| Purpose:To observe the activation of signal pathways in Knee cartilage and ligament and the expression of related genes in this pathway in perimenopausal model rats with Knee Osteoarthritis after treatment by Zuogui Wan,and to explore the mechanism of prevention and treatment of knee osteoarthritis by Zuogui Wan.Materials and methods:A total of 40 SPF 8-month-old female rats were fed rat diet without special additives twice a day,with free diet,adequate water and 12-hour circadian rhythm.The formal experiment began one week after the adaptive feeding observation in the laboratory.Ten female rats were randomly selected as normal group(Group A),the remaining30 female rats were randomly divided into three groups: model group(B),Zuoguiwan group(C),and Western medicine group(D),all of which were treated with KOA model.First,the perimenopausal model was established by natural aging method,and then the model was constructed with 0.1ml papain solution with 10%concentration.The normal control group was injected with equal volume sterilization water for injection in the same way,and the model was constructed once every three days for a total of three times for nine days.After each modeling,the rats were forced to exercise for 0.5h,and no human intervention was performed during the rest time.After successful modeling,the four groups were given 1ml distilled water,distilled water,Zuogui Wan suspension and glucosamine sulfate suspension,respectively,by continuous gavage for 4 weeks.After the last intragastric administration,abdominal aorta blood was collected from rats after fasting for 12 hours,and serum was stored at 80℃ after resting,centrifugation and separation.The rats were sacrificed by neck breaking the subcutaneous joints were exposed,soft tissues were obtained.All and part of the knee cartilage and ligaments were collected.The morphologic changes of knee cartilage were observed by HE staining under light microscope;Detection of NF in cartilage and ligament of knee joint in rats by RT-PCR and Western blot-κB inhibitor IκB.The expression level of NALP3 in inflammatory corpuscles,and the content of matrix metalloproteinase-3(MMP-3)was detected by ELISA.Results:1.After HE staining of the rat cartilage on the modeling side,the morphological observation results showed that the model group had more cartilage deprivation,significantly thinning,fewer and scattered chondrocytes,while the Zuoguiwan group and glucosamine sulfate group had less cartilage stripping and slightly reduced chondrocytes.The Zuoguiwan group had less cartilage stripping than glucosamine sulfate group,and the number of chondrocytes was roughly normal.The observation results of the overall motion state of rats showed that Zuoguiwan group > glucosamine sulfate group > simulation group.2.PCR and Western blot results showed that compared with the normal group,the expression level of IκB in the knee cartilage and ligament of the other three groups was decreased,but the expression level of Zuoguiwan group was higher than the other two groups.The expression level of NALP3 in the cartilage and ligament of model mice increased in the other three groups compared with the normal group,but decreased in Zuoguiwan group compared with the other two groups,the differences were statistically significant(P<0.05).3.ELISA results showed that compared with the normal group,the expression level of MMP-3 in the knee cartilage and ligament of the other three modules was increased,and the differences were statistically significant(P<0.05);The expression level of MMP-3 in cartilage and ligament of Zuoguiwan group was down-regulated compared with glucosamine sulfate group,and the difference was statistically significant(P<0.05).Conclusion:1.Zuogui pill can reduce the inflammatory reaction of female rats with knee osteoarthritis during menopause,and can effectively improve the knee cartilage injury of knee osteoarthritis rat model.2.Zuogui pill can reduce the expression level of MMP-3,and the lower its concentration is,the smaller the degree of injury of articular cartilage is.3.Zuogui Pill can reduce cartilage injury and ligament inflammation in knee osteoarthritis rat model,and its therapeutic mechanism may be related to the increase of I κ B.Decreasing the expression level of NALP3 m RNA and protein is related. |
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