目的:探讨蠲痹方含药血清对绝经后膝骨关节炎(knee osteoarthritis，KO A)大鼠软骨细胞自噬的影响及其作用机制。方法:①绝经后KOA动物模型的建造和蠲痹方含药血清的制备。采用摘取大鼠卵巢，并切断膝关节内侧副韧带、前交叉韧带，摘除膝关节内侧半月板的方法，建造绝经后KOA大鼠模型。造模成功后，对模型大鼠进行蠲痹方浓缩液灌胃，制备蠲痹方含药血清。②大鼠软骨细胞的提取。分别取正常大鼠和模型大鼠的膝关节软骨分离、提取软骨细胞。③蠲痹方含药血清最佳作用浓度筛选。将模型大鼠软骨细胞分为模型组及1。25％、2。5％、5％、10％、20％含药血清组6组，除模型组外，其余各组分别加入相应体积分数的蠲痹方含药血清干预24 h。检测各组软骨细胞的活力，筛选蠲痹方含药血清最佳作用浓度。④蠲痹方含药血清对大鼠软骨细胞G蛋白耦联受体30(G protein-coupled receptor 30，GPR30)表达影响的检测。将模型大鼠软骨细胞分为模型组及1。25％、2。5％、5％、10％含药血清组，并取正常大鼠软骨细胞设为空白组。除模型组和空白组外，其他各组软骨细胞用相应体积分数的蠲痹方含药血清干预24 h。检测各组大鼠软骨细胞GPR30的相对表达量。⑤蠲痹方含药血清对大鼠软骨细胞自噬相关蛋白表达和 AMP 活化蛋白激酶(AMP-activated protein kinase，AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)信号通路影响的检测。将模型大鼠软骨细胞分为模型组、含药血清组、含药血清+Compound C组、Compound C组，并取正常大鼠软骨细胞设为空白组。除模型组和空白组外，含药血清组、含药血清+Compound C组、Compound C组分别用10％蠲痹方含药血清、10％蠲痹方含药血清+Compound C及Compound C干预24 h。检测各组软骨细胞中自噬相关蛋白微管相关蛋白轻链3β(microtu-bule-associated protein light chain 3 beta，MAPLC3β)、Beclin-1、p62，及 AMPK/mTOR 信号通路相关蛋白磷酸化 AMP 活化蛋白激酶(phosphorylated AMP-activated protein kinase，p-AMPK)、磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapam-ycin，p-mTOR)的相对表达量。结果:①动物模型鉴定结果。造模8周后，可见造模大鼠膝关节表面软骨毛糙或缺损，关节间隙变窄，滑膜增生，绝经后KOA大鼠模型造模成功。②软骨细胞鉴定结果。甲苯胺蓝染色及Ⅱ型胶原蛋白免疫荧光鉴定结果显示，所提取的细胞符合软骨细胞特征。③蠲痹方含药血清最佳作用浓度筛选结果。5％、10％、20％含药血清组细胞活力均高于1。25％、2。5％含药血清组和模型组(LSD-t=6。767，P=0。003，LSD-t=7。666，P=0。002，LSD-t=5。091，P=0。007;LSD-t=5。080，P=0。007，LSD-t=6。690，P=0。003，LSD-t=3。433，P=0。027;LSD-t=7。590，P=0。002，LSD-t=8。200，P=0。001，LSD-t=6。031，P=0。004);10％含药血清组细胞活力高于5％含药血清组和20％含药血清组(LSD-t=3。204，P=0。033，LSD-t=4。671，P=0。010)。④蠲痹方含药血清对大鼠软骨细胞GPR30表达影响的检测结果。模型组GPR30相对表达量低于空白组及5％、10％含药血清组(LSD-t=5。695，P=0。005，LSD-t=5。400，P=0。006，LSD-t=9。006，P=0。001)，5％、10％含药血清组 GPR30 相对表达量均高于1。25％含药血清组(LSD-t=2。782，P=0。049，LSD-t=4。473，P=0。011)，10％含药血清组GPR30相对表达量高于2。5％含药血清组(LSD-t=4。544，P=0。011)。⑤蠲痹方含药血清对大鼠软骨细胞自噬相关蛋白表达影响的检测结果。模型组、含药血清+Compound C 组、Compound C 组 MAPLC3β 相对表达量均低于空白组、含药血清组(LSD-t=6。855，P=0。002，LSD-t=8。675，P=0。001;LSD-t=5。096，P=0。007，LSD-t=5。931，P=0。004;LSD-t=5。560，P=0。005，LSD-t=5。354，P=0。006);模型组、Compound C 组 MAPLC3β 相对表达量均低于含药血清+Compound C 组(LSD-t=4。627，P=0。010，LSD-t=8。677，P=0。001)。模型组、Com-pound C组Beclin-1相对表达量低于空白组、含药血清组、含药血清+Compound C(LSD-t=12。912，P=0。000，LSD-t=7。401，P=0。002，LSD-t=5。360，P=0。006;LSD-t=2。950，P=0。042，LSD-t=5。484，P=0。005，LSD-t=3。903，P=0。018);含药血清+Com-pound C组Beclin-1相对表达量低于空白组(LSD-t=26。840，P=0。000)。含药血清组、空白组p62相对表达量低于模型组、含药血清+Compound C 组、Compound C 组(LSD-t=3。925，P=0。017，LSD-t=3。985，P=0。019，LSD-t=0。016，P=0。001;LSD-t=3。149，P=0。035，LSD-t=5。094，P=0。007，LSD-t=8。740，P=0。001)，含药血清+Compound C 组 p62 相对表达量低于 Compound C组(LSD-t=3。455，P=0。026)。⑥蠲痹方含药血清对大鼠软骨细胞AMPK/mTOR信号通路影响的检测结果。含药血清组p-AMPK相对表达量高于模型组、Compound C 组(LSD-t=3。623，P=0。022，LSD-t=6。537，P=0。003)，空白组 p-AMPK 相对表达量高于模型组、含药血清+Compound C 组、Compound C 组(LSD-t=4。149，P=0。014，LSD-t=2。791，P=0。049，LSD-t=5。734，P=0。004)，含药血清+Compound C组p-AMPK相对表达量高于Compound C组(LSD-t=5。958，P=0。004)。空白组、含药血清组、含药血清+Compound C 组 p-mTOR 相对表达量均低于模型组(LSD-t=8。722，P=0。001，LSD-t=8。849，P=0。001，LSD-t=5。558，P=0。005)，含药血清组、空白组p-mTOR相对表达量均低于含药血清+Compound C组、Compound C组(LSD-t=4。201，P=0。014，LSD-t=10。030，P=0。001;LSD-t=4。879，P=0。008，LSD-t=9。782，P=0。001)，含药血清+Compound C 组 p-mTOR 相对表达量低于Compound C组(LSD-t=6。934，P=0。002)。结论:蠲痹方含药血清作用于绝经后KOA大鼠软骨细胞，可增加细胞活力，并通过上调MAPLC3β、Beclin-1的表达和抑制p62的表达增强细胞自噬能力;其作用机制可能与促进GPR30表达，上调AMPK磷酸化水平，下调mTOR磷酸化水平，激活GPR30和AMPK/mTOR信号通路有关。
Effects and mechanism of Juanbi Fang(蠲痹方)medicated serum on autophagy of chondrocytes in postmeno-pausal rats with knee osteoarthritis:an experimental study
