CN114404434B - 一种治疗骨关节炎的化合物及其用途 - Google Patents
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Abstract
本申请公开一种治疗骨关节炎的化合物及其用途,包括紫花前胡苷,小鼠动物实验中,紫花前胡苷浓度:10mg/kg,用0.5%羧甲基纤维素钠配置,软骨细胞实验中,紫花前胡苷浓度:5‑150μM,用DMSO配置,紫花前胡苷通过Drp1/ROS/NLRP3信号轴延缓膝骨关节炎的分子机制来达到治疗的目的。本发明构建了C57小鼠及原代软骨细胞KOA模型,研究紫花前胡苷对KOA小鼠关节软骨及滑膜炎症的作用,以及对体外软骨细胞中Drp1、ROS和NLRP3等相关指标的影响,以此探究紫花前胡苷通过Drp1/ROS/NLRP3信号轴干预KOA的潜在疗效分子机制。
Description
技术领域
本申请涉及治疗骨关节炎的领域,尤其涉及一种治疗骨关节炎的化合物及其用途。
背景技术
骨关节炎(osteoarthritis,OA)是一种最常见的骨关节疾病,常常累及膝、髋、脊柱等关节部位,膝关节是最主要的受累关节。膝骨关节炎(kneeosteoarthritis,KOA)是整个关节的疾病,以软骨丢失、软骨下骨硬化、骨赘形成及滑膜炎症等为主要病理表现。流行病学调查显示,全球有超过2.4亿人受OA影响,大于45岁的人中约30%有OA影像学改变,OA造成的直接医疗费用超过1000亿美元。随着世界人口老龄化,OA发病率越来越高,而OA是导致老年人残疾最主要的原因之一,给社会和患者带来沉重的经济负担,严重影响了人民的健康生活。目前,OA仍然缺乏有效的干预治疗药物,现有的临床一线用药也只能减轻临床症状,缓解患者痛苦,从而延缓疾病进程,全膝关节置换术仍然是终末期KOA唯一有效的治疗方式。然而,OA发病机制尚不完全清楚,给研究工作带来很大的挑战,也是研究者们探索研究的热点方向。
线粒体是细胞的能量中枢,源源不断的产生能量以维持生命活动的正常进行。线粒体为高度动态的网管状细胞器,通过有序地***、融合以维持能量供应及细胞稳态。线粒体***/融合动态平衡受到诸多蛋白调控,其中Drp1(Dynamin-relatedprotein1,Drp1)为调控线粒体***的关键蛋白,Drp1主要位于细胞质中,受线粒体外膜分子Fisl招募而转位至线粒体外膜潜在断裂点,低聚形成环状复合体,逐渐压缩促使线粒体***。***后,Drp1重新回到胞浆,反复循环。当细胞内Drp1高表达时,可加速线粒体***,引起线粒体碎片化,同时出现线粒体功能紊乱,如膜电位降低、内源性活性氧簇(Reactive oxygen species,ROS)水平上升等。
ROS主要来源于线粒体,有证据表明,KOA患者软骨细胞线粒体功能紊乱,ROS生成增多,细胞抗氧化能力降低,而抗氧化能力不足与基质降解和软骨细胞死亡密切相关。软骨细胞中ROS生成与消耗失衡不仅会导致氧化损伤,还会诱导氧化还原调节的信号通路异常,造成软骨细胞功能紊乱,形成细胞损伤的微环境,加速KOA疾病进程。软骨细胞内ROS亦能激活紫花前胡苷样受体蛋白3(紫花前胡苷-like receptor family pyrin domain-containing 3,NLRP3)炎症小体,核转录因子-κB(Nuclear transcription factor-κB,NF-κB)等炎症相关信号分子。NLRP3炎症小体是炎症性、心血管、神经退行性等疾病研究的热点,由NLRP3、ASC和Caspase-1前体组成。研究表明,NLRP3炎症小体能激活toll样受体和NF-κB信号通路触发滑膜炎症反应,加剧KOA疾病的发展。因此,Drp1/ROS/NLRP3轴可能是干预KOA的潜在靶点。
发明内容
