补肾壮筋汤对实验性骨关节病NO和软骨细胞凋亡的影响

目的:探讨补肾壮筋汤对兔膝骨关节病的血清和关节液一氧化氮(nitric oxide,NO)含量,软骨细胞凋亡和增殖的影响及其内在关系.方法:青紫蓝兔,50只分5组,参照Hulth's法造模.7周末测定血清和膝关节液NO值;光镜,透射电镜下观察软骨形态学变化;免疫组化法观察软骨细胞凋亡和增殖情况.结果:血清和关节液NO值有相关性(P＜0.01),模型组NO值和其他所有组比较有统计学意义(P＜0.05);中药组的凋亡细胞指数、增殖指数与模型组比较有统计学意义(P＜0.05).结论:补肾壮筋汤能降低兔膝骨关节病血清和关节液的NO的浓度,减少软骨细胞的凋亡,促进软骨细胞的增殖.     

龟甲胶、鹿角胶调控MKK基因表达促进豚鼠OA软骨细胞增殖的研究

目的通过观察龟甲胶、鹿角胶含药血清对豚鼠膝骨关节炎软骨细胞增殖及其丝裂原活化蛋白激酶激酶(MKK)基因表达的影响,探讨龟甲胶、鹿角胶对膝骨关节炎的治疗机制。方法采用Ⅱ型胶原酶消化法获取3月龄豚鼠膝关节软骨细胞,建立体外培养系,将龟甲胶组、鹿角胶组、盐酸氨基葡萄糖组、对照组4组含药血清分别对其进行干预,采用MTT法检测含药血清干预后软骨细胞增殖情况,荧光定量PCR检测含药血清干预对软骨细胞MKK基因表达的影响。结果 5%含药血清干预72 h后,软骨细胞的MTT的检测结果为龟甲胶组(0.315±0.048)、鹿角胶组(0.236±0.029)、盐酸氨基葡萄糖组(0.190±0.022)、对照组(0.146±0.023),差异有统计学意义(P0.05);荧光定量PCR检测MKK表达量结果为龟甲胶(3.287±0.675)、鹿角胶(2.147±0.204)、盐酸氨基葡萄糖组(1.137±0.123)及对照组(0.627±0.102),差异有统计学意义(P0.05)。结论龟甲胶、鹿角胶对软骨细胞的促增殖作用强于盐酸氨基葡萄糖,这一作用可能与其能有效上调关节软骨细胞MKK的基因表达有关。     

补肾壮筋汤对兔早期实验性膝骨关节炎软骨细胞凋亡及增殖细胞核抗原表达的影响

目的:探讨以补肝益肾为治疗手段的补肾壮筋汤对兔早期膝关节实验性OA的软骨细胞凋亡和增殖细胞核抗原(PCNA)表达的影响。方法:40只青紫蓝兔随机分为4组,每组10只,分别为中药组、假模组、模型组、正常组,参照Hulth法造成早期骨关节炎模型。术后第4周开始,中药组、假模组口服补肾壮筋汤,模型组、正常组口服生理盐水。7周末取兔膝关节软骨,肉眼、光镜、透射电镜观察形态学变化,用原位末端标记法(TUNEL法),免疫组化法分别观察软骨细胞凋亡和PCNA的表达。结果:中药组的软骨细胞凋亡指数小于模型组(P<0.05);而中药组的PCNA表达要高于其他各组(P<0.05)。结论:补肾壮筋汤减少兔早期膝关节实验性OA软骨细胞的凋亡,促进软骨细胞的增殖,对早期OA有治疗作用。     

中药舒筋止痛液经离子导入治疗兔膝骨关节炎

目的:探讨中药舒筋止痛液经离子导入治疗兔膝骨关节炎的疗效和机制。方法:采用前交叉韧带切断 内侧半月板切除方法,构建家兔膝骨关节炎模型。24只骨关节炎家兔随机分为3组,Ⅰ组为药透组,家兔患侧膝关节放置中药舒筋止痛液药垫,应用电脑中频电疗仪进行离子导入治疗;Ⅱ组为阳性对照组,家兔饲服盐酸氨基葡萄糖胶囊;Ⅲ组为空白对照组。分别于治疗前、治疗4周和8周时进行关节周径和关节活动度测量、血清一氧化氮检测、关节软骨细胞凋亡检测等。结果:Ⅰ组和Ⅱ组家兔膝关节周径小于Ⅲ组,关节活动度大于Ⅲ组;Ⅰ组血清一氧化氮含量小于Ⅱ组和Ⅲ组,8周时尤为明显;Ⅰ组和Ⅱ组的软骨细胞凋亡率明显低于Ⅲ组。结论:中药舒筋止痛液配合离子导入对骨关节炎有一定的治疗作用,其作用机制与该疗法抑制软骨细胞凋亡进程和炎性因子分泌有关。     

