目的 通過(guò)蛋白組學(xué)聯(lián)合分子生物學(xué)技術(shù)解析荊防顆粒對(duì)博來(lái)霉素誘導(dǎo)的急性肺損傷(acute lung injury,ALI)小鼠腸道屏障的改善作用及機(jī)制。方法 利用博來(lái)霉素氣管滴注建立小鼠ALI模型,并給予荊防顆粒進(jìn)行干預(yù)。檢測(cè)小鼠肺指數(shù)和肺組織病理學(xué)變化;ELISA檢測(cè)血清中二胺氧化酶(diamine oxidase,DAO)、脂多糖特異性免疫球蛋白A抗體(lipopolysaccharide specific immunoglobulin A antibody,LPS-SIgA)和D-乳酸水平;試劑盒檢測(cè)血清中丙二醛(malondialdehyde,MDA)、活性氧(reactive oxygen species,ROS)、谷胱甘肽(glutathione,GSH)水平及過(guò)氧化氫酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)活性;Lebel-free蛋白組學(xué)技術(shù)檢測(cè)結(jié)腸組織蛋白表達(dá)譜,尋找荊防顆粒調(diào)控的差異表達(dá)蛋白和關(guān)鍵信號(hào)通路;Western blotting檢測(cè)結(jié)腸組織閉合蛋白(Occludin)、閉鎖小帶蛋白1 (zonula occludens 1,ZO-1)、激活轉(zhuǎn)錄因子4(activating transcription factor 4,ATF4)、C/EBP同源蛋白(C/EBP-homologous protein,CHOP)、磷酸化真核翻譯起始因子2α(phosphorylated eukaryotic initiation factor 2α,p-EIF2α)、磷酸化蛋白質(zhì)激酶RNA樣內(nèi)質(zhì)網(wǎng)激酶(phosphorylated protein kinase RNA-like endoplasmic reticulum kinase,p-PERK)的表達(dá);Western blotting檢測(cè)肺組織核因子E2相關(guān)因子2(nuclear factor E2-related factor 2,Nrf2)、血紅素加氧酶-1(heme oxygenase-1,HO-1)和NAD(P)H醌氧化還原酶1(NAD(P)H: quinone oxidoreductase 1,NQO-1)蛋白表達(dá)。結(jié)果 與模型組比較,荊防顆粒顯著降低ALI小鼠肺指數(shù)(P＜0.05、0.01),改善肺組織病理?yè)p傷,降低血清中DAO、LPS-SIgA和D-乳酸水平(P＜0.05、0.01),上調(diào)結(jié)腸組織Occludin和ZO-1的表達(dá)(P＜0.05)。蛋白組學(xué)和Western blotting分析發(fā)現(xiàn),荊防顆粒調(diào)控了內(nèi)質(zhì)網(wǎng)蛋白加工紊亂,進(jìn)而下調(diào)p-PERK、p-EIF2α、ATF4及CHOP的表達(dá)(P＜0.05),抑制結(jié)腸組織內(nèi)質(zhì)網(wǎng)應(yīng)激。同時(shí),荊防顆粒降低了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),并上調(diào)肺組織Nrf2、HO-1及NQO-1的表達(dá)(P＜0.01)。結(jié)論 荊防顆粒通過(guò)抑制內(nèi)質(zhì)網(wǎng)應(yīng)激維持腸道屏障功能,抑制肺組織氧化應(yīng)激,改善ALI小鼠肺損傷。

Objective To evaluate the improvement effect and mechanism of Jingfang Granules (荊防顆粒) 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

山東省重點(diǎn)研發(fā)計(jì)劃（2024CXPT074）