| Objective: osteoarthritis(OA)is the most common reasons for chronic disability in elderly patients that seriously reduce the quality of patients' life,consume a large amount of family and social resources.Because of human activities and natural living habits,the frequency of knee osteoarthritis is much higher than other human joints.Based on the status of OA,there is no special drugs for OA treatment.Knee joint replacement surgery for advanced osteoarthritis not only increases the treatment cost,but also has great potential problems(postoperative infection,prosthesis aging,etc.).All these bring great difficulty and resistance to the osteoarthritis treatment.However,the cause of OA is still unknown,it is certain that the cause of the disease is complicated by a variety of factors.Many studies have shown that the typical features of osteoarthritis is degeneration of articular cartilage.As the only cell component in articular cartilage,chondrocytes play a role in supporting and maintaining the normal morphology of cartilage and metabolic balance of the cartilage microenvironment.The pathological changes of chondrocytes are usually the most obvious pathological features of osteoarthritis.Abnormal activation of chondrocyte apoptosis and autophagy dysregulation are typical characteristics of osteoarthritis.The new calcium cation channel,transient receptor potential vanilloid subfamily member 5(TRPV5)is hot protein to calcium ion gated mediated extracellular calcium aion(Ca2+)influx in recent years.It plays a vital role in maintaining the balance of intracellular calcium ions.In my previous study,TRPV5 was expressed in chondrocytes widely.Ca2+,as an important inorganic substance in life,is not only involved in maintaining the stability of osmotic pressure inside the cell,at the same time Ca2+ as the second messenger of the cell,participates in the vital processes of cell proliferation,differentiation,apoptosis and autophagy.A large number of evidences also showed that intracellular free calcium ions were significantly increased in Osteoarthritis chondrocytes.Therefore,it is conjectured that up-regulated TRPV5 mediating extracellular calcium influx induces chondrocytes apoptosis and inhibits chondrocytes autophagy.The relationship between exercise and osteoarthritis has always been the focus of the debate.A lot of evidences showed that exercise can change the apoptosis and autophagy of cartilage cells and then change the process of osteoarthritis.Therefore,it is conjectured that different exercise loads affect the process of osteoarthritis by changing the expression of TRPV5 in osteoarthritis.Based on previous evidences and hypothesis,this study is divided into four parts:firstly,to investigate the changes of expression of TRPV5 in rat osteoarthritis chondrocytes;the second part: To investigate the mechanism of TRPV5 mediated by extracellular calcium influx inhibition of autophagy chondrocytes;the third part: To investigate the mechanism of TRPV5 mediated by the extracellular calcium influx induced apoptosis in chondrocyte;lastly: To investigate the changes of expression level of TRPV5 in cartilage of rat osteoarthritis in different exercise load.The mechanism of osteoarthritis and the effect of exercise load on osteoarthritis will be described from a new perspective.Methods:1.SD rat knee osteoarthritis model:Total 75 healthy 2 month old male 220g-230 g rats were randomly divided into 5groups.The knee joint experimental group,3 groups were sterilized with 5mg/ml monoiodoacetic acid 50?L,and fourth groups were injected with ruthenium red(TRPV5 inhibitor).The control group was injected with normal saline 50?L.2.Evaluation of osteoarthritis of the knee joint in each group:(1)The rats of knee joint X-ray shooting and scores;(2)Knee in each group observed evaluation;(3)HE and toluidine blue staining of rat knee joint tissue in rats;(4)Knee joint Mankin scores.3.The expression of TRPV5 in the cartilage of the knee joint of rats was detected.(1)TRPV5 m RNA levels were detected by real-time RCR in cartilage tissue;(2)the expression of TRPV5 protein was detected in cartilage tissue by western-blotting.4.To detect the autophagy level of the cartilage tissue of the knee joint in each group(1)Localization and expression of LC3 B was detected by immunohistochemical staining in articular cartilage tissues;(2)Expression levels of LC3 II protein was detected by western-blotting method in cartilage tissue.5.The chondrocytes of the knee joint were isolated and chondrocyte was stimulated like osteoarthritis in vitro,and the level of autophagy was detected.