补骨颗粒含药血清对大鼠软骨细胞凋亡及Trx2信号通路的影响

目的观察补骨颗粒含药血清对体外培养的大鼠膝关节软骨细胞凋亡及对Trx2、ASK1及Caspase3表达的影响,从而探讨补骨颗粒预防骨性关节炎发生发展的作用机制。方法采用两步酶消化法分离培养大鼠软骨细胞,并进行传代培养。应用膜联蛋白V-FITC/PI染色后经流式细胞仪检测软骨细胞的凋亡情况。同时,通过电泳分离蛋白并通过蛋白质印迹分析Trx2、ASK1及Caspase3的表达情况。结果软骨细胞培养15 d左右铺满80%～90%的培养皿,大部分细胞呈梭形。流式细胞检测结果显示空白血清组的细胞凋亡率为22.80%,明显高于含药血清组(P0.05),而20%含药血清组(15.91%)与30%含药血清组(17.93%)的细胞凋亡率又明显低于10%含药血清组(21.58%),各组间比较差异具有统计学意义(P0.05)。蛋白质印迹分析结果显示含药血清组Trx2的表达量都明显增多,以10%含药血清组的表达量最多;空白组与10%含药血清组的ASK1与Caspase3的表达量比20%与30%含药血清组多。结论补骨颗粒可以通过激活Trx2信号通路而抑制软骨细胞的凋亡,从而起到预防骨性关节炎发生发展的作用。     

温阳补肾方含药血清对破骨细胞凋亡率及RANK蛋白的影响

目的:探讨温阳补肾方含药血清对破骨细胞凋亡率及RANK蛋白的影响。方法:体外诱导破骨前体细胞系RAW264.7向破骨细胞分化,分别用温阳补肾方低、中、高剂量含药血清和生理盐水血清干预,通过流式细胞术检测各组凋亡率,Western Blot检测各组RANK蛋白水平。结果:与空白对照组比较,温阳补肾方含药血清可诱导破骨细胞凋亡(P=0.000),降低破骨细胞RANK蛋白的表达(P=0.000)。结论:温阳补肾方含药血清可诱导破骨细胞凋亡,温阳补肾方高剂量效果最佳。     

补肾通络颗粒对膝骨关节炎大鼠血清细胞因子及关节软骨含水率的影响

目的:观察补肾通络颗粒高、中、低剂量对膝骨关节炎大鼠血清白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)水平以及关节软骨含水率的影响。方法:将60只大鼠随机分为空白对照组,模型对照组,补肾通络颗粒高、中、低剂量组,美洛昔康组,每组10只。除空白对照组外,其余各组均予膝关节腔注射木瓜蛋白酶造模,并予药物干预后,用ELISA法测定大鼠血清IL-1β、IL-6、TNF-α的水平,同时光镜下观察关节软骨并检测软骨含水率。结果:实验结束后,根据病理组织学检测发现模型对照组大鼠膝关节软骨退行性改变明显。与空白对照组比较,模型对照组膝骨关节炎大鼠血清IL-1β、IL-6、TNF-α水平及关节软骨含水率均升高(P0.05)。与模型对照组比较,补肾通络颗粒高、中剂量组与美洛昔康组膝骨关节炎大鼠血清IL-1β、IL-6、TNF-α水平均降低(P0.05),且补肾通络颗粒高、中剂量组关节软骨含水率亦较模型对照组降低(P0.05),而补肾通络颗粒低剂量组仅IL-1β、TNF-α水平低于模型对照组(P0.05)。与补肾通络颗粒低剂量组及美洛昔康组比较,补肾通络颗粒高剂量组膝骨关节炎大鼠血清IL-6水平及关节软骨含水率均降低(P0.05),补肾通络颗粒中剂量组仅关节软骨含水率降低(P0.05)。补肾通络颗粒高、中剂量组膝骨关节炎大鼠血清IL-1β、IL-6、TNF-α水平及关节软骨含水率差异均无统计学意义(P0.05)。结论:补肾通络颗粒可能通过调节大鼠膝骨关节炎动物模型血清细胞因子水平及降低关节软骨含水率达到治疗作用,尤以中、高剂量组作用显著。     

