目的 本实验拟探讨虾青素纳米乳液对SD大鼠全层皮肤缺损创面的愈合和愈合后瘢痕增生的影响及其初步机制。方法 24只雄性SD大鼠,用随机数字表法分为空白对照组、模型组、积雪苷霜软膏阳性对照组和虾青素纳米乳液实验组,每组6只,采用全层皮肤缺损法进行动物模型制作,其中空白对照组仅做禁食、麻醉及剃毛处理,而不进行实际的造模手术。药物干预阳性对照组和实验组在术后次日分别涂覆500 mg积雪苷霜软膏和等量虾青素纳米乳液于每个创面,每日2次,并在术后第1、3、6、7、9、11、14、18、21、28天测量其创面或瘢痕面积,第2、3、4周时进行稍大于造模范围皮肤全层的取材,利用苏木精-伊红(HE)、马苏(Masson)染色分别观察不同时期皮肤组织中成纤维细胞和胶原纤维的形态、分布、含量等;蛋白质免疫印迹(Western blot,WB)检测第4周皮肤组织中α-平滑肌肌动蛋白(α-SMA)、白介素6(IL-6)以及凋亡自噬相关因子。对数据行单因素方差分析处理。结果 在整个实验过程中,给药组较模型组愈合更快,瘢痕愈合质量更高;第4周时,虾青素纳米乳液给药组成纤维细胞数密度低于积雪苷霜组(P＜0.05);胶原纤维面密度较积雪苷霜组降低也更为显著(P＜0.01);WB实验结果显示第4周时,虾青素组与积雪苷霜组的α-SMA无明显差异,但均低于模型组水平,且差异具有统计学意义;虾青素组IL-6蛋白表达低于模型组水平(P＜0.01),但积雪苷霜组IL-6蛋白表达水平却明显高于其他两组(P＜0.001);虾青素组的促凋亡因子Bcl-2相关X蛋白(Bax)和磷脂酰肌醇3激酶/丝氨酸/苏氨酸蛋白激酶/雷帕霉素靶蛋白(PI3K/Akt/mTOR)通路相关蛋白的表达显著增加(P＜0.05)。结论 虾青素纳米乳液可促进大鼠皮肤创面愈合、减少愈合后成纤维细胞的增殖和胶原生成、抑制纤维化、减轻炎症、抑制瘢痕挛缩,从而改善皮肤创面增生性瘢痕的发生发展,其机制可能与诱导凋亡、激活PI3K/Akt/mTOR通路下调自噬有关。
Abstract
OBJECTIVE To investigate the effect and preliminary mechanism of astaxanthin-nano-emulsion on the healing and post-healing scar hyperplasia of full-thickness skin defect wounds in SD rats. METHODS Twenty-four male SD rats were divided into blank control group, model group, cumene cream ointment positive control group and astaxanthin-nano-emulsion experimental group using the random number table method. Animal models were made using the whole skin defect method, in which the blank control group was treated with fasting and anesthetic shaving only, without the actual modeling procedure. In the positive control group and experimental group, 500 mg of cumene cream ointment and equal amount of astaxanthin-nano-emulsion were applied to each wound twice daily on the next postoperative day, and the wound or scar area was measured on the 1st, 3rd, 6th, 7th, 9th, 11th, 14th, 18th, 21st, and 28th postoperative days. At weeks 2, 3, and 4, the whole skin layer was taken slightly larger than the extent of the mold, HE and Masson staining were conducted to observe the morphology, distribution and contents of fibroblasts and collagen fibers in the skin tissues at different time; and the alpha-smooth muscle actin (α-SMA), interleukin 6 (IL-6), apoptotic and autophagy-related factors in the skin tissues at week 4 were detected by protein immunoblotting (Western blot, WB). One-way ANOVA was performed on the data. RESULTS During the whole experiment, the administration group was observed to have faster healing and higher quality of scar healing than the model group. At week 4, the number of fibroblasts in the astaxanthin-nano-emulsion group was significantly lower than that in the cumene cream group (P<0.05), the collagen fiber area density was also significantly lower than that in the cumene cream group (P<0.01). The results of WB experiments showed that the α-SMA in the astaxanthin and cumene cream groups were not significantly different at week 4, but both were significantly lower than the model group (P<0.01). The IL-6 protein expression in the astaxanthin group was lower than the model group (P<0.01), but the IL-6 protein expression level in the cumene cream group was significantly higher than the other two groups (P<0.001). The expressions of pro-apoptotic factors Bcl-2-associated X protein (Bax) and phosphatidylinositol 3 kinase/serine/threonine protein kinase/rapamycin (PI3K/Akt/mTOR) pathway-related proteins were significantly increased in the astaxanthin group (P<0.05). CONCLUSION Astaxanthin-nano-emulsion can promote skin wound healing, reduce fibroblast proliferation and collagen production, inhibit fibrosis, reduce inflammation, and inhibit scar contracture in rats, thus improving the development of hypertrophic scars on skin wounds after healing, and the mechanism may be related to the induction of apoptosis and the activation of PI3K/Akt/mTOR pathway down-regulates autophagy.
关键词
虾青素 /
创面愈合 /
增生性瘢痕 /
凋亡 /
自噬
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Key words
astaxanthin /
wound healing /
hypertrophic scar /
apoptosis /
autophagy
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参考文献
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脚注
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基金
国家自然科学基金委员会项目资助(82060349);兴滇英才支持“万人计划”名医专项资助(RSC2019MY018);云南省科技计划-生物医药重大专项资助(2018ZF002)
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