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Traditional Chinese Medicine, Qingfei
Paidu Decoction and Xuanfei Baidu
Decoction, Inhibited Cytokine
Production via NF-κB Signaling
Pathway in Macrophages:
Implications for Coronavirus Disease
2019 (COVID-19) Therapy
Yujia Li
1
,
2
, Bin Li
2
, Pan Wang
3
*
†
and Qinghua Wang
3
*
†
1
Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China,
2
The
Joint Laboratory on Transfusion-transmitted Diseases (TTD) Between Institute of Blood Transfusion, Chinese Academy of
Medical Sciences and Nanning Blood Center, Nanning, China,
3
The Traditional Chinese Medicine Hospital of Wenjiang District,
Chengdu, China
Background and Aims: Qingfei Paidu decoction (QPD) and Xuanfei Baidu decoction
(XBD) are two typical traditional Chinese medicines with proven efficacy for the treatment of
SARS-CoV-2, although the underlying mechanism is not well defined. Blunted immune
response and enhanced production of pro-inflammatory cytokines (cytokine storm) are
two main features observed in patients infected with SARS-CoV-2. Analysis based on
network pharmacology has revealed that both QPD and XBD played an important role in
the regulation of host immunity. We therefore investigated the role of QPD and XBD in the
modulation of innate immunity in vitro, focusing on the type 1 interferon (IFN) signaling
pathway in A549 cells and pro-inflammatory cytokine production in macrophages.
Methods: A549 cells were treated with QPD or XBD and the production of
endogenous IFNαand IFNβas well as the expression levels of some interferon-
stimulated genes (ISGs) were detected by reverse transcriptase-quantitative PCR (RT-
qPCR). Macrophages derived from THP-1 cells were treated with QPD or XBD and their
pro-inflammatory cytokine expression levels were measured by RT-qPCR, 6 h post LPS
stimulation. In addition, the expression levels of some pro-inflammatory cytokines were
further analyzed by ELISA. The effect of QPD and XBD on the NF-κB signaling pathway and
the pinocytosis activity of THP-1-derived macrophages were evaluated by Western blot
and neutral red uptake assay, respectively.
Results: Although QPD and XBD showed very little effect on the type 1 IFN signaling
pathway in A549 cells, either QPD or XBD markedly inhibited the production of pro-
inflammatory markers including interleukin-6, tumor necrosis factor-α, monocyte
chemotactic protein-1, and chemokine ligand 10 in THP-1-derived M1 macrophages.
In addition, the phosphorylation of IκBαand NF-κB p65 during the process of macrophage
polarization was significantly suppressed following QPD or XBD treatment. QPD and XBD
Edited by:
Luca Rastrelli,
University of Salerno, Italy
Reviewed by:
Sofia Viana,
University of Coimbra, Portugal
Rita Celano,
University of Salerno, Italy
*Correspondence:
Qinghua Wang
qinghuawang2015@126.com
Pan Wang
wangpan761@163.com
†
These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Ethnopharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 08 June 2021
Accepted: 27 September 2021
Published: 26 October 2021
Citation:
Li Y, Li B, Wang P and Wang Q (2021)
Traditional Chinese Medicine, Qingfei
Paidu Decoction and Xuanfei Baidu
Decoction, Inhibited Cytokine
Production via NF-κB Signaling
Pathway in Macrophages: Implications
for Coronavirus Disease 2019 (COVID-
19) Therapy.
Front. Pharmacol. 12:722126.
doi: 10.3389/fphar.2021.722126
Frontiers in Pharmacology | www.frontiersin.org October 2021 | Volume 12 | Article 7221261
ORIGINAL RESEARCH
published: 26 October 2021
doi: 10.3389/fphar.2021.722126
also suppressed the pinocytosis activity of macrophages.
Conclusion: QPD and XBD have been shown to have robust anti-inflammatory activities
in vitro. Our study demonstrated that both QPD and XBD decreased pro-inflammatory
cytokine expression, inhibited the activation of the NF-κB signaling pathway, and blunted
pinocytosis activity in THP-1-derived macrophages.
Keywords: COVID-19, traditional Chinese medicine, macrophage, NF-κB signaling pathway, cytokine storm
INTRODUCTION
Globally, there are more than 154 million confirmed cases of
coronavirus disease 2019 (COVID-19), including 3.2 million
deaths as of May 6, 2021 (Available online: https://covid19.who.
int/).TheCOVID-19pandemiciscausedbyinfectionwithanon-
enveloped RNA beta coronavirus, specifically the severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2
infection, like most other virus infections, triggers the host’s innate
immune response which constitutes the first line of defense against
invading pathogens. The dysregulation of the innate immune
response is closely associated with morbidity and mortality of
COVID-19 patients. For example, although it is part of the first
line of defense against virus infections, production of type 1
interferon (IFN), one of the key antiviral mediators, is blunted in
patients infected with SARS-CoV, which is in contrast to the fact that
high levels of type 1 IFN have been detected in patients infected with
SARS-CoV (Acharya et al., 2020). Moreover, the potential use of
IFNs in COVID-19 therapy (Park et al., 2020) also highlights that
impaired systemic IFN production is a crucial determinant in the
pathogenesis of SARS-CoV-2 infection. Another characteristic of
severe COVID-19 patients is the cytokine storm: over-production of
numerous cytokines and chemokines such as interleukin-6 (IL-6),
tumor necrosis factor-α(TNF-α), monocyte chemotactic protein-1
(CCL2), and chemokine (C-X-C motif) ligand 10 (CXCL10) (Merad
et al., 2020). As one of the most enriched immune cell types in the
lungs of COVID-19 patients, macrophages have been shown to
contribute to hyper-inflammation that leads to cytokine storms in
patients with severe COVID-19. However, the exact contribution of
macrophages in the pathogenesis of SARS-Cov-2 remains to be
elucidated (Wang C. et al., 2020;Booz et al., 2020;Merad et al., 2020).
Various traditional Chinese medicines (TCM) have been used
to treat patients infected with SARS-CoV-2 in China, mainly
including oral medication, such as Lianhua Qingwen capsules,
Jinhua Qinggan granules, different kinds of decoctions, and TCM
injections such as Xuebijing injections and Shenfu injections
[reviewed in (Al-Romaima et al., 2020;Luo et al., 2020;Wang
et al., 2021b;Luo et al., 2021)]. Among them, two decoctions, the
Qingfei Paidu decoction (QPD) and the Xuanfei Baidu decoction
(XBD), have been shown to have significant efficacy against
SARS-CoV-2 infection (Shi et al., 2020;Xiong et al., 2020;
Huang et al., 2021). With an effective rate of over 90% (Al-
Romaima et al., 2020), QPD was officially recommended for the
treatment of mild, medium, severe, and critical COVID-19
patients in the 7th version of the diagnosis and treatment
guidelines issued by the National Health Commission (NHC)
of China (Available online: http://www.nhc.gov.cn/xcs/zhengcwj/
202003/46c9294a7dfe4cef80dc7f5912eb1989.shtml). XBD
granules are also recommended for the treatment of moderate
patients (Huang et al., 2021). Network pharmacology analysis
revealed that both QPD (Niu et al., 2021) and XBD (Wang Y. et al.