传统中医药补肾固筋方对中老年膝骨关节炎患者 IL-1、iNOS水平的影响和临床意义

目的 观察补肾固筋方治疗膝骨关节炎IL4、iNOS水平的研究。方法 实验分两组进行,每组病例各92例,治疗8周,中药组口服补肾固筋方,西药组口服美洛昔康胶囊。收集患者治疗前后关节滑液及空腹血清,采用酶联免疫吸附法检测 IL4、iNOS的水平,观察治疗效果和不良反应。结果 治疗前中药组和对照组IL4、iNOS水平较治疗前显著增高;治疗后中药组关节液和血清IL4、iNOS相比对照组显著降低(P < 0. 05);治疗后两组均未见明显血常规和肝肾功能异常。结论 补肾固筋方在治疗中降低了IL4、iNOS的水平及比值,不良反应少见,提示中药补肾固筋方通过调节细胞因子的表达水平来调控细胞凋亡和减轻软骨破坏,改善膝关节骨关节炎发生及发展。     

消肿止痛合剂对大鼠膝骨性关节炎软骨中AMPK/mTOR信号通路影响的实验研究

目的观察消肿止痛合剂对大鼠膝骨性关节炎软骨中LC3、Beclin1、caspase-9、单磷酸腺苷活化蛋白激酶(p-AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)表达的影响,探讨消肿止痛合剂干预膝骨性关节炎(knee osteoarthritis,KOA)形成的作用机制。方法将80只Wistar大鼠随机分为4组:观察组(消肿止痛合剂组)、对照组(硫酸氨基葡萄糖片组)、模型组、假手术组,每组20只,除假手术外,其余3组通过改良Hulth法行膝骨关节炎造模。药物连续干预8周后,取各组大鼠膝关节软骨,采用ELISA方法检测血清中白细胞介素-6 (IL-6)、基质金属蛋白酶-3(MMP-3)、环氧化酶-2(COX-2)水平,实时荧光定量聚合酶链式反应检测各组软骨中LC3、Beclin1、caspase-9 mRNA的表达情况,蛋白质印迹法(Western blot)检测各组软骨细胞中pAMPK、mTOR蛋白的表达。结果与假手术组比较,模型组IL-6、MMP-3、COX-2水平明显升高,下调LC3、Beclin1、p-AMPK的表达量,上调caspase-9、mTOR的表达量,差异有统计学意义(P0. 05);与模型组比较,观察组、对照组降低IL-6、MMP-3、COX-2水平,显著上调LC3、Beclin1、p-AMPK的表达量,下调caspase-9、mTOR的表达量,差异有统计学意义(P0. 05);其中观察组优于对照组,差异有统计学意义(P0. 05)。结论消肿止痛合剂对KOA关节软骨损伤的保护可能与AMPK/mTOR信号通路相关。     

加味补肾壮筋汤颗粒剂对去卵巢骨质疏松大鼠血清NO、SOD及MDA的影响

目的探讨去卵巢骨质疏松大鼠血清中NO、SOD、MDA的变化及中药加味补肾壮筋汤颗粒剂对其的影响。方法选用6月龄清洁级Wistar雌性大鼠45只,随机分为正常对照组、模型组和中药组,每组15只。模型组和中药组行双侧卵巢切除术,术后正常对照组及模型组分别给予生理盐水67ml·kg-1·d-1,中药组给予加味补肾壮筋汤颗粒混悬液67ml·kg-1·d-1,分别于造模前及造模后3个月进行血清NO、SOD、MDA含量的测定。结果造模后3个月模型组血清中NO和SOD含量明显低于正常对照组,MDA含量明显高于正常对照组,且差异有统计学意义(P<005);而使用加味补肾壮筋汤颗粒剂3个月后血清中NO和SOD的含量明显升高,MDA含量明显降低,且差异与模型组比较有统计学意义(P<005)。结论血清中NO、SOD、MDA可能参与了绝经后骨质疏松症的病理进程,而加味补肾壮筋汤颗粒剂可以调节血清中NO、SOD、MDA含量,这可能是其防治骨质疏松症的机制之一。     

