关键词: 水蛭素类, 结核, 胸膜, 纤维化, 小鼠

Abstract:

Objective: To investigate the effect of hirudin on tuberculous pleural fibrosis in mice by regulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Methods: Sixty male C57 mice weighing 20-25 g were randomly divided into the following groups: control group, model group, low-dose hirudin group (L group), medium-dose hirudin group (M group), high-dose hirudin group (H group), and high-dose hirudin+ML385 (an Nrf2 transcription inhibitor) group (H+ML385 group). Except for the control group, all other groups were established as models by injection of a bacillus Calmette-Guérin (BCG) suspension. After 28 days, the distribution of pleural effusion was observed in each group, and the volume of pleural effusion was collected and recorded. Pleural adhesion was observed; the adhesiveness of pleural effusion was assessed using fibrinogen (FBG) levels and the score of pleural adhesive bands number. Pleural thickness was measured. Histopathological changes of pleural tissues were observed by hematoxylin-eosin (HE) staining. The protein expressions of Nrf2 and HO-1 in pleural tissues were detected by western blot. The contents of matrix metalloproteinases (MMP-1, MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) in pleural tissues were measured by enzyme-linked immunosorbent assay (ELISA). Results: After 28 days, the H group showed significantly lower values compared to the model group in the following parameters: pleural effusion volume ((666.00±87.62) μl vs. (1144.00±15.00) μl), pleural thickness ((16.50±1.17) μm vs. (21.11±2.98) μm), pleural adhesion band score (2.60±0.51 vs. 3.90±0.31), FBG ((0.30±0.07) g/L vs. (0.48±0.07) g/L), MDA ((74.51±6.53) mmol/mg vs. (102.70±14.06) mmol/mg), MMP-1 ((14.08±0.85) μg/L vs. (18.54±1.20) μg/L), MMP-9 ((4.84±0.38) μg/L vs. (7.65±1.02) μg/L), and TIMP-1 ((2.63±0.13) μg/L vs. (3.62±0.36) μg/L). Conversely, SOD ((134.00±8.07) U/mg vs. (90.79±4.68) U/mg) and GSH ((350.80±15.73) nmol/g vs. (247.30±22.28) nmol/g) levels were significantly higher in the H group than those in the model group. All these differences were statistically significant (t=8.48, P<0.001; t=4.54, P<0.001; t=6.79, P<0.001; t=5.53, P<0.001; t=5.73, P<0.001; t=9.57, P<0.001; t=8.16, P<0.001; t=8.10, P<0.001; t=14.64, P<0.001; t=12.00, P<0.001, respectively). HE staining revealed that hirudin intervention groups (especially in the high-dose group) markedly alleviated pathological fibrotic changes in the pleura compared with the model group. Western blot results indicated that Nrf2 protein expression in the H group (0.83±0.07) was significantly higher than that in the model group (0.57±0.06) and also significantly higher than that in the H+ML385 group (0.67±0.50) (t=8.36, P<0.001; t=4.09, P=0.017, respectively). HO-1 protein expression in the H group (0.72±0.03) was significantly higher than that in the model group (0.31±0.02), with a statistically significant difference (t=8.36, P<0.001). Conclusion: hirudin can ameliorate tuberculous pleural fibrosis by inhibiting oxidative stress through the regulation of the Nrf2/HO-1 signaling pathway.

Key words: Hirudins, Tuberculosis, Pleura, Fibrosis, Mice