| BackroundIn recent years,with the gradual aging of the world,osteoarthritis(OA)has become a major problem that seriously affects people's quality of life.The main characteristic manifestation of OA is the gradual degeneration of articular cartilage and inflammation of surrounding tissues.At present,the cause of OA is not clear,and most of its treatments are the use of non-steroidal anti-inflammatory drugs to relieve pain and joint replacement.Studies have shown that the synthesis and catabolism of normal articular cartilage cells,cartilage matrix,and subchondral bone are in a steady state.When this steady state is out of balance,the extracellular matrix is degraded and proliferated poorly,and cartilage cells are excessively apoptosis,which eventually causes the entire Degeneration of articular cartilage.Therefore,maintaining and improving the activity of articular cartilage cells and inhibiting the inflammatory response around the joints are the key points for the prevention and non-surgical treatment of OA.Inflammatory reaction is an important feature of OA,which occurs throughout all stages of OA.The increased expression of pro-inflammatory cytokines in cartilage,synovium and subchondral bone is believed to be closely related to the occurrence and development of OA.When the inflammatory signal is stimulated,the related inflammatory-dependent response and caspase(mainly caspase-1,caspase-1)are activated,which eliminates the inactive precursors of IL-1 and IL-18,And stimulate its secretion.Recent studies have found that Autophagy plays an important role in the inflammatory response of osteoarthritis.Autophagy is a key mechanism for eukaryotic cells to maintain cell homeostasis.Its mode of action is to complete the physiological degradation of cytoplasmic proteins and organelles through lysosomes.At present,it is known that the autophagy process can be divided into three types according to its delivery mode:microautophagy,macroautophagy and chaperone-mediated autophagy.Chaperone-mediated autophagy only occurs in mammals,and it participates in the degradation of a single soluble protein.Macroautophagy and microautophagy occur in a wide range,including the degradation of the cytoplasm of eukaryotic organisms including mammals,plants and fungi,as well as the degradation of organelles.Among them,macroautophagy is considered to be the main type of autophagy,and it is also the most widely studied type of autophagy.It is a highly conserved process mediated by double(or multiple)membrane-bound vacuoles.It is mainly used to remove damaged organelles or unused proteins.This process is highly conservative under normal physiological conditions,but it can also occur when induced by cellular factors such as oxidative stress and stress stimulation.Recent studies have found that in OA animal models,maintaining a stable level of autophagy is an important factor for chondrocyte survival.Autophagy in normal adult articular cartilage is an important mechanism to maintain cell homeostasis.People found high expression of autophagy-related proteins BECN1 and MAP1LC3 in cells in the surface area of normal cartilage.In the early stage of cartilage degradation,autophagy in OA chondrocytes and cartilage increases.More importantly,autophagy can affect the expression of OA risk genes by regulating the effects of cell apoptosis and reactive oxygen species(ROS).Autophagy can promote the synthesis of polyprotein inflammatory complexes caused by oxidative stress,that is,the synthesis of inflammasomes.O3 can be quickly transformed into O2 and oxygen atoms with oxidizing ability at room temperature,and then react with biological factors to generate reactive oxygen species(ROS)and lipid peroxides(LOPs).ROS is believed to have anti-inflammatory and analgesic effects,and has a relieving effect on diseases such as osteoarthritis and soft tissue pain.Studies have reported that low-concentration O3(?30 ?g/mL)injection into the articular cavity can inhibit the production of inflammatory factors IL-1? and TNF-?,and play a protective effect on chondrocytes,but the specific mechanism of action is still unclear.In addition to inflammatory stimuli,mechanical load is another major factor in the occurrence of OA.Stress is one of the main external signals received by the organism,and it affects the organism all the time.Therefore,it is necessary to study the role of biomechanical signals in the process of cartilage degeneration.Biomechanics constitutes an important external environment for cartilage.On the one hand,appropriate mechanical signals can help the maintenance and repair of cartilage.On the other hand,abnormal mechanical signals may cause cartilage development disorders or cartilage damage.In clinical practice,people have found that for patients with knee OA,an obvious phenomenon is that the inner side of the tibial plateau bears excessive pressure,the inner side of the knee joint is painful,and the inner articular cartilage is worn.Therefore,the current clinical surgical method for OA knee-saving treatment is mainly tibia or fibula osteotomy to compensate for the imbalanced stress.The influence of mechanical stress load on OA is very complicated.Stress