The Modified JiuWei QiangHuo Decoction Alleviated Severe Lung Injury Induced by H1N1 Influenza Virus by Regulating the NF- κ B Pathway in Mice

Abstract

A new approach to treat infections of highly pathogenic influenza virus is to inhibit excessive innate immune response. JiuWei QiangHuo decoction has been used for centuries for the treatment of pulmonary disorders in China. In this study, we evaluated the anti-inflammatory activities of the modified JiuWei QiangHuo (MJWQH) decoction in the treatment of influenza A (H1N1) virus-induced severe pneumonia in mice. The results showed that MJWQH significantly increased the survival rate of H1N1-infected mice and suppressed the production of TNF-α, IL-1, IL-6, MCP-1, RANTES, and IFN-α on day 4 after infection. Moreover, oral administration of MJWQH efficiently inhibited virus replication and alleviated the severity of lung injuries. The results also showed that MJWQH may have potential therapeutic effect on severe lung injury induced by H1N1 virus by regulating the NF-κB pathway. Our study suggested that MJWQH might be an alternative therapy for the treatment of viral pneumonia.

Figure 1

HPLC chromatogram of MJWQH (280 nm). Calycosin-7-O-β-D-glucopyranoside (1) and baicalin (2) were separated from MJWQH at a retention time of 31.6 and 43.4 min, respectively.

Figure 2

Experimental protocol. All BALB/c mice except the NC ones were challenged intranasally with influenza A/FM/1/47 (H1N1) virus (10 LD₅₀ per mouse) and then treated with 0.5% CMC solution (model control, MC), MJWQH, or Rib one hour later for 2, 4, or 7 days, respectively.

Figure 3

MJWQH treatment protected mice from lethal influenza challenge. BALB/c mice (n = 12 mice/group) were treated as described in Figure 2 for 7 days and monitored daily for signs and symptoms, body weight, and survival for 14 consecutive days. (a) Body weight (means ± SEM); (b) survival (number of survivors/total number of mice).  ^** P < 0.01 versus the MC group.

Figure 4

MJWQH alleviated the severity of H1N1-induced lung injuries. BALB/c mice (n = 6 mice/group) were treated as described in Figure 2 for 4 days. (a) Macroscopic appearance of lungs; (b) pathological changes of lung tissues (HE, ×200); (c) pathological scores; and (d) lung index. Data were presented as mean ± SD.  ^** P < 0.01 versus the MC group.

Figure 5

MJWQH inhibited virus replication. BALB/c mice (n = 6 mice/group) were treated as described in Figure 2 for 4 days. (a) HA titers of lung homogenates; (b) relative quantitation of influenza A virus. Data were presented as mean ± SD.  ^* P < 0.05;  ^** P < 0.01 versus the MC group.

Figure 6

MJWQH suppressed innate immune responses. Proinflammatory cytokine and chemokine levels were analyzed in lung homogenates of mice on day 4 after infection by ELISA. Data were presented as mean ± SD.  ^* P < 0.05;  ^** P < 0.01 versus the MC group.

Figure 7

MJWQH inhibited the NF-κB signaling pathway. BALB/c mice (n = 6 mice/group) were treated as described in Figure 2 and killed on day 2 or 4 after infection. Cytosolic fractions and nuclear extracts were prepared from lung homogenates. NF-κB p65, IκB-α, TLR7, MyD88, and TRAF6 levels were detected by specific antibodies in cytosolic fractions using Western blot analysis. Phosphorylation of IKKα/β and p65 was determined in the nuclear extracts. GAPDH protein was used as a loading control.

Figure 8

MJWQH inhibited the NF-κB signaling pathway. BALB/c mice (n = 6 mice/group) were treated as described in Figure 2 and killed on day 4 after infection. Lungs were processed for total RNA and subjected to real-time PCR for detection of TLR7, MyD88, and NF-κB p65 gene expressions, using GAPDH as a housekeeping gene. Data were presented as mean ± SD.  ^* P < 0.05;  ^** P < 0.01 versus the MC group.

Figure 9

MJWQH inhibited the NF-κB expression in lung tissue (×200). BALB/c mice (n = 6 mice/group) were treated as described in Figure 2 and killed on day 4 after infection. Lung sections were stained with rabbit anti-p65 (brown) and counterstained with hematoxylin.