| Research backgroundOsteoarthritis(OA)is a common bone and joint disease in clinic,which is characterized by the ulceration of articular cartilage as a significant pathological change,and involves in various tissues of the joint,including articular cartilage,synovial membrane,subchondral bone,surrounding muscles and ligaments.Its main pathological characteristics include progressive aggravation of articular cartilage breakdown,sclerosis,cystic degeneration of osteophyte formation of subchondral bone,and clinical manifestations are mainly pain,stiffness and dysfunction[1].Knee osteoarthritis is one of the main reasons for the loss of labor force among middle-aged and elderly population.According to previous surveys,nearly half of the population is affected by it,which greatly affects the quality of life of the people,brings huge economic burden and social pressure Therefore,the diagnosis and treatment of osteoarthritis has been the focus of bone and joint surgery.Wnt/?-catenin signaling pathway plays an important role in the pathogenesis of osteoarthritis,which can activate and inhibit a variety of proteins and inflammatory factors that play an important role in osteoarthritis Previous animal studies have shown that Wnt/?-catenin plays an important role in the differentiation of osteocytes.Over-activation of Wnt/?-catenin can result in the differentiation of bone marrow mesenchymal stem cells into osteoblasts,thus reducing the number of chondrocytes and promoting the onset of osteoarthritis[2].Wnt/?-catenin can stimulate Fas gene transcription and induce cell apoptosis.Abnormal activation of Wnt signaling pathway can express a variety of proteases in chondrocytes and degrade cartilage matrix[3].Meanwhile,the expression of Wnt3A,Wnt7A and BMP-2 increased in patients with knee osteoarthritis,which was closely related to the pathogenesis of osteoarthritis[4].Vitamin D is a derivative of cholesterol,of which main active ingredients in vivo include 25-hydroxy-vitamin D and 1,25(OH)2D3.The former is the main form,but the latter is more active.It can affect bone metabolism through regulating the signaling pathways of various cytokines and growth factors.Previous experiments have shown that 1,25(OH)2 D3can promote osteoblasts to convert to osteocalcin and osteopontin,accelerate their maturation and function.By increasing the synthesis of RANKL and inhibiting the function synthesis in osteoclasts,it plays a very important role in the differentiation of mature osteoblasts[5].The therapeutic effect of vitamin D on osteoarthritis still remains to be unclear.There is still a lack of research on whether appropriate supplementation can improve the symptoms of osteoarthritis and alleviate the pa(?)hogenesis of osteoarthritis,as well as how does 1,25(OH)2 D3,active metabolite of vitamin D,achieve this process.Previous studies have shown that vitamin D plays an important role in many diseases such as cancer,renal failure and digestive tract diseases by interfering with Wnt/?-catenin signaling pathway.So far,there is no study on the mechanism of 1,25(OH)2 D3 regulating Wnt/?-catenin signaling pathway on articular cartilage.ObjectivePrevious reports indicate that patients with knee osteoarthritis generally lack vitamin D,but there have been no studies on the relationship between 25-hydroxyvitamin D levels in vivo and inflammatory indicators of synovial fluid in patients,whether appropriate supplementation of vitamin D can alleviate the symptoms of knee osteoarthritis and delay the progression of osteoarthritis,and the pathways through which vitamin D can achieve this process.This study innovatively studied the relationship between synovial inflammatory factors and clinical symptoms and serum 25-hydroxy-vitamin D in patients with osteoarthritis,and the effects of 1,25(OH)2 D3 on osteoarthritis in animal models,as well as the mechanism of 1,25(OH)2 D3 regulating Wnt/?