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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 investigate the effect of topical application of cinnamic aldehyde (CA) on wound healing in diabetic mice and its mechanism. Methods Eight 8-week-old female Nrf2 wild type (Nrf2 ~ (+ / +)) and Nrf2 knockout (Nrf2 ~ (-) -) male hairless mice were injected intraperitoneally with streptozotocin (Streptozotocin, STZ, 50 mg / kg) to establish a diabetic mouse model. (Nrf2 ~ (+ / +) Veh group); (2) Nrf2 ~ (+ / +) mice CA treatment group (Nrf2 ~ (Nrf2 ~ (- / -) Veh group); (4) Nrf2 ~ (- / -) CA group (Nrf2 ~ (- / -) CA group) (n = 4). Four weeks after STZ injection, a full-thickness skin excision wound 6 mm in diameter was established on the back of the mouse. From the day after the operation, 20 μL polyethylene glycol 400 (PEG400) containing 4 μmol / L CA was topically applied to the Nrf2 ~ (+ / +) CA group and the Nrf2 ~ (- On the 14th day, 20% PEG400 (Vehicle, Veh) was used as a blank control to collect wounds on the wounds. Nrf2 ~ (+ / +) Veh group and Nrf2 ~ (- / -) The wound healing area was monitored every other day for 2 weeks postoperatively to assess the healing rate. The expression of Nrf2 protein, downstream target protein HO-1 and oxidative stress-related indicator 8-oxo-d G were compared with immunohistochemical staining in each wound. Results Topical application of CA significantly accelerated wound healing in Nrf2 ~ (+ / +) diabetic mice (P <0.05), but did not affect wound healing in Nrf2 ~ (- / -) diabetic mice (P> 0.05). Immunohistochemistry showed that the expression of Nrf2 in wounds of Nrf2 ~ (+ / +) diabetic mice after local application of CA [(2.42 ± 0.29) vs (7.42 ± 0.73), P <0.05] and downstream target protein HO (9.46 ± 0.28) vs (8.46 ± 0.23), respectively. The level of 8-oxo-d G was significantly lower than that of the control group (5.92 ± 0.36 vs 8.88 ± 0.53, P 0.05) P <0.05]. Compared with Nrf2 ~ (- / -) Veh group, the expression of Nrf2, HO-1 and 8-oxo-d G in Nrf2 ~ (- / -) diabetic mice after local topical CA application obvious change. Conclusion Topical application of CA can promote diabetic wound healing by activating Nrf2 pathway in the skin of diabetic mice and alleviating oxidative stress injury.