| Objective: Osteoarthritis(OA)is a common degenerative disease of the knee joint,the second most disabling disease in the world,and causes heavy economic burden in the society.The incidence of OA is related to age,obesity,gender,trauma and so on.In recent years,with the increase in the prevalence of obesity and the aging of the population,the number of patients with OA has also increased significantly,which has become a public health issue of great concern.However,the pathogenesis of OA is still unclear.Autophagy is a complex process involved in senescence or the degradation of malfunctioning organelles within cells.Dysregulation of autophagy is associated with certain cancers,neurodegenerative diseases,immune dysfunction,and aging,which is a potential therapeutic target for many diseases.Numerous studies have shown that autophagy plays a crucial role in the development of OA.micro RNA-33a(miR-33a)is an intronic micro RNA located in the sterol regulatory element-binding protein gene and is the target of some key autophagy genes,suggesting that miR-33 a may play an important regulatory role in autophagy.The PI3K/AKT/mTOR pathway is closely related to the development of OA.Inhibiting the PI3K/AKT/mTOR signaling pathway can promote autophagy and reduce inflammation in articular chondrocytes of OA rats.The AMPK/mTOR pathway can also affect the development of OA.It was found to effectively alleviate cartilage degeneration and aging by regulating the AMPK/mTOR signaling pathway.In this study,through animal and clinical sample studies combined with in vitro experiments,we explored the mechanism by which miR-33 a regulates PI3K/AKT/mTOR and AMPK/mTOR signaling pathways to mediate autophagy,thereby affecting the occurrence and development of OA.New therapeutic targets provide a rationale.Methods: 1.In animal experiment part,male C57BL/6 mice were randomly divided into the control group(sham group)and the medial meniscus destabilization surgery(DMM)group,and the mice in the sham group were subjected to sham surgery at 18 weeks of age.The joint capsule was incised and sutured,and the incision was sutured after the medial meniscus instability operation in the DMM group.All the mice were sacrificed 8weeks after the operation,and the medial and lateral condyle cartilage of the mouse femur was taken for further experimental research.qRT-PCR was used to detect the expression level of miR-33 a in the articular cartilage of mice;Safranin O-fast green staining was used to observe the damage of cartilage tissue in mice;immunofluorescence staining and Western Blot were used to detect the relative expression levels of autophagy-related proteins Beclin1,p62 and LC3;immunofluorescence staining and Western Blot were used to detect PI3K/AKT The expression levels of proteins related to the /mTOR and AMPK/mTOR signaling pathways were altered.2.Clinical part.Knee articular cartilage samples from OA and non-OA patients were collected,and qRT-PCR was used to detect the expression level of miR-33 a in human articular cartilage;Safranin O-fast green staining was used to observe the damage of human cartilage tissue;Immunofluorescence staining and Western Blot method were used to detect the related expression levels of autophagy-related proteins Beclin1,p62 and LC3;immunofluorescence staining and Western Blot method were used to detect the changes in the expression levels of related proteins in PI3K/AKT/mTOR and AMPK/mTOR signaling pathways.3.In vitro experiments,using SW1353 cell line,treated cells with IL-1β at concentrations of 10,20,and 40 n M,and collected cell samples at 24 h,48 h,and 72 h;qRT-PCR was used to detect the expression level of miR-33 a in SW1353 cells The expression levels of autophagy-related proteins Beclin1,P62,and LC3 were detected by Western Blot;after determining the optimal treatment time and treatment concentration,SW1353 cells treated with 20 n M IL-1β were transfected with miR-33 a mimics and inhibitors.Cell samples were collected at 48 h and 72 h to detect the changes in the expression levels of autophagy-related proteins and the expression levels of PI3K/AKT/mTOR and AMPK/mTOR signaling pathways-related proteins;the mTOR activator MHY1485 was used to inhibit autophagy,and then The mimics of miR-33 a were subjected to recovery experiments,and cell samples were collected at 48 h;Western Blot was used to detect the expression levels of autophagy-related proteins Beclin1,p62,and LC3 after high and low expression of miR-33a;Western Blot was used to detect PI3K/ Changes in AKT/mTOR and AMPK/mTOR signaling pathways.Result: 1.Results of animal experiments: The knee joint cartilage tissue of mice in sham group was intact,with uniform thickness,and the boundary between cartilage tissue and bone tissue was clear.The cartilage tissue of the knee joint of mice in the DMM group showed severe