| Background:Diabetic patients are prone to peripheral vascular lesions and neuropathy,which impair the skin barrier function,and are prone to skin wounds which are not easy to heal and even develop into chronic ulcers,the most representative of which is diabetic foot ulcer.The treatment and care of chronic ulcers creates a huge psychological burden for patients,is a huge economic burden for individuals and society,and seriously affects the quality of life.It is urgent to study the pathological mechanism of delayed wound healing in diabetes mellitus.The role of calcium ion in wound healing as the second messenger is emphasized.As an important member of wound healing in epithelialization,keratinocytes play an important role.As the second messenger in the cell,the role of calcium ion in wound healing is attracting more and more attention.At present,the literature reports on calcium ion and keratinocyte function at home and abroad mainly focus on the effect of calcium ion on keratinocyte differentiation,while there are few literatures on calcium ion and keratinocyte migration function.According to our previous study,the expression of TRPV6 was abnormally reduced under the condition of high glucose.TRPV6 protein is a highly selective epithelial constitutive calcium channel that plays an important role in controlling intracellular calcium ion concentration.The purpose of this study was to explore the effect of TRPV6 on keratinocyte migration and its mechanism.Objective:1 To verify that high glucose inhibits the expression of TRPV6 in keratinocytes,and to explore the effect of high glucose on keratinocyte migration.2 To explore the effect of TRPV6 on keratinocyte function.3 To explore the mechanism of TRPV6 affecting keratinocyte function.Methods:1 Scratch test was used to observe the migration of keratinocytes after high glucose treatment,PCR and WB were used to verify the expression of TRPV6 after high glucose treatment,and calcium imaging was used to observe the calcium influx of keratinocytes after high glucose treatment.2 After knockdown and overexpression of TRPV6 in keratinocytes,the changes in cell migration function were observed by scratch assay and calcium ion flow was observed by calcium imaging.3 RNA-seq was used to detect differential gene expression between TRPV6 group and control group,and was verified by WB and q PCR.4 WB and PCR were used to detect the expression of FN1 in keratinocytes after TRPV6 knockdown,TRPV6 overexpression and high glucose treatment.5 On the basis of overexpression of TRPV6,calmodulin inhibitor(W-7)was added to observe the cell function by scratch,and the expression of FN1 was detected by WB.6 Cell migration was detected by scratch assay after FN1 was knocked down.Results:1 Compared with the normal group,the expression of TRPV6 was decreased under the condition of high glucose,the function of cell migration was inhibited,the influx of calcium ions was reduced,and the expression of FN1 was decreased.2 Compared with the control group,TRPV6 knockdown group showed decreased expression of TRPV6,decreased calcium inflow,decreased expression of FN1,and inhibited cell migration.3 Compared with the control group,calcium influx and FN1 expression increased in the TRPV6 group,which promoted cell migration.4 Compared with the overexpression group,the expression of FN1 in the overexpression plus W-7 group was reduced and the cell migration promoted by overexpression of TRPV6 was restored.5 Keratinocyte migration was impaired after FN1 knockdown compared with control group.Conclusions:High glucose reduces the intracellular calcium influx by inhibiting the expression of TRPV6 in keratinocytes,and reduces the expression of FN1 by inhibiting the activation of calmodulin,thereby inhibiting the migration of keratinocytes. |
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