load can change the bone microenvironment.On the one hand,proper stress load helps maintain bone homeostasis.On the other hand,abnormal stress load can cause the balance of osteogenesis and bone resorption to be broken,which will cause pathological changes.In osteoarthritis,abnormal angiogenesis can lead to the progression of OA.Abnormal angiogenesis of subchondral bone and invasion of blood vessels into the bone-cartilage junction are one of the pathological changes of human osteoarthritis.Using the rabbit osteoarthritis model,some scholars have observed that the angiogenic activity of the subchondral bone reaches its peak in the early stage to the advanced stage of OA,and gradually decreases to the normal level in the late stage of OA,indicating that there is a close relationship between OA and angiogenesis.Blood vessels are composed of many types of cells,among which endothelial cells play an important regulatory role in the process of blood vessel formation and angiogenesis.Recent studies have identified a new type of blood vessel subtype in the mouse skeletal system,called H-type blood vessel,which is characterized by high expression of CD31(platelet endothelial cell adhesion molecule)and Endomucin(cell surface mucin)(CD31hiEmcnhi).It has been observed that this specific vascular subtype can maintain the bone progenitor cells in the bone,couple angiogenesis and osteogenesis in the development and repair process,and the density will decrease with age.Platelet derived growth factor(Platelet derived growth factor,PDGF)was first reported in 1974.It is a peptide regulator stored in platelet alpha particles.It is named because it is derived from platelets and can stimulate tissue cell growth.Common PDGF is formed by two polypeptide chains connected by disulfide bonds,and is a dimer structure with various forms,including PDGF-AA,PDGF-BB,PDGF-AB,PDGF-CC and PDGF-DD.PDGF-BB is a chemokine and mitogenic factor in the PDGF family.It promotes the migration,proliferation and differentiation of various mesenchymal cells(such as endothelial progenitor cells and mesenchymal stem cells)to promote angiogenesis and osteogenesis Vital.Some scholars have found that osteoclast precursor cells are the main source of PDGF-BB in bone marrow and peripheral blood.This finding supports previous reports that PDGF-BB is secreted by immature non-resorbable osteoclasts.PDGF-BB derived from osteoclast precursor cells promotes the migration and differentiation of mesenchymal stem cells and bone marrow endothelial cells by binding to its receptor PDGF receptor(3(PDGFR?),and triggers mitogen-activated kinase(mitogen-activated kinase)and phosphoinositide-3 kinase-Akt signaling cascade(phosphoinositide-3 kinase-Akt signaling cascade).Studies by scholars have confirmed that PDGF-BB secreted by osteoclasts plays an important role in the process of coupling blood vessels to bone formation.They found that in a mouse model of osteoporosis(ovariectomized),the PDGF-BB secreted by osteoclast precursor cells was reduced and the coupling caused the number of H-type blood vessels to decrease,which resulted in the disruption of bone balance and bone loss.The use of cathepsin K inhibitors to interfere with osteoporosis(ovariectomized)mice can increase the number of osteoclast precursor cells and increase the formation of H-type blood vessels to promote bone formation.In summary,as two important factors affecting the occurrence of osteoarthritis,we designed the following study to initially explore the relationship between autophagy caused by inflammatory response and angiogenic osteogenesis changes caused by stress load and osteoarthritis.Part I The effect of oxidative reaction on autophagy of cartilage in osteoarthritis ratsObjectiveIn this study,Monoiodoacetic Acid(MIA)-induced rat OA model was established and observed the effect of medical ozone on autophagy of cartilage and cartilage degeneration of OA.Methods1.Experimental grouping and establishing an OA model.Female Wistar rats were randomly divided into 3 groups.Sham surgery group:50 ?L saline solution was injected into the right knee joint;MIA group:50 ?L of MIA was injected into the right knee joint to establish the OA model;MIA+O3 group:0.5 mL ozone(30?g/mL)was injected into the right knee joint on the 7th day,the 14th day and the 21st day of injection MIA.2.Detection of the general behavior,the mechanical withdrawal threshold(MWT)and the thermal withdrawal latency(PWL)of the rats.The general behavior,MWT(up-down method,Von Frey Hairs test)and PWL were observed and measured in each group on 1 day before MIA injection,and 1,4,7,14,21,28 days after injection.To observe the effect of medical ozone on pain behavior of OA rats.3.HE staining and Safranine O-fast green staining.28 days after MIA injection,rats were sacrificed and 4%paraformaldehyde was perfused to fixate.And then the right knee joint was taken down,fixed,decalcified,paraffin embedded,sliced and so on.HE staining and Safranine O-fast green staining were performed,and then Mankin score was measured to observe the effect of medical ozone on the degeneration of OA cartilage.4.The expression of cartilage MMP-13,collagen 2,LC3II and p62 