Metabonomic Analysis of Wenfei Huaxian Granules Against Idiopathic Pulmonary Fibrosis in Mice

LI Shanshan; HUANG Jun; LI Longxue; LI Shaofeng; ZHANG Cheng; YU Xiao; ZHANG Yuanbing

doi:10.13422/j.cnki.syfjx.20220849

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Metabonomic Analysis of Wenfei Huaxian Granules Against Idiopathic Pulmonary Fibrosis in Mice

更新时间：2023-03-30

- Metabonomic Analysis of Wenfei Huaxian Granules Against Idiopathic Pulmonary Fibrosis in Mice
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 9, Pages: 166-178(2023)
- 作者机构：
  
  1.江西中医药大学实验动物科技中心，中医学院，药学院，南昌 330004
  2.江西省中医院，南昌 330006
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220849
  CLC： R22;R28;R96;R563
- Received：30 May 2022，
  
  Published Online：22 August 2022，
  
  Published：05 May 2023
- 稿件说明：
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李姗姗,黄俊,李龙雪等.温肺化纤颗粒抗小鼠特发性肺纤维化的代谢组学分析[J].中国实验方剂学杂志,2023,29(09):166-178.

LI Shanshan,HUANG Jun,LI Longxue,et al.Metabonomic Analysis of Wenfei Huaxian Granules Against Idiopathic Pulmonary Fibrosis in Mice[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(09):166-178.
李姗姗,黄俊,李龙雪等.温肺化纤颗粒抗小鼠特发性肺纤维化的代谢组学分析[J].中国实验方剂学杂志,2023,29(09):166-178. DOI： 10.13422/j.cnki.syfjx.20220849.

LI Shanshan,HUANG Jun,LI Longxue,et al.Metabonomic Analysis of Wenfei Huaxian Granules Against Idiopathic Pulmonary Fibrosis in Mice[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(09):166-178. DOI： 10.13422/j.cnki.syfjx.20220849.

摘要

目的

探讨硫酸博莱霉素（BLM）诱导肺纤维化形成不同阶段和给药小鼠血清中差异代谢物的变化，以及温肺化纤颗粒抗特发性肺纤维化的作用机制。

方法

小鼠随机分为空白组、14 d空白组、模型组、14 d模型组及温肺化纤颗粒低、高剂量组。模型组、14 d模型组和温肺化纤颗粒低、高剂量组气管注入BLM（0.04 U/只）造模，空白组和14 d空白组气管注入等量无菌生理盐水；温肺化纤颗粒低、高剂量组气管注入BLM次日起每天分别灌胃给予不同剂量温肺化纤颗粒，空白组、14 d空白组、模型组和14 d模型组每天灌胃给予等量无菌水；14 d空白组和14 d模型组小鼠气管注入BLM后14 d取外周血制备血清，其余各组小鼠连续给药28 d后取外周血制备血清并取材。采集小鼠支气管肺泡灌洗液（BALF）进行白细胞分类计数；小鼠肺组织进行病理学检测及羟脯氨酸（HYP）含量测定；采用超高效液相色谱-质谱法（UHPLC-MS）分析各组小鼠血清样本小分子代谢产物，通过主成分分析（PCA）、正交偏最小二乘法-判别分析（OPLS-DA）等筛选差异代谢物并进行代谢通路分析。

结果

与空白组比较，模型组小鼠肺组织出现大量连续纤维化病灶，肺泡炎评分、纤维化程度评分及HYP含量显著上升（

0.01），BALF中白细胞总数、巨噬细胞总数和淋巴细胞总数明显上升（

0.05）。14 d模型组较14 d空白组共筛选出33个差异代谢物，以脂质代谢物为主，主要涉及氧化损伤和炎症过程；模型组较空白组共筛选出34个差异代谢物，以氨基酸代谢物为主，主要涉及核酸损伤和炎症过程。与模型组比较，温肺化纤颗粒高剂量组小鼠肺组织的HYP含量、纤维化评分及肺泡炎评分明显下降（

0.05，

0.01），BALF中淋巴细胞总数明显下降（

0.05）；与模型组比较，温肺化纤颗粒高、低剂量组分别筛选出40、27个差异代谢物；温肺化纤颗粒低、高剂量组各自与模型组的共有差异代谢物共计9个，主要涉及炎症相关脂质、精氨酸和色氨酸代谢途径，在温肺化纤颗粒低、高剂量组中的变化趋势较模型组均明显回调。

结论

温肺化纤颗粒可减轻BLM所致的小鼠肺纤维化，减少胶原沉积和炎症反应，其抗纤维化作用可能与影响炎症因子、一氧化氮及氧化应激相关代谢通路有关。

Abstract

Objective

To investigate the changes of differential metabolites in the serum of mice at different stages of bleomycin sulfate（BLM）-induced pulmonary fibrosis modeling and administration， and the mechanism of Wenfei Huaxian granules（WHG）against idiopathic pulmonary fibrosis.

Method

Mice were randomly divided into control group， control group of 14 days， model group， model group of 14 days， low-dose WHG group and high-dose WHG group. BLM（0.04 U per mouse）was injected into the trachea of mice in the model group， model group of 14 days， low-dose WHG group and high-dose WHG group， and sterile normal saline was injected into the trachea of mice in the control group and control group of 14 days. Mice of low-dose WHG group and high-dose WHG group were given different doses of WHG by gavage every day after injection of BLM， and mice of control group， control group of 14 days， model group and model group of 14 days were given sterile water by gavage every day. The peripheral blood of mice in the control group of 14 days and model group of 14 days were taken to prepare serum after injection of BLM for 14 days， and the peripheral blood and other materials of mice in the other groups were taken after continuous administration for 28 days. The bronchoalveolar lavage fluid（BALF）was collected for leucocyte differential count， the pathological examination and hydroxyproline（HYP）content determination of lung tissues of mice were performed， and the small molecule metabolites in serum samples of mice in each group were determined by ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS）. Principal component analysis（PCA）and orthogonal partial least squares-discriminant analysis（OPLS-DA）were conducted to screen differential metabolites and their biological functions were analyzed.

Result

Compared with the control group， a large number of continuous fibrotic foci appeared in the lung tissue of mice in the model group， the alveolitis score， fibrosis degree score and HYP content increased significantly（

0.01）， and the total number of leukocytes， macrophages and lymphocytes in BALF increased significantly（

0.05）. A total of 33 differential metabolites were screened between the control group of 14 days and model group of 14 days， mainly lipid metabolites， which were mainly involved in oxidative damage and inflammatory process. A total of 34 differential metabolites， mainly amino acid metabolites， were screened between the control group and model group， mainly involving nucleic acid damage and inflammatory process. Compared with the model group， the HYP content， fibrosis score and alveolitis score in the lung tissue of mice from high-dose WHG group decreased significantly（

0.05，

0.01）， and the total number of lymphocytes in BALF decreased significantly（

0.05）. Compared with the model group， 27， 40 differential metabolites were identified in the serum of mice from the low-dose WHG group and high-dose WHG group separately. There were totally 9 common differential metabolites between the model group and low-dose WHG group/high-dose WHG group， which mainly involved in the metabolic pathways of inflammation related lipids metabolism， arginine and tryptophan metabolism， and the change trends in low-dose WHG group and high-dose WHG group were significantly back-regulated compared with the model group.

Conclusion

WHG can alleviate BLM-induced pulmonary fibrosis， collagen deposition and inflammatory reaction in mice， and its anti-fibrotic effect may be related to the adjusting of inflammatory factors， nitric oxide and oxidative stress related metabolic pathways.

关键词

Keywords

references

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