己酮可可碱腹腔注射对1型糖尿病小鼠足溃疡的改善作用及其机制

Effect and mechanism of intraperitoneal injection of pentoxifylline on foot ulcer in T1DM mice

目的观察己酮可可碱腹腔注射对1型糖尿病(T1DM)小鼠足溃疡的改善作用,并探讨其作用机制。方法18只健康雄性SPF级小鼠随机分成3组:模型组、己酮可可碱组和贝复济组,每组6只。3组小鼠均制备T1DM足溃疡模型。制备成功后,己酮可可碱组小鼠腹腔注射己酮可可碱连续14 d,贝复济组小鼠外喷贝复济连续14 d,模型组小鼠外用50%乙醇溶液连续14 d。取各组小鼠,在制备T1DM足溃疡模型当天和药物干预14 d时分别拍摄足溃疡创面处照片,计算创面愈合率。取各组小鼠,用血糖仪与医用血糖试纸检测尾静脉血FPG。取各组小鼠足溃疡处皮损组织,采用免疫荧光法测算皮损组织中中性粒细胞数量(以MPO阳性细胞数表示),采用免疫组化法测算皮损组织中NETs形成标志物Cit-H3阳性细胞占比,采用免疫荧光法评估皮损组织中NETs释放情况(以MPO+NE双阳性细胞数表示),采用ELISA法检测皮损组织中NETs相关标志物MPO、NE、LL37,采用流式细胞法检测皮损组织中ROS,采用Western blot法检测皮损组织中p38 MAPK、p-p38 MAPK、ERK1/2、p-ERK1/2蛋白。结果己酮可可碱组和贝复济组小鼠,足溃疡创面愈合率均高于模型组(P均<0.05)FPG水平、皮损组织中中性粒细胞数均低于模型组。己酮可可碱组和贝复济组小鼠皮损组织中Cit-H3阳性细胞占比、MPO+NE双阳性细胞数、ROS荧光强度和皮损组织中MPO、NE、LL37水平均低于模型组,且己酮可可碱组低于贝复济组(P均<0.05)。己酮可可碱组皮损组织中p38 MAPK、p-p38 MAPK、ERK1/2、p-ERK1/2蛋白相对表达量均高于模型组(P均<0.05),贝复济组皮损组织中p-p38 MAPK、p-ERK1/2蛋白相对表达量均高于模型组(P均<0.05),且己酮可可碱组皮损组织中p-p38 MAPK、ERK1/2、p-ERK1/2蛋白相对表达量均高于贝复济组(P均<0.05)。结论己酮可可碱腹腔注射可显著改善T1DM小鼠的足溃疡,其作用机制可能与降低FPG水平和中性粒细胞数、抑制NETs形成和释放、抑制ROS生成、激活p38MAPK/ERK信号通路有关。

Objective To observe the alleviating effect of intraperitoneal injection of pentoxifylline on foot ulcers in type 1 diabetes mellitus(T1DM)mice and to explore its mechanism of action.Methods Eighteen healthy male SPFgrade mice were randomly divided into three groups:model group,pentoxifylline group,and Beifuji group,with six mice in each group.All mice in the three groups were induced to develop T1DM foot ulcer models.After successful model prep‑aration,mice in the pentoxifylline group were intraperitoneally injected with pentoxifylline for 14 consecutive days,mice in the Beifuji group were externally sprayed with Beifuji for 14 consecutive days,and mice in the model group were exter‑nally treated with 50%ethanol solution for 14 consecutive days.Photographs of the foot ulcer sites were taken on the day of T1DM foot ulcer model preparation and after 14 days of drug intervention to calculate the wound healing rate.FPG levels from the tail vein blood of mice in each group were measured using the glucometer and medical glucose test strips.The number of neutrophils(represented by the number of MPO positive cells)was measured by immunofluorescence method,and the proportion of NETS-forming marker Cit-H3 positive cells was measured by immunohistochemistry method.Immuno‑fluorescence was used to evaluate the release of NETs in the skin tissues(represented by the number of MPO+NE double positive cells),ELISA was used to detect MPO,NE and LL37,and flow cytometry was used to detect ROS in the skin tis‑sues.Western blotting was used to detect p38 mitogen-activated protein kinase(p38 MAPK),phosphorylation-p38 mito‑gen-activated protein kinase(p-p38 MAPK),extracellular signal regulated kinase 1/2(ERK1/2)and p-ERK1/2 proteins in the skin lesions.Results The healing rates of foot ulcer wound in the pentoxifylline group and Beifuji group were high‑er than that in the model group(P<0.05).The level of FPG and the number of neutrophils in skin lesions were lower than that in the model group.The proportion of Cit-H3 positive cells,the number of MPO+NE positive cells,ROS fluorescence intensity and MPO,NE and LL37 levels in the skin tissues of mice in the pentoxifylline group and Beifuji group were lower than those in the model group,and those in the pentoxifylline group were lower than those in the Beifuji group(all P<0.05).The relative expression levels of p38 MAPK,P-P38 MAPK,ERK1/2 and P-ERK1/2 proteins in skin lesions of the pentoxifylline group were higher than those of the model group(all P<0.05),and the relative expression levels of p-p38 MAPK and p-ERK1/2 proteins in the skin lesions of the Beifuji group were higher than those of the model group(all P<0.05).The relative expression levels of p-p38 MAPK,ERK1/2 and p-ERK1/2 proteins in the skin tissues of the pent‑oxifylline group were higher than those of the Beifuji group(all P<0.05).Conclusion Intraperitoneal injection of pent‑oxifylline can significantly alleviate foot ulcers in T1DM mice,and its mechanism may be related to reducing FPG level and neutrophil count,inhibiting NETs formation and release,inhibiting ROS production,and activating p38 MAPK/ERK signaling pathway.