| BackgroundOsteoarthritis(OA)is a chronic degenerative disease that mainly occurs in the middle-aged and the elderly,with its clinical manifestations mainly including joint pain and stiffness,swelling,deformity and joint dysfunction,which affect a large number of people in the world,and is one of the main reasons leading to human disability and inability to work.As people live longer and their lifestyles change,the number of people suffering from osteoarthritis is increasing.More than half of the people over 65 years old worldwide suffer from knee arthritis.It is estimated that osteoarthritis will become the fourth most disabling disease in the worldby 2020.The prevalence of symptomatic knee osteoarthritis among the middle-aged and the elderly in China is 8.8%,and the "disability loss of life years" caused by knee osteoarthritis is about 968 per 100,000 population,ranking 10th among all diseases.While causing great pain to patients,it also brings heavy burden to the society.This disease is characterized by progressive joint damage and degeneration,characterized by destruction of articular cartilage.Chondrocytes are the only cells in articular cartilage.Chondrocytes can produce and maintain articular cartilage matrix mainly composed of collagen and proteoglycan,which is responsible for maintaining the health of human and animal cartilage and joints.Numerous studies have shown that increased chondrocyte loss caused by apoptosis is a major feature of osteoarthritis.With the progressive development of OA,chondrocytes undergo apoptosis,and chondrocyte apoptosis is the main reason for the gradual decrease of chondrocytes in articular cartilage.All these changes lead to the irreversible progressive degeneration of articular cartilage.Endoplasmic Reticulum Stress(ERS)is a sub-organelle pathological process of er physiological dysfunction,which reflects the imbalance between the load of unfolded or misfolded proteins in ER and the processing capacity of ER.ERS is involved in the pathological process of many diseases.Recent studies have shown that,with the development of OA,er stress runs through many signaling pathways that cause chondrocyte apoptosis.Taurine is a sulfur-containing free amino acid.In recent years it has been found that it has a wide range of biological effects such as immune regulation and anti-lipid peroxidation damage.The human body mainly depends on the intake of taurine in food to meet the needs of the body.Taurine is an endogenous antioxidant,which has an important effect on apoptosis of myocardial cells,nerve cells,smooth muscle cells,tumor cells and other cells,and has a certain protective effect on cells.However,no studies have examined the protective effect of taurine on human chondrocytes.Therefore,we hypothesized that taurine treatment may protect chondrocytes by reducing endoplasmic reticulum stress,reduce chondrocyte apoptosis,protect articular cartilage,and slow down the development of osteoarthritis.Objective1.To investigate the therapeutic effect of taurine on rat osteoarthritis model and to find out its related mechanism:2.To investigate the effect of taurine on endoplasmic reticulum stress in human osteoarthritis chondrocytes and to explore the mechanism of its effect on osteoarthritis.Material and methodsPart one:1.A rat model of osteoarthritis was established by anterior cruciate ligament amputation and medial meniscus resection.2.Different concentrations(100 mg/kg)were given by tail vein injection.200 mg/kg;Taurine treatment at 400 mg/kg.3.Osteoarthritis and the effects of taurine were assessed every two weeks before and after surgery.4.Two-channel weight sensor was used to measure the weight distribution in the hind limbs of rats.5.The secondary mechanical heterotopic pain was measured using Von Frey Filaments to stimulate the posterior limb contractile response in rats.6.The width of the knee joint was measured to reflect the swelling of the knee joint.7.At week 14,the knee cartilage was histopathologically analyzed.8.Protein expressions of mmp-3 and CHOP were evaluated by western blot.Part two:1.Samples of osteoarthritis cartilage tissue.Extraction of cartilage tissue RNA,through the qRT-quantitative PCR in cartilage collagen type ?(collagen ?)and endoplasmic reticulum stress and apoptosis markers(GRP78,GADD153 and Caspase-12)mRNA expression level.2.Extraction of cartilage tissue protein by Western blot detection in cartilage collagen type ?(collagen ?)and endoplasmic reticulum stress and apoptosis markers(GRP78,GADD1 53 and Caspase-12)protein expression level.3.Cultured articular chondrocytes were tested by H2O2 and taurine gradient assay,and the cell viability of chondrocytes was evaluated by cell counting kit cck-8 to determine the culture conditions that induced endoplasmic reticulum stress.4.Chondrocytes were cultured in three groups:1.blank control group;2.0.3mmH2O2 treatment induced Erstress group;3.After 25mm taurine pre-culture,0.3mmH2O2 treatment group.5.Cell viability of chondrocytes was assessed by cellcounting kit CCK-8,and apoptosis was detected by Annexin v-fitc/PI flow cytometry6.Through the protein imprinting technology assessment ? type collagen in cartilage and chondrocytes,endoplasmic reticulum stress and apoptosis markers of protein expression levels.ResultsPart one:1.Osteoarthritis rat modelsThe medial meniscus resection(MMx)on the anterior cruciate ligament transaction(ACLT)can quickly induce osteoarthritis in rats.The disease progression after surgical modeling is faster and more consistent,which well simulates the mechanical and biological characteristics of osteoarthritis.2.Taurine treatment alleviated symptoms in rat models of osteoarthritisIn the allodynia assessment test,paw withdrawal latency(PWL)showed a significant