荣筋拈痛方对膝关节软骨细胞外基质代谢的作用及机制

Role and mechanism of Rongjin Niantong Fang for extracellular matrix metabolism of chondrocytes

背景:课题组前期动物实验研究表明,荣筋拈痛方可通过LncRNA GAS5/miR-21影响软骨基质分解和合成代谢,以达到防治膝骨关节炎的作用。LncRNA GAS5/miR-21能否作为防治膝骨关节炎的靶点,仍需从细胞水平研究验证。目的:从细胞水平探讨荣筋拈痛方防治膝骨关节炎的作用机制。方法:(1)8周龄SPF级雄性SD大鼠40只,随机分组后给予生理盐水、荣筋拈痛方(0.45 g/mL)、盐酸氨基葡萄糖胶囊(0.015 g/mL)灌胃,制备含药血清。(2)分离培养SD大鼠膝关节软骨细胞,采用白细胞介素1β干预软骨细胞,诱导退变软骨细胞模型,采用CCK-8法明确含药血清干预软骨细胞的量效时效关系。(3)将第2代软骨细胞分为空白组(空白血清)、模型组(白细胞介素1β+空白血清)、治疗组(白细胞介素1β+荣筋拈痛方含药血清)、对照组(白细胞介素1β+盐酸氨基葡萄糖胶囊含药血清)诱导干预48 h,Real-time PCR法和Western blot法检测各组软骨细胞血清干预后LncRNA GAS5、miR-21、基质金属蛋白酶抑制物3(tissue inhibitor of metalloproteinases 3,TIMP-3)、基质金属蛋白酶(matrix metalloproteinase,MMP)3、MMP-9、MMP-13和解聚蛋白样金属蛋白酶5(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS-5)基因及蛋白表达。(4)慢病毒转染软骨细胞过表达LncRNAGAS5,分为LncRNAGAS5空载体+空白血清组、LncRNAGAS5空载体+荣筋拈痛方含药血清组、LncRNA GAS5过表达+空白血清组、LncRNA GAS5过表达+荣筋拈痛方含药血清组,各组干预后Real-time PCR检测LncRNA GAS5、miR-21、TIMP-3、MMP-9、MMP-13、ADAMTS-5基因表达。结果与结论:(1)荣筋拈痛方含药血清体积分数为10%、培养48 h;盐酸氨基葡萄糖胶囊含药血清体积分数为15%、培养48 h,能显著提高软骨细胞活性;(2)干预48 h后,与模型组相比,荣筋拈痛方含药血清和盐酸氨基葡萄糖胶囊含药血清均能抑制Lnc RNA GAS5、MMP-3、MMP-9、MMP-13、ADAMTS-5基因和蛋白的表达(P<0.05),促进miR-21、TIMP-3基因和蛋白表达(P<0.05);(3)与空载体组相比,LncRNAGAS5过表达组LncRNA GAS5、MMP-9、MMP-13、ADAMTS-5基因表达量明显升高(P<0.05),miR-21、TIMP-3基因表达量明显降低(P<0.05);与LncRNAGAS5过表达+空白血清组相比,LncRNAGAS5过表达+荣筋拈痛方含药血清组MMP-9、MMP-13和ADAMTS-5mRNA表达量均降低(P<0.05),miR-21、TIMP-3基因表达量升高(P<0.05);(4)结果表明,荣筋拈痛方通过调控LncRNA GAS5/miR-21,促进TIMP-3的表达,抑制MMP-3、MMP-9、MMP-13、ADAMTS-5等表达,从而延缓退变软骨细胞外基质降解,起到防治膝骨关节炎的作用。

BACKGROUND:Our previous animal experiments have shown that Rongjin Niantong Fang can affect the decomposition and anabolism of cartilage matrix through long non-coding RNA(LncRNA)growth arrest-specific transcript 5(GAS5)/miR-21,there by preventing and treating knee osteoarthritis.Whether LncRNA GAS5/miR-21 can be used as a target for the prevention and treatment of knee osteoarthritis still needs to be verified at the cellular level.OBJECTIVE:To explo re the mechanism of Rongjin Niantong Fang in the treatment of knee osteoarthritis at the cellular level.METHODS:(1)Forty 8-week-old SPF male Sprague-Dawley rats we re randomly divided into blank serum group,Rongjin Niantong Fang-containing serum group,and glucosamine hydrochloride capsule-containing serum group.0.9% normal saline,Rongjin Niantong Fang(0.45 g/mL),and glucosamine hydrochlo ride capsule(0.015 g/m L)were intragastrically given to the rats in the corresponding groups to collect drug containing serum.(2)Cells were treated with interleukin-1βto induce degenerative chondrocyte model.(3)Chondrocytes from the knee joint of Sprague-Dawley rats were isolated and cultured.Passage2 chondrocytes were obtained and divided into blank group(blank serum),and model group(interleukin-1β+blank serum),treatment group(inte rleukin-1β+Rongjin Niantong Fang-containing serum),and control group(inte rleukin-1β+glucosamine hydrochloride capsule-containing serum).After 48 hours of intervention,the gene and protein expressio ns of LncRNA GAS5,miR-21,tissue inhibitor of metalloproteinases 3(TIMP-3),matrix metalloproteinase(MMP)-3,MMP-9,MM P-13,and a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS-5)were detected by real-time PCR and western blot,respectively.(4)Chondrocytes were transfected with LncRNA GAS5 ove rexpression lentiviral vector.Then,the transfected cells were divided into LncRNA GAS5empty vector+blank serum group,LncRNA GAS5 empty vector+Rongjin Niantong Fang-containing serum group,LncRNA GAS5 overexpression+blank serum group,and LncRNA GAS5 overexpression+Rongjin Niontong Fong-containing serum group.The gene expressions of LncRNA GAS5,miR-21,TIM P-3,MM P-9,MMP-13,and ADAMTS-5 were detected using real-time PCR.RESULTS AND CONCLUSION:Culture with the serum containing 10%Rongjin Niantong Fang and 15% glucosamine hydrochloride capsule for 48 hours significantly increased the activity of chondrocytes.Compared with the model group,Rongjin Niontong Fang-containing serum and glucosamine hydrochloride capsule-containing serum could inhibit the gene expression of LncRNA GAS5,MMP-3,MM P-9,MM P-13,and ADAMTS-5(P<0.05)and promote the gene and protein expression of miR-21 and TIMP-3(P<0.05).Compared with the blank group,the gene expression of LncRNA GAS5,MMP-3,MMP-9,MM P-13,and ADAMTS-5 in the LncRNA GAS5 overexpression group increased significantly(P<0.05),while the gene expression of miR-21 and TIMP-3 decreased significantly(P<0.05).Compared with the LncRNA GAS5 overexpressio n+blank serum group,the gene expression of MMP-9,MMP-13,and ADAMTS-5 was significantly decreased in the LncRNA GASS ove rexpressio n+Rongjin Niantong Fang-containing serum group(P<0.05),while the gene expression of miR-21 and TIMP-3were significantly increased(P<0.05).To conclude,Rongjin Niantong Fang could delay the degradation of cartilage extracellular matrix by promoting TIMP-3expression and inhibiti ng the expression of MMP-3,MMP-9,MMP-13 and ADAMTS-5 through regulating LncRNA GAS5/miR-21,there by preventing and treating knee osteoarthritis.