Objective To investigate the mechanism by which Bushen Zhuangjin Decoction (BZD) regulates the SDF-1/CXCR4 axis to promote the homing of bone marrow mesenchymal stem cells (BMSCs) and protect articular cartilage in mice, thereby providing experimental basis for the rehabilitation treatment of knee osteoarthritis (KOA). Methods (1) In the animal experiment, 30 SPF male C57BL/6 mice, 8 weeks old, were selected and randomly divided into sham group, model group and BZD group, with 10 mice in each group. Intervention in each group lasted for 12 weeks. The morphological changes of the cartilage in each group were observed by micro-CT and hematoxylin-eosin staining. The fluorescence intensity of SDF-1 in bone tissue was observed by laser confocal microscopy. qPCR and Western blot were used to detect mRNA transcription level and protein relative expression level of homing-related regulatory factors in each group. (2) In the cell experiment, 4-week-old SPF male C57BL/6 mice were selected and the primary BMSCs were extracted by the whole bone marrow adherence method. After the extracted cells were identified by flow cytometry, the optimal lentivirus MOI value was screened. The cells were randomly divided into five groups: blank group, empty vector group, BZD group, sh-SDF-1 group and sh-SDF-1+BZD group. The migration of BMSCs in each group was observed by cell scratch test. The chondrogenic differentiation ability of cells in each group was observed by immunocytological staining of Collagen Ⅱ and Alcian blue. The fluorescence intensity of SDF-1 and CXCR4 in each group was observed by confocal laser microscope. mRNA transcription level of homing-related regulatory factors were detected by qPCR. Results (1) In the animal experiment, the joint histomorphologic findings (micro-CT and hematoxylin-eosin staining) showed that compared with the sham group, there was a circular defect between the femoral condyles, with cortical separation and loss of chondrocytes in the model group (P<0.05); compared with the model group, the annular defect between the femoral condyles was improved and the arrangement of chondrocytes was slightly disordered in the BZD group. The immunofluorescence staining of joint tissues showed that compared with the sham group, the relative expression of SDF-1 protein increased in the model group (P<0.05); compared with the model group, the relative expression level of SDF-1 increased in the BZD group (P<0.05). qPCR and Western blot results showed that compared with the sham group, the mRNA transcription level and protein relative expression level of key homing regulatory factors (SDF-1, CXCR4, MIP-1α, MCP-1, MIP-1β, RANTES, VEGF, G-CSF, NCAM-1, MMP-2) increased in the model group (P<0.05); compared with the model group, the mRNA transcription level and relative protein expression level of key homing regulatory factors increased in the BZD group (P<0.05). (2) In the cell experiment, BMSCs were identified by flow cytometry: CD44 and CD105 were positively expressed, while CD34 was negatively expressed. When the MOI value was 100, the infection rate of SDF-1 gene was the highest. The results of BMSCs migration and chondrogenic differentiation showed that the number of cells migrating to the scratched area, the relative expression of Collagen Ⅱ protein, and acid mucosaccharide of sh-SDF-1 cells were significantly reduced compared with the blank group. The number of migrated cells, Alcian blue staining and the relative expression of Collagen Ⅱ protein significantly increased in the BZD group and the sh-SDF-1+BZD group (P<0.05). Immunofluorescence finding showed that compared with the blank group, the relative protein expression of SDF-1 and CXCR4 in the sh-SDF-1 group was significantly reduced (P<0.05), while the relative protein expression of SDF-1 and CXCR4 significantly increased in the BZD group and the sh-SDF-1+BZD group (P<0.05). qPCR results showed that compared with the blank group, the mRNA transcription level of key homing regulatory factors decreased in the sh-SDF-1 group (P<0.05), while those increased in the BZD group (P<0.05). Conclusion BZD can promote the homing of BMSCs to protect articular cartilage in mice by upregulating the SDF-1/CXCR4 axis.

