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Nanocurcumin Potently Inhibits SARS-CoV‑2 Spike Protein-Induced
Cytokine Storm by Deactivation of MAPK/NF-κB Signaling in
Epithelial Cells
Vivek K. Sharma,
§
Prateeksha,
§
Shailendra P. Singh, Brahma N. Singh,*Chandana V. Rao,
and Saroj K. Barik*
Cite This: ACS Appl. Bio Mater. 2022, 5, 483−491
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sıSupporting Information
ABSTRACT: Interleukin-mediated deep cytokine storm, an
aggressive inflammatory response to SARS-CoV-2 virus infection
in COVID-19 patients, is correlated directly with lung injury,
multi-organ failure, and poor prognosis of severe COVID-19
patients. Curcumin (CUR), a phenolic antioxidant compound
obtained from turmeric (Curcuma longa L.), is well-known for its
strong anti-inflammatory activity. However, its in vivo efficacy is
constrained due to poor bioavailability. Herein, we report that
CUR-encapsulated polysaccharide nanoparticles (CUR−PS-NPs)
potently inhibit the release of cytokines, chemokines, and growth
factors associated with damage of SARS-CoV-2 spike protein
(CoV2-SP)-stimulated liver Huh7.5 and lung A549 epithelial cells.
Treatment with CUR−PS-NPs effectively attenuated the interaction of ACE2 and CoV2-SP. The effects of CUR-PS-NPs were
linked to reduced NF-κB/MAPK signaling which in turn decreased CoV2-SP-mediated phosphorylation of p38 MAPK, p42/44
MAPK, and p65/NF-κB as well as nuclear p65/NF-κB expression. The findings of the study strongly indicate that organic NPs of
CUR can be used to control hyper-inflammatory responses and prevent lung and liver injuries associated with CoV2-SP-mediated
cytokine storm.
KEYWORDS: nanocurcumin, SARS-CoV-2, spike protein, cytokine storm, MAPK/NF-κB signaling, epithelial cells
■INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS-
CoV-2), the causal organism of coronavirus disease-19
(COVID-19), emerged in December, 2019, and became the
most calamitous pandemic of the 21st century.
1
The very high
rate of mutation of SARS-CoV-2 continues to pose challenges
to the scientific and medical professionals worldwide to
effectively control the disease. SARS-CoV-2 relates to the β-
coronavirus genus with around 79.5% sequence similarity with
the SARS-CoV that appeared in southern China in 2002.
2
The
mechanisms so far understood for the COVID-19 progress are
characterized by a quick viral replication, resulting in elevated
cytolysis of host cells and a hyper-inflammatory state due to
excessive production of pro-inflammatory cytokines known as
a“cytokine storm”inducing multiple organ damage.
3,4
The
cytokine storm is a life-threatening systemic inflammatory
syndrome that involves elevated levels of circulating cytokines
and immune-cell hyperactivation leading to secondary organ
dysfunction, particularly renal, hepatic, or pulmonary. Various
pathogens, therapies, cancers, autoimmune conditions, and
monogenic disorders have already been reported to trigger
such a syndrome in humans.
3
Angiotensin-converting enzyme 2 (ACE2), a member of
dipeptidyl carboxypeptidase group, is widely expressed in
different human organs including lungs, kidneys, liver, gut, and
vascular systems. It is recognized as a key entry receptor for
SARS-CoV-2.
5
The binding of SARS-CoV-2 surface spike
protein (CoV2-SP) to human ACE2 through its receptor
binding domain triggers a series of physiopathological events
including the cytokine storm through activation of nuclear
factor κB(NF-κB) and mitogen-activated protein kinase
(MAPK) by IL-6 trans-signaling.
6,7
This storm induces several
pathological complications, particularly acute respiratory
distress syndrome (ARDS), often found in serious COVID-
19 patients.
8,9
The cytokine storm caused by SARS-CoV-2 is
characterized by enhanced levels of IL-6, tumour necrosis
factor α(TNF-α), and C−C motif chemokine ligand (CCL2).
Received: August 6, 2021
Accepted: January 16, 2022
Published: February 3, 2022
Articlewww.acsabm.org
© 2022 American Chemical Society 483
https://doi.org/10.1021/acsabm.1c00874
ACS Appl. Bio Mater. 2022, 5, 483−491
This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use
and analyses in any form or by any means with acknowledgement of the original source.
These permissions are granted for the duration of the World Health Organization (WHO)
declaration of COVID-19 as a global pandemic.
The patients with COVID-19-associated ARDS suffer from
more organ and tissue injuries, and have greater mortality than
the ARDS not related to COVID-19.