were detected by immunohistochemistry.28 days after MIA injection,rats were sacrificed and 4%paraformaldehyde was perfused to fixate.And then the right knee joint was taken down,fixed,decalcified,paraffin embedded and sliced.Then,after dewaxing,hydration,antigen retrieval,blocking,primary and secondary antibody incubation,DAB color development,hematoxylin counterstaining and sealing,the expressions of MMP-13,collagen 2,LC3II and p62 were observed.To explore the effect of medical ozone on cartilage degeneration and cartilage autophagy.5.The expression of p-AMPK in the joint cartilage and spinal cord was detected by immunohistochemistry.28 days after MIA injection,rats were sacrificed and 4%paraformaldehyde was perfused to fixate,and then the right knee joint and L2-L4 spinal cord were cut from rats.After fixation,decalcification,paraffin embedding,sectioning,and then baked,dewaxed,hydrated,antigen repair,blocking,immune response,DAB color development,hematoxylin counterstaining,and sealing,the expression of p-AMPK in the cartilage and spinal cord was observed.To explore the possible therapeutic mechanism and analgesic mechanism of medical ozone on OA.6.The concentration of TNF-? and IL-6 in peripheral blood of rats was determined by EILSA.28 days after MIA injection,5 mL of peripheral blood was collected after anesthesia,and centrifuged at 3000 rpm for 10 min to obtain serum for testing.The standard samples,experimental samples and the antibody were added to the well of the test plate,respectively.And then the TMB color agent and the terminating agent were successively added after washing 3 times.In the end,the concentration of TNF-? and IL-6 were detected by a microplate reader.To explore the effect of medical ozone on the inflammation of OA.ResultsWe observed that for rats in the MIA group,1 day after MIA injection,compared with the Sham group,the activity of the rats in the MIA group was significantly reduced.The landing time of his right lower limb was also significantly shorter than that of Sham.At the same time,the frequency of licking his feet increased,and the frequency of lameness increased.The range of motion of the knee joint was lower than that of the Sham group,and the flexibility was reduced.One day before modeling,there was no significant difference in MWT of rats in each group(P>0.05).After MIA injection(1,4,7 d,14 d,21 d,28 d),the MWT in the MIA group was significantly lower than that in the Sham group(P<0.01).After the initial O3 intervention(1d-7 d),the MWT changes in the MIA+O3 group and the MIA group were similar,but after O3 intervention(7 d-21 d),the MWT of the MIA+O3 group recovered and was significantly higher after the 21st day In the MIA group(P<0.01),but still lower than the Sham group.The results of PWL are similar to those of MWT.The articular surface of the right knee of rats in the Sham group was intact and smooth,and there was no obvious proliferation of soft tissue around,and no obvious bleeding points were seen.The articular surface of the right knee of rats in the MIA group was rough and defective,cartilage destruction was seen,the surrounding synovial soft tissue hyperplasia,and multiple scattered bleeding points were seen on the articular surface.The articular surface of the right knee of the rats in the MIA+O3 group was relatively complete,with no synovial-like soft tissue hyperplasia around it,and no obvious cartilage destruction.Compared with the cartilage layer of rats in the Sham group,the cartilage surface of rats in the MIA group was thinner,unevenly colored,chondrocytes were arranged irregularly,and the number of cells was reduced.The cartilage layer thickness of the rats in the MIA+O3 group was increased compared with that in the MIA group,the coloring was still uniform,the chondrocytes were arranged in an orderly sequence,and the number of cells was not significantly reduced compared with Sham.Safranine O results showed that the articular cartilage of the Sham group was clearly stained,chondrocytes were arranged neatly,and the cell staining did not significantly decrease.In the MIA group,Safranine O staining decreased,cartilage structure was disordered,and the number of joint surface cells increased.The Safranine O staining of rats in the MIA+O3 group did not significantly decrease,the articular cartilage structure was not significantly disordered,and the number of joint surface cells did not change significantly.The Mankin score results showed that the Mankin score(8.0±1.1038)of the MIA group was significantly higher than that of the Sham group(P<0.01).Although the Mankin score(4.9±0.8944)of the MIA+O3 group was higher than that of the Sham group,it was significantly lower than that of the MIA group(P<0.01).Compared with the Sham group,the expression of collagen 2 in the MIA group was significantly reduced(P<0.01).However,after O3 intervention,the expression of collagen 2 was higher than that of the MIA group(P<0.05).The expression of MMP-13 in the MIA group was significantly higher than that in the Sham group(P<0.01).At the same time,O3 intervention could reduce this increase,and the expression of MMP-13 was significantly lower than that in the MIA group(P<0.01).After