Background
Influenza A virus (IAV) is a respiratory tract pathogen that causes a high number of deaths and hospitalizations, including approximately 49,000 deaths and up to 35,600,000 morbidity cases annually in the USA alone [
1,
2]. IAV recruits host cell machinery to support their replication and transportation inside the cell [
3,
4]. In this regard, targeting cellular proteins in virus-host interaction pathways could be effective against influenza infection. The benefit of this approach is to reduce virus drug resistance. However, this strategy requires more understanding of the intracellular pathways that influenza A virus uses to replicate [
3].
Several studies have suggested an association between inflammation and severe cases of IAV infection [
5‐
7]. The host inflammatory response following influenza virus infection presents the host cells and the immune system with somewhat overwhelming effects. Host inflammatory response is a key factor in controlling virus proliferation but is also associated with lung damage, morbidity and death in the case of overwhelming inflammation [
8]. Uncontrolled and exacerbated response to the virus may be associated with intense lung injury and death [
9,
10].
In view of the foregoing, a growing number of studies have suggested that immunomodulatory strategies may improve disease outcome without affecting the ability of the host to deal with infection [
11,
12]. Herbal medications have equally attracted tremendous attention [
13,
14], as complementary therapies and preventive medicine [
15‐
18].
Rho GTPase molecules, which can be modified post-translationally, control a wide variety of signal transduction pathways. They can affect cell polarity, microtubule dynamics, membrane transport and transcription factor activities [
19]. They are well-known proteins to regulate the intracellular signal transducers [
20‐
22]. RhoA is one of the best-studied members of this family which is widely documented as a key regulator of cytoskeletal dynamics reorganization on membrane trafficking [
23,
24]. Increasing evidence suggests a link between Rho proteins and apoptosis [
25‐
27]. Several studies have also focused on the therapeutic effects of flavonoid compounds through Rho proteins expression and apoptosis pathway [
28‐
30]. Different types of flavonoids have been identified as antiviral agents [
31‐
34]. The reports of antiviral activity of flavonoids and derivatives against several viral infections through different mechanisms are increasing such as Adenovirus, Herpes Simplex Virus, Japanese Encephalitis Virus, Respiratory Syncytial Virus, HIV-1, Dengue Virus, Hepatitis C Virus and Zika Virus [
35‐
41]. The potent antiviral effect of flavonoids against influenza virus infection [
42‐
44] and immunomodulatory effects of flavonoids in different viral infections [
45‐
47] have been reported as well.
Quercetin from the flavonoid group of plant compounds has been studied in small clinical trials [
48]. There are limited studies on immunomodulatory effects of quercetin on influenza infection. One such study indicated the inhibitory activity of quercetin on influenza infection in the early stage of entry [
49]. Q3R from
Houttuynia cordata demonstrated strong anti-influenza A/WS/33 virus activity, reducing the formation of visible CPE, and inhibited virus replication in the initial stage of virus infection [
50].
The biological activity of flavonoids depends on the configuration, the total number of hydroxyl groups, and substitution of functional groups about their nuclear structure [
34].
Quercetin belongs to the class called flavonols that cannot be produced in the human body but only in plant material and products [
51]. It is one of the important flavonoid compounds isolated from more than twenty plant material from USA, Europe, and eastern countries which is known for its different properties especially anti-inflammatory activities [
52,
53]. We also reported quercetin isolation from
Rapanea melanophloeos (Myrsinaceae family) an indigenous South African plant for the first time [
54].
In continuation of our previous study [
54], this research was designed to confirm and reveal the additional immunomodulatory activity of Q3R and its effect on the apoptosis pathway, in controlling influenza infection. Computational molecular docking was also performed to screen the potential binding ability of Q3R with neuraminidase/hemagglutinin glycoproteins and M2 transmembrane from H1N1, and Human RhoA.
