调控水通道蛋白表达延缓膝骨关节炎模型大鼠软骨退变

Delaying cartilage degeneration by regulating the expression of aquaporins in rats with knee osteoarthritis

背景:下调水通道蛋白表达能延缓膝骨关节炎软骨退变,但其具体机制不明确。目的:探讨p38 MAPK信号通路调控水通道蛋白表达对大鼠膝骨关节炎软骨退变的影响。方法:60只SD大鼠随机分为假手术组、模型组、SB203580低浓度组(7.5 mg/kg)和SB203580高浓度组(30 mg/kg),每组15只。除假手术组外其余3组均采用改良Hulth法构建大鼠膝骨关节炎模型;假手术组大鼠仅从右膝关节内侧打开关节腔,不破坏韧带和半月板,并保留关节软骨面。SB203580低、高浓度组大鼠于术后1周开始腹腔注射不同浓度p38 MAPK抑制剂SB203580进行干预;假手术组和模型组大鼠膝关节注射等体积生理盐水,1次/周,共8周。测量各组大鼠膝骨关节肿胀程度;苏木精-伊红染色观察大鼠软骨组织病理改变并进行Mankin′s评分;ELISA检测大鼠滑膜组织中白细胞介素1β、肿瘤坏死因子α和基质金属蛋白酶13表达水平;RT-PCR检测软骨组织中水通道蛋白1、水通道蛋白3 mRNA水平;Western blot检测软骨组织中水通道蛋白1、水通道蛋白3、cleaved-caspase-3、Bax、Bcl-2、p38MAPK和p-p38MAPK等蛋白表达水平。实验方案经广东省第二中医院动物实验伦理委员会批准(批准号为20180125004)。结果与结论:①与假手术组比较,模型组大鼠软骨组织损伤严重,Mankin′s评分及骨关节肿胀度显著增加(P<0.05);滑膜组织中细胞介素1β、肿瘤坏死因子α和基质金属蛋白酶13水平、软骨组织中cleaved-caspase-3、Bax和p-p38MAPK蛋白表达及水通道蛋白1、水通道蛋白3的mRNA和蛋白表达均显著上升(P<0.05),而Bcl-2蛋白表达水平显著降低(P<0.05);②与模型组比较,SB203580高浓度组大鼠软骨组织损伤得到明显改善,Mankin′s评分、骨关节肿胀度、滑膜组织中白细胞介素1β、肿瘤坏死因子α和基质金属蛋白酶13水平、软骨组织中cleaved-caspase-3、Bax和p-p38MAPK蛋白表达及水通道蛋白1、水通道蛋白3的mRNA和蛋白表达均显著降低(P<0.05),而Bcl-2蛋白表达显著增加(P<0.05);③SB203580低浓度组与模型组比较,相关指标差异均无显著性意义(P>0.05);④结果推测,阻断p38MAPK信号通路,可通过抑制水通道蛋白1和水通道蛋白3表达从而延缓膝骨关节炎关节软骨退变。

BACKGROUND:Down-regulation of aquaporins(AQPs)can delay cartilage degeneration in knee osteoarthritis,but its specific mechanism is undefined.OBJECTIVE:To investigate the effect of p38 MAPK signaling pathway regulating AQP expression on cartilage degeneration in a rat model of knee osteoarthritis.METHODS:Sixty Sprague-Dawley rats were randomly divided into sham operation group(sham),model group,SB203580 low concentration group(SB203580-L,7.5 mg/kg)and high concentration group(SB203580-H,30 mg/kg),with 15 rats in each group.The modified Hulth method was used to construct the rat model of knee osteoarthritis.In the sham group,the joint cavity was only opened from the medial side of the right knee joint,without damage to the ligament and meniscus,and with preservation of the articular cartilage surface.Intraperitoneal injection of SB203580 with different concentrations was started at 1 week after surgery,once per week,for 8 weeks in total.The degree of knee joint swelling was measured.The pathological changes of cartilage tissue were observed by hematoxylineosin staining,and graded by the Mankin’s score.The expression levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and matrix metalloproteinase-13(MMP-13)in synovial tissue were detected by ELISA.The AQP1,AQP3 mRNA levels in cartilage tissue were detected by RT-PCR,and the protein levels of AQP1,AQP3,cleaved caspase-3,Bax,Bcl-2,p38MAPK and p-p38MAPK in cartilage tissue were detected by western blot.An approval for this study was obtained from the Animal Experimental Ethics Committee of Guangdong Second Traditional Chinese Medicine Hospital With an approval No.20180125004 RESULTS AND CONCLUSION:Compared with the sham group,the rats in the model group had severe cartilage injury,the Mankin’s score and the degree of joint swelling were increased significantly(P<0.05).Compared with the sham group,the levels of IL-1β,TNF-αand MMP-13 in synovial tissues,the cleaved caspase-3,Bax,and p-p38MAPK protein expression levels in chondrocytes,the mRNA and protein expression levels of AQP1 and AQP3 all increased significantly in the model group(P<0.05),whereas the expression level of Bcl-2 protein decreased significantly(P<0.05).Compared with the model group,the cartilage injury of rats in the SB203580-H group was improved significantly(P<0.05);the Mankin’s score and the degree of joint swelling were decreased significantly(P<0.05);the levels of IL-1β,TNF-αand MMP-13 in synovial tissues,the cleaved caspase-3,Bax,and p-p38MAPK protein expression levels in chondrocytes,and the mRNA and protein expression levels of AQP1 and AQP3 all decreased significantly(P<0.05),whereas the expression level of Bcl-2 protein increased significantly(P<0.05).There was no significant difference between the SB203580-L group and model group(P>0.05).Therefore,blocking the p38MAPK signaling pathway is deduced to delay the degeneration of articular cartilage in knee osteoarthritis by inhibiting the expression of AQP1 and AQP3.