厚朴麻黄汤对哮喘患者血清代谢及TRPV1/Ca2+/TJs的影响
2022/12/21
摘要
民族药物相关性：厚朴麻黄汤（HPMHD）是用于治疗哮喘的经典中药方剂之一。HPMHD在加重哮喘中的治疗作用和机制仍有待探索，特别是从代谢组学和瞬时受体电位香草醇-1（TRPV1）/Ca2+/紧密连接（TJ）调节的角度。
研究目的：研究HPMHD对哮喘大鼠的治疗和代谢调节作用及其潜在机制。
实验材料与方法：哮喘大鼠给予相应的HPMHD（剂量为5.54、11.07、22.14 mg/kg）。然后计数外周血和支气管肺泡灌洗液（BALF）中的炎性细胞，测定BALF中白介素（IL）-4和IL-13的水平，并检测增强暂停（Penh）的变化和肺组织的病理损伤，以评估HPMHD的保护作用。使用超高效液相色谱质谱仪（UHPLC-MS）研究了哮喘大鼠HPMHD的血清代谢谱，并通过Western blotting分析检测了HPMHD对哮喘大鼠TRPV1和TJs的调节作用。体外，用IL-4加SO2衍生物刺激16HBE细胞，然后给予HPMHD。然后通过钙成像和蛋白质印迹检测TRPV1调节的细胞内Ca2+，以及TRPV1和TJ蛋白（TJ）的表达水平。通过抑制TRPV1和短发夹RNA（shRNA）介导的TRPV1沉默细胞，证实了这种作用。
结果：HPMHD显著减轻了哮喘大鼠的气道炎症，并降低了外周血和BALF中炎性细胞的水平以及BALF中IL-4和IL-13的水平。此外，气道高反应性和肺部病理损害得到缓解。血清代谢组学分析显示，在生理盐水、模型和HPMHD处理的大鼠中，31种代谢产物的表达存在差异。途径富集分析表明，代谢产物参与了45条途径，其中TJs调节相关途径与TRP香草类通道介导的Ca2+浓度变化有关。体内和体外实验表明，HPMHD通过抑制TRPV1的表达和激活来降低细胞内Ca2+浓度，增加ZO-1、Occludin和Claudin-3的表达，并保护TJ的完整性
结论：目前的研究表明，HPMHD减轻了大鼠哮喘，并参与了血清代谢的调节。HPMHD的抗哮喘作用可能与通过TRPV1抑制细胞内Ca2+浓度来保护TJs有关。
Effects of Houpo Mahuang Decoction on serum metabolism and TRPV1/Ca2++/TJs in asthma
Zhou, L., Hao, M., Fan, X., Lao, Z., Li, M., & Shang, E
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE:Houpo Mahuang Decoction (HPMHD is one of the classic traditional Chinese prescriptions that has been used in the treatment of asthma. The therapeutic effects and mechanism of HPMHD in aggravated asthma remain to be explored, especially from the perspective of metabolomics and Transient Receptor Potential Vanilloid-1 (TRPV1)/Ca2++/Tight junction (TJ) regulation.
AIM OF THE STUDY:To investigate the therapeutic and metabolic regulatory effects and the underlying mechanism of HPMHD in asthmatic rats.
MATERIALS AND METHODS:The asthmatic rats were administered with the corresponding HPMHD (at dosages of 5.54, 11.07, 22.14 mg/kg). Then inflammatory cells in peripheral blood and bronchoalveolar lavage fluid (BALF) were counted, the levels of interleukin (IL)-4 and IL-13 in BALF were measured, and the changes in enhanced pause (Penh) and pathological damage of lung tissues were also detected to evaluate the protective effects of HPMHD. The serum metabolic profile of HPMHD in asthmatic rats was explored using Ultra-High-Performance Liquid Chromatography-mass spectrometer (UHPLC-MS), and the regulatory effects on TRPV1 and TJs of HPMHD in asthmatic rats were detected by Western blotting analysis. In vitro, 16HBE cells were stimulated with IL-4 plus SO2 derivatives and then administered HPMHD. The intracellular Ca2++ regulated by TRPV1, and the expression levels of TRPV1 and TJ proteins (TJs) were then detected by calcium imaging and Western blotting. The effects were verified by inhibition of TRPV1 and in short hairpin RNA (shRNA)-mediated TRPV1 silencing cells.
RESULTS:HPMHD significantly attenuated the airway inflammation of asthmatic rats, and reduced the levels of inflammatory cells in peripheral blood and BALF as well as the levels of IL-4 plus IL-13 in BALF. In addition, the airway hyperresponsiveness and lung pathological damage were alleviated. Serum metabolomic analysis showed that 31 metabolites were differentially expressed among the normal saline-, model-, and HPMHD-treated rats. Pathway enrichment analysis showed that the metabolites were involved in 45 pathways, among which, TJs regulation-relevant pathway was associated with the Ca2++ concentration change mediated by the TRP Vanilloid channel. In vivo and in vitro experiments indicated that HPMHD reduced the concentration of intracellular Ca2++ via suppressing the expression and activation of TRPV1, increased the expression of ZO-1, Occludin, and Claudin-3, and protected the integrity of TJs.
CONCLUSION:The current study indicates that HPMHD alleviates rat asthma and participates in the regulation of serum metabolism. The anti-asthma effects of HPMHD might be related to the protection of TJs by inhibiting the intracellular Ca2++ concentration via TRPV1.
