Inhibition Effects of Patchouli Alcohol, Carvacrol, p-Cymene, Eucalyptol and Their Formulations Against Influenza Virus Pneumonia Through TLR4/NF-κB/NLRP3 Signaling Pathway

Abstract

As a kind of drug mostly used historically to treat epidemics, aromatic botanicals have volatile oils as active components. The study aims to evaluate the anti-influenza viral pneumonia effects of volatile monomers patchouli alcohol (PA), carvacrol (CV), p-Cymene (PC), eucalyptol (EC) and their formulations from various aspects through the influenza virus A/PR/8/34 (H1N1) infection experiment in vivo and in vitro and carry out in-depth studies on the anti-inflammatory mechanisms. In this study, we found that all four volatile monomers mentioned above could exert antiviral effects by suppressing pulmonary viral load and lung index and improving lung lesions in mice with influenza pneumonia. In addition, elevated levels of cytokines and chemokines in the serum were suppressed, the proportion of T-lymphocytes in the peripheral blood was altered, and antioxidative stress indices were improved, whose mechanism of action related to anti-inflammation, possibly acting on the Toll-Like Receptor 4/Nuclear Factor-κB/nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (TLR4/NF-κB/NLRP3) pathway. The study provides an experimental basis for volatile monomers and their formulations of aromatic herbs for treating influenza virus pneumonia.

Keywords: TLR4/NF‐κB/NLRP3 signaling pathway; antiviral activity; carvacrol; eucalyptol; inflammation; influenza virus type a; patchouli alcohol; p‐cymene.

FIGURE 1

The scheme of exploring the anti‐influenza virus effect mechanisms of patchouli alcohol (PA), carvacrol (CV), p‐Cymene (PC), eucalyptol (EC) and their formulations. The experiments in vitro demonstrated the administration time and operation steps of cytotoxicity and antiviral assays, while the experiments in vivo showed the administration days and sampling nodes of mice, as well as the categories of detection indicators.

FIGURE 2

In vitro assays. Cytotoxicity and antiviral activity of (a) Oseltamivir (positive control drug), (b) PA, (c) CV, (d) PC and (e) EC in Madin‐Darby canine kidney (MDCK) cells (n = 3).

FIGURE 3

Survival test results of monomers. (a) Effect of different groups on the survival rate of H1N1‐infected mice (n = 10). (b) Average body weight changes of mice among different groups during the survival test.

FIGURE 4

Effects of PA, CV, PC and EC on lung in H1N1‐infected mice. (a) Gross macroscopic examination of lungs in different groups. Changes of (b) lung index (n = 10) and (c) relative expression of Influenza Virus A nucleoprotein (IVA‐NP) mRNA (n = 3) in lung tissue among groups. (d) Pathological changes of lung tissues following H&E staining. The bright yellow circle shows pulmonary hemorrhage. The blue circle shows the dilated and congested blood vessels. The dark yellow circle shows a pink transparent film formed by serous exudation. The orange circle shows normal alveoli. The purple circle shows collapsing alveoli. The purple arrow points to the exudation of inflammatory cells. The green circle shows collapsed alveoli. Data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the control group.

FIGURE 5

Effects of PA, CV, PC, and EC on levels of cytokine/chemokine and the expression of the T lymphocyte immunophenotype in blood in H1N1‐infected mice. Levels of (a) Tumor Necrosis Factor‐α (TNF‐α), (b) Interferon‐γ (IFN‐γ), (c) Interleukin‐6 (IL‐6), (d) Interferon‐inducible Protein‐10 (IP‐10), (e) Monocyte Chemoattractant Protein‐1 (MCP‐1), (f) Macrophage Inflammatory Protein‐1α (MIP‐1α) in serum were determined using ELISA kits (n = 7). Changs in (g) CD3⁺CD4⁺/CD3⁺CD8⁺, the percentage of (h) CD3⁺CD4⁺, (i) CD3⁺CD8⁺ and (j) CD3⁺ in blood between groups detected by flow cytometry assay (n = 3). Data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the control group.

FIGURE 6

In vitro assays. Cytotoxicity and antiviral activity of (a) the formulation of PA, EC, and CV (PEC), (b) the formulation of PA, EC, CV, and PC (PECP) in MDCK cells (n = 3).

FIGURE 7

Survival test results of formulations. (a) Effect of different groups on the survival rate of H1N1‐infected mice (n = 10). (b) Average body weight changes of mice among different groups during the survival test.

FIGURE 8

Effects of PEC and PECP on lung in H1N1‐infected mice. (a) Gross macroscopic examination of lungs in different groups. Changes of (b) lung index (n = 10) and (c) relative expression of IVA‐NP mRNA (n = 3) in lung among groups. (d) Pathological changes of lung tissues in mice following H&E staining. Data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the control group.

FIGURE 9

Effects of PECP on levels of cytokine/chemokine and the expression of the T lymphocyte immunophenotype in blood in H1N1‐infected mice. Levels of (a) TNF‐α, (b) IFN‐γ, (c) IL‐6, (d) IP‐10, (e) MCP‐1, (f) MIP‐1α in serum were determined using ELISA kits (n = 7). Changs in (g) CD3⁺CD4⁺/CD3⁺CD8⁺ and the percentage of (h) CD3⁺CD4⁺, (i) CD3⁺CD8⁺ and (j) CD3⁺ in blood between groups were detected by flow cytometry assay (n = 3). Data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the control group.

FIGURE 10

Effects of PA, CV, PC, EC, and PECP on antioxygenic ability and protein expression of TLR4/NF‐κB/NLRP3 signaling pathway in lung tissue in H1N1‐infected mice. Levels of (a) total superoxide dismutase (T‐SOD), (b) glutathione peroxidase (GSH‐Px), (c) Malondialdehyde (MDA), (d) reactive oxygen species (ROS) in lung tissue were determined using ELISA kits (n = 7). (e) Protein expression of TLR4, MyD88, p‐NF‐κB p65, NF‐κB p65, NLRP3, Caspase1, ASC in lung tissue determined by western blotting (n = 3). Quantification of (f) TLR4, (g) MyD88, (i) NLRP3, (j) Caspase1, (k) ASC protein expression relative to GAPDH. Quantification of (h) p‐NF‐κB p65 protein expression relative to NF‐κB p65 (n = 3). Data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus the model group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the control group.