| Background:As one of the most common complications of diabetes,diabetic wound is characterized by low cure rate,high recurrence rate,and high amputation risk.Astragaloside Ⅳ(AS-Ⅳ)is the primary active constituent of Astragalus membranaceus(Huangqi)[2],which is widely used for tissue regeneration[1].Studies have demonstrated the role of AS-Ⅳ in lowering blood glucose,immunomodulatory,anti-inflammatory,anti-oxidant,anti-aging,anti-fibrotic,anti-tumor,and so on[3-6].Nevertheless,the effects and underlying mechanisms of AS-Ⅳ in high glucoseinduced skin wound repair cells have not been fully elucidated.Objective:To investigate the effects and underlying mechanisms of AS-Ⅳ on the biological behavior of high glucose-induced keratinocytes(HaCaT),vascular endothelial cells(HUVEC),and fibroblasts(HSF),and provides a new idea for the prevention and control of chronic diabetic wound.Methods:This experiment was divided into four parts.In the first part,AS-Ⅳ group(HG+ASⅣ group),high glucose group(HG+DMSO group),and normal control group(NG+DMSO group)were set up.The cell viability,apoptosis and migration were detected by CCK-8,flow cytometry,and scratch assay.In the second part,the level of ROS,MDA,SOD,IL-6,IL-8,and IL-10 were detected by fluorescent probes and ELISA.In the third part,the expression of TGF-β1,TβRⅠ,TβRⅡ,Smad2,p-Smad2,Smad3,p-Smad3,and Smad7 were detected by WB and qRT-PCR.In the fourth part,according to the results from part three,inhibitor group(HG+AS-Ⅳ+SB431542 group)or activator group(HG+AS-Ⅳ+SRI011381 group),AS-Ⅳ group(HG+AS-Ⅳgroup),and high glucose group(HG+DMSO group)were set up.The cell viability,apoptosis,migration,the level of ROS,MDA,SOD,IL-6,IL-8,and IL-10 were detected by CCK-8,flow cytometry,scratch assay,fluorescent probes,and ELISA to verify the role of TGF-β1/Smad2/3 pathway in the regulation of high glucose-induced skin wound repair cells with AS-Ⅳ.Results:1.AS-Ⅳ affect the biological behavior of high glucose-induced HaCaT,HUVEC,and HSFThe cell viability and the scratch healing rate in the HG+DMSO group were lower than that in the NG+DMSO group(P<0.01),but the apoptosis rate of cells was higher(P<0.01).The cell viability and the scratch healing rate in the HG+AS-Ⅳgroup were higher than that in the HG+DMSO group(P<0.01),but the apoptosis rate of cells was lower(P<0.01).2.AS-Ⅳ affect the oxidative stress and inflammatory response of high glucoseinduced HaCaT,HUVEC,and HSFThe concentration of SOD in the HG+DMSO group was lower than that in the NG+DMSO group(P<0.01),but the ROS-positive cells and the concentration of MDA were higher(P<0.01);the concentration of IL-10 in the HG+DMSO group was lower than that in the NG+DMSO group(P<0.01),but the concentration of IL-6 and IL-8 were higher(P<0.01).The concentration of SOD in the HG+AS-Ⅳ group was higher than that in the HG+DMSO group(P<0.05),but the ROS-positive cells and the concentration of MDA were lower(P<0.05);the concentration of IL-10 in the HG+AS-Ⅳ group was higher than that in the HG+DMSO group(P<0.01),but the concentration of IL-6 and IL-8 were lower(P<0.01).3.AS-Ⅳ affect the TGF-β1/Smad2/3 pathway of high glucose-induced HaCaT,HUVEC,and HSF(1)HaCaT part:The expression of TGF-β1 at both the protein and RNA level,and the phosphorylation of Smad2 and Smad3 in the HG+DMSO group were lower than that in the NG+DMSO group(P<0.05),but the expression of Smad7 at both the protein and RNA level was higher(P<0.05).The expression of TGF-β1 at both the protein and RNA level,and the phosphorylation of Smad2 and Smad3 in the HG+AS-Ⅳgroup were higher than that in the HG+DMSO group(P<0.05),but the expression of Smad7 at both the protein and RNA level was lower(P<0.01).