为解决上述技术问题,本申请提供一种治疗骨关节炎的化合物及其用途。本发明构建了C57小鼠及原代软骨细胞KOA模型,研究紫花前胡苷对KOA小鼠关节软骨及滑膜炎症的作用,以及对体外软骨细胞中Drp1、ROS和NLRP3等相关指标的影响,以此探究紫花前胡苷通过Drp1/ROS/NLRP3信号轴干预KOA的潜在疗效分子机制。
第一方面,提供一种治疗骨关节炎的化合物,包括紫花前胡苷。
作为优选,小鼠动物实验中,紫花前胡苷浓度:10mg/kg,用0.5%羧甲基纤维素钠配置。
作为优选,软骨细胞实验中,紫花前胡苷浓度:5-150μM,用DMSO配置。
作为优选,所述紫花前胡苷制成散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。
第二方面,提供上述的治疗骨关节炎的化合物在制备防治骨关节炎的药物中的用途。
作为优选,治疗骨关节炎的化合物为紫花前胡苷,紫花前胡苷通过Drp1/ROS/NLRP3信号轴延缓膝骨关节炎的分子机制。
本发明有益效果:
本发明构建了C57小鼠及原代软骨细胞KOA模型,研究紫花前胡苷对KOA小鼠关节软骨及滑膜炎症的作用,以及对体外软骨细胞中Drp1、ROS和NLRP3等相关指标的影响,以此探究紫花前胡苷通过Drp1/ROS/NLRP3信号轴干预KOA的潜在疗效分子机制。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1:小鼠膝关节Mico-CT三维重建图。A膝关节三维重建正面、背面和侧面图。B膝关节内侧软骨下骨矢状位图。C相关参数定量分析统计图。
图2:小鼠膝关节番红/快绿染色和免疫组化染色图。A关节软骨番红/快绿染色图。B关节软骨II型胶原免疫组化染色图。C关节软骨OARSI评分统计图。D关节软骨II型胶原阳性面积统计图。
图3:小鼠血清中炎症因子和滑膜HE染色图。A小鼠体重变化统计图。B小鼠血清中IL-1β和TNF-α统计图。C关节滑膜HE染色图。D滑膜炎症评分统计图。
图4:关节软骨免疫荧光染色图。A关节软骨p-Drp1免疫荧光染色图。B关节软骨NLRP3免疫荧光染色图。C阳性p-Drp1统计图。D阳性NLRP3统计图。
图5:关节软骨蛋白印迹图。A关节软骨Collagen II、NLRP3和p-Drp1蛋白印迹图。BCollagen II、NLRP3和p-Drp1统计分析图。
图6:原代软骨细胞鉴定及细胞中MitoSOX水平图。A不同浓度紫花前胡苷对软骨细胞活性的影响统计图。B软骨细胞II型胶原免疫荧光染色图。C软骨细胞中MitoSOX染色图。DMitoSOX统计分析图。
图7:软骨细胞聚合酶链式反应和蛋白印迹图。A软骨细胞中COX2、IL-1β和TNF-αmRNA水平统计图。B软骨细胞Collagen II、NLRP3、p-Drp1和MMP13蛋白印迹图。CCollagenII、NLRP3、p-Drp1和MMP13统计分析图。
图8:软骨细胞免疫荧光染色图。Ap-Drp1免疫荧光染色图。B NLRP3免疫荧光染色图。C Collagen II免疫荧光染色图。D MMP13免疫荧光染色图。E统计分析图。
具体实施方式
下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
实施例
一种治疗骨关节炎的化合物,包括紫花前胡苷。
本实施例中,小鼠动物实验中,紫花前胡苷浓度:10mg/kg,用0.5%羧甲基纤维素钠配置。
本实施例中,软骨细胞实验中,紫花前胡苷浓度:5-150μM,用DMSO配置。
本实施例中,所述紫花前胡苷制成散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。
上述的治疗骨关节炎的化合物在制备防治骨关节炎的药物中的用途。
本实施例中,治疗骨关节炎的化合物为紫花前胡苷,紫花前胡苷通过Drp1/ROS/NLRP3信号轴延缓膝骨关节炎的分子机制。
本发明构建了C57小鼠及原代软骨细胞KOA模型,研究紫花前胡苷对KOA小鼠关节软骨及滑膜炎症的作用,以及对体外软骨细胞中Drp1、ROS和NLRP3等相关指标的影响,以此探究紫花前胡苷通过Drp1/ROS/NLRP3信号轴干预KOA的潜在疗效分子机制。
