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骨關節炎|牛膝有效成分（ABE）干預BMSCs路徑治療骨關節炎獲得重大( 二 )

廣州醫科大學預防回家過年泌尿系結石結石胃結石曾國華一多三少腎盂結石


方法：將20只健康實驗兔按照隨機數字表法分為空白組、模型組及牛膝醇提物低、中、高劑量組，每組4只，分別予等量蒸餾水及牛膝醇提物低、中、高劑量灌胃1周后采血制備牛膝醇提物含藥血清，用牛膝醇提物不同濃度含藥血清連續培養骨髓間充質干細胞7 d，在第7天時從骨髓間充質干細胞上清液中提取外泌體，再將外泌體通過關節穿刺的方法注射到不同組別兔膝骨關節炎模型內，1次/周，連用3周，第6周后進行膝關節軟骨組織病理HE染色、關節活動度評分及關節粘連改善度評分，用microCT檢測膝關節局部周圍骨組織超微結構及骨量，采用qRT-PCR檢測滑膜組織中炎癥小體關鍵分子NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA表達水平。
結果：實驗兔膝關節腔注射外泌體第6周后，與模型組比較，低、中、高劑量組兔膝關節活動度和粘連改善度評分均明顯升高（P＜0.01），且與劑量呈正相關。模型組兔膝關節軟骨基質染色變淺，軟骨細胞數量明顯減少，關節面凹凸不平，血管入侵潮線結構；低、中、高劑量組兔膝關節軟骨基質染色逐漸恢復正常，軟骨細胞數量較模型組增多，軟骨細胞呈卵圓形，關節面恢復平整，潮線結構依次恢復正常。microCT檢測提示，與模型組比較，低、中、高劑量組兔膝關節的Mean、BMD、Conn.Dn.、BV/TV均明顯升高（P＜0.05），BS均明顯降低（P＜0.05），而BS/BV、BS/TV、Tb.N、Tb.Sp、DA組間比較，差異均無統計學意義（P>0.05），其中Mean、BMD、Conn.Dn.、BV/TV、BS與劑量呈相關性。qRT-PCR檢測結果顯示，模型組兔滑膜組織NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA相對表達量均明顯高于空白組（P＜0.01），低、中、高劑量組兔滑膜組織NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA相對表達量均明顯低于模型組（P＜0.05），且與劑量呈正相關。
結論：牛膝醇提物能改善OA模型局部骨組織骨含量及骨微結構，能降低炎癥小體通路關鍵分子NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA表達，其作用機理有待進一步研究。
骨關節炎|牛膝有效成分（ABE）干預BMSCs路徑治療骨關節炎獲得重大
文章插圖
圖8 外泌體（exosomes）提取與鑒定
骨關節炎|牛膝有效成分（ABE）干預BMSCs路徑治療骨關節炎獲得重大
文章插圖
圖9 各組兔膝關節周圍骨組織microCT檢測超微結構圖
圖10 各組兔膝關節滑膜組織中NLRP3 mRNA、Caspase-1 mRNA、Toll4 mRNA、NF-kB mRNA相對表達量比較（x(—)±s，n=8）
三、牛膝有效組分通過花生四烯酸途徑減輕骨關節炎的炎癥反應
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.