(2)HUVEC part:The expression of TGF-β1 at both the protein and RNA level,and the phosphorylation of Smad2 and Smad3 in the HG+DMSO group were higher than that in the NG+DMSO group(P<0.01),but the expression of Smad7 at both the protein and RNA level was lower(P<0.05).The expression of TGF-β1 at both the protein and RNA level,and the phosphorylation of Smad2 and Smad3 in the HG+ASⅣ group were lower than that in the HG+DMSO group(P<0.05),but the expression of Smad7 at both the protein and RNA level was higher(P<0.05).(3)HSF part:The expression of TGF-β1,TβR1,and TβR2 at both the protein and RNA level,and the phosphorylation of Smad2 and Smad3 in the HG+DMSO group were lower than that in the NG+DMSO group(P<0.05).The expression of TGF-β1,TβR1,and TβR2 at both the protein and RNA level,and the phosphorylation of Smad2 and Smad3 in the HG+AS-Ⅳ group were higher than that in the HG+DMSO group(P<0.05).4.AS-Ⅳ attenuates high glucose-induced HaCaT,HUVEC,and HSF injury via TGFβ1/Smad2/3 signaling pathway.(1)HaCaT part:The cell viability and the scratch healing rate at 24 h and 48 h in the HG+AS-Ⅳ+SB group were lower than that in the HG+AS-Ⅳ group(P<0.01),but the apoptosis rate of cells was higher(P<0.01);the concentration of SOD in the HG+AS-Ⅳ+SB group was lower than that in the HG+AS-Ⅳ group(P<0.05),but the ROS-positive cells and the concentration of MDA were higher(P<0.05);the concentration of IL-10 in the HG+AS-Ⅳ+SB group was lower than that in the HG+AS-Ⅳ group(P<0.05),but the concentration of IL-6 and IL-8 were higher(P<0.05).(2)HUVEC part:The cell viability and the scratch healing rate at 24 h and 48 h in the HG+AS-Ⅳ+SRI group were lower than that in the HG+AS-Ⅳ group(P<0.05),but the apoptosis rate of cells was higher(P<0.01);the concentration of SOD in the HG+AS-Ⅳ+SRI group was lower than that in the HG+AS-Ⅳ group(P<0.01),but the ROS-positive cells and the concentration of MDA were higher(P<0.05);the concentration of IL-10 in the HG+AS-Ⅳ+SRI group was lower than that in the HG+AS-Ⅳ group(P<0.01),but the concentration of IL-6 and IL-8 were higher(P<0.01).(3)HSF part:The cell viability and the scratch healing rate at 12 h and 24 h in the HG+AS-Ⅳ+SB group were lower than that in the HG+AS-Ⅳ group(P<0.01),but the apoptosis rate of cells was higher(P<0.05);the concentration of SOD in the HG+AS-Ⅳ+SB group was lower than that in the HG+AS-Ⅳ group(P<0.01),but the ROS-positive cells and the concentration of MDA were higher(P<0.05);the concentration of IL-10 in the HG+AS-Ⅳ+SB group was lower than that in the HG+AS-Ⅳ group(P<0.01),but the concentration of IL-6 and IL-8 were higher(P<0.01).Conclusion:AS-Ⅳ exerts anti-oxidant,anti-inflammatory,and anti-apoptotic effects to protect the proliferation and migration of high glucose-induced HaCaT,HUVEC,and HSF by regulating the TGF-β1/Smad2/3 signaling pathway,which may benefit for reepithelialization and granulation tissue formation of chronic diabetic wound. |
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