| BackgroundOsteoarthritis(OA)is one of the most common bone and joint diseases in hospitals.Pathological changes are manifested as obvious damage to articular cartilage,and various joints and tissues of the body will be affected.The pathological symptoms of osteoarthritis are mainly the progressive destruction of articular cartilage,chondrosclerosis,cystic degeneration and the formation of osteophyte.Pain,stiffness and dysfunction are the main clinical symptoms.Among them,the most vulnerable part of the body is the knee joint,which is also one of the main causes of knee pain in the elderly.At present,the diagnosis of bone joint diseases is mainly based on the comprehensive analysis of physical examination,symptoms,X-rays,etc.The severity of the disease is mainly the imaging performance,and the damage of related structures is observed to evaluate its severity.However,in actual clinical diagnosis and laboratory research,the severity of clinical symptoms and imaging manifestations of patients with osteoarthritis are different in many cases,so the correct diagnosis of the disease is very important.difficult.The main drug therapy for the clinical treatment of osteoarthritis is non-steroidal anti-inflammatory drugs.Some patients use traditional Chinese medicine acupuncture and moxibustion to relieve symptoms.If conservative treatment fails,surgical intervention is used.However,surgical treatment is affected by many factors,the treatment cost is high,the trauma is large,and it is more difficult to deal with postoperative complications.Even for individual patients,it may only have the effect of relieving local pain and cannot fully satisfy the therapeutic effect.Therefore,it is very important to find a more effective and conservative treatment for knee osteoarthritis.Many studies have shown that interleukin-1(IL-1)plays an important role in the occurrence and development of osteoarthritis.IL-1? and IL-1? are two subtypes of IL-1,of which IL-1? is the main subtype.IL-1? is related to the inflammation of the synovium,which can affect the normal metabolism of chondrocytes and change the structure and function of bone cells.In addition,it promotes the cell death of chondrocytes and the decomposition of cartilage matrix.Therefore,IL-1? is inextricably linked with the occurrence and development of osteoarthritis.Calcitonin(calcitonin,CT)is an amino acid peptide with many other biological functions.In clinical practice,calcitonin is often used to treat abnormal bone metabolism diseases such as hypercalcemia?osteoporosis,and Paget's disease.Many studies have confirmed that calcitonin can regulate the synthesis and metabolism of important components in the cartilage matrix,protects important joint structures such as cartilage and subchondral bone,and has great potential for the treatment of osteoarthritis.In the occurrence and development of osteoarthritis,the Wnt/?-catenin signaling pathway plays a crucial role.It can regulate the proliferation of chondrocytes and maintain cartilage homeostasis,which is necessary for the expression of ?-catenin.of.However,the current research has not fully explained how calcitonin protects chondrocyte damage caused by IL-1? through the Wnt/?-catenin signaling pathway.Obviously,the specific mechanism needs to be further explored.ObjectiveThis study explored the protective mechanism of calcitonin(CT)on chondrocyte damage induced by IL-1?,and the purpose was to explore the role and significance of IL-1? in the pathogenesis of clinical knee arthritis,and the purpose was to clarify the effect of calcitonin on bone and joint The protective mechanism of inflammation provides a theoretical basis for the clinical application of calcitonin.Method(1)Take 20 cases of knee osteoarthritis patients and 20 cases of healthy knee joint synovial fluid,and detect the level of IL-1? by ELISA.The changes in the IL-1?value of joint fluid at different stages of knee osteoarthritis were analyzed,and whether the IL-1? value of knee joint fluid was related to the clinical evaluation of patients and the severity of knee osteoarthritis was analyzed.(2)Separate and culture SD rat knee joint chondrocytes,observe the cell morphology,and use immunohistochemical methods to confirm the expression of type II collagen and transcription factor SOX9 in the chondrocytes.(3)Apply various concentrations of IL-1? to rat chondrocytes to determine the expression of proliferation activity in each cell group.The most suitable experimental concentration of IL-1? was established.The rat chondrocytes were grouped by random combination method.Divided into:?Normal group(Normal),?IL-1?(10 ng/mL),?IL-1?+CT(10 nM)group,?IL-1?+CT(50 nM)group,?IL-1?(10 ng/mL))+IWR-1-endo group,?IL-1 ?+CT(50 nM)+IWR-1-endo group.The proliferation activity and apoptosis rate of each group were tested separately.Covers the detection of the degree of activation of Cleaved caspase-3 in cells,and the expression of apoptosis-related proteins Cleaved caspase-3,Bax,and Bcl-2.In addition,the upstream factor DKK-1 of the Wnt/?-catenin signaling pathway,as well as the cartilage matrix degrading enzymes MMP-3,MMP-9,MMP-13,ADAMTS-5,ADAMTS-4,and their mRNA and protein expressions are also detected and analyzed.Result(1)The level of IL-1? in the joint fluid of patients with knee arthritis was significantly higher than that of healthy individuals(P<0.05).Compared with healthy people,the level of IL-1? in the knee joint fluid of patients with different stages of knee arthritis was significantly higher(P<0.05);the level of IL-1? in the knee joint fluid of patients with intermediate and advanced stages was similar to that in early patients.The ratio increased significantly,and the level of IL-1? in knee synovial fluid was significantly increased in late-stage patients and mid-stage patients(P<0.05);The level of IL-1? in patients with knee arthritis and the evaluation of the clinical pathological parameters of the patient,the pain score,function score,stiffness score and WOMAC score of the patient are all shown to be correlated with their IL-1? level.