Expression Of Inlfuenza A Virus Nucleoprotein (NP) Interfere With Scutellaria Baicalensis Georgei And Its Extracts In Vitro

OBJECTIVE:To investigate the effect of Scutellaria baicalensis Georgei and its extracts against influenza A virus NP gene, and the effect of the expression of NP to provide experimental basis for the search of new antiviral drugs which can anti-influenza.METHODS:First, used cytopathic effect (CPE) method to determine the concentration of drugs subsequent experiments; then eukaryotic recombinant plasmid pcDNA3.1(+)/NP by fat-plasmid transfection method was transfected into HeLa cells.The successfully transfected HeLa cells as target cells,48hours after transfection, the supernatant was used to test the expression of NP measured by colloidal gold immunoassay chromatography;at the same time, RNA was extracted from cells after transient transfection, afterwards, synthetised cDNA, the starting amount of NP gene was tested by means of fluorescent quantitative RT-PCR. In our research, installed eight groups as follows:HeLa cell group, empty plasmid group (transfected pcDNA3.1(+)), liposome group, and eukaryotic recombinant plasmid group (transfected pcDNA3.1(+)/NP), skullcap water decoction group, total flavonoids group, baicalin group, baicalein group.RESULTS:Used cytopathic effect method to get the toxic boundaries of various drugs on HeLa cells, the skullcap water decoction:3906.25μg·ml-1, total flavonoids:0.976562525μg·ml-1, baicalin:62.525μg·ml-1, baicalein:31.2525μg·ml-1,respectively; so, the experimental concentrations of each drug was:the skullcap water decoction:1953.125μg·ml-1, total flavonoids:0.48828125μg·ml-1, baicalin:31.25μg·ml-1, baicalein:15.625μg·ml-1.48hours after transfection, the supernatant was used testing the expression of NP by colloidal gold immunoassay chromatography:the eukaryotic recombinant plasmid group、total flavonoids group、baicalin group and baicalein group was positive, to the contrary,the HeLa cell group、liposome group、empty plasmid group and skullcap water decoction group was negative. At the same time, we used the cDNA that synthesized from the total RNA which extracted from HeLa cells to measure the the starting amount of NP gene by means of fluorescent quantitative RT-PCR. We got a standard curve:Y=-3.455X+48.119. According to the formula, we calculated the starting amount of NP gene of each group:HeLa cell group:(0.6613±0.1020) X105copies·μl-1, empty plasmid group:(0.2520±0.0946) X105copies·μl-1, liposome group:(0.4750±0.1943)×105opies·μl-1, eukaryotic recombinant plasmid group:(81.4500±29.6597) X105copies·μl-1, skullcap water decoction group:(29.6250±7.6596) X105copies·μl-1, total flavonoids group:(61.0250±25.3261)×105copies·μl-1, baicalin group:(27.7250±12.8484) X105copies·μl-1, baicalein group:(31.8250±8.2152)×105copies·μl-1, respectively. Student t test was used for statistical analysis, in comparison with eukaryotic recombinant plasmid group, the HeLa cell group with statistically significant,t=5.448,P=0.012<0.05; statistically significant of empty plasmid group,t=5.475,P=0.012<0.05; liposome group also with statistically significant,t=5.460,P=0.012<0.05; statistically significant of skullcap water decoction group,t=3.384, P=0.015<0.05; without statistically significant of eukaryotic recombinant plasmid group,t=1.047, P=.335>0.05; statistically significant of baicalin group,t=3.324,P=0.016<0.05; baicalein group with statistically significant,t=3.225,P=0.018<0.05.CONCLUTIONS:The skullcap water decoction could inhibit the expression of influenza A virus NP and decrease the starting amount of influenza A virus NP gene; total flavonoids have no effect on the expression of influenza A virus NP and NP gene; baicalin and baicalein could decrease the starting amount of influenza A virus NP gene.