MEBT/MEBO对小鼠烧伤创面组织中K19表达的影响及作用机制

Effects and Action Mechanism of MEBT/MEBO on the Expression of K19 in Burn Wounds of Mouse

目的探究烧伤创疡再生医疗技术(moist exposed burn therapy/moist exposed burn ointment, MEBT/MEBO)对烧伤创面组织中角蛋白19 (keratin 19, K19)表达的影响及可能的作用机制。方法选取15只健康雄性昆明小鼠,按照随机数表法随机选取5只作为对照组,10只建立深Ⅱ度烫伤模型后随机分为模型组与治疗组,其中治疗组小鼠创面采用湿润烧伤膏进行处理、模型组小鼠创面采用生理盐水进行处理、对照组小鼠背部脱毛后不做任何处理,1周后取皮肤或创面组织并通过免疫组织化学染色、蛋白质印迹(Western blot)及定量聚合酶链反应(quantitative polymerase chain reaction, Q-PCR)等技术检测自噬相关蛋白Beclin-1、LC3及K19的表达水平。结果治疗第1-7天,治疗组小鼠创面清洁,并始终保持湿润状态,且随着治疗时间的延长逐渐呈现上皮化状态;模型组小鼠创面逐渐加深,并出现感染征象。治疗1周后,免疫组织化学染色法及Western blot法检测结果显示,3组小鼠皮肤或创面组织中Beclin-1、LC3及K19表达水平对比,治疗组>模型组>对照组,P均<0.01,差异具有统计学意义;除Western blot法检测结果显示治疗组与模型组小鼠创面组织中LC3表达水平对比,P>0.05外,其余各检测结果均显示小鼠皮肤或创面组织中Beclin-1、LC3及K19表达水平两两对比,P<0.01,差异具有统计学意义。Q-PCR技术检测结果显示,3组小鼠皮肤或创面组织中K19 mRNA表达水平对比,治疗组>模型组>对照组,P <0.01,差异具有统计学意义;3组小鼠皮肤或创面组织中K19mRNA表达水平两两对比,P均<0. 01,差异具有统计学意义。结论 MEBT/MEBO促进烧伤创面愈合可能与增加创面组织中K19的表达有关,而诱导自噬相关蛋白的高表达可能是其具体机制之一。

Objective To explore the effects and possible action mechanism of MEBT/MEBO(moist exposed burn therapy/moist exposed burn ointment) on the expression of keratin 19(K19) in burn wounds. Methods Fifteen healthy male Kunming mice were selected as experimental subjects, of which, 5 mice were randomly assigned into a control group according to the random number table, and the other 10 mice were randomly divided into a model group and a treatment group after the models of deep second degree burn wounds were established. The wounds in the treatment group were managed with MEBO while the wounds in the model group were managed with normal saline. In contrast, no intervention was given to the mice in the control group after their back hair was removed. One week later, skin or wound tissues were taken in all subjects, and the expression levels of autophagy-related proteins including Beclin-1, LC3 and K19 were tested with methods such as immunohistochemical staining, Western blot, quantitative polymerase chain reaction(Q-PCR) and etc. Results During the treatment duration of day 1-7, the wound surface was clean and always at a moist status in the treatment group, gradually presenting epithelialization with time, while the wounds in the model group deepened in depth gradually, with signs of infection. One week after treatment, the results of immunohistochemical staining and Western blot showed statistically significant differences as the expression levels of Beclin-1, LC3 and K19 in mouse skin or burn wounds were respectively compared among the three groups, with highest levels in the treatment group, then the model group and lowest in the control group, all P <0. 01. Except the Western blot results showed no statistically significant difference when the LC3 expression level in wound tissues was compared between the treatment group and the model group(P > 0. 05), all the other pairwise comparisons in terms of the expression levels of Beclin-1, LC3 and K19 in skin or wound tissues among the three groups showed statistically significant differences based on the other test results, P < 0. 01. According to the Q-PCR test results, the comparison of K19 mRNA expression levels in mouse skin or wound tissues among the three groups showed statistically significant difference, with highest level in the treatment group, and then the model group and lowest in the control group, P <0. 01. The pairwise comparisons of K19 mRNA expression levels in skin or wound tissues among the three groups all showed statistically significant difference, P <0. 01. Conclusion The effect of MEBT/MEBO in promoting burn wound healing may be related to the increased expression of K19 in wound tissues, and inducing the high expression of autophagy related proteins may be one of its specific mechanisms.