蠲痹胶囊对膝骨关节炎豚鼠关节软骨组织形态及滑膜中Toll样受体4、NF-κB p65及肿瘤坏死因子-α表达的影响

Impact of Juanbi Jiaonang(蠲痹胶囊,JBJN) on articular cartilage tissue form and expression of Toll- like receptor 4,NF-kB p65 and tumor necrosis factor- alpha in synovium in guinea pigs with knee osteoarthritis

目的:观察蠲痹胶囊对膝骨关节炎(knee osteoarthritis,KOA)豚鼠关节软骨组织形态及滑膜中Toll样受体4(Toll-like receptor 4,TLR4)、NF-κB p65及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达的影响,探讨其治疗KOA的可能作用机制。方法:将40只1月龄豚鼠随机分为正常组、3月龄组、5月龄组、氨基葡萄糖组和蠲痹胶囊组,每组8只。分组后正常组豚鼠直接进行标本采集,其余4组饲养至3月龄建立自发性KOA模型。造模结束后,3月龄组豚鼠进行标本采集;5月龄组、氨基葡萄糖组和蠲痹胶囊组分别以纯水、盐酸氨基葡萄糖胶囊和蠲痹胶囊灌胃,每天1次,连续干预2个月后进行标本采集。采集标本为豚鼠左后下肢膝关节髌下韧带表面的滑膜和胫骨内侧平台软骨,制成石蜡切片。软骨组织切片以番红固绿染色后光镜下观察软骨组织形态,采用Mankin’s病理评分标准进行评分;滑膜组织切片进行免疫组织化学染色(SP法),用Image-pro Plus 6.0图像分析软件对阳性染色的平均光密度值进行半定量分析,测定指标包括TLR4、NF-κB p65及TNF-α的表达水平。结果:5组豚鼠关节软骨Mankin’s评分比较,差异有统计学意义[(0.132±0.125)分,(5.011±0.180)分,(7.324±0.293)分,(4.250±0.250)分,(2.500±0.327)分,F=108.400,P=0.000];3月龄组评分高于正常组(P=0.000),5月龄组评分高于3月龄组(P=0.000),氨基葡萄糖组评分低于5月龄组(P=0.000),蠲痹胶囊组评分低于氨基葡萄糖组(P=0.000)。5组豚鼠关节滑膜中TLR4、NF-κB p65及TNF-α表达量比较,组间差异均有统计学意义(0.000±0.000,0.268±0.021,0.454±0.043,1.879±0.013,0.116±0.015,F=97.000,P=0.000;0.023±0.019,0.251±0.142,0.525±0.103,0.217±0.087,0.211±0.019,F=40.293,P=0.000;0.047±0.023,0.386±0.142,0.812±0.174,0.289±0.151,0.204±0.108,F=78.242,P=0.000);3月龄组TLR4、NF-κB p65及TNF-α表达量均高于正常组(P=0.000,P=0.000,P=0.000),5月龄组TLR4、NF-κB p65及TNF-α表达量均高于3月龄组(P=0.000,P=0.000,P=0.000),氨基葡萄糖组TLR4、NF-κB p65及TNF-α表达量均低于5月龄组(P=0.000,P=0.000,P=0.000),蠲痹胶囊组TLR4、NF-κB p65及TNF-α表达量均低于氨基葡萄糖组(P=0.011,P=0.019,P=0.008)。结论:蠲痹胶囊和盐酸氨基葡萄糖均可延缓KOA豚鼠关节软骨退变,蠲痹胶囊的作用更加明显;其作用机制可能是通过激活TLR4/NF-κB信号转导通路来抑制炎性因子TNF-α表达,从而减轻组织炎症反应。

Objective：To observe the impact of Juanbi Jiaonang（蠲痹胶囊,JBJN）on articular cartilage tissue form and expression of Toll-like receptor 4（TLR4）,NF-κB p65 and tumor necrosis factor-alpha（TNF-a）in synovium in guinea pigs with knee osteoarthritis （KOA）,and to explore the possible mechanism of action of JBJN in the treatment of KOA.Methods：Forty 1 -month-old guinea pigs were randomly divided into normal group,3-month-old group,5 -month-old group,glucosamine group and JBJN group,8 cases in each group.After grouping,the specimens were collected directly in normal group,while the guinea pigs in the other 4 groups were bred continu-ously and spontaneous KOA models were built when they were 3 months old.After modeling,the specimens were collected in 3 -month-old group,while the guinea pigs in 5 -month-old group,glucosamine group and JBJN group were intragastric administrated with pure wa-ter,glucosamine hydrochloride capsules and JBJN respectively,once a day for consecutive 2 months,and then the specimens were collected. The superficial synovium of infrapatellar ligament and the medial tibial plateau cartilage were collected from the knee of left posterior-infe-rior extremity and were made into paraffin sections.The cartilage tissue sections were received safranin-fast green staining and the cartilage tissue forms were observed under optical microscope and were evaluated according to Mankin score.The synovium tissue sections were re-ceived immunohistochemical staining（SP method）,and the semiquantitative analysis were carried out on the average optical density value of the positive staining by using Image-pro Plus 6.0 image analysis software,and the main measuring indexes included the expression level of TLR4,NF-kB p65 and TNF -a.Results：There was statistical differences in Mankin scores of articular cartilage between the 5 groups （0.1 32＋/-0.1 25,5.01 1 ＋/-0.1 80,7.324 ＋/-0.293,4.250 ＋/-0.250,2.500 ＋/-0.327 points,F=1 08.400,P=0.000）,and 3 -month-old group surpassed normal group（P=0.000）,and 5 -month-old group surpassed 3 -month-old group（P=0.000）and glu-cosamine group（P=0.000）,and glucosamine group surpassed JBJN group（P=0.000）.There was statistical differences in the expression level of TLR4,NF-kB p65 and TNF-a in the articular synovium between the 5 groups（0.000＋/-0.000,0.268＋/-0.021 ,0.454＋/-0.043,1 .879＋/-0.01 3,0.1 1 6 ＋/-0.01 5,F=97.000,P=0.000;0.023 ＋/-0.01 9,0.251 ＋/-0.1 42,0.525 ＋/-0.1 03,0.21 7 ＋/-0.087,0.21 1 ＋/-0.01 9,F=40.293,P=0.000;0.047 ＋/-0.023,0.386 ＋/-0.1 42,0.81 2 ＋/-0.1 74,0.289 ＋/-0.1 51 ,0.204 ＋/-0.1 08,F=78.242,P=0.000）,and 3-month-old group surpassed normal group（P=0.000,P=0.000,P=0.000）,and 5 -month-old group surpassed 3-month-old group（P=0.000,P=0.000,P=0.000）and glucosamine group（P=0.000,P=0.000,P=0.000）, and glucosamine group surpassed JBJN group（P=0.01 1 ,P=0.01 9,P=0.008）.Conclusion：Both JBJN and glucosamine hydrochloride can delay the articular cartilage degeneration in guinea pigs with KOA and the former has more significant effect.JBJN can inhibit the ex-pression of inflammatory factor TNF-a through activating LR4/NF-kB signaling pathway,which may be the mechanism of action.