Effects of Freund′s complete adjuvant on autophagy protein expression in rat tuberculous wound model
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摘要:
目的 在大鼠结核性创面模型中探讨弗氏完全佐剂对自噬蛋白表达的影响。 方法 采用实验研究方法。第1批取12只6周龄雄性SD大鼠，臀部皮下注射弗氏完全佐剂致敏，3周后于大鼠背部脊柱两侧皮下注射牛分枝杆菌减毒株(BCG)感染，成功建立结核性创面大鼠模型后，按照随机数字表法(分组方法下同)分为感染8 d组、感染15 d组、感染32 d组、感染43 d组，每组3只，继续正常喂养至感染后相应天数。第2批取23只6周龄雄性SD大鼠，分成空白对照组3只(正常喂养未行任何处理)、单纯BCG组5只、BCG＋雷帕霉素组6只、BCG＋3-甲基腺嘌呤组6只、BCG＋饥饿组3只。后4组大鼠同前致敏，1周后同前感染。单纯BCG组大鼠感染后正常喂养未行任何处理；BCG＋雷帕霉素组大鼠感染后腹腔注射雷帕霉素隔日1次，正常喂养；BCG＋3-甲基腺嘌呤组大鼠感染后腹腔注射3-甲基腺嘌呤隔日1次，正常喂养；BCG＋饥饿组大鼠感染后饥饿48 h后正常喂养。将第1批4组所有大鼠分别于感染后相应天数处死，取臀部注射弗氏完全佐剂处的组织；将第2批单纯BCG组、BCG＋雷帕霉素组、BCG＋3-甲基腺嘌呤组、BCG＋饥饿组大鼠于感染后7 d同前取材，空白对照组所有大鼠于相同时间点取相同部位组织。行苏木精-伊红染色，观察取材部位组织细胞结构形态；采用免疫组织化学法观察取材部位组织中Beclin-1、微管相关蛋白1轻链3B(LC3B)蛋白表达情况。对数据行Kruskal-Wallis检验及Bonferroni校正。 结果 感染8 d组大鼠注射弗氏完全佐剂部位组织可见炎症细胞浸润，感染15 d组可见肉芽肿形成，感染32 d组、感染43 d组可见部分组织细胞坏死，感染43 d组细胞坏死较感染32 d组加重。感染后7 d，单纯BCG组、BCG＋雷帕霉素组、BCG＋3-甲基腺嘌呤组、BCG＋饥饿组大鼠注射弗氏完全佐剂部位组织均可见炎症细胞浸润，空白对照组细胞排列规整且未见炎症细胞浸润。感染8 d组、感染15 d组、感染32 d组、感染43 d组大鼠注射弗氏完全佐剂部位组织Beclin-1、LC3B蛋白表达，组间总体比较差异无统计学意义(
H ＝1.923、5.821，
P >0.05)。感染后7 d，空白对照组、单纯BCG组、BCG＋雷帕霉素组、BCG＋3-甲基腺嘌呤组、BCG＋饥饿组大鼠注射弗氏完全佐剂部位组织Beclin-1和LC3B蛋白表达分别为0.325%(0.250%，0.360%)、3.225%(1.340%，3.987%)、4.823%(2.630%，6.559%)、4.216%(1.790%，5.969%)、1.765%(0.865%，2.649%)和0.301%(0.264%，0.516%)、2.865%(1.455%，5.768%)、1.033%(0.398%，1.873%)、1.168%(0.429%，1.907%)、0.655%(0.283%，1.652%)，其中BCG＋雷帕霉素组大鼠注射弗氏完全佐剂部位组织Beclin-1蛋白表达明显高于空白对照组(
Z ＝4.796，
P <0.05)，单纯BCG组大鼠注射弗氏完全佐剂部位组织LC3B蛋白表达明显高于空白对照组(
Z ＝4.953，
P <0.05)。 结论 结核性创面大鼠模型中弗氏完全佐剂可以增强局部组织自噬相关蛋白Beclin-1、LC3B蛋白的表达水平。
Abstract:Objective To explore the effects of Freund′s complete adjuvant on autophagy protein expression in rat tuberculous wound model. Methods The experimental research method was used. In the first batch, twelve 6-week-old male Sprague-Dawley (SD) rats were sensitized by subcutaneous injection of Freund′s complete adjuvant into the hips. Three weeks later, the rats were infected with attenuated Bacille Calmette-Guérin (BCG) subcutaneously on both sides of the back spine. After establishing the tuberculosis wound rat model, according to the random number table (the same grouping method below), the rats were divided into 8 d infection group, 15 d infection group, 32 d infection group, and 43 d infection group, with 3 rats in each group, with continuous normal feeding to the corresponding days after infection. In the second batch, twenty-three 6-week-old male SD rats were divided into blank control group (
n =3, normal feeding without any treatment), BCG alone group (
n =5), BCG+ rapamycin group (
n =6), BCG+ 3-methyladenine group (
n =6), and BCG+ starvation group (
