The Effect And Mechanism Of HDAC3 In The Physical Therapy Of Osteoarthritis

Objectives:Osteoarthritis(OA)is a global disease with a high incidence.It dosenot only increase the disability rate of the people,but also causes a huge economic burden on society.The main clinical manifestations of osteoarthritis are joint swelling and pain,osteophyte hyperplasia,joint deformation accompanied by different degrees of movement disorders,which greatly impaired the patient’s ability to move.The pathological changes of osteoarthritis are mainly the massive death of chondrocytes,the excessive degradation of the cartilage matrixleading to dramatic changes in the microenvironment of articular cartilage,the metabolism of cartilage is broken,and the subchondral bone invasion,which cause a series of clinical symptom.At present,the pathogenesis of osteoarthritis is very complex and unclear.Many factors such as obesity,mechanical damage,and genetic factors play an important role in the occurrence and development of osteoarthritis.Inflammation is a core role in pathogenesis of OA,many factors will cause the release of inflammatory factors in cartilage tissue,thereby further destroying cartilage.Histone deacetylase 3(HDAC3)is an important transcriptional regulatory protein,which can not only regulate the acetylation status of histones,but also interact with certain non-histone proteins to regulate corresponding signaling pathways.HDAC3 plays an important role in the formation and development of cartilage.At the same time,in some inflammatory diseases,HDAC3 can promote the release of inflammatory factors and aggravate disease damage.However,the role of HDAC3 in OA is unclear.At present,the treatment of osteoarthritis is relatively limited,and can only delay the development of OA.Exercisetherapy is an effectiveand costless method for OA.Exercise stress could treat OA through action on chondrocytes.However,the research on the mechanism of exercise therapy is not enough.Exercise can regulate the content and activity of HDAC3,thereby regulating the expression of inflammatory factors and inflammatory proteins in diseases;NF-kappaB,as a classic inflammatory transcription factor,has an important role in various inflammatory diseases,and HDAC3 can regulate transcriptional activityof NF-KappaB and promote the expression of downstream inflammatory molecules and proteins of NF-Kappa B.Current research shows that,under the stimulation of IL-1β,the HDAC3 in chondrocytes increased,andthe elevated expression of a large number of pro-inflammatory factors causes chondrocyte damage.However,it is not clear whether exercise therapy is achieved through the HDAC3 / NF-kappa B pathway.Based on the previous work of our research group,we firstly established OA rat models at different periods,and observed the changes in the content of HDAC3 in knee joints of OA rats of different severity and the correlation between HDAC3 and cartilage injury.Secondly,different exercise loads were applied to OA rats through rat treadmill,and the effects of different exercise loads on the changes of HDAC3 content in OA rats and their correlation with different therapeutic effects were explored.At the same time,HDAC3 inhibitor(RGFP966)was injected into the joint cavity to determine the role of HDAC3 in OA exercise therapy.Furthermore,we verified the results of in vivo experiments in animals,and isolated the chondrocytes from the knee joints of normal SD rats for primary culture..By adding RGFP966,we observed its effect on the HDAC3 / NF-kappaB pathway and inflammatory factors in chondrocytes stimulated by IL-1β.This project finally confirmed the role of HDAC3 in OA,clarified the mechanism of OA exercise therapy and provided a potential new target for the treatment of OA.Methods:1.Establish an OA model in SD ratby intra-articular injection of monosodium iodoacetate(MIA).2.Establish OA rat models with different weeks,observe the RNA and protein levels of HDAC3 and related proteins in rat knee joints,and perform histological observation and scoring of knee joints.3.According to related literature,we apply different intensity of treadmill exercise to OA rats: different treadmill speeds,same time and same slope.The expression of HDAC3 and related proteins in rat knee joints at RNA and protein levels was observed,and the knee joints were histologically observed and scored.4.OA rat model was prepared,and the HDAC3 inhibitor RGFP966 was injected into the joint cavity to investigate the role of HDAC3 inhibitor in OA inflammation in vivo;the changes of related inflammatory proteins and pathway proteins in the knee joint of rats were measured.Meanwhile,Histological score of the knee joint was performed.Detection of related protein expression in rat knee joint cartilage:(1)Detection of RNA expression of HDAC3 and related molecules in tissues by PCR method;(2)Detection of HDAC3 and related molecules in tissues by histochemical staining and Western-Blot method at the protein level.5.Isolate primary chondrocytes from SD rat knee joints for primary culture and use type II collagen staining to identify chondrocytes.Furthermore,add IL-1β to simulate an inflammation modelin vitro6.To investigate the inflammatory effects of HDAC3 in IL-1β-stimulated chondrocytes and the effect of HDAC3 inhibitor RGFP966 on chondrocyte inflammation and related pathways: Select 3th generation chondrocytes and use IL-1β for simulate inflammatory model in vitro.The optimal effect concentration and time of RGFP966 were selected by CCK-8 viability experiment.To observe the effects of RGFP966 on chondrocyte inflammation and related pathways,Western-Blot method was used to detect the expression of related proteins in chondrocytes.Result:1.Injectiong of MIA into the knee joint cavity of SD rats can cause obvious OA symptoms(1)Macroscopic observation of knee joints in OA rats: In the normal control group,the surface of knee cartilage was smooth and intact,and a small amount of translucent joint fluid was seen in the joint cavity;in the OA model group(1 week group),the interarticular cartilage ruined.The cartilage injury aggravated along with time,at 4 weeks,cartilage was obviously damaged,accompanied by thick turbidity and even blood type synovial fluid.