| Background and Objective:Skin is often affected by external irritants.Wound healing is a pivotal physiological process to maintain the integrity of skin after trauma.The incidence of unnormal wound healing is continually increasing,which has become a serious public health problem.In recent years,studies have found that vitamin D plays a role in regulating immune function,inhibiting the excessive inflammatory response,and enhancing the antiinfection ability,which are related with skin wound healing.In this study,male C57BL/6J mice with full-thickness excisional wound were used to explore the effects of different vitamin D statuses on skin wound healing.In vitro,we investigated the effects of Vitamin D Receptor(VDR)knockdown or active vitamin D[1α,25(OH)2D3]intervention on the proliferation and migration ability of HaCaT cells,and further explored the potential mechanism.Methods:This study was divided into the following two parts.(1)Effects of Vitamin D deficiency and Vitamin D supplementation on skin wound healing in mice:A total of 104 male C57BL/6J mice were randomly divided into four groups as follows:Control group(CON group,Dietary vitamin D3 level was 1000 IU/kg,AIN93G diet,n=26),Vitamin D-deficient group(VDD group,Dietary vitamin D3 level was 25 IU/kg,n=26),Vitamin D supplementary group(HVD group,Dietary vitamin D3 level was 10000 IU/kg,n=26),and intraperitoneal injection of active vitamin D group(IHVD group,Dietary vitamin D3 level was 1000 IU/kg,and intraperitoneal injection of active vitamin D 1000 ng/kg every other day,n=26).Both feeding and intraperitoneal injection lasted for 10 weeks.The body weight and feed intake of mice were monitored.In the 11th week,the skin would was made on the back of each mouse.The skin wounds were photographed everyday.Image J was used to quantify the size of each wound area,and the wound closure of each group was calculated.On the 4th,7th,11th,and 14th day post-wound,blood samples,and skin wound tissues were collected for subsequent analysis.1)Serum 25(OH)D3 concentration was measured by Liquid Chromatograph Mass Spectrometry and the protein expression level of VDR of the wound tissue was detected by Western Blot.2)Inflammation:H&E staining was used to observe inflammatory cell infiltration.The levels of Interleukin-6(IL-6),Tumor necrosis factor alpha(TNF-α),and Interleukin-10(IL10)were determined by Western Blot and Real-time PCR.3)Epithelial-mesenchymal transition(EMT):EMT-related markers were analyzed by Western Blot and Real-time PCR,including E-cadherin,Zonula occludens-1(ZO-1),Ncadherin,Vimentin,and EMT transcription factors Snaill,Slug,ZEB1,ZEB2,and Twist.The protein levels of E-cadherin,N-cadherin,and Vimentin were further confirmed by Immunohistochemical assay.4)Extracellular matrix(ECM)deposition:Collagen deposition was assessed by Masson’s trichrome staining.The levels of Fibronectin(FN),Collagen Ⅰ(COL Ⅰ),CollagenⅢ(COL Ⅲ),Matrix metalloproteinase 9(MMP9),Matrix metalloproteinase 1(MMP1),and Tissue inhibitors of metalloproteinase 1(TIMP1)were detected by Western Blot and Realtime PCR.TGF-β1/Smad2/3 pathway-related markers:The level of Transforming growth factor beta(TGF-β1)was detected by Immunohistochemistry,Western Blot,and Real-time PCR.The protein levels of Smad2/3 and phosphorylated Smad2/3 were further determined.(2)Effects of VDR knockdown or active Vitamin D intervention on proliferation and migration of HaCaT cells:HaCaT cells were transfected with VDR-shRNA plasmid by lentivirus to construct a VDR-knockdown HaCaT cell model,including shNC group and shVDR group.In addition,HaCaT cells were incubated under different concentrations of active vitamin D(100 nM and 200 nM)which includes CON group,DMSO group,100VD group,and 200VD group.The following markers were measured:1)VDR:The expression level of VDR after VDR knockdown or active vitamin D intervention was determined by Western Blot and Real-time PCR.2)Cell proliferation ability:The effects of VDR knockdown or active vitamin D intervention on cell proliferation were analyzed by these methods:CCK-8 assay for cell viability,Western Blot for protein expression of proliferating cell nuclear antigen PCNA,and Real-time PCR for mRNA expression of proliferation marker Ki67.3)Cell migration ability:Scratch and Transwell assays were used to test the migration ability of HaCaT cells.4)EMT:The expression levels of EMT markers and EMT transcription factors were detected by Western Blot and Real-time PCR.5)MMPs:The expression levels of MMP9,MMP1,and TIMP1 were measured by Western Blot and Real-time PCR.6)TGF-β1/Smad2/3 pathway-related markers:The expression level of TGF-β1 was detected by Western Blot and Real-time PCR,and the protein levels of its downstream target gene Smad2/3 and phosphorylated Smad2/3 were further analyzed.Results:1.Effects of Vitamin D deficiency and Vitamin D supplementation on skin wound healing in mice:(1)Body weight,food intake,skin wound healing rate,serum 25(OH)D3 level,and VDR expression level in wound tissue of mice1)The body weight of mice in each group increased steadily.However,from the fourth week,the body weight of mice in the IHVD group grew more slowly than the other groups,and their body weight were significantly lower than that of the other groups(P<0.05).This phenomenon persisted until the end