Study On The Anti-influenza Virus Mechanism Of GM-CSF And Anti-HBV Effects Of Entecavir-baicalin Combination

Part 1 Study on the anti-influenza virus mechanism of GM-CSFGranulocyte-macrophage colony-stimulating factor (GM-CSF) was initially identifiedas an activity present in lung cell-conditioned medium, capable of stimulatinggrowth of granulocytes and macrophages from cultured hematopoietic progenitors. However, GM-CSF also has pleiotropic activating effects on mature leukocytes, which may improve leukocyte function and augment protective immunity against microbial infections. Our previous study show for the first time that prophylactic treatment with GM-CSF resulted in reduced viral titer, minimal pulmonary pathology and decreased mortality of the mice challenged with lethal dose of influenza virus, which evidenced that GM-CSF protects against lethal influenza virus infection.The findings were published in the 2010 of the journal Cytokine.To further elucidate the the anti-influenza virus mechanism of GM-CSF, on the first part of this study, the regulation of GM-CSF on inflammation and apoptosis was studied. Mice were treated intranaslly daily with 1.34 mg/Kg GM-CSF for 7 days prior to lethal infction with FM1 influenza A virus, and the mice were sacrificed on day 0, day 2 and day 4 post infection respectively, the lungs were harvested and the pulmonary leukocyte population were analysis by flowcytometry, the cytokines in lung homogenates were measured by ELISA assay, the pulmonary viral burden, NF-kB, caspase 3, bax, and bcl-2 mRNA wre quantified by real time RT PCR and NF-κB, phospho-NF-κB, IκB, caspase-3, cleaved caspase-3, bax, and bcl-2 protein expression were determined by Western blot, also, localization of phospho-NF-KB and cleaved caspase-3 in deparaffinized sections of formalin-fixed tissue were detected by immunohistochemistry.The data showed that mice prophylactic treated with GM-CSF had high levels of TNF-αand IFNα/β, as while as increased leukocyte recruitment at early stages of infection compared with the vehicle treated mice, whereas these proinflammatory cytokines and percentage of neutrophil fell,and were much lower than vehicle treated mice at the late stages of infection, which suggest that GM-CSF treated mice mount an early protective innate immune response and controlled later inflammatory response. Accordingly, the GM-CSF pretreatment resulted in a significant reduction in viral burden, here represented by decreased influenza A virus M gene expression in lung on day 2 and day 4 post infection. Further findings show that levels of NF-κB mRNA and phosphate NF-κB protein were upgraded in mice after they were treated intranaslly with GM-CSF for 7 days and the levels of NF-κB and phosphate NF-κB did not change greatly after infection. In contrast, NF-κB mRNA and phosphate NF-κB protein expression in vehicle treated mice rose markedly from very low levels to much higher than GM-CSF treated mice after infection. In addition, GM-CSF pretreatment significantly upregulated the expression of antiapoptosis agent bcl-2, while downregulated the expression of proapoptosis agents, bax, caspase 3 and cleaved caspase 3.The data presented here indicate that by activing the NF-κB pathway, GM-CSF promots the production of antiviral cytokines, TNF-a and IFNa/p, as where as leukocyte recruitment at early stages of viral infection, which facilitates the rapid clearance of the invading virus. Consistent with the control of viral burden, immune response maintain in moderate levels, which is important in limiting pulmonary inflammation and tissue damage. Additionally, by increasing of bcl-2 expression and decreasing of bax expression, GM-CSF inhibits activation of caspase pathway, which is critical in prevention of influenza virus induced apoptosis and lung injury.In a word, innate immune defence were up-ragulated after GM-CSF pretreatment, which facilitates the rapid clearance of the invading virus, and excessive inflammation and apoptosis were avoided due to the low viral burden, so that the influenza virus infected mouse were protected.Part 2 Anti-HBV effects of entecavir-baicalin combinationBaicalin(BA) is a flavonoid