Objective:To observe the effects of Juanbi Fang(蠲痹方,JBF)medicated serum on autophagy of chondrocytes in postm-enopausal rats with knee osteoarthritis(KOA),and to explore its mechanism.Methods:①Twenty rats were subjected to ovariectomy,tran-section of knee medial collateral ligament(MCL)and anterior cruciate ligament(ACL),followed by knee medial meniscectomy for inducing postmenopausal KOA.After successful modeling,15 model rats were intragastric administrated with JBF concentrate for making JBF medica-ted serum.②The normal rats and model rats were sacrificed and their knee cartilages were harvested for isolating and extracting chondro-cytes.③The chondrocytes extracted from the model rats were divided into model group,1.25％medicated serum group,2.5％medicated serum group,5％medicated serum group,10％medicated serum group,and 20％medicated serum group.Except for the model group,the chondrocytes in the other groups were intervened with JBF medicated serum in their corresponding volume fraction for consecutive 24 hours.After the end of intervention,the viability of chondrocytes in each group was detected,and the optimal acting concentration of the JBF medi-cated serum was screened.④The chondrocytes extracted from the model rats were assigned into model group,1.25％medicated serum group,2.5％medicated serum group,5％medicated serum group,and 10％medicated serum group,and the chondrocytes from the normal rats were taken as blank group.Except for the model group and blank group,the chondrocytes in other 4 groups were intervened with JBF medicated serum in their corresponding volume fraction for consecutive 24 hours.After the end of intervention,the relative expression level of G protein-coupled receptor 30(GPR30)in chondrocytes was detected by using Western blotting assay.⑤The chondrocytes extracted from the model rats were divided into model group,medicated serum group,medicated serum+Compound C group,and Compound C group,and the chondrocytes from the normal rats were taken as blank group.Except for the model group and blank group,the chondrocytes in medica-ted serum group,medicated serum+Compound C group,and Compound C group were intervened with 10％JBF medicated serum,10％JBF medicated serum combined with Compound C,and Compound C,respectively,for consecutive 24 hours.After the end of intervention,the rel-ative expression levels of autophagy-related protein microtubule-associated protein light chain 3 beta(MAPLC3β),Beclin-1 and p62,as well as the relative expression levels of AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)signaling pathway-relat-ed protein phosphorylated AMP-activated protein kinase(p-AMPK)and phosphorylated mammalian target of rapamycin(p-mTOR)in chon-drocytes was detected by using Western blotting assay.Results:①Eight weeks after the modeling,the coarse or defective cartilage on the knee surface,narrowed joint space,and proliferated synovial membrane were observed in the model rats,which suggested the models were successfully built.②The primary cells were stained with toluidine blue,and the intracellular type Ⅱ collagen was detected by immunofluores-cence staining,the results showed that the extracted cells were indicated as chondrocytes.