Therapeutic Effect Of Betaine On Osteoarthritis In Mice And Its Mechanism

Background:Osteoarthritis is a common disease of motor system.In China,about 5.7% of men and10.3% of women suffer from different degrees of osteoarthritis.Osteoarthritis often occurs in knee and hip joints,mainly manifested in joint pain,stiffness,limited movement and endstage deformity.At present,osteoarthritis mainly adopts symptomatic treatment such as nonsteroidal drugs for pain relief,weight loss and functional exercise.Joint replacement surgery is performed when necessary in the end-stage,which brings a serious burden to patients,families and society.In joint activities,cartilage and subchondral bone share the weight of the joint.It is found that the changes of subchondral bone are earlier than those of cartilage in the process of osteoarthritis.Subchondral bone is a dynamically balanced tissue.Under the influence of hormone signals and physical stimulation,subchondral bone is also undergoing remodeling.When mechanical force changes,such as joint instability,the balance of bone coupling is broken,and osteocytes feel the changes of mechanical force,induce osteoclast activation,absorb subchondral bone matrix,and release TGF-β1 et al in bone matrix,stimulate mesenchymal stem cell(MSc)differentiation and promote abnormal bone island formation and abnormal angiogenesis.After the formation of bone islands and blood vessels caused by abnormal bone coupling,it can affect the articular cartilage covered on the subchondral bone,damage the articular cartilage,and promote the occurrence of arthritis.It is found that inhibiting the activity of osteoclasts can inhibit the abnormal bone remodeling of subchondral bone,so as to inhibit the progress of osteoarthritis.In the process of OA,angiogenesis also increased significantly.It is found that angiogenesis inhibitors also play an important role in inhibiting the progression of OA.Betaine is an alkaloid that is very soluble in water.The chemical formula is C5H11NO2.It has a slight characteristic smell(sweet taste).Betaine is contained in Lycium barbarum,legumes and other plants.Studies have shown that betaine can reduce the levels of NF-κB,MAPK,TGF-β1,matrix metalloproteinase-2(MMP-2)and VEGF in tissues.It has antiinflammatory,anti-fibrosis,anti-cysteinemia and other pharmacological effects.Because inflammation,NF-κB and MAPK are closely related to the activation of osteoclasts,MMP-2 and VEGF are closely related to angiogenesis,we speculate that betaine may have the potential to inhibit the activity of osteoclasts and inhibit angiogenesis.The aim of this study was to observe the effect of betaine on the osteoarthritis induced by ACLT in mice,and to study the possible molecular mechanism of its inhibition on the progression of osteoarthritis.Objective:1.To study the effect of betaine on OC differentiation and its mechanism in vitro;2.To study the protective effect of betaine on the articular cartilage of OA mice induced by ACLT;3.To study the effect of betaine on abnormal bone remodeling of subchondral bone in OA mice induced by ACLT;4.To study the effect of betaine on abnormal angiogenesis in subchondral bone of OA mice induced by ACLT.Methods:Part 1: To study the effect of betaine on OC differentiation and its mechanism in vitroRAW264.7 cells and BMMCs were induced to differentiate into OC.Different concentrations of Betaine(0 μM,125 μM,250 μM,500 μM)were added to the betaine treatment group.After induction,the level of osteoclast differentiation was detected by TRAP staining.BMMCs cells were divided into control group and betaine treatment group to induce them to differentiate into osteoclasts.Different concentrations of Betaine(0 μM,500 μM)were added to the betaine treatment group.After induction,DCFH-DA staining was used to detect the production of ROS,and Western blot was used to detect the activation level of downstream MAPK pathway.Part 2: To study the protective effect of betaine on the articular cartilage of OA mice induced by ACLTMice were randomly split into three groups: sham group,ACLT group and ACLT +betaine group.ACLT group and ACLT + betaine group were treated with anterior cruciate ligament transection of the right knee to establish OA model caused by mechanical instability.In the sham group,the skin and capsule of the right knee joint were cut without breaking the anterior cruciate ligament.The concentration of betaine was 2% W/V in the daily drinking water of mice,and betaine was given from the second day after operation.On the 14 th,30th and 60 th day,the knee joints of mice were harvested and sectioned.The pathological damage of articular cartilage was detected by safranine green staining;calcified cartilage and hyaline cartilage changes were detected by HE staining;immunofluorescence staining was used to detect the changes of chondrocyte related marker.Part 3: To study the effect of betaine on abnormal bone remodeling of subchondral bone in OA mice induced by ACLTMouse grouping and surgical modeling are the same as the second part.The concentration of betaine was 2% W/V in the daily drinking water of mice,and betaine was given from the second day after operation.On the 14 th,30th and 60 th day,the knee joints of mice were harvested and sectioned.Micro-CT was used to detect the changes of related indexes in subchondral bone tissue of mouse knee joint;the activation of osteoclasts in subchondral bone was detected by TRAP staining;Part 4: To study the effect of betaine on abnormal angiogenesis in subchondral bone of OA mice induced by ACLTMouse grouping and surgical modeling are the same as the second part.The concentration of betaine was 2% W/V in the daily drinking water of mice,and betaine was given from the second day after operation.On the 30 th day,the knee joints of mice were harvested and sectioned.The angiogenesis in subchondral bone of knee joint was detected by angiography,and the changes of angiogenesis related marker factors were detected by immunofluorescence.Results:1.Betaine could inhibit the differentiation of osteoclasts.When RANKL+M-CSF was added to induce BMMCs to differentiate into osteoclasts,the production level of ROS increased.Betaine treatment could inhibit the production of ROS and inhibit the activation of MAPK pathway;2.Compared with the sham groups,the degree of articular cartilage injury in ACLT group was significantly increased,the OARSI score was significantly increased,the thickness of hyaline cartilage in tibial plateau was significantly decreased,and the thickness of calcified cartilage was significantly increased.The levels of cartilage protective markers,such as Lubricin,were significantly decreased,while the levels of cartilage injury markers,such as Col X and MMP-13,were significantly increased.Betaine treatment can reduce the degree of cartilage damage,protect the thickness of hyaline cartilage and reduce the thickness of calcified cartilage.Betaine also decreased the levels of Col X and MMP-13,and increased the level of Lubricin;3.Compared with the sham groups,subchondral bone remodeling in ACLT group was abnormal,which was manifested by increase of trabecular bone pattern factor(Tb.Pf)and decrease of the thickness of subchondral bone plate(SBP Th).The activity of osteoclasts in subchondral bone increased significantly.Betaine can inhibit abnormal bone remodeling and osteoclast activity in subchondral bone;4.Compared with the sham groups,angiogenesis in the subchondral bone of ACLT group was increased,which showed that the total volume and number of vessels were significantly increased,the number of CD31 and Endomucin positive vessels was significantly increased,and the expression level of MMP-2 was significantly increased.Betaine can inhibit the formation of abnormal blood vessels in subchondral bone,reduce the number and total volume of blood vessels,reduce the number of CD31 and Endomucin positive blood vessels,and reduce the expression level of MMP-2.Conclusion:1.Betaine can inhibit osteoclast differentiation.For mechanism,betaine can inhibit RANKL induced ROS production and subsequent activation of MAPK pathway;2.Betaine can reduce OA progression and protect articular cartilage;3.Betaine can inhibit abnormal subchondral bone remodeling and osteoclast activity in OA mice;4.Betaine can inhibit abnormal angiogenesis in subchondral bone of OA mice.