of the experiment.There was no significant difference in the average daily food intake among all groups.During the whole wound healing process,the skin wound healing rate of VDD group was significantly lower than that in the other three groups(P<0.05),and the healing rates of CON,HVD,and IHVD groups were basically equal.On the 14th day,the skin wounds of mice in the CON,HVD and IHVD groups were almost completely healed,while the wounds of VDD group were not completely closed.2)Serum 25(OH)D3 and VDR:Compared with the CON group,serum 25(OH)D3 concentration was significantly lower in the VDD group(3.8 ± 0.60 vs 33.6 ± 4.08 ng/mL,P=0.031).While the HVD group had a higher 25(OH)D3 concentrations(50.4 ± 7.65 ng/mL),but the difference was not statistically significant(P=0.641).No significant difference of 25(OH)D3 concentrations existed between IHVD group(27.7 ± 2.40 ng/mL,P=1.000)and the CON group.Compared with the CON group,VDD group had a significantly decreased VDR protein level(P<0.05).Although the protein level of VDR in HVD group and IHVD group was increased,the difference did not reach statistical significance.(2)Inflammation:H&E staining results showed that on the 4th and 7th day,the skin wound margin tissue of each group was highly infiltrated with neutrophils.On the 11th day,the inflammatory cell infiltration of the HVD and IHVD groups was significantly reduced,but the inflammatory cells in VDD group continued to infiltrate the surroundings of the wound at this time.Until the 14th day,the inflamatory state of each group returned to normal levels.On the 4th day,the protein level of pro-inflammatory cytokine IL-6 in the VDD group was significantly lower than that in the HVD and IHVD groups,and the mRNA level of Tnfa was significantly lower than that in the other three groups(CON group,HVD group,and IHVD group).The mRNA level of anti-inflammatory cytokine Il-10 in the VDD group was significantly higher than that of HVD group and IHVD group(P<0.05).On the 7th and 11th day,the protein levels of IL-6 and TNF-α in the VDD group were significantly higher than that in the other three groups.The mRNA level of Il-10 in the VDD group was significantly lower than that in the HVD and IHVD groups on the 7th day,and lower than that in the CON and IHVD groups on the 11th day(P<0.05).On the 14th day,the mRNA level of Il-6 in the VDD group was significantly higher than that in the HVD and IHVD groups,the level of TNF-α was not significantly different among all groups,and the mRNA level of Il-10 in the VDD group was significantly lower than that in HVD and IHVD groups(P<0.05).(3)EMT:On the 4th,7th,and 11th day,vitamin D deficiency enhanced the protein and mRNA levels of epithelial markers E-cadherin and the mRNA expression level of Zo-1,and inhibited the protein levels of mesenchymal markers N-cadherin and Vimentin.The levels of EMT transcription factors Snail1,Slug,ZEB1,ZEB2,and Twist in the VDD group were also down-regulated.In comparison to the other three groups,the mRNA level of E-cadherin and Zo-1 in the VDD group were significantly decreased,and the protein levels of Ncadherin and Vimentin were significantly increased on the 14th day.The mRNA levels of Snail1 and Twist in the VDD group were also significantly up-regulated(P<0.05)on the 14th day.On the 11th day,Immunohistochemical results further confirmed that the protein expression level of E-cadherin in the VDD group was significantly higher than that in the CON,HVD,and IHVD groups,and the protein level of N-cadherin and Vimentin in the VDD group were significantly lower than that in the other three groups.The protein level of Vimentin in the HVD group was also significantly higher than that in the CON group.(4)ECM deposition1)FN:The mRNA level of Fn in the VDD group was significantly lower than that in the HVD and IHVD groups on the 4th day,and the protein level was lower than that in the other three groups on the 7th and 11th day.And on the 14th day,the mRNA level of Fn was significantly higher than that in the other three groups(P<0.05).2)Collagens:Masson’s trichrome staining revealed that the VDD group had less collagen fibers than the other three groups.In addition,collagen deposition in the IHVD group was significantly higher than that of CON and HVD groups on the 4th day.The collagen deposition of HVD group and IHVD group were also significantly higher than that of CON group on the 7th and 11th day(P<0.05).The ratio of Ⅰ/Ⅲ collagen in the HVD and IHVD groups were significantly higher than that in the VDD group on the 11th day.On the 14th day,the ratio of Ⅰ/Ⅲ collagen in IHVD group was significantly higher than that in the other three groups,and the HVD group had a significantly increased the ratio of Ⅰ/Ⅲ collagen than the VDD group(P<0.05).3)MMPs:The results demonstrated that vitamin D deficiency resulted in a decreased expression of MMP9 on the 4th,7th,and 11th day.But on the 14th day,the protein level of MMP9 in the VDD group was significantly higher than that in the other three groups(P<0.05).Although no significant difference was detected,the protein expression level of MMP1 in the VDD group showed a decreasing trend when compared with the other three groups during the whole wound healing process.On the 4th and 7th day,the mRNA level of Timpl in the VDD group was significantly higher than that in the other three groups.And on the 14th day,the mRNA level of Timpl in the VDD group was significantly lower than that in the other three groups(P<0.05).