【作者】 高航；

【导师】 刘建国；

【作者基本信息】 吉林大学 ， 外科学， 2017， 博士

【摘要】 研究背景骨性关节炎(osteoarthritis,OA)是一种临床常见的退行性关节疾患,多发生于老年人。临床上主要表现为关节僵硬疼痛、压痛、僵硬、活动障碍。骨关节炎主要以关节软骨细胞的变性为特征,但其具体发病机制尚未完全阐明。近年来研究发现炎症在骨关节炎发病过程中发挥重要作用,炎性介质在骨关节炎的发生过程中作用备受关注。通过对炎性介质在骨关节炎发病中的作用机制和作用环节的调控,对骨关节炎的防治及相关药物研发具有重要意义。柴胡皂苷A是从中药柴胡中提取的化合物,研究报道具有重要的抗炎作用。本课题以骨关节炎为主要研究对象,选择我国传统中药材柴胡中所含有的天然结构化合物柴胡皂苷A为治疗药物,首先在木瓜蛋白酶诱导的大鼠骨关节炎模型中证实柴胡皂苷A的治疗效果,其次在人骨关节炎软骨细胞上验证柴胡皂苷A的抗炎效果,最后在人骨关节炎软骨细胞上探索柴胡皂苷A的抗炎机制,并对其作用靶标进行初步确认,为骨关节炎治疗药物的研发提供实验依据及参考。实验方法首先,建立木瓜蛋白酶诱导的大鼠骨关节炎模型,在上述模型中通过灌胃给予不同剂量的柴胡皂苷A。实验分为空白对照组、骨关节炎模型组、柴胡皂苷A(25、50、100mg/kg体重)治疗组。通过关节肿胀度观察,平均关节炎指数(MAI)的测定,关节组织中炎性细胞因子IL-1β和MMP1,MMP3,MMP13的检测,及关节组织中核转录因子NF-κB的检测,验证柴胡皂苷A对骨关节炎的治疗效果。其次,在人骨关节炎软骨细胞上验证柴胡皂苷A的抗炎效果,选择IL-1β刺激人骨关节炎软骨细胞炎症模型作为工具,通过MTT法检测柴胡皂苷A的细胞毒性效果,确定在低于15μM浓度范围内,柴胡皂苷A对人骨关节炎软骨细胞没有细胞毒性。在细胞培养基中加入5,10,15μM柴胡皂苷处理2小时,然后添加IL-1β刺激,ELISA和q RT-PCR检测柴胡皂苷A对IL-1β刺激的人骨关节炎软骨细胞MMP1,MMP3,和MMP13表达影响;对IL-1β诱导的PGE2和NO表达进行检测;Western Blot检测柴胡皂苷A对IL-1β刺激的人骨关节炎软骨细胞NF-κB信号通路的影响。最后,探索柴胡皂苷A的抗炎作用靶点,通过Western blot检测柴胡皂苷A对核受体LXRα表达的影响。选用LXRα特异性拮抗剂GGPP阻断LXRα,检测柴胡皂苷A对IL-1β诱导的炎性介质NO,PGE2,MMP1,MMP3,and MMP13的影响。实验结果(1)柴胡皂苷A对大鼠骨关节炎模型的治疗效果。(1)与骨关节炎模型组相比,柴胡皂苷A治疗组关节肿胀度明显减轻;(2)与骨关节炎模型组相比,柴胡皂苷A治疗组关节组织中炎性细胞因子IL-1β和MMP1,MMP3,MMP13的表达明显降低,并呈计量依赖性;(3)与骨关节炎模型组相比,柴胡皂苷A治疗组关节组织中核转录因子NF-κB激活明显降低,以上实验初步证实柴胡皂苷A对木瓜蛋白酶诱导的大鼠骨关节炎具有治疗效果;(2)柴胡皂苷A对人骨关节炎软骨细胞抗炎效果研究。(1)柴胡皂苷A在低于15μM浓度范围内对人骨关节炎软骨细胞没有细胞毒性;(2)柴胡皂苷A可以抑制IL-1β诱导的PGE2和NO的表达,此外,IL-1β诱导的MMP1,MMP3,和MMP13表达被柴胡皂苷A显著抑制,并呈剂量依赖性;(3)Western blot结果显示柴胡皂苷A可以抑制IL-1β诱导的NF-κB蛋白表达,上述结果显示柴胡皂苷A通过抑制IL-1β诱导的炎性介质及NF-κB激活来发挥其抗炎作用;(3)柴胡皂苷A对人骨关节炎软骨细胞抗炎机制研究。(1)柴胡皂苷可以增加LXRα表达,并呈计量依赖性;(2)柴胡皂苷A对IL-1β诱导的NO,PGE2,MMP1,MMP3,and MMP13表达的影响可以被GGPP(LXRα抑制剂)阻断,表明柴胡皂苷A通过激活LXRα发挥抗炎作用;(3)敲低LXRα后,柴胡皂苷A的抗炎作用被抑制。上述结果显示柴胡皂苷A通过激活核受体LXRα发挥其抗炎作用,初步确认LXRα可能是柴胡皂苷的作用靶点;结论(1)研究结果表明柴胡皂苷A对木瓜蛋白酶诱导的大鼠骨关节炎模型具有治疗作用,主要是通过抑制炎性介质的产生发挥作用。(2)柴胡皂苷A可以抑制IL-1β诱导NO,PGE2,MMP1,MMP3,and MMP13表达及NF-κB激活,发挥体外抗炎作用。(3)柴胡皂苷A发挥其抗炎作用是通过激活核受体LXRα,从而抑制IL-1β诱导的NF-κB激活及下游炎性介质的表达,来发挥抗骨关节炎作用。以上结果为柴胡皂苷A在骨关节炎防治中的作用提供理论依据,同时为以LXRα为靶点的骨关节炎抗炎药物研发提供初步的实验数据和参考。更多还原

