Study On The Efficacy And Mechanism Of Lianhua Qingwen Granule Against Influenza Virus And Regulating Related Immune Response

Objective:Lianhuaqingwen granule (LH-G) is a novel prescription composed of Chinese medicinal herb such as zhimahuang, jinyinhua, bohe, guanzhong, hongjingtian, shigao and guanghuoxiang. It commonly used in the prevention and treatment disease related to respiratory tract infection (RTI).Lianhuaqingwen Capsule (LH-C), which extend from two TCM prescriptions that are used as a Chinese medicine in treating regular seasonal influenza. Yinqiao powders comes from Wen Bing Tiao Bian in Qing dynasty, is used in treating onset of Wen-Bing, Another component of LH-C, Maxing Shigan decoction comes from Shang Han Lun in Han dynasty is used in against plague. Previous studies have shown that LH-C could reduce time to fever resolution in patients with H1N1 influenza virus infection and effective reduce the duration of viral shedding [15,16]. In the present study, we addressed the effect of LH-C on different influenza virus strain including HPAI A(H7N9) virus and further addressed the impact of LH-C on influenza A(PR/8) virus inoculated cell line and BALB/c mice, with particular focus on cytokine production, inflammatory damage and viral titer.Method:1. We used using MTT and plaque reduction method to investigate the antiviral activity of LH-G on different type of influenza virus, and its pharmacodynamic effects in different mode of administration.2. Time of addition studies used single cycle virus growth conditions was used to determine which stage of virus replication was blocked,3. Hemagglutination inhibition assay, Hemolysis inhibition assay and sialic-acid inhibition assay are performed to explore the inhibition mechanism of LH-G in the early phase of virus replication cycle.4. LH-G’s effects on regulates different signaling transductions was evaluated by Western blotting assay.5. LH-G’s ability to regulates cytokine/chemokine expression was evaluated by qRT-PC.6. We performed western blotting assay to examined the activity of LH-G in inhibit of cell apoptosis7. Experiment in mice model was performed to identify the antiviral activity of LH-G in vivo.7.1. Groups of mice were orally administered with LH-G in different concerntration for 5 days after Influenza challenge. The weight and survival data were record after infection.7.2. At 2,4,6 and 8 day post infection, mice lungs were collected from infected mice for detection of virus titer.7.3. We examined the level of cytokines in the lung of infected mice at 4,6 and 8 day post infection by means of end-point titration and real-time RT-PCR for mRNA expression of cytokines expression.7.4. Histopathological analysis was also performed at the same time points.Results:1. LH-C exhibited inhibitory activities against several influenza viruses from different human isolates and avian influenza viruses with 50% inhibitory concentration (IC50) ranging from 0.35 to 2 mg/mL. And selective index (SI) ranging from 1.56 to 15.6. LH-C showed the best inhibitory effect in A/Aichi/2/68(H3N2) and less inhibitory activity against B/Lee/1940.2. The hemagglutination inhibition assay, Hemolysis inhibition assay are both negative in H1N1、H3N2、H9N2 virus treated with different concentration of LH-G.3. The antiviral mechanism of LH-G was via inhibition of the early stage of influenza virus replication and the nuclear export of viral RNPs was effectively inhibited by LH-G.4. LH-C treatment suppressed influenza A virus-induced NF-kB activation but not Raf/MEK/ERK cascade in the virus-infected cell.5. High concentration of LH-G can inhibit the express of TRAIL in the cell. The suppress effect to both Caspase-3 and PARP indicate its activity in inhibit of cell apoptosis.6. Virus infection induce robust increase in the gene expression of IL-6, IL-8, TNF-α, IP-10, MCP-1, whereas LH-C treament exhibit prominent inhibitory effect in does-depentdent manner.7. Significant decrease (>2 log) of viral titers in mice lung by TCID50 was found in groups administrated with LH-C(1300mg/kg/day) and placebo at days 6 and 8 post challenge. Low dosage LH-C treated groups also presented reduced lung viral titers, though values did not appear to be significantly different from those of the control group. LH-C treated groups presented reduced mRNA expression of TNF-α, IL-6, MCP-1, IP-10, LH-C treatment significant eradication of perivascular inflammation and fewer cell exudates were observed. In view of previous results, which showed viral loads following LH-C administration, these results suggested that LH-C treatment improved the lung pathology of influenza-infected mice.Conclusions:1. LH-C exhibited inhibitory activities against several influenza viruses from different human isolates and avian influenza viruses2. The antiviral mechanism of LH-G was via inhibition of the early stage of influenza virus replication and the nuclear export of viral RNPs was effectively inhibited by LH-G.3. LH-C treatment suppressed influenza A virus-induced NF-kB activation and prominent inhibitory effect in the gene expression of IL-6, IP-10 in dose-dependent manner. It also presents activity in inhibit of cell apoptosis4. Significant decrease (>2 log) of viral titers in mice lung by TCID50 was found in groups administrated with LH-G and at days 6 and 8 post challenge.5. LH-C treatment improved the lung pathology of influenza-infected mice by reduce the early expression of inflammatory foctors.