曲安奈德通过AMPK/mTOR信号通路对小鼠皮肤纤维化的自噬水平的调控作用

Triamcinolone Acetonide Regulates Autophagy Levels in Mouse Skin Fibrosis Through the AMPK/mTOR Signaling Pathway

目的 探究曲安奈德对小鼠皮肤纤维化的自噬水平的调控作用,并分析可能的机制。方法 将40只大鼠随机分为对照组、模型组及曲安奈德低、高剂量组,每组10只。模型组及曲安奈德低、高剂量组小鼠采用博莱霉素[30μg/(kg·d),连续4周]皮下注射法建立皮肤纤维化模型,对照组于同时间背部皮下注射等量生理盐水,建模期间观察各组小鼠于博莱霉素注射7 d、14 d、21 d、28 d时的一般情况;模型建立结束后,曲安奈德低、高剂量组小鼠分别腹腔注射20 g/(L·d)、80 g/(L·d)(连续给药8周),对照组、模型组小鼠于同时间腹腔注射等量生理盐水。常规HE染色并于光学显微镜下测量小鼠皮肤组织厚度;免疫印迹法测量小鼠皮肤组织中自噬相关蛋白[微管相关蛋白1轻链3-Ⅰ(LC3-Ⅰ)、LC3-Ⅱ、家蚕隔离体蛋白1(SQSTM1,又名p62)]、AMP激活蛋白激酶/雷帕霉素靶蛋白(AMPK/mTOR)通路蛋白(AMPK、p-AMPK、mTOR、p-mTOR)水平变化。结果 建模期间模型组及曲安奈德低、高剂量组小鼠分别死亡1只、2只、1只。曲安奈德给药结束后,与对照组比较,模型组及曲安奈德低、高剂量组小鼠皮肤组织厚度、p62蛋白水平升高,LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK水平降低(均P<0.05);与模型组比较,曲安奈德低、高剂量组小鼠皮肤组织厚度、p62蛋白水平降低,LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK水平升高(均P<0.05),呈剂量依赖性。结论 曲安奈德可抑制小鼠皮肤纤维化发生发展,可能是通过促进AMPK-mTOR通路促进皮肤组织自噬有关。

Objective In order to investigate the role of triamcinolone acetonide in the regulation of autophagy levels in mice with skin fibrosis and to analyze the possible mechanisms. Methods Forty rats were randomly divided into control group,model group and triamcinolone acetonide low and high dose groups with 10 rats in each group. Mice in model group and triamcinolone acetonide low and high dose groups were injected subcutaneously [30 μg/(kg·d) for four weeks] to establish skin fibrosis model. Mice in control group were injected with the same amount of normal saline subcutaneously at the same time,the general condition of the mice in each group was observed on the 7th,14th,21th and 28th days after Bleomycin injection,mice in the low and high dose groups of Triamcinolone Acetonide were intraperitoneally injected with 20 g/(L·d)and 80 g/(L·d) for eight weeks,while mice in the control group and model group were intraperitoneally injected with the same amount of normal saline at the same time. The thickness of mouse skin tissue was measured by hematoxylin-eosin(HE)staining and light microscope. The changes of autophagy associated protein [ microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅰ),microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ),bombyx Mori isolate protein 1(P62)],Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)/Sirolimus pathway protein [AMPK,phospho-AMPK(p-AMPK),mammalian target of rapamycin(mTOR),phospho-mTOR( p-mTOR)] in mouse skin were measured by Western blotting.Results One,two and one of the mice in the model group and the low and high dose groups of triamcinolone acetonide died respectively during the modeling period. After triamcinolone acetonide administration,compared with the control group,the thickness and p62 protein level of skin tissue in the model group and the low and high dose groups of triamcinolone acetonide were increased,while the LC3-Ⅱ/LC3-Ⅰand p-AMPK/AMPK levels were decreased(P<0.05),the skin tissue thickness and p62 protein level of mice in triamcinolone acetonide low and high dose groups decreased,while LC3-Ⅱ/LC3-Ⅰand pAMPK/AMPK levels increased(P<0.05),in a dose-dependent manner. Conclusion Triamcinolone acetonide inhibits the development of fibrosis in mice,possibly by promoting autophagy in skin tissue through the AMPK/mTOR pathway.