8,10
Anti-COVID-19
pharmacological strategy using anti-inflammatory approaches
via modulating IL-6
11
and IL-8
12
has been quite effective.
Therefore, discovery of compounds having the ability to inhibit
cytokine storms, and to understand the mechanisms of their
anti-inflammation activity can help development of effective
anti-COVID-19 drugs.
Curcumin (CUR), a key dietary polyphenolic compound
predominantly present in the rhizome of turmeric plant
(Curcuma longa L.), exhibits a range of biological activities and
medicinal properties for the treatment of cancer, atheroscle-
rosis, diabetes, obesity, and microbial infections.
13,14
CUR
provides strong anti-inflammatory effects against the SARS-
CoV-2-induced cytokine storm.
15,16
Several pre-clinical and
clinical studies have revealed that CUR and its analogues (e.g.,
diarylpentanoids) significantly attenuate the levels of pro-
inflammatory cytokines viz., IL-1, IL-6, IL-8, and TNF-α.
17
However, the anti-inflammatory effectiveness of CUR is
restricted because of its poor bioavailability.
16
Numerous
approaches have been used to enhance the bioavailability of
CUR including the use of piperine as an adjuvant agent,
liposome-based CUR, and phospholipid CUR complexes.
18
Organic nanoparticle (NP)-mediated CUR delivery could be
an effective approach to increase its bioavailability, and also
sustained and controlled release.
19−21
Organic NPs may
enhance the anti-inflammatory potential of CUR and also
minimize the quantity of CUR required. The large surface area
and small size of NPs provide greater stability and can readily
internalize into the cells without compromising its efficacy and
integrity. Several recent investigations using cell lines and
animal models suggest that inorganic NPs of CUR as a
therapeutic agent is more powerful than bulk CUR .
16,18,22
Since organic NPs are nontoxic, biocompatible, biodegradable,
and non-immunogenic, the organic NPs of CUR have the
potential to be used as a safe and effective drug against
COVID-19.
Therefore, we investigated the anti-inflammatory efficacy of
CUR-encapsulated polysaccharide NPs (CUR−PS-NPs) tar-
geting the SARS-CoV-2 spike protein (CoV2-SP)-induced
cytokine storm, and compared it with bulk CUR (B-CUR) in
liver and lung epithelial cells. We deciphered the mecha-
nism(s) of actions underlying the NF-κB signaling inhibition
and MAPK deactivation by CUR that inhibit CoV2-SP-
induced cytokine storms. We also investigated the role of CUR
in reducing CoV2-SP-mediated phosphorylation of p38
MAPK, p42/44 MAPK, p65/NF-κB, and nuclear p65/NF-κB
expression, and release of cytokines, chemokines, and growth
factors linked with the liver and lung epithelial cell injury.
■RESULTS AND DISCUSSION
Preparation and Characterization of CUR−PS-NPs
and In Vitro Release Kinetics of CUR. We used an emulsion
solvent evaporation process to prepare CUR−PS-NPs (Figure
1a−c).
23
Scanning electron microscopy (SEM) images showed
the uniform spherical shape of the CUR−PS-NPs with particle
size in the range of 18−27 nm (Figure 1d). The morphology of
these NPs was observed using transmission electron
microscopy (TEM). The average size of CUR−PS-NPs was
22 ±4 nm, and the particles distributed uniformly in PS matrix
in spherical shapes (Figure 2a). The mean hydrodynamic size
and zeta potential of CUR−PS-NPs measured through the
dynamic light scattering (DLS) technique revealed that the
mean size was 43 ±5 nm, and the particles exibited a low
(0.52) polydispersity index (PDI), confirming the formation of
monodispersed CUR-PS-NPs (Figure 2b). Zeta potential/
surface charge of the CUR−PS-NPs was −18 ±1.6 mV
(Figure 2b). We determined the entrapment efficiency of CUR
in a PS matrix, which was ∼25 ±2%. In other words, 1 mg of
CUR−PS-NPs encapsulated 250 μg of CUR (Figure 2b). All
these data confirmed that the size of CUR−PS-NPs was within
the nanomaterial range. The observed bigger size in DLS
Figure 1. CUR−PS-NPs fabrication scheme. (a) Mixing of CUR and polysaccharide-rich fraction isolated from the rhizome of turmeric under
stirring conditions at 250 rpm for 120 min. (b) Addition of polyvinyl alcohol solution (1%) to the mixture of CUR and PS. (c) Prepared CUR−PS-
NPs. (d) SEM image of CUR−PS-NPs.