, 2020) play an important role in regulating host immunity to
prevent hyperinflammation, which may result in cytokine storm.
Clinical data (Xiong et al., 2020) also showed that C-reactive
protein, a non-specific marker of inflammation, was significantly
decreased in the XBD-treated group compared to that in the
control group. However, little is known about the underlying
molecular mechanisms.
Therefore, in the present study, we aimed to investigate the
effect of QPD and XBD on the host’s innate immunity, focusing
on the type 1 IFN signaling pathway and inflammatory pathway
in macrophages.
MATERIAL AND METHODS
Cells
Human adenocarcinomic alveolar basal epithelial cell line (A549)
and human myeloid leukemia mononuclear (THP-1) cells were
purchased from the West China Medical Center of Sichuan
University and routinely preserved in our laboratory. The
A549 cells were maintained in Dulbecco’s Modified Eagle
Medium (DMEM) (Hyclone, United States) supplemented
with 10% fetal bovine serum (Gibco, United States), 100 IU/
ml ampicillin, and 100 mg/ml streptomycin (Gibco,
United States) at 37°C in a 5% CO2 humidified incubator. The
THP-1 cells were maintained in RPMI-1640 (Hyclone,
United States) medium supplemented with 10% fetal bovine
serum (Gibco, United States), 10 mmol/L HEPES (Cellgro,
United States), 100 IU/ml ampicillin, and 100 mg/ml
streptomycin (Gibco, United States) at 37°C in a 5% CO2
humidified incubator. The THP-1 cells were differentiated into
M0 macrophages by 100 ng/ml phorbol-12-myristate-13-acetate
(PMA) (Sigma, United States) stimulation for 48h, followed by
24 h rest in RPMI-1640 medium without PMA. The M0
macrophages were primed with fresh culture medium with
20 ng/ml IFN-γ(Peprotech, United States) and 1 µg/ml
Escherichia coli 0111:B4 lipopolysaccharide (LPS) (Sigma,
United States) for M1 polarization as previously reported by
Chanput et al. (2014).
Decoction Preparation
The preparation processes for QPD and XBD are exactly the
same. The drugs (raw materials) were soaked in 500 ml of pure
Frontiers in Pharmacology | www.frontiersin.org October 2021 | Volume 12 | Article 7221262
Li et al. Traditional Chinese Medicine and COVID-19
water for 30 min and then boiled until 300 ml of liquid remained,
which was collected by filtration as the first part. Another 300 ml
of pure water was added, to the dregs and then the mixture was
boiled slowly until 200 ml of liquid remained, which was collected
by filtration as the second part and mixed well with the first part
to obtain an approximately 500 ml decoction. The decoction was
centrifuged at 5,000 rpm (4,109 ×g) for 30 min at room
temperature and the supernatant was collected and filtered by
0.22 µm polypropylene microporous membrane and stored at
−80°C until use. The QPD was concentrated to a density of 1.02 g/
ml and the XBD was concentrated to 0.98 g/ml. The raw materials
of QPD and XBD are listed in Table 1 and Table 2, respectively.
Previous studies reported that 129 compounds have been
identified in QPD by liquid chromatography quadrupole-time
of flight mass spectrometry analysis (Yang et al., 2020;Wang
et al., 2021b). Among them, eight specific compounds were
identified as potential candidates which may directly interact
with the SARS-CoV-2 viral proteins.
Cytotoxicity Assay
The cytotoxic effect of QPD and XBD on A549 and THP-1 cells
were evaluated with Cell Counting Kit-8 (CCK-8) (Biosharp,
China), following the manufacturer’s instructions. Briefly,
monolayers of A549 cells or M0 THP-1 macrophages in 96-
well plates were incubated with indicated concentrations of QPD
or XBD. The cells were rinsed with phosphate-buffered saline
(PBS) (Hyclone, United States) at 0, 24, 48, 72, and 96h, followed
by staining with 10ul of CCK8 solution per well. The absorbance
was measured at 450 nm using a Multiskan Spectrum reader
(Thermo Fisher, United States).
RNA Isolation and Reverse Transcriptase-quantitative PCR
Analysis (RT-qPCR).
TABLE 1 | Raw materials of Qingfei Paidu decoction.
Name Chinese name Medicinal parts Quantities (g)
Ephedra sinica Stapf Ma Huang Stem 9
Glycyrrhiza uralensis Fisch.ex DC. Zhi Gan Cao Root 6
Prunus armeniaca L Xing Ren Seed 9
Gypsum fibrosum Sheng Shi Gao * 30
Atractylodes macrocephala Koidz Bai Zhu Root 9
Bupleurum chinense DC. Chai Hu Root 16
Scutellaria baicalensis Georgi Huang Qin Root 6
Pinellia ternate (Thunb.) Makino Jiang Ban Xia Root 9
Aster tataricus L.F. Zi Wan Root 9
Tussilago farfara L. Kuan Dong Hua Flower 9
ris domestica (L.) Goldblatt and Mabb She Gan Root 9
Asarumsieboldii Miq Xi Xin Whole plant 6
Dioscorea opposite Thunb Shan Yao Root 12
Citrus ×aurantium L Zhi Shi Fruit 6
Pogostemon cablin (Blanco) Benth Huo Xiang Whole plant 9
Zingiber officinale Rosc Sheng Jiang Root 15
Poria cocos (Schw.) Wolf Fu Ling whole 15
Citrus ×aurantium L Chen Pi Fruit 6
Cinnamomum cassia (L.) J.PreslGui Zhi Stem 9
Alisma orientalis (Sam.) Juzep Ze Xie Root 9
Polyporus umbellatus (Pers.) Fries Zhu Ling whole 9
*, Sheng Shi Gao (Gypsum fibrosum) is an inorganic substance. All botanical drugs in Qingfei Paidu decoction were fully validated using http://www.plantsoftheworldonline.org/
TABLE 2 | Raw materials of Xuanfei Baidu decoction
Name Chinese name Medicinal parts Quantities (g)
Ephedra sinica Stapf Ma Huang Stem 6
Prunus armeniaca L Xing Ren Seed 15
Gypsum fibrosum Sheng Shi Gao * 30
Coix lacryma-jobi L Yi Yi Ren Seed 30
Atractylodes lancea (Thunb.) DC. Cang Zhu Root 10
Pogostemon cablin (Blanco) Benth Huo Xiang Whole plant 15
Artemisia annua L Qing Hao Whole plant except root 12
Citrus ×reticulata Blanco Ju Hong Fruit 15
Glycyrrhiza uralensis Fisch.ex DC. Zhi Gan Cao Root 10
Phragmites communis Trin Lu Gen Root 30
Lepidium apetalum Willd Ting Li Zi Seed 15
Verbena officinalis L Ma Bian Cao Whole plant except root 30
Reynoutria japonica Houtt Hu Zhang Root 20
*, Sheng Shi Gao (Gypsum fibrosum) is an inorganic substance. All botanical drugs in Xuanfei Baidu decoction were fully validated using http://www.plantsoftheworldonline.org/
Frontiers in Pharmacology | www.frontiersin.org October 2021 | Volume 12 | Article 7221263
Li et al. Traditional Chinese Medicine and COVID-19
The total intracellular RNA was extracted using Trizol
(Invitrogen, United States) and quantified by NanoDrop
(Thermo, United States). Reverse-transcription was carried out
using Rever TraAceq PCR RT Master Mix (TOYOBO, Japan)
following the manufacturer’s recommended protocol. The
resulting cDNA was amplified by NovoStart SYBR qPCR
SuperMix Plus (Novoprotein, China). Primers for quantitative
PCR are listed in Table 3.
