[Anti-inflammatory mixture alleviates acute lung injury induced by sepsis in rats by modulating Beclin-1-mediated autophagy]

Abstract

Objective: To investigate the protective effects of an anti-inflammatory mixture on acute lung injury (ALI) induced by sepsis in rats, as well as its possible mechanisms.

Methods: A total of 40 Sprague-Dawley (SD) rats were randomly divided into the sham group, septic ALI model group (model group), 3-methyladenine (3-MA) control group, and anti-inflammatory mixture pretreatment group, with 10 rats in each group. Cecal ligation and perforation (CLP) was performed to reproduce a septic ALI model. The rats in the sham group only underwent opening and closing the abdomen without perforation and ligation. Both groups were given saline gavage and intraperitoneal injection for 3 consecutive days before surgery. The 3-MA control group was given intraperitoneal injection of saline and autophagy inhibitor 3-MA 15 mg/kg for 3 consecutive days before modeling. The anti-inflammatory mixture pretreatment group was given 8.8 mL/kg of anti-inflammatory mixture by gavage [the composition of anti-inflammatory mixture: rhubarb 15 g (after the next), coptis chinensis 15 g, baical skullcap root 12 g, magnoliae cortex 12 g, dahurian patrinia herb 30 g] and saline intraperitoneal injection for 3 consecutive days before modeling. The rats in each group were anesthetized 24 hours after surgery and died due to abdominal aortic blood collection. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum inflammatory cytokines interleukins (IL-1β and IL-6). Lung tissue was taken and then the bronchoalveolar lavage fluid (BALF) was collected, and the levels of IL-1β and IL-6 were detected by ELISA. Lung wet/dry weight (W/D) ratio was measured. After hematoxylin-eosin (HE) staining, the histopathological changes of the lungs were observed under light microscopy. Western blotting was used to detect the expression of autophagy markers microtubule-associated protein 1 light chain 3- II/I (LC3- II/I) and Beclin-1 protein in lung tissue. Autophagosomes in lung tissue were observed with transmission electron microscopy.

Results: Compared with the sham group, the rats in the model group exhibited severe destruction of lung tissue structure, with significant infiltration of inflammatory cells, the lung W/D ratio and the levels of IL-1β and IL-6 in serum and BALF were significantly increased, the expressions of LC3- II/I and Beclin-1 protein were down-regulated, the autophagosomes were more. The rats in the 3-MA control group exhibited more severe lung tissue injury as compared with the model group, the lung W/D ratio and the levels of inflammatory cytokines in serum and BALF were further increased, the expressions of LC3- II/I and Beclin-1 protein still showed a decrease tendency as compared with the sham group, and the autophagosomes were less than that in the model group. Compared with the model group, the anti-inflammatory mixture pretreatment group showed milder lung tissue injury with a minimal amount of inflammatory cell infiltration, the lung W/D ratio was significantly reduced (7.07±1.02 vs. 11.33±1.85, P < 0.05), the levels of IL-1β and IL-6 in both serum and BALF were significantly decreased [IL-1β (ng/L): 26.04±3.86 vs. 40.83±5.46 in serum, 17.75±2.02 vs. 26.86±4.32 in BALF; IL-6 (ng/L): 91.28±10.15 vs. 129.44±13.05 in serum, 76.06±7.51 vs. 120.91±7.47 in BALF, all P < 0.05], and the ratio of LC3- II/I and Beclin-1 protein expression were significantly increased [LC3- II/I ratio: 1.23±0.02 vs. 0.60±0.02, Beclin-1 protein (Beclin-1/GAPDH): 2.37±0.33 vs. 0.62±0.05, both P < 0.05]. Furthermore, an increase in the number of autophagosomes was observed.

Conclusions: The anti-inflammatory mixture improves lung injury in rats with sepsis induced by CLP and reduce inflammation levels, potentially through upregulation of Beclin-1-mediated autophagy.