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ACS Appl. Bio Mater. 2022, 5, 483−491
484
analysis than that in TEM was attributed to the hydrodynamic
size measurement in the former and the absolute size
measurement in the later.
18
The Fourier-Transform Infrared Spectroscopy (FT-IR)
analysis revealed that OH, C−H, and CO stretching
vibrations of PS fraction peaked at 3429 ±4, 2923 ±3 and
1637 ±3cm
−1, respectively, which got shifted to 3531 ±5,
3014 ±4, and 1703 ±3cm
−1, respectively, in case of CUR−
PS-NPs, indicating that electrostatic interactions took place
between CUR and PS of C. longa rhizome.
24
CUR−PS-NPs
remained stable even after 12 months of preparation at room
temperature.
We assessed the release kinetics of CUR from the CUR−PS-
NPs for 170 h in a water and ethanol solution (1:1 ratio) at 37
±1°C. We observed an exponential release up to 72 h
achieving 55% of CUR liberation, and in total ∼63% of the
entrapped CUR was released in 170 h (Figure 2c). These
results confirmed the sustained release of CUR from the
CUR−PS-NPs.
CUR−PS-NPs Inhibit Interaction between Human
ACE2 and CoV2-SP. To examine if CUR−PS-NPs attenuate
the interaction between human ACE2 and CoV2-SP, we used
an ELISA-based assay in which biotinylated purified human
ACE-2 protein binds with the immobilized CoV2-SP.
25
ACE2,
a cellular receptor present on the outer surface of a range of
human cells and tissues, is the first host cell target of CoV2-
SP.
26
Thus, disrupting the interaction between CoV2-SP and
ACE2 can be an effective strategy to design potential drugs.
27
We used different doses (0.001, 0.01, 0.1, 0.2, 0.5, and 5.0 μM)
of B-CUR and CUR−PS-NPs for this study. CUR−PS-NPs
and B-CUR exhibited a dose-dependent inhibitory effect on
the interactions between ACE2 and CoV2-SP, while PSNPs
did not show any significant effect. However, CUR-PS-NPs at
5μM concentration showed a significantly greater inhibitory
effect (69.3%) than B-CUR (21.7%) (Figure 3a). This firmly
establishes the strong inhibitory effect of CUR−PS-NPs on the
interactions between ACE2 and CoV2-SP.
Inhibition of the CoV2-SP and ACE2 interaction by CUR−
PS-NPs was further assessed in epithelial cells such as Huh7.5
(liver) and A549 (lung), exposed to 5 nM of CoV2-SP for 24 h
following the method given by Gasparello et al.
28
We analyzed
the expression of ACE2 by RT-PCR. The mRNA expression of
ACE2 was significantly decreased in CoV2-SP-exposed lung
epithelial cells, while the ACE2 level was enhanced significantly
in CUR−PS-NPs-treated Huh7.5 and A549 cells (Figure 3b,c).
This confirms that CoV2-SP induces inhibition of ACE2
expression in lung epithelial cells, and CUR-NS-NPs enhance
mRNA expression of ACE2.
7
These results further confirm
that the CUR−PS-NPs have significantly greater potential to
inhibit the interactions between human ACE2 receptor and
CoV2-SP than B-CUR and PSNPs.
Effects of CUR−PS-NPs on Cell Viability and Internal-
ization of CUR. In order to assess if the cell viability is a factor
for the observed inhibitory effect of CUR−PS-NPs on the
interaction between human ACE2 and CoV2-SP in epithelial
cells, we evaluated the effects of CUR−PS-NPs and B-CUR
(0.1, 0.2, 0.5, 5, and 10 μM) on cell viability by the Alamar
blue assay. After 24 h of exposure to CUR−PS-NPs and B-
CUR, we found no significant effect on the cell viability of both
liver Huh7.5 and lung A549 epithelial cells up to 5 μM
concentration. However, at 10 μM dose, the viability of cells
got reduced significantly (Figure 4a,b). We further confirmed if
5μM concentration produces non-lethal effects of CUR−PS-
NPs and B-CUR by FACS analysis and fluorescence
microscopy using Annexin V-FITC-PI in both Huh7.5 and
A549 cells. At 5 μM concentration, CUR−PS-NPs and B-CUR
did not reduce cell viability in both the cells (Figure 4c−e;
Figure S2a,b). Thus, we selected 5 μM concentrations of
CUR−PS-NPs and B-CUR for all subsequent cell-based
experiments. We prepared the stock (50 μM) of the CUR−
PS-NPs and B-CUR in water and ethanol solution (1:1 ratio),
and further diluted (1:10 ratio) it for all subsequent
experiments.