实验研究资料如下:
研究本发明治疗骨关节炎的化合物的依据:
1实验动物、造模及给药
SPF级8周龄雄性C57BL/6小鼠,饲养于12h明暗交替、温度、湿度恒定环境,所有小鼠均能自由获取食物与水源。购买于湖南斯莱克景达实验动物有限公司,合格证号SCXK(湘)2019-0004,所有动物实验均符合湖南中医药大学实验动物伦理委员会的规定。所有小鼠随机分为3组,假手术组、模型组、模型+紫花前胡苷组。适应性饲养1周后,采用内侧半月板失稳手术制备KOA模型,模型组:首先,2%异氟烷麻醉小鼠,切开皮肤及关节囊,然后用显微手术剪剪断内侧半月板胫骨韧带;假手术组只切开皮肤及关节囊,缝合切口,碘伏擦拭后放入笼内待小鼠苏醒。紫花前胡苷购买于江苏永健医药科技有限公司(纯度≧98%),溶解于0.5%羧甲基纤维素钠,浓度1mg/mL,按10mg/kg剂量灌胃给药,每日一次,持续至第8周,假手术组与模型组给予等量生理盐水。
2micro-CT检测
小鼠膝关节采用micro-CT仪器(Quantum GX,PerkinElmer)扫描并进行三维重建,设定扫描电压90kV,电流80μA,分辨率10μm。使用分析软件(CTAn v1.9)和三维可视化软件(CTVol v2.0)对数据进行分析。胫骨生长板和胫骨平台之间确定为感兴趣的区域,基于三维形态测量的断层数据对骨体积(BV,mm3)、骨体积分数(BV/TV,%)、骨小梁厚度(Tb.Th,mm),骨小梁分离度(Tb.Sp,mm)和骨小梁数量(Tb.N,1/mm)进行统计分析。
3组织形态学检测
小鼠膝关节样本用10%多聚甲醛固定24h后,20%EDTA脱钙1个月,梯度乙醇脱水,石蜡包埋,矢状位切成4.0μm备用。苏木精-伊红(HE)染色:软骨标本用明矾苏木精染色40min,伊红染色40s,封片后显微镜下观察滑膜形态,并进行滑膜炎症评分。番红O-固绿染色:固绿染色30min,1%醋酸洗10秒,番红O染色3min,封片后显微镜下观察软骨形态,进行OARSI评分和统计胫骨侧软骨面积,两名独立的研究人员对研究数据进行统计分析。
4血清ELISA检测
IL-1β(EK0391,Boster)和TNF-α(FEK0527,Boster)使用ELISA试剂盒按使用说明进行检测。
5免疫组化、免疫荧光染色
免疫组化染色:切片脱蜡,3%过氧化氢孵育10min去除内源性过氧化物酶,5%BSA孵育20min,II型胶原(1:100,28459-1-AP,Proteintech)4℃孵育过夜,HRP标记的二抗孵育40min,二氨基联苯胺(DAB)显色,苏木精染色,封片后显微镜下拍片。免疫荧光染色:切片脱蜡,5%BSA孵育20min,NLRP3(1:50,MA5-32255,ThermoFisher)和p-Drp1(1:50,AF5791,碧云天)4℃孵育过夜,荧光二抗孵育40min,DAPI封片后荧光显微镜下观察拍片。
6软骨组织Western blot
软骨组织与加入磷酸酶抑制剂的RIPA裂解液混合冰上研磨10min,裂解10min,离心后收集上清,BCA测定总蛋白浓度。10%的SDS-PAGE凝胶电泳并将分离的蛋白条带转移到PVDF膜,5%的脱脂牛奶室温下孵育1h。一抗4℃孵育过夜(Col II,1:1000,28459-1-AP,Proteintech;NLRP3,1:1000,MA5-32255,ThermoFisher;p-Drp1,1:1000,AF5791,Beyotime;β-actin,1:5000,66009-1-Ig,Proteintech),TBST洗膜,二抗室温孵育1h,使用化学发光检测***(Bio-Rad)将信号可视化。
7原代软骨细胞分离、培养与鉴定
