中药复方防感汤对SARS-CoV-2刺突蛋白引起的肺和结肠上皮细胞损伤的保护作用与TGF-β/Smad2/3和NF-κB通路的调节有关

doi:10.24920/004198

摘要/Abstract

摘要：

背景与目的 探讨中药复方防感汤对SARS-CoV-2刺突蛋白诱导的肺、肠损伤的体外和体内保护作用及其机制。
方法用重组SARS-CoV-2刺突蛋白刺激BALB/c雌性小鼠和3株经防感汤预处理的细胞系。检测各组组织苏木精-伊红（HE）染色、病理评分、细胞通透性和活力以及肺、结肠ACE2表达。酶联免疫吸附测定法（ELISA）检测小鼠血清和细胞上清中炎症因子水平。蛋白质印迹法（Western blotting）检测NF-κB p65、p-NF-κB p65、p- IκB α、p-Smad2/3、TGF-β1、Caspase3、Bcl-2的表达。
结果从病理评分以及细胞通透性和活力来看，中草药在体内和体外均对刺突蛋白诱发的肺和结肠损伤均有保护作用（P<0.05）。中草药上调肺和结肠中被刺突蛋白降低的ACE2表达，显著改善刺突蛋白引起的炎症标志物的分泌紊乱，同时调节TGF-β/Smads和NF-κB通路的活性。
结论中药复方防感汤对SARS-CoV-2刺突蛋白刺激的肺和肠组织损伤具有一定的保护作用，其机制基于对可能具有组织类型特异的NF-κB和TGF-β1/Smad通路的调控作用。

关键词: COVID-19, SARS-CoV-2, 中草药, 预防, 转化生长因子-β

Abstract:

Objective To explore the effects and mechanisms of a traditional Chinese medicine (TCM) prescription, “Fang-gan Decoction” (FGD), in protecting against SARS-CoV-2 spike protein-induced lung and intestinal injuries in vitro and in vivo.
Methods Female BALB/c mice and three cell lines pretreated with FGD were stimulated with recombinant SARS-CoV-2 spike protein (spike protein). Hematoxylin-eosin (HE) staining and pathologic scoring of tissues, cell permeability and viability, and angiotensin-converting enzyme 2 (ACE2) expression in the lung and colon were detected. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of inflammatory factors in serum and cell supernatant. The expression of NF-κB p65, p-NF-κB p65, p-IκBα, p-Smad2/3, TGF-β1, Caspase3, and Bcl-2 was evaluated by Western blotting.
Results FGD protected against the damage to the lung and colon caused by the spike protein in vivo and in vitro according to the pathologic score and cell permeability and viability (P<0.05). FGD up-regulated ACE2 expression, which was reduced by the spike protein in the lung and colon, significantly improved the deregulation of inflammatory markers caused by the spike protein, and regulated the activity of TGF-β/Smads and NF-κB signaling.
Conclusion Traditional Chinese medicine has a protective effect on lung and intestinal tissue injury stimulated by the spike protein through possible regulatory functions of the NF-κB and TGF-β1/Smad pathways with tissue type specificity.

Key words: COVID-19, SARS-CoV-2, traditional chinese medicine, prevention, transforming growth factor-β

基金资助: 深圳市宝安中医药发展基金会“新冠肺炎治疗和疫情防控技术研究及应用中医药专项”(2020KJCX-KTYJ-302)

Chao Huang, Hao-Sheng Liu, Bing-Jun Liang, Sheng-Rong Liao, Wei-Zeng Shen. A Chinese Herb Prescription “Fang-gan Decoction” Protects Against Damage to Lung and Colon Epithelial Cells Caused by the SARS-CoV-2 Spike Protein by Regulating the TGF-β/Smad2/3 and NF-κB Pathways[J].Chinese Medical Sciences Journal, 2023, 38(3): 206-217.

图/表 7

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Markers	Time	Normal group	Model Gorup	NS group	TCM group	P value
IL-6	3d	644.61	717.36^*	696.89^*	702.85^*	0.009
	5d	268.02	342.09^*	344.88^*	298.89^#?	<0.001
IL-10	3d	25.14	82.12^*	74.86^*	44.7^#	0.009
	5d	87.61	145.79^*	123.84^*	121.52	0.019
IL-13	3d	352.69	344.69	362.46	351.09	0.998
	5d	91.9	102.87	92.42	100.23	0.931
TNF-α	3d	566.19	853.87	840.04	633.38	0.141
	5d	575.77	1042.36^*	1236.73^*#	864.43^*#?	<0.001
CRP	3d	18175.8	21129.27^*	21550.72^*	19786.15	0.042
	5d	5933.09	7413.08^*	6567.13^#	6093.33^#	0.001

Markers	Control group	S model group	S model+10% TCM group	S model+25% TCM group	S model+50% TCM group	P value
IL-6 (ng/L)	20.24	33.01^*	29.81^*#	27.95^*#	20.01^#	<0.001
IL-10 (ng/L)	428.32	435.40	435.54	435.38	418.36	0.762
IL-13 (ng/L)	41.33	27.17^*	30.75^*#	33.67^*#	35.04^*#	<0.001
TNF-α (ng/L)	212.14	309.29^*	277.51^*	263.84	222.83	<0.001
NF-κB (ng/L)	215.69	187.85	183.57	206.98	213.29	0.154
TGF-β1 (ng/L)	285.49	156.97^*	185.03^*#	196.94^*#	272.26^#	<0.001

Cells	Markers	Control group	S model group	S model+10% TCM group	S model+25% TCM group	S model+50% TCM group	P value
HsIMECs	IL-6 (ng/L)	22.40	30.49^*	27.62^*	25.39^#	22.11^#	<0.001
	IL-10 (ng/L)	348.14	307.22^*	318.12^*	327.84^*#	339.72^#	0.001
	IL-13 (ng/L)	30.47	30.59	28.49	31.09	29.16	0.757
	TNF-α (ng/L)	121.90	273.29^*	271.30^*	234.02^*	202.70^*#	<0.001
	NF-κB (ng/L)	178.52	177.08	175.88	178.74	180.86	0.859
	TGF-β1 (ng/L)	149.71	146.78	143.00	141.65	145.37	0.986
HCoEpiCs	IL-6 (ng/L)	24.13	28.28^*	25.26^#	23.78^#	21.95^#	0.001
	IL-10 (ng/L)	372.10	384.02	378.86	388.56	388.92	0.910
	IL-13 (ng/L)	23.69	15.99^*	20.01	22.77^#	21.53^#	0.021
	TNF-α (ng/L)	168.63	286.16^*	281.62^*	230.50^*#	221.83^*#	<0.001
	NF-κB (ng/L)	133.02	131.88	134.09	126.03	134.28	0.201
	TGF-β1 (ng/L)	177.31	173.80	176.35	177.77	171.37	0.452

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