摘要
目的:探讨五福健膝方治疗膝骨关节炎(knee osteoarthritis,KOA)的作用机制。方法:①网络药理学研究。采用网络药理学方法筛选五福健膝方治疗KOA的靶点。②动物实验。将18只10周龄SPF级雄性C57BL/6小鼠随机分为3组,每组6只。模型组和五福健膝方组小鼠采用内侧半月板失稳术在右后肢构建KOA模型,假手术组小鼠仅切开对应区域皮肤和关节囊后缝合。造模手术后第2天,五福健膝方组小鼠以五福健膝方汤剂(生药浓度1.1 g·mL^(-1))灌胃,每次0.6 mL,每天1次,连续灌胃8周;模型组和假手术组小鼠以等量生理盐水灌胃。药物干预结束后,收集小鼠右后肢膝关节,分别进行股骨远端Micro-CT扫描、组织病理学观察(阿尔新蓝-苏木素/橙黄G染色)及免疫组织化学染色。免疫组织化学染色主要针对Ⅱ型胶原蛋白与网络药理学研究确定的核心靶点蛋白,测量Ⅱ型胶原蛋白表达阳性区域的厚度(视为软骨厚度),同时计算核心靶点蛋白阳性细胞百分比。结果:①网络药理学研究结果。通过网络药理学研究确定的五福健膝方治疗KOA的关键靶点基因15个,其中TP53为核心靶点基因,其对应的蛋白为P53蛋白。②动物实验结果。股骨远端Micro-CT检查结果显示,模型组较假手术组骨表面积骨体积比值降低(P=0.028)、骨体积分数升高(P=0.003),五福健膝方组较模型组骨表面积骨体积比值升高(P=0.004)、骨体积分数降低(P=0.048)。膝关节软骨组织阿尔新蓝-苏木素/橙黄G染色显示,模型组小鼠软骨缺失明显,五福健膝方组小鼠软骨丢失较模型组明显改善。免疫组织化学染色结果显示,模型组小鼠膝关节软骨厚度较假手术组减小(P=0.001),五福健膝方组小鼠膝关节软骨厚度较模型组增加(P=0.048);模型组小鼠膝关节软骨组织中P53阳性细胞百分比较假手术组增加(P=0.000),五福健膝方组小鼠膝关节软骨组织中P53阳性细胞百分比较模型组减少(P=0.000)。结论:五福健膝方能够有效延缓KOA模型小鼠的软骨退变,其机制可能与其抑制P53蛋白表达有关。
Objective:To explore the mechanism of Wufu Jianxi Fang(五福健膝方,WFJXF)against knee osteoarthritis(KOA).Methods:①Network pharmacology research.The action targets of WFJXF against KOA were screened by using the network pharmacology approach.②Animal experimentation.Eighteen 10-week-old specific pathogen-free(SPF)-grade male C57BL/6 mice were selected and randomized into model group,WFJXF group and sham-operated group,6 cases in each group.The mice in model group and WFJXF group were subjected to destabilization of the medial meniscus(DMM)on the right hindlimbs for inducing KOA,while the ones in sham-operated group were merely incised the skin and joint capsule at the corresponding site and then sutured.On day 2 after the modeling surgery,the mice in WFJXF group were intervened by intragastric administration with WFJXF decoction(the crude drug concentration was 1.1 g/mL),once a day,0.6 mL at a time for consecutive 8 weeks;while the ones in model group and sham-operated group with the same dosage of normal saline.After the end of drug intervention,the mice were executed,and the knee joints were harvested from their right hindlimbs for scanning the distal femur by using Micro-CT,and observing the histopathological changes of knee articular cartilage tissues via alcian blue-hematoxylin/orange G(ABH/OG)staining,furthermore,the immunohistochemistry(IHC)staining was performed for detecting the typeⅡcollagen and core target proteins determined by network pharmacology analysis,and then the thickness of typeⅡcollagen-positive areas(regarded as the cartilage thickness)was measured,and the proportion of core target protein-positive cells was calculated.Results:①The results of network pharmacology research.As revealed by the network pharmacology analysis,15 key target genes of WFJXF against KOA were obtained,among which the TP53 was identified as the core target gene,with its corresponding protein being recognized as the P53.②The results of animal experimentation.As revealed by Micro-CT examination on distal femur,the ratio of bone surface(BS)to bone volume(BV)decreased,while the bone volume fraction(BVF)increased in model group compared to sham-operated group(P=0.028;P=0.003);whereas,the ratio of BS to BV increased and the BVF decreased in WFJXF group compared to model group(P=0.004;P=0.048).The ABH/OG staining results showed that the cartilages obviously lost in mice of model group,however,the cartilage loss was significantly improved in mice of WFJXF group compared with that of model group.Moreover,the results of IHC staining revealed that the thickness of knee cartilage decreased in mice of model group compared with that of sham-operated group(P=0.001),and it increased in mice of WFJXF group compared with that of model group(P=0.048);besides,the proportion of P53 positive cells in the knee articular cartilage tissues increased in mice of model group compared with that of sham-operated group(P=0.000),and it decreased in mice of WFJXF group compared with that of model group(P=0.000).Conclusion:WFJXF can effectively delay the cartilage degeneration in KOA model mice,which may work by inhibiting the expression of P53 protein.
作者
胡松峰
郭蔓岑
金红婷
袁文华
HU Songfeng;GU Mancen;JIN Hongting;YUAN Wenhua(Shaoxing Hospital of Traditional Chinese Mediceine,Shaoxing 312000,Zhejiang,China;Zhejiang Chinese Medical Univ ersity,Hangzhou 310053,Zhejiang,China;Zhejiang Provincial Hospital of Traditional Chinese Medicine,Hangzhou 310006,Zhejiang,China)
出处
《中医正骨》
2024年第10期18-24,共7页
The Journal of Traditional Chinese Orthopedics and Traumatology
基金
浙江省中医药科技计划项目(2020ZA117)
浙江省基础公益研究计划项目(LTGY23H270008)
浙江省创伤性骨病诊治中医药传承创新团队项目(浙卫发[2023]31号)。
关键词
骨关节炎
膝
五福健膝方
肿瘤抑制蛋白p53
网络药理学
动物实验
osteoarthritis,knee
w ufu Jianxi Fang
tumor suppressor protein p53
network pharmacology
animal experimentation