无菌条件下,将大鼠膝关节软骨组织切成小块,用PBS洗涤后,37℃下0.25%的胰酶中消化1h,然后与0.2%II型胶原酶培养基孵育4h。1200rpm离心5min后,弃上清将软骨细胞接种到10cm培养皿中,37℃,5%CO2恒温培养箱中培养。细胞约90%时用胰酶消化,进行传代和用于后续实验。细胞鉴定:细胞爬片PBS清洗后4%多聚甲醛固定30min,0.2%的TritonX-100通透10min,5%BSA封闭1h,II型胶原(1:50,28459-1-AP,Proteintech)4℃孵育过夜,PBS清洗后荧光二抗室温孵育1h,DAPI封片后荧光显微镜下观察。
8CCK8检测
使用CCK8试剂盒测定紫花前胡苷对软骨细胞活力的影响。将细胞以每孔1×104个细胞的密度接种到96孔板的孔中,加入不同浓度的紫花前胡苷培养基,培养12h后每孔分别10μL的CCK-8溶液,细胞培养箱培养4h后用酶标仪测定其波长为450nm处的吸光度。
9细胞内ROS检测
以1×105个细胞的密度接种于铺有15mm爬片的12孔板内,培养12h后去除培养基,PBS轻洗后加入2mLMitoSOX探针工作液充分覆盖爬片生长的细胞,于37℃避光孵育10min,用预热的PBS轻洗细胞,加入适量4%多聚甲醛进行固定,使用DAPI对细胞进行复染,取出爬片封片后进行荧光检测。
10细胞免疫荧光染色
软骨细胞用4%多聚甲醛固定30min,0.2%的TritonX-100通透10min,5%BSA封闭1h,一抗(Col II,1:50,28459-1-AP,Proteintech;NLRP3,1:50,MA5-32255,ThermoFisher;p-Drp1,1:50,AF5791,Beyotime;MMP13,1:50,18165-1-AP,Proteintech)4℃孵育过夜,然后用荧光二抗室温孵育1h,DAPI封片后用荧光显微镜观察拍片。
11Realtime PCR
用Trizol试剂盒(Takara)从软骨细胞中提取总RNA,并使用试剂盒(Takara)逆转录成cDNA。使用CFX96 Touch实时PCR***(BioRad)进行PCR扩增,使用2-ΔΔCt计算基因的相对表达量。所用的引物序列如下:β-actin:forward 5′-ACATCCGTAAAGACCTCTATGCC-3′,reverse 5′-TACTCCTGCTTGCTGATCCAC-3′;COX2:forward 5′-CTGATGACTGCCCAACTCCC-3′,reverse 5′-GGTCCTCGCTTCTGATCTGTC-3′;IL-1β:forward 5′-CAGCAGCATCTCGACAAGAG-3′,reverse 5′-AAAGAAGGTGCTTGGGTCCT-3′;TNF-α:forward 5′-CCCCTCTATTTATAATTGCACCT-3′,reverse 5′-CTGGTAGTTTAGCTCCGTTT-3′.
12Westernblot
每孔中加入200uLRIPA裂解液,用细胞刮刀刮下细胞,收集悬液,超声破碎1.5min,冰上裂解10分钟,4℃,12000rpm离心10min后的上清转移1.5mL离心管中,BCA法测定总蛋白浓度。10%的SDS-PAGE凝胶电泳并将分离的蛋白条带转移到PVDF膜,5%的脱脂牛奶室温下孵育1h。一抗4℃孵育过夜(Col II,1:1000,28459-1-AP,Proteintech;NLRP3,1:1000,MA5-32255,ThermoFisher;p-Drp1,1:1000,AF5791,Beyotime;MMP13,1:1000,18165-1-AP,Proteintech;β-actin,1:5000,66009-1-Ig,Proteintech),TBST洗膜,二抗室温孵育1h,使用化学发光检测***(Bio-Rad)将信号可视化。
13统计分析
