| Objectives: Osteoarthritis(OA)is a common age-related chronic joint disease that affects a significant number of middle-aged and elderly people.It mainly manifested as severe pain and movement disorders.The complicated pathogenesis and limited treatment methods result in immeasurable decreased quality of life of patients and socio-economic burden.At present,with the aging of population in our country,people's requirements for quality of life are increasing.The incidence and visiting rate of OA are getting higher and higher.On the basis of the current research,the pathogenesis of the disease needs to be further explored,and it is of great significance to seek cost-effective treatment means.Chondrocytes are poorly regenerated and repaired,so maintaining the homeostasis of chondrocytes plays a key role in the process of joint degeneration and OA.Autophagy plays an important role as an important cellular homeostasis mechanism in eukaryotic cells.Currently,the study of OA and autophagy remains on non-selective autophagy.In recent years,BAG3-mediated chaperone-assisted selective autophagy(CASA)has been shown to be a novel selective autophagy pathway.Its main role is to identify the misfolded protein for degradation and reuse,which has been proven to play a key role in many diseases.The treatment effect of moderate exercise to OA has been proved in many documents,but the molecular mechanism is not yet completely revealed.To explore the changes of CASA expression in chondrocytes and OA models with or without exercise intervention will help us to further understand the pathogenesis of OA and the molecular mechanism of exercise in the treatment of OA and provide a theoretical basis for further study.Methods: Part One: The role of BAG3-mediated selective autophagy pathway in rat primary chondrocytesRat primary chondrocytes were used to study the CASA pathway-related protein expression and gene transcription level changes by Western blot and RT-qPCR after monosodium iodoacetate(MIA)intervention at different concentrations and different times.The same method was used to detect the above changes in protein and gene transcription levels after Bafilomycin A1(Baf A1)and MIA intervention.The changes of each marker under different treatment conditions were compared and analyzed.Part Two: The effect of exercise treatment on CASA pathway in cartilage of knee articular of OA model rats.The animal model of OA was established by MIA injecting in the knee joint of SD rats,and then the moderate-intensity exercise training was performed.After 4 weeks,the rats were sacrificed,and the degree of cartilage damage was analyzed by histological staining and scoring.Inflammatory cytokine changes in serum and intra-articular lavage fluid(IALF)were detected by ELISA assay.Changes of key proteins of cartilage matrix were detected by Western blot and immunohistochemistry.Western blot and RT-qPCR methods were used to detect the expression of CASA pathway-related proteins and the changes of gene transcription.Finally,the differences among groups were compared and analyzed.Results: Part One: The role of BAG3-mediated selective autophagy pathway in rat primary chondrocytes(1)In the immunofluorescence staining of MIA,type II collagen were significantly reduced in both MIA intervention groups compared with the control group,whereas the ADAMTS5 was significantly increased.There's no significant difference between the MIA intervention groups.(2)SQSTM1 and LC3 B expression increased in both intervention groups compared with the control group,but the difference was not obvious between the two groups.The expression of HSPA8 and HSPB8 did not change significantly in the control and MIA short term intervention groups but decreased in the MIA long term intervention group.BAG3 in MIA short term intervention group was significantly reduced and this decrease was alleviated in the MIA long term intervention group.There was no significant change in FLNA expression among groups.(3)Compared to the control group,changes of CASA-related mRNA in two intervention groups were similar except for HSPA8.BAG3,SQSTM1 and LC3 B were significantly higher than the control group.Compared with the MIA short term intervention group and the control group,HSPA8 was increased statistically in the MIA long term intervention group.(4)CASA-related proteins were significantly increased in the group containing Baf A1 except for FLNA.Compared with Baf A1 with MIA co-intervention group,HSPA8,HSPB8 and SQSTM1 have an increasing trend but not statistically significant in MIA group,whereas expression of LC3 II was significantly increased.(5)All CASA-related genes were significantly increased in MIA group and decreased when Baf A1 added.The FLNA was not increased statistically.Part Two: The effect of exercise treatment on CASA pathway in cartilage of knee articular of OA rats.(1)HE and toluidine blue staining suggested that the OA group had more severe arthritis than the OAE group.The OARSI and Modified Mankin score suggested that the pathology of OA was aggravated in the OA group and was relieved in the OAE group with statistically significant differences.(2)The level of IL-1? in OA group was the highest in serum and IALF,and decreased in OAE group,while the expression of IL-4 was opposite.The differences were statistically significant.(3)The expression of type II collagen was the strongest in the control group,weakest in the OA group,and relieved in the OAE group.The level of ADAMTS5 is opposite.All differences were statistically significant.(4)LC3B was widely expressed in the superficial zone(SZ)and deep zone(DZ)of the normal cartilage,but significantly decreased in the OA group and increased in the OAE group.These changes were statistically significant.SQSTM1 were gradually increased in the C group,OA group and OAE group both in SZ and DZ,and statistically significant.Compared with the control group,the expression of BECLIN1 of different regions in OA and OAE groups increased significantly,but there was no significant difference between the two groups.(5)The expression of HSPA8,HSPB8 and LC3 II in the OA group decreased obviously and alleviated in the OAE group.There's no significant difference between the OA group and control about BAG3 expression,but significantly increase showed in the OAE group.SQSTM1 and FLNA were significantly elevated in the OA and OAE groups.(6)The mRNA levels of BAG3,FLNA,SQSTM1 and LC3 B increased significantly in OA and OAE groups,and LC3 B and SQSTM1 increased more obviously in OAE group.In the OA and OAE group,HSPA8 and HSPB8 had an increased trend but no statistically significant existence.Conclusions:(1)MIA stimulates of certain concentration cause changes in the phenotype of chondrocytes,especially for the synthesis and secretion of cartilage matrix components and the functional changes of degradation.(2)MIA stimulates of certain concentration can increase the activity of BAG3-mediated CASA pathway.And the autophagic activity caused by different concentration and time of intervention are not exactly the same.(3)The intervention of Baf A1 can result in protein accumulation about BAG3-mediated CASA pathway.This effect has an enhanced tendency when co-intervention by Baf A1 and MIA.(4)Moderate exercise intervention can relieve the injury of articular cartilage caused by injection of MIA in the knee joint in rats.(5)Moderate exercise intervention can upregulate the expression of classic autophagy markers after intra-articular injection of MIA at different zones of articular cartilage.(6)Long-term stimulation of MIA in the knee joint can decrease the level of BAG3-mediated CASA pathway activity in articular cartilage,while moderate exercise intervention could improve this effect.(7)Long-term stimulation of MIA in the knee joint can cause the expression of FLNA rise in articular cartilage,and there's no significant effect of moderate exercise intervention. |
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