| Objective:To explore the inhibitory effect of geniposide on the influenza A (H1N1) virus-induced cytopathic effects (CPE) in Madin-Darby Canine Kidney (MDCK) cells; and to investigate the suppression effects of geniposide on airway inflammation and virus replication in a murine model of influenza A (H1N1) virus-associated pneumonia.Methods:In vitro, geniposide was administrated as a precaution drug, a direct deactivation drug or a treatment drug at different doses. Peramivir was applied as a positive control.The MTT assay was applied to test both the cytotoxicity of geniposide on MDCK cells and the cytopathogenic effect (CPE) of geniposide on MDCK cells infected by Influenza A/Jiangsu/1/2009(NT0901). The inhibition rates of geniposide on NT0901 were also calculated. In vivo, six to eight weeks old female ICR mice were randomly divided into seven groups(n=10):placebo group, NT0901 group, geniposide treatment(5,10, or 20 mg/kg) group, peramivir treatment(30 mg/kg) group and combined treantment group(20 mg/kg geniposide+30 mg/kg peramivir). On Day 0, the mice were intranasal administrated with NT0901 to induce lung injury after anesthetized with diethyl ether, while sterile normal saline was nasally instilled instead in placebo group. From Day 1, mice in drug-treated groups were intraperitoneally(i.p.) administrated with geniposide, peramivir or combination respectively twice a week, as the ones in placebo group and NT0901 control group with sterile normal saline instead. The general condition, body weight and survival were observed daily. Lung tissues were collected at different time points. The lung index and survival rate were calculated. Paraffin sections of lung tissues were stained with Haematoxylin and eosin(H&E) to access the pathological alterations. Histological immunostaining was performed to evaluate nucleoprotein(NP) of NT0901 in lung tissues and the positive stained area was compared by the software of Image pro plus 6.0. Sandwich enzyme-linked immunosorbent assay(ELISA) was used to measure the concentrations of tumour necrosis factor-a(TNF-a), interferon-y(IFN-y), interleukin-4(IL-4), interleukin-6(IL-6) and interleukin-10(IL-10) in the supernatants of the lung tissue homogenates.Results:The results showed that the TDso(median toxic dose) of geniposide in MDCK cells was higher than 1040 μmol/L. Meanwhile, the ECso(concentration for 50% of maximal effect) of geniposide for pretreatment, simultaneous treatment or post-treatment in NT0901-infected MDCK cells were 91.90,96.25,87.68 μmol/L respectively. Besides, geniposide delivered in different ways performed significant inhibitory effect on virus-induced CPE in MDCK cell in a dose-dependent way(P<0.05). The murine model of influenza A (H1N1) virus-associated ALI was successfully established. The lung index and virus titration of mice treated with geniposide significantly declined compared with NT0901 group(P<0.05). The virus-infected lungs were typically characterized by interstitial pneumonia, including intact structures damage, congestion and oedema of bronchia wall, bronchiole wall and their surrounding tissues and the interlobular septum as well as the infiltration of abundant inflammatory cells, which could be alleviated by geniposide(20 mg/kg). The area stained by nucleoprotein(NP) antibody was reduced by geniposide(10 and 20 mg/kg) treatment compared with NT0901 group(P<0.05). In addition, levels of TNF-a, IFN-y, and IL-6 were also markedly decreased in geniposide-treated groups compared with NT0901 group(P<0.05), while IL-4 and IL-10 elevated significantly(P<0.05).Conclusions:Our investigation suggested that geniposide effectively inhibited pandemic A/Jiangsu/1/2009 (H1N1) influenza virus-induced CPE in MDCK cells. Furthermore, geniposide mitigated virus-induced acute inflammation, reduced virus titration and protected lung tissue from virus-medicated injury in vivo. |
PDF Full Text Request