采用GraphPadPrism7.0软件(GraphPad Software)进行统计分析。所有数据以均数±标准差表示,通过正态性检验的数据采用单因素分析,采用Tukey多重比较检验,非正态分布但包含方差相等的数据采用非参数Mann-WhitneyU检验,P<0.05表示差异具有统计学意义。
14结果
(1)紫花前胡苷改善KOA小鼠软骨下骨骨吸收
软骨下骨硬化吸收,骨赘形成是KOA典型的病理改变,为了评估紫花前胡苷对KOA模型小鼠软骨下骨的保护作用,本实验采用micro-CT扫描及三维成像对膝关节进行检测。实验结果发现,紫花前胡苷显著抑制了骨赘形成,改善了软骨下骨骨硬化、吸收,如图1A和1B所示。骨形态学指标定量分析发现,与模型组比较,紫花前胡苷治疗后显著改善了KOA小鼠胫骨侧软骨下骨骨体积(BV)、骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数量(Tb.N),也能降低骨小梁分离度(Tb.Sp)(P<.05)。
(2)紫花前胡苷减轻KOA小鼠软骨损伤
关节软骨厚度丢失、细胞外基质降解是伴随KOA发生发展,是疾病进展的敏感性指标。本实验番红O-固绿染色结果发现,与假手术组比较,模型组小鼠膝关节关节软骨表面粗糙、不完整,软骨厚度明显丢失;而紫花前胡苷治疗后,关节软骨厚度增加,OARSI评分发现,模型组与紫花前胡苷组之间差异具有统计学意义(P<.01)(如图2A、2C所示)。关节软骨II型胶原免疫组化染色发现,假手术组II型胶原染色均匀,颜色较深,说明软骨中含量丰富;胫骨侧关节软骨II型胶原阳性染色统计发现,紫花前胡苷能显著增加软骨中II型胶原的含量(P<.01)(如图2B、2D所示)。实验结果表明,紫花前胡苷能够减轻软骨损伤,延缓软骨基质降解。
(3)紫花前胡苷抑制KOA小鼠炎症反应
滑膜炎症既是KOA的诱发因素,又是其主要的病理表现,在疾病过程中发挥重要作用。本实验ELISA结果发现,紫花前胡苷能够显著降低KOA小鼠血清中炎症因子IL-1β和TNF-α水平(P<.001)(如图3B所示)。膝关节滑膜HE染色发现,模型组滑膜区域炎症细胞浸润,滑膜增生明显,而紫花前胡苷减轻了细胞浸润,两组之间滑膜炎症评分具有显著差异(P<.05)(如图3C、3D所示)。实验结果表明,紫花前胡苷能够降低KOA小鼠血清中炎症因子水平,抑制关节滑膜炎症。
(4)紫花前胡苷抑制KOA小鼠关节软骨p-Drp1、NLRP3表达
线粒体依靠***/融合动态平衡为细胞提供能量,Drp1是促使线粒体***最主要的蛋白分子,而细胞内Drp1大量磷酸化会造成线粒体过度***、碎片化,使线粒体功能紊乱,进而造成细胞损伤。本实验IF染色发现,紫花前胡苷能显著减低关节软骨细胞中p-Drp1表达,抑制线粒体过度***(P<.01)(如图4A、4C所示)。NLRP3炎症小体参与多种病理过程,是诱发炎症反应的重要信号分子。实验结果发现,与模型组比较,紫花前胡苷治疗后关节软骨细胞中NLRP3表达明显降低(P<.01)(如图4B、4D所示)。
(5)紫花前胡苷调控KOA小鼠关节软骨Collagen II、p-Drp1和NLRP3表达
关节软骨中II型胶原减少,与软骨细胞内Drp1磷酸化、线粒体过度***及NLRP3炎症小体激活密切相关。本实验结果证明,紫花前胡苷能够逆转DMM诱导的KOA模型小鼠膝关节关节软骨中II型胶原减少和p-Drp1、NLRP3增多。Western blot发现,模型组与紫花前胡苷组Collagen II、p-Drp1和NLRP3具有显著性差异(P<.05)(如图5A、5B所示)
(6)紫花前胡苷抑制软骨细胞ROS表达
为了探究紫花前胡苷通过Drp1/ROS/NLRP3信号轴干预KOA的疗效分子机制。首先,本实验分析了紫花前胡苷对原代软骨细胞活性的影响,CCK8实验结果发现,紫花前胡苷(5-75μM)对细胞活性无明显影响,浓度继续升高后细胞活性降低(如图6A所示)。然后,本实验选择了75μM浓度进行后续细胞实验。线粒体是ROS最主要的来源,线粒体过度***会造成ROS大量生成,进而对软骨细胞不仅造成氧化应激的直接损伤,还会激活上下游信号通路,恶化软骨细胞损伤的微环境。本实验中ROS探针检测发现,紫花前胡苷组中软骨细胞内ROS明显减少(P<.01)(如图6C、6D所示),说明紫花前胡苷能够减轻软骨细胞氧化应激损伤,改善局部微环境。