-catenin signaling pathway on articular cartilage through in vitro cell experiments,aims to reveal the vitamin D in prevention and treatment of osteoarthritis,improve clinical symptoms,provide more direct experimental basis for clinical prevention and treatment.Methods1 In this study,patients with knee osteoarthritis admitted to the orthopaedic department of Rizhao People's Hospital were selected as the experimental group,and healthy people without knee osteoarthritis were selected as the control group after physical examination in Rizhao People's Hospital.The experimental group was divided into three groups according to Kellgren-Lawrence grading:experimental group A(K/L grade II),experimental group B(K/L grade ?),experimental group C(K/L grade IV).Serum 25-hydroxy-vitamin D level,TNF-a level in knee joint fluid,and WOMAC score were measured in each group,respectively.Rocaltrol(1,25(OH)2 D3)intervention therapy was conducted for 4 weeks.Serum 25-hydroxy-vitamin D level,knee joint fluid TNF-a level and WOMAC score were detected again in each group.The relationship between oral administration of 1,25(OH)2 D3 and clinical symptoms in patients with osteoarthritis was analyzed.2 Male SD rats with similar size and body mass were selected to establish the rat osteoarthritis model by means of joint instability(cutting the medial collateral ligament and removing the medial meniscus).Another 12 SD rats were taken and the joint instability model was established according to the previous method,which were randomly divided into experimental group A and experimental group B.The experimental group A was given rocaltrol(1,25(OH)2 D3 0.1ug kg-1)daily for intragastric intervention,while the experimental group B was given the same volume of peanut oil for intragastric administration.LequesneMG score was made for each group 4 weeks after the operation,and knee joint specimens were taken to observe the cartilage of the knee joint in rats.The relationship between oral administration of 1,25(OH)2 D3 and the progression of osteoarthritis was analyzed.3 Chondrocyte of rat knee joint was isolated and cultured,of which morphology of chondrocyte was observed.At the same time,the expression of the chondrocyte markers ? type collagen and transcription factor SOX9 was detected by immunohistochemistry.Chondrocytes were treated with different concentrations of TNF-?(5,10,25,50,75 and 100 ng/ml)for 24,48 and 72 hours respectively.Then the proliferation of chondrocytes was measured by CCK-8 method to determine the optimal concentration of TNF-? to inhibit chondrocyte proliferation.The cultured rat chondrocytes were divided into blank group and experimental group.The experimental group included:TNF-? group,TNF-?±1,25(OH)2 D3(10nM),TNF-?,25(OH)2 D3(100nM),TNF-?±?-catenin inhibitor(5M IWR-l-endo).After chondrocyte treatment,CCK8 was used to detect cell proliferation,flow cytometry was used to detect apoptosis,colorimetry was used to detect Casp se 3 activity,Real-time PCR and Western blot were used to detect the expression of ?-catenin,COL-?,SOST and MMP13.Results1 Control group:Serum 25-hydroxy-vitamin D level was 27.13 ±4.51 ng/ml and synovial fluid TNF-? level was 268.27 ± 54.76pg/ml Experimental group A(K/L grade ?):Serum 25-hydroxy-vitamin D level was 22.33 ± 4.81 ng/ml,synovial fluid TNF-? level was 388.72 ± 50.65 pg/ml,WOMAC score was 24.83 ± 6.30 points.After rocaltrol(1,25(OH)2 D3)intervention treatment,serum 25-hydroxy-vitamin D level was 28.26 ± 2.99 ng/ml,synovial fluid TNF-? level was 273.78 ± 37.35 pg/ml,WOMAC score was 15.17 ±3.87 points.Experimental group B(K/L grade 111):Serum 25-hydroxy-vitamin D level was 19.]2 ± 4.61 ng/ml,synovial fluid TNF-? level was 483.09 ± 54.92pg/ml,WOMAC score was 52.00 ± 6.80 points.After rocaltrol(1,25(OH)2 D3)intervention treatment,serum 25-hydroxy-vitamin D level was 27.78 ± 5.11ng/ml,synovial fluid TNF-? level was 398.59 ±58.75pg/ml,WOM AC score was 44.13 ± 7.49 points.Experimental group C(K/L grade ?):Serum 25-hydroxy-vitamin D level was 11.93 ± 3.23ng/ml,synovial fluid TNF-? level was 600.50± 66.81 pg/ml,WOMAC score was 85.50± 5.36 points.After rocaltrol(1,25(OH)2 D3)intervention treatment,serum 25-hydroxy-vitamin D level was 23.46 ± 4.92ng/ml,synovial fluid TNF-? level was 460.85 ± 38.16pg/ml,WOMAC score was 82.17 ± 5.71 points.We found that vitamin D deficiency was common in patients with knee osteoarthritis,and the degree of deficiency was positively correlated with the severity of knee osteoarthritis.The level of TNF-? in synovial fluid of