From the perspective of lncRNA MALAT1 regulating cholesterol metabolism in chondrocytes, this paper explores the effect and mechanism of Tougu Xiaotong Capsules(TGXTC) in delaying the degeneration of osteoarthritis. After one week of adaptive feeding, 48(8-week-old) C57BL/6 mice were randomly divided into a blank group(12 mice) and a model group(36 mice) by random number table method. The mice in the model group were anesthetized by inhalation of 5% isoflurane, and the OA model was induced by Hulth method. The experiment randomly divided the mice into a model group(12 mice), a drug-positive group(taururso-deoxycholic acid)(12 mice), and a TGXTC group(12 mice). The drug-positive group was given 500 mg·kg~(-1) taurodeoxycholic acid by intragastric administration. TGXTC group was given TGXTC 368 mg·kg~(-1) by gavage. The blank group and model group were given the same amount of normal saline for four weeks. After the intervention, the mice in each group were killed under anesthesia, and the knee cartilage tissue was separated and collected. The morphologic changes of knee cartilage were observed. The level of lncRNA MALAT1 in the cartilage tissue was detected by real-time PCR. The protein expressions of ABCA1, ApoA1, LXRβ, CHOP, and caspase-3 in mouse articular cartilage were detected by Western blot. Lentivirus-coated plasmid was used to transfect mouse chondrocytes with sh-MALAT1. The gene levels of lncRNA MALAT1 in mouse chondrocytes transfected with sh-MALAT1 were detected by real-time PCR. Western blot was used to detect the effect of TGXTC on the protein content of ABCA1, ApoA1, LXRβ, CHOP, and caspase-3 in thapsigargin(TG)-induced mouse chondrocytes after lncRNA MALAT1 knockdown. Flow cytometry was used to detect the effect of TGXTC on apoptosis of TG-induced mouse chondrocytes after lncRNA MALAT1 knockdown. The results of HE and saffranine O staining showed that compared with the model group, the structure of the cartilage layer was basically intact; the damage degree of joint structure was significantly improved, and the cartilage matrix was significantly enhanced by saffranine O staining in the TGXTC group and drug-positive group. Compared with the model group, the lncRNA MALAT1 level was significantly decreased in the TGXTC group and drug-positive group. Compared with the model group, the protein content of ABCA1, ApoA1, and LXRβ was significantly increased, while that of CHOP and caspase-3 in the TGXTC group and drug-positive group significantly decreased. Compared with the TG group, the lncRNA MALAT1 level in the TG+sh-MALAT1 group was decreased. The lncRNA MALAT1 level in the TG+sh-MA-LAT1+TGXTC group was increased compared with the TG+TGXTC group. Western blot results showed that compared with the model group, protein expressions of ABCA1, ApoA1, LXRβ, CHOP, and caspase-3 in the TGXTC group were significantly decreased, after lncRNA MALAT1 knockdown, the regulation and apoptosis of ABCA1, ApoA1, LXRβ, CHOP, and caspase-3 in TG-induced mouse chondrocytes were weakened by TGXTC. TGXTC can improve the disorder of cholesterol metabolism in OA chondrocytes and delay OA degeneration, which is closely related to the regulation of lncRNA MALAT1.

目的:从分子水平可视化探讨丹参和降香配伍在改善骨关节炎炎症微环境中的协同作用.方法:①从北京大学天然产物库中,检索180个丹参化学成分和119个降香化学成分,构建各自的化学成分数据集;从化学结构角度进行聚类分析,并计算两者的全局指纹相似度分值.②依托定量构效关系及主成分分析平台,分析丹参和降香化学成分数据集的化学空间.③以白细胞介素1β(Interleukin-1β,IL-1β)、IL-6、肿瘤坏死因子α(Tumor necrosis factor-α,TNF-α)、基质金属蛋白酶1(Matrix Metallopeptidase 1,MMP-1)、MMP-3、MMP-9和MMP-13为改善骨关节炎炎症微环境的靶点,利用分子对接和生物网络等平台,研究其与丹参、降香中化学成分的相互作用,并构建丹参和降香的化合物-靶点网络,分析丹参和降香配伍在改善骨关节炎炎症微环境中的意义.结果:①对丹参和降香化学成分数据集进行聚类,发现丹参和降香化学成分数据集在第1、2、3、4、5、6、8、9、10类存在交集,但在第7类仅出现丹参化学成分;同时,对两个数据集的全局指纹进行比较,发现其相似度分值为0.637 5.②丹参和降香化学成分数据集在化学空间的后底部存在相近的分布.③丹参化合物-靶点网络显示,丹参的潜在活性化合物个数达到32个,其核心化合物为酚酸类成分,核心作用靶点为IL-1β、IL-6、TNF-α、MMP-1和MMP-9;降香化合物-靶点网络显示,降香中的潜在活性化合物个数达到64个,其核心化合物为黄酮类成分,核心作用靶点为IL-1β、IL-6、TNF-α、MMP-1、MMP-3和MMP-9.结论:从化学结构特征及化学空间分布看,丹参和降香存在相似之处,配伍后具有更多的潜在活性成分种类和数量、更广的化学空间分布,可通过对应的特定靶点和共同靶点在改善骨关节炎炎症微环境中相互作用发挥互补协同效应.即计算机模拟实现丹参和降香配伍协同改善骨关节炎炎症微环境作用的可视化分析.

