| Background:Spinal cord injury often results in permanent disability,which imposes significant pain and financial burden on patients but remains a lack of effective therapeutic strategies.The formation of fibrotic scar in the lesion site following spinal cord injury constitutes a major impediment to axonal regeneration and functional recovery,with fibroblasts and inflammatory response playing a crucial role in this process: under the stimulation of inflammation response,fibroblasts undergo extensive proliferation and differentiation,leading to excessive secretion of extracellular matrix(ECM)proteins;consequently,a dense fibrotic scar tissue forms at the lesion core,significantly hindering axon regeneration and functional recovery of the spinal cord.CD44,a type I transmembrane glycoprotein,is expressed in the fibroblasts and plays a vital role in inflammation and tissue fibrosis.However,the role and underlying mechanism of CD44 in fibrotic scar formation following spinal cord injury remain unclear,and whether CD44 can be a potential therapeutic target to improve spinal cord regeneration is of great research value.Objective:To investigate whether CD44 regulates fibrotic scar formation after spinal cord injury and its underlying mechanisms,and the potential use of CD44 as a therapeutic target for improving spinal cord regeneration,thereby offering a new idea for the management of spinal cord injury.Methods:1.Spinal cord crush injury models were constructed in female C57 BL/6 mice: Mice were anesthetized and fixed in the prone position on the operating table.After T10 laminectomy,the spinal cord was completely clamped with a No.5 Dumont forceps for 5 seconds.After clamping,a transverse hyperemic trace promptly emerged at the site of clamping,indicating successful construction of the model.2.Western blot and immunohistochemical staining were used to assess the expression of fibrosis-related proteins(collagen-I,fibronectin,laminin,andα-SMA)and CD44 of the injury site.3.IM7 was intrathecal injected to inhibited the function of CD44.Then the changes of the expression of fibrosis-related proteins were detected at 2 and 12 weeks after spinal cord injury,respectively.4.Biotinylated dextran amine(BDA)was injected into the cortex at the 10 th week after spinal cord injury.Two weeks later,samples of the injured spinal cord were fixed and stained to examine the existence of regenerated CST in the distal part of the injury site by confocal microscopy.5.Mouse motor function(Basso Mouse Scale,BMS)and sensory function(Tail-flick test)were evaluated weekly from the 2nd week after spinal cord injury to characterize the functional recovery from spinal cord injury.6.The macrophage polarization in vitro was induced by lipopolysaccharide(LPS)to produce inflammatory supernatant,which was used to simulate the inflammatory microenvironment in vivo.7.The differentiation,secretion,proliferation,and migration abilities of BJ-1fibroblasts under the inflammatory environment after IM7 treatment were assessed using Western blot analysis,Ed U cell proliferation assay,Transwell cell migration assay,and scratch assay.8.The same method was used to explore the effects of JAK2/STAT3 inhibitors(WP1066 and AG490)on fibroblast function.Results:1.Fibrotic scar tissue appeared at the core area of injury site after spinal cord injury in mice.Immunohistochemistry and Western blot revealed that the expression of ECM proteins including collagen-I,fibronectin,laminin and α-SMA was up-regulated with the progression of the injury,accompanied by the elevation of the expression of CD44.Additionally,CD44 was markedly co-labelled with collagen-I and fibroblasts.2.After inhibition of CD44 by intrathecal injection of IM7,immunohistochemistry and Western blot showed significant downregulation of collagen-I,fibronectin,laminin and α-SMA expression in both the acute(2 weeks)and chronic(12 weeks)phases of spinal cord injury.3.BDA cerebral cortex injection showed that the BDA-labelled CST axons in the control group were mainly located in the rostral side of the injury site,while no obvious regenerated axons were observed crossing the scar region;in contrast,the IM7 administration group showed that a large number of BDA-labelled CST axons existed in the scar region with misdirection,and at the same time,regenerated CST axons could be seen crossing the scar region and growing into the caudal side of the spinal cord.4.Behavioral results of 10 consecutive weeks after spinal cord injury showed that mice in the IM7 administration group exhibited remarkable functional recovery in the BMS score and the tail-flick test compared with those in the control group.5.Upregulation of expression of collagen-I,α-SMA and CD44 in fibroblasts could be induced by the inflammatory supernatant of macrophages.6.After inhibiting the function of CD44 by IM7,the expression of collagen-I,fibronectin,laminin,and α-SMA in fibroblasts exhibited a subsequent down-regulation upon stimulation with inflammatory supernatant.Additionally,both the average proliferation rate and average migration rate of fibroblasts returned to the levels of those observed in the control group.7.Inhibition of the JAK2/STAT3 signalling pathway by WP1066 or AG490 also inhibited the functional promotion of fibroblasts in inflammatory supernatant but did not affect CD44 expression.Conclusions:1.The expression of fibrosis-associated proteins was up-regulated after spinal cord injury,predominantly localized within the core region of the injury site;meanwhile,the expression of CD44 was also up-regulated,which was closely related to fibrosis-associated proteins.2.The inhibition of CD44 function by IM7 can effectively suppress the expression of fibrosis-related proteins during both acute and chronic stages of spinal cord injury in mice.3.Inhibition of CD44 can promote the growth of regenerating CST axons across the scar region to the caudal side of the spinal cord,and improve the recovery of motor and sensory functions.4.Inhibition of CD44 can block the promotion of fibroblast differentiation,secretion,proliferation and migration induced by the inflammatory microenvironment stimulation.5.CD44 regulated fibroblast differentiation,secretion,proliferation,and migration in the inflammatory microenvironment through the JAK2/STAT3 signalling pathway. |
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