patients with knee osteoarthritis was significantly higher than that of healthy people,and the level of TNF-? in synovial fluid of patients with knee osteoarthritis was positively correlated with the severity of knee osteoarthritis.Vitamin D supplementation in patients with knee osteoarthritis could reduce the level of TNF-? in synovial fluid of knee joint,reduce the clinical score of patients and improve the clinical symptoms of patients.2 The medial collateral ligament was cut off and the medial meniscus was excised to establish joint instability in rats.At the 6th week after operation,the anatomical results showed obvious cartilage erosion and ulcer in the medial femoral condyle of rats,a large number of osteophyte formation and the serious wear and tear on the articular surface of medial tibial plateau and cartilage ulcer,which proved that the model of knee osteoarthritis was made successfully.Another 12 SD rats were randomly divided into experimental group A and experimental group B.The experimental group A was given rocaltrol(1,25(OH)2D3 0.1ug kg-1)daily for intragastric intervention,while the experimental group B was given the same volume of peanut oil for intragastric administration.Observation at 4 weeks after the operation:the LequesneMG scores of experimental group A and experimental group B were significantly increased,and Lequesne MG score decreased in experimental group A compared with experimental group B(p<0.05).Through the general observation of knee joint specimens in experimental group A and B,it was found that in experimental group A,there were a few erosion ulcers on the articular surface of the medial femoral condyle,mild articular cartilage wear and a few ulcers on the tibial plateau,osteoporosis and osteophyte formation were observed.Experimental group B:The medial femoral condyle had obvious cartilage erosion and ulcer,a large number of osteophyte formation,serious wear and tear on the articular surface of medial tibial plateau,and cartilage ulcer.3 Under the microscope,a large number of triangular and spindle-shaped rat knee chondrocytes were observed,which were irregular in shape and grew well.It was found that pseudopods were connected with each other,the cells were filled with cytoplasm,the transparent nucleus was located in the center with round shape and big nuclei.The larger the cell was,the clearer the nucleus was.Immunohistochemical staining of type ? collagen and transcription factor SOX9 in primary rat chondrocytes showed that the collected cells strongly expressed type ? collagen and transcription factor SOX9(P<0.01),suggesting that ch ndrocyte culture was successful.Chondrocytes were treated with different concentrations of TNF-?,and the inhibition of chondrocyte proliferation was dose-dependent.Especially,compared with the control group,the optimal concentration of TNF-? was 50ng/ml.Meanwhile,it was also found that 1,25(OH)2 D3 significantly inhibited TNF-?-mediated chondrocyte proliferation and apoptosis(P<0.01).In addition,1,25(OH)2 D3(10 and 100 nM)treatment significantly inhibited the increase of caspase-3 activity mediated by TNF-a,with inhibition rates of 16.1%and 45.8%,respectively(P<0.01).The expressions of SOST,?-cateninCOL-? and MMP13 in chondrocytes induced by TNF-? were also determined by real-time PCR and Western blot.The results showed that 50 ng/ml TNF-a treatment significantly increased the expression of ?-catenin and MMP13,but decreased the expression of SOST and COL-?(P<0.01).However,1,25(OH)2 D3 or IWR-1-endo(5mM)treatment significantly inhibited the expression of SOST,?-catenin,COL-? and MM 13 mediated by TNF-? in chondrocytes(P<0.01).ConclusionThis study makes up for the blank of vitamin D in alleviating knee osteoarthritis symptoms and delaying the progression of osteoarthritis,as well as the mechanism of 1,25(OH)2D3 regulating Wnt/?-catenin signaling pathway on articular cartilage.It is concluded that the active ingredient of VitD,1,25(OH)2 D3,block Wnt/?-catenin signaling pathway to protect articular chondrocytes,thus delaying articular cartilage degeneration and providing theoretical basis for the prevention and treatment of osteoarthritis. |
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