目的:观察荣筋拈痛方对膝骨关节炎大鼠软骨组织自噬的影响,探讨其防治膝骨关节炎的作用机制.方法:将24只SD大鼠按照随机分层法分为假手术组、模型对照组、荣筋拈痛方组,每组8只.除假手术组外,其他组采用改良Hulth法复制膝骨关节炎模型.给药12周后,从假手术组和模型对照组中随机抽取3只大鼠拍摄膝关节MRI,观察模型是否成功.采集大鼠膝关节组织,HE染色法观察软骨组织形态结构,采用Mankin病理评分对关节软骨进行组织形态学评分;免疫组织化学法观察软骨组织酵母自噬相关基因6同源物(Beclin1)及微管相关蛋白Ⅰ轻链3B(LC3B)的定位与表达.结果:MRI显示,假手术组关节间隙正常,关节面平整;模型对照组关节间隙增宽,关节软骨面不平整、局部缺损,结缔组织增生,骨赘形成.荣筋拈痛方组大鼠骨关节炎模型关节软骨的大体外观及HE染色均改善明显,Mankin软骨评分高于模型对照组,差异有统计学意义(P<0.01).荣筋拈痛方组自噬蛋白Beclin1及LC3B表达均呈上升趋势.结论:荣筋拈痛方可能通过提高自噬相关蛋白Beclin1、LC3B的表达,改善软骨细胞状态,延缓关节软骨退变.

背景:前期研究发现荣筋拈痛方能有效抑制软骨基质降解.长链非编码RNA GAS5可作为竞争性内源性RNA吸附miR-21-5p调控基质金属蛋白酶的表达,进而影响关节软骨基质代谢.但荣筋拈痛方是否通过长链非编码RNA GAS5/miR-21调控软骨基质代谢,发挥防治骨关节炎的作用机制有待深入研究.目的:从长链非编码RNA GAS5/miR-21调控软骨基质合成与分解代谢角度,探讨荣筋拈痛方治疗SD大鼠膝骨关节炎的作用机制.方法:SPF级8周龄雄性SD大鼠60只,随机分为4组,即空白组、模型组、治疗组及对照组,每组15只.后3组采用改良Hulth法建立大鼠膝骨关节炎模型.造模后2周,空白组和模型组予生理盐水,治疗组予荣筋拈痛方汤剂,对照组予盐酸氨基葡萄糖胶囊水溶液,每天灌胃一次,共干预12周.药物干预结束后取材,采用苏木精-伊红染色、改良番红O-固绿染色观察软骨组织显微结构形态变化,实时荧光定量PCR法检测长链非编码RNA GAS5、miR-21的表达及基质代谢因子mRNA的表达,Western blot法检测软骨组织中基质代谢因子蛋白的表达.结果 与结论:①软骨显微结构观察,模型组软骨4层结构紊乱,排列不规律,潮线及黏合线不规则或消失,软骨细胞增生肥大且排列紊乱,软骨下骨增生,胶原、蛋白多糖大量丢失;治疗组和对照组软骨4层结构、潮线可辨,软骨细胞排列较规律,软骨下骨结构较完整,胶原、蛋白多糖部分丢失;②与空白组相比,模型组软骨组织中长链非编码RNA GAS5及基质金属蛋白酶3、基质金属蛋白酶9、基质金属蛋白酶13、血小板反应蛋白解整合素金属肽酶5 mRNA表达量升高(P<0.01或P<0.05),治疗组、对照组与模型组相比表达量降低(P<0.01或P<0.05);模型组软骨组织中miR-21及组织金属蛋白酶抑制因子3、Ⅱ型胶原和聚集蛋白聚糖mRNA表达量降低(P<0.01或P<0.05),治疗组、对照组与模型组相比表达量升高(P<0.05);③与空白组相比,模型组软骨组织中组织金属蛋白酶抑制因子3、Ⅱ型胶原和聚集蛋白聚糖蛋白表达降低(P<0.01或P<0.05),治疗组和对照组中表达升高(P<0.01或P<0.05);模型组软骨组织中基质金属蛋白酶3、基质金属蛋白酶9、基质金属蛋白酶13、血小板反应蛋白解整合素金属肽酶5蛋白表达升高(P<0.01或P<0.05),治疗组和对照组中表达降低(P<0.01或P<0.05);④提示荣筋拈痛方下调膝骨关节炎大鼠软骨组织中长链非编码RNA GAS5、基质金属蛋白酶3、基质金属蛋白酶9等的表达,上调miR-21、组织金属蛋白酶抑制因子3、Ⅱ型胶原和聚集蛋白聚糖的表达,影响软骨基质分解和合成代谢,减少软骨基质中胶原、蛋白多糖的丢失,延缓软骨基质降解,减轻软骨组织形态结构的破坏.

