| Tendinopathy is very common in our daily life and exercises,especially it has become the most challenging problem in orthopedics and sports medicine.Because its lacking in the number of cells,blood supply and innervation,damaged tendons heal slowly,rarely retain the structural integrity and mechanical strength of the healthy tendon.At the same time,injury tendons are commonly replaced by scar tissues,and complications such as ectopic calcification,fat infiltration and persistent pain often occur in the injury site,thus the incidence of re-injury/-rupture is very high.In recent years,the incidence of tendinopathy increased year by year,which accounted for over 50% of all sports related diseases,and 14% of the professional athletes was plagued with these diseases.In addition to long-term and repeated overuse of tendons,genetic factors and gene mutation often make some individuals more vulnerable to tendinopathy,and first line conservative treatment was often followed by 3-6 months of recovery time.For the limitation of treatment strategy and curative effect,there were about 24 to 45.5% of patients who will develope into refractory tendinopathy,which greatly influenced the achievement of professional athletes and the patient’s quality of daily life and also brought huge economic burden for families and the whole society.So to carry out in-depth research for the treatment of tendinopathy is imperative.Tendon stem cells(TSCs),as the precursors of tendon cells,have the similar biological function with that of mesenchymal stem cells(MSCs),but they have specific surface markers.They have multidirectional differentiation potential and strong differentiation ability,which can make them differentiate into tendon cells,adipocytes,osteocytes and chondrocytes.Currently,many scholars have reported that TSCs can directly participate in the regeneration and repair process of tendon injury.How to inhibit the non-tenogenic differentiation(adipogenesis,osteogenesis and chondrogenesis)under pathological conditions and to promote the tenogenic differentiation of TSCs are effective strategies for the treatment of tendinopathy.Based on the problems and current situation above,we can conclude’ that the proliferation,apoptosis,differentiation and changes in the microenvironment of TSCs are of great significance in the occurrence,development and healing of tendinopathy.Currently,the treatment for tendinopathy includes reducing physical activities and changing inappropriate exercises,non-steroidal anti-inflammatory drugs(NSAIDs),local injection of hormones,extracorporeal shock wave therapy,cytology(platelet rich plasma injection,stem cells injection)and tissue engineering therapy(biomaterials+ stem cells transplantation).Because of anti-inflammatory and analgesic effect of NSAIDs,they have become most commonly used in clinical treatment for tendinopathy.But,the effect of NSAIDs on the cell biology of TSCs,repair and healing of injury tendon are not yet clear.Based on these,we believe that the elaboration of the scientific problems and specific mechanisms above can provide new ideas and basis for the clinical treatment of NSAIDs for tendinopathy.As the most classic representative of NSAIDs,aspirin(ASA)has been widely used in many fields such anti-tumor and anti-coagulation.At the same time,aspirin has been reported to have effects on differentiation of stem cells.So we want to make clear that whether aspirin has effects on differentiation of TSCs and promotes healing of injury tendon or not.Whether it promotes injury tendon healing process through regulating of multidirectional differentiation of TSCs or not.We carry out this study around the problems above.Firstly,the present study screened the relatively safe concentration of aspirin,then we observed the effect of aspirin on injury tendon healing,and finally we expounded the mechanism of aspirin promoting injury tendon healing is to regulate the multidirectional differentiation of TSCs.1 Effect and mechanism of aspirin on apoptosis of TSCs1.1 Methods1.1.1 Concentration gradient of aspirin was set at 0,0.25,0.5,1,2 and 5m M respectively,and treated with TSCs for 24 h.Time-dependent experiment was set at 0,1,2,4,8,12,16,20,and 24 h respectively with the concentration of 5m M.1.1.2 Nuclear staining of Hoechst33342 and flow cytometry analysis of FITC/PI were used to detect the apoptosis of TSCs.1.1.3 Expression changes of apoptosis-related mitochondrial pathway proteins Bcl2,Bax and Cleavaged(Cl).Caspase-3 were observed by Western blot.1.1.4 Expression changes of canonical Wnt/β-catenin pathway related