Enzyme-Linked Immunosorbent Assay
The THP-1 monocytes were differentiated into M0 macrophages
as described above and treated with decoctions at indicated
concentrations for 24 h. The cells were then washed with PBS
three times and supplied with phenol-red free RPMI-1640 media
with 1 µg/ml LPS and 20 ng/ml IFN-γ. Twenty-four hours later,
the culture supernatant was collected for ELISA analysis. The
levels of IL-6 and NF-κB were detected by manual IL-6 and NF-
κB ELISA kits (Elabscience, China) respectively, following the
manufacturer’s instructions.
Western Blot
The M0 THP-1 macrophages were treated with 5% QPD or 5%
XBD for 24 h. Then the cells were stimulated with or without
1 µg/ml LPS and 20 ng/ml IFN-γ. The total intracellular protein
was collected at 1 h and 2 h post-stimulation. IκBα, phospho-
IκBα, NF-κB p65, and phospho-NF-κB p65 protein levels were
assessed by western blot using IκBα(L35A5) mouse mAb,
phospho-IκBα(Ser32/36)(5A5) mouse mAb, NF-κB p65
(D14E12) XP
®
rabbit mAb, and phospho-NF-kB p65 (Ser536)
(93H1) rabbit mAb (Cell Signaling Technology, United States),
respectively. Secondary antibodies were HRP-labeled goat anti-
mouse or anti-rabbit IgG (Proteintech, China). The protein bands
were visualized using an ECL chemiluminescent detection kit
(Millipore, United States) in an ImageQuant LAS 4000mini (GE,
United States).
Pinocytic Activity Assay
The THP-1 monocytes were seeded in a 96-well plate at the
density of 4 ×10
5
cells/ml with 200 μL/well, and the cells were
differentiated into M0 macrophages and polarized into M1
macrophages as described above. A neutral red uptake assay
was employed to evaluate the pinocytosis function of the
macrophages as previously described (Jacobo-Salcedo Mdel
et al., ).
Statistical Analyses
The experiments were repeated three times. The significance of
the differences between the grops was assessed using ANOVA
(data normality and homogeneity of variance) or the Kruskal-
Wallis rank test, where appropriate. p<0.05 was considered
statistically significant.
RESULTS
Qingfei Paidu Decoction and Xuanfei Baidu
Decoction Have Little Effect on the
Activation of the Type 1 IFN Signaling
Pathway in A549 Cells
Activation of the IFN signaling pathway in host cells is one of
the important immune responses to viral infections. The fact
that SARS-CoV-2 blunts the host’sinnateimmuneresponse
and is characterized by weak IFN production indicates that
SARS-CoV-2 may target the IFN pathway as part of its
strategy to avoid being eliminated by innate immunity.
Thus, we first investigated the effect of QPD and XBD on
type 1 IFN signaling. Cells expressing both angiotensin-
converting enzyme 2 (ACE2) and transmembrane serine
protease (TMPRSS)-2 are the main targets during SARS-
CoV-2 infection. Therefore, the A549 cell, a human lung
epithelial cell line with both ACE2 (Figure 1A) and TMPRSS-
2(Figure 1B)expression,wasselectedasthecellmodelfor
this study. Consistent with Qi et al. (2020), the endogenous
expression level of ACE2 was very low, although it could be
expressed in multiple organs and tissues. The cell viability
after QPD or XBD treatment was determined with CCK-8
kits. Both QPD (Figure 2A,left)andXBD(Figure 2A,right)
TABLE 3 | Primers used for real-time PCR.
Gene name Nucleotide sequence Gene name Nucleotide sequence
GAPDH F: 5′-GCCTCCTGCACCACCAACTG-3′IFIT-1 F: 5′- GCAGCCAAGTTTTACCGAAG-3′
R: 5′-ACGCCTGCTTCACCACCTTC-3 R: 5′- GCCCTATCTGGTGATGCAGT-3′
ACE2 F: 5′-AACTGCTGCTCAGTCCACC-3′IL-6 F: 5′-ATGCCTGACCTCAACTCCACT-3′
R: 5′-AAAAGGCAGACCATTTGTCCC-3 R: 5′-GCCACCCAGCTGCAAGATTTC-3′
TMPRSS2 F: 5′-CCTGTGTGCCAAGACGACTG-3′TNF-αF: 5′-AGCTGCCAGGCAGGTTCTCTTCC-3′
R:5′-TTATAGCCCATGTCCCTGCAG-3′R: 5′-GGTTATCTCTCAGCTCCACGCCA-3′
IFNαF: 5′-TCGCCCTTTGCTTTACTGAT-3′CCL2 F: 5′-TCTGTGCCTGCTGCTCATAG-3′
R: 5′- GGGTCTCAGGGAGATCACAG-3′R: 5′-TGGAATCCTGAACCCACTTC-3′
IFNβF: 5′-AAACTCATAGCAGTCTGCA-3′CXCL10 F: 5′-GCCTTGGCTGTGATATTGTG-3′
R: 5′-AGGAGATCTTCAGTTTCGGAGG-3′R: 5′-TAAGCCTTGCTTGCTTCGAT-3′
MxA F: 5′-GTGCATTGCAGAAGGTCAGA-3′NF-kB F: 5′- ATGTGGAGATCATTGAGCAGC-3′
R: 5′-CTGGTGATAGGCCATCAGGT-3′R: 5′-CCTGGTCCTGTGTAGCCATT-3′
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ACE2, angiotensin-converting enzyme 2; TMPRSS2, transmembrane serine protease; IFN, interferon; MxA, myxovirus resistance
1; IFIT-1, Interferon-induced tetrapeptide repeat protein 1; IL-6, Interleukin-6; TNF-α, tumor necrosis factor-α; CCL2, monocyte chemotactic protein-1; CXCL10, chemokine (C-X-C motif)
ligand 10; NF-kB, nuclear factor kappa-B.