目的 基于lncRNA NEAT1和IRE1α/XBP1信号通路,探讨透骨消痛胶囊延缓膝骨关节炎(KOA)软骨退变的机制.方法 48只8周龄小鼠适应性喂养1周后,采用随机数字表法分为空白组12只、造模组36只,造模组动物经5%异氟醚吸入麻醉后,采用Hulth法诱导KOA模型,随机数字表法分为模型组12只、透骨消痛胶囊组12只和对照组12只.透骨消痛胶囊组予以透骨消痛胶囊 368 mg/kg进行灌胃;对照组使用牛磺熊去氧胆酸500 mg/kg进行灌胃;空白组和模型组给予等量生理盐水灌胃,连续4周.干预结束后,麻醉状态下处死各组小鼠,分离与收集膝关节软骨组织,Micro-CT观察小鼠膝关节软骨组织形态变化;Western blot检测关节软骨IRE1α/XBP1通路相关蛋白表达;qPCR检测软骨组织中lncRNA NEAT1表达水平.结果 与空白组比较,模型组半月板结构缺失,胫骨平台软骨面呈现凹凸不平状、伴有明显的骨赘增生,软骨组织IRE1α、XBP1、GRP78、CHOP蛋白含量、lncRNA NEAT1表达均显著升高(P＜0.05);透骨消痛胶囊组、对照组小鼠内侧半月板结构缺失,2组软骨组织的IRE1α、XBP1、GRP78、CHOP蛋白含量均显著降低(P＜0.05),lncRNA NEAT1表达水平均显著降低(P＜0.05).结论 透骨消痛胶囊可通过下调KOA小鼠关节软骨组织中lncRNA NEAT1水平,抑制软骨组织与内质网应激有关的IRE1α/XBP1通路蛋白表达发挥作用.

Endoplasmic reticulum stress (ERS) has been identified to be an important factor leading to chondrocyte apoptosis in osteoarthritis (OA). Previous studies have confirmed that Achyranthes bidentata polysaccharides (ABPS) can inhibit chondrocyte apoptosis; however, the mechanism of action of ABPS on chondrocyte ERS remains unclear. Thus in this study, we aim to investigate whether ABPS could inhibit OA-associated chondrocyte apoptosis by regulating ERS, especially by observing the relationship between the lncRNA NEAT1 and miR-377-3p, to explore further the protective mechanism of ABPS in OA. In vitro and in vivo experiments showed that ABPS inhibited chondrocyte ERS by regulating the expression of lncRNA NEAT1 and miR-377-3p. Moreover, both lncRNA NEAT1 silencing and miR-377-3p inhibition could attenuate the therapeutic effect of ABPS on ERS. Dual-luciferase results indicated that miR-377-3p targets the lncRNA NEAT1 gene in mouse chondrocytes. Therefore, we concluded that ABPS could inhibit thapsigargin (TG)-induced chondrocyte ERS through the lncRNA NEAT1/miR-377-3p axis.

目的:探讨荣筋拈痛方调控NOD样受体热蛋白结构域相关蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白酶1(caspase-1)/消皮素D(GSDMD)通路减轻软骨细胞焦亡,改善骨关节炎炎性病变的作用机制.方法:将二代软骨细胞随机分为正常组、模型组[脂多糖(LPS)+三磷酸腺苷(ATP)]、荣筋拈痛方组(LPS+ATP+荣筋拈痛方)和抑制剂组(LPS+ATP+MCC950)oHoechst 33342/PI双染检测软骨细胞膜的完整性;双转盘活细胞共聚焦实时成像分析系统观察NLRP3和caspase-1的定位与表达;ELISA检测白细胞介素(IL)-1 β和IL-18的水平;RT-PCR检测NLRP3、caspase-1、GSDMD、Ⅱ 型胶原蛋白(collagen Ⅱ)和聚集蛋白聚糖(aggrecan)mRNA水平;Western Blot检测NLRP3、pro-caspase-1、caspase-1、GSDMD、GSDMD-N、collagen Ⅱ、aggrecan、基质金属蛋白酶(MMP)-3和MMP-13蛋白表达.结果:与模型组比较,荣筋拈痛方可降低因细胞膜完整性丧失而染上的PI红色荧光强度;降低NLRP3和caspase-1蛋白表达的绿色荧光强度(P＜0.01);降低软骨细胞上清液中炎症因子IL-1 β和IL-18水平(P＜0.01);降低NLRP3、caspase-1、GSDMD mRNA水平(P＜0.05,P＜0.01),提高collagen Ⅱ和aggrecan mRNA水平(P＜0.01);降低NLRP3、pro-caspase-1、caspase-1、GSDMD、GSDMD-N、MMP-3和MMP-13蛋白表达(P＜0.05,P＜0.01),提高collagen Ⅱ和aggrecan蛋白表达(P＜0.05).结论:荣筋拈痛方可通过调控软骨细胞NLRP3/caspase-1/GSDMD途径,减轻因细胞焦亡而导致的炎症反应,改善骨关节炎炎性病变.

