| Objective:Osteoarthritis(OA)is the most common chronic degenerative joint disease.It is a whole-joint disease involving multiple skeletal parts.The knee joint is the most serious,and the incidence is increasing year by year.Pathological features of OA include total articular cartilage destruction,synovial inflammation,osteophyte formation,subchondral bone sclerosis,and ligamentous and meniscal degeneration.These pathological processes are the main cause of joint stiffness and pain,leading not only to limited mobility and reduced quality of life,but also to huge socioeconomic losses.Although OA is more than 250 years old,the exact complex molecular mechanisms of its progression remain unclear.The occurrence and development of OA is related to a variety of pathological factors,such as overload,trauma,imbalance of the inflammatory system,and impaired anti-inflammatory pathways.Currently,there are still no effective treatments to address joint degeneration and inflammation,only symptomatic therapies aim to reduce pain,and there are currently no globally approved disease-modifying drugs to slow the onset and progression of osteoarthritis.Therefore,joint replacement is often the last effective treatment.Extracellular signal-regulated kinase 1/2(ERK1/2)is a serine/threonine protein kinase,which is a signal transduction protein that transmits mitogen signal.A member of the Mitogen-activated protein kinase(MAPK)family,MAPK plays a role in signaling cascades,transporting extracellular signals to intracellular targets.Therefore,MAPK cascades are central signaling elements that regulate basic processes such as cell proliferation,differentiation and stress response.These cascades transmit signals through sequential activation of three to five layers of protein kinases(MAP4K,MAP3 K,MAPKK,MAPK,and MAPK-activated protein kinase(MAPKAPK)).The first three layers of the center are considered to be a basic core unit,while the last two are now in some cascade.Four MAPK cascdes have been defined based on the composition of the MAPK layer :ERK1/2,C-Jun N-terminal kinase(JNK),P38 MAPK,and ERK5.ERK cascades are highly regulated cascades responsible for basic cellular processes,including cell proliferation and differentiation.These regulators influence bispectic phosphatases,scaffold proteins,signal duration and intensity,and dynamic subcellular localization of cascade fractions.Due to the importance of the ERK cascade,ERK disorders are harmful to cells and ultimately to the body.MAPK signaling pathway participates in the occurrence and development of OA.Inflammatory cytokines such as IL-1β activate MAPK(ERK1/2,JNK1,and P38)signaling pathways,leading to the expression of catabolic and inflammatory event-related genes.In addition,MAPK activation increases the expression of transcription factors such as RUNX-2,HIF-2α,and C/EBPβ,which further activates the NF-κB signaling pathway.Activated NF-κB p65 subunit migrates to the nucleus and is involved in the regulation of matrix degradation enzymes and proinflammatory factors,affecting the synthesis and remodeling of extracellular matrix.TESC,also known as Tescalcin or calcineurin B homologous protein 3(CHP3),was first discovered during analysis of mouse embryonic testes and contains an EF-hand motif that features a superfamily of calcium-binding proteins,members of which play key roles in several cellular processes.TESC play an important role in megakaryocyte differentiation and maturation by regulating the gene expression of E26transformation-specific transcription factors.Several recent studies have reported that overproduction of cellular TESC is associated with the progression of several types of cancers and tumors,including radiation-induced papillary thyroid cancer,acute myeloid leukemia,renal cell carcinoma,and human colorectal cancer.However,the regulatory signals and mechanisms by which TESC mediates cells are not well understood.On the basis of the previous work of the research group,this project first established in vivo and in vitro OA models of articular cartilage of SD rats,and verified the expression changes of TESC in OA cartilage of knee joint of SD rats as well as the correlation between TESC and the severity of articular cartilage.Secondly,the TESC gene sequence of primary chondrocytes of SD rats was knocked down by adenovirus to observe the relief degree of OA articular chondrocytes.Similarly,the TESC gene sequence of cartilage tissue was injected into the knee cavity of rats by adenovirus to observe the improvement degree of knee cartilage of SD rats made by operation of OA model.To explore the role of TESC in OA articular cartilage in rats.Finally,ERK 1/2pathway activator Honokiol was used to repeatedly study the regulation method and mechanism of TESC on rat OA chondrocytes through the recovery