目的:探讨补肾壮筋汤抑制膝骨关节炎软骨退变的作用机制.方法:选取2月龄SPF级雄性SD大鼠共45只,随机数字表法分为空白组(15只)与造模组(30只),空白组行假手术,造模组采用改良Hulth法建立动物模型.造模组随机数字表法分为模型组(15只,0.9％氯化钠溶液灌胃)和补肾壮筋汤组(15只,补肾壮筋汤10.5g·kg-1·d-1灌胃);空白组(15只,0.9％氯化钠溶液灌胃)1次/d,连续干预12周.HE染色观察关节软骨组织形态结构的变化;Mankin's评分法评估关节软骨退变程度;ELISA法检测关节滑膜组织中白细胞介素1 β(IL-1β)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶13(MMP-13)的含量;Western Blot检测软骨组织中解聚蛋白样金属蛋白酶4(ADAMTS-4)、解聚蛋白样金属蛋白酶5(ADAMTS-5)、基质金属蛋白酶3(MMP-3)、MMP-13的表达量.结果:①HE染色显示空白组软骨结构清晰,潮线清晰可见,软骨表面光滑完整,软骨细胞分布均匀,骨小梁宽度和密度适中.模型组结构分辨不清,形态不规整,软骨变薄,潮线模糊,扭曲或中断,骨小梁稀疏.补肾壮筋汤组软骨及软骨下骨的改变介于空白组和模型组之间.②关节软骨的Mankin's评分,模型组显著高于空白组(P＜0.01),补肾壮筋汤组显著低于模型组(P＜0.05).③膝关节滑膜组织中IL-1β、TNF-α、MMP-13的表达量,模型组显著高于空白组(P＜0.05,P＜0.01),补肾壮筋汤组显著低于模型组(P＜0.05,P＜0.01).④关节软骨组织中ADAMTS-4、ADAMTS-5、MMP-3、MMP-13蛋白表达量,模型组显著高于空白组(P＜0.01),补肾壮筋汤组显著低于模型组(P＜0.05,P＜0.01).结论:补肾壮筋汤通过降低滑膜组织中TNF-α、IL-1β、MMP-13含量,抑制关节软骨组织中ADAMTS-4、ADAMTS-5、MMP-3、MMP-13蛋白的表达,从而延缓软骨退变.

目的 探讨补肾壮筋汤抑制脂多糖(LPS)介导软骨细胞表达基质紊乱的作用机制. 方法 体外培养大鼠软骨细胞,采用甲苯胺蓝染色法、Ⅱ型胶原酶免疫组化法鉴定.采用10 ng/mL LPS建立软骨细胞炎症模型,将软骨细胞分为正常组、模型组、抑制剂组、氨基葡萄糖组和补肾壮筋汤组,分别进行相应干预后,酶联免疫法(ELISA)检测各组细胞上清液中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的表达,Western blot法及qRT-PCR法检测各组基质金属蛋白酶(MMP)-3、MMP-13、解聚蛋白样金属蛋白酶(ADAMTS)4、ADAMTS5及RAF蛋白、mRNA的表达,并用qRT-PCR法检测miR-140基因的表达. 结果 ①甲苯胺蓝染色胞浆内可见蓝紫色异染颗粒,Ⅱ型胶原酶免疫组化可见胞浆呈棕黄色阳性染色,鉴定为软骨细胞.(②与正常组比较,模型组IL-1β、TNF-α明显升高(P＜0.05),MMP-3、MMP-13、ADAMTS4、ADAMTS5、RAF蛋白及mRNA的表达明显升高(P＜0.01,P＜0.05),miR-140表达下降(P＜0.01);与模型组比较,抑制剂组、氨基葡萄糖组和补肾壮筋汤组IL-1β、TNF-α明显降低(P＜0.05),MMP-3、MMP-13、ADAMTS4、ADAMTS5、RAF蛋白及mRNA的表达明显降低(P＜0.01,P＜ 0.05),miR-140表达升高(P＜0.01). 结论 补肾壮筋汤通过抑制炎症相关的关键调控因子表达,从而抑制LPS介导软骨细胞表达基质紊乱,保护软骨细胞的功能.

