基于TNF-α/Caspase-8/MLKL信号通路探讨湿润烧伤膏对糖尿病小鼠创面组织细胞坏死性凋亡影响的研究
Effects of MEBO on Necrotic Apoptosis of Wound Tissue in Diabetes Mice Based on TNF-α/Caspase-8/MLKL Signaling Pathway
劳日玲 1李杰辉 2范琨悦 1王亚亚 1倪宝灵 1甘丽琰 3王丽3
作者信息
- 1. 530021 广西壮族自治区南宁市，广西中医药大学
- 2. .530023 广西壮族自治区南宁市，广西中医药大学第一附属医院创面修复外科
- 3. 530023 广西壮族自治区南宁市，广西壮族自治区中医药研究院
- 折叠
摘要
背景 糖尿病性创面在过度炎症反应和氧化应激的条件下，极易发展成为慢性难愈性创面。而坏死性凋亡在炎症性疾病的发生发展中具有重要的作用，抑制坏死性凋亡的发生可以减轻炎症反应。湿润烧伤膏（MEBO）是由中药制成的膏剂，目前常用于临床多种创面的治疗，可减轻炎症反应，缩短创面愈合时间。但MEBO治疗糖尿病性创面的作用靶点或通路还需进一步研究。目的 探讨MEBO对糖尿病性创面中肿瘤坏死因子ɑ（TNF-α）/非半胱天冬蛋白酶8（Caspase-8）/混合谱系激酶结构域样蛋白（MLKL）信号通路的影响及其治疗作用机制。方法 选用54只8~9周龄的SPF级自发性2型糖尿病模型db/db雄性小鼠，随机分为模型（Model）组、MEBO和重组牛碱性成纤维细胞生长因子外用凝胶（bFGF）组，每组18只；另选取18只杂合db/m雄性小鼠作为对照（Control）组。MEBO组通过用无菌纱布与MEBO混匀后制成药物纱条进行创面外敷，bFGF组将bFGF凝胶浸透无菌纱布制备药物纱条敷到小鼠创面，Model组和Control组用凡士林与无菌纱布混匀后敷到小鼠创面。分别于第3、6、9天对各组小鼠进行过度麻醉处死取材。采用实时荧光定量PCR（RT-qPCR）及免疫印迹法（Western blot）检测TNF-α、Caspase-8、MLKL、RIPK1、RIPK3 mRNA和蛋白表达情况。结果 造模第3、6、9天Model组创面愈合率较Control组降低，MEBO组、bFGF组创面愈合率较Model组升高，差异均有统计学意义（P<0.05）。RT-qPCR结果显示，干预第6、9天，Model组创面组织中TNF-α、MLKL、RIPK1、RIPK3 mRNA表达水平较Control组显著升高，Caspase-8 mRNA表达水平显著降低；与Model组比较，MEBO组和bFGF组TNF-α、MLKL、RIPK1、RIPK3 mRNA表达水平均显著降低，Caspase-8 mRNA表达水平显著升高，差异均有统计学意义（P<0.05）。Western blot 结果显示，干预第6、9天，Model组TNF-α、MLKL、RIPK3蛋白表达升高，Caspase-8蛋白表达降低；MEBO干预后可显著下调TNF-α、MLKL、RIPK1、RIPK3蛋白表达，并上调Caspase-8蛋白表达，差异均有统计学意义（P<0.05）。结论 MEBO可能通过调控TNF-α/Caspase-8/MLKL信号通路抑制创面组织细胞坏死性凋亡，促进糖尿病小鼠创面愈合。
Abstract
BackgroundUnder conditions of excessive inflammation and oxidative stress, diabetic wounds can easily develop into chronic and difficult-to-heal wounds. Necroptosis plays an important role in the occurrence and development of inflammatory diseases, and inhibiting the occurrence of necroptosis can reduce the inflammatory response. Moist exposed burn ointment (MEBO) is an ointment made from traditional Chinese medicine. It is currently commonly used in the treatment of various clinical wounds. It can reduce inflammatory reactions and shorten wound healing time. However, the target or pathway of MEBO in treating diabetic wounds requires further study. ObjectiveTo explore the effect of MEBO on the TNF-/Caspase-8/MLKL signaling pathway in diabetic wounds and its therapeutic mechanism. Methods Fifty-four 89-week-old specific pathogen-free male db/db mice with spontaneous type 2 diabetes were randomly assigned to the Model group, MEBO group, and recombinant bovine basic fibroblast growth factor topical gel (bFGF) group, with 18 mice in each group. In addition, 18 heterozygous male db/m mice were included as the Control group. In the MEBO group, sterile gauze was mixed with MEBO to prepare medicated gauze strips, which were applied externally to the wounds. In the bFGF group, sterile gauze was saturated with bFGF gel to prepare medicated gauze strips, which were then applied to the wounds. In the Model and Control groups, sterile gauze mixed with Vaseline was applied to the wounds. Mice in each group were euthanized by overdose anesthesia on days 3, 6, and 9 after modeling, and wound tissue samples were collected. The mRNA and protein expression levels of TNF-, caspase-8, MLKL, receptor-interacting protein kinase 1 (RIPK1), and receptor-interacting protein kinase 3 (RIPK3) were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Results On days 3, 6, and 9 after modeling, the wound healing rate was significantly lower in the Model group than in the Control group, whereas the wound healing rates in the MEBO and bFGF groups were significantly higher than those in the Model group; all differences were statistically significant (P<0.05). RT-qPCR analysis showed that, on days 6 and 9 after intervention, the mRNA expression levels of TNF-, MLKL, RIPK1, and RIPK3 in wound tissues were significantly increased in the Model group compared with the Control group, whereas caspase-8 mRNA expression was significantly decreased. Compared with the Model group, the MEBO and bFGF groups showed significantly decreased mRNA expression levels of TNF-, MLKL, RIPK1, and RIPK3 and significantly increased caspase-8 mRNA expression; all differences were statistically significant (P<0.05). Western blot analysis showed that, on days 6 and 9 after intervention, protein expression levels of TNF-, MLKL, and RIPK3 were increased, whereas caspase-8 protein expression was decreased in the Model group. MEBO intervention significantly downregulated the protein expression levels of TNF-, MLKL, RIPK1, and RIPK3 and upregulated caspase-8 protein expression; all differences were statistically significant (P<0.05). Conclusion MEBO may inhibit necroptosis of wound tissue cells and promote wound healing in diabetic mice by regulating the TNF-/Caspase-8/MLKL signaling pathway.关键词
糖尿病/糖尿病并发症/糖尿病性创面/湿润烧伤膏Key words
Diabetes/Diabetic complications/Diabetic wounds/Moist burn ointment引用本文复制引用
劳日玲,李杰辉,范琨悦,王亚亚,倪宝灵,甘丽琰,王丽.基于TNF-α/Caspase-8/MLKL信号通路探讨湿润烧伤膏对糖尿病小鼠创面组织细胞坏死性凋亡影响的研究[EB/OL].(2026-09-22)[2026-09-27].https://chinaxiv.org/abs/202609.00311.学科分类
临床医学