(7)紫花前胡苷抑制软骨细胞IL-1β、NLRP3等炎症因子mRNA和蛋白表达
原代软骨细胞验证紫花前胡苷对炎症因子和II型胶原的影响。如图7A所示,紫花前胡苷干预软骨细胞后,LPS诱导的COX2、IL-1β、TNF-αmRNA水平明显降低(P<.01),这与血清ELISA结果一致。提取软骨细胞总蛋白进行Westernblot检测,实验结果发现,LPS诱导软骨细胞p-Drp1、MMP13和NLRP3蛋白表达上升,而II型胶原表达降低,但75μM紫花前胡苷显著抑制了上述现象的发生(P<.001)(如图7B、7C所示)。同时,软骨细胞IF染色结果与Westernblot结果一致(P<.05)(如图8所示)。因此,紫花前胡苷不仅能减少软骨细胞IL-1β、TNF-α等炎症因子产生、减轻滑膜炎症,还能增加II型胶原表达、抑制MMP13表达,减轻细胞外基质降解,从而延缓KOA疾病进程。
本发明采用DMM手术诱导小鼠KOA模型,证实了紫花前胡苷对软骨和软骨下骨有明显的保护作用,并且通过原代软骨细胞验证了紫花前胡苷通过Drp1/ROS/NLRP3信号轴延缓KOA的分子机制。软骨下骨为关节软骨提供稳定的生理环境和机械支持,同时,软骨下骨经历着破骨细胞和成骨细胞调控的骨吸收和形成过程。相关研究表明,由于机械负荷的改变,KOA早期破骨细胞活性和骨吸收显著增加。本实验中micro-CT分析显示,紫花前胡苷可以减轻软骨下骨骨吸收、增加骨小梁的数量、减少骨赘形成,从而逆转软骨下骨病理改变。保护关节软骨是KOA防治最主要的目的,也是评估疾病进程的重要指标,本实验中紫花前胡苷对软骨有明显的保护作用,不仅降低了关节软骨OARSI评分,还增加了细胞外基质中II型胶原的含量。研究结果与众多的实验研究一致,表明紫花前胡苷可通过抑制软骨下骨异常的骨重建来保持与软骨的稳态环境,从而延缓疾病进展。
滑膜炎症普遍存在于KOA病程中,主要表现为滑膜增生、肥厚和炎症细胞浸润。KOA患者滑膜、血清中IL-1β、TNF-α和IL-6等炎症因子水平明显升高,它们不仅抑制软骨细胞合成胶原和蛋白聚糖等基质成分,还参与促进基质金属蛋白酶的合成,如MMP13、MMP3等,从而加速细胞外基质降解。形成炎症因子-基质金属蛋白酶-软骨降解恶性循环的微环境,促进KOA疾病的进展。因此,滑膜炎症通过多种途径参与KOA的发生发展,是抗炎治疗的重要靶点,也是KOA防治研究的热点。本实验中,DMM手术诱导的KOA模型小鼠滑膜部位出现明显的增生肥厚及大量的炎症细胞浸润,体外软骨细胞中COX2、IL-1β和TNF-α等炎症因子mRNA水平升高,紫花前胡苷干预后明显改善了滑膜炎症,降低了滑膜炎症评分,另外,KOA小鼠血清中IL-1β和TNF-α水平也明显降低。同时,体外实验也证明,紫花前胡苷能够抑制软骨细胞MMP13表达且促进II型胶原的合成和分泌。说明紫花前胡苷能通过抑制炎症反应、抑制基质降解发挥延缓KOA作用。
线粒体是细胞的能量中枢,对细胞稳态的维持发挥至关重要的作用。线粒体功能紊乱与多种疾病密切相关,如癌症、神经退行性疾病、退行性关节疾病等。研究表明,线粒体自噬可清除细胞内受损或功能紊乱的线粒体,在骨关节炎的防治中发挥积极作用。Drp1是线粒体***的重要蛋白,与线粒体***/融合平衡稳态密切相关。有研究发现,Drp1过度磷酸化导致的线粒体异常***会激活神经胶质细胞NF-κB信号通路和NLRP3炎症小体,诱发脊髓型颈椎病大鼠炎症反应。另有研究发现,OA患者软骨细胞凋亡和线粒体过度***与细胞中Drp1高表达相关,而抑制ERK1/2激活可以阻断Drp1磷酸化和线粒体过度***。本实验体内体外实验结果均发现,紫花前胡苷显著抑制软骨细胞中Drp1蛋白磷酸化,减少线粒体***和碎片化,维持其正常的生理功能,并调控ROS及其下游NLRP炎症小体的形成,从而减少炎症细胞因子的产生,改善KOA局部微环境。