damage,different thicknesses,and unclear demarcation between cartilage and bone.Compared with the sham group,the expression level of miR-33 a in knee articular cartilage of mice in DMM group was significantly increased(p<0.05);the expression levels of autophagy-related proteins Beclin1 and LC3 were significantly increased(p<0.01),and the expression level of p62 protein was significantly increased(p<0.01).Significantly decreased(p<0.01);the expression levels of p-PI3 K,p-AKT,and p-AMPK were significantly increased(p<0.01),and the expression level of p-mTOR was significantly decreased(p<0.01).2.Experimental results of clinical samples: The surface cells of the articular cartilage in the CON group were neatly arranged,the surface of the cartilage tissue was smooth and flat,and the extracartilage matrix was evenly stained.However,in the OA group,the surface cells of the cartilage tissue were chaotically arranged,the surface of the cartilage tissue was rough and uneven,and the staining of the extracartilage matrix was uneven.Compared with the CON group,the expression level of miR-33 a in knee articular cartilage of OA patients was significantly increased(p<0.05);the expression levels of autophagy-related proteins Beclin and LC3 were significantly increased(p<0.01),and the expression level of p62 protein was significantly decreased(p<0.01,p<0.05);the expression levels of p-PI3 K,p-AKT,and p-AMPK were significantly increased(p<0.05),and the expression level of p-mTOR was significantly decreased(p<0.05).3.In vitro experimental results: 3.1 After treating SW1353 cells with different concentrations of IL-1β(10,20,40 n M)for 24 h,48h,and 72 h,it was found that compared with the CON group,the autophagy-related proteins Beclin1 and LC3 in the IL-1β group were higher than those in the CON group.The expression increased significantly at 48h(p<0.05);the expression decreased significantly at 72h(p<0.05).The expression of p62 protein showed a completely opposite trend.Compared with the CON group,the expression of p62 protein in the IL-1β group decreased significantly at 48h(p<0.05)and increased significantly at 72h(p<0.05).3.2 Compared with the CON group,the expression level of miR-33 a in SW1353 cells treated with IL-1β was significantly increased at 48h(p<0.05)and decreased significantly at 72h(p<0.05).3.3 The effects of miR-33 a mimic and inhibitor on SW1353 cells treated with IL-1β for 48 h,it was found that the expression level of miR-33 a in the IL-1β+mimic group was significantly higher than that in the IL-1β group(p<0.05);autophagy-related proteins The expression level of LC3 was significantly increased(p<0.05),and the expression level of p62 was significantly decreased(p<0.05).The expression level of miR-33 a in the IL-1β+inhibitor group was significantly lower than that in the IL-1β group(p<0.01);the expressions of autophagy-related proteins Beclin1 and LC3 were significantly decreased(p<0.05).3.4After IL-1β treatment of cells for 48 h,the protein expression levels of p-PI3 K,p-AKT and p-AMPK in the IL-1β group were significantly increased(p<0.05),and the expression of p-mTOR was significantly decreased(p<0.05).After treating cells with IL-1β for 48 h,the low expression of miR-33 a caused by miR-33 a inhibitor can cause corresponding changes in the expression levels of PI3K/AKT/mTOR pathway and AMPK/mTOR pathway.However,overexpression of miR-33 a using miR-33 a mimic did not cause changes in the corresponding level of the pathway.3.5 After 72 h of IL-1βtreatment,the expression levels of p-PI3 K,p-AKT and p-AMPK in the IL-1β group were significantly decreased(p<0.05),and the expression level of p-mTOR was significantly increased compared with the CON group(p<0.05,p<0.05);after treating cells with IL-1βfor 72 h,the overexpression of miR-33 a caused by miR-33 a mimic can cause changes in PI3K/AKT/mTOR pathway and AMPK/mTOR pathway.However,the low expression of miR-33 a by miR-33 a inhibitor did not cause the corresponding level change of the pathway.3.6 After using the mTOR activator MHY1485,the expressions of mTOR and p62 proteins were significantly increased(p<0.05),and the expressions of Beclin1 and LC3 proteins were significantly decreased(p<0.05).After adding mimic of miR-33 a,the expressions of mTOR and p62 The expression of Beclin1 and LC3 protein increased(p<0.05).Conclusion: 1.In human cartilage and mouse cartilage,the expression of miR-33 a in OA tissue was significantly higher than that in normal tissue.2.In the OA model induced by IL-1β,the expression of miR-33 a in chondrocytes increased first and then decreased;3.The expression of miR-33 a in chondrocytes was positively correlated with autophagy;Regulation of PI3K/AKT/mTOR and AMPK/mTOR signaling pathways regulates chondrocyte autophagy,which in turn affects the progression of OA. |
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