(5)TGF-β1/Smad2/3 pathway related markers:The VDD group had a lower TGF-β1 expression than the other three groups on the 4th and 14th day,and the protein level of TGF-β1 in the VDD group was significantly lower than that in the HVD and IHVD groups on the 7th and 11th day(P<0.05).Results from Immunohistochemical analysis confirmed that the protein expression level of TGF-β1 in the VDD group was significantly decreased on the 11th day.On the 7th and 14th day,the proportion of phosphorylated Smad2/3 in the VDD group was significantly lower than that in the other three groups,and the proportion of phosphorylated Smad2/3 in the VDD group was only significantly lower than that in the HVD and IHVD groups on the 4th and 11 th day(P<0.05).2.Effects of VDR knockdown or active Vitamin D intervention on proliferation and migration of HaCaT cells:(1)The expression level of VDR in HaCaT cells:Compared with shNC group,the expression level of VDR in shVDR group was significantly down-regulated(P<0.05).After the intervention of different doses of active vitamin D,the expression level of VDR was significantly up-regulated(P<0.05).For HaCaT cells transfected with VDR-shRNA,treatment with active vitamin D had no effect on VDR mRNA level.(2)Cell proliferation ability1)CCK-8 assay:HaCaT cell viability in shVDR group was significantly decreased(P<0.05).In contrast,after different doses of active vitamin D intervention,the cell viability was significantly higher than that of DMSO group(P<0.05).2)PCNA:The protein level of PCNA in shVDR group was significantly higher than that in the shNC group(P<0.05).After active vitamin D intervention,the protein level of PCNA was increased when compared with DMSO group,but the difference was not statistically significant.3)Ki67:VDR knockdown significantly inhibited the mRNA expression level of Ki67,and the mRNA level of Ki67 was significantly upregulated after treatment with different doses of active vitamin D(P<0.05).(3)Cell migration ability1)Scratch test:The migration area of cells in shVDR group at 24 h was significantly reduced when compared with shNC group(P<0.05).After intervention with active vitamin D,the migration area of 100VD group was significantly increased at 12 h,24 h and 48 h when compared with DMSO group.The cell migration of 200VD group at 12 h and 48 h was also significantly up-regulated when compared with DMSO group(P<0.05).2)Transwell migration test:The number of cells migrated through Transwell membrane at 24 h in shVDR group was significantly decreased compared with the shNC group(P<0.05).After 24 h,treatment with active vitamin D increased the number of cells that traversed the filter(P<0.05).(4)MMPs-related indicators:When compared with the shNC group,the mRNA level of MMP9 in shVDR group was significantly decreased and the mRNA level of TIMP1 in shVDR group was significantly increased,and the protein levels of MMP9 and MMP1 in shVDR group were significantly decreased(P<0.05).Compared with DMSO group,the mRNA level of MMP9 in the 100VD group was significantly increased(P<0.05).The protein levels of MMP9 and MMP1 in the 100VD group and 200VD group were significantly increased(P<0.05).(5)EMT:VDR knockdown significantly enhanced the protein and mRNA levels of E-cadherin and the mRNA level of ZO-1,and the protein and mRNA expression level of N-cadherin and the mRNA level of Vimentin were significantly attenuated.The mRNA levels of ZEB1,ZEB2,Slug,and Twist in shVDR group were significantly decreased,and the protein level of ZEB1 was also significantly decreased(P<0.05).Active vitamin D significantly reduced the protein level of E-cadherin and the mRNA levels of ZO-1,and upregulated the mRNA expression levels of N-cadherin,Vimentin,ZEB2,Slug,and Twist.And the protein level of Snail1 was upregulated after active vitamin D intervention.The protein level of Slug in 100VD group was also significantly increased(P<0.05).(6)TGF-β1/Smad2/3 pathway-related markers:VDR knockdown significantly suppressed the protein and mRNA expression of TGFβ1,and reduced the proportion of phosphorylated Smad2/3(P<0.05).After intervention with active vitamin D,the mRNA level of TGF-β1 in 100VD group,and the protein and mRNA levels of TGF-β1 in the 200VD group were significantly higher than that in the DMSO group.Active vitamin D also induced Smad2/3 phosphorylation(P<0.05).For HaCaT cells transfected with shVDR,active vitamin D treatment had no effect on TGF-β1 mRNA level.Conclusions:(1)Vitamin D deficiency leads to delayed skin wound healing in mice,and additional supplementation of active vitamin D could improve wound healing quality.(2)Vitamin D might affect skin wound healing by regulating EMT progression via TGF-β1/Smad2/3 pathway.(3)VDR knockdown may inhibit the EMT process through TGF-β1/Smad2/3 pathway,and thus impaired the proliferation and migration ability of HaCaT cells.And active vitamin D treatment promoted the EMT process by activating this pathway,and then promoted the proliferation and migration of HaCaT cells.Active vitamin D may regulate TGF-β1/Smad2/3 pathway by binding to VDR. |
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