Objective:To explore the mechanism of action of Tiaogu Pian(跳骨片,TGP)medicated serum in inhibiting inflammatory reaction induced by lipopolysaccharides in chondrocytes.Methods:Ten 8-week-old male SD rats were randomly divided into TGP group and blank group.The rats in TGP group were intragastric administrated with TGP in dosage of 0.32 g/kg,while the others in blank group were intragastric administrated with the same dose of normal saline,once per day for 7 consecutive days.At 1 hour after the last intragastric administration,their blood were fetched out from abdominal aorta and were made into TGP medicated serum and blank serum respectively and the serum were reserved at low temperature for future use.The chondrocytes of ten 4-week-old SD rats were isolated from knee articular cartilage and were cultured.The chondrocytes morphology were observed under optical microscope,and immunohistochemical identification were carried out by using type Ⅱ collagenase.The second-generation chondrocytes were randomly divided into blank serum group,model group and TGP medicated serum group.The chondrocytes in blank serum group were cultured in dulbecco modified eagle medium (DMEM) supplemented with 10％ blank serum.The chondrocytes in model group were cultured in DMEM supplemented with lipopolysaccharide(LPS)with concentration of 10 ng/ml and 10％ blank serum.The chondrocytes in TGP medicated serum group were cultured in DMEM supplemented with LPS with concentration of 10 ng/ml and 10％ TGP medicated serum.The chondrocytes in the 3 groups were intervened and cultured for continuous 8 hours.The content of matrix metalloproteinase(MMP) 3 and MMP9 in chondrocytes were detected by using enzyme-linked immunoadsordent assay (ELISA).The expression levels of gene related to Wnt/β-catenin signaling pathway in chondrocytes were detected by using fluorescence quantitative RT-PCR method.The protein expressions of β-catenin and Frizzled-2 in chondrocytes were detected by using Western blot method.The protein expressions of β-catenin,glycogen synthasc kinase-3β (GSK-3β) and proteoglycans 1 (PGS1) in chondrocytes were detected by using immunofluorescence assay(IFA).Results:The second-generation chondrocytes had typical biological characteristics of chondrocytes and their endochylemas and cytomembranes presented with brown-yellow positive staining.After 8-hour intervention by LPS,there was statistical difference in the content of MMP3 and MMP9 in chondrocytes between blank serum group,model group and TGP medicated serum group (34.019 +/-1.036,44.645 +/-2.473,32.941 +/-1.792 ng/ml,F =36.060,P =0.000;1.348 +/-0.038,1.562 +/-0.112,1.331 +/-0.015 ng/ml,F =1 1.319,P =0.000).The content of MMP3 and M MP9 in chondrocytes were higher in model group compared to blank serum group (LSD-t =-7.016,P =0.000;LSD-t =-3.768,P =0.003) and were lower in TGP medicated serum group compared to model group (LSD-t =7.652,P =0.000;LSD-t =4.066,P =0.002).There was no statistical difference in the content of MMP3 and MMP9 in chondrocytes between blank serum group and TGP medicated serum group (LSD-t =0.635,P =0.549;LSD-t =0.299,P =0.770).After 8-hour intervention by LPS,there was statistical difference in gene expression levels of β-catenin,GSK-3β,Frizzled-2,Wnt-4 and CKI-ε between blank serum group,model group and TGP medicated serum group (1.000 +/-0.275,2.258 +/-0.206,1.431 +/-0.304,F =36.709,P =0.000;1.000 +/-0.133,0.417 +/-0.104,0.842 +/-0.094,F=29.259,P=0.000;1.000 +/-0.191,1.737 +/-0.238,1.445 +/-0.337,F=7.027,P=0.015;1.000 +/-0.341,3.801 +/-0.579,1.876 +/-0.388,F =71.903,P =0.000;1.000 +/-0.309,2.208 +/-0.708,1.441 +/-0.421,F =64.178,P =0.000).The gene expression levels of β-catenin,Frizzled-2,Wnt-4 and CKI-εin chondrocytes were higher and the gene expression levels of GSK-3βwere lower in model group compared to blank serum group (LSD-t =-8.431,P =0.000;LSD-t =-3.723,P =0.005;LSD-t =8.062,P =0.000;LSD-t =-11.235,P =0.000,LSD-t =7.397,P =0.000).The gene expression levels of β-catenin,Frizzled-2,Wnt-4 and CKI-ε in chondrocytes were lower and the gene expression levels of GSK-3β were higher in TGP medicated serum group compared to model group (LSD-t =5.541,P =0.000;LSD-t =1.477,P =0.017;LSD-t =8.062,P =0.000;LSD-t =6.882,P =0.000;LSD-t =-5.387,P =0.000).The gene expression levels of β-catenin,Wnt-4 and CKI-ε in chondrocytes were lower in blank serum group compared to TGP medicated serum group (LSD-t =-2.289,P =0.018;LSD-t =-3.658,P =0.005;LSD-t =-4.352,P =0.002).There was no statistical difference in gene expression levels of GSK-3β and Frizzled-2 between blank serum group and TGP medicated serum group (LSD-t =2.009,P =0.075;LSD-t =-3.658,P =0.051).After 8-hour intervention by LPS,there was statistical difference in protein expressions of β-catenin and Frizzled-2 between blank serum group,model group and TGP medicated serum group (0.449 +/-0.063,0.746 +/-0.156,0.549 +/-0.056,F =5.323,P =0.026;1.348 +/-0.038;1.562 +/-0.112;1.331 +/-0.015,F =6.291,P =0.034).The protein expressions of β-catenin and Frizzled-2 in chondrocytes were higher in model group compared to blank serum group(LSD-t =-11.235,P =0.005;LSD-t =-3.104,P =0.021).The protein expressions of β-catenin and Frizzled-2 in chondrocytes were lower in TGP medicated serum group compared to model group(LSD-t =6.883,P =0.037;LSD-t =3.039,P =0.023).The protein expressions of β-catenin in chondrocytes were lower in blank serum group compared to TGP medicated serum group(LSD-t =-4.352,P =0.002).There was no statistical difference in protein expressions of Frizzled-2 between blank serum group and TGP medicated serum group (LSD-t =-0.065,P =0.950).After 8-hour intervention by LPS,the chondrocytes presented with obvious green staining of β-catenin,GSK-3β and PGS1 protein.There was statistical difference in protein expressions of β-catenin,GSK-3β and PGS1 between blank serum group,model group and TGP medicated serum group(0.014 +/-0.002,0.029 +/-0.006,0.018 +/-0.002,F =9.910,P =0.013;0.380 +/-0.011,0.237 +/-0.015,0.287 +/-0.002,F =56.639,P =0.000;0.034 +/-0.003,0.022 +/-0.002,0.029 +/-0.003,F =27.232,P =0.001).The protein expressions of β-catenin were higher and the protein expressions of GSK-3β and PGS1 were lower in model group compared to blank serum group (LSD-t =-4.103,P =0.006;LSD-t =1.048,P =0.000;t =7.365,P =0.000).The protein expressions of β-catenin were lower and the protein expressions of GSK-3β and PGS1 were higher in TGP medicated serum group compared to model group(LSD-t =-3.548,P =0.012;LSD-t =-3.657,P =0.011;LSD-t =-3.273,P =0.017).There was no statistical difference in protein expressions of β-catenin between blank serum group and TGP medicated serum group (LSD-t =-0.554,P =0.599).The protein expressions of GSK-3 βand PGS1 were higher in blank serum group compared to TGP medicated serum group (LSD-t =6.827,P =0.000;LSD-t =4.092,P =0.010).Conclusion:TGP medicated serum can inhibit inflammatory reaction induced by LPS in chondrocytes and delay the articular cartilage degeneration.The mechanisms of action may be related to the regulation of Wnt/β-catenin signaling pathway,in which β-catenin gene,Frizzled-2 gene,GSK-3β gene,Wnt-4 gene and CKI-ε gene may be the important action targets.However,many factors can cause OA and there are many complicated ingredients in TGP medicated serum,further studies are needed to confirm the specific action targets.