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Li et al. Traditional Chinese Medicine and COVID-19
FIGURE 1 | ACE2 and TMPRSS-2 expression in different cell lines. Seed cells to be 80–90% confluent in 6-well plate until cells were harvested and total RNAs were
extracted. 1ug total RNA was applied for reverse transcription. ACE2 and TMPRSS-2 expression was assessed using real-time PCR (normalized to GAPDH). Data are
presented as mean ±SD (n ≥3).
FIGURE 2 | Effect of QPD and XBD on IFN signaling pathway, ACE2, and TMPRSS-2 in A549 cells. (A): Cytotoxic effect of QPD (left) and XBD (righ t) on A549 cells.
A549 cells were seeded at 6 ×10
5
/ml, 2 ml per well in 6-well plates for 24 h before QPD or XBD was added into each well at indicated concentrations (%, v/v). 48 h post
QPD or XBD treatment, total RNA was extracted to detect IFNα,IFNβ, MxA, IFIT-1 mRNAs (B) or ACE2 and TMPRSS-2 mRNAs (C) by RT-qPCR. UN, untreated control.
Data are presented as mean ±SD (n≥3).
Frontiers in Pharmacology | www.frontiersin.org October 2021 | Volume 12 | Article 7221265
Li et al. Traditional Chinese Medicine and COVID-19
showed no apparent cytotoxicity for A549 cells at
concentrations up to 15% (v/v). However, neither QPD
(Figure 2B,left)norXBD(Figure 2B, right) showed any
effect on IFNα,IFNβ, or ISG expression (RT-qPCR),
indicating that the endogenous production of type 1 IFNs
and subsequent (down-stream) ISGs expression were not
activated by these two decoctions. Moreover, no significant
difference of ACE2 nor TMPRSS-2 expression was found
between the decoction-treated group and the control group
(Figure 2C).
FIGURE 3 | QPD and XBD inhibited inflammatory cytokines expression in LPS-stimulated THP-1 macrophages. Cytotoxic effect of QPD (left) and XBD (right) on
THP-1 macrophages (A). THP-1 monocytes were seeded at 0.5 ×10
6
/ml, 2 ml per well in 6-well plates and differentiated into M0 macrophages before QPD or XBD was
added into each well at indicated concentrations (%, v/v). 24 h post QPD or XBD treatment, the supernatant was removed and the cells were washed with PBS three
times and incubated in the medium supplied with or without 1 µg/ml LPS and 20 ng/ml IFN-γ. 6 h later, the total RNA was extracted to detect IL-6, TNF-α, CCL2,
and CXCL10mRNA levels by RT-qPCR (B). 24 h later, the supernatant was collected to analyze IL-6 and TNF-αby ELISA (C). M0 THP-1 macrophages were treated with
or without 1 µg/ml LPS and 20 ng/ml IFN-γfor 6 h and then treated with QPD or XBD at indicated concentrations for 24 h. Total RNA was extracted to detect IL-6, TNF-
α, CCL2, and CXCL10 mRNA levels by RT-qPCR (D). C and UN, untreated control. LPS+, treated with LPS and IFN-γ; LPS-, treated without LPS and IFN-γ. Data are
presented as mean ±SD (n≥3). *p<0.005; **p<0.001; ***p<0.001 compared with C or UN + LPS group or as indicated.
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Li et al. Traditional Chinese Medicine and COVID-19
Qingfei Paidu Decoction and Xuanfei Baidu
Decoction Significantly Inhibit Cytokine
Production in THP-1-Derived Macrophages
THP-1 is a cell model widely used to explore macrophage function
and inflammatory response pathways. THP-1 cells can be
differentiated into mature macrophages with relatively high
similarity to human peripheral blood mononuclear cells
(PBMCs) and monocyte-derived macrophages (Chanput et al.,
2014). It was reported that SARS-CoV-2 could infect peripheral
blood monocytes and promote ACE2 expression (Codo et al.,
2020). Consistent with that study, THP-1-derived macrophages
also showed significant levels of ACE2 and TREMPSS-2 expression
in the M0, M1, and M2 stages (Figure 1). The results from CCK-8
tests indicated that both QPD (Figure 3A, left) and XBD
(Figure 3A, right) showed no apparent cytotoxicity for THP-1-
derived macrophages at concentrations up to 10% (v/v). To
determine the effect of QPD and XBD on the expression of
cytokines and chemokines in M1-like inflammatory
macrophages, we treated the M0 THP-1 macrophages with
QPD or XBD for 24 h and analyzed the mRNA expression
levels of some typical cytokines and chemokines 6 h after LPS
stimulation. The results from RT-qPCR analysis showed that both
QPD and XBD significantly inhibited the expression of IL-6, TNF-
α, CCL2, and chemokine (C-X-C motif) ligand 10 (CLCX10)
(Figure 3B), and this inhibition effect was further confirmed by
ELISA (Figure 3C). Similar results were obtained when the M0
THP-1-derived macrophages were stimulated by LPS for 6 h first
and then treated with QPD or XBD for 24 h (Figure 3D).
Qingfei Paidu Decoction and Xuanfei Baidu
Decoction Inhibited the Activation of the
NF-κB Signaling Pathway and the
Pinocytosis Activity of THP-1-Derived
Macrophages
Activation of the transcription factor nuclear factor kappa B (NF-κB)
is a key step in mediating the expression of various cytokines. To
investigate whether the inhibition effect of QPD and XBD on
cytokine production was related to the modulation of the NF-κB
signaling pathway, we measured total IκBα,NF-κB p65, phospho-
IκBα, and phospho-NF-κB p65 protein levels by western blot in
THP-1 macrophages treated with LPS and IFN-γ.Asshownin
Figure 4A, the phosphorylation levels of IκBαand NF-κBp65were
elevated in the LPS-stimulated control groups, indicating successful
activation of the NF-κB pathway following LPS stimulation. Both
QPD and XBD treatments suppressed phosphorylation of IκBαand
NF-κB p65 at 1 h or 2 h post LPS stimulation. The levels of total
IκBαwere not changed following QPD or XBD treatment. However,
both QPD and XBD treatments slightly inhibited the total level of
NF-κB mRNA expression in the LPS-free group, which was further
confirmed by RT-qPCR (Figure 4B).
Soluble antigens have been proven to directly enter macrophages
and thereby induce signaling activation to mediate macrophage
polarization. We therefore tested whether QPD and XBD affect the
pinocytosis activity of THP-1-derived macrophages using neutral
red uptake assay. As shown in Figure 5, the pinocytosis activity was
significantly inhibited by QPD or XBD treatment, especially in the
LPS-free group. Moreover, the suppression effect of QPD or XBD on
pinocytosis was less significant in the M1 polarized macrophages,
which might be due to the blunted pinocytosis activity induced by
LPS stimulation.
