| ObjectiveIn this study,Yanghe decoction were used to intervene in knee osteoarthritis(KOA)model rabbits,and we observed the effects on cartilage degeneration and PI3K/Akt/Bcl-2signaling pathway in cartilage of Yang deficiency and cold coagulation knee osteoarthritis rabbits,and the mechanism of Yanghe decoction inhibiting KOA cartilaged degeneration was explored,which provided a theoretical basis for clinical application and subsequent research.Methods1 Establishment of model of rabbit knee osteoarthritis with yang deficiency cold coagulation: In this experiment,the combination model of disease evidence was used,papain injection was injected into the joint cavity to establish knee osteoarthritis,and peppermint oil was superimposed on a 4℃ artificial climate box to establish the yang deficiency cold coagulation type.Except for the blank control group,the same molding method was adopted for the 27 New Zealand white rabbits in the module.On the 1st,4th and 7th days of the first week,papain was injected to establish the KOA model,and continuously placed in a constant temperature and humidity incubator at 4 ℃ for 1.5 hours every day for 4 weeks,and0.65 m L/(kg·d)peppermint oil was given for 2 weeks of continuous gavage for 2 weeks to establish a yang deficiency and cold coagulation model.2 Model identification: After 4 weeks of molding,3 specimens were selected from the blank control group and 3 from the model group to evaluate the TCM syndrome and the modified Mankin’s score of knee cartilage,and the articular cartilage showed pathological changes and 1 or more rabbit deficiency symptoms and cold symptoms,or 2 or more ldelayed symptoms and mild symptoms.3 Experimental grouping: After determining the success of molding,the modules were randomly divided into model group,inhibitor group,Yanghe decoction+inhibitor group,Yanghe decoction group,with 6 animals in each group.4 Drug intervention: blank control group and model group were given gavage saline;Yanghe decoction groups are given gavage Yanghe decoction;The inhibitor group was given intraperitoneal injection of PI3 K inhibitor LY294002 in rabbits;The Yanghe decoction+inhibitor groups were first subjected to intraperitoneal injection of PI3 K inhibitor LY294002,followed by Yanghe decoction gavage.5 TCM symptom assessment: On the 28 th day of the first modelling intervention and 1day after the end of medication,each group of rabbits were evaluated for TCM symptoms.6 Morphological observation:HE staining was used to observe the cartilage tissue damage of knee joints in each group;The TUNEL method detects chondrocytes apoptosis.7 Cartilage tissue detection: RT-q PCR detected the expression of PI3 K,AKT,Bcl-2,Bax,Caspase-3 and Caspase-9 m RNA in rabbit cartilage tissues in each group;Western Blot method detected the expression of PI3 K,p-AKT,Bcl-2,Bax,Caspase-3 and Caspase-9 proteins in cartilage tissues.The data groups were compared and analyzed.Results1 Model identification results of rabbit knee osteoarthritis with yang deficiency and cold coagulation28 days after the first modeling,the total score of Mankin’s score in the model group was≥7 points,and the average score of Mankin’s score was 7.67 points,and there were 1 or more false symptoms and cold symptoms,and 2 or more delayed symptoms and mild symptoms,and the average score of TCM symptom assessment was 88.83.2 TCM syndrome scoring results in each groupAfter the intervention of Yanghe decoction,the TCM syndrome score of Yanghe decoction and the Yanghe decoction+inhibitor Group were significantly improved.There was no significant difference from the normal group,while the symptoms of yang deficiency and cold coagulation were not improved in the inhibitor group and the model group,and the TCM symptom score was significantly higher than that of the blank control group(P<0.05),and the Yanghe decoction+inhibitor group had limited lower limb mobility,improved spirit,and slightly curled up and quiet,and compared with the model group,the symptoms of yang deficiency and cold coagulation were significantly improved(P<0.05),indicating that Yanghe decoction could significantly improve the symptoms of yang deficiency and cold coagulation.3 Effects of Yanghe decoction on pathological morphology