ROS主要由线粒体产生,存在于所有类型的细胞中,在细胞信号转导、基因调控和细胞周期中充当重要的信使分子。细胞对ROS的反应取决于信号传递的强度、持续时间和性质,以及细胞的氧化还原状态。生理情况下,当细胞中氧化还原状态平衡时,ROS参与细胞中信号传递等多种生理过程。而当细胞中存在大量ROS时,细胞的抗氧化能力不足,出现氧化应激,ROS与DNA、蛋白质和脂质发生反应,并破坏它们的正常结构,损害他们的正常功能而导致细胞毒性反应。氧化应激还能诱导细胞凋亡,使细胞内容物释放到细胞外环境中,含有氧化分子的降解产物和细胞内容物相互影响,形成恶性循环进一步促进ROS生成和细胞成分降解。本实验中,为了检测KOA环境中软骨细胞ROS的生成以及紫花前胡苷对ROS产生的影响,我们使用了mitoSOX探针发现,LPS诱导的软骨细胞ROS增多被紫花前胡苷抑制,紫花前胡苷组中ROS阳性率明显降低。Drp1促使线粒体过度***产生的ROS能够激活下游NLRP3炎症小体和NF-κB信号通路,诱发滑膜炎症反应。我们的Westernblot和IF结果发现,与模型组比较,体内体外实验中紫花前胡苷组NLRP3的蛋白水平及阳性率均明显减低。因此,我们的研究结果表明,抑制Drp1引起的线粒体过度***可以通过调节ROS诱发的氧化应激和下游NLRP3信号分子来缓解DMM手术诱导的KOA小鼠疾病进展。
紫花前胡苷是名老中医经验方加味独活寄生合剂君药独活中主要的香豆素类活性成分,其药理学和分子特性显示:口服生物利用度为(oral bioavailability,OB)57.12%,类药性(drug-likeness)0.69,具有很好的药物转化潜力。本文通过体内体外实验证了其潜在的疗效物质基础,紫花前胡苷能够通过调控软骨细胞Drp1/ROS/NLRP3信号轴减轻KOA模型小鼠软骨下骨破坏吸收、骨赘形成,抑制关节滑膜炎症及保护关节软骨,从而延缓疾病进展,为中药复方的临床疗效提供了证明基础。
15结论
综上所述,软骨细胞中依赖Drp1产生的ROS及其下游NLRP3炎症小体在KOA病程中发挥重要作用。实验证明,紫花前胡苷能够减轻软骨损伤、软骨下骨重建,抑制滑膜炎症从而延缓DMM手术诱导的KOA模型小鼠疾病进展,而Drp1/ROS/NLRP3是其潜在的疗效分子机制。因此,紫花前胡苷是治疗KOA的潜在药物。
以上结合具体实施方式和范例性实例对本申请进行了详细说明,不过这些说明并不能理解为对本申请的限制。本领域技术人员理解,在不偏离本申请精神和范围的情况下,可以对本申请技术方案及其实施方式进行多种等价替换、修饰或改进,这些均落入本申请的范围内。本申请的保护范围以所附权利要求为准。
Claims (6)
1.紫花前胡苷作为唯一活性成分在制备防治骨关节炎的药物中的用途;其中,所述骨关节炎为关节软骨厚度丢失、细胞外基质降解的骨关节炎,紫花前胡苷通过Drp1/ROS/NLRP3信号轴延缓膝骨关节炎。
2.根据权利要求1所述的用途,其特征在于:紫花前胡苷增加软骨中II型胶原的含量。
3.根据权利要求1所述的用途,其特征在于:小鼠动物实验中,紫花前胡苷浓度:10mg/kg,用0.5%羧甲基纤维素钠配置。
4.根据权利要求1所述的用途,其特征在于:软骨细胞实验中,紫花前胡苷浓度:5-150μM,用DMSO配置。
5.根据权利要求1所述的用途,其特征在于:所述紫花前胡苷制成散剂、膏剂、粉剂、水剂、肠溶缓释制剂或注射剂。
6.紫花前胡苷作为唯一活性成分在制备防治骨关节炎的药物中的用途;其中,所述骨关节炎为关节软骨厚度丢失、细胞外基质降解的骨关节炎,所述药物用于Drp1阳性和/或NLRP3阳性的患者。
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