FIGURE 4 | QPD and XBD prevented LPS-induced activation of NF-κB
signaling pathway in THP-1 macrophages. M0 THP-1 macrophages were
treated with QPD or XBD at indicated concentrations for 24 h and then
stimulated with or without 1 µg/ml LPS and 20 ng/ml IFN-γfor 1 h or2 h.
Total proteins were extracted to detect IκBα, phospho-IκBα, NF-κB p65, and
phospho-NF-κB p65 by western blot (A). Total RNA was extracted to detect
NF-κB by RT-qPCR (B). LPS+, treated with LPS and IFN-γ; LPS-, treated
without LPS and IFN-γ. ***p<0.001.
FIGURE 5 | QPD and XBD inhibited pinocytosis activity of THP-1
macrophages. M0 THP-1 macrophages were treated with QPD or XBD (5%,
v/v) for 24 h and then stimulated with or without 1 µg/ml LPS and 20 ng/ml
IFN-γfor another 24 h before neutral red uptake assay. LPS+, treated
with LPS and IFN-γ; LPS-, treated without LPS and IFN-γ.**p<0.001;
***p<0.001 compared with UN or as indicated.
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Li et al. Traditional Chinese Medicine and COVID-19
DISCUSSION
Cytokine storm is closely associated with the severity and mortality
of patients with COVID-19 (Hu et al., 2021;Kim et al., 2021).
Accordingly, anti-inflammatory therapies are of great importance in
the management of patients with severe COVID-19. In our current
study, we focused our investigation on macrophages because they
play a key role in cytokine storms and are the major source of pro-
inflammatory cytokines (Wang J. et al., 2020) including IL-6 and
TNF-α. Although the detailed immune modulation mechanisms
vary among viruses, the activation of multiple Toll-like receptors
(TLRs) is involved in the induction of a cytokine storm. Recent
studies revealed that SARS-CoV-2 induced inflammation via TLR2/
4activation(Bhattacharya et al., 2020;Choudhury et al., 2020;Khan
et al., 2021;Zheng et al., 2021). Similarly, LPS stimulation could also
activate TLR2/4 signaling in macrophages (Orecchioni et al., 2019;
FengTT.etal.,2020), which mimic the activation status induced by
SARS-CoV-2 to some extent. Moreover, the expression profiles of
the pro-inflammatory cytokines (e.g. IL-6, TNF-a, CXCL10, and so
on) in THP-1 are very similar in both the LPS-stimulated group and
the SARS-CoV-2 envelope protein-stimulated group (Chiok et al.,
2021;Pantazi et al., 2021;Shirato et al., 2021). Based on this, we
utilized LPS-stimulated THP-1 macrophages in the present study.
Our present data show that both QPD and XBD could directly
suppress the production of IL-6 and TNF-αin THP-1-derived
macrophages, indicating a TCM-induced inflammatory modulation
effect. As a cytokine critical to mediate inflammation, IL-6 has
pleiotropic activity (Tanaka et al., 2014)andmayplayanopposing
role in the immune response to different viral infections
(Gubernatorova et al., 2020). Clinical data (Feng X. et al., 2020;
Chen et al., 2020;Wang et al., 2021a)haveshownthatCOVID-19
patients, especially severe patients, experienced significantly elevated
systemic levels of IL-6 compared to healthy controls. Therefore, IL-6 is
considered to be a useful biomarker for predicting the severity of a
SARS-Cov-2 infection, although the exact mechanism remains to be
elucidated. In line with this, the therapeutic potential of IL-6 inhibitors
(Gritti et al., 2020;Liu et al., 2020;Xu et al., 2020), such as Tocilizumab
and Siltuximab, have been investigated clinically. At the same time, the
effect of TCM on IL-6 has also been explored. Pharmacological assays
in vitro demonstrated the effects of some TCM, such as Liu Shen
capsules (Ma et al., 2020), ReDuNing injections (Ma et al., 2021), and a
novel formula NRICM101 (Tsai et al., 2021), in suppressing the
expression of IL-6, as well as TNF-α.QPDhasalsobeenshownto
contribute to IL-6 production. Recently, Y Ren et al. (2020) have
found that QPD inhibited the arachidonic acid (AA) metabolic
pathway which was closely involved in IL-6 production. Ruocong
Yang and colleagues (Yang et al., 2020)reportedthatonemajor
compound in QPD, glycyrrhizic acid, could inhibit IL-6 production
via Toll-like receptor signaling. In the current study, we present
further evidence to support these earlier observations. It has been
reported that LPS could induce the expression of IL-6 and TNF-αvia
the activation of the NF-κB signaling pathway (Koch et al., 2014;Lee
et al., 2017). Accordingly, we tested the activation of the NF-κB
signaling pathway in macrophageswithorwithoutQPDorXBD
treatment, following LPS stimulation. We found that both QPD and
XBD suppressed NF-κB signaling, to a striking degree. Moreover,
network pharmacology studies (Li et al., 2021;Niu et al., 2021;Xia
et al., 2021) have revealed that numerous active compounds in TCM
have significant molecular binding affinities with IL-6 or could block
IL-6 mediated JAK-STAT signaling pathway, raising another
possibility for the anti-inflammatory activity of TCM.
Interestingly, our data also showed that QPD and XBD could
inhibit the pinocytosis activity of THP-1-derived macrophages. It is
already known that pinocytosis is involved in macrophage activation
and polarization, and contributes to different immune responses. M
Hashimoto et al. (2014) reported that soluble HIV-1 Nef protein
entered M2 macrophages by macro-pinocytosis, driving them towards
M1-like macrophages by activating the transforming growth factor
(TGF)-β-activated kinase 1 (TAK1) cascade. Abraxane, a first-line drug
for the treatment of pancreatic cancer, could exploit macro-pinocytosis
for its entry into the macrophages to facilitate the differentiation into
proinflammatory M1 phenotype (Cullis et al., 2017). Although more
robust scientific evidence is needed, the possibility does exist that QPD
and XBD may change macrophages’response to the
microenvironment via regulation of pinocytosis, leading to a more
favorable prognosis in COVID-19 patients.
The diverse impacts of QPD and XBD on SARS-CoV-2 are
consistent with the complicated constituents and compounds in
the decoctions. In addition to immune regulation, TCM may also
affect SARS-CoV-2 infection in other ways. 1) QPD exerts anti-viral
effects via acting on several ribosomal proteins, resulting in suppressed
viral replication (Alshaeri et al., 2020). 2) Lianhua Qingwen capsules
could directly inhibit viral replication and lead to abnormal virus
morphology (Runfeng et al., 2020). In addition, SARS-CoV-2 hijacked
ACE2 to enter host cells (Zamorano Cuervo et al., 2020), which is very
important for virus replication. The SARS-CoV-2 spike (S) protein is
composed of two functional units: S1 which directly binds to ACE2,
and S2 which is responsible for the fusion of virus and cellular
membranes after being cleaved by TMPRSS2 (Zhang et al., 2020).
