Effect and mechanism of intraperitoneal injection of pentoxifylline on foot ulcer in T1DM mice
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 SPF-grade 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.
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