局部应用肉桂醛通过激活Nrf2通路促进糖尿病小鼠创口愈合

目的 探讨局部应用肉桂醛(cinnamic aldehyde,CA)对糖尿病小鼠创口愈合的影响及其机制.方法 8周龄Nrf2野生型(Nrf2+/+)和Nrf2敲除型(Nrf2-/-)雄性SKH-1无毛小鼠各8只,持续5d腹腔注射链脲霉素(streptozotocin,STZ,50 mg/kg),建立糖尿病小鼠模型.采用随机数字表法分为:①Nrf2+/+鼠对照组(Nrf2+/+Veh组);②Nrf2+/+鼠CA治疗组(Nrf2+/+ CA组);③Nrf2-/-鼠Veh组(Nrf2-/-Veh组);④Nrf2-/-鼠CA组(Nrf2-/-CA组)(n=4).STZ注射后第4周,在小鼠背部建立直径6 mm的全层皮肤切除创口.Nrf2+/+CA组和Nrf2-/-CA组术后当日开始局部应用20 μL含4μmol/LCA的聚乙二醇400(polyethylene glycol400,PEG400),每隔1天干预直至第14天,收集创缘处皮肤,Nrf2+/+Veh组和Nrf2-/-Veh组采用20 μL PEG400(Vehicle,Veh)局部应用作为空白对照.术后2周内每隔1天监测皮肤创口愈合面积评估愈合速率.免疫组化染色对比各组创缘处皮肤组织Nrf2蛋白、下游靶蛋白HO-1和氧化应激损伤相关指标8-oxo-dG的表达水平.结果局部应用CA明显加速Nrf2+/+糖尿病小鼠创口愈合(P＜0.05),但是并不影响Nrf2-/-糖尿病小鼠创口愈合(P＞0.05).免疫组化示Nrf2+/+糖尿病溃疡小鼠局部应用CA后,其创缘皮肤Nrf2的表达水平(2.42±0.29) vs (7.42±0.73),P＜0.05]和下游靶蛋白HO-1的表达水平(5.92 ±0.36) vs (8.88 ±0.53),P＜0.05]显著增强,8-oxo-dG的表达水平则明显减少(9.46 ±0.28)vs(8.46 ±0.23),P＜0.05].Nrf2-/-糖尿病小鼠创口局部外用CA后,其创缘处皮肤Nrf2、HO-1及8-oxo-dG的表达水平与Nrf2-/-Veh组相比并无明显变化.结论 局部应用CA通过激活糖尿病小鼠皮肤组织Nrf2通路,减轻氧化应激损伤,从而促进糖尿病创口愈合.

Topical administration of cinnamic aldehyde accelerates wound healing in diabetic mice by activation of Nrf2 pathway

Objective To determine the effect of topical administration of cinnamic aldehyde (CA) on wound healing in diabetic mice,and investigate the underlying mechanism.Methods A total of 8 Nrf2+/+ and 8 Nrf2-/-male hairless SKH-1 mice (8 weeks old) were intraperitoneally injected with streptozotocin (STZ,50 mg/kg) for 5 consecutive days to establish diabetic model.The mice were randomly divided into 4 groups:control group of Nrf2 +/+ mice (Nrf2 +/+ Veh),CA treatment group of Nrf2 +/+ mice (Nrf2+/+ CA),control group of Nrf2-/-mice (Nrf2-/-Veh),and CA treatment group of Nrf2-/-mice (Nrf2-/-CA) (n =4 for each group).In 4 weeks after STZ injection,2 full-thickness wounds (6 mm in size) were made on the back of the mice.From the day after surgery,Nrf2+/+ CA and Nrf2-/-CA groups received topical administration with 20 μL 4 μmol/L CA diluted by polyethylene glycol 400 (PEG 400) every other day until skin tissues were harvested at the 14th day post-surgery,and Nrf2 +/+ Veh and Nrf2-/-Veh groups were given 20 μL PEG 400 as control.Meanwhile,the wound closure rates were monitored and assessed in 2 weeks post-surgery.The expression levels of Nrf2,downstream target protein HO-1,and 8-oxo-2'-deoxyguanosine (8-oxo-dG,a sensitive parameter for oxidative stress) in the skin of each group were assessed by immunohistochemistry.Results Topical administration of CA significantly accelerated the wound healing of Nrf2+/+ diabetic mice (P ＜0.05),but had no such effect on the Nrf2-/-CA group (P ＞0.05).Moreover,the expression levels of Nrf2 (2.42 ± 0.29 vs 7.42 ±-0.73,P ＜ 0.05) and HO-1 (5.92 ± 0.36 vs 8.88-± 0.53,P ＜ 0.05) were increased,while the level of 8-oxo-dG (9.46 ± 0.28 vs 8.46 ± 0.23,P ＜0.05) was decreased at the wound site after CA treatment in Nrf2 +/+ diabetic mice.However,in the Nrf2-/-diabetic mice,CA treatment showed no effect on the expression levels of Nrf2,HO-1 and 8-oxo-dG.Goncltsion Topically application of CA promotes diabetic wound healing via activation of Nrf2 pathway and alleviation of oxidative damage.