目的：观察荣筋拈痛方对白细胞介素-1β（IL-1β）诱导的体外大鼠软骨细胞自噬相关基因表达的影响，探讨其防治膝骨关节炎的作用机制。方法：取4周龄SD大鼠膝关节软骨，进行软骨细胞的体外分离培养，选取第2代软骨细胞甲苯胺蓝染色进行细胞鉴定。经IL-1β 10 ng·mL -1 诱导建立体外软骨细胞炎症模型，诱导成功后，给予荣筋拈痛方(50，100，200 μg·mL -1 )孵育48 h。将细胞分为空白对照组，模型对照组，荣筋拈痛方低、中、高剂量组。分别采用CCK-8法检测荣筋拈痛方对软骨细胞活性的影响，qPCR法检测软骨细胞中LC3Ⅱ、Beclin 1的mRNA表达，Western Blot法观察自噬蛋白LC3Ⅱ/Ⅰ比值、Beclin 1的蛋白表达水平。结果：与模型对照组比较，荣筋拈痛方低、中、高剂量组均显著提高细胞活力，呈剂量依赖性增加（P ＜ 0.01）；模型对照组较空白对照组LC3Ⅱ、Beclin 1的mRNA及LC3Ⅱ/Ⅰ、Beclin 1的蛋白表达水平升高（P ＜ 0.05）；经100 μg·mL -1 荣筋拈痛方干预后LC3Ⅱ、Beclin 1的mRNA及LC3Ⅱ/Ⅰ、Beclin 1的蛋白表达水平降低（P ＜ 0.05）。结论：荣筋拈痛方可能通过抑制自噬水平，从而具有延缓膝骨关节炎退变的作用。

目的:观察荣筋拈痛方对白细胞介素-1β(IL-1β)诱导的体外大鼠软骨细胞自噬相关基因表达的影响,探讨其防治膝骨关节炎的作用机制.方法:取4周龄SD大鼠膝关节软骨,进行软骨细胞的体外分离培养,选取第2代软骨细胞甲苯胺蓝染色进行细胞鉴定.经IL-1β10 ng·mL-1诱导建立体外软骨细胞炎症模型,诱导成功后,给予荣筋拈痛方(50,100,200μg·mL-1)孵育48 h.将细胞分为空白对照组,模型对照组,荣筋拈痛方低、中、高剂量组.分别采用CCK-8法检测荣筋拈痛方对软骨细胞活性的影响,qPCR法检测软骨细胞中LC3Ⅱ、Beclin 1的mRNA表达,Western Blot法观察自噬蛋白LC3Ⅱ/Ⅰ比值、Beclin 1的蛋白表达水平.结果:与模型对照组比较,荣筋拈痛方低、中、高剂量组均显著提高细胞活力,呈剂量依赖性增加(P<0.01);模型对照组较空白对照组LC3Ⅱ、Beclin 1的mRNA及LC3Ⅱ/Ⅰ、Beclin 1的蛋白表达水平升高(P<0.05);经100μg·mL-1荣筋拈痛方干预后LC3Ⅱ、Beclin 1的mRNA及LC3Ⅱ/Ⅰ、Beclin 1的蛋白表达水平降低(P<0.05).结论:荣筋拈痛方可能通过抑制自噬水平,从而具有延缓膝骨关节炎退变的作用.

骨关节炎病位在体为筋骨,在脏为肝肾,两者相辅相成;肝肾相济则骨坚筋强,肝肾不足则骨痿筋弱.研究表明临床运用补肝肾、强筋骨类中药,可促进软骨细胞增殖,延缓软骨退变,但其具体作用机制尚不明确.故以补肝肾、强筋骨类中药及相关活性成分促进软骨细胞增殖为切入点,查阅文献并综述认为,其作用机制可能与促进细胞外基质合成、调控细胞周期相关蛋白、促进转化生长因子-β相关受体表达、调控细胞外调节激酶、Wnt信号通路及miRNA有关,可为进一步研究中药靶向治疗骨关节炎提供思路.

目的 基于PERK通路探讨荣筋拈痛方(RJNTD)对毒胡萝卜素(TG)诱导的软骨细胞内质网应激反应的抑制作用.方法 将30只4周龄SPF级雄性C57BL/6小鼠处死后,体外分离双膝软骨细胞置于低糖DMEM培养基培养,经Ⅱ型胶原免疫细胞化学染色法鉴定后将软骨细胞分成空白组、对照组、模型组和中药组.空白组常规培养4 h,对照组常规培养4 h后更换300μg/mL RJNTD培养液培养12 h,模型组用25μmol/L TG培养液培养4 h后改为常规培养,中药组用25μmol/L TG培养液培养4 h后更换300μg/mL RJNTD培养液培养12 h.干预后采用Real-time PCR检测各组软骨细胞中miRNA-377-3p相对表达水平;Western blot检测各组软骨细胞中内质网降解增强子(EDEM1)、蛋白激酶R样内质网激酶(PERK)、转录激活子4(ATF4)、免疫球蛋白重链结合蛋白(BIP)与DNA损伤诱导基因153(GADD153)蛋白表达水平.结果 对照组各指标与空白组比较均无统计学意义(P均>0.05);与空白组比较,模型组及中药组miRNA-377-3p相对表达水平显著降低(P<0.05),且中药组较模型组降低更不明显(P<0.05);与空白组比较,模型组及中药组EDEM1、PERK、ATF4、BIP、GADD153蛋白表达水平均升高(P均<0.05),且中药组上述各指标较模型组升高更不明显(P均<0.05).结论 RJNTD可通过PERK通路抑制内质网应激反应,进而发挥延缓TG诱导的软骨细胞退变作用,其潜在作用机制可能与调节miRNA-377-3p相关.