compound purified from the medicinal plant Scutellaria baicalensis Georgi, Its structure has been determined to be 7-D-glucuronicacid -5-6-dihydroxy-flavon.Previous studies have demonstrated that baicalin has multiple pharmacological effects, such as anti-inflammatory, antipyretic, antibacterial, anti-viral, antitumor, antioxidant, anti-allergic, tranquilizing and diuretic activities, and it has been wildely used in oriential medicine and show to alleviate the progress of a number of inflammatory and infection disease, including hepatitis B. Also, baicalin has been observed to inhibit the replication of HBV in vitro. Despite the evidence of its anti-HBV activity in vivo and in vitro, the molecular mechanism of action of baicalin remains to be determined.This report is the first to describe anti-HBV mechanism of baicalin. HepG2 2.2.15 cells were treated with 50uM、25μM 和 12.5μM baicalin,and it was found that HBsAg, HBeAg and HBV DNA in culture supernatant were both inhibited. Also, Northern blot and real time PCR analysis revealed that baicalin significantly decreased HBV 2.1/2.4kb and 3.5kb RNA levels,as where as downregulated the expression of HNF 1 aand HNF4α,which have been shown to bind HBV promoter/enhancer elements and to be activating and regualating HBV transcription. So we hypothesis that baicalin suppresses HBV replication by downregulation of HNF 1αand HNF4α,thus HBV RNAs expression was blocked first, followed by viral proteins,and then DNA.To assess the antiviral effect of entecavir-baicalin combination on wild type HBV and lamivudine-resistant HBV, HepG2 2.2.15 cell line and the cell line expressing rtM204V+rtL180M mutant HBV were used as cell models.The results showed Entecavir-baicalin combination inhibited HBsAg, HBeAg and HBV DNA production by either or wild-type HBV and lamivudine-resistant HBV in a dose-dependent manner.In HepG2 2.2.15 cell line expressing wild type HBV, ETV3nM+BA50μM、 ETV0.75nM+BA50μM 和ETV0.19nM+BA50μM increased the HBsAg inhibitory rate by 30.00%、30.00%、29.58% and 32.92%, respectively, increased the HBsAg inhibitory rate by 30.00%、30.00%、29.58% and 32.92%, respectively, increased the HBV DNA inhibitory rate by 10.66%、13.19% and 12.17%, respectively, compared with that of ETV mono treatment. And in the cell line expressing rtM204V+rtL180M mutant HBV, ETV 48nM+BA 50μM, ETV 16nM+BA 50μM,ETV 3nM+BA50μM and ETV 0.75nM+BA50μM elevated the HBsAg inhibitory-rate by 30.00%、30.00%、29.58% and 32.92%,respectively, elevated the HBsAg inhibitory rate by 28.88%、31.08%、32.67% and 30.94%, respectively, elevated the HBV DNA inhibitory rate by 37.60%、41.00%、35.80% and 33.88%.respectively, compared with that of ETV mono treatment.To further evaluate the antiviral activity of Entecavir-baicalin combination in vivo, duck DHBV model were used. The dot bolt and the southern blot analysis revealed the potent inhibition of the combination on plasma and liver DHBV DNA production after treatment, respectively. Compared with that of ETV monothreapy, ETV 100ng/kg/d+BA 250 mg/kg/d and ETV 25ng/kg/d+BA 250 mg/kg/d dosage combination increased the inhibitory rate of plasma DHBV DNA by 14.74% and 15.03%,respectively, and increased the inhibitory rate of liver DHBV DNA by 8.60% and 19.24%,respectively.These results elucidated that ETV-BA combination elevated the antiviral effect on either wild type HBV or YMDD mutant HBV compared with that of ETV mono treatment. ETV has been proved to inhibit HBV replication by blocking viral DNA synthesis, while we found that BA downregulated the viral RNA prodution, hence complementation between these two different antiial mechanism may occur when they are combinated, which contribute to the increase of anti-HBV activity. Addtionly, we found that ETV-BA combination elevated the antiviral effect more significantly on rtM204V+rtL180M mutant HBV than that on wild type HBV.The reason may lie in that the YMDD mutant HBV showed a marked insensitivity to ETV, while because BA inhibited HBV replication by targeting on HNF of host cells, the mutantion had no effect on antiviral activity of BA. It is suggested that ETV-BA combination perhaps have a great advantage in drug resistant mutants therapy.