连翘苷对感染性休克小鼠急性肺损伤的作用与机制

doi:10.13418/j.issn.1001-165x.2024.2.12

摘要/Abstract

摘要： 目的探讨连翘苷通过腺苷酸活化蛋白激酶（adenosine monophosphate activated protein kinase，AMPK）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）/p70核糖体S6蛋白激酶（p70 S6 kinase，p70S6K）信号通路介导的自噬对感染性休克小鼠急性肺损伤（Acute lung injury，ALI）的影响。方法随机选择12只小鼠作为对照组，其余小鼠通过腹腔注射20 mg·kg-1脂多糖(Lipopolysaccharide, LPS)构建感染性休克小鼠模型，将感染性休克小鼠随机平分为模型组、低、中、高剂量实验组（5 mg·kg-1、10 mg·kg-1、20 mg·kg-1连翘苷）、高剂量+抑制剂组（20 mg·kg-1连翘苷+20 mg·kg-1 AMPK抑制剂compound C），每组均12只小鼠。称量肺干重及湿重，计算W/D比值；ELISA法检测BALF中炎性因子肿瘤坏死因子-α（tumor necrosis factor -α，TNF-α）、白细胞介素1β（interleukin-1β, IL-1β）、白细胞介素6（interleukin-6, IL-6）水平、血清内毒素（endotoxin，ET)含量、肺组织髓过氧化物酶（myeloperoxidase，MPO）活性；HE染色检测肺组织病理变化；Western blot检测自噬蛋白微管相关蛋白-轻链3（microtubule-associated protein -light chain 3，LC3）-II/I、Beclin 1、Ras相关GTP结合蛋白7（Ras-associated GTP binding protein 7，Rab7）、溶酶体关联膜蛋白2（lysosomal associated membrane protein 2，LAMP2）、AMPK/mTOR/p70S6K信号通路蛋白表达。结果对照组、模型组、低、中、高剂量实验组和高剂量+抑制剂组小鼠肺组织LC3-II/I比值分别为1.43±0.14、0.73±0.07、0.81±0.07、1.12±0.10、1.39±0.13、0.76±0.08，Beclin1蛋白水平分别为1.05±0.11、0.43±0.05、0.50±0.05、0.76±0.08、0.98±0.10、0.46±0.05，Rab7蛋白水平分别为1.53±0.17、0.67±0.06、0.70±0.07、1.04±0.10、1.41±0.14、0.69±0.06，LAMP2蛋白水平分别为1.47±0.15、0.72±0.07、0.81±0.08、1.09±0.11、1.35±0.13、0.74±0.07，p-AMPK/AMPK蛋白水平分别为0.95±0.05、0.33±0.03、0.39±0.04、0.68±0.07、0.91±0.09、0.36±0.04，p-mTOR/mTOR蛋白水平分别为0.28±0.02、0.94±0.06、0.88±0.07、0.57±0.05、0.30±0.03、0.87±0.09，p70S6K蛋白水平分别为0.32±0.07、0.96±0.04、0.90±0.07、0.69±0.06、0.38±0.04、0.92±0.06。上述指标：模型组与对照组比较，差异均有统计学意义（均P<0.05）；中、高剂量实验组与模型组比较，差异均有统计学意义（均P<0.05）；高剂量+抑制剂组与高剂量实验组比较，差异均有统计学意义（均P<0.05）。结论连翘苷可能通过调控AMPK/mTOR/p70S6K信号通路介导的自噬对感染性休克小鼠ALI起到改善作用。