Therefore, the ACE2/TMPRSS2 pathway is a promising target to
block the early stages of SARS-CoV-2 infections (Monteil et al., 2020;
Ragia et al., 2020). Our present data show that there was no significant
difference in ACE2 and TMPRSS2 mRNA expression between the
TCM-treated group and the control group. However, we failed to
evaluate ACE2 or TMPRSS2 at the protein level, and the cellular
location and enzymatic activity should be also considered. More
evidence will be needed to reveal whether other phases of SARS-
CoV-2 replication can be blocked by QPD and XBD.
TCM has been developed “from the clinic to the laboratory”,
which is opposite to the “laboratory to the clinic”process in
Western medicine. There is still a long way to go to understand
the mechanisms underlying TCM, including the action of QPD
and XBD in COVID-19 therapy. In vivo animal studies are
needed to complement the in vitro cell-based experiments, and
the roles of the individual components of the decoctions should
be evaluated, while the synergistic effects of different compounds
also need to be explored in the future.
In conclusion, we demonstrated a significantly decreased IL-6
and TNF-αproduction in response to LPS stimulation in QPD-
and XBD-treated macrophages, where NF-κB signaling may be
the key regulator in the present study. Moreover, both QPD and
XBD inhibited the pinocytosis function of THP-1-derived
macrophages. Because macrophages are one of the most
important effectors involved in the process of cytokine storms,
Frontiers in Pharmacology | www.frontiersin.org October 2021 | Volume 12 | Article 7221268
Li et al. Traditional Chinese Medicine and COVID-19
we speculate that the QPD and XBD can inhibit the inflammatory
phenotype of macrophages, reducing the risk of a deleterious,
hyper-activated inflammatory response. Our current results
partly explain the efficacy of QPD and XBD in the treatment
of COVID-19 patients, especially in severe patients.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be
directed to the corresponding authors.
AUTHOR CONTRIBUTIONS
Conceptualization and Formal analysis: QW and PW.
Investigation and Methodology: YL and BL. Writing—original
draft: YL. Writing—review and Editing: QW and PW. Funding
acquisition: QW.
FUNDING
This work was supported by the Chengdu Municipal Health
Commission, Chengdu, China (grant number 2020179) to QW.
REFERENCES
Acharya, D., Liu, G., and Gack, M. U. (2020). Dysregulation of Type I Interferon
Responses in COVID-19. Nat. Rev. Immunol. 20 (7), 397–398. doi:10.1038/
s41577-020-0346-x
Al-Romaima, A., Liao, Y., Feng, J., Qin, X., and Qin, G. (2020). Advances in the
Treatment of Novel Coronavirus Disease (COVID-19) with Western Medicine
and Traditional Chinese Medicine: a Narrative Review. J. Thorac. Dis. 12 (10),
6054–6069. doi:10.21037/jtd-20-1810
Alshaeri, H. K., and Natto, Z. S. (2020). A Contemporary Look at COVID-19
Medications: Available and Potentially Effective Drugs. Eur. Rev. Med.
Pharmacol. Sci. 24 (17), 9188–9195. doi:10.26355/eurrev_202009_22870
Bhattacharya, M., Sharma, A. R., Mallick, B., Sharma, G., Lee, S. S., and
Chakraborty, C. (2020). Immunoinformatics Approach to Understand
Molecular Interaction between Multi-Epitopic Regions of SARS-CoV-
2 Spike-Protein with TLR4/MD-2 Complex. Infect. Genet. Evol. 85, 104587.
doi:10.1016/j.meegid.2020.104587
Booz, G. W., Altara, R., Eid, A. H., Wehbe, Z., Fares, S., Zaraket, H., et al. (2020).
Macrophage Responses Associated with COVID-19: A Pharmacological
Perspective. Eur. J. Pharmacol. 887, 173547. doi:10.1016/j.ejphar.2020.173547
Chanput, W., Mes, J. J., and Wichers, H. J. (2014). THP-1 Cell Line: an In Vitro Cell
Model for Immune Modulation Approach. Int. Immunopharmacol 23 (1),
37–45. doi:10.1016/j.intimp.2014.08.002
Chen, R., Sang, L., Jiang, M., Yang, Z., Jia, N., Fu, W., et al. (2020). Longitudinal
Hematologic and Immunologic Variations Associated with the Progression of
COVID-19 Patients in China. J. Allergy Clin. Immunol. 146 (1), 89–100.
doi:10.1016/j.jaci.2020.05.003
Chiok, K., Hutchison, K., Miller, L. G., Bose, S., and Miura, T. A. (2021).
Proinflammatory Responses in SARS-CoV-2 Infected and Soluble Spike
Glycoprotein S1 Subunit Activated Human Macrophages. bioRxiv.
doi:10.1101/2021.06.14.448426
Choudhury, A., and Mukherjee, S. (2020). In Silico studies on the Comparative
Characterization of the Interactions of SARS-CoV-2 Spike Glycoprotein with
ACE-2 Receptor Homologs and Human TLRs. J. Med. Virol. 92 (10),
2105–2113. doi:10.1002/jmv.25987
Codo, A. C., Davanzo, G. G., Monteiro, L. B., de Souza, G. F., Muraro, S. P.,
Virgilio-da-Silva, J. V., et al. (2020). Elevated Glucose Levels Favor SARS-CoV-
2 Infection and Monocyte Response through a HIF-1α/Glycolysis-dependent
Axis. Cell Metab 32 (3), 498–499. e5. doi:10.1016/j.cmet.2020.07.007
Cullis, J., Siolas, D., Avanzi, A., Barui, S., Maitra, A., and Bar-Sagi, D. (2017).
Macropinocytosis of Nab-Paclitaxel Drives Macrophage Activation in
Pancreatic Cancer. Cancer Immunol. Res. 5 (3), 182–190. doi:10.1158/2326-
6066.cir-16-0125
Feng, T. T., Yang, X. Y., Hao, S. S., Sun, F. F., Huang, Y., Lin, Q. S., et al. (2020a).
TLR-2-mediated Metabolic Reprogramming Participates in Polyene
Phosphatidylcholine-Mediated Inhibition of M1 Macrophage Polarization.
Immunol. Res. 68 (1), 28–38. doi:10.1007/s12026-020-09125-9
Feng, X., Li, P., Ma, L., Liang, H., Lei, J., Li, W., et al. (2020b). Clinical
Characteristics and Short-Term Outcomes of Severe Patients with COVID-
19 in Wuhan, China. Front. Med. (Lausanne) 7, 491. doi:10.3389/
fmed.2020.00491
Gritti, G., Raimondi, F., Ripamonti, D., Riva, I., Landi, F., Rambaldi, A., et al.
(2020). Use of Siltuximab in Patients with COVID-19 Pneumonia Requiring
Ventilatory Support. medRxiv. doi:10.1101/2020.04.01.20048561
Gubernatorova, E. O., Gorshkova, E. A., Polinova, A. I., and Drutskaya, M. S.