n =3). The last 4 groups of rats were sensitized as before, and infected as before 1 week later. Rats in BCG alone group were fed normally without any treatment. Rats in BCG+ rapamycin group or BCG+ 3-methyladenine group were intraperitoneally injected with rapamycin or 3-methyladenine once every other day and fed normally. Rats in BCG+ starvation group were fasted for 48 hours after infection and then fed normally. All the rats in the first batch of 4 groups were sacrificed on the corresponding days after infection, and the tissue where the buttocks were injected with Freund′s complete adjuvant was harvested; the tissue of rats in the second batch of BCG alone group, BCG+ rapamycin group, BCG+ 3-methyladenine group, and BCG+ starvation group were harvested the same as before 7 days after infection, and all the rats in blank control group were taken the same tissue at the same time point. Hematoxylin-eosin staining was performed to observe the structure and morphology of cells in the tissue harvested; immunohistochemistry was used to observe the protein expressions of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B) in the tissue harvested. Data were statistically analyzed with Kruskal-Wallis test and Bonferroni correction. Results Inflammatory cell infiltration was observed in the tissue of rats where the Freund′s complete adjuvant was injected in 8 d infection group, granuloma formation was seen in 15 d infection group, part of tissue cell necrosis was seen in 32 d infection group and 43 d infection group, and cell necrosis in 43 d infection group was worse than that in 32 d infection group. Seven days after infection, inflammatory cell infiltration was seen in the tissue of rats where the Freund′s complete adjuvant was injected in BCG alone group, BCG+ rapamycin group, BCG+ 3-methyladenine group, and BCG+ starvation group, while regular arrangement of cells and no inflammatory cell infiltration were observed in blank control group. There were no statistically significant differences in the protein expressions of Beclin-1 or LC3B in the tissue of rats where the Freund′s complete adjuvant was injected in 8 d infection group, 15 d infection group, 32 d infection group, and 43 d infection group (
H =1.923, 5.821,
P >0.05). Seven days after infection, the protein expressions of Beclin-1 and LC3B in the tissue of rats where the Freund′s complete adjuvant was injected in blank control group, BCG alone group, BCG+ rapamycin group, BCG+ 3-methyladenine group, and BCG+ starvation group were respectively 0.325% (0.250%, 0.360%), 3.225% (1.340%, 3.987%), 4.823% (2.630%, 6.559%), 4.216% (1.790%, 5.969%), 1.765% (0.865%, 2.649%), and 0.301% (0.264%, 0.516%), 2.865% (1.455%, 5.768%), 1.033% (0.398%, 1.873%), 1.168% (0.429%, 1.907%), 0.655% (0.283%, 1.652%). The protein expression of Beclin-1 in the tissue of rats where the Freund′s complete adjuvant was injected in BCG+ rapamycin group was significantly higher than that of blank control group (
Z =4.796,
P <0.05). The protein expression of LC3B in the tissue of rats where the Freund′s complete adjuvant was injected in BCG alone group was significantly higher than that of blank control group (
Z =4.953,
P <0.05). Conclusions Freund′s complete adjuvant can enhance the expression levels of local tissue autophagy-related proteins Beclin-1 and LC3B in rat tuberculous wound model.
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Key words:
- Tuberculosis, cutaneous /
- Wound healing /
- Autophagy
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