自噬相关基因在博来霉素诱导小鼠皮肤纤维化中的表达及作用

何香; 李洁; 刘佳琦; 郑朝; 胡大海

doi:10.3760/cma.j.cn501120-20200210-00047

自噬相关基因在博来霉素诱导小鼠皮肤纤维化中的表达及作用

doi: 10.3760/cma.j.cn501120-20200210-00047

空军军医大学第一附属医院全军烧伤中心,烧伤与皮肤外科,西安 710032

基金项目:

国家自然科学基金（81530064）

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出版历程
- 收稿日期: 2020-02-10
- 网络出版日期: 2021-10-28
- 刊出日期: 2020-05-20

Expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice

Department of Burns and Cutaneous Surgery, Burn Center of PLA, the First Affiliated Hospital, Air Force Medical University, Xi′an 710032, China

摘要

摘要: 目的探讨自噬相关基因在博来霉素诱导的小鼠皮肤纤维化中的表达及作用。方法 (1)取72只6周龄雄性BALB/c小鼠，采用随机数字表法分为空白对照组、单纯磷酸盐缓冲液(PBS)组和博来霉素组，每组24只。空白对照组小鼠不予任何处理；单纯PBS组和博来霉素组小鼠背部皮肤分别皮下注射100 μL PBS、博来霉素(1 mg/mL)，每天1次，连续注射28 d。注射7、14、21、28 d，每组分别取6只小鼠，肉眼观察小鼠背部皮肤变化，观察结束后处死小鼠，取背部皮肤组织。取注射28 d皮肤组织，行常规苏木精－伊红染色，测量皮肤组织厚度；Masson染色行皮肤组织形态学观察。取注射7、14、21、28 d皮肤组织，酶联免疫吸附测定法检测羟脯氨酸含量，实时荧光定量反转录PCR法和蛋白质印迹法分别检测p62、微管相关蛋白1轻链3 Ⅱ(LC3 Ⅱ)和Beclin－1的mRNA和蛋白表达。(2)取实验(1)中空白对照组小鼠皮肤组织，收集第3～6代成纤维细胞(Fb)，采用随机数字表法分为空白对照组、单纯PBS组、博来霉素组，每组6孔。空白对照组细胞不予任何刺激，单纯PBS组、博来霉素组细胞分别加入20 μL PBS、博来霉素(1 mg/mL)刺激72 h，细胞免疫荧光染色观察LC3 Ⅱ的表达。对数据行析因设计方差分析、单因素方差分析、t检验及Bonferroni校正。结果 (1)空白对照组和单纯PBS组小鼠注射7、14、21、28 d背部皮肤菲薄、红润、静脉血管清晰，单纯PBS组从注射14 d起穿刺部位可见数个隆起的皮丘。博来霉素组小鼠注射7 d皮肤红润，穿刺点处可见数个隆起的皮丘；注射14 d，皮肤轻微变白；注射21 d，皮肤明显变白，周围血管不清晰；注射28 d，皮肤变白，周围血管无法辨认。(2)注射28 d，空白对照组与单纯PBS组小鼠皮肤组织厚度相近(t＝0.79，P>0.05)，博来霉素组小鼠皮肤组织厚度较空白对照组和单纯PBS组明显增加(t＝0.50、0.50，P<0.01)。(3)注射28 d，空白对照组与单纯PBS组小鼠皮肤组织结构相似，均可见少量胶原，胶原排列整齐有序，毛囊分布均匀；博来霉素组小鼠皮肤胶原数目显著增多，但排列杂乱无序，毛囊数明显减少。(4)注射7、14、21、28 d，博来霉素组小鼠皮肤组织中羟脯氨酸含量明显高于空白对照组、单纯PBS组(t＝0.99、0.98、0.50、0.51，0.50、0.50、0.52、0.51，P<0.05或P<0.01)。(5)注射7 d，博来霉素组小鼠皮肤组织p62的mRNA表达明显低于单纯PBS组(t＝0.93，P<0.05)。注射14、21 d，博来霉素组小鼠皮肤组织p62、LC3 Ⅱ、Beclin－1的mRNA表达明显高于空白对照组(t＝0.74、0.70、0.58，0.49、0.51、0.74，P<0.05)和单纯PBS组(t＝0.94、0.65、0.65，0.77、0.49、0.51，P<0.05)。注射28 d，博来霉素组小鼠皮肤组织p62、Beclin－1的mRNA表达明显低于空白对照组(t＝0.50、0.44，P<0.05)和单纯PBS组(t＝0.97、0.55，P<0.05)，而LC3 Ⅱ的mRNA表达仍显著高于空白对照组和单纯PBS组(t＝0.51、0.98，P<0.01)。(6)注射7、14、21、28 d，空白对照组、单纯PBS组、博来霉素组小鼠皮肤组织LC3 Ⅱ的蛋白表达为0.167±0.042、0.122±0.016、0.553±0.078、0.118±0.035，0.120±0.023、0.117±0.061、0.581±0.039、0.159±0.065，0.233±0.027、0.304±0.031、1.020±0.010、0.089±0.045。注射14 d，博来霉素组小鼠皮肤组织p62和Beclin－1的蛋白表达明显高于空白对照组(t＝0.86、0.89，P<0.05)和单纯PBS组(t＝0.42、0.89, P<0.05)。注射21 d，博来霉素组小鼠皮肤组织p62、LC3 Ⅱ和Beclin－1的蛋白表达明显高于空白对照组和单纯PBS组(t＝0.82、0.45、0.50，0.79、0.51、0.50，P<0.01)。注射28 d，博来霉素组小鼠皮肤组织p62、LC3 Ⅱ和Beclin－1的蛋白表达明显低于空白对照组和单纯PBS组(t＝0.77、0.54、0.52，0.50、0.51、0.50，P<0.05)。(7)培养72 h，博来霉素组Fb中LC3 Ⅱ的表达明显低于空白对照组、单纯PBS组。结论博来霉素刺激皮肤纤维化过程中，自噬相关基因先升高后降低，自噬过程被激活，有望逆转皮肤纤维化进程。
- 自噬 /
- 纤维化 /
- 博来霉素 /
- 增生性瘢痕
Abstract: Objective To investigate the expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice. Methods (1) Totally 72 male BALB/c mice aged 6 weeks were divided into blank control group, simple phosphate buffer solution (PBS) group, and bleomycin group according to the random number table, with 24 mice in each group. Mice in blank control group received no treatment, and 100 μL of PBS and bleomycin (1 mg/mL) were respectively injected subcutaneously in the back skin of mice in simple PBS and bleomycin group, once a day for 28 days. On injection day (ID) 7, 14, 21, and 28, 6 mice in each group were collected to observe the skin change on the back of mice with naked eyes. After the observation, the mice were sacrificed and skin tissue on the back was taken. Skin tissue of mice on ID 28 was collected to measure the thickness of skin tissue by routine hematoxylin-eosin staining and observe skin tissue morphology by Masson staining. Skin tissue on ID 7, 14, 21, and 28 was taken to detect content of hydroxyproline by enzyme linked immunosorbent assay, and mRNA and protein expressions of p62, microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and Beclin-1 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. (2) Skin tissue of mice in blank control group in experiment (1) was taken to culture fibroblasts (Fbs) in 3rd-6th passages. The cells were divided into blank control