experiment.Methods:1.Use NCBI GEO database expression profile chip(GSE117999;GSE169077)to analyze the m RNA expression level of TESC in normal articular cartilage and OA articular cartilage.2.The primary chondrocytes of SD rats were extracted and cultured.The chondrocyte OA model was established by using rat recombinant IL-1β.All cells were selected from the first generation chondrocytes.The expression changes of TESC and related inflammatory molecules at the m RNA and protein levels in primary chondrocytes of SD rats,and their correlation with the severity of OA chondrocyte degeneration were observed by presetting the concentration gradient and time gradient model of IL-1β.and determine the modeling concentration and action time of IL-1β.3.The SD rat knee joint OA surgery model was established by anterior cruciate ligament transection(ACLT).The expression changes of TESC and related inflammatory molecules at the protein level were detected by histochemical staining method.And its correlation with the severity of OA cartilage degeneration in SD rats.At the same time,the X-ray film of the knee joint and the histological score of the knee joint were carried out,and the modeling time of the SD rat OA model was finally determined.4.The effects of empty adenovirus and adenovirus with TESC knock down motif on chondrocyte activity and cytotoxicity were observed by CCK-8 assay.Knockdown the TESC gene sequence of SD rat primary chondrocytes to prepare a chondrocyte OA model,and detect the expression levels of TESC m RNA and protein to verify the knock down efficiency.Role of OA articular chondrocytes and determination of optimal sequences for TESC knockdown adenovirus.5.Knock down the TESC gene sequence of SD rat primary articular chondrocytes to prepare a chondrocyte OA model,and use immunofluorescence to detect the expression level of TESC protein and further verify the efficiency of adenovirus knockdown.At the same time,the expression level of related inflammatory molecular proteins was detected.To explore the effect of TESC on OA articular chondrocytes.6.Knock down the TESC gene sequence of SD rat primary articular chondrocytes to prepare a chondrocyte OA model,and use flow cytometry,q RT-PCR,and WB to detect apoptosis-related factors to explore the effect of TESC on OA articular chondrocyte apoptosis.influence and role.7.One week before the modeling,SD rats were injected with adenovirus into the knee joint to knock down the TESC gene sequence of articular cartilage,and then ACLT was performed to prepare the SD rat knee joint OA surgery model.The efficiency of TESC adenovirus knockdown was verified by immunohistochemistry(IF),and the improvement of OA by knockdown of TESC was judged by knee X-ray and histological score.The effect of TESC on OA articular cartilage degeneration was explored by immunohistochemical staining of related inflammatory factors.8.Knock down the TESC gene sequence of primary articular chondrocytes of SD rats,create a chondrocyte OA model,explore the effect of TESC on the downstream signaling pathways of OA chondrocytes,and apply the ERK1/2 pathway activator Honokiol,using the reverse experiment,reverse Verify the relationship between TESC and ERK1/2 signaling pathway.The recovery of chondrocyte degeneration was observed by q RT-PCR,WB and IF detection of related inflammatory molecules,and the recovery of OA chondrocyte apoptosis rate was observed by flow cytometry and the detection of related apoptosis indicators.Results:1.Under the stimulation of rat recombinant IL-1β,the expression of TESC in SD rat primary chondrocytes increased,and with the increase of stimulation concentration,the effect time was prolonged,showing a positive correlation.(1)Primary chondrocytes were stimulated with 1ng/ml,2.5ng/ml and 5ng/ml IL-1βfor 24 h respectively to simulate in vitro OA model.(2)Through q RT-PCR and WB,we found that chondrocytes showed inflammatory changes.COL2A1,AGGRECAN,etc.represented a decrease in anabolic indexes,and MMP-3,MMP-13,etc.represented an increase in catabolic indexes.increased,the change increased,and the change was significantly different at 1ng/ml concentration.The m RNA and protein expression levels of TESC increased with the increase of IL-1βconcentration,and the changes were significantly different at 1ng/ml concentration.(3)The primary chondrocytes were stimulated with 1ng/ml IL-1β for 6h,12 h and24h to simulate the OA model in vitro.(4)Through q RT-PCR and WB,we found that chondrocytes showed inflammatory changes.COL2A1,AGGRECAN,etc.represented a decrease in anabolic indexes,and MMP-3,MMP-13,etc.represented an increase in catabolic indexes.With the prolongation of time,the change increased,and the most significant difference was found at 24 h.The m RNA and protein expression levels of TESC increased with the prolonged stimulation time of IL-1β,and the difference was most significant at 24 h.2.In SD rat knee joint model of ACLT simulating OA operation,the protein expression level of TESC showed a positive correlation with the prolongation of modeling time.