③The chondrocyte viability was higher in 5％,10％,and 20％medicated serum group compared to 1.25％,2.5％medicated serum group and model group(LSD-t=6.767,P=0.003,LSD-t=7.666,P=0.002,LSD-t=5.091,P=0.007;LSD-t=5.080,P=0.007,LSD-t=6.690,P=0.003,LSD-t=3.433,P=0.027;LSD-t=7.590,P=0.002,LSD-t=8.200,P=0.001,LSD-t=6.031,P=0.004),and was higher in 10％medicated serum group com-pared to 5％and 20％medicated serum group(LSD-t=3.204,P=0.033,LSD-t=4.671,P=0.010).④The relative expression level of GPR30 in chondrocytes was lower in model group compared to blank group,5％,and 10％medicated serum group(LSD-t=5.695,P=0.005,LSD-t=5.400,P=0.006,LSD-t=9.006,P=0.001),and was higher in 5％and 10％medicated serum group compared to 1.25％medicated serum group(LSD-t=2.782,P=0.049,LSD-t=4.473,P=0.011),and was higher in 10％medicated serum group compared to 2.5％medicated serum group(LSD-t=4.544,P=0.011).⑤The relative expression level of MAPLC3β was lower in model group,medicated serum+Compound C group,and Compound C group compared to blank group and medicated serum group(LSD-t=6.855,P=0.002,LSD-t=8.675,P=0.001;LSD-t=5.096,P=0.007,LSD-t=5.931,P=0.004;LSD-t=5.560,P=0.005,LSD-t=5.354,P=0.006),and was lower in model group and Compound C group compared to medicated serum+Compound C group(LSD-t=4.627,P=0.010,LSD-t=8.677,P=0.001).The relative expression level of Beclin-1 was lower in model group and Compound C group compared to blank group,medicated serum group,and medicated serum+Compound C group(LSD-t=12.912,P=0.000,LSD-t=7.401,P=0.002,LSD-t=5.360,P=0.006;LSD-t=2.950,P=0.042,LSD-t=5.484,P=0.005,LSD-t=3.903,P=0.018),and was lower in medicated serum+Compound C group compared to blank group(LSD-t=26.840,P=0.000).The relative expression level of p62 was low-er in medicated serum group and blank group compared to model group,medicated serum+Compound C group,and Compound C group(LSD-t=3.925,P=0.017,LSD-t=3.985,P=0.019,LSD-t=0.016,P=0.001;LSD-t=3.149,P=0.035,LSD-t=5.094,P=0.007,LSD-t=8.740,P=0.001),and was lower in medicated serum+Compound C group compared to Compound C group(LSD-t=3.455,P=0.026).⑥The relative expression level of p-AMPK was higher in medicated serum group compared to model group and Compound C group(LSD-t=3.623,P=0.022,LSD-t=6.537,P=0.003),and was higher in blank group compared to model group,medicated serum+Com-pound C group,and Compound C group(LSD-t=4.149,P=0.014,LSD-t=2.791,P=0.049,LSD-t=5.734,P=0.004),and was higher in medicated serum+Compound C group compared to Compound C group(LSD-t=5.958,P=0.004).The relative expression level of p-mTOR was lower in blank group,medicated serum group and medicated serum+Compound C group compared to model group(LSD-t=8.722,P=0.001,LSD-t=8.849,P=0.001,LSD-t=5.558,P=0.005),and was lower in medicated serum group and blank group com-pared to medicated serum+Compound C group and Compound C group(LSD-t=4.201,P=0.014,LSD-t=10.030,P=0.001;LSD-t=4.879,P=0.008,LSD-t=9.782,P=0.001),and was lower in medicated serum+Compound C group compared to Compound C group(LSD-t=6.934,P=0.002).Conclusion:JBF medicated serum can increase the viability of chondrocytes and enhance the autophagy abil-ity of chondrocytes via up-regulating the expression of MAPLC3β and Beclin-1 and inhibiting the expression of p62 in postmenopausal rats with KOA.It may work by promoting the expression of GPR30,up-regulating the phosphorylation level of AMPK,down-regulating the phos-phorylation level of mTOR,and activating signaling pathways of GPR30 and AMPK/mTOR.
万方数据
维普