Objective To observe the effect of Tiaogu Tablets on the inflammatory response mediated by the Wnt /β‐catenin signaling pathway in rat knee osteoarthritis .Methods A total of 30 healthy male SD rats of 8 weeks old were randomly divided into a sham group ,a model group and a Tiaogu Tablet group ,10 rats in each .Rats in the model and Tiaogu Tablet groups were induced into knee osteoarthritis by the modified Hulth method .The Tiaogu Tablet group was given Tiaogu Tablets 0 .32 g/kg every day ,while the sham group and the model group were given the same dose of saline .After treatment for 12 weeks ,the bilateral knee joints of the rats in all 3 groups were taken ,fixed , decalcified ,embedded and sliced ;then hematoxylin‐eosin (HE) staining and toluidine blue staining were used to observe the morphology of the tissue .The levels of interleukin (IL)‐1β ,matrix metalloproteinases (MMP)‐13 and tumor necrosis factor (TNF)‐α in the synovium were measured by enzyme linked immunosorbent assay (ELISA) .The protein level of β‐catenin and Wnt‐4 was detected by Western blot .Results In the synovium ,the levels of IL‐1β ,MMP‐13 and TNF‐α in the model group were significantly increased (P< 0 .05) compared with the sham group and the Tiaogu Tablet group .Compared with the sham group and Tiaogu Tablet group ,the protein levels of β‐catenin and Wnt‐4 in the model group were significantly increased (P<0 .05) .HE staining showed that cartilage destruction in Tiaogu Tablet group was less severe than&nbsp;that in the model group . The toluidine blue staining showed the proteoglycan content in chondrocytes in the Tiaogu Tablet group was significantly higher than that in the model group (P<0 .01) .Conclusion Tiaogu Tablets suppress the expression of inflammatory cytokines in the synovium , inhibit the inflammation‐mediated cartilage matrix degradation , and delay the degradation of articular cartilage by down‐regulating the Wnt/β‐catenin signaling pathway .

