Effects of Freund's complete adjuvant on autophagy protein expression in rat tuberculous wound model

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.