(2020). IL-6: Relevance for Immunopathology of SARS-CoV-2. Cytokine
Growth Factor. Rev. 53, 13–24. doi:10.1016/j.cytogfr.2020.05.009
Hashimoto, M., Nasser, H., Chihara, T., and Suzu, S. (2014). Macrop inocytosis and
TAK1 Mediate Anti-inflammatory to Pro-inflammatory Macrophage
Differentiation by HIV-1 Nef. Cell Death Dis 5 (5), e1267. doi:10.1038/
cddis.2014.233
Hu, B., Huang, S., and Yin, L. (2021). The Cytokine Storm and COVID-19. J. Med.
Virol. 93 (1), 250–256. doi:10.1002/jmv.26232
Huang, K., Zhang, P., Zhang, Z., Youn, J. Y., Wang, C., Zhang, H., et al. (2021).
Traditional Chinese Medicine (TCM) in the Treatment of COVID-19 and
Other Viral Infections: Efficacies and Mechanisms. Pharmacol. Ther. 225,
107843. doi:10.1016/j.pharmthera.2021.107843
Jacobo-Salcedo, Mdel. R., Juárez-Vázquez, Mdel. C., González-Espíndola, L. Á.,
Maciel-Torres, S. P., García-Carrancá, A., and Alonso-Castro, A. J. (2013).
Biological Effects of Aqueous Extract from Turkey Vulture Cathartes aura
(Cathartidae) Meat. J. Ethnopharmacol 145 (2), 663–666. doi:10.1016/
j.jep.2012.11.014
Khan, S., Shafiei, M. S., Longoria, C., Schoggins, J., Savani, R. C., and Zaki, H.
(2021). SARS-CoV-2 Spike Protein Induces Inflammation via TLR2-
dependent Activation of the NF-Κb Pathway. bioRxiv. doi:10.1101/
2021.03.16.435700
Kim, J. S., Lee, J. Y., Yang, J. W., Lee, K. H., Effenberger, M., Szpirt, W., et al. (2021).
Immunopathogenesis and Treatment of Cytokine Storm in COVID-19.
Theranostics 11 (1), 316–329. doi:10.7150/thno.49713
Koch, L., Frommhold, D., Buschmann, K., Kuss, N., Poeschl, J., and Ruef, P. (2014).
LPS- and LTA-Induced Expression of IL-6 and TNF-Ain Neonatal and Adult
Blood: Role of MAPKs and NF-Κb. Mediators Inflamm. 2014, 1–8. doi:10.1155/
2014/283126
Lee, S. B., Lee, W. S., Shin, J. S., Jang, D. S., and Lee, K. T. (2017). Xanthotoxin
Suppresses LPS-Induced Expression of iNOS, COX-2, TNF-α, and IL-6 via AP-
1, NF-Κb, and JAK-STAT Inactivation in RAW 264.7 Macrophages. Int.
Immunopharmacol 49, 21–29. doi:10.1016/j.intimp.2017.05.021
Li, Y., Chu, F., Li, P., Johnson, N., Li, T., Wang, Y., et al. (2021). Potential Effect of
Maxing Shigan Decoction against Coronavirus Disease 2019 (COVID-19)
Revealed by Network Pharmacology and Experimental Verification.
J. Ethnopharmacol 271, 113854. doi:10.1016/j.jep.2021.113854
Liu, B., Li, M., Zhou, Z., Guan, X., and Xiang, Y. (2020). Can We Use Interleukin-6
(IL-6) Blockade for Coronavirus Disease 2019 (COVID-19)-Induced Cytokine
Release Syndrome (CRS)?. J. Autoimmun. 111, 102452. doi:10.1016/
j.jaut.2020.102452
Luo, E., Zhang, D., Luo, H., Liu, B., Zhao, K., Zhao, Y., et al. (2020). Treatment
Efficacy Analysis of Traditional Chinese Medicine for Novel Coronavirus
Pneumonia (COVID-19): an Empirical Study from Wuhan, Hubei Province,
China. Chin. Med. 15, 34. doi:10.1186/s13020-020-00317-x
Luo, H., Yang, M., Tang, Q. L., Hu, X. Y., Willcox, M. L., and Liu, J. P. (2021).
Characteristics of Registered Clinical Trials on Traditional Chinese Medicine
for Coronavirus Disease 2019 (COVID-19): A Scoping Review. Eur. J. Integr.
Med. 41, 101251. doi:10.1016/j.eujim.2020.101251
Frontiers in Pharmacology | www.frontiersin.org October 2021 | Volume 12 | Article 7221269
Li et al. Traditional Chinese Medicine and COVID-19
Ma, Q., Pan, W., Li, R., Liu, B., Li, C., Xie, Y., et al. (2020). Liu Shen Capsule Shows
Antiviral and Anti-inflammatory Abilities against Novel Coronavirus SARS-
CoV-2 via Suppression of NF-Κb Signaling Pathway. Pharmacol. Res. 158,
104850. doi:10.1016/j.phrs.2020.104850
Ma, Q., Xie, Y., Wang, Z., Lei, B., Chen, R., Liu, B., et al. (2021). Efficacy and Safety
of ReDuNing Injection as a Treatment for COVID-19 and its Inhibitory Effect
against SARS-CoV-2. J. Ethnopharmacol 279, 114367. doi:10.1016/
j.jep.2021.114367
Merad, M., and Martin, J. C. (2020). Pathological Inflammation in Patients with
COVID-19: a Key Role for Monocytes and Macrophages. Nat. Rev. Immunol.
20 (6), 355–362. doi:10.1038/s41577-020-0331-4
Monteil, V., Kwon, H., Prado, P., Hagelkrüys, A., Wimmer, R. A., Stahl, M., et al.
(2020). Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues
Using Clinical-Grade Soluble Human ACE2. Cell 181 (4), 905–913. e7.
doi:10.1016/j.cell.2020.04.004
Niu, W. H., Wu, F., Cao, W. Y., Wu, Z. G., Chao, Y. C., and Liang, C. (2021).
Network Pharmacology for the Identification of Phytochemicals in Traditional
Chinese Medicine for COVID-19 that May Regulate Interleukin-6. Biosci. Rep.
41 (1), BSR20202583. doi:10.1042/bsr20202583
Orecchioni, M., Ghosheh, Y., Pramod, A. B., and Ley, K. (2019). Macrophage
Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and
M2(LPS-) vs. Alternatively Activated Macrophages. Front. Immunol. 10,
1084. doi:10.3389/fimmu.2019.01084
Pantazi, I., Al-Qahtani, A. A., Alhamlan, F. S., Alothaid, H., Matou-Nasri, S.,
Sourvinos, G., et al. (2021). SARS-CoV-2/ACE2 Interaction Suppresses IRAK-
M Expression and Promotes Pro-inflammatory Cytokine Production in
Macrophages. Front. Immunol. 12, 683800. doi:10.3389/fimmu.2021.683800
Park, A., and Iwasaki, A. (2020). Type I and Type III Interferons - Induction,
Signaling, Evasion, and Application to Combat COVID-19. Cell Host Microbe
27 (6), 870–878. doi:10.1016/j.chom.2020.05.008
Qi, F., Qian, S., Zhang, S., and Zhang, Z. (2020). Single Cell RNA Sequencing of 13
Human Tissues Identify Cell Types and Receptors of Human Coronaviruses.
