups(the difference had no statistical significance).Compared with the sham operation group,M1/M2 ratio in the model group was significantly increased(P<0.01).Compared with the model group,M1/M2 ratio in all FHDP groups was significantly decreased(P<0.01),and M1/M2 ratio in the high and medium-dose FHDP groups was lower than that in the loxoprofen sodium group(P<0.05).Compared with the sham operation group,the levels of TNF-α,IL-1β,and MMP-13 in synovium and cartilage of the model group were significantly increased(P<0.01),the level of IL-10 was significantly decreased(P<0.01).Compared with the model group,the levels of TNF-αand IL-1βin synovium were decreased in all administration groups(P<0.05),but the difference of the levels of MMP-13 and IL-10 in synovium had no statistical significance.The level of inflammatory mediators in cartilage was not affected in all administration groups.Compared with the sham operation group,the levels of TNF-αand IL-βin serum of the model group were significantly increased(P<0.01),the level of IL-10 was decreased(P<0.05).Compared with the model group,the level of TNF-αin the high-dose FHDP group was decreased(P<0.05),and the level of IL-10 was increased in all administration groups(P<0.05,P<0.01).The difference of the level of IL-βin all administration groups had no statistical significance.Conclusion:FHDP attenuated the synovitis of KOA rats.FHDP exert the effect on the releasing of proinflammatory cytokines and MMP by inhibiting the polarization of M1 macrophages in synovium,and had no significant effect on the polarization of M2 macrophages.Modulating the imbalanced polarization of synovial macrophages was a possible mechanism of FHDP on attenuating synovitis and treating KOA.
作者
危一飞
程桯
肖潇
展嘉文
蒋科卫
银河
白天宇
梁佩文
赵勇
WEI Yi-fei;CHENG Ting;XIAO Xiao;ZHAN Jiawen;JIANG Kewei;YIN He;BAI Tianyu;LIANG Peiwen;ZHAO Yong(Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China;Beijing Traditional Chinese Medicine(TCM)Hospital Affiliated to Capital Medical University,Beijing 100010,China;Beijing Key Laboratory of TCM Orthopedics Technology,Beijing 100102,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第13期112-122,共11页
Chinese Journal of Experimental Traditional Medical Formulae
基金
中国中医科学院骨伤科学重大攻关项目(C12021A02001)
国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-C-202003)。
关键词
防己黄芪消肿方
骨关节炎
巨噬细胞极化
滑膜炎
炎症
Fangji Huangqi detumescence prescription(FHDP)
osteoarthritis
polarization of macrophages
synovitis
inflammation
