| Achilles tendon is the strongest and thickest tendon in the human body,but it is also the most easily broken.Therefore,Achilles tendon rupture is a clinically common tendon injury.The process of tendon healing occurs in three different stages: inflammation,proliferation and tendon reconstruction.During the healing period,fibroblasts of Achilles tendon multiply,and the composition of the extracellular matrix(ECM),mainly type III collagen,is increased and deposited on the lesion.During the remodeling period,the cell structure becomes smaller and the synthesis rate becomes slower.The healing process after tendon injury includes internal healing and external healing.External healing is characterized by a distinct inflammatory response followed by specialized fibroblast recruitment and proliferation.The repair of acute Achilles tendon rupture usually works well,but tendon fibrosis adhesion is one of the most common complications after tendon injury.Thus,inhibition of external healing and promotion of internal healing helps to inhibit the formation of tendon adhesions and improve the healing quality of tendon repair.Fibroblast migration in the para-sacral tissue plays an important role in adhesion formation.A number of studies have confirmed that inhibition of inflammation and fibroblast proliferation can significantly inhibit the surrounding area of adhesion after injury.It is well-known that,TGF-?1 is one of the factors of pathological fibrosis in various tissues after injury.TGF-?1/Smad3 is an important signaling pathway in the process of wound repair.This signaling pathway can stimulate the DNA synthesis of sputum cells during tendon injury,and promote the recruitment of fibroblasts and macrophages.It also promotes the proliferation of tendon cells and the repair of tendons by participating in angiogenesis and stimulation of collagen production.TGF-?1 also regulates the expression of matrix metalloproteinases MMP-2 and MMP-9,and mediates the synthesis and degradation of extracellular matrix(ECM)and collagen by promoting the cleavage of collagen fragments or gelatin.Other studies have shown that TGF-?1 activates both the standard Smad2/3 transcription factor pathway and the non-standard p38 MAPK intracellularsignaling pathways to induce collagen expression in fibroblasts.In recent years,the anti-inflammatory and anti-fibrotic effects of calcium channel blockers have received increasing attention.The calcium channel blocker verapamil inhibits the production of inflammatory cytokines such as TNF-?,IL-1 and IL-6,increases the levels of inflammatory cytokines such as plasma IL-10,and inhibits fibroblast adhesion.And proliferation to reduce scar formation,participate in extracellular matrix degradation and reduce scar formation.Studies have shown that,topical verapamil can reduce the formation of type I and type III collagen in the sciatic nerve at the injury site,reduce the formation of scar tissue after peripheral nerve repair,and promote the growth of axons.However,there is no report on whether verapamil has a therapeutic effect on achilles tendon injury.Therefore,clarifying whether verapamil can be applied to the treatment of sachilles tendon injury,and clarifying its possible mechanisms will provide a theoretical basis for us to improve or develop a more effective program on repairing achilles tendon injury.Purpose:In this study,an acute injury model of rat achilles tendon traversal cut was established.The efficacy of topical verapamil was investigated to evaluate the reparative effect of verapamil on achilles tendon injury and its role in preventing adhesions.And to explore whether its underlying mechanism of action is associated with TGF-?1/smad3 and ERK1/2/p38 MAPK signaling pathways,which provides a new direction to obtain more effective treatment of achilles tendon injury and recovery of achilles tendon function.Methods:An acute injury model was established for the transverse ablation of the Achilles tendon in rats.After the achilles tendon transection,the modified "Kessler" method was used to suture the achilles tendon with a 5-0 non-invasive suture.The model control group used 100 ul physiological The gelatin sponge of the salt water(area 0.5cm×0.5cm)wrapped around the wound of the Achilles tendon and sutured the skin;the intervention group used a gelatin sponge with verapamil drug(concentration 2.5mg/ml)to encapsulate the damaged achilles tendon Surrounded and intermittently sutured the skin,the sham operation group opened the surface of the achilles tendon of the rat,freed the soft tissue around the achilles tendon,and then sutured the skin wound.Adhesion scores and inflammatory grading were performed by histomorphological observation(HE,Masson staining),and the effects of calcium channel blocker verapamil on the formation of Achilles tendon adhesion were evaluated by biomechanical measurementand gait analysis.Immunohistochemical method was used to investigate the effect of verapamil on the expression of matrix metalloproteinase(MMP-2,MMP-9)in the injured tendon.The actin(?-SMA)and vimentin were examined by immunofluorescence.Western blot was used to investigate the effect of verapamil on the expression of extracellular matrix proteins,col I and col III,related pathways TGF-?1/smad3,ERK1/2 and p38 MAPK signals related to fibrosis adhesion.Results:1.Compared with the control group,the tendon of VP group was fuller and smoother,and the degree of separation of the depression and the iliac crest was lower than that of control group.The healing and maximum breaking force of VP group was better than that of control group.The adhesion classification of VP group was lower than control group.2.There was significant difference in the inflammation grade between the control group and the intervention group(P<0.05),and the achilles tendon function(AFI)was significantly improved in VP group compared with control group.3.Compared with the control group,the expression of MMP-2 and MMP-9 was significantly decreased in the first week and the second week after operation(P<0.05),and the expression of Vimentin and ?-SMA in the first week,2 weeks and 4 weeks decreased significantly(P<0.05).4.Compared with the control group,VP intervention significantly inhibited the expressions of col I and col III,and the expression levels of TGF-?1,p-smad3,p-p38 and p-ERK1/2 protein were significantly decreased with VP intervention(P< 0.05).Conclusion:1.Verapamil has anti-adhesive injury after adhesion,it can reduce the deposition of extracellular matrix(ECM)in the tissue around the tendon,effectively balance the synthesis and degradation of collagen,reduce the distribution of inflammatory cells;2.Verapamil may inhibit the activation of TGF-?1 by inhibiting smad3 phosphorylation,thereby inhibiting the synthesis of MMP-2 and MMP-9,reducing the expression of collagen col I and col III,slowing the excessive proliferation of fibroblasts and inhibiting the process of fibrosis.;3.Verapamil inhibits the proliferation of p38 and ERK1/2 pathways by reducing the intracellular calcium concentration and inhibiting the excessive proliferation of fibroblasts. |
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