Biochem. Biophys. Res. Commun. 526 (1), 135–140. doi:10.1016/
j.bbrc.2020.03.044
Ragia, G., and Manolopoulos, V. G. (2020). Inhibition of SARS-CoV-2 Entry
through the ACE2/TMPRSS2 Pathway: a Promising Approach for Uncovering
Early COVID-19 Drug Therapies. Eur. J. Clin. Pharmacol. 76 (12), 1623–1630.
doi:10.1007/s00228-020-02963-4
Ren, Y., Yao, M. C., Huo, X. Q., Gu, Y., Zhu, W. X., Qiao, Y. J., et al. (2020). [Study
on Treatment of "cytokine Storm" by Anti-2019-nCoV Prescriptions Based on
Arachidonic Acid Metabolic Pathway]. Zhongguo Zhong Yao Za Zhi 45 (6),
1225–1231. doi:10.19540/j.cnki.cjcmm.20200224.405
Runfeng, L., Yunlong, H., Jicheng, H., Weiqi, P., Qinhai, M., Yongxia, S., et al.
(2020). Lianhuaqingwen Exerts Anti-viral and Anti-inflammatory Activity
against Novel Coronavirus (SARS-CoV-2). Pharmacol. Res. 156, 104761.
doi:10.1016/j.phrs.2020.104761
Shi, N., Liu, B., Liang, N., Ma, Y., Ge, Y., Yi, H., et al. (2020). Association between
Early Treatment with Qingfei Paidu Decoction and Favorable Clinical
Outcomes in Patients with COVID-19: A Retrospective Multicenter Cohort
Study. Pharmacol. Res. 161, 105290. doi:10.1016/j.phrs.2020.105290
Shirato, K., and Kizaki, T. (2021). SARS-CoV-2 Spike Protein S1 Subunit Induces
Pro-inflammatory Responses via Toll-like Receptor 4 Signaling in Murine and
Human Macrophages. Heliyon 7 (2), e06187. doi:10.1016/j.heliyon.2021.e06187
Tanaka, T., Narazaki, M., and Kishimoto, T. (2014). IL-6 in Inflammation,
Immunity, and Disease. Cold Spring Harb Perspect. Biol. 6 (10), a016295.
doi:10.1101/cshperspect.a016295
Tsai, K. C., Huang, Y. C., Liaw, C. C., Tsai, C. I., Chiou, C. T., Lin, C. J., et al. (2021).
A Traditional Chinese Medicine Formula NRICM101 to Target COVID-19
through Multiple Pathways: A Bedside-To-Bench Study. Biomed.
Pharmacother. 133, 111037. doi:10.1016/j.biopha.2020.111037
Wang, C., Xie, J., Zhao, L., Fei, X., Zhang, H., Tan, Y., et al. (2020a). Alveolar
Macrophage Dysfunction and Cytokine Storm in the Pathogenesis of Two
Severe COVID-19 Patients. EBioMedicine 57, 102833. doi:10.1016/
j.ebiom.2020.102833
Wang, J., Jiang, M., Chen, X., and Montaner, L. J. (2020b). Cytokine Storm and
Leukocyte Changes in Mild versus Severe SARS-CoV-2 Infection: Review of
3939 COVID-19 Patients in China and Emerging Pathogenesis and Therapy
Concepts. J. Leukoc. Biol. 108 (1), 17–41. doi:10.1002/jlb.3covr0520-272r
Wang, T., Tang, R., Ruan, H., Chen, R., Zhang, Z., Sang, L., et al. (2021a). Predictors
of Fatal Outcomes Among Hospitalized COVID-19 Patients with Pre-existing
Hypertension in China. Clin. Respir. J. 15, 915–924. doi:10.1111/crj.13382
Wang, Y., Li, X., Zhang, J. H., Xue, R., Qian, J. Y., Zhang, X. H., et al. (2020c).
[Mechanism of Xuanfei Baidu Tang in Treatment of COVID-19 Based on
Network Pharmacology]. Zhongguo Zhong Yao Za Zhi 45 (10), 2249–2256.
doi:10.19540/j.cnki.cjcmm.20200325.401
Wang, Z., and Yang, L. (2021b). Chinese Herbal Medicine: Fighting SARS-CoV-2
Infection on All Fronts. J. Ethnopharmacol 270, 113869. doi:10.1016/
j.jep.2021.113869
Xia, S., Zhong, Z., Gao, B., Vong, C. T., Lin, X., Cai, J., et al. (2021). The Important
Herbal Pair for the Treatment of COVID-19 and its Possible Mechanisms.
Chin. Med. 16 (1), 25. doi:10.1186/s13020-021-00427-0
Xiong, W. Z., Wang, G., Du, J., and Ai, W. (2020). Efficacy of Herbal Medicine
(Xuanfei Baidu Decoction) Combined with Conventional Drug in Treating
COVID-19:A Pilot Randomized Clinical Trial. Integr. Med. Res. 9 (3), 100489.
doi:10.1016/j.imr.2020.100489
Xu, X., Han, M., Li, T., Sun, W., Wang, D., Fu, B., et al. (2020). Effective Treatment
of Severe COVID-19 Patients with Tocilizumab. Proc. Natl. Acad. Sci. U S A.
117 (20), 10970–10975. doi:10.1073/pnas.2005615117
Yang, R., Liu, H., Bai, C., Wang, Y., Zhang, X., Guo, R., et al. (2020). Chemical
Composition and Pharmacological Mechanism of Qingfei Paidu Decoction and
Ma Xing Shi Gan Decoction against Coronavirus Disease 2019 (COVID-19): In
Silico and Experimental Study. Pharmacol. Res. 157, 104820. doi:10.1016/
j.phrs.2020.104820
Zamorano Cuervo, N., and Grandvaux, N. (2020). ACE2: Evidence of Role as Entry
Receptor for SARS-CoV-2 and Implications in Comorbidities. Elife 9, e61390.
doi:10.7554/eLife.61390
Zhang, X., Li, S., and Niu, S. (2020). ACE2 and COVID-19 and the Resulting
ARDS. Postgrad. Med. J. 96 (1137), 403–407. doi:10.1136/postgradmedj-2020-
137935
Zheng, M., Karki, R., Williams, E. P., Yang, D., Fitzpatrick, E., Vogel, P., et al.
(2021). TLR2 Senses the SARS-CoV-2 Envelope Protein to Produce
Inflammatory Cytokines. Nat. Immunol. 22 (7), 829–838. doi:10.1038/
s41590-021-00937-x
Conflict of Interest: The authors declare that the research was conducted in the
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