To test if the delivery of CUR into lung A549 epithelial cells
is enhanced using PSNPs, internalization of CUR from CUR-
PS-NPs was examined by fluorescence microscopy (FM) as
curcumin inherently yields green fluorescence under FM.
Greater internalization of CUR was noticed in A549 cells,
when exposed to 5 μM of CUR−PS-NPs for 2 h in comparison
to B-CUR (Figure S3). This confirms the superior
bioavailability of nano-CUR than B-CUR .
CUR−PS-NPs Inhibit CoV2-SP-Mediated Activation of
MAPK/NF-κB Axis. Patra et al. reported that CoV2-SP
exposure triggers the activation of MAPK and NF-κB signaling
in epithelial cells viz., Huh7.5 and A549 cells7. This was
Figure 2. Characterization of CUR−PS-NPs. (a) TEM image of
CUR−PS-NPs. Scale bar: 100 nm. (b) Measurement of size by TEM
and DLS, entrapment efficiency, PDI and zeta-potential of CUR−PS-
NPs. (c) Percent release kinetics of CUR from the CUR−PS-NPs.
Results are expressed as mean ±SEM of three individual experiments.
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concluded based on the higher expression of phosphorylated
p38 MAPK (Thr180/Tyr182) and p42/44 MAPK (Thr202/
Tyr204) proteins.
7
The elevated MAPK controls p65/NF-κB
activation for the production of cytokines.
29
Phosphorylation
of NF-κB (Ser276) and IκBαdegradation have been reported
to trigger transcriptional activation of nuclear p65/NF-κB.
30,31
In our study, we obtained the increased level of phosphory-
lated NF-κB (Ser276) and reduced IκBαlevel in Huh7.5 and
A549 cells at 5 nM concentration of CoV2-SP (Figure S2).
CUR−PS-NPs at the dose of 5 μM led to greater reduction in
CoV2-SP-induced phospho-p38 MAPK (Thr180/Tyr182),
phospho-p42/44 MAPK (Thr202/Tyr204), phosphorylation
of p65/NF-κB, and nuclear p65/NF-κB expression in both the
epithelial cells compared to B-CUR at similar dose (Figure
S4a,b). No significant effect of PSNPs on the levels of these
proteins was observed (data not shown). Several investigations
have concluded that CoV2-SP is a leading factor in the
increased cytokines−inflammatory reaction linked with
COVID-19 through activation of MAPK/NF-κB signaling.
Some clinical studies with COVID-19 patients have indicated
that administration of MAPK/NF-κB blocker medicines results
in less chance of hospitalization and admission to the intensive
care unit.
32,33
The findings of our study clearly indicate that
the CUR−PS-NPs can be used as potential inhibitors of CoV2-
SP-induced activation of a MAPK/NF-κB pathway.
CUR−PS-NPs Block CoV2-SP-Induced IL-6 and IL-8
Production. IL-6 and IL-8 are two important pro-inflamma-
tory cytokines which are linked to the development of chronic
inflammatory diseases.
34
The synthesis of these cytokines is
controlled via MAPK/NF-κB activation that plays a major role
in inducing a cytokine storm in COVID-19 patients.
28,35
To
establish the role of CUR−PS-NPs in blocking CoV2-SP-
induced IL-6 and IL-8 production, we first determined the
levels of IL-6 and IL-8 in the culture supernatant of Huh7.5
and A549 cells exposed to 5 nM of CoV2-SP for 24 h by
ELISA.
28
An increase in the extracellular IL-6 and IL-8 release
was observed in CoV2-SP-stimulated cells compared to
unstimulated cells (Figure 5a,b). The stimulated cells were
also used to isolate RNA for RT-PCR analysis. The elevated
levels of IL-6 and IL-8 were detected in CoV2-SP-stimulated
cells which was not the case with unstimulated cells (Figure
5c,d). When CUR−PS-NPs were applied to CoV2-SP-
stimulated cells, a significant inhibitory effect on IL-6 and
IL-8 levels was detected. Although B-CUR treatment also
caused reduction in IL-6 and IL-8 levels it was far less than that
of CUR−PS-NPs (Figure 5e−h). This confirms that the
inhibition of cytokines production was greater in CUR−PS-
NPs-treated Huh7.5 and A549 cells than those treated with B-
CUR. The PSNPs treatment alone had no significant
inhibitory effects on IL-6 and IL-8 release in CoV2-SP-
stimulated Huh7.5 and A549 cells. This suggests that the
inhibition of IL-6 and IL-8 levels was due to CUR only, not the
PSNPs per se. These data indicate that organic NPs of CUR
effectively inhibit cytokine production induced by CoV2-SP in
epithelial cells.
