骨关节炎|牛膝有效成分（ABE）干预BMSCs路径治疗骨关节炎获得重大( 二 ) 曾国华|一多三少|胃结石|广州

方法：将20只健康实验兔按照随机数字表法分为空白组、模型组及牛膝醇提物低、中、高剂量组，每组4只，分别予等量蒸馏水及牛膝醇提物低、中、高剂量灌胃1周后采血制备牛膝醇提物含药血清，用牛膝醇提物不同浓度含药血清连续培养骨髓间充质干细胞7 d，在第7天时从骨髓间充质干细胞上清液中提取外泌体，再将外泌体通过关节穿刺的方法注射到不同组别兔膝骨关节炎模型内，1次/周，连用3周，第6周后进行膝关节软骨组织病理HE染色、关节活动度评分及关节粘连改善度评分，用microCT检测膝关节局部周围骨组织超微结构及骨量，采用qRT-PCR检测滑膜组织中炎症小体关键分子NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA表达水平。
结果：实验兔膝关节腔注射外泌体第6周后，与模型组比较，低、中、高剂量组兔膝关节活动度和粘连改善度评分均明显升高（P＜0.01），且与剂量呈正相关。模型组兔膝关节软骨基质染色变浅，软骨细胞数量明显减少，关节面凹凸不平，血管入侵潮线结构；低、中、高剂量组兔膝关节软骨基质染色逐渐恢复正常，软骨细胞数量较模型组增多，软骨细胞呈卵圆形，关节面恢复平整，潮线结构依次恢复正常。microCT检测提示，与模型组比较，低、中、高剂量组兔膝关节的Mean、BMD、Conn.Dn.、BV/TV均明显升高（P＜0.05），BS均明显降低（P＜0.05），而BS/BV、BS/TV、Tb.N、Tb.Sp、DA组间比较，差异均无统计学意义（P>0.05），其中Mean、BMD、Conn.Dn.、BV/TV、BS与剂量呈相关性。qRT-PCR检测结果显示，模型组兔滑膜组织NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA相对表达量均明显高于空白组（P＜0.01），低、中、高剂量组兔滑膜组织NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA相对表达量均明显低于模型组（P＜0.05），且与剂量呈正相关。
结论：牛膝醇提物能改善OA模型局部骨组织骨含量及骨微结构，能降低炎症小体通路关键分子NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA表达，其作用机理有待进一步研究。

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图8 外泌体（exosomes）提取与鉴定

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图9 各组兔膝关节周围骨组织microCT检测超微结构图
图10 各组兔膝关节滑膜组织中NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA相对表达量比较（x(—)±s，n=8）
三、牛膝有效组分通过花生四烯酸途径减轻骨关节炎的炎症反应
Achyranthes bidentate is a common traditional Chinese medicine (TCM) used in treating osteoarthritis (OA). The compatibility between effective components has now become a breakthrough in understanding the mechanism of TCM. This study aimed at determining the optimal compatibility and possible mechanism of Achyranthes bidentate for OA treatment. Results showed that the adhesion score of the OA group is higher than NC group, and showed a trend of down-regulation in the intervention group. The CHI3L1 and IL-1β in joint fluid of the OA group was significantly increased compared to the sham operation group (NC group). Group G, I, and L exhibited significantly down-regulated CHI3L1, while groups C, F, I, K, and L exhibited reduced IL-1β. Joint adhesion, damage in cartilage, and synovial tissue was found in the OA model, cartilage tissue was found recovered in groups I, J, and L, and synovial tissue was recovered in group G, I, and L. Thus, group I and L were chosen for metabolite analysis, and indole-3-propionic acid was slightly up-regulated, while koeiginequinone A, prostaglandin H2, and 1-hydroxy-3-methoxy-10-methylacridonew were down-regulated in group I and L. According to functional analysis, the arachidonic acid (AA) metabolic pathway is enriched. Down-regulated expression of vital proteins in the AA metabolism pathway, such as PGE2 and COX2 in group I and L were verified. In conclusion, Hydroxyecdysone, Oleanolic acid, Achyranthes bidentata polysaccharide at a compatibility of 0.03-μg/mg, 2.0-μg/mg, 20.0-μg/mg or 0.03-μg/mg, 2.0-μg/mg, 10.0-μg/mg, respectively, may be the optimal compatibility of Achyranthes bidentate.