褪黑素对烫伤大鼠毛囊细胞氧化应激损伤的保护作用

Protective effect of melatonin on oxidative stress inducing hair follicle injury in scald rat

目的了解早期应用褪黑素对烫伤大鼠皮肤残留毛囊细胞的保护作用。方法将18只雄性SD大鼠分为烫伤组、治疗组和假伤组，每组6只。在烫伤组和治疗组大鼠背部造成30％TBSA的深Ⅱ度烫伤，假伤组浸入37℃水中25S致假伤。烫伤组和治疗组大鼠在伤后1h补充平衡盐液抗休克，假伤组不补液。治疗组大鼠伤后1min和8、16h腹腔注射褪黑素溶液（10mg／kg）。烫伤组和假伤组腹腔注射相同剂量的乙醇-等渗盐水溶液（两者体积比为1：99）。于伤后6、12、24h采集各组大鼠致伤区皮肤组织，检测丙二醛（MDA）和还原型谷胱甘肽（GSH）含量。采用原位缺口末端标记法和半胱氨酸天冬氨酸蛋白酶3（caspase-3）免疫组织化学法，检测致伤区皮肤组织残留毛囊细胞凋亡情况。结果烫伤组大鼠伤后各时相点皮肤组织MDA含量均显著高于假伤组（P〈0．01），且伤后12h升高达峰值；治疗组MDA含量较烫伤组明显降低（P〈0．05）。GSH的变化情况与MDA相反。在荧光显微镜下可见残留的毛囊细胞呈蓝色，凋亡细胞呈绿色。烫伤组伤后6、12、24h细胞凋亡率分别为（20．2±3．4）％、（31．2±3．6）％、（22．4±2．7）％，均明显高于假伤组[（4．3±2．3）％、（5．1±2．5）％、（4．1±2．4）％，P〈0．01]。治疗组伤后6、12、24h细胞凋亡率分别为（10．9±3．2）％、（19．1±3．7）％、（13．1±3．4）％，均明显低于烫伤组（P〈0．05）。烫伤组各时相点caspase-3阳性细胞评分均高于假伤组（P〈0．01），而治疗组均低于烫伤组（P〈0．05）。结论大面积深Ⅱ度烫伤后，大鼠氧化应激水平与毛囊细胞凋亡率关系密切；早期应用褪黑素，可以改善机体的氧化应激程度，进而对皮肤残留的毛囊细胞产生抗凋亡作用。

Objective To investigate the protective effect of melatonin on residual hair follicle cells of scald rats at early stage. Methods Eighteen male Sprague-Dawley rats were randomly divided into scald group, treatment group, sham group , with 6 rats in each group. The rats in scald group and treatment group were subjected to 30% TBSA partial thickness scald on the back, and were resuscitated with balanced solution after 1 hour, while those in sham group were immersed in water at 37 ℃ for 25 s to simulate scald, and did not receive fluid replacement. Rats in treatment group were intraperitoneally injected with 10 mg/kg melatonin solution at 1 minute, 8 hours and 12 hours after scald, while those in sham group and scald group were given equal volume of 1% alcohol sodium-isotonic saline instead. Tissue samples were harvested at 6, 12 and 24 post scald hours（PSH） for determination of MDA and GSH levels. Apoptosis of residul hair follicle was detected by TUNEL method and immunohistochemistry of caspase-3. Results The level of MDA in scald group at each time point was much higher than that in sham group（ P 〈 0.01 ）and treatment group （ P 〈 0.05 ） , and it peaked at 12 PSH. The changes in GSH were just opposite to that of MDA. Under fluorescence microscope, the residual hair follicle ceils were blue, and the apoptotic cells appeared green. The apoptosis rate in scald group at 6,12,24 PSH was obviously higher than that in sham （ P 〈 0.01 ） and treatmentgroups（ P 〈0.05）, which was （20.2±3.4）%vs（4.3±2.3）% vs （10.9±3.2）%, （31.2±3.6）% vs（5.1±2.5） % vs（19.1±3.7）%, （22.4±2.7）% vs （4.1±2.4）% vs（13.1±3.4）%, respectively. The score of caspase-3 positive cell in scald group was higher than those in sham group （ P 〈 0.01 ） and treatment group （ P 〈 0.05 ）. Conclusions There is obvious correlation between oxidative stress and apoptosis rate of hair follicle cells in rats with partial thickness scald. Early administration of melatonin may have anti-apoptosis ability for residual hair follicle ceils by attenuation of oxidative stress.