关键词: 连翘苷, , , AMPK/mTOR/p70S6K信号通路, , , 自噬, , , 感染性休克, , , 急性肺损伤

Abstract: Objective To investigate the impact of phillyrin on acute lung injury (ALI) in septic shock mice through autophagy mediated by adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/p70 S6 kinase(p70S6K) signal pathway. Methods Twelve mice were randomly selected as the control group, and the rest of the mice were injected intraperitoneally with 20 mg·kg-1 LPS to construct the model of septic shock, the mice with septic shock were randomly divided into a model group, an Experimental-L, -M, -H group (5 mg·kg-1, 10 mg·kg-1, 20 mg·kg-1 phillyrin), and an Experimental -H+compound C group (20 mg·kg-1 phillyrin+20 mg·kg-1 AMPK inhibitor compound C), there were 12 mice in each group. The lung dry weight and wet weight were weighed, and the W/D ratio was calculated; the levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) in BALF, serum endotoxin (ET) and myeloperoxidase (MPO) of lung tissue were detected by ELISA; HE staining was applied to detect pathological changes of lung tissue; Western blot was applied to detect the expression of autophagic proteins microtubule-associated protein -light chain 3 (LC3-II/I), Beclin 1, Ras-associated GTP binding protein 7 (Rab7), lysosomal associated membrane protein 2 (LAMP2) and AMPK/mTOR/p70S6K signaling pathway proteins. Results In control group, model group, Experimental-L,-M,-H group and Experimental-H+compound C group LC3-II/I ratios were 1.43±0.14, 0.73±0.07, 0.81±0.07, 1.12±0.10, 1.39±0.13, 0.76±0.08, respectively. Beclin1 protein levels were 1.05±0.11, 0.43±0.05, 0.50±0.05, 0.76±0.08, 0.98±0.10, 0.46±0.05, respectively. Rab7 protein levels were 1.53±0.17, 0.67±0.06, 0.70±0.07, 1.04±0.10, 1.41±0.14, 0.69±0.06, respectively. LAMP2 protein levels were 1.47±0.15, 0.72±0.07, 0.81±0.08, 1.09±0.11, 1.35±0.13, 0.74±0.07, respectively. p-AMPK/AMPK protein levels were 0.95±0.05, 0.33±0.03, 0.39±0.04, 0.68±0.07, 0.91±0.09, 0.36±0.04, respectively. p-mTOR/mTOR protein levels were 0.28±0.02, 0.94±0.06, 0.88±0.07, 0.57±0.05, 0.30±0.03, 0.87±0.09, respectively. The protein levels of p70S6K were 0.32±0.07, 0.96±0.04, 0.90±0.07, 0.69±0.06, 0.38±0.04, 0.92±0.06, respectively. There were statistical differences between the model group and the control group (all P<0.05). There were statistical differences between Experimental-M, -H group and model group (all P<0.05). There were significant differences between Experimental-H+compound C group and Experimental-H group (all P<0.05). Conclusions Phillyrin may improve ALI in septic shock mice by regulating autophagy mediated by AMPK/mTOR/p70S6K signaling pathway.

Key words: Phillyrin, , , AMPK/mTOR/p70S6K signal pathway, , , Autophagy, , , Septic shock, , , Acute lung injury

中图分类号:

R563.1

张凡, 韦焕杰, 李龙, 欧阳涛, 蔡娟, 梁秋玲, 曾育辉. 连翘苷对感染性休克小鼠急性肺损伤的作用与机制[J]. 中国临床解剖学杂志, 2024, 42(2): 186-190.

Zhang Fan, Wei Huanjie, Li Long, Ouyang Tao, Cai Juan, Liang Qiuling, Zeng Yuhui. Effect and mechanism of Phillyrin on acute lung injury in septic shock mice[J]. Chinese Journal of Clinical Anatomy, 2024, 42(2): 186-190.

参考文献

[1] Liu XW, Ma T, Cai Q, et al. Elevation of serum PARK7 and IL-8 levels vs associated with acute lung injury in patients with severe sepsis/septic shock[J]. J Intensive Care Med, 2019, 34(8):662-668. DOI: 10.1177/0885066617709689.

