Evaluation Of Kidney-yang Deficiency Knee Osteoarthritis Rat Model And Changes In Chondrocyte Autophagy Level

Osteoarthritis(OA)is mainly characterized by cartilage degeneration and destruction,which often involves weight-bearing joints,and knee joint is one of the most commonly involved joints.The prevalence of OA increased with age.Studies have shown that the autophagy level of cells in normal people decreases with age,while the occurrence and progression of OA is accompanied by the decrease of autophagy level of chondrocytes.Therefore,the older the age.the lower the autophagy level of chondrocytes,the higher the risk of OA disease or the more serious the disease.OA belongs to the category of "gu bi" in traditional Chinese medicine.With aging,kidney-yang deficiency gradually appears in the human body.Therefore.we consider whether kidney-yang deficiency is related to the decreased autophagy level of chondrocytes.The purpose of this study was to establish a Knee Osteoarthritis(KOA)rat model by Hulth method:?To establish and evaluate a compound model of KOA rats with kidney-yang deficiency;?To explore the relationship between the change of autophagy level of chondrocytes and kidney Yang deficiency.The first research part Establishment and evaluation of KOA rat model of kidney-yang deficiencyObjective To establish and evaluate of KOA rat model of kidney-yang deficiencyMethods Male SD rats were randomly divided into normal group,KOA group.castrated group(Hulth+castrate)and adenine group(Hulth+adenine gavage).with 6 rats in each group.KOA model was established by Hulth method and kidney-yang deficiency model was established by castrating method and adenine intragastric administration.The KOA model was evaluated by Mankin's score and OARSI grading.Evaluation table of symptoms and signs of kidney-yang deficiency,hormone levels of thyroid axis,gonad axis and adrenal axis were used to evaluate kidney-yang deficiency model.Results The KOA group.castration group,and adenine group were all in line with the KOA pathological manifestations.Among them,the Mankin's score in the adenine group was the highest(P<0.05),and the OARSI classification was level 3;the Mankin's score in the castration group was in the middle(P<0.05),and the OARSI grade was 2;KOA group Mankin's score was the lowest(P<0.05),and the OARSI grade was 1.Compared with the other three groups,the adenine group had the highest score of symptoms and signs of kidney-yang deficiency,the least weight gain,the lowest anal temperature,the lowest levels of T3,T4,FSH,LH,T,and the highest level of E2(P<0.05);There was no significant difference in CORT level among all groups(P>0.05).Conclusion ?Compared with Hulth+castrate method.Hulth+adenine gavage is a more suitable method to construct a compound model of KOA combined with syndrome of kidney-yang deficiency.?Kidney-yang deficiency may aggravate the degeneration of KOA cartilage,and the more severe the kidney-yang deficiency is,the more serious the degeneration of KOA cartilage is.The second research part The relationship between the change of chondrocyte autophagy and the deficiency of kidney-yangObjective To explore the relationship between autophagy level of chondrocytes and kidney-yang deficiency in KOA ratsMethods Male SD rats were randomly divided into normal group,KOA group,castrated group(Hulth+castrate)and adenine group(Hulth+adenine gavage),with 6 rats in each group,KOA model was established by Hulth method and kidney-yang deficiency model was established by castrating method and adenine intragastric administration.Quantitative Real-time PCR(qPCR),Western Blot(WB)and immunohistochemistry(IHC)methods were used to detect chondrocyte autophagy-related genes(Beclin1,Lc3,P62)and protein(Beclin1,LC3,P62)expression.Results qPCR:?P62 gene:Compared with the normal group and adenine group,the expression level of P62 gene in the KOA group was the lowest(P<0.01);compared with the castration group,the expression level of P62 in the KOA group was not statistically significant.?Beclin1 gene:Compared with the castration group and adenine group,the expression level of Beclin1 gene in the KOA group was the highest(P<0.01);there was no significant difference in Beclin1 expression between the KOA group and the normal group.?Lc3 gene:Compared with the castration group and adenine group,Lc3 expression level was the highest in the KOA group(P<0.05);there was no significant difference in Lc3 gene expression between the KOA group and the normal group.WB:?P62 protein:There was no significant difference in P62 expression among all groups.?Beclin1 protein:Compared with normal group and adenine group,the expression level of Beclin1 in KOA group was the highest(P<0.01);compared with the castration group,the Beclin1 protein expression level in the KOA group was higher(P<0.05).?LC3-?/LC3-?:Compared with the normal group and adenine group,the level of LC3-?/LC3-? in the KOA group was the highest(P<0.01):compared with the castration group,LC3-?/LC3-? level is higher in the KOA group(P<0.05).IHC:?P62 protein:There was no significant difference in the expression of P62 protein among all groups.?Beclin1 protein:Compared with the normal group,the castration group and the adenine group,the expression of Beclin1 in the KOA group was the highest(P<0.05).?LC3 protein:Compared with the normal group,castration group,and adenine group,the expression level of LC3 protein in the KOA group was the highest(P<0.01).Conclusion ?Compared with the castration group and adenine group,the knee joint chondrocyte autophagy level in the KOA group was the highest;?The change in the knee joint chondrocyte autophagy level may be related to the kidney-yang deficiency,and with the aggravation of the kidney-yang deficiency,the autophagy level of chondrocytes decreased.