increase in the taurine treatment groups,compared with the OA group.After daily 1× and 2×taurine treatment for six weeks,PWL significantly increased(1.25 ± 0.12 g and 1.6± 0.1 g,respectively)compared with the OA group(P<0.05 and P<0.01,respectively).In addition,significant pain alleviation began from the eighth week after surgery in OA + 0.5×taurine group(P<0.05).Thus,higher doses of taurine had much more rapid anti-allodynic activity.Taurine treatment alleviated secondary mechanical allodynia of OA rats in a dose-dependent and time-dependentmanner.The hind limb weight distribution had no change in the sham group before and after surgery.In contrast,the weight-bearing difference between the contralateral and ipsilateral hind limbs noticeably increased by the second week aftersurgery.After a daily 2×taurine treatment for four weeks after surgery,the weight-distribution differential significantly decreased(P<0.05)compared to that in the OA group.Higher doses of taurine induced more rapid activity.However,at week 14,there was little difference in the weight-distribution differential between OA+1×taurine and OA+2×taurine groups.Thus,taurine treatment had anti-swelling activity in OA ratjoints after daily injection.In thesham group,the surface of the cartilage was smooth and the chondrocytes were evenly distributed.The ACLT plusMMx surgery caused severe chondrocyte death and severe cartilage degeneration compared to the sham group.The area and depth of cartilage degeneration obviously decreased in the taurine treatment groups compared to the OA group.The expressions of MMP-3 and CHOP in the rats' cartilage in the different groups were analyzed by western blot.Protein levels of MMP-3 and CHOP in the OA group showed a significant rising trend compared to that of the sham group(P<0.001).However,the protein expressions of MMP-3 and CHOP were obviously lower in taurine groups compared to the OAgroup(P<0.05,P<0.01 and P<0.01,respectively),which indicated that the effect of taurine was dose-dependent.Part two:1.Chondrocyte endoplasmic reticulum stress and apoptosis were enhanced with the progression of osteoarthritisAs osteoarthritis progresses,chondrocytes die and the cartilage matrix decreases.Collagen type ? mRNA and protein expression level is reduced,and the endoplasmic reticulum stress and apoptosis markers(GRP78,GADD153,Caspase-12)mRNA and protein expression increased significantly.2.Taurine inhibits H2O2 induced endoplasmic reticulum stress in chondrocytesH2O2 induced the chondrocytes to produce endoplasmic reticulum stress.Through the gradient experiment of cck-8 using the cell technology kit,the treatment of chondrocytes with 0.3mm H2O2 for 4 hours resulted in significant cytotoxicity,and about 60%of chondrocytes remained alive.After treatment with 0.4,0.5,1 and 2mM H2O2 for 4 hours,the survival rate of chondrocytes was reduced to 42%,33%,23%and 11%,respectively.0.3mMH2O2 was the highest dose without significant cytotoxicity,and 0.3mMH2O2 was used for the experiment.After the gradient experiment,25mM taurine dose was selected for the experiment.Compared with the control group,the expression levels of proteins such as GRP78,GADD153 and caspase-12 were significantly decreased after the treatment with taurine.3.Taurineprotects chondrocytes cultured in vitro and alleviates chondrocyte apoptosisH2O2 can induce endoplasmic reticulum stress on cultured cartilage in vitro,thereby initiating ERS mediated chondrocyte apoptosis.H202 can induce endoplasmic reticulum stress on cultured cartilage in vitro,thereby initiating ERS mediated chondrocyte apoptosis.Conclusion1.In this study,we found that the rats with osteoarthritis showed secondary mechanical heterotopic pain after surgery compared with the control group,and postural changes resulted in changes in weight bearing of the hind limbs and significant swelling of the joints.After treatment with different doses of taurine tail intravenous injection,the secondary mechanical ectopic pain was relieved in the rat model of osteoarthritis,which alleviated the abnormal weight bearing of the hind limbs caused by osteoarthritis and reduced the bone and joint swelling.Taurine administration showed a correlation with dose and time.2.With the development of osteoarthritis severity,cartilage cell apoptosis increases,cartilage showed the rise of endoplasmic reticulum stress markers GRP78,reflects the increase of the cartilage cells endoplasmic reticulum stress and apoptosis markers GADD153 and Caspase-12 also increases,illustrates the cartilage cells endoplasmic reticulum stress is likely to make progress and osteoarthritis chondrocytes apoptosis.3.This study found that taurine injection treatment for osteoarthritis in rats,taurine can inhibit the expression of apoptosis factorCHOP cartilage cells apoptosis,inhibition of MMP-3 type reduce ? loss of collagen and cartilage destruction,alleviate symptoms of osteoarthritis.4.This study of cartilage cells in vitro using taurine treatment for the first time,for the first time confirmed that taurine reduced bone arthritis chondrocytes in the endoplasmic reticulum stress,clarify taurine can alleviate the cells induced by H202 endoplasmic reticulum stress,reduce the apoptosis,promote the synthesis of cartilage cell vitality and collagen type ?,to protect cartilage cells.In summary,this study confirmed the therapeutic effect of taurine on the progression of osteoarthritis.It is suggested that taurine may be used as a therapeutic agent to limit the development of osteoarthritis by alleviating endoplasmic reticulum stress in chondrocytes,inhibiting the apoptosis of chondrocytes caused by endoplasmic reticulum stress and reducing the loss of cartilage matrix. |
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