Discussion
The modification and manupulation of cytokines production is a critical step in influenza pathogenesis, which can recruit a variety of innate immune cells [
59]. In this study, in completing our previous study [
54,
60], interleukin-6 (IL-6) and chemokine C-C motif ligand 2 (CCL-2) from the category of pro-inflammatory cytokines and interferon-β (IFN-β) from the category of anti-inflammatory cytokines were tested at the genome and protein levels.
The cytokine production was affected by quercetin during influenza course. Q3R was able to significantly decrease the IL-6 production to 0.0004, 0.0202 and 0.0733 fold in co-, pre- and post-penetration treatments, respectively at the genome level (Fig.
1). This decrement was observed in protein level as well. However, the co-penetration treatment showed more elimination to − 76.66% (Table
2). This cytokine is highly correlated with high body temperature during influenza illnesses and infection [
61,
62], and strong up-regulation of this cytokine can predict the severity of the infection [
63]. Consequently, Q3R has a high potential to decrease the fever and severity of influenza illnesses by regulating the excessive innate inflammatory reaction.
Chemokine (C-C motif) ligand 2 (CCL-2) was one of the affected chemokines evaluated in this study. This chemokine recruits memory T cells, exudate macrophages (exMACs) and monocytes to the site of the infection [
60,
63,
64]. Attraction of these CCR2
+ inflammatory cells to the site would lead to naive T cell proliferation, NO synthesis 2 (NOS2) and TNF-α production [
63]; however, the extreme recruitment of these cells causes extra cytokine production and apoptosis induction by tumor necrosis factor-related apoptosis inducing legend (TRAIL) activation [
65]. In the children with deadly acute encephalopathy associated with IAV, high concentrations of CCL-2 and CSF have been reported [
66]. Decrement in CCL-2 and mononuclear cells in the infection site can effectively control the virus infection by improving the condition without affecting the CD8
+ T cell expansion [
67]. In the current study, Q3R showed the ability to significantly decrease the CCL-2 protein production and gene expression especially in the co-penetration treatment to − 52.488% (Table
2) and 5.2221 fold (Fig.
1), respectively compared to H1N1 inoculation which can decrease the mortality associated with the highly pathogenic influenza viruses.
IFN-β is a type I interferon and inhibitor of inflammation [
68]. It can shift the cytokine networks in favor of anti-inflammatory effects [
69]. In the study of protein level, H1N1 showed a mild increase in IFN-β protein compared to the negative control, however, no significant difference was observed at the protein level between H1N1 inoculation and combined treatments. The gene expression of IFN-β decreased in H1N1 sample by 0.0004 fold but in all combined treatments it showed increments at the same level (Fig.
1). This can be referred to excessive intracellular viral NS1 protein that prevents the induction of beta interferon [
70]. In addition, referred to our previous study which showed elevated levels in TNF-α production in H1N1 sample and its significant decrement when subjected to Q3R [
54], it is reported that increase of TNF-α can inhibit the production of IFN α/β [
54]. This outcome might be the reason for this effect of Q3R which shows that Q3R does not act through IFNs.
Thus, the compound Q3R could interrupt the effect of the influenza virus on the cytokines which could decrease pro-inflammatory and increase anti-inflammatory cytokines.
It has been studied that active RhoA prevents endothelial apoptosis. Therefore, inhibition of Rho in endothelial cells not only reduced the expression of anti-apoptotic Bcl-2 and Mcl-1 and increased proapoptotic Bid protein levels, but also, activated caspase-9- and − 3-dependent programmed cell death (apoptosis) [
25]. A recent study highlighted that RhoA might be important for the proliferation and apoptosis in lung cancer cells. Alterations in caspase-3 may be the underlying molecular mechanisms associated with the effect of RhoA on cell proliferation and apoptosis [
71].