CUR−PS-NPs Regulate CoV2-SP-Induced Expression
of Cytokines, Chemokines, and Growth Factors.
Emerging evidences suggest that an excessive production of
Figure 3. Impact of CUR−PS-NPs, B-CUR and PSNPs on human ACE2-CoV2-SP. (a) Concentration dependent effects of CUR−PS-NPs, B-
CUR and PSNPs on interaction of ACE2 and CoV2-SP were assessed by ELISA and the results were presented as % inhibition. (b,c) Impact of 24
h treatment of different doses of CUR−PS-NPs and B-CUR on ACE2 mRNA expression in (b) Huh7.5 cells and (c) A549 cells was measured by
RT-qPCR analysis. Results are presented as mean ±SEM of six individual experiments. “*p< 0.05”,“**p< 0.01”and “***p< 0.001”for
unstimulated vs CoV2-SP-stimulated. “@p< 0.05”,“#p< 0.01”and “$p< 0.001”for CoV2-SP-stimulated vs CUR−PS-NPs/B-CUR treated.
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Figure 4. Impact of CUR−PS-NPs and B-CUR on cell viability and apoptosis. Cells were exposed to the indicated concentrations of CUR−PS-NPs
and B-CUR for 24 h and determined the cell viability using Alamar blue technique in (a) Huh7.5 cells and (b) A549 cells. (c) Detection of cell
viability and apoptosis in treated or untreated Huh7.5 cells and A549 cells by flow cytometry using FITC Annexin V and PI staining. Treated or
untreated (d) Huh7.5 cells and (e) A549 cells were also analyzed by fluorescence microscope for the detection of cell viability and apoptosis.
Results are expressed as mean ±SEM of three individual experiments. “*p< 0.05”for untreated vs CUR−PS-NPs/B-CUR-treated.
Figure 5. Effect of CUR−PS-NPs, B-CUR and PSNPs on CoV2-SP-induced IL-6 and IL-8 mediated storm in epithelial cells. Measurement of IL-6
and IL-8 protein release after 24 h exposure of 5 nM CoV2-SP to (a) Huh7.5 cells and (b) A549 cells. Quantification of IL-6 and IL-8 mRNA
expression after 24 h exposure of 5 nM CoV2-SP to (c) liver epithelial Huh7.5 cells and (d) lung A549 epithelial cells. Cells were exposed to 5 nM
CoV2-SP for 24 in the presence or absence of CUR−PS-NPs, B-CUR and PSNPs (5 μM). Measurement of IL-6 and IL-8 protein release by ELISA
in (e) Huh7.5 cells and (f) A549 cells. Quantification of IL-6 and IL-8 mRNA by RT-qPCR in (g) Huh7.5 cells and (h) A549 cells. Results are
presented as mean ±SEM of six individual experiments. (1) Unstimulated cells; (2) CoV2-SP-stimulated cells; (3) CoV2-SP-stimulated plus
CUR−PS-NPs (5 μM); (4) CoV2-SP-stimulated plus B-CUR (5 μM); (5) CoV2-SP-stimulated plus PSNPs (5 μM). “#p< 0.05”and “$p< 0.01”
for unstimulated vs CoV2-SP-stimulated. “*p< 0.05”,“**p< 0.01”and “***p< 0.001”for CoV2-SP-stimulated vs CUR−PS-NPs/B-CUR.
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circulatory biomarkers of inflammation including cytokines
(IL-1, IL-6, and IL-12), chemokines (CXCL8, MCP-1, and IP-
10), and growth factors (CCL3 and VEGF) is responsible for
the occurrence of ARDS in COVID-19 patients.
36
IP-10
(CXCL10) is also used as a key prognostic marker for SARS
disease development.
37
Although the levels of circulatory
VEGF remain high in SARS-CoV-2 infected patients, no
difference has been observed between severe and mild
patients.