骨髓间充质干细胞向成骨细胞分化中CXCR4蛋白表达的研究

目的 探究中医补肾阴、补肾阳、健脾、补肾健脾不同治法对骨髓间充质干细胞向成骨细胞分化过程中CXCR4蛋白表达的影响,从而为中药防治骨质疏松症提供依据。方法 体外分离培养大鼠BMSCs,将SPF雌性SD大鼠用随机数字表法随机分成6组,依次分别为：正常组、诱导液组、补肾阴组、补肾阳组、健脾组、补肾健脾组。对大鼠进行7 d灌胃,制备含药血清,各组成骨诱导18 d后,进行茜素红染色检测矿化情况,采用酶联免疫吸附法(ELISA)检测上述每组细胞上清液CXCR4、BMP2蛋白表达水平。结果 诱导18 d后,与正常组相比,补肾阳组与补肾健脾组BMP-2含量均显著升高(P<0.01),诱导组、补肾阴组BMP-2含量有所升高,但差异不具有统计学意义(P>0.05);与诱导组相比,补肾健脾组BMP-2含量均显著升高(P<0.01),补肾阴组与补肾阳组BMP-2含量有所升高,但差异无统计学意义(P>0.05),各含药组BMP-2蛋白浓度比较依次为：补肾健脾组>补肾阳组>补肾阴组>健脾组;与正常组相比,各组CXCR4含量均显著升高(P<0.01),且补肾健脾组上...     

单味中药对膝骨性关节炎软骨细胞凋亡的作用述略

目的 粗略汇总单味中药对膝骨性关节炎软骨细胞凋亡的文献,探究单味中药其作用机理。方法 检索1990年至2015年期间CNKI,维普,pubmed数据库上有关单味中药对膝骨性关节炎软骨细胞凋亡的国内外中英文文献,从中筛选出所发表期刊具有影响力的、所选中药临床常用并具有代表性的、研究量大且具有针对性的文献,将筛选出的文献进行分类,总结提炼出核心结论并进行分析。结果 国内外已经存在大量研究单味中药作用于软骨细胞机制的文献。国内文献多根据中医对本病的认识,辨证虚实,对应中药性味施治,集中研究单味中药的临床疗效研究。国外的中医理论知识薄弱,将中药抽离中医基础,更关注中药内主要有效成分,注重有效成分在软骨细胞中的作用靶点,故对其机理研究较多。其中大部分中药的治疗作用与调节软骨细胞的凋亡密切相关。结论 总体上,国内外承认中药对本病的有效作用,大量研究认为中药能够抑制软骨细胞的凋亡,对软骨细胞的增殖有促进作用,可以通过不同途径改善软骨损伤。     

补肾健骨方含药血清对ROBs和rBMSCs增殖、分化和矿化的影响

目的观察补肾健骨方含药血清对大鼠颅骨成骨细胞(rat calvarial osteoblasts,ROBs)和大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,r BMSCs)细胞活性及分化、矿化的影响。方法制备补肾健骨方含药血清,将ROBs和r BMSCs分为5%、10%、15%、20%、25%浓度含药血清组、无药血清组及传统诱导组,分别对各组ROBs和r BMSCs进行成骨诱导。MTT法检测ROBs和r BMSCs细胞的增殖情况;用试剂盒检测ROBs和r BMSCs的碱性磷酸酶(ALP)活性;茜素红染色法检测矿化结节形成。结果不同浓度含药血清组与空白无药血清组、传统诱导组相比,均不同程度地促进ROBs和r BMSCs的增殖,使ALP活性提高,增加钙化结节数量和面积。其中含药血清浓度为20%时促进ROBs增殖分化作用最佳,浓度为10%时促进r BMSCs增殖分化效果最为显著。结论不同浓度补肾健骨方含药血清均能够促进ROBs和r BMSCs的成骨增殖、分化和矿化,分别在浓度为20%和10%时最佳。     

Rat Chondrocyte-Associated Antigen Identified as Sialylated Transmembrane Protein Tmp21 Belonging to the p24 Protein Family

