Interaction between fibroblasts and keratinocytes in the wound edge skin tissue of a diabetic foot patient and the mechanism
Objective To investigate the interaction between fibroblasts(Fb)and keratinocytes(KC)in the wound edge skin tissue of a diabetic foot patient and the mechanism.Methods This was an experimental research.The wound edge skin tissue from a diabetic foot patient(male and 33 years old)admitted to the Department of Wound Repair of Liyuan Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology in August 2021 and from an acute foot injury patient(male and 50 years old)admitted to the Department of Hand Surgery of the hospital in September 2021 was collected.The single-cell transcriptome sequencing was performed to analyze the interaction between chemokine ligands of Fb subgroup and chemokine receptors of KC subgroup.The supernatant was collected after human foreskin fibroblast(HFF)was cultured routinely and with high concentration of glucose for 7 days as normal conditioned medium(CM)and high glucose CM,respectively.HaCaT cells were collected and divided into normal CM group cultured with normal CM and high glucose CM group cultured with high glucose CM,the scratch test was performed to calculate the cell migration rates at 24 and 48 h after scratch(n=3).The content of cytokines in the two kinds of CM was detected by liquid suspension chip(n=5).HFF was collected and divided into normal group cultured routinely and high glucose group cultured with high concentration of glucose for 7 days,and the mRNA expressions of C-X-C motif chemokine ligand 1(CXCL1),CXCL2,CXCL8,and CXCL12 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction(n=6).HaCaT cells in normal CM group and high glucose CM group were collected to detect the protein expressions of C-X-C motif chemokine receptor 4(CXCR4)in cells cultured for 48 h by Western blotting(n=3).HaCaT cells were collected and divided into normal CM group,high glucose CM group,normal CM+CXCL12 group,and high glucose CM+CXCL12 group.The first two groups of cells were treated as before,and the latter two groups of cells were cultured with normal CM and high glucose CM containing recombinant human CXCL12,respectively.Scratch test was performed,and cell migration rates were calculated at 24 and 48 h after scratch(n=3);the protein expression of CXCR4 in cells cultured for 48 h was detected by Western blotting(n=3).Results Compared with those in the wound edge skin tissue of acute foot injury,the interactions between chemokine ligands(CXCL1,CXCL2,CXCL3,CXCL8,and CXCL12)of Fb subgroup and chemokine receptors(CXCR2 and CXCR4)of KC subgroup were significantly weakened in the wound edge skin tissue of diabetic foot.At 24 and 48 h after scratch,the migration rates of HaCaT cells in high glucose CM group were significantly lower than those in normal CM group(with t values of 23.50 and 15.65,respectively,P＜0.05).Compared with that in normal CM,the content of CXCL1 in high glucose CM was significantly increased(P＜0.05),and the content of CXCL12 was significantly decreased(P＜0.05).After 7 days of culture,compared with those in normal group,the mRNA expressions of CXCL1,CXCL2,and CXCL8 in HFF in high glucose group were significantly increased(with t values of 4.25,4.98,and 10.04,respectively,P＜0.05),while the mRNA expression of CXCL12 was significantly decreased(t=4.10,P＜0.05).After 48 h of culture,the CXCR4 protein expression in HaCaT cells in high glucose CM group was significantly lower than that in normal CM group(t=5.13,P＜0.05).At 24 and 48 h after scratch,the migration rates of HaCaT cells in high glucose CM group were significantly lower than those in normal CM group and high glucose CM+CXCL12 group(with P values all＜0.05);at 24 h after scratch,the migration rate of HaCaT cells in normal CM+CXCL12 group was significantly lower than that in normal CM group(P＜0.05);at 48 h after scratch,the migration rate of HaCaT cells in normal CM+CXCL12 group was significantly higher than that in high glucose CM+CXCL12 group(P＜0.05).At 48 h of culture,the CXCR4 protein expression of HaCaT cells in high glucose CM+CXCL12 group was 0.446±0.050,which was significantly higher than 0.247±0.010 in high glucose CM group(P＜0.05)and similar to 0.522±0.082 in normal CM+CXCL12 group(P＞0.05);the CXCR4 protein expression in HaCaT cells in normal CM group was 0.509±0.055,which was significantly higher than that in high glucose CM group(P＜0.05).Conclusions The interactions between chemokine ligands of Fb subgroup and chemokine receptors of KC subgroup were significantly weakened in the wound edge skin tissue of diabetic foot.High glucose can inhibit CXCL12 secretion of HFF,and the stimulation of its cell culture supernatant can decrease HaCaT cell migration ability and CXCR4 expression.Exogenous CXCL12 protein can increase the CXCR4 protein expression in HaCaT cells and enhance the cell migration ability.
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