(2)Most of the SD knee chondrocytes isolated and cultured under an optical microscope are polygonal,and some are fusiform or irregular.It can be observed that there are many cytoplasm in the cell,and the larger and circular shape is the nucleus(Figure 2-1 A).In order to stain the chondrocyte markers type II collagen and SOX 9(transcription factor),the method uses immunohistochemical staining.Brown-yellow particles can be observed in the cytoplasm and nucleus.As the cell density increases,the color Gradually become thicker.The above research results indicate that the cartilage cells from the knee joint of rats were successfully isolated and can be used in the next experimental study.IL-1? has the effect of inhibiting the proliferation of mouse chondrocytes at 24,48 and 72 hours,and it is related in time and concentration.IL-1? at a concentration of 10ng/ml has a significant anti-proliferative effect on chondrocytes,and the cell proliferation activity cannot be inhibited to>50%.Therefore,this concentration was chosen for the following experimental studies.The results of the experiment showed that the cell proliferation activity was higher than that of the normal group,and compared with the normal group(Normal group),the proliferation activity of rat chondrocytes in the other groups decreased,and the decrease in the IL-1? group was particularly significant(P<0.01).The cells in the IL-1 ?+CT(10nM)group,IL-1?+CT(50nM)group,IL-1?+IWR-1-endo group,IL-1?+CT(50nM)+IWR-1-endo group are proliferating The activity expression increased significantly(P<0.05).Compared with IL-1?+CT(50nM)group and IL-1 ?+IWR-1-endo group,IL-1?+CT(50nM)+IWR-1-endo cell proliferation activity expression was particularly enhanced(P<0.01).The results of cell apoptosis detection in each control group showed that compared with the normal group(Normal group),the apoptosis expression of rat chondrocytes in each group increased.Among them,the degree of increase in IL-1? group It became more obvious(P<0.01).The IL-1?+CT(10nM)group,IL-1?+CT(50nM)group,IL-1?+IWR-1-endo group,IL-1?+CT(50nM)+IWR-1-endo group were in apoptosis The rate of expression was significantly reduced(P<0.05).Compared with IL-1?+CT(50nM)group and IL-1?+IWR-1-endo group,the apoptotic rate of IL-1?+CT(50nM)+IWR-1-endo group decreased significantly(P<0.01).The measurement results of Cleaved casspase-3 activation showed that compared with the normal group(Normal group),the Cleaved casspase-3 activation level of rat chondrocytes in each group increased,while the IL-1? group Cleaved caspase-3 activation level increased.High is the most obvious.Compare with IL-1? group,in IL-1?+CT(10nM)group,IL-1?+CT(50nM)group,IL-1?+IWR-1-endo group,IL-1?+CT(50nM)+IWR In the-1-endo group,the activation of Cleaved casspase--3 was significantly reduced.Compared with IL-1?+CT(50nM)group and IL-1?+IWR-1-endo group,the activity of Cleaved casspase-3 in IL-1?+CT(50nM)+IWR-1-endo group was significantly reduced,especially(P<0.01).The detection results of proteins related to apoptosis were compared with the normal group(Normal group).The expression of Cleaved caspase-3 protein and the ratio of Bax/Bcl-2 both increased significantly,while the expression of apoptosis rate was IL-1? The increase in the group is particularly obvious.Compared with IL-1? group,IL-1?+CT(10nM)group,IL-1?+CT(50nM)group,IL-1?+IWR-1-endo group,IL-1?+CT(50nM)+IWR-The expression of Cleaved casspase-3 protein and the ratio of Bax/Bcl-2 in the 1-endo group decreased significantly(P<0.05).Compared with IL-1?+CT(50nM)group and IL-1?+IWR-1-endo group,IL-1?+CT(50nM)+IWR-1-endo group had the most significant decrease(P<0.01).The test results showed that compared with the normal group(Normal group),the expression levels of Dkk-1 mRNA and protein in the changes in the mRNA and protein of the Wnt/?-catenin signaling pathway and matrix decomposition-related genes after the addition of calcitonin treatment The chondrocytes in the other groups were higher than those in the normal group.However,?-catenin,MMP-3,MMP-9,MMP-13,ADAMTS-5,ADAMTS-4 mRNA and protein expression levels have increased to varying degrees.In the IL-1? group,the mRNA and protein expression levels of the above cell genes decreased and increased significantly(P<0.01).Compared with IL-1? group,the expression levels of Dkk-1 mRNA and protein in cells were in IL-1?+CT(10nM)group,IL-1?+CT(50nM)group,IL-1?+IWR-1-endo In the IL-1?+CT(50nM)+IWR-1-endo group,the mRNA and protein of ?-catenin,MMP-3,MMP-9,MMP-13,ADAMTS-5,and ADAMTS-4 were significantly increased.Decrease(P<0.01).Compared with IL-1?+CT(50nM)group and IL-1?+IWR-1-endo group,the expression level of Dkk-1 mRNA and protein in cells is IL-1? CT(50nM)+IWR-1-endo The expression of the group increased significantly,on the contrary,?-catenin,MMP-3,MMP-9,MMP-13,ADAMTS-5,ADAMTS-4 mRNA and protein decreased significantly(P<0.01).Conclusion(1)The level of IL-1? in the joint fluid of healthy people is significantly lower than that of patients with knee arthritis,and the severity of knee joint bone and joint disease is positively correlated with the level of IL-1?;(2)Calcitonin can protect rat chondrocyte damage induced by IL-1? by blocking the Wnt/?-catenin signaling pathway. |
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