group, simple PBS group, and bleomycin group according to the random number table, with 6 wells in each group. Cells in blank control group were not stimulated, and cells in simple PBS group and bleomycin group were stimulated with 20 μL of PBS and bleomycin (1 mg/mL) for 72 h, respectively. Cellular immunofluorescence staining was used to observe the expression of LC3 Ⅱ. Data were statistically analyzed with analysis of variance of factorial design, one-way analysis of variance, t test, and Bonferroni correction. Results (1) Skin on the back of mice in blank control group and simple PBS group was thin and ruddy, and the veins were clear on ID 7, 14, 21, and 28. Several raised ridges were visible on the puncture site of mice in simple PBS group from ID 14. Skin on the back of mice was ruddy, with several raised ridges visible on the puncture site of mice in bleomycin group on ID 7, the skin turned slightly white on ID 14, the skin turned white obviously with unclear surrounding blood vessels on ID 21, and the skin turned white and the surrounding blood vessels could not be recognized on ID 28. (2) On ID 28, the skin thicknesses of mice in blank control group and simple PBS group were similar (t=0.79, P>0.05). Compared with that in blank control group and simple PBS group, the skin thickness of mice in bleomycin group was significantly increased (t=0.50, 0.50, P<0.01). (3) On ID 28, the skin tissue structure of mice in blank control group and simple PBS group was similar, with a small amount of orderly arranged collagen and evenly distributed hair follicle; the number of collagen of skin in mice of bleomycin group was increased obviously and arranged disorderly, and the number of hair follicle was decreased significantly. (4) On ID 7, 14, 21, and 28, the content of hydroxyproline in the skin tissue of mice in bleomycin group was significantly higher than that in blank control group and simple PBS group (t=0.99, 0.98, 0.50, 0.51, 0.50, 0.50, 0.52, 0.51, P<0.05 or P<0.01). (5) On ID 7, p62 mRNA expression in the skin tissue of mice in bleomycin group was significantly lower than that in simple PBS group (t=0.93, P<0.05). On ID 14 and 21, the mRNA expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group (t=0.74, 0.70, 0.58, 0.49, 0.51, 0.74, P<0.05) and simple PBS group (t=0.94, 0.65, 0.65, 0.77, 0.49, 0.51, P<0.05). On ID 28, the mRNA expressions of p62 and Beclin-1 in the skin tissue of mice in bleomycin group were significantly lower than those in blank control group (t=0.50, 0.44, P<0.05) and simple PBS group (t=0.97, 0.55, P<0.05), and that of LC3 Ⅱ was significantly higher than that in blank control group and simple PBS group, respectively (t=0.51, 0.98, P <0.01). (6) On ID 7, 14, 21, and 28, the protein expressions of LC3 Ⅱ in blank control group, simple PBS group, and bleomycin group were 0.167±0.042, 0.122±0.016, 0.553±0.078, 0.118±0.035, 0.120±0.023, 0.117±0.061, 0.581±0.039, 0.159±0.065, 0.233±0.027, 0.304±0.031, 1.020±0.010, 0.089±0.045. On ID 14, the protein expressions of p62 and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group (t=0.86, 0.89, P<0.05) and simple PBS group (t=0.42, 0.89, P<0.05). On ID 21, the protein expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group and simple PBS group (t=0.82, 0.45, 0.50, 0.79, 0.51, 0.50, P<0.01). On ID 28, the protein expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly lower than those in blank control group and simple PBS group (t=0.77, 0.54, 0.52, 0.50, 0.51, 0.50, P<0.05). (7) After culture for 72 h, the expression of LC3 Ⅱ in Fbs of bleomycin group was significantly lower than that of blank control group and simple PBS group, respectively. Conclusions In the process of bleomycin stimulating skin fibrosis, autophagy-related genes increase firstly and then decrease. When the autophagy process is activated, it is expected to reverse the process of skin fibrosis.
- Autophagy /
- Fibrosis /
- Bleomycin /
- Hypertrophic scar