(1)The rat articular primary chondrocyte was isolated and cultured;(2)The si RNA interference knockdown TRPV5,and knockdown efficiency was detected by real-time RCR and Western-blotting;(3)Expression level of LC3 B and calmodulin was detected by immunofluorescence in chondrocytes.6.Determination of calcium ion concentration in chondrocytes in each group Fluo-3AM cell intracellular calcium staining was used.Background dye was used as the background color F0,and the average fluorescence intensity of all cells was randomly selected from the field to be measured.The average fluorescence intensity of was F,and the final calculated fluorescence intensity was F/ F0.7.Determination of protein in the autophagy inhibition pathway(1)The expression levels of TRPV5,calmodulin,CAMKII and p-CAMKII,Beclin1,p-Beclin1,LC3?and Bcl-2 were measured by Western-blotting;(2)The co-immunoprecipitation binding assay for combination detection of Beclin1 and Bcl-2.8.Detection of the level of cartilage tissue apoptosis and the detection of calmodulin in the knee joint of rats in each group(1)T Localization and expression of calmodulin was detected by immunohistochemical staining in articular cartilage tissues;(2)The expression level of calmodulin and cleaved caspase-8 were detected by Western-blotting in cartilage tissue protein.9.Detection of chondrocyte apoptosis rate in each group The apoptosis of chondrocytes was detected by flow cytometry after staining with Annexin V-FITC & PI.10.Determination of protein in the apoptosis pathway.The expression level of TRPV5,calmodulin,DAP,FADD,cleaved caspase-8,cleaved caspase-3,cleaved caspase-6 and cleaved caspase-7 was used to detect by western-blotting in chondrocytes.11.Construction of rats in exercise load model with knee osteoarthritis(1)Injection monoiodoacetic acid to induce osteoarthritis;(2)Different exercise treadmill training(divided into low,meddile and high intensity training),each exercise intensity was divided into 10 days,20 days,40 days for each sport cycle.12.Evaluation of knee osteoarthritis after exercise load in rats with osteoarthritis(1)The X-ray scores of the knee joint in each group of rats.(2)The knee joints in each group were observed and evaluated.13.The expression of TRPV5 in the cartilage of the knee joint after exercise load in rats with osteoarthritis TRPV5 protein expression is detected by western-blotting in cartilage.Results:1.Evaluation of osteoarthritis of the knee joint(1)X-ray showed that the joint space was normal,the cartilage surface was smooth and round,and no osteophyte was formed in the control group.However,osteoarthritis by monoiodoacetic acid induced in rats showed a slight narrowing of the joint space.The cartilage surface was not smooth and had osteophyte formation.The injection time is longer,the osteoarticular degenerative disease is much severe.However,the 21 day combined injection of ruthenium red(TRPV5 inhibitor)group showed that the joint was narrowed slightly and the cartilage surface was smooth,but the osteophyte formation was reduced compared with the 21 day injection of monoiodoacetic acid alone.The results of the X-ray scores were in accordance with the X-ray results.(2)The total knee joint assessment of rats showed that the cartilage was smooth and round,and the color and lustre were bright and without cartilage destruction in normal control group.The knee joint of the rats injected with monoiodoacetic acid showed that the cartilage was slightly dark and the cartilage was damaged.The injection time is longer,the osteoarticular degenerative disease is much severe.However,the cartilage destruction of the knee joint in the rats with ruthenium red was reduced.(3)The HE and toluidine blue staining was found: the microstructure shows normal cartilage cartilage with no degeneration.In the monoiodoacetic acid injection,the cartilage surface appear fracture,mild loss of staining with toluidine blue and cartilage cells lost more obvious loss of staining and staining with injection time.There was no obvious fissure and chondrocytes were relatively neatly arranged.No obvious dyed dyeing was found.The severity of osteoarthropathy is specifically expressed by the Mankin score.2.Detection of TRPV5 expression in the cartilage tissue of different groups of knee joints(1)TRPV5 m RNA increased gradually along with the progression of disease(prolonged MIA injection time).The maximum was reached in the 21 day group,and ruthenium red did not significantly reduce the expression of TRPV5 m RNA.