Recombinant Expression Of Human IL-33 And Its Effect On Skin Wound Healing In Diabetic Mice

Objective:Diabetic foot ulcer(DFU)is a prevalent complication of diabetes.The ulceration and unhealing of the wound seriously affect the daily life and health of patients.A lot of researches are committed to solving the problem of chronic wounds,a large number of study about different factors that affect wound healing have been carried out.However,no existing drugs that can achieve expeditious wound healing.This study intends to establish a model of wound healing in diabetic mouse to explore the mechanism of IL-33 promoting wound healing and provide a new therapy for diabetic foot ulceration.Method:1.Expression of rhmat IL-33:The c DNA sequence of human mature IL-33 was obtained from the Gene Bank database.The fragment was inserted into the prokaryotic expression vector p ET-28a(+)to construct the recombinant human mature IL-33(rhmat IL-33)plasmid.Then the constructed vector was transformed into E.coli BL21(DE3)to express recombinant protein,while the temperature and induction time by IPTG were optimized.Finally,recombinant protein was purified by Ni-NTA affinity chromatography,and SEC-HPLC was used to detect the purity of the recombinant protein.2.The activity assay of rhmat IL-33 on cells :Biological activity of rhmat IL-33 was detected on cells: including the induction of inflammatory cytokine TNF-α(tumor necrosis factor-α)secretion in mouse Raw 264.7cells,the promotion of cell migration of HSF(human skin fibroblast)and Haca T(human immortalized keratinocytes)cells by scratch wound healing assay.3.Effect of rhmat IL-33 on wound healing in diabetic mice:The model of diabetic mice was established using STZ(streptozotocin),mice with a glucose concentration exceeding 16.7 mmol/L were considered diabetic.Full-thickness dorsal wounds were performed on the back of wild type(WT)and diabetic model(DM)mice.Mice were divided into WT,WT+rhmat IL-33,DM,DM+rhmat IL-33 groups,rhmat IL-33(10 μl,1 μg)or PBS(10 μl)was administrated locally at the wound site after wound preparation once a day for 7 consecutive days.Then compared the wound area to evaluate the effect of rhmat IL-33 on the wound healing;The effect of rhmat IL-33 on the keratinocytes proliferation around the wound was observed by HE staining.4.The mechanism of rhmat IL-33 on promoting wound healing:The proportion of ILC2(Group 2 innate lymphoid cell)in wound tissue was compared by flow cytometry assay to investigate the effect of rhmat IL-33 treatment on ILC2s;the expression of canonical M2 macrophage activators IL-13 and IL-4,M2macrophage-associated genes such as ArgⅠ and YM1 and extracellular matrix(ECM)synthesis-associated genes Collagen Ⅲa in skin wound were detected by RT PCR to explore the effects of rhmat IL-33.Result:1.Expression of rhmat IL-33:After optimizing expression conditions of rhmat IL-33,the recombianant Ecoli was cultured at 37℃ and induced for 6 h,rhmat IL-33 expressed in soluble form,and the yields of recombinant protein beyonded 70% of total cell lysate;rhmat IL-33 was eluted with PBS buffer containing 100 m M and 300 m M imidazole using Ni-NTA affinity chromatography,the purity rhmat IL-33 was close to 100%.2.The activity assay of rhmat IL-33 on cells :The rhmat IL-33 could induced inflammatory cytokine TNF-α secretion from Raw264.7 cells in a dose-dependent manner,and rhmat IL-33 accelerated HSF and Haca T cells migration.3.Effect of rhmat IL-33 on wound healing in diabetic mice:Administration of rhmat IL-33 obviously accelerated wounds gaping narrower in WT+rhmat IL-33 group and DM+rhmat IL-33 group(P < 0.05),as assessed by measuring the rate of wound closure.HE stained of the wounds demonstrated a significant increase in keratinocytes in rhmat IL-33 treated mice.4.The mechanism of rhmat IL-33 on promoting wound healing:Rhmat IL-33 treatment resulted in a significant increase in the frequency of ILC2 s in both WT+rhmat IL-33 group(P < 0.001)and DM+rhmat IL-33 group(P < 0.05)at day 7;after rhmat IL-33 treatment,the expression of IL-13 and IL-4 were upregulated,but not significantly;and the expression of ArgⅠ,YM1(P < 0.05)and CollagenⅢa(P < 0.05)were upregulated.Conclusion:1)The recombinant human mature IL-33 protein was obtained with the purity closeing to 100%.2)The rhmat IL-33 was identified to increase TNF-α secretion and cell migration.3)The rhmat IL-33 was fould to accelerate skin wound healing in mice,especially in diabetic mice.3)The increase of ILC2 cells porption and the expression of canonical M2 macrophage activators IL-13,IL-4;M2 macrophages markers YM1,ArgⅠ and ECM synthesis-associated genes Collagen Ⅲa in wound by rhmat IL-33 may be the potential mechanism of rhmat IL-33 promoting wound healing.