(2)Histological staining results: HE staining in the normal control group showed that the joint surface was smooth and intact,the tide-marker was normal,and the chondrocytes were arranged regularly.Toluidine blue staining showed that the cartilage matrix was uniform Ly dense blue;HE staining in the OA model group showed joints surface was rough,and the chondrocytes were disorderly arranged.Meanwhile,along with time,the cartilage is severely damaged,and a large area ofdefects appeared.The toluidine blue staining showedthat the chondrocytes disappeared,and only the cartilage pits existed.(3)X-ray examination of rat knee joint: normal knee joint structure was clear and complete,and joint space was normal;OA rat knee joint structure was blurred,joint destruction was severe,and normal structure was lost.2.HDAC3 and relative inflammation factors in OA rats were higher than those in the control group,and showed a time-dependent manner.(1)Histological images of OA rat models in different periods: X-ray showed knee joint damage and blurred structure in the OA model group,and the damage was more obvious in the OA group at 4 weeks;HE and toluidine blue staining showed that cartilage increased along with time.The cartilage damage gradually increased,and even lost the normal structure;Mankin and OARSI scores showed that the knee score increased along with time,and the difference was more obvious in the model group at 4 weeks.(2)Changes of HDAC3 content in OA models in different periods: The differences in HDAC3 content were detected by Western-Blot,PCR,and immunohistochemistry.We found that HDAC3 increased in protein and RNA levels along with time,and there were significant differences at 4 weeks.(3)Changes in type II collagen and related inflammatory factors in OA models at different periods: Using Western-Blot,PCR,and immunohistochemical methods,we found that type II collagen decreased,and MMP13,NF-kappaB increased along with time.=the difference was most obvious at 4 weeks.3.Moderate intensity exercise has better cartilage repair effect,which can significantly inhibit the inflammatory response in knee joints of OA rats and the intranuclear content of HDAC3(1)The knee joint sections of OA rats were stained with toluidine blue.The results of HE staining showed that the cartilage refreshed well in the moderate-intensity group,the cartilage structure was relatively complete,and the cartilage matrix was well stained.In contrast,the OA symptoms in the high-intensity group were more severe,The Mankin and OARSI scores had the same trend as the tissue staining results with significant differences.(2)The moderate-intensity exercise group had the best therapeutic effect among the different intensity exercise loads.The Western-Blot results showed that compared with the OA group,the type-2 collagen increased and the MMP13 content decreased in moderate-intensity group,while translocation of HDAC3 and NF-kappa B was also inhibited;PCR results showed that inflammatory factors such as MMP13,and HDAC3 in the moderate-intensity exercise group were also significantly reduced at the RNA level compared with the OA group;the immunohistochemical results showed the same trend as the Western-Blot results.All three experimental methods have Statistical significance.4.Injection of HDAC3 inhibitor RGFP966 into the joint cavity of OA rats can inhibit the inflammatory response and treat OA.(1)Histological staining and scoring of knee joints of OA rats: HE and toluidine blue staining of knee joint sections of rats showed that the cartilage refreshed in the RGFP966 injection group,the joint structure was more complete and the cartilage matrix staining was significantly increased;The trend of Mankin and OARSI score was consistent with the observation of histological staining.The score of RGFP966 group was lower than that of OA group,and the difference was significant.(2)Expression of HDAC3 and related proteins in knee joints of OA rats: Using immunohistochemical staining,we observed that under the intervetion of RGFP966,the nuclear translocation of HDAC3 and NF-kappaB in chondrocytes was inhibited,while type II collagen increased,inflammatory factors decreased.And the difference was significant.5.Under the stimulation of IL-1β,HDAC3 can promote the inflammatory response mediated by NF-kappaB in chondrocytes,and RGFP966 has the same anti-inflammatory effect in vitro.(1)Select 3rd generation primary chondrocytes and stimulate the chondrocytes with IL-1β(20ng/m L)to simulate in vitro inflammation.(2)IL-1β(20ng/m L)stimulated chondrocytes.Using Western-Blot method,we found thatinflammatory changes,type II collagen content decreased,and MMP13 expression increased in chondrocyte.(3)IL-1β(20ng/m L)stimulated chondrocytes.Using Western-Blot method,we found that IL-1β can promote the nuclear translocationof HDAC3 and NF-kappaB.(4)On the basis of IL-1β(20ng/mL)stimulation of chondrocytes,RGFP966(10μM)was added to culture medium.Western-Blot showed that RGFP966 can inhibit the expression of MMP13.(5)On the basis of IL-1β(20ng/mL)stimulation of chondrocytes,RGFP966(10μM)was added to culture medium.Western-Blot showed that RGFP966 can inhibit the nuclear translocation of HDAC3 and NF-kappaB.Conclusion: 1.HDAC3 is involved in the pathogenesis of OA,and HDAC3 can be used as an indicator of the severity of OA.2.Moderate-intensity treadmill exercise has better cartilage repair effect,and the nuclear expression of HDAC3 in cartilage tissue is inhibited.HDAC3 is an important target for exercise to treat osteoarthritis.3.In vivo and in vitro experiments,the HDAC3 inhibitor RGFP966 can inhibit the nuclear translocation of HDAC3 and NF-kappaB,confirming that HDAC3 can promote NF-kappaB-mediated inflammation in cartilage tissue.4.The whole experiment verified that OA exercise therapy achieved the therapeutic effect on OA through the HDAC3 / NF-kappaB pathway.