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

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

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5 d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:(1)Nrf2^(+/+)鼠对照组(Nrf2^(+/+)Veh组);(2)Nrf2^(+/+)鼠CA治疗组(Nrf2^(+/+)CA组);(3)Nrf2^(-/-)鼠Veh组(Nrf2^(-/-)Veh组);(4)Nrf2^(-/-)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20μL含4μmol/L CA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20μL 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-d G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-d G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-d G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。

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. Conclusion Topically application of CA promotes diabetic wound healing via activation of Nrf2 pathway and alleviation of oxidative damage.