Rabbit serum produced after transplantation of isolated rat chondrocytes [sensitized rabbit serum (SRS)] demonstrated M _r ~ 74- and ~23-kDa (western blot analysis) antigens in rat chondrocyte extracts. Only the latter remained after reduction in 2-mercaptoethanol. Protein sequence analysis of 23-kDa chondrocyte-associated antigen (CAA) revealed that it corresponds to transmembrane Tmp21 protein belonging to the p24 protein family. These proteins mainly participate in the traffic between the endoplasmic reticulum and Golgi complex and in some cells appear also in the membrane of secretory granules and plasmalemma. Tmp21 extracted from chondrocytes was sialylated and ceased to bind SRS after deglycosylation. A previous study from our laboratory indicated that expression of CAA, now identified as sialylated Tmp21, decreased in cultured chondrocytes concomitantly with the decline of collagen type II and aggrecan and the rise of collagen type I and versican expression. Since the sialylated form of Tmp21 (also known as emp24) was not described in other tissues and seems to be specific for chondrocytes, we assume that CAA may be considered a chondrocyte differentiation antigen.     

Osteoarthritic cartilage fibrillation is associated with a decrease in chrondrocyte adhesion to fibronectin

OBJECTIVE: Cartilage destruction in osteoarthritis (OA) is generally accepted as a failed repair process. Cell adhesion is implicated in tissue repair. Therefore, adhesion of OA chondrocytes to extracellular matrix proteins was investigated. DESIGN: Using chondrocytes from human OA femoral head cartilage, adhesion to fibronectin and type II collagen of cells from distinct areas showing an intact cartilage surface or a fibrillated cartilage surface was studied. Modulation of chondrocyte adhesion by both protein kinase C (PKC) inhibitors and glucosamine sulfate (GS) was also investigated. RESULTS: A significant (P < 0.05) decrease in adhesion to fibronectin of chondrocytes from fibrillated cartilage, relative to those from grossly normal OA cartilage, was demonstrated. Adhesion to type II collagen was not modified by the chondrocyte origins (either from normal or fibrillated OA cartilage). Adhesion to fibronectin of cells from grossly intact cartilage was decreased by the addition of PKC and calmodulin-dependent kinase inhibitors, W7 and sphingosine, to the cell culture. Adhesion to fibronectin of chondrocytes from fibrillated cartilage was significantly (P < 0.05) increased after glucosamine sulfate treatment. CONCLUSION: Fibrillation of cartilage from OA femoral head is associated with a defective adhesion of chondrocytes to fibronectin. The process is suggested to be dependent of PKC and/or calmodulin-dependent kinases and potentially reversible. Conceivably, it could play a role in OA cartilage destruction.     

Transplants of rat chondrocytes evoke strong humoral response against chondrocyte-associated antigen in rabbits

Rat chondrocytes transplanted intramuscularly in rabbits produced cartilage. In 1-day-old transplants chondrocytes remained viable. After 1 week peripheral chondrocytes of the transplant were dead and the cartilage was surrounded and resorbed by macrophages. In 2-week-old transplants cartilage deteriorated and was invaded by fibroblast-like cells and macrophages. Sera of rabbits that received two or three consecutive transplants of rat chondrocytes with 2-week intervals contained high titer of antichondrocyte cytotoxic antibodies. A part of the cytotoxic activity could be removed by absorption with rat splenocytes. Western blot analysis of lysates from fresh or 24-h cultured chondrocytes with absorbed sera detected antigen with M(r), of approximately 74 and approximately 23 kDa. Only the latter remained after reduction in 2-mercaptoethanol. In lysates of fibroblasts and endotheliocytes the 23-kDa antigen was not found but the serum reacted with M(r) 39-kDa antigen. In lysates of thymocytes a weak band corresponding to M(r) of 35 kDa was present. Serum from rabbits receiving transplants of living chondrocytes followed by chondrocytes suspended in complete Freund's adjuvant contained antibodies directed against components of crude collagenase used for cell isolation. Such antibodies could not be detected in sera of rabbits receiving transplants of living chondrocytes only. Molecular weight of detected antigen differs from that of collagen type II, core of aggrecan, link proteins, and several other macromolecules of cartilage matrix. It could represent either a component of chondrocyte membrane or a membrane-bound substance resistant to enzymes used for isolation. Availability of antibodies against presumably chondrocyte-specific antigen produced during transplant rejection may help to characterize it more precisely and to ascertain whether its presence may influence results of autogenous chondrocyte transplants in humans.