38
We used ProcartaPlex analysis to examine the
effects of CUR−PS-NPs, B-CUR, and PSNPs (μM) on the
expression of inflammatory biomarkers (Table S3). Biomarkers
having more than 1 pg/mL concentration in the culture
medium of CoV2-SP-induced cells were considered for further
assessment. After incubation of 24 h, CoV2-SP-induced
Huh7.5 cells showed an elevated secretion of nine proteins
viz., IFNγ, IL-1β, IL-6, IL-8, CCL2 (MCP-1), CCL3 (MIP-
1α), CCL4 (MIP-1β), CCL5 (RANTES), and TNFα, while 14
proteins viz., CSF-3 (G-CSF), GM-CSF, IFNγ, IL-1β, IL-
12p70, IL-6, IL-8, IP-10 (CXCL10), CCL2, CCL3, CCL5,
TNFα, VEGF-A, and FGF-2 got elevated secretion in A549
cells (Figure 6a). In addition to IL-6 and IL-8, we found
greater inhibition in respect of these proteins in CUR−PS-
NPs-treated Huh7.5 cells (Figure 6b−j) and A549 cells
(Figure 6k−x) as compared to B-CUR and PSNPs-treated
cells, with an exception of CCL10, CCL5 and VEGF-A in
A549 cells. Treatment of CUR−PS-NPs, B-CUR and PSNPs
had no effect on unstimulated Huh7.5 cells (Figure S5a−i) and
A549 cells (Figure S6a−n). These results confirm that organic
NPs of CUR have a high potential to reduce COVID-19-
induced cytokine storm-related inflammation and organ
injuries, particularly the lungs and liver.
■CONCLUSIONS
We prepared the organic NPs of CUR using polysaccharide-
rich fraction of turmeric rhizome in this study, which inhibited
the cytokine storm induced by human CoV2-SP in liver
Huh7.5 and lung A549 epithelial cells. Treatment of CoV2-SP-
stimulated epithelial cells with CUR−PS-NPs potently
inhibited the release of cytokines, chemokines, and growth
factors that cause epithelial cell damage through deactivation
of NF-κB/MAPK signaling pathway. However, further in vivo
studies are required to confirm the potential of CUR-PS-NPsas
Figure 6. Impact of CUR−PS-NPs, B-CUR and PSNPs on the release of cytokines, chemokines and growth factors by CoV2-SP-stimulated
epithelial cells. (a) Profile of 37 cytokines, chemokines and growth factors in 24 h stimulation of Huh7.5 cells and A549 cells with 5 nM of CoV2-
SP. Released protein levels exceeding the content of 1 pg/mL in the culture medium are presented in the graph. The results are expressed as fold
change (CoV2-SP-stimulated cells vs untreated control cells). (b−j). Impacts of CUR−PS-NPs, B-CUR and PSNPs on the inflammation-related
cytokines, chemokines and growth factors induced by CoV2-SP in Huh7.5 cells. (k−x) Impacts of CUR−PS-NPs, B-CUR and PSNPs on the
inflammation-related cytokines, chemokines and growth induced by CoV2-SP in A549 cells. (1) Untreated and unstimulated cells; (2) CoV2-SP-
stimulated cells; (3) CoV2-SP-stimulated plus CUR−PS-NPs (5 μM); (4) CoV2-SP-stimulated plus B-CUR (5 μM); (5) CoV2-SP-stimulated plus
PSNPs (5 μM). Results are presented as mean ±SEM of six individual experiments. “*p< 0.05”,“**p< 0.01”and “***p< 0.001”for unstimulated
vs CoV2-SP-stimulated. “@p< 0.05”,“#p< 0.01”and “$p< 0.001”for CoV2-SP-stimulated vs CUR−PS-NPs/B-CUR.
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inhibitors of cytokine storm induced by CoV2-SP in liver and
lung epithelial cells.
■EXPERIMENTAL PROCEDURES
Materials. We bought CUR with more than 97% purity from
Sigma-Aldrich, St. Louis, MO, the USA. We obtained purified SARS-
CoV-2 spike recombinant glycoprotein and biotinylated recombinant
human ACE2 from Abcam (Cambridge, UK). All other analytical
grade reagents were used as received without additional purification.
Isolation and Characterization of PS-Rich Fraction. We
grinded the dried rhizomes of C. longa (250 g) which are collected
from Shillong, Meghalaya to make a coarse size powder and soaked it
in 500 mL of water for 60 min. After refluxing for 120 min, the
mixture was centrifuged at 8000 rpm for 30 min. We fractionated the
supernatant with ethyl acetate and n-butanol. The remaining water
layer was mixed properly with ethanol in the ratio of 1:1 and
centrifuged to obtain a precipitated material.
39
The yield of fraction
was 0.74%. The composition of fraction was also determined by gas
chromatography−mass spectrometry (GC−MS) equipped with a TR
50-MS capillary column (30 m ×0.32 mm) and flame ionization
detector (Thermo ScientificDSQIIGC−MS system) in a
temperature gradient of 100−280 °Cat10°C/min. The fraction
comprised D-glucose (49%), L-rhamnose (14%), D-galacturonic acid
(27%), L-arabinose (4%), and D-galactose (6%) (Table S1). The
fraction was hydrolyzed at 100 °C for using 2 M of sulphuric acid,
followed by acetylation as reported by Huang and colleagues.
