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

Zhonghua Yi Xue Za Zhi. 2021 Jun 8;101(21):1598-1605. doi: 10.3760/cma.j.cn112137-20201017-02859.
[Article in Chinese]


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.

目的: 探讨十二指肠结扎对大鼠胃食管反流及博莱霉素肺纤维化的影响。 方法: 将Wistar大鼠随机分为对照(Ctrl)组、十二指肠结扎(GER)组、博莱霉素(BLM)组和十二指肠结扎+博莱霉素(BLM+GER)组。于d0,GER组和BLM+GER组开腹后在幽门下方1.0 cm处将十二指肠结扎30%;Ctrl组和BLM组行假手术。于d14,BLM组和BLM+GER组经气管滴入BLM 5 mg/kg,Ctrl组和GER组滴入生理盐水。d28和d42处死动物,肺组织行HE、Masson和TUNEL染色,碱水解法检测肺组织羟脯氨酸(HYP)含量,硫代巴比妥酸比色法检测肺泡灌洗液(BALF)丙二醛(MDA)含量,ELISA法检测BALF细胞因子和胃蛋白酶原含量,蛋白印迹法和RT-PCR法分别检测肺组织蛋白和mRNA表达。 结果: GER组肺组织轻度炎性细胞浸润。BLM组表现为明显的肺泡炎和纤维化,而BLM+GER组肺泡炎和纤维化较之更为严重。Ctrl组、GER组、BLM组和BLM+GER组d28平均每个200倍视野下肺组织凋亡细胞数分别为(5.6±3.0)、(6.4±5.3)、(15.4±5.3)、(18.4±9.1)(P=0.008)。d42时肺组织Masson蓝染区域比例(%)分别为(21.5±2.8)、(23.4±2.5)、(34.0±5.8)、(41.3±2.9) (P<0.05),HYP(μg/ml)分别为(0.77±0.01)、(1.26±0.01)、(2.02±0.01)、(2.39±0.01) (P<0.01),MDA(μmol/L)分别为(0.51±0.09)、(0.87±0.12)、(1.40±0.31)、(1.71±0.12) (F=23.448,P<0.01),Ctrl或GER组与BLM组或BLM+GER组相比,d42这三项指标差异均有统计学意义(均P<0.05)。与Ctrl组相比,GER组d28 和d42时胃蛋白酶、pH、白细胞介素(IL)-1β、转化生长因子(TGF)-β1、MDA、HYP的差异均有统计学意义(均P<0.05)。与BLM组相比,BLM+GER组d42时BALF的胃蛋白酶及pH值以及d28和d42时TGF-β1、HYP、磷酸化细胞信号转导分子3(p-Smad3)、波形蛋白、磷酸化的细胞外调节蛋白激酶(p-ERK1/2)、活化caspase-3表达差异有统计学意义(均P<0.05)。BLM与Ctrl组相比、BLM+GER组与GER组相比,d28和d42时胃蛋白酶和pH的差异均有统计学意义(均P<0.05)。 结论: 结扎十二指肠引起胃食管反流,活化肺部炎症和纤维化信号,显著加重BLM肺损伤和纤维化。同时,肺纤维化也可能诱发或加重反流。.

MeSH terms

  • Animals
  • Bleomycin
  • Bronchoalveolar Lavage Fluid
  • Duodenum
  • Gastroesophageal Reflux*
  • Lung
  • Pulmonary Fibrosis* / chemically induced
  • Rats
  • Rats, Wistar


  • Bleomycin