of knee cartilage in KOA model rabbits and Mankin’s score resultsIn the blank control group,the knee cartilage surface was smooth,the chondrocytes were arranged neatly,the cartilage tide line was complete,and the cartilage thickness was uniform.The surface of knee cartilage in the model group and inhibitor group was uneven,the number of chondrocytes decreased,the tide line receded significantly,and the cartilage thickness was uneven.The surface of chondrocytes in the Yanghe decoction groups was uneven,there was no obvious destruction,the thickness of cartilage was uniform,a small amount of collagen fibers in the cartilage matrix was hyperplasia,the number of chondrocytes was not significantly reduced,the cartilage tide line moved back,and the cartilage thinned;The cartilage surface of the Yanghe decoction+inhibitor group was uneven,the thickness of cartilage was uneven,the arrangement of chondrocytes was still neat,the number of chondrocytes decreased,the cartilage tide line shifted back,and the cartilage thinned.Compared with the blank control group,the Mankin’s score in the model group and inhibitor group was significantly increased(P<0.05),and the score in the inhibitor group was more obvious(P<0.05).Compared with the model group and inhibitor group,the Mankin’s score of Yanghe decoction and Yanghe decoction+inhibitor group was reduced(P<0.05),and the decrease was more obvious in the Yanghe decoction group(P<0.05),indicating that Yanghe decoction could effectively delay the degenerative changes of articular cartilage.4 TUNEL method to detect chondrocytes apoptosisAfter staining by the TUNEL method,apoptotic chondrocytes show yellow-green fluorescence under the microscope and normal chondrocytes show blue fluorescence.The ratio of the number of apoptotic cells to the total number of cells is the chondrocytes apoptosis rate.The results showed that there was a very small amount of yellow-green fluorescence in the blank control group,a significant increase in blue-green fluorescence in the model group and inhibitor group,and a small amount of blue-green fluorescence in cartilage tissue in the Yanghe decoction group and the Yanghe decoction+ inhibitor groups.Compared with the blank control group,the apoptosis rate of chondrocytes in the model group and inhibitor group increased significantly(P<0.05),and the increase in the inhibitor group was more obvious(P<0.05).Compared with the model group and inhibitor group,the apoptosis rate of chondrocytes in the Yanghe decoction group and the Yanghe decoction+inhibitor groups was significantly reduced(P<0.05).5 RT-q PCR observed the expression levels of PI3 K,AKT,Bcl-2,Bax,Caspase-3 and Caspase-9 m RNA in the knee cartilage tissues of each groupCompared with the blank group,the expression of PI3 K,AKT and Bcl-2 m RNA of cartilage in the model group and inhibitor group was significantly reduced,the expression of Bax,Caspase-3 and Caspase-9 m RNA was significantly increased(P<0.05),the expression of PI3 K,AKT and Bcl-2 m RNA in the rabbit articular cartilage tissue of the Yanghe decoction group and the Yanghe decoction+inhibitor groups were significantly increased,and the expression of Bax,Caspase-3 and Caspase-9 m RNA was significantly reduced(P<0.05)。6 Western Blot detected the expression levels of PI3 K,p-AKT,Bcl-2,Bax,Caspase-3 and Caspase-9 proteins in the knee cartilage tissues of each groupCompared with the normal group,the expression of PI3 K,p-AKT and Bcl-2 proteins in cartilage in the model group and inhibitor group was significantly reduced,the expression of Bax,Caspase-3 and Caspase-9 proteins was significantly increased(P<0.05),the expression of PI3 K,p-AKT and Bcl-2 proteins in the rabbit articular cartilage tissue in the Yanghe decoction group and the Yanghe decoction+inhibitor groups were significantly increased,and the expression of Bax,Caspase-3 and Caspase-9 proteins decreased(P<0.05).ConclusionsYanghe Decoction can effectively inhibit the degenerative changes of cartilage tissue in a rabbit model of yang deficiency and cold coagulation knee osteoarthritis,and its mechanism is related to the regulation of PI3K/Akt/Bcl-2 signaling pathway to inhibit chondrocytes apoptosis. |
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