40
Preparation and Characterization of CUR−PS-NPs. We used
an emulsion solvent evaporation technique to prepare CUR−PS-NPs
with minor changes.
23
For this, we dissolved CUR (50 mg) in acetone
(1.25 mL) and 250 mg of the fraction in dichloromethane (4 mL) and
mixed it together under stirring conditions at 250 rpm for 120 min.
We added polyvinyl alcohol solution (1%) to the mixture and stirred
for 8 h for removal of organic solvents. Afterward, centrifugation at
9500 rpm for 45 min under 4 °C was carried out. We re-suspended
pallets in water and centrifuged, and the process was repeated thrice.
Eventually, to achieve a solid dry powder, NPs were freeze-dried using
a lyophilizer (Labconco, USA) and stored at 4 °C under anhydrous
conditions for until use.
We recorded optical extinction spectra of CUR−PS-NPs by a UV−
Vis spectrophotometer (Evolution 201, Thermo, USA) with the help
of cuvettes (2 ×2 mm). We determined the hydrodynamic size and
zeta potential and PDI of CUR−PS-NPs by performing DLS analysis
using a Zetasizer system (MAL1010294 Malvern, UK). We calculated
the size and PDI from three individual analyses through intensity
distribution. We also examined the morphology and size distribution
of CUR−PS-NPs at 80 kV using carbon-coated copper grids by a
TEM (JEM-2100, JEOL).
Assessment of Interaction between Human ACE2 and
CoV2-SP. We assessed the interactions between human ACE2 and
CoV2-SP using an ELISA kit (Biosystems, USA), according the
manufacturer’s instructions. Briefly, we coated each well of the
microtiter plate (Genaxy Scientific, India) with CoV2-SP (25 ng) for
12 h, followed by careful three washings with phosphate-buffered
saline (PBS) (pH 7.2). We added different concentrations of CUR−
PS-NPs and B-CUR (5−25 μg/mL) to each well, followed by
addition of biotinylated recombinant human ACE2 (62.5 ng),
incubated at 37 °C for 30 min, and maintained a total volume of
100 μL ineach well. The sample without inhibitor was considered as a
negative control. For the detection of interactions between CoV2-SP
and ACE2, we added streptavidin-HRP (horse-radish peroxidase) and
peroxidise substrate (3.3′,5,5′-tetramethylbenzidine). We recorded
the absorbance at 450 nm using a Synergy/HTX microplate reader
(BioTek, Germany).
Cell Culture. We procured lung epithelial A549 cells and liver
epithelial Huh7.5 cells from American Type Culture Collection
(ATCC)-recognized cell repository at National Centre for Cell
Sciences, Pune, India. We cultured cells in a humidified atmosphere at
37 °C with 5% CO2in Dulbecco’s modified Eagle’s medium
(DMEM) (Gibco, Thermo Fisher Scientific) supplemented with
heat-inactivated fetal bovine serum (10%) (MP Biomedicals),
penicillin (100 U/mL), and streptomycin (100 mg/mL) (MP
Biomedicals).
Analysis of Cell Viability. We evaluated cell viability by the
Alamar blue assay, and FACS analysis and fluorescence microscopy
using Annexin V-FITC and PI that measured cell apoptosis in treated
or untreated epithelial cells.
41
After treatment of 24 h, 25 μL of the
Alamar blue dye (Thermo Fisher Scientific) was added to each well,
and cells were incubated for 2 h in CO2incubator at 37 °C. We
measured the absorbance at 570 and 600 nm using a Synergy/HTX
microplate reader (BioTek, Germany). For FACS analysis, trypsinized
cells were washed twice with 1×PBS. After addition of 100 μL
binding buffer (BF) containing 5 μL of Annexin V-FITC and PI, we
incubated cells in the dark for 15 min. We added 400 μL of BF to the
cells and analyzed using Attune NxT flow cytometry with Attune NxT
version 2.6 software (Thermo Fisher Scientific) and fluorescence
microscope (Leica DCF 700 T, Germany).
Stimulation of Epithelial Cells with CoV2-SP. We prepared a
stock solution of 7.2 μM CoV2-SP in urea (9%), Tris-HCl (0.32%;
pH 7.2) and 50% glycerol, and diluted in DMEM medium (200 μL)
to attain the final doses applied to treat epithelial cells as recently
reported by Gasparello et al.