目的:探讨独活寄生汤治疗大鼠膝骨关节炎(knee osteoarthritis,KOA)的作用机制.方法:从66只大鼠中随机选取48只,采用改良Hulth法建立大鼠KOA模型,随机选择3只正常大鼠和3只建模大鼠进行鉴定.证实建模成功后,将剩余的15只正常大鼠纳入正常对照组,采用随机数字表将剩余的45只KOA模型大鼠分为KOA模型组、独活寄生汤治疗组、盐酸氨基葡萄糖治疗组,每组15只.正常对照组、KOA模型组、独活寄生汤治疗组、盐酸氨基葡萄糖治疗组大鼠均按每天10 mL·kg-1分别以0.9％生理盐水、0.9％生理盐水、独活寄生汤(浓度为0.93 g·mL-1)、盐酸氨基葡萄糖溶液(浓度为0.015 g·mL-1)灌胃,连续干预12周.12周后,切取大鼠双侧胫骨近端和股骨下端的软骨组织.采用实时定量PCR检测大鼠胫骨近端软骨组织中miR-675-3p、长链非编码RNA(long non-coding RNA,lncRNA)H19及聚集蛋白聚糖(Aggrecan)mRNA、Ⅱ型胶原蛋白(CollagenⅡ)mRNA的表达量,采用蛋白印迹法检测大鼠股骨远端软骨组织中Aggrecan、CollagenⅡ的蛋白表达量.结果:①大鼠饲养结果.KOA模型组大鼠因肺栓塞死亡2只,独活寄生汤治疗组大鼠因腹泻死亡1只,盐酸氨基葡萄糖治疗组大鼠因肺栓塞死亡1只.②大鼠胫骨近端软骨组织中KOA相关基因的RNA表达结果.正常对照组、KOA模型组、独活寄生汤治疗组、盐酸氨基葡萄糖治疗组大鼠胫骨近端软骨组织中miR-675-3p、lncRNA H19、Aggrecan mRNA、CollagenⅡmRNA的表达量比较,组间差异均有统计学意义(F=83.050,P=0.000;F=436.003,P=0.000;F=129.212,P=0.000;F=135.844,P=0.000).KOA模型组、独活寄生汤治疗组、盐酸氨基葡萄糖治疗组大鼠胫骨近端软骨组织中miR-675-3p、Aggrecan mRNA、CollagenⅡmRNA的表达量均低于正常对照组(miR-675-3p:P=0.000;P=0.003;P=0.002;Aggrecan mRNA:P=0.000;P=0.000;P=0.000;CollagenⅡmRNA:P=0.000;P=0.000;P=0.000);KOA模型组大鼠胫骨近端软骨组织中lncRNA H19的表达量低于正常对照组(P=0.002),独活寄生汤治疗组、盐酸氨基葡萄糖治疗组大鼠胫骨近端软骨组织中lncRNA H19的表达量与正常对照组比较,差异无统计学意义(P=0.058,P=0.069);独活寄生汤治疗组、盐酸氨基葡萄糖治疗组大鼠胫骨近端软骨组织中miR-675-3p、Aggrecan mRNA、CollagenⅡmR-NA的表达量均高于KOA模型组(miR-675-3p:P=0.000;P=0.000;Aggrecan mRNA:P=0.000;P=0.000;CollagenⅡmRNA:P=0.000;P=0.000);独活寄生汤治疗组大鼠胫骨近端软骨组织中miR-675-3p、lncRNA H19、Aggrecan mRNA、CollagenⅡmR-NA的表达量与盐酸氨基葡萄糖治疗组比较,组间差异均无统计学意义(P=0.922;P=0.778;P=0.857;P=0.904).③大鼠股骨远端软骨组织中软骨组成相关基因的蛋白表达结果.正常对照组、KOA模型组、独活寄生汤治疗组、盐酸氨基葡萄糖治疗组的大鼠股骨远端软骨组织中Aggrecan和CollagenⅡ的蛋白表达量比较,组间差异均有统计学意义(F=58.118,P=0.002;F=16.625,P=0.001).KOA模型组、独活寄生汤治疗组大鼠股骨远端软骨组织中Aggrecan的蛋白表达量均低于正常对照组(P=0.003;P=0.004),盐酸氨基葡萄糖治疗组大鼠股骨远端软骨组织中Aggrecan的蛋白表达量与正常对照组相比,差异无统计学意义(P=0.078),KOA模型组、独活寄生汤治疗组、盐酸氨基葡萄糖治疗组大鼠股骨远端软骨组织中CollagenⅡ的蛋白表达量均低于正常对照组(P=0.000;P=0.028;P=0.018),独活寄生汤治疗组、盐酸氨基葡萄糖治疗组大鼠股骨远端软骨组织中Aggrecan和Colla-genⅡ的蛋白表达量均高于KOA模型组(Aggrecan:P=0.025;P=0.034;CollagenⅡ:P=0.003;P=0.004),独活寄生汤治疗组大鼠股骨远端软骨组织中Aggrecan和CollagenⅡ的蛋白表达量与盐酸氨基葡萄糖治疗组比较,组间差异均无统计学意义(P=0.999;P=0.775).结论:独活寄生汤治疗大鼠KOA的作用机制,可能与其能够上调miR-675-3p、lncRNA H19的表达进而促进Aggrecan、CollagenⅡ的表达有关.

