Effects of duodenal ligation on gastroesophageal reflux and bleomycin-induced pulmonary fibrosis in rats

Abstract

Objective: To investigate the effects of duodenal ligation on gastroesophageal reflux and bleomycin-induced pulmonary fibrosis in rats. Methods: Wistar rats were randomized into the control (Ctrl) group, bleomycin (BLM) group, duodenal ligation (GER) group and duodenal ligation plus bleomycin treatment (BLM+GER) group. At day 0 (d0), duodenum ligation was performed in the GER and the BLM+GER group through an open-abdomen surgery at 1.0 cm below the pylorus by about 30% of the circumference. Meanwhile, sham operation was performed in the Ctrl and the BLM group with similar procedures to the above without ligation of the duodenum. At day 14, bleomycin solution (5 mg/kg, for the BLM and BLM+GER groups) or saline (for the Ctrl and GER groups) was intratracheally instilled. Rats were sacrificed at d28 or at d42. HE, Masson's trichrome or TUNEL staining was performed on lung sections of the groups. The levels of hyrdoxyproline (HYP) or malondialdehyde (MDA) were measured respectively by alkaline hydrolysis or thiobarbituric acid colorimetry. The levels of pepsin and cytokines in bronchoalveolar lavage fluid (BALF) samples were assessed by ELISA. Western blot or RT-PCR was used to quantify relative lung expression of proteins or mRNA, respectively. Results: Lungs of the GER group rats were presented with mild inflammatory cell infiltration. Alveolitis and lung fibrosis was prominent in the BLM group but even more severe in the BLM+GER group. Of the Ctrl, GER, BLM and BLM+GER group, the average numbers of apoptotic cells per each magnified field (×200) on d28 lung sections was (5.6±3.0), (6.4±5.3), (15.4±5.3) and (18.4±9.1), respectively (P=0.008); the proportion (%) of blue-stained area under Masson's trichrome at d42 was (21.5±2.8), (23.4±2.5), (34.0±5.8) and (41.3±2.9) (P<0.05); the HYP contents (mg/L) at d42 of each group was (0.77±0.01), (1.26±0.01), (2.02±0.01) and (2.39±0.01) (P<0.01); the BALF levels of MDA (μmol/L) at d42 were (0.51±0.09), (0.87±0.12), (1.40±0.31) and (1.71±0.12) (P<0.001), and differences of these three indices at d42 reached statistical significance when comparing the Ctrl or GER group with the BLM or BLM+GER group (all P<0.05). The levels of pepsin, pH, interleukin (IL)-1β, transforming growth factor (TGF)-β1 and HYP at d28 and d42 were statistically different between the GER group and the Ctrl group (all P<0.05). As compared with the BLM group, the values of TGF-β1, HYP, p-Smad3, vimentin, p-ERK1/2 and cleaved caspase-3 at d28 and d42 were different in the BLM+GER group (all P<0.05). At both d28 and d42, the BALF levels of pepsin and pH were statistically different between the BLM and the Ctrl group, or between the BLM+GER group and the GER group (all P<0.05). Conclusions: Gastroesophageal reflux is induced through duodenal ligation, which activates proinflammatory and profibrotic signals in the lungs and significantly aggravates bleomycin-induced lung injury and fibrosis. In addition, pulmonary fibrosis may induce or worsen the extent of reflux.