proteins DKK1,P-GSK-3β,P-β-catenin,C-myc and Cyclin D1 were observed by Western blot.1.1.5 After Wnt pathway activators of Wnt3 a and Li Cl were added into TSCs,the expression changes of Wnt pathway related proteins and apoptosis-related mitochondrial pathway proteins were detected by Western blot.1.1.6 After Wnt pathway activators of Wnt3 a and Li Cl were added to TSCs,the changes of apoptosis were observed by Hoechst33342 staining and flow cytometry.1.1.7 The effect of aspirin on the expression of COX-2 was observed by Western blot,and the expression of the apoptosis-related protein Cl.Caspase-3 after the addition of COX-2 inhibitor NS398 was also observed.1.1.8 We observed the changes of apoptosis after adding NS398 under aspirin treatment through Hoechst33342 staining and flow cytology analysis.1.2 Results1.2.1 From the perspective of concentration dependence and time dependence,aspirin increased the number of Hoechst33342(+)cells and FITC(+)/PI(+)cells respectively,and the number of positive cells above was decreased after adding Wnt pathway activators of Li Cl and Wnt3 a.1.2.2 From the perspective of concentration dependence and time dependence,the expressions of the apoptotic marker proteins Bcl2 and Cl.caspase-3 was decreased and the expression of the apoptotic antagonistic protein Bax was increased after the addition of aspirin.After the addition of Wnt pathway activators of Li Cl and Wnt3 a,the protein expression above was reversed.1.2.3 The expression of Wnt pathway-related proteins DKK1 and P-β-Catenin was increased,while the expression of P-GSK-3β,Cyclin D1 and C-Myc was decreased,and the expression of proteins above was reversed after the addition of Wnt pathway-related activators of Li Cl and Wnt3 a.1.2.4 Aspirin promoted the expression of COX-2.After the addition of NS398,the number of Hoechst33342(+)cells and the number of FITC(+)/PI(+)cells was decreased,while the expression of Cl.Caspase-3 was increased.1.3 Summary1.3.1 We concluded that the relative safe dosage of administration for aspirin was 0~2m M.2 Effect of aspirin on tendon injury healing in tendinopathy2.1 Methods2.1.1 Establishment of the animal model of rat Achilles tendon micro-injuryAfter intraperitoneal anesthesia,24 male rats were injected with 10mg/ml type I collagenase at the midpoint of the right Achilles tendon,and PBS was injected on the left side as the control group.2.1.2 Treatment of aspirinAfter 1 week of acute inflammation,aspirin was given at 30mg/d for 4 weeks.The experimental groups were divided into control group,natural healing with tendinopathy group and aspirin treatment with tendinopathy group.8 rats in each group were collected for the following experiments after 4 weeks of treatment.2.1.3 Establishment of cell inflammatory model in vitroTSCs cells were seeded in 6-well plates(60000 /well),and IL-1(10ng/ml)was added to establish inflammatory cell model in vitro.The experimental groups were divided into control group,IL-1β group,ASA group and IL-1β +ASA(2m M)group.The duration of treatment was 24 h.2.1.4 Effects of aspirin on type of macrophages and expression of inflammatory cytokines in Achilles tendinopathyFor the animal experiments,the Achilles tendons were collected after 4 weeks of treatment.Immunohistochemistry was used to observe the changes of type I macrophage marker i NOS,monocyte marker CD14 and type II macrophage marker CD206.The changes of inflammatory cytokines IL-6 and anti-inflammatory cytokines IL-10 were also observed.For the cell experiments,the changes of IL-6 and IL-10 were observed by q RT-PCR after treatment for 24 h.2.1.5 Effect of aspirin on the synthesis and degradation of extracellular matrix of injury tendonFor the animal experiments,the Achilles tendons were collected after treatment for 4 weeks.The expression changes of MMP-3,TIMP-3 and Col-I were observed by immunohistochemistry.For the cell experiments,the expression changes of MMP-3,TIMP-3 and Col-I were analyzed by q RT-PCR.2.1.6 effect of aspirin on scar formation after tendon injuryFor the animal experiments,Achilles tendons were collected after treatment for 4 weeks,and the expression changes of the scar related markers of Biglycan,Comp,Fibronectin and TGF-1 were observed by immunofluorescence.For the cell experiments,the expression changes of ACAN,COMP,EGR-1 and FMOD were observed by q RT-PCR.2.1.7 Effect of aspirin on proliferation and migration of TSCsThe changes of migration ability of TSCs induced by IL-1β treatment with aspirin were observed by scratch test.The changes of proliferation ability of TSCs induced by IL-1βtreatment with