Based On AMPK/mTOR/ULK-1 Signaling Pathway The Effect Of Gulu Erxian Jiao On Autophagy Of Chondrocytes In Mice Was Investigated

PurposeThe effect of Gulu Erxian Jiao decoction on AMPK/m TOR/ULK-1 signaling pathway in mouse knee chondrocytes was observed in the treatment of knee osteoarthritis(KOA)model mice,and the related mechanism of this formula on KOA was explored,providing theoretical basis for its clinical application.Methods1.Establishment and grouping of KOA model mice:The medial meniscus instability(DMM)operation was used to construct The KOA model.Thirty-six C57BL/6 mice were randomly divided into sham operation group and model group by random number table method.The KOA mouse model was identified by knee mri.After the model was successfully constructed,the model group was randomly divided into operation group and Gulu Erxian jiao group.2.Model identification:3 weeks later,3 mice were selected from the sham operation group and the model group respectively for knee mri scanning.Combined with imaging images,MOAKS score was used to determine whether the modeling was successful.3.Drug intervention:Mice in sham operation group and operation group were intragastric with normal saline,and mice in Gulu Erxian Jiao group were intragastric with Gulu Erxian Jiao decoction.4.Morphological observation of knee joint in mice:The degree of cartilage damage was evaluated by ferro red solid green staining;TUNEL staining was used to detect cell apoptosis in the cartilage tissue of the knee joint.5.Detection and observation of cartilage tissue indicators:MRNA expression levels of AMPK,m TOR,ULK-1,Atg5,LC3 B and PARP-P85 in cartilage tissues of mice in each group were detected by RT-QPCR.The protein expression levels of AMPK,m TOR,ULK-1,Atg5,LC3 B and PARP-P85 in cartilage tissues of mice in each group were detected by Western-blot,and the data were analyzed.Results1.Identification of KOA mouse modelIn the sham operation group,the surface of knee cartilage was smooth and neat,the joint space was equally wide,the rectus femoris tendon was normal without edema,there was a small amount of synovial fluid in the joint cavity,and no soft tissue swelling around the joint.In the model group,there was fluid in the knee cavity and soft tissue swelling around the joint.The semi-quantitative MRI score of knee joint in model group was significantly higher than that in normal group,and the difference was statistically significant(P < 0.05).2.Effect of Gulu Erxian Jiao Decoction on knee cartilage in KOA miceThe results showed that the surface of knee cartilage tissue of mice in the operation group was incomplete,the color of the knee cartilage was not uniform,the arrangement of chondrocytes was disorderly and irregular,and the proliferation of chondrocytes appeared.The surface of knee cartilage of mice in Gulu Erxian Jiao group was relatively intact,with uniform color of Muscovy,orderly arrangement of chondrocytes and no proliferation of chondrocytes.Tunel staining showed that there were more Tunel positive cells(apoptotic chondrocytes)in the knee tissues of the operation group.There was only a small amount of apoptosis in Gulu Erxian Jiao group,and the difference was statistically significant.3.Effects of Gulu Erxian Jiao Decoction on mTOR and PARP-P85 factors in knee cartilage of KOA miceReal-time quantitative PCR and Western blot showed that compared with the sham operation group,the expression levels of m TOR and PARP-P85 in knee cartilage tissues in the operation group were significantly increased.Compared with the operation group,the m TOR and PARP-P85 of knee cartilage in Gulu Erxian Jiao group were significantly decreased,and the difference was statistically significant.4.Effects of Gulu Erxian Jiao Decoction on AMPK,ULK-1,Atg5 and LC3 B factors in knee cartilage of KOA miceReal-time quantitative PCR and Western blot showed that the expression levels of AMPK,ULK-1,Atg5 and LC3 B in knee cartilage tissues in the operation group were significantly lower than those in the sham operation group.Compared with the operation group,AMPK,ULK-1,Atg5 and LC3 B in the knee cartilage tissue of Gulu Erxian Jiao group were significantly increased,and the difference was statistically significant.Conclusion1.Gulu Erxian Jiao can enhance the autophagy of chondrocytes,inhibit the apoptosis of chondrocytes and protect articular cartilage.2.The mechanism of Treatment of KOA by Gulu Erxian Jiao is to inhibit the e xpression of m TOR and enhance the activities of AMPK and ULK-1,therebyactivating AMPK/m TOR/ULK-1 signaling pathway and causing chondrocyte autophagy.