Effects of different principles of Traditional Chinese Medicine treatment on TLR7/NF-κB signaling pathway in influenza virus infected mice

Abstract

Background: Influenza virus is a single-stranded RNA virus that causes influenza in humans and animals. About 600 million people around the world suffer from influenza every year. Upon recognizing viral RNA molecules, TLR7 (Toll-like receptor) initiates corresponding immune responses. Traditional Chinese Medicines (TCMs), including Yinqiao powder, Xinjiaxiangruyin and Guizhi-and-Mahuang decoction, have been extensively applied in clinical treatment of influenza. Although the therapeutic efficacy of TCMs against influenza virus in vivo was reported previously, its underlying mechanisms are not clearly understood. This study aimed to investigate the immunological mechanisms in the treatment of influenza virus infected mice with three Chinese herbal compounds as well as the effect on TLR7/NF-κB signaling pathway during recovery.

Methods: Wild type and TLR7 KO C57BL/6 mice were infected with influenza virus FM1 and then treated with three TCMs. The physical parameters of mice (body weight and lung index) and the expression levels of components in TLR7/NF-κB signaling pathway were evaluated.

Results: After viral infection, Guizhi-and-Mahuang decoction and Yinqiao powder showed better anti-viral effect under normal condition. Compared to the viral control group, expression levels of TLR7, MyD88, IRAK4 and NF-κB were significantly reduced in all treatment groups. Furthermore, the three TCM treatment groups showed poor therapeutic efficacy and no difference in viral load compared to the viral control group in TLR7 KO mice.

Conclusion: Our study indicated that Guizhi-and-Mahuang decoction and Yinqiao powder might play a crucial role of anti-influenza virus by regulating TLR7/NF-κB signal pathway.

Keywords: Guizhi-and-Mahuang decoction; Influenza virus; TLR7 gene knockout; TLR7/NF-κB signaling pathway; Traditional Chinese Medicine; Xinjiaxiangruyin; Yinqiao powder.

Fig. 1

The fingerprints of TCMs. a The fingerprint of Yinqiao powder; peak number and identity, 1: 3,4-dihydroxybenzoic acid; 2: chlorogenic acid; 3: liquiritin; 4: forsythin; 5: arctiin. b The fingerprint of Xinjiaxiangruyin; peak number and identity, 1: chlorogenic acid; 2: forsythin; 3: thymol; 4: magnolol. c The fingerprint of Guizhi-and-Mahuang decoction; peak number and identity, 1: ephedrine hydrochloride; 2: amygdalin; 3: paeoniflorin; 4: liquiritin

Fig. 2

Effects of three TCM compounds on body weight loss and lung index of mice infected with influenza virus. C57BL/6 wild type (a) and TLR7 KO (b) mice were infected with 50 μL FM1 virus and then were gavaged with distilled water, containing Oseltamivir, Xinjiaxiangruyin, Guizhi-and-Mahuang decoction or Yinqiao powder daily 24 h after virus infection, respectively. Changes in body weight of each mouse were recorded. Each group was compared to the model group. c The body weight of infected mice and blank were monitored every day for 6 days. The mice were sacrifice on day 6, and the total lung were completely removed and weighed. Lung index = lung weight/body weight × 100%. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

The influenza FM1 viral load in the lung tissue of wild type and TLR7 KO mice infected with influenza virus on day 6. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Lung histopathology in wild type and TLR7 KO mice infected with influenza FM1. The lungs were collected on day 6 postinfection, and sections were prepared for histopathological analysis. a, b Indicate C57BL/6 wild mice and TLR7 KO mice, respectively. This figure shows representative results of experiments with ten mice in each group. Bars = 100 μm

Fig. 5

Profiling of Th17 cells and Treg cells in splenocytes of mice. C57/6 wild type mice (n = 10/group) and TLR7 KO mice (n = 10/group) were infected with 50 μL of influenza FM1 virus. Splenocytes were isolated from 3 mice out of each group on day 6 post-infection and Th17 cell and Treg cell response were assayed in wild type mice (a) and TLR7 KO mice (b). Data are presented as the mean ± SEM. Th17 cell response was determined by intracellular CD4, IL-17 staining. Treg cell response was determined by intracellular CD4, CD25 and Foxp3 staining. Data are presented as representative density plots in wild type mice (c) and TLR7 KO mice (d). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

The effect of different drugs on the mRNA expression of TLR7 and NF-κB signaling pathway in lung tissue of influenza virus infected mice. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

The effect of different drugs on the protein expression of TLR7 and NF-κB signaling pathway in lung tissue of influenza virus infected mice. a Proteins were evaluated by western blotting assay. b Quantification of TLR7, MyD88, IRAK4, NF-κB protein was detected by densitometric analysis. *P < 0.05, **P < 0.01, ***P < 0.001