aspirin were observed by Brd U staining.2.1.8 Effects of aspirin on injury tendon healing and biomechanical propertiesFor the animal experiments,the expression changes of Col-I/Col-III after aspirin treatment were observed by immunofluorescence staining.The changes of biomechanical properties after treatment were observed by biomechanical tests.2.2 Results2.2.1 For the animal experiments,immunohistochemical results showed that the number of type I macrophages and monocytes decreased after aspirin treatment compared with the injury group,while the number of type II macrophages increased.The proinflammatory cytokine of IL-6 decreased and the anti-inflammatory cytokine of IL-10 increased.The expression of matrix metalloproteinase-3(MMP-3)was decreased,the expression of metalloproteinase inhibitor TIMP-3 was increased,and the expression of tenogenesis marker of Col-1a1 was increased.2.2.2 For the cell experiments,PCR showed that the expressions of IL-6 and MMP-3 in the aspirin group decreased compared with the IL-1 group,while the expressions of IL-10,TIMP-3 and Col1a1 were increased.2.2.3 For the animal experiments,immunostaining showed that the expressions of scar related markers of Biglycan,COMP,Fibronectin and TGF-1 decreased after aspirin treatment compared with the injury group.For the cell experiments,PCR showed that aspirin decreased the expressions of ACAN,COMP and EGR-1,while the expression of scar formation inhibitor FMOD was increased compared with the IL-1β group.The ratio of Col-I/III was significantly higher than that of the injury group.2.2.4 Scratch assay showed that IL-1β promoted the migration of TSCs,and the migration ability of TSCs was reversed after aspirin treatment.The result of Brd U experiment on cell proliferation showed that IL-1β promoted the proliferation of TSCs,while the addition of aspirin inhibited the proliferation of TSCs.2.2.5 The biomechanical test of Achilles tendon indicated that the maximum breaking force and young’s modulus were significantly increased after aspirin treatment.2.3 Summary2.3.1 Aspirin accelerated the healing of Achilles tendon injury.2.3.2 Aspirin improved the biomechanical properties of injury Achilles tendon and decreased the risk of re-rupture.3 Effect and mechanism of aspirin on tenogenic differentiation of TSCsIn this part,we want to explore whether aspirin improved the healing process of injury tendon through regulation of tenogenic differentiation of TSCs or not.3.1 Methods3.1.1 In vitro,under tenogenic induction medium,the concentrations of aspirin were set at 0,0.25,0.5,1,and 2m M,and Sirius red staining was used to observe the changes of tenogenic differentiation of TSCs.3.1.2 Under 2m M aspirin,tenogenic differentiation was induced for 3,7 and 14 days respectively.The changes of tenogenic differentiation markers including SCX,TNMD and TNC were observed by PCR and Western blot.3.1.3 The induction group and the induction group with aspirin were observed through m RNA sequencing,and the molecular changes between the two groups were screened out.Western blot and PCR were then used to verify whether the screened molecules were consistent with results of m RNA sequencing or not.3.1.4 The target molecules were filtrated by m RNA sequencing,Western blot and PCR.After that,the target molecules were added into TSCs.The expression changes of Col-I,TNMD,SCX and TNC were observed by Sirius red staining,Western blot and PCR.3.1.5 We observed the expression changes of P-Smad1/5 between the two groups of cells(Induction group and Induction group+aspirin)at 3 days,7 days and 14 days by Western blot,and verify the effects of screening molecules on P-Smad1/5 and the changes of p-smad1/5 after adding their inhibitor.Expression changes of TNMD and SCX under tenogenic differentiation were observed by Sirius red staining,Western blot and PCR.3.1.6 For the animal experiments,aspirin was used for the treatment after Achilles tendinopathy model was established by collagenase I injection.HE staining was used to verify the animal model and effect of the treatment,and histological score was measured at the same time.The expression changes of tenogenic related markers of SCX,TNMD and TNC were observed by immunostaining.The biomechanical properties including the maximum breaking force,the final breaking pressure and the elongation rate were analyzed through biomechanical tests.3.2 Results3.2.1 For the cell experiments,with the increasing concentration of aspirin,the positive rate of cells stained by Sirius red and spindle-like cells increased.PCR and Western blot results showed that the