Psoralen (PSO), the active ingredient of Fructus Psoraleae (FP) the dried ripe fruit of Psoralea corylifolia L., has been commonly used in traditional Chinese medicine (TCM) for the treatment of osteoarthritis (OA). We found that PSO activates cartilaginous cellular functions of rat chondrocytes in vitro. However, the effect of PSO on chondrocyte proliferation and the precise mechanisms involved remain to be elucidated. We investigated the effects of PSO on chondrocytes isolated from Sprague-Dawley (SD) rats and evaluated involvement of the Wnt/beta-catenin signaling pathway. The viability of chondrocytes treated with PSO was increased in a dose-and time-dependent manner, as assessed by MTT assay. We found that the gene expression and protein levels of Wnt-4, Frizzled-2, beta-catenin and cyclin D1 in the PSO-treated chondrocytes were significantly upregulated, while the gene expression and protein level of glycogen synthase kinase-3 beta (GSK-3 beta) were downregulated, compared with the untreated chondrocytes. By immunofluorescence, we also found that PSO induced beta-catenin nuclear translocation. Importantly, the expression of beta-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/beta-catenin signaling pathway. Additionally, Col-II expression in chondrocytes was increased after treatment with PSO. Taken together, these results indicate that PSO promotes chondrocyte proliferation by activating the Wnt/beta-catenin signaling pathway, and it may play an important role in the treatment of OA.