随着全社会人口老龄化的加剧,绝经后女性罹患骨质疏松性骨关节炎的风险亦呈现高发趋势,严重危害中老年人的健康.研究证实血管形成及功能异常是加剧骨质疏松、骨关节炎病理退变的重要事件.H型血管(内皮细胞)在骨与软骨损伤修复中兼具成骨和成血管偶联功能,其形态与功能异常将影响骨质疏松、骨关节炎的损伤修复.铁死亡作为近年来发现的一种新型细胞死亡模式,其主要由铁依赖性脂质过氧化和活性氧诱导损伤引起细胞膜断裂,线粒体变小等改变.而H型血管铁死亡对骨质疏松性骨关节炎病程的影响途径及调节机制尚需进一步探讨,旨在为今后以H型血管铁死亡为切入点进而防治骨质疏松性骨关节炎开辟新方向.

目的:探讨荣筋拈痛方对白细胞介素-1β (IL-1β)诱导退变软骨细胞增殖的作用机制.方法:CCK8法筛选荣筋拈痛方对退变软骨细胞最佳干预条件后,将软骨细胞随机分为正常组、模型组、抑制剂组和荣筋拈痛方组,分组干预后采用激光共聚焦观察各组Ⅱ型胶原蛋白表达;流式细胞术检测各组周期分布情况;RT-PCR与Western Blot检测各组G1/S检测点相关调节因子p16、Cyclin D1、CDK4、pRB基因和蛋白表达.结果:荣筋拈痛方100μg/mL干预退变软骨细胞48h可显著增强软骨细胞活力(P＜0.01).与正常组比较,模型组和抑制剂组Ⅱ型胶原蛋白表达量显著降低(P＜0.01);与模型组和抑制剂组比较,荣筋拈痛方组Ⅱ型胶原蛋白表达量显著增加(P＜0.01).与正常组比较,模型组和抑制剂组G0/G1期比例升高,G2/M期、S期比例显著降低(P＜0.01);与模型组和抑制剂组比较,荣筋拈痛方组G0/G1期比例显著降低,G2/M期、S期比例显著升高(P＜0.01).与正常组比较,模型组和抑制剂组Cyclin D1、CDK4、pRB基因和蛋白表达量显著降低,p16基因和蛋白表达量显著增加(P＜0.01,P＜0.05);与模型组和抑制剂组比较,荣筋拈痛方组Cyclin D1、CDK4、pRB基因和蛋白表达量显著增加,p16基因和蛋白表达量显著降低(P＜0.01).结论:荣筋拈痛方可调控软骨细胞G1/S期的转换,促进Ⅱ型胶原蛋白表达和软骨细胞增殖.

目的 观察独活寄生汤对膝骨关节炎(KOA)大鼠软骨LncRNA GAS5/miR-21及其下游MMP-3、MMP-9、MMP-13和ADAMTS-5的影响,探讨独活寄生汤治疗KOA的作用机制.方法 2月龄SD大鼠66只,适应性喂养1周后,随机分为正常组(18只)和造模组(48只).应用改良Hulth法复制大鼠KOA模型,造模2周后,从正常组和造模组中随机抽取3只大鼠进行小动物MRI观察,鉴定造模是否成功.造模成功后,采用抽签法分为模型组、治疗组和西药组各15只.正常组和模型组给予0.9％生理盐水灌胃,治疗组给予独活寄生汤灌胃,西药组给予盐酸氨基葡萄糖胶囊灌胃.治疗12周后,切取右侧胫骨平台软骨组织,Real-time PCR法检测LncRNA GAS5、miRNA-21、MMP-3、MMP-9、MMP-13、ADAMTS-5基因表达.结果 Real-time PCR结果显示,与模型组相比,独活寄生汤和盐酸氨基葡萄糖胶囊均能抑制LncRNA GAS5、MMP-3、MMP-9、MMP-13、ADAMTS-5基因表达(P<0.05),促进miR-21基因表达(P<0.05),但2组间无统计学意义(P>0.05).结论 独活寄生汤可能通过调控LncRNA GAS5/miR-21,延缓软骨细胞外基质降解,起到治疗KOA的作用.