expressions of SCX,TNMD and TNC were increased at 3,7 and 14 days.3.2.2 Results of m RNA sequencing showed that GDF6,GDF7 and GDF11 were filtrated between the two groups.Further verification by Western blot and PCR indicated that the expressions of GDF7 and GDF11 were increased but not in GDF6.3.2.3 The effect of GDF7 and GDF11 on tenogenic differentiation of TSCs were further verifed.According to the results of Sirius red staining,Western blot and PCR,GDF7 promoted the tenogenic differentiation,while GDF11 has little effect on tenogenic differentiation.3.2.3 GDF7 increased the phosphorylation level of P-smad1/5,and its inhibitor LDN193189 reversed this trend,which was further verified by Sirius red staining,Western blot and PCR.3.2.4 In vivo,histological score showed that the Achilles tendon score significantly increased after aspirin treatment,and the Achilles tendon was more orderly under HE staining.Immunostaining results suggested that SCX,TNMD and TNC increased respectively in the treatment group.The Achilles tendon pull test indicated that biomechanical properties were improved obviously after aspirin treatment.3.3 Summary3.3.1 Aspirin promoted tenogenic differentiation through the GDF7/Smad1/5 pathway.3.3.3 Aspirin promoted the healing process of injury Achilles tendon through its role in promoting tenogenic differentiation.4 Effect and mechanism of aspirin on adipogenic differentiation of TSCsIn this part,we want to explore whether aspirin improved the healing process of injury tendon through regulation of adipogenic differentiation of TSCs or not.4.1 Methods4.1.1 In vitro,under adipogenic induction medium,the concentrations of aspirin were set at 0,0.25,0.5,1 and 2m M for 24 h,and oil red O staining was performed to observe the adipogenic differentiation level of each group.4.1.2 With 2m M aspirin,adipogenic differentiation markers of ap2,PPAR and C/EBPα were observed by PCR at 3,7 and 14 days under adipogenic induction,and expression changes of ap2,PPAR and C/EBPα were observed by Western blot.4.1.3 We filtrated the differentially expressed molecules between induction group and induction group+aspirin group by m RNA sequencing.Western blot was used to verify whether the selected signal pathway was consistent with the results of m RNA sequencing.4.1.4 The targeting signal pathway was filtrated by m RNA sequencing and Western blot.The activators or inhibitors of the signal pathway were added,and the adipogenic differentiation and expression of PPARγ were observed by oil red O staining and Western blot respectively.4.1.5 For the animal experiments,after the tendinopathy model of Achilles tendon was constructed by injection of collagenase I,aspirin was used for the treatment.HE staining was used to observe the changes of the injury model,treatment effect and fatty formation.Histological score was made at the same time.The expressions of adipogenic differentiation markers of ap2 and PPARγ were observed by immunostaining.The biomechanical properties of the three groups,including the maximum breaking force,the final breaking pressure and the elongation rate,were analyzed.4.2 Results4.2.1 For the cell experiments,with the increasing concentration of aspirin,the fat formation was significantly reduced.PCR and Western blot showed that expressions of ap2,PPAR and C/EBP were decreased at 3,7 and 14 days.4.2.2 m RNA sequencing showed that PTEN/PI3K/AKT pathway was filtrated between the two groups.After addition of pathway inhibitors of VO-Ohpic and IGF-1 into TSCs,western blot indicated that aspirin could activate the signaling pathway.4.2.3 Oil red O staining and Western blot were used to show whether aspirin inhibited the adipogenic differentiation of TSCs by activating the PTEN/PI3K/AKT pathway or not.After the addition of pathway inhibitors VO-Ohpic and IGF-1,the adipogenic inhibition was reversed.4.2.3 For the animal experiments,histological score showed that the Achilles tendon score after aspirin treatment was significantly increased.HE staining showed that the Achilles tendons were more orderly,and lipid accumulation was significantly reduced.Immunostaining suggested that expression of ap2 and PPAR decreased respectively in the treatment group.The biomechanical properties were improved obviously after aspirin treatment.4.3 summary4.3.1 Aspirin inhibited adipogenic differentiation through activating PTEN/PI3K/AKT pathway and inhibited osteogenic differentiation.4.3.3 Aspirin promoted the healing level by inhibiting adipogenic differentiation and lipids accumulation. |
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