目的：探讨跳骨片水提物对体外大鼠关节软骨细胞活性的影响。方法：4周龄SPF级SD大鼠5只，采用机械-Ⅱ型胶原酶消化法获取膝关节软骨细胞，倒置相差显微镜观察软骨细胞的形态结构，Ⅱ型胶原免疫组化染色鉴定软骨细胞。0，25，50，100μg·mL-1跳骨片水提物对第3代软骨细胞，分别干预24，48，72 h后，MTT法检测细胞活性。结果：①Ⅱ型胶原免疫组化染色后软骨细胞胞浆呈棕黄色，为阳性；②以0μg·mL-1跳骨片水提物干预的软骨细胞作为对照组，干预24 h后，50μg·mL-1组软骨细胞活性明显高于对照组（P <0.05）；干预48h后，25，50μg·mL-1组明显高于对照组（P <0.05）；干预72h后，25，50，100μg·mL-1组与对照组比较，差异无统计学意义（P >0.05）。结论：跳骨片水提物在25，50，100μg·mL-1时均可以促进软骨细胞的增殖，具有剂量和时间依赖性，其中50μg·mL-1干预24 h后最为明显。

疼痛是膝骨关节炎的主要临床表现之一,严重影响患者的生活质量.肝肾亏虚、筋骨失衡是膝骨关节炎的中医病理特点,也是引起膝骨关节炎疼痛的关键因素.筋骨相连,筋束骨,骨张筋,骨衰筋伤,而为痹.筋与骨结构上密不可分,功能上相互协调,筋伤内动于肝,肝血不足,筋失所养,筋病难愈.筋损骨失所束,则影响骨之生理,从而加重膝骨关节炎的病理进程.以筋骨失衡为切入点,以膝骨关节炎的疼痛为研究对象,查阅国内外文献,采用综合-分析-综合的方法,探讨筋骨失衡与膝骨关节炎疼痛之间的内在关系.

Objective To explore the mechanism of Tougu Xiaotong capsule (TXC)inhibiting lipopolysaccharide (LPS)-stimulated inflammation in chondrocytes.Methods The chondrocytes harvested from the knee joints of 4-week-old SD rats were cultured in vitro and divided into 3 groups:the sham group,model group and TXC group.The sham group was cultured in 2 mL low-glucose Dulbecco Modified Eagle Medium (DMEM/LOW)containing 10% fetal bovine serum (FBS);the model group was cultured in 2 mL DMEM/LOW containing 10% FBS and 10 ng/mL LPS;the TXC group was cultured in 2 mL DMEM/LOW containing 10%FBS,10 ng/mL LPS and 300μg/mL TXC. After chondrocytes being treated for 8 h,the microRNA (miRNA)-140 expression was detected by quantitative real-time polymerase chain reaction (qPCR ), and the expressions of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS )4 and matrix metalloproteinase (MMP)-3 were observed by immunofluorescence staining,the average optical density of each group was analyzed by Image J. Results Compared with the sham group,the expression of MMP-3 (P<0.05 )and ADAMTS4 (P<0.01 )in the model group were significantly increased.Compared with the model group,the expression of MMP-3 (P<0.05)and ADAMTS4 (P<0.01)in the TXC group were significantly decreased.The expression of miRNA-140 in the model group was lower than that in the control group (P<0.01)and the&nbsp;TXC group (P<0.01).Conclusion TXC may delay the cartilage degeneration in osteoarthritis (OA)by promoting the expression of miRNA-140 to reduce the secretion of inflammatory cytokines such as ADAMTS4 and MMP-3 and thus inhibit the inflammation-mediated cartilage matrix degradation.