[2] Zhou C, Lu M, Cheng J, et al. Review on the pharmacological properties of phillyrin[J]. Molecules, 2022, 27(12):3670-3682. DOI: 10.3390/molecules27123670.
[3] Zhong WT, Wu YC, Xie XX, et al. Phillyrin attenuates LPS-induced pulmonary inflammation via suppression of MAPK and NF-κB activation in acute lung injury mice[J]. Fitoterapia, 2013, 90:132-139. DOI: 10.1016/j.fitote.2013.06.003.
[4] Shao BZ, Wang SL, Fang J, et al. Alpha7 nicotinic acetylcholine receptor alleviates inflammatory bowel disease through induction of AMPK-mTOR-p70S6K-mediated autophagy[J]. Inflammation, 2019, 42(5):1666-1679. DOI: 10.1007/s10753-019-01027-9.
[5] Zhao H, Chen H, Meng XY, et al. Autophagy activation improves lung injury and inflammation in sepsis[J]. Inflammation, 2019, 42(2):426-439. DOI: 10.1007/s10753-018-00952-5.
[6] Shao F, Zhou L, Zhang Y, et al. Gastrodin alleviates inflammatory injury of cardiomyocytes in septic shock mice via inhibiting NLRP3 expression[J]. In Vitro Cell Dev Biol Anim, 2021, 57(5):571-581. DOI: 10.1007/s11626-021-00593-3.
[7] 王小青, 王剑, 赵诗云, 等. 穿心莲内酯对小鼠早期感染性休克的影响[J]. 实用中西医结合临床, 2016, 16(8):76-78.
[8] Di S, Wang Z, Hu W, et al. The protective effects of melatonin against LPS-induced septic myocardial injury: A potential role of AMPK-mediated autophagy[J]. Front Endocrinol (Lausanne), 2020, 11:162-181. DOI: 10.3389/fendo.2020.00162.
[9] 侯良绢, 李晓朋, 赵春娥, 等. 黄芪注射液对肺结核大鼠免疫功能、自噬和凋亡的影响[J]. 中国临床药理学杂志, 2022, 38(10):1092-1096. DOI: 10.13699/j.cnki.1001-6821.2022.10.016.
[10] Shi X, Li T, Liu Y, et al. HSF1 protects sepsis-induced acute lung injury by inhibiting NLRP3 inflammasome activation[J]. Front Immunol, 2022, 13:781003. DOI: 10.3389/fimmu.2022.781003.
[11] 常保超, 潘艳, 郭亚玲, 等. 白芍总苷通过增加糖尿病大鼠肾组织自噬活性减轻肾损伤的研究[J]. 中国临床药理学杂志, 2023, 39(7):984-988. DOI: 10.13699/j.cnki.1001-6821.2023.07.016.
[12] 田华, 郭连军, 刘富, 等. 葛根素下调Atg5、LC3Ⅰ/Ⅱ蛋白对缺血再灌注脑损伤小鼠的神经保护作用[J]. 中国临床药理学杂志, 2023, 39(4):508-512. DOI: 10.13699/j.cnki.1001-6821.2023.04.011.
[13]辛颖, 员丽培, 王峰, 等. 麦冬皂苷D调控磷酸酶及张力蛋白同源物诱导激酶1/帕金蛋白通路对脓毒症心肌损伤的保护作用[J]. 中国临床药理学杂志, 2023, 39(7):971-975. DOI: 10.13699/j.cnki.1001-6821.2023.07.013.
[14] Sun J, Mu Y, Jiang Y, et al. Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates[J]. Cell Death Dis, 2018, 9(3):407-422. DOI: 10.1038/s41419-018-0441-0.
[15]Hu Y, Tao Y, Hu J. Cannabinoid receptor 2 deletion deteriorates myocardial infarction through the down-regulation of AMPK-mTOR-p70S6K signaling-mediated autophagy[J]. Biosci Rep, 2019, 39(4):BSR20180650. DOI: 10.1042/BSR20180650.