(2)The expression level of TRPV5 protein in the cartilage of rats osteoarthrosis is in accordance with the expression of TRPV5 m RNA.3.Changes of the autophagy level in the knee joint cartilage tissue induced by MIA(1)LC3B immunohistochemical staining showed that the expression of LC3 B was weak in the control group.In the 7 days group,the LC3 B increased with the injection of MIA.On the 14 days,the brown positive results of LC3 B decreased significantly and the LC3 B decreased significantly in the 21 days group.However,the expression level increased after injection of TRPV5 inhibitor.(2)Calmodulin and LC3? protein quantitative finding: after MIA stimulation,the expression level of LC3 II in articular cartilage decreased,and the expression level gradually decreased with the disease progression.However,the expression level of LC3 II increased after the TRPV5 inhibitor addition.The ratio of LC3II/LC3 I is in accordance with the trend of LC3 II expression.At the same time,it was found that the expression of calmodulin was opposite to the expression of LC3B-II protein.4.The changes of autophagy in the primary chondrocytes in vitro after MIA induction(1)TRPV5-si RNA lentivirus infection of the primary chondrocytes in rats: the expression of TRPV5 in the interference group decreased significantly,and the interference efficiency was about 75%.(2)The chondrocytes stimulated with different concentrations in the immunofluorescence showed that with the increase of MIA concentration,the expression of LC3 B appeared first increased and then decreased,adding TRPV5 inhibitor ruthenium or red knockdown of TRPV5 revealed that the expression of LC3 B increased again;however,the expression of calmodulin increased gradually,but the addition of the TRPV5 inhibitor ruthenium red or knockdown of TRPV5 found that calmodulin expression was decreased.5.Detection of the function of TRPV5 mediated calcium influx in each group(1)The intensity of the fluorescence reflects the number of intracellular calcium ions,and then reflects the function of the TRPV5 ion channel.Found that: with the increase of the concentration of MIA(0 ?M,1.5?M,3?M,6 ?M),the fluorescence intensity of calcium staining deepened gradually,the fluorescence intensity is the strongest at 6?M MIA,but the fluorescence intensity was significantly weakened in the addition of the TRPV5 inhibitor and TRPV5 si RNA interference.(2)The fluorescence intensity was also decreased after adding calcium chelating agent.The intracellular calcium concentration was MIA concentration dependent and time dependent.6.Changes in the expression of protein molecules in the autophagic inhibition pathway(1)found that: TRPV5,calmodulin,p-CAMKII,p-Beclin1 and Bcl-2 was increased gradually along with the increase of the MIA concentration(0 ?M,1.5?M,3?M,6?M),and there was no significant change in the amount of total protein of Beclin1,CAMKII.However,the amount of LC3 II protein decreased with the increase of MIA concentration(0 ?M,1.5?M,3?M,6 ?M),and the ratio of LC3 II / LC3? decreased significantly.(2)We also found that TRPV5 inhibitors or knockdown of TRPV5 stimulation,calmodulin,p-CAMK?,P-Beclin1 and Bcl2 was significantly reduced,but LC3 II protein expression was significantly increased.Combination Bcl-2 and Beclin1 increased After 6?M MIA stimulation.7.Changes in expression of protein molecules in the apoptosis pathway(1)The level of expression of cleaved caspase-8 protein in the rat osteoarthritis cartilage:after MIA stimulation,the expression of cleaved caspase-8 in articular cartilage with the progression of the disease gradually increased,but the addition of the TRPV5 inhibitor ruthenium red,cleaved caspase8 expression decreased.(2)Immunofluorescence showed that the expression of cleaved-caspase8 increased gradually with the increase of MIA concentration,(3)but calmodulin expression increased gradually after TRPV5 inhibitor ruthenium red or knocking down TRPV5.