Quercetin is a flavanol compound occurring naturally in plant materials. It increased the expressions of RhoA and Rho-associated, coiled-coil containing protein kinase-1 (ROCK1), but inhibited the expression of NF-κB p65 in SAS Human oral cancer cells. This role of quercetin was mentioned to be associated with the down-regulation of PKC and RhoA by blocking MAPK and PI3K/AKT signaling pathways and NF-κB [
29]. Rho GTPase-mediated mechanisms of isoquercitrin were tested in a study. It was shown that RhoA underwent translocation to the cytoplasm upon treatment with isoquercitrin. Thus, it affected RhoA localization preventing the translocation to the plasma membrane [
28]. Flavonoids have also shown protection against apoptosis on neurons involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3 [
30].
Different quercetin derivatives may play different roles in signaling pathways depending on the type of cell or disease. The effect of quercetin-3-O-(2″-galloyl)-α-l-rhamnopyranoside (QGR) was investigated on TRAIL-induced apoptosis in human keratinocytes. Treatment with QGR prevented TRAIL-induced apoptosis-related protein activation, but quercetin had an additive effect on TRAIL-induced apoptosis-related protein activation and cell death [
72]. In a recent study, quercetin-3-O-α-L-rhamnopyranoside (Q3R) decreased the oxidative stress in human umbilical vein endothelial cells (HUVECs) by promoting the nuclear transfer of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 by activating autophagy. This study supported the use of quercetin-3-O-α-L-rhamnopyranoside (Q3R) as a health supplement to alleviate oxidative stress [
73].
In regards to apoptosis pathway and RhoA and apoptosis interaction [
25], it was found in this study that in combination treatment of Q3R with H1N1, RhoA expression decreased and caspase-3 activity increased compared to the H1N1 sample. Caspase-3, a member of the interleukin-1β converting enzyme (ICE) family of cysteine proteases, is one of the principal caspases found in the apoptotic cells. The highest values of caspase-3 activity in Q3R sample compared to H1N1 correlates with the RhoA decrement in Q3R treatment. And intermediate value of caspase-3 activity in combination treatment of Q3R with H1N1 was indicative of regulatory role of Q3R on RhoA activity against viral molecular pathway.
The molecular docking results showed strong binding ability of Q3R with M2 transmembrane, Neuraminidase of 2009 pandemic H1N1, N1 neuraminidase, PR/8/1934 Human strain H1 and Human RhoA, with docking energy of − 10.81, − 10.47, − 9.52, − 9.24 and − 8.78 Kcal/mol, respectively. Regarding the docking study, there are three essential types of interactions with the enzyme active site including hydrogen bond, Pi-Pi staking, and hydrophobic interactions. The Q3R showed strong interactions with M2 transmembrane (2KQT) residues, namely with ALA A30, ILE B33, VAL D27, VAL C27, ALA D30, phenolic and sugar moieties (Fig.
5a). The Q3R showed several hydrogen-bonding interactions with 3TI6 receptors, namely with ASP151, ASN347, and ILE149, together with Pi-Pi staking with ILE149, ILE427, PRO431, TRP403 and TYR406 from phenolic and sugar moieties. This implies that the presence of both parts is essential for high affinity with ligand and receptor.
Conclusions
In conclusion, while our previous studies showed the safety effects of Q3R over amantadine and oseltamivir, and also supporting data from the current study, in vitro evaluation of the consequences of Q3R revealed this natural compound has the potential to alleviate influenza infection by modulating the inflammatory response, affecting the apoptosis pathway and efficiently improve the outcome of the influenza disease.
Further details on the docking results showed that the structural features of Q3R might be helpful for further drug design and development. We concluded that Q3R can inhibit the virus affecting the virus penetration/adsorption directly. Correspondingly, it showed the ability to indirectly dominate the severity of the disease by changing the cytokines pattern and affecting the apoptosis pathway. Thus, the concurrent application of quercetin-3-O-α-L-rhamnopyranoside with influenza A virus infection is highly effective in influenza infection modulation. Therefore, understanding and targeting the cellular proteins and intracellular pathways required for influenza replication are valuable and beneficial to prevent or treat this infection more efficiently. Further in vivo evaluation can also assist in understanding the benefits of quercetin against influenza disease in a perceptible way.
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