目的 观察荣筋拈痛方对骨关节炎(OA)大鼠SDF-1/CXCR4信号通路相关调节因子的影响,探讨荣筋拈痛方治疗OA的作用机制.方法 采用4％木瓜蛋白酶注射关节腔复制大鼠OA模型,Ⅱ型胶原酶消化法获取正常滑膜细胞与OA滑膜细胞,正常滑膜细胞即空白组,OA滑膜细胞分为模型组和治疗组.空白组和模型组细胞用10％空白血清培养,治疗组用10％荣筋拈痛方含药血清培养;各组干预24 h后,采用ELISA法检测滑膜细胞上清液中SDF-1含量,Real-time PCR和Western blot法检测滑膜细胞SDF-1 mRNA和蛋白表达.收集的滑膜细胞上清液干预退变软骨细胞24 h后,Real-time PCR和Western blot法检测退变软骨细胞中CXCR4、MMP-3、MMP-9和MMP-13 mRNA和蛋白表达.结果 与空白组比较,模型组大鼠滑膜细胞上清液中SDF-1含量明显增加(P<0.05),大鼠滑膜细胞SDF-1 mRNA和蛋白表达明显升高(P<0.05),退变软骨细胞CXCR4、MMP-3、MMP-9和MMP-13 mRNA和蛋白表达明显升高(P<0.05);与模型组比较,治疗组大鼠滑膜细胞上清液中SDF-1含量明显减少(P<0.05),大鼠滑膜细胞SDF-1 mRNA和蛋白表达明显降低(P<0.05),退变软骨细胞CXCR4、MMP-3、MMP-9和MMP-13 mRNA和蛋白表达明显降低(P<0.05).结论 荣筋拈痛方可通过调控SDF-1/CXCR4信号通路,抑制OA炎症反应,起到治疗OA作用.

目的:基于生物信息学和网络药理学方法,筛选荣筋拈痛方君药牛膝相关活性成分,通过动物体内体外实验观察其对骨关节炎相关治疗靶点的影响.方法:通过中药系统药理学数据库与分析平台(TCMSP)数据库筛选中药牛膝主要活性成分和治疗靶点;通过GEO数据库获得与疾病的差异表达基因,并进行交叉分析;采用实时荧光定量聚合酶链式反应(Real-time PCR),蛋白免疫印迹法(Westemblot)检测牛膝主要活性成分对骨关节炎大鼠体内体外富集治疗靶点的影响.结果:牛膝作用骨关节炎相关活性成分共20种,槲皮素为其中重要成分之一;靶基因共3个,其中骨桥蛋白(OPN),纤维蛋白溶解抑制剂-1(PAI-1)等为该网络中的关键基因;基因本体(GO)分析共获得相关条目227条,涉及到的功能包括创伤生理反应调节(GO:1903034),创伤反应负调节(GO:1903035)等.京都基因与基因组百科全书(KEGG)通路分析获得相关通路12条,涉及细胞外基质受体相互作用(hsa04512)等.动物实验中,与正常组比较,模型组OPN,PAI-1 mRNA和蛋白表达量增加与模型组比较,槲皮素组OPN,PAI-1 mRNA和蛋白表达量降低(P＜0.05).细胞实验中,与正常组比较,模型组OPN,PAI-1蛋白表达量增加,Ⅱ型胶原(CollagenⅡ)蛋白表达量降低;与模型组比较,槲皮素组和抑制剂组OPN,PAI-1蛋白表达量降低,CollagenⅡ蛋白表达量明显增加(P＜0.05).结论:牛膝可能主要通过其活性成分(槲皮素)下调软骨细胞OPN,PAI-1靶mRNA表达,上调CollagenⅡ蛋白表达,实现抑制软骨退变,防治骨关节炎.

细胞焦亡、铁死亡是区别于传统细胞凋亡、坏死、自噬的两种新型细胞程序性死亡方式,两者具有不同的生物学特征,对骨关节炎软骨基质稳态的调控方式、作用特点亦有区别.通过细胞焦亡、铁死亡对骨关节炎软骨基质稳态的影响及可能作用途径进行初步探讨,为进一步防治骨关节炎开辟新方向.