目的 通过蛋白组学联合分子生物学技术解析荆防颗粒对博来霉素诱导的急性肺损伤(acute lung injury,ALI)小鼠肠道屏障的改善作用及机制。方法 利用博来霉素气管滴注建立小鼠ALI模型,并给予荆防颗粒进行干预。检测小鼠肺指数和肺组织病理学变化;ELISA检测血清中二胺氧化酶(diamine oxidase,DAO)、脂多糖特异性免疫球蛋白A抗体(lipopolysaccharide specific immunoglobulin A antibody,LPS-SIgA)和D-乳酸水平;试剂盒检测血清中丙二醛(malondialdehyde,MDA)、活性氧(reactive oxygen species,ROS)、谷胱甘肽(glutathione,GSH)水平及过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)活性;Lebel-free蛋白组学技术检测结肠组织蛋白表达谱,寻找荆防颗粒调控的差异表达蛋白和关键信号通路;Western blotting检测结肠组织闭合蛋白(Occludin)、闭锁小带蛋白1 (zonula occludens 1,ZO-1)、激活转录因子4(activating transcription factor 4,ATF4)、C/EBP同源蛋白(C/EBP-homologous protein,CHOP)、磷酸化真核翻译起始因子2α(phosphorylated eukaryotic initiation factor 2α,p-EIF2α)、磷酸化蛋白质激酶RNA样内质网激酶(phosphorylated protein kinase RNA-like endoplasmic reticulum kinase,p-PERK)的表达;Western blotting检测肺组织核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)、血红素加氧酶-1(heme oxygenase-1,HO-1)和NAD(P)H醌氧化还原酶1(NAD(P)H: quinone oxidoreductase 1,NQO-1)蛋白表达。结果 与模型组比较,荆防颗粒显著降低ALI小鼠肺指数(P＜0.05、0.01),改善肺组织病理损伤,降低血清中DAO、LPS-SIgA和D-乳酸水平(P＜0.05、0.01),上调结肠组织Occludin和ZO-1的表达(P＜0.05)。蛋白组学和Western blotting分析发现,荆防颗粒调控了内质网蛋白加工紊乱,进而下调p-PERK、p-EIF2α、ATF4及CHOP的表达(P＜0.05),抑制结肠组织内质网应激。同时,荆防颗粒降低了ALI小鼠血清中ROS和MDA水平(P＜0.01),升高了GSH水平及CAT、SOD活性(P＜0.05),并上调肺组织Nrf2、HO-1及NQO-1的表达(P＜0.01)。结论 荆防颗粒通过抑制内质网应激维持肠道屏障功能,抑制肺组织氧化应激,改善ALI小鼠肺损伤。

Objective To evaluate the improvement effect and mechanism of Jingfang Granules (荆防颗粒) on intestinal barrier in mice with acute lung injury (ALI) induced by bleomycin through proteomics combined with molecular biology techniques. Methods The mice model of ALI was established using bleomycin tracheal instillation and then intervened with Jingfang Granules. Lung index and pathological changes in lung tissue of mice were detected. ELISA was used to detect the levels of diamine oxidase (DAO), lipopolysaccharide specific immunoglobulin A antibody (LPS-SIgA) and D-lactic acid in serum. The corresponding assay kits were used to measure the levels of malondialdehyde (MDA), reactive oxygen species (ROS), glutathione (GSH) and activities of catalase (CAT), superoxide dismutase (SOD) in serum. The lebel-free proteomics technology was used to detect the protein expression profile of colon tissue, the differential expressed proteins and key signaling pathways regulated by Jingfang Granules were screened. Western blotting was used to detect the expressions of Occludin, zonula occludens 1 (ZO-1), activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), phosphorylated eukaryotic initiation factor 2α (p-EIF2α) and phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK) in colon tissue. Western blotting was used to detect the expressions of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1) proteins in lung tissue. Results Compared with model group, Jingfang Granules significantly reduced the lung index of ALI mice (P < 0.05, 0.01), improved the pathological damage of lung tissue, decreased the levels of DAO, LPS-SIgA and D-lactic acid in serum (P < 0.05, 0.01), up-regulated the expressions of Occludin and ZO-1 in colon tissue (P < 0.05). Proteomics and Western blotting analysis revealed that Jingfang Granules regulated the disorder of protein processing in endoplasmic reticulum, thereby down-regulating the expressions of p-PERK, p-EIF2α, ATF4 and CHOP to inhibit endoplasmic reticulum stress in colon tissue of ALI mice (P < 0.05). Meanwhile, Jingfang Granules reduced the levels of ROS and MDA in serum of ALI mice (P < 0.01), increased GSH level and activities of CAT, SOD (P < 0.05), and up-regulated the expressions of Nrf2, HO-1 and NQO-1 in lung tissues (P < 0.01). Conclusion Jingfang Granules alleviate lung injury in ALI mice by inhibiting endoplasmic reticulum stress to preserve intestinal barrier function and suppressing pulmonary oxidative stress.

R285.5

山东省重点研发计划（2024CXPT074）