MIA intervention,the expression of apoptosis-related factor p62 in rats was significantly higher than that in the Sham group(P<0.01).After O3 intervention,the expression of p62 was significantly lower than that in the MIA group(P<0.01).Compared with the Sham group,the expression of autophagy-related protein LC3? in the MIA group was significantly reduced(P<0.05).Compared with the rats in the Sham group,the expression of p-AMPK in the MIA group was significantly decreased(P<0.01).The level of p-AMPK in the cartilage of rats in the MIA+O3 group was significantly higher than that in the MIA group(P<0.01).In the spinal cord,compared with the Sham group,the expression of p-AMPK in the MIA group decreased(P<0.05),while the expression of p-AMPK in the spinal cord of the MIA+O3 group was significantly increased(P<0.05).Compared with the Sham group,the IL-6 concentration in peripheral blood of rats in the MIA group was significantly increased(P<0.01),and the concentration of TNF-? was also significantly increased(P<0.01).After O3 intervention,the IL-6 concentration in peripheral blood was significantly lower than that in the MIA group(P<0.01)and also lower than that in the Sham group.For the concentration of TNF-?,the MIA group was significantly higher than that of the Sham group(P<0.01),and significantly decreased after the application of O3 intervention(P<0.01).ConclusionOxidative stimulation(O3 intervention in the knee joint cavity)can reduce the serum inflammatory-related factors IL-6 and TNF-? concentration in rats,reduce the expression of MMP-13 in articular cartilage and reduce the degradation of collagen-2,and delay the degeneration of OA cartilage.At the same time,it can up-regulate the expression of chondrocyte autophagy-related protein LC3II and inhibit the expression of p62,and its effect may be related to AMPK signaling pathway.Part ? Mechanical Loading Induces CD31 hiEmcnhi Vessel Formation by Preosteoclast Secretion of PDGF-BBObjectiveIn this study,mechanical loading and unloading mouse model was established and observed its effect on CD31hiEmcnhi Vessel Formation by osteoclast Secretion of PDGF-BBMethodKnockout Mice:We use TRAP-Cre mice and Pdgfbfl/fl mice to generate:Trap-Cre;Pdgfbfl/fl mice(Pdgfb-/-).The genotype of the mice was determined by PCRanalysis.Establish loading/unloading animal model:loading tests on WT,Pdgfbfl/fl and Pdgfb-/-groups of mice.The groups the right hind leg of mice mounted on a specially designed fixture for axial compression.Established an animal model of unloading.Using the hindlimb suspension method,the mice were divided into 3 groups:normal load group,hindlimb suspension(HLS)group and HLS+cathepsin K(CatK)inhibitor L-006235 group(HLS+L-235),suspended for 4 weeks.For the HLS+L-235 group,use vehicle or L-235(50mg kg-1 bw)daily.The mice were treated for 4 weeks.Micro-CT analysis of bone changes:Micro-CT scan of mouse bone tissue,and reconstruction and analysis by Skyscan software.Measure volume fraction(BV/TV),trabecular bone thickness(Tb.Th),number of trabecular bone(Tb.N),trabecular bone separation(Tb.Sp),cortical bone thickness(Ct.Th),bone Intimal circumference(Es.Pm)and outer circumference(Ps.Pm).Dynamic bone formation experiment:The growth of the bone tissue of each group of mice was observed by the calcein fluorescent labeling method.Immunofluorescence staining(Immunofluorescence):Immunofluorescence of bone tissue in each group of CD31,endomucin,the TRAP,and PDGF-BB expression.ResultsWe have successfully established a mechanical loading and unloading model in mice.The results show that compared with unstressed bones,bones after stress stimulation have significantly increased mineral attachment rate(MAR)and bone formation rate(BFR).Cortical bone thickness(Ct.Th)and periosteum circumference(Ps.Pm)also increased significantly,confirming that stress stimulation can promote bone growth.We analyzed the number of osteoclasts in the periosteum of bones expressing PDGF-BB by immunostaining TRAP and PDGF-BB,and the results showed that stress stimulation increased the number of TRAP/PDGF-BB cells in the tibial periosteum.In addition,the immunostaining of CD31 and Emcn identified H-type blood vessels,and the results showed that compared with unstressed limbs,the number of H-type blood vessels in the periosteum increased after stress.For Pdgfb selective gene knockout mice,the bone mass cannot be increased significantly after stress load,and the number of H-type blood vessels has not changed significantly.At the same time,I also conducted a stress loss experiment(HLS).The reduction in bone mass after stress loss is not only reflected in the mineral attachment rate(MAR),bone formation rate(BFR),cortical bone thickness(Ct.Th)is reduced,and there is a significant reduction in trabecular bone score(Tb.BV/TV),trabecular bone thickness(Tb.Th)and trabecular bone number(Tb.N).Using L-235 can avoid this change.ConclusionMechanical stress can cause changes in bone mass in mice.This change is related to the increase of H-type blood vessels induced by the secretion of PDGF-BB by osteoclasts.Application of cathepsin K inhibitors can restore bone loss caused by stress loss. |
PDF Full Text Request