Objectives: To observe the effect of Guasha on inflammation factors, apoptosis and autophagy in the cartilage tissue of knee joint in rats with knee osteoarthritis (KOA), so as to explore its mechanisms underlying improvement of KOA.
Methods: A total of 51 male SD rats were randomized into three groups：blank control, KOA model and Guasha (n= 17 in each group) . The rats in the blank control group received intra-articular injection of 0.9% NaCl solution in the right knee joint. The KOA model was established by intraarticular injection of glutamate sodium iodoacetic acid in the right knee joint. For rats of the Guasha group, Guasha (at a frequency of 1 time/s, and an applied pressure of 0.3-0.5 kgf) was applied to "Yanglingquan" (GB34) and "Xuehai"(SP10) areas of the right leg, once every other day, for 7 consecutive sessions. The circumference of the right knee was measured, The histopathological changes of right knee cartilage were observed after H.E. staining. The contents of inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the right knee articular cartilage tissue were assayed using ELISA. The expression levels of autophagy-related key molecule Beclin-1 (homologous series of yeast Atg6), light chain protease complication 3 type II/I (LC3II/LC3 I), ubiquitin binding factor 62 (P62) and cysteine aspartate protease-3 (Caspase-3) mRNAs and proteins of the right knee articular cartilage tissue were measured using real-time fluorescent quantitative PCR and Western blot, separately. The apoptosis of chondrocytes was assayed using TUNEL staining, and the immunoactivity of LC3 determined using immunofluorescence staining.
Results: After modeling, the right knee circumfe-rence of the model and Guasha groups was significantly increased compared with the blank control group (P<0.01), and after the intervention, the knee circumference of the Guasha group was markedly decreased in comparison with that of the model group (P<0.05). Results of H.E. staining showed obvious degeneration and defects in the cartilage tissue, necrosis of a large number of chondrocytes, fibrous hyperplasia, accompanied by inflammatory cell infiltration, osteoclast increase, fibroplasia and bone trabecular destruction in the model group, which was relatively milder in the Guasha group. Compared with the blank control group, the expression of Beclin-1 and LC3 mRNAs and proteins, and LC immunofluorescence intensity in the right knee articular cartilage tissue were significantly down-regulated (P<0.01, P<0.001), whereas the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, contents of IL-1β and TNF-α in the right knee articular cartilage tissue considerably increased (P<0.01, P<0.001) in the model group. In contrast to the model group, the Guasha group had an apparent increase in the expression levels of Beclin-1 and LC3 mRNAs and proteins and LC immunofluorescence intensity in the right knee articular cartilage tissue (P<0.05), and a pronounced decrease in the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, and contents of IL-1β and TNF-α in the right knee articular cartilage tissue (P<0.05, P<0.01).
Conclusions: Guasha stimulation of GB34 and SP10 can improve joint cartilage damage in KOA rats, which may be associated with its functions in inhibiting the excessive release of inflammatory factors and apoptosis, possibly by down-regulating the expression of P62 and Caspase-3 mRNAs and proteins and up-regulating the expression of Beclin-1 and LC3 mRNAs and proteins, and by promoting autophagy of chondrocytes.
目的: 观察刮痧对膝关节骨关节炎（KOA）大鼠膝关节软骨组织炎性因子、细胞凋亡及自噬的影响，探讨刮痧治疗KOA的作用机制。方法: SD大鼠随机分为空白组、模型组和刮痧组，每组17只。采用右膝关节腔内注射谷氨酸钠碘乙酸复制KOA大鼠模型。刮痧组选择右侧“阳陵泉”“血海”进行刮痧治疗，频率为1次/s，按压力度在0.3~0.5 kgf，隔日干预1次，连续干预7次。记录各组大鼠右膝关节周径变化；HE染色法观察大鼠右膝关节软骨病理形态变化；ELISA法检测大鼠右膝关节软骨组织中白细胞介素（IL）-1β及肿瘤坏死因子（TNF）-α含量；荧光定量PCR法和Western blot法分别检测大鼠膝关节软骨组织中自噬性关键小分子酵母Atg6的同素系物（Beclin-1）、微管相关结合蛋白1轻链蛋白酶复合物3 Ⅱ/Ⅰ型（LC3Ⅱ/LC3Ⅰ）、泛素相关结合蛋白62（P62）、半胱天冬氨酸蛋白酶-3（Caspase-3）mRNA和蛋白的表达水平；TUNEL染色观察大鼠膝关节软骨细胞的凋亡情况；免疫荧光法检测大鼠右膝关节软骨中LC3的表达水平。结果: 造模后，与空白组比较，模型组右侧膝关节周径明显增大（P<0.01）；软骨层有明显的紊乱和缺损，软骨细胞大量坏死，有纤维增生及大量骨小梁破坏；软骨组织细胞凋亡率、IL-1β和TNF-α含量及Caspase-3、P62 mRNA和蛋白表达水平均升高（P<0.01，P<0.001），Beclin-1、LC3 mRNA和蛋白表达水平和LC3平均荧光强度降低（P<0.01，P<0.001）。治疗后，与模型组比较，刮痧组右侧膝关节周径缩小（P<0.05）；软骨层排列趋于正常，炎性细胞浸润程度较低；软骨组织细胞凋亡率、IL-1β和TNF-α含量降低（P<0.05），Caspase-3、P62 mRNA和蛋白表达水平降低（P<0.01，P<0.05），Beclin-1、LC3 mRNA和蛋白表达水平及LC3平均荧光强度升高（P<0.05）。结论: 刮痧能够改善KOA大鼠关节软骨损伤，抑制炎性因子过量释放，减少软骨细胞凋亡率。这可能与上调Beclin-1、LC3的表达，降低P62、Caspase-3的表达，促进KOA大鼠软骨细胞自噬有关。.
Keywords: Apoptosis; Autophagy; Guasha; Knee osteoarthritis.