Effect And Mechanism Of Resveratrol On Skin Wound Healing In Diabetic Mice

【Objective】To investigate the effect of resveratrol（RSV）on skin wound healing in diabetic mice and the polarization of THP-1 monocyte-macrophages,and to explore its mechanism preliminarily.【Methods】（1）C57/B6 mice induced by intraperitoneal injection of streptozotocin（STZ）were established as diabetic mice models.Trauma models with a diameter of 1 cm were made on the back of normal and diabetic mice,respectively.Animal experiments were randomly divided into three groups:normal group（NC）,diabetic group（DM）,and diabetic+RSV group（DR）.The experimental period was 15 days.Mice in DR group were injected with 100μl RSV（10μmol/l）intradermally into the wound edge on days 0,1,2,3,5,7,9,11,13,and 15 after the wound modeling.NC and DM groups were injected with the same amount of PBS.The wound healing and the change of the wound area in each group were observed,and the wound’s healing rate was compared.To evaluate the effect of RSV on the quality and process of diabetic wound healing.At the same time,the skin wound tissues of the three groups of mice were collected on the 10^th and 15^th day of wound healing,and the epidermal thickness and collagen deposition were evaluated by HE and Masson staining.The expression levels of M2 macrophage-related markers and inflammatory factors were detected by quantitative real-time fluorescence PCR（q RT-PCR）and protein immunoblotting（Western bolt,WB）.Immunofluorescence detected the proportion of M2 macrophages and the number of neovascularization.（2）THP-1 cells were cultured in high glucose medium in vitro,and THP-1 mononuclear macrophages induced by Phorbol 12-myristate 13-acetate were used to construct cell models in vitro.The CCK-8 assay was used to detect the effect of RSV on macrophage activity and to determine the cellular experimental RSV concentration.Cell experiments were divided into LPS group and LPS+RSV group.Flow cytometry,q RT-PCR,and WB were used to detect the differentiation of macrophages induced by RSV and its effect on the secretion of pro-inflammatory factors.LY294002,a specific PI3K/Akt signaling pathway inhibitor,was pretreated before RSV treatment.The expression levels of key molecules in PI3K/Akt signaling pathway were detected by WB and flow cytometry was used to detect the proportion of M2macrophages after LY294002 pretreatment.【Results】（1）Local injection of RSV did not affect the blood glucose level of diabetic mice（P>0.05）;The skin wound healing rate of mice in DM group was significantly lower compared with NC group（P<0.05）,and the duration of inflammation was longer and granulation tissue was lacking in DM group;Local injection of RSV was able to improve the wound healing rate and shorten the healing time（P<0.05）,and the duration of inflammation was shorter and granulation tissue was abundant in diabetic mice.HE and Masson staining were used to observing the histological structure and collagen deposition.The results showed that compared with NC group on the 15^thday after the wound modeling,the epidermis of DM group mice was thinner,the collagen deposition was less and arranged in disorder.After local injection of RSV,the epidermis around the wound was significantly thickened,the collagen deposition was significantly increased and the collagen arrangement was tight.The results of WB experiments revealed that on day 15 after wound mapping,the expression levels of the markers of myofibroblastsα-SMA and Collagen I were significantly lower in the wound tissue of DM group compared with NC group on day 15 of wound mapping（P<0.01）,and the expression ofα-SMA and Collagen I in the wound rim of the mice was significantly increased after local treatment with RSV（P<0.05）;Immunofluorescent staining of wound tissue showed that the number of new blood vessels in DM group was significantly lower compared with NC group（P<0.01）,while the number of neovascularization in the traumatic surface of DR group was significantly increased（P<0.05）.（2）The q RT-PCR results showed that the levels of M2 macrophage markers Arg-1 and CD206 m RNA were significantly decreased（P<0.01）,and the levels of pro-inflammatory cytokine markers TNF-α,i NOS,and IL-1βwere significantly increased（P<0.001）in the traumatic surfaces of mice in DM group,compared with NC group.Arg-1 and CD206 gene expression were significantly increased in DR group（P<0.05）,and the pro-inflammatory cytokine markers TNF-α,i NOS,and IL-1βwere significantly decreased（P<0.01）;WB results further confirmed that the expression of CD206 and Arg-1 protein in DM group was significantly lower than that in NC group（P<0.01）,while the pro-inflammatory cytokine TNF-αand i NOS protein expression was significantly increased（P<0.01）.CD206 and Arg-1 protein expression was significantly increased（P<0.05）,while TNF-αand i NOS protein expression was decreased（P<0.01）in the trabecular tissues of mice in DR group.Immunofluorescence staining showed that the number of CD206⁺cells in DM group was significantly less than that in NC group（P<0.01）,while the number of CD206⁺cells in DR group was significantly increased（P<0.01）.（3）In vitro,q RT-PCR results showed that compared with LPS group,RSV significantly increased the expression of Arg-1 and CD206（P<0.01）,and decreased the expression of IL-1βand IL-6 genes（P<0.05）.WB results showed that compared with LPS group,RSV treatment group significantly increased the expression of Arg-1 and CD206 protein（P<0.05）,and decreased the expression of TNF-αand IL-1βprotein（P<0.01）.Flow cytometry results showed an increased percentage of M2 macrophages（CD206⁺）in RSV group compared to LPS group（P<0.001）.（4）WB results showed that the level of p-Akt in THP-1 mononuclear macrophages after RSV administration was significantly higher than that in the control group（P<0.01）,while the level of p-Akt in RSV after pretreatment with LY294002 decreased again（P<0.001）.The results of flow cytometry showed that the proportion of M2 macrophages（CD206⁺）in RSV+LY294002 group was significantly lower than that in RSV group（P<0.01）.In the wound tissue of mice,the expression level of p-Akt protein in the wound of mice in DM group was significantly lower than that in NC group（P<0.001）,and the p-Akt protein expression level of mice in the DR group was higher than that in the DM group（P<0.05）.【Conclusion】（1）RSV improved collagen deposition and neovascularization of wound surface,and induced macrophages to polarize to M2;RSV promote wound healing in diabetic mice.（2）RSV induce M2 macrophage polarization and reduce inflammation by activating PI3K/Akt signaling pathway,thus accelerating wound healing in diabetic mice.