28
We seeded cells (5 ×105cells/mL)
and incubated until 50% of confluence. Afterward, cells were exposed
with CoV2-SP (5 nM). To achieve maximum spike protein
interaction with the receptor, we incubated these cells for 30 min at
4°C as reported by Wang and colleagues.
42
Then, the final volume of
500 μL was made up by adding DMEM medium. We further
incubated cells at 37 °C for 24 h. We treated cells with DMSO
(Sigma-Aldrich, USA) for the consideration as unstimulated cells and
used these cells as reference controls.
RNA Extraction and RT-qPCR Reactions. Trypsinized cells
were washed thrice with 1×PBS. We isolated total RNA from
obtained cell pallets using an RNeasy mini kit (Qiagen, USA)
according to the manufacturer’s protocol. We washed the isolated
RNA with cold ethanol (75%) oncde, and after drying RNA pallets
were re-dissolved in nuclease-free water.
We prepared cDNA using a Verso complementary DNA (cDNA)
synthesis kit (Applied Biosystem, Thermo Fischer Scientific)
according to manufacturer’s instructions. We amplified 2 μLof
cDNA in the presence of a SYBR green PCR master mix (Thermo
Fisher, USA) and 800 nM primer for 40 cycles according to
manufacturer’s instructions using a real-time PCR system (7900HT;
Applied Biosystems, USA). We calculated relative expression of each
gene using the comparative cycle threshold ΔΔCtmethod. We used
β-actin as an internal reference control to normalize the gene
expression. No template cDNA as a negative control was also used in
each experiment to study specificity and to exclude contamination.
We carried out RT-qPCR experiments in triplicate for both target and
normalize genes. The gene-specific primers were designed using
Primer 3 version 0.4.0 and used for the amplification of target genes
(Table S2).
Statistical Analysis. The results are presented as mean ±
standard error of the mean (SEM). GraphPad Prism 8 was used to
analyze the data. Comparison among treatments was evaluated using
analysis of variances (ANOVA). Differences were defined with */@p<
0.05, **/#p< 0.01 and ***/$p< 0.001.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsabm.1c00874.
FT-IR spectra of PS fraction and CUR−PS-NPs, impact
of B-CUR on cell viability and apoptosis, internalization
of CUR−PS-NPs and B-CUR inside the lung A549
epithelial cells, impact of CUR−PS-NPs and B-CUR on
MAPK/NF-κB signaling in epithelial cells, effect of
CUR−PS-NPs and B-CUR on the release of cytokines,
chemokines and growth factors in liver epithelial Huh7.5
ACS Applied Bio Materials www.acsabm.org Article
https://doi.org/10.1021/acsabm.1c00874
ACS Appl. Bio Mater. 2022, 5, 483−491
489
cells, effect of CUR−PS-NPs and B-CUR on the release
of cytokines, chemokines and growth factors in lung
epithelial A549 cells, PS composition of isolated fraction,
list of primers, and list of used cytokines/chemokines/
growth factors and methodologies of immunoblot and
profiling of cytokines/chemokines/growth factors
(PDF)
■AUTHOR INFORMATION
Corresponding Authors
Brahma N. Singh −Pharmacology Division, CSIR-National
Botanical Research Institute, Lucknow 226001, India;
orcid.org/0000-0002-9296-1380;
Email: singhbrahmanand99@gmail.com
Saroj K. Barik −Pharmacology Division, CSIR-National
Botanical Research Institute, Lucknow 226001, India;
Email: sarojkbarik@gmail.com
Authors
Vivek K. Sharma −Pharmacology Division, CSIR-National
Botanical Research Institute, Lucknow 226001, India
Prateeksha −Pharmacology Division, CSIR-National
Botanical Research Institute, Lucknow 226001, India
Shailendra P. Singh −Department of Botany, Banaras Hindu
University, Varanasi 221005, India
Chandana V. Rao −Pharmacology Division, CSIR-National
Botanical Research Institute, Lucknow 226001, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsabm.1c00874
Author Contributions
§
Vivek K. Sharma and Prateeksha contributed equally. Vivek K.
Sharma: Performed experiments, data analysis. Prateeksha:
Performed experiments, Ddata analysis. Sailendra P. Singh:
Writing. Brahma N. Singh. Conceptualization, funding, friting
−feview and editing. Chandana V. Rao: Data analysis. Saroj K.
Barik: Conceptualization, funding, writing review and editing.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This work was supported by Council of Scientificand
Industrial Research (CSIR), New Delhi, India under in-
house project OLP-0106 and Department of Biotechnology
(DBT), New Delhi, India project No. BT/01/17/ 835 NE/
TAX.
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