(3)Detection of the apoptosis of chondrocytes apoptosis kit with the increase of MIA concentration,apoptosis rate increased from 3.19% to 18%,but adding TRPV5 inhibitors or knockdown of TRPV5,even at the highest concentration of MIA when stimulated cartilage cell apoptosis rate significantly decreased.8.Changes in the expression of protein molecules in the apoptosis pathway of chondrocytes TRPV5 induction We extracted the cartilage cells stimulated by MIA protein,we found: TRPV5,calmodulin,DAP,FADD,cleaved-caspase-8,cleaved-caspase-3,cleaved-caspase-6,cleaved-caspase-7 along with the increase of the MIA concentration(0 M,1.5 M,3 M,6 M),the expression of these proteins increased gradually,and the caspase-8, Caspase-3,caspase-6,caspase-7 total protein did not change significantly.These up regulated proteins are down regulated by knocking down TRPV5 or adding TRPV5 inhibitors or adding calcium ion chelating agents.9.Evaluation of osteoarthritis of the knee after exercise load in rats with osteoarthritis(1)The assessment score of X-ray of the knee joint of exercise load showed that the X-ray of knee joint of rats showed significant difference with different exercise load.Compared with the non exercise osteoarthritis group,in the high intensity exercise load,the joint space narrowed and the formation of osteophyte was obvious,and the longer the exercise time,the more obvious the formation of osteophyte.In medium exercise strength,the formation of osteophyte is relatively light.(2)There were significant differences in the articular cartilage damage of the knee joint in rats with different exercise loads.Compared with the control group,osteoarthritis of the control group,in high intensity and moderate exercise load,cartilage damage was obvious,the cartilage surface became more uneven,and the longer the exercise time,the more obvious the damage.However,in the lower mild exercise,the cartilage is relatively smooth and round,and the color and lustre are bright and without cartilage destruction.(3)The articular cartilage was observed by light microscope after the staining of red and fast green: in high intensity and medium exercise load,the articular cartilage structure was lost,and chondrocytes were further stained.10.The changes in the expression level of TRPV5 in the cartilage of the knee joint after exercise load in osteoarthritis rats After exercise training,there was a significant difference in the expression of TRPV5 protein in articular cartilage.Among them,the expression level of TRPV5 protein in low intensity training showed a low expression level.The longer the time was,the lower the expression level was.TRPV5 showed a high level of expression in moderate intensity training and high intensity training.The longer the time was,the higher the expression level was.Conclusions:1.TRPV5 expression was found in articular chondrocytes.The expression of TRPV5 increased in osteoarthritis,and positively correlated with the progression of osteoarthritis.2.Intraarticular injection of ruthenium red(TRPV5 ion channel inhibitor)can significantly delay and improve the progression of osteoarthritis.It is suggested that TRPV5 is a function of the ion channel in the pathogenesis of osteoarthritis.3.The up-regulated TRPV5 increased extracellular calcium influx in the occurrence of osteoarthritis.4.Increased calcium ion influx can combined with calmodulin to form a Ca2+/Ca M complex,Ca2+/Ca M complex phosphorylated CAMK II to form p-CAMK II.p-CAMK II phosphorylated Beclin1,and p-Beclin1 inhibited the formation of autophagosomes,inhibiting chondrocyte autophagy.Increased calcium influx increased Bcl-2,the complex of Bcl-2 and Beclin1 formation can be further synergy inhibition of chondrocyte autophagy,resulting in osteoarthritis.5.The Ca2+/Ca M complex level makes caspase-8 self sheared to form cleaved caspase-8.Cleaved caspase-8 can shear caspase-3,6 and 7 to induce apoptosis of chondrocytes,leading to osteoarthritis.6.Different exercise loads have different effect on osteoarthritis in rats.Low intensity has a protective effect on osteoarthritis,the longer of time is,the more obvious of protective effect is.The moderate intensity and high intensity can worsen osteoarthritis.The longer of time is,the more serious of osteoarthritis is.7.The expression of TRPV5 in the osteoarthritis rats articular was different under different exercise loads,and the low intensity decrease the expression of TRPV5,Medium and high intensity can increase the expression of TRPV5. |
PDF Full Text Request