【Abstract】 Background:Osteoarthritis(OA)is a common degenerative joint disease in clinic that often occurred in the elderly.The main clinical manifestations were joint stiffness,pain,tenderness,stiffness,and movement disorders.Osteoarthritis is characterized mainly by degeneration of articular chondrocytes.However,the specific mechanism has not yet been fully elucidated.In recent years,studies showed that inflammation played an important role in the development of OA.Through the regulation of inflammatory mediators in the development of OA,the prevention and treatment of OA and related drug research and development is of great significance.Saikosaponin A is a compound extracted from the root of Chinese medicine Bupleurum chinense and has been reported to have anti-inflammatory effects.In the present study,we investigated the effects of Saikosaponin A in the treatment of OA.Firstly,we confirm the therapeutic effects of Saikosaponin A on OA in the model of papain-induced OA in rats.Secondly,we investigate the anti-inflammatory effects of Saikosaponin A in human osteoarthritis chondrocytes.Finally,we clarify the anti-inflammatory mechanism of Saikosaponin A in human osteoarthritis chondrocytes.We provide experimental basis and reference for the treatment of osteoarthritis.Methods:Firstly,we established the model of OA induced by papain in rats and the rats were given different doses of Saikosaponin A.The rats were divided into five groups: normal control group,model group,and Saikosaponin A(25,50,100mg/kg)groups.To investigate the therapeutic effects of Saikosaponin A on OA,the mean arthritis index(MAI)and joint swelling were detected.Furthermore,the levels of IL-1β,MMP1,MMP3,and MMP13,as well as the activation of NF-κB were measured.Secondly,we investigated the anti-inflammatory effect of Saikosaponin A on IL-1β-stimulated human osteoarthritis chondrocytes.The cells were pretreated with Saikosaponin A 12 h before IL-1β treatment.The production of PGE2 and NO were detected by ELISA and Griess method.The levels of MMP1,MMP3,and MMP13 were measured by ELISA and qRT-PCR.The expression of NF-κB and LXRα were tested by western blot analysis.Results:(1)The therapeutic effects of Saikosaponin A on OA.(1)Compared with OA group,Saikosaponin A significantly attenuated joint swelling;(2)Compared with OA group,the levels of IL-1β,MMP1,MMP3,and MMP13 were significantly inhibited by the treatment of Saikosaponin A.(3)Compared with OA group,the activation of NF-κB was significantly inhibited by the treatment of Saikosaponin A.These results suggested that Saikosaponin A had therapeutic effects against OA.(2)Anti-inflammatory effects of Saikosaponin A on IL-1β-stimulated human osteoarthritis chondrocytes.(1)Saikosaponin A did not affect the viability of human osteoarthritis chondrocytes at the doses of 15μM.(2)Saikosaponin A inhibited IL-1β-induced PGE2 and NO production in a concentration-dependent manner.Saikosaponin A also suppressed IL-1β-induced MMP1,MMP3,and MMP13 production.(3)Saikosaponin A significantly attenuated IL-1β-induced phosphorylation levels of NF-κB p65 and IκBα.These results suggested that Saikosaponin A could inhibit the inflammatory response in human chondrocytes in vitro by inhibiting NF-κB activation.(3)Anti-inflammatory mechanism of Saikosaponin A on IL-1β-stimulated human osteoarthritis chondrocytes.(1)Saikosaponin A up-regulated the expression of LXRα in a dose-dependent manner;(2)The inhibition of Saikosaponin A on PGE2,NO,MMP1,MMP3,and MMP13 production were reversed by GGPP,the inhibitor of LXRα.(3)The anti-inflammatory effects of Saikosaponin A were blocked when LXRαwas knockdown.The results demonstrated that Saikosaponin A inhibited inflammatory responses in human chondrocytes by activating LXRα and LXRα was the target of Saikosaponin A.Conclusions:(1)The results showed that Saikosaponin A had therapeutic effects against OA and the therapeutic effects was due to its ability to inhibit inflammatory mediators production.(2)Saikosaponin A exhibited its anti-inflammatory effects in human osteoarthritis chondrocytes by inhibiting IL-1β-induced NO,PGE2,MMP1,MMP3,and MMP13 production,as well as NF-κB activation.(3)Saikosaponin A exhibited its anti-inflammatory effects in human osteoarthritis chondrocytes by activating LXRα,which subsequently inhibited IL-1β-induced NF-κB activation and inflammatory mediators production.The above results provide a theoretical basis for the role of Saikosaponin A in the prevention and treatment of OA.Meanwhile,we provide preliminary experimental data and reference for the research and development of anti-inflammatory drugs for osteoarthritis targeting LXRα.更多还原