牛膝醇提物对实验兔膝骨关节炎模型AMPK/Wnt/MAPK信号通路串话的影响

Effect of Alcohol Extract of Niuxi(Achyranthis Bidentatae Radix)on Crosstalk of AMPK/Wnt/MAPK Signaling Pathway in Experimental Rabbit Knee Osteoarthritis Model

目的:探究牛膝醇提物对实验兔膝骨关节炎(OA)模型AMPK/Wnt/MAPK信号通路串话的影响。方法:将64只实验兔按照随机数字表法分为造模组50只、空白对照组(未造模)14只,造模6周后,从造模组和空白对照组各随机取2只实验兔进行OA模型验证;造模成功后,将造模组48只实验兔随机分为模型对照组、牛膝醇提物低剂量组、牛膝醇提物中剂量组、牛膝醇提物高剂量组,每组12只。各组连续灌胃给药6周后,HE染色法观察软骨组织及病理学改良Mankin’s评分;免疫组化检测膝关节软骨AMPK、ERK、JNK、p38、COX-2、β-Catenin蛋白表达变化;Western blotting检测关节软骨AMPK、ERK、JNK、p38、COX-2、β-Catenin蛋白表达情况。根据https://string-db.org/蛋白互作网址预测牛膝醇提物对AMPK/Wnt/MAPK信号通路关键蛋白互作网络机制图。结果:HE染色提示模型对照组血管入侵潮线结构、软骨细胞数量明显减少,软骨细胞排列纹理、基质染色变浅,牛膝醇提物低、中、高剂量组局部软骨表面可见不规则裂隙,达钙化层,软骨细胞数量增多,基质染色呈浅红色,潮线结构稍正常。病理学改良Mankin’s评分显示,模型对照组改良Mankin’s评分高于空白对照组(P<0.01),牛膝醇提物低、中、高剂量改良Mankin’s评分低于模型对照组(P<0.05)。免疫组化和Western blotting检测显示,模型对照组兔膝关节软骨ERK、JNK、p38、COX-2、β-Catenin蛋白表达明显高于空白对照组(P<0.05),AMPK蛋白表达与空白对照组比较,差异无统计学意义(P>0.05);牛膝醇提物低、中、高剂量组兔膝关节软骨ERK、JNK、p38、COX-2、β-Catenin蛋白表达明显低于模型对照组(P<0.05);牛膝醇提物高剂量组兔膝关节软骨AMPK蛋白表达明显高于模型对照组(P<0.05),牛膝醇提物低、中剂量组兔膝关节软骨AMPK蛋白表达与模型组比较,差异均无统计学意义(P>0.05)。String蛋白互作预测图显示Akt家族蛋白节点最多,Mapk家族蛋白节点次之。结论:牛膝醇提物能改善兔膝骨关节炎模型病理评分,其中牛膝醇提物高剂量组能有效上调AMPK蛋白表达,下调ERK、JNK、p38、COX-2、β-Catenin蛋白表达;Akt家族蛋白、Mapk家族蛋白在牛膝醇提物对AMPK/Wnt/MAPK信号通路网络互作之间的联系起到了关键桥接蛋白作用,其作用机理有待进一步研究。

Objective:To explore the effect of alcohol extracts of Niuxi(Achyranthis Bidentatae Radix)on crosstalk of AMPK/Wnt/MAPK signaling pathway in experimental rabbit knee osteoarthritis(OA)model.Methods:According to the random number table method,64 rabbits were randomly divided into model group(n=50)and blank control group(n=14).After 6 weeks of modeling,2 rabbits were randomly selected from the model group and blank control group for OA model validation.After successful modeling,48 experimental rabbits were randomly divided into model control group,alcohol extract of Niuxi(Achyranthis Bidentatae Radix)low-dose group,alcohol extract of Niuxi(Achyranthis Bidentatae Radix)medium-dose group,and alcohol extract of Niuxi(Achyranthis Bidentatae Radix)high-dose group,with 12 rabbits in each group.After 6 weeks of continuous intragastric administration,the cartilage tissue and pathological modified Mankin's score were observed by HE staining method.The expression of AMPK,ERK,JNK,p38,COX-2,β-Catenin protein in knee articular cartilage was detected by immunohistochemistry.Western blotting was used to detect the expression of AMPK,ERK,JNK,p38,COX-2,β-Catenin protein in articular cartilage.The network mechanism diagram of key protein interactions of AMPK/Wnt/MAPK signaling pathway was predicted by using the website https://string-db.org/protein interaction.Results:HE staining showed that the vascular invasion tidal line structure,the number of chondrocytes decreased significantly,the arrangement texture of chondrocytes and the staining of matrix became shallow in the model control group.Irregular fissures were seen on the cartilage surface,reaching the calcified layer,the number of cartilage cells increased,the matrix staining was light red,and the tide line structure was slightly normal in alcohol extract of Niuxi(Achyranthis Bidentatae Radix)low,medium and high-dose group.Pathological modified Mankin's score showed that the modified Mankin's score of the model control group was higher than that of the blank control group(P<0.01).The modified Mankin's score of alcohol extract of Niuxi(Achyranthis Bidentatae Radix)low,medium and high-dose group was lower than the model control group(P<0.05).Immunohistochemistry and Western blotting showed that the expressions of ERK,JNK,p38,COX-2,and β-Catenin in rabbit knee cartilage in the model control group were significantly higher than those in the blank control group(P<0.05),and the expression of AMPK protein had no significant difference between the two groups(P>0.05).The expression of ERK,JNK,p38,COX-2 and β-Catenin protein in rabbit knee articular cartilage in alcohol extract of Niuxi(Achyranthis Bidentatae Radix)low,medium and high-dose group was significantly lower than that of the model control group(P<0.05).The AMPK protein expression of rabbit knee articular cartilage in alcohol extract of Niuxi(Achyranthis Bidentatae Radix)high-dose group was significantly higher than that of the model control group(P<0.05).There was no significant difference in the expression of AMPK protein among the model control group,alcohol extract of Niuxi(Achyranthis Bidentatae Radix)medium-dose group and alcohol extract of Niuxi(Achyranthis Bidentatae Radix)high-dose group.According to the String protein interaction prediction graph,Akt family protein nodes are the most,followed by Mapk family protein nodes.Conclusion:The alcohol extract of Niuxi(Achyranthis Bidentatae Radix)can improve the pathological score of rabbit knee osteoarthritis model.The high-dose alcohol extract of Niuxi(Achyranthis Bidentatae Radix)can effectively up-regulate the expression of AMPK protein and down-regulate the expression of ERK,JNK,p38,COX-2,and β-Catenin protein.Akt family proteins and Mapk family proteins play a key role in bridging the relationship between the alcohol extracts of Niuxi(Achyranthis Bidentatae Radix)and AMPK/Wnt/MAPK signaling pathway network interaction.The mechanism of action needs to be further studied.