民族药物相关性：厚朴麻黄汤（HPMHD）是用于治疗哮喘的经典中药方剂之一。HPMHD在加重哮喘中的治疗作用和机制仍有待探索，特别是从代谢组学和瞬时受体电位香草醇-1（TRPV1）/Ca2+/紧密连接（TJ）调节的角度。
研究目的：研究HPMHD对哮喘大鼠的治疗和代谢调节作用及其潜在机制。
实验材料与方法：哮喘大鼠给予相应的HPMHD（剂量为5.54、11.07、22.14 mg/kg）。然后计数外周血和支气管肺泡灌洗液（BALF）中的炎性细胞，测定BALF中白介素（IL）-4和IL-13的水平，并检测增强暂停（Penh）的变化和肺组织的病理损伤，以评估HPMHD的保护作用。使用超高效液相色谱质谱仪（UHPLC-MS）研究了哮喘大鼠HPMHD的血清代谢谱，并通过Western blotting分析检测了HPMHD对哮喘大鼠TRPV1和TJs的调节作用。体外，用IL-4加SO2衍生物刺激16HBE细胞，然后给予HPMHD。然后通过钙成像和蛋白质印迹检测TRPV1调节的细胞内Ca2+，以及TRPV1和TJ蛋白（TJ）的表达水平。通过抑制TRPV1和短发夹RNA（shRNA）介导的TRPV1沉默细胞，证实了这种作用。
结果：HPMHD显著减轻了哮喘大鼠的气道炎症，并降低了外周血和BALF中炎性细胞的水平以及BALF中IL-4和IL-13的水平。此外，气道高反应性和肺部病理损害得到缓解。血清代谢组学分析显示，在生理盐水、模型和HPMHD处理的大鼠中，31种代谢产物的表达存在差异。途径富集分析表明，代谢产物参与了45条途径，其中TJs调节相关途径与TRP香草类通道介导的Ca2+浓度变化有关。体内和体外实验表明，HPMHD通过抑制TRPV1的表达和激活来降低细胞内Ca2+浓度，增加ZO-1、Occludin和Claudin-3的表达，并保护TJ的完整性
结论：目前的研究表明，HPMHD减轻了大鼠哮喘，并参与了血清代谢的调节。HPMHD的抗哮喘作用可能与通过TRPV1抑制细胞内Ca2+浓度来保护TJs有关。
(中日友好医院呼吸与危重症医学科 沈焜路 摘译 林江涛 审校)
(J Ethnopharmacol. 2023 Feb 10. DOI: 10.1016/j.jep.2022.115873)
(J Ethnopharmacol. 2023 Feb 10. DOI: 10.1016/j.jep.2022.115873)
Effects of Houpo Mahuang Decoction on serum metabolism and TRPV1/Ca2++/TJs in asthma
Zhou, L., Hao, M., Fan, X., Lao, Z., Li, M., & Shang, E
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE:Houpo Mahuang Decoction (HPMHD is one of the classic traditional Chinese prescriptions that has been used in the treatment of asthma. The therapeutic effects and mechanism of HPMHD in aggravated asthma remain to be explored, especially from the perspective of metabolomics and Transient Receptor Potential Vanilloid-1 (TRPV1)/Ca2++/Tight junction (TJ) regulation.
AIM OF THE STUDY:To investigate the therapeutic and metabolic regulatory effects and the underlying mechanism of HPMHD in asthmatic rats.
MATERIALS AND METHODS:The asthmatic rats were administered with the corresponding HPMHD (at dosages of 5.54, 11.07, 22.14 mg/kg). Then inflammatory cells in peripheral blood and bronchoalveolar lavage fluid (BALF) were counted, the levels of interleukin (IL)-4 and IL-13 in BALF were measured, and the changes in enhanced pause (Penh) and pathological damage of lung tissues were also detected to evaluate the protective effects of HPMHD. The serum metabolic profile of HPMHD in asthmatic rats was explored using Ultra-High-Performance Liquid Chromatography-mass spectrometer (UHPLC-MS), and the regulatory effects on TRPV1 and TJs of HPMHD in asthmatic rats were detected by Western blotting analysis. In vitro, 16HBE cells were stimulated with IL-4 plus SO2 derivatives and then administered HPMHD. The intracellular Ca2++ regulated by TRPV1, and the expression levels of TRPV1 and TJ proteins (TJs) were then detected by calcium imaging and Western blotting. The effects were verified by inhibition of TRPV1 and in short hairpin RNA (shRNA)-mediated TRPV1 silencing cells.
RESULTS:HPMHD significantly attenuated the airway inflammation of asthmatic rats, and reduced the levels of inflammatory cells in peripheral blood and BALF as well as the levels of IL-4 plus IL-13 in BALF. In addition, the airway hyperresponsiveness and lung pathological damage were alleviated. Serum metabolomic analysis showed that 31 metabolites were differentially expressed among the normal saline-, model-, and HPMHD-treated rats. Pathway enrichment analysis showed that the metabolites were involved in 45 pathways, among which, TJs regulation-relevant pathway was associated with the Ca2++ concentration change mediated by the TRP Vanilloid channel. In vivo and in vitro experiments indicated that HPMHD reduced the concentration of intracellular Ca2++ via suppressing the expression and activation of TRPV1, increased the expression of ZO-1, Occludin, and Claudin-3, and protected the integrity of TJs.
CONCLUSION:The current study indicates that HPMHD alleviates rat asthma and participates in the regulation of serum metabolism. The anti-asthma effects of HPMHD might be related to the protection of TJs by inhibiting the intracellular Ca2++ concentration via TRPV1.
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