骨赘是在生物力学和生物化学因素的共同影响下，发生于关节边缘的骨和软骨性新生组织，是骨关节炎病理过程中的一个特征性表现。骨赘的形成是用于临床诊断骨关节炎的一个特征性结构变化，其本质是关节组织的代偿性修复；但因骨赘形成改变了关节的力学结构，使关节面不适应，结果却引起了关节畸形和破坏，加重了关节退变。因此，深入研究骨赘形成机制、骨赘与骨关节炎病理进展的内在联系，为骨关节炎的防治研究提供新的思路。

Objective:To investigate the effect of Achyranthes bidentata polysaccharides(ABPS) on the expressions of typeⅡcollagen and proteoglycan in cartilage cells.Methods:Ten 4-week-old healthy SD rats were used to isolate the knee joints,scrape cartilaginous tissue off the surface of the knee joint,and get knee joint cartilage cells with the mechanical typeⅡcollagenase digestion method.An in vitro culture system was established and the cell morphology was observed with an inverted phase contrast microscope.TypeⅡ immunohistochemistry method was used to identify the chondrocytes.By the second generation of in-vitro-cultured chondrocytes,ABPS(0,50,100,200 μg·mL-1 respectively)was used to interfere in the cartilage cells. After intervention,Western blot was used to detect the expression changes of typeⅡcollagen and proteoglycan of each group,and immunolfuorescence was used to observe the expression changes of typeⅡcollagen respectively in the blank control group and the dosing group.Results:The morphology of the second generation chondrocytes showed that cartilage cells contained rich typeⅡcollagen.Western blot showed that ABPS promoted chondrocyte typeⅡcollagen and the expression of typeⅡcollagen was the highest when there was 100 μg·mL-1 ABPS,the difference being statistically signiifcant(P< 0.05).After intervention with 100 μg·mL-1 ABPS,the expression of proteoglycan in cartilage cells increased,but the difference was not statistically significant(P> 0.05). Compared with the blank control group,the expression of intervened collagen typeⅡcollagen was stronger. Conclusion:ABPS promotes the expression of typeⅡcollagen and proteoglycan.

炎症是导致骨关节炎发生发展的关键因素之一。乌头汤是治疗痹证的常用方剂，具有抑制白细胞介素-1β、肿瘤坏死因子-α等炎症因子的作用。通过深入了解骨关节炎与炎症反应的关系，探讨乌头汤治疗骨关节炎的作用机制，为乌头汤治疗骨关节炎的应用研究提供新思路。

骨关节炎病在筋骨，筋骨失养为主要病理表现，常见于中老年人，软骨退变在骨关节炎筋骨失养过程中发挥重要作用。水占关节软骨湿重的80%，是软骨所需营养物质的重要媒介，在关节软骨生化组成中所占比重最大，水与胶原纤维及蛋白多糖相结合共同提供了软骨细胞生物学功能的内环境，与软骨生物力学功能密切相关。调节含水率可能是延缓或阻止软骨退变的重要机制之一，提示有效调控关节软骨含水率在骨关节炎形成和发展中的作用，对骨关节炎筋骨失养的防治研究具有重要意义。

Objective:To investigate the molecular mechanism of the development of temporomandibular joint.Methods:Heads of five BAL B/C newlyborn rats were taken,fixed,decalcified,dehydrated and embedded. Immunohistochemical technique was used to detect the expressions of CollagenⅡ(ColⅡ),CollagenⅠ(ColⅠ),Aggrecan and Ihh.Results:In the temporomandibular joint disk,the expressions of ColⅠ and ColⅡwere positive,the expression of Ihh was strongly positive and Aggrecan had no obvious expression;while in condylar cartilage,the expression of ColⅡ was positive,the expression of Aggrecan was strongly positive,and Ihh and ColⅠ had a little expression.Conclusion:ColⅡ,ColⅠ,Aggrecan and Ihh have normal expressions,playing an important role in the formation and development of the temporomandibular joint.