(1)The OA model was simulated by cutting off the anterior cruciate ligament of the SD rat knee joint,and the rat knee joint was detected at 2,4,and 8 weeks after the operation.(2)By analyzing the X-ray plain film and histological score of SD rat knee joint,with the prolongation of modeling time,the OA became more serious.(3)By immunohistochemical staining of the knee joints of SD rats,COL2A1,AGGRECAN,etc.,representing the decrease of anabolic indexes,MMP-3,MMP-13,etc.representing the increase of catabolic indexes,and with the prolongation of modeling time,the changes changed.increased,and showed a significant trend of change at 4weeks after surgery.The protein expression level of TESC increased with the prolongation of modeling time,and showed a significant change trend at 4 weeks after operation.3.Using adenovirus to knock down the TESC gene sequence of primary chondrocytes of SD rats,after modeling with IL-1β(1ng/ml,24h),the degeneration of chondrocytes was relieved.(1)It was found by q RT-PCR,WB and IF that the TESC gene sequence of SD rat primary chondrocytes was successfully knocked down,and the knockdown efficiency was more than 40%.The CCK-8 experiment proved that it has no toxicity and activity on chondrocytes.(2)IL-1β was used to simulate the chondrocyte OA model.It was found by q RT-PCR,WB and IF that after knocking down TESC,the changes of COL2A1,AGGRECAN,MMP-3,MMP-13 and other indicators were reversed,and the catabolism was reduced,increased anabolism and alleviated OA chondrocyte degeneration.(3)The apoptosis rate of primary chondrocytes in SD rats decreased after TESC knockdown by flow cytometry and detection of related apoptosis factors.4.Using adenovirus to knock down the TESC gene sequence of SD rat knee articular cartilage,after the establishment of a surgical model(ACLT,4w),the articular cartilage degeneration was improved compared with the control group.(1)By immunohistochemical staining on the knee joints of SD rats,the protein expression level of TESC was significantly reduced,confirming that the knockdown was successful.(2)OA was improved by X-ray plain film and histological score analysis of the knee joint of SD rats.(3)By immunohistochemical staining on the knee joints of SD rats,the changes of COL2A1,AGGRECAN,MMP-3,MMP-13 and other indicators were reversed,the catabolism was reduced,and the anabolism was increased,which alleviated the articular cartilage degeneration of OA.5.Under the stimulation of IL-1β,primary chondrocytes with knockdown of TESC can reduce the degeneration of chondrocytes by reducing the activation of ERK1/2signaling pathway,and under the action of the ERK1/2 pathway activator Honokiol,the degeneration is obtained.replied.(1)Under the stimulation of IL-1β(1ng/ml,24h),the phosphorylation degree of ERK1/2 in primary chondrocytes with knockdown of TESC was significantly lower than that in the control group.(2)The phosphorylation of ERK1/2 in primary chondrocytes with knockdown of TESC was significantly increased compared with the control group by stimulation with the ERK1/2 pathway activator Honokiol.(3)After the primary chondrocytes with knockdown of TESC were stimulated by Honokiol(5μM),COL2A1,AGGRECAN and other anabolic indexes decreased,MMP-3,MMP-13 and other catabolic indexes increased,and the change trend was reversed.(4)After TESC knockdown primary chondrocytes were stimulated by Honokiol,the apoptosis rate of SD rat primary chondrocytes increased.Conclusion:1.In the OA model of SD rat chondrocytes simulated by IL-1β,TESC expression increased and was positively correlated with the occurrence and development degree of OA.2.TESC expression was increased in the knee OA model of SD rats simulated by anterior cruciate ligament dissection,and was positively correlated with the occurrence and development of OA.3.Adenovirus knockdown of TESC expression in primary chondrocytes of SD rats can alleviate OA degeneration of chondrocytes treated with IL-1β,including increased extracellular matrix anabolic index,decreased catabolic index and inhibition of apoptosis.4.TESC expression in articular cartilage tissue of SD rats was reduced by intra knee injection of adenovirus,and OA degeneration of articular cartilage simulated by ACLT was improved,including increased anabolic indexes of cartilage matrix,decreased catabolic indexes and decreased cartilage destruction.5.In primary chondrocytes of SD rats treated with IL-1β,TESC promoted OA degeneration of chondrocytes by activating ERK1/2 signaling pathway and activating it. |
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