| ObjectiveBy detecting the level of genes expression of Th1/Th2 immune factors, TLR2/TLR4 and MYD88 in influenza infected mice to explore the immune mechanisms of Bing-Xing Powder on influenza prevention and treatment. And through chicken embryos experiments and the early research of the Bing-Xiang Powder to discuss the pharmacodynamics base material of the Bing-Xiang Powder.Methods1、The extraction of essential oil:Extract the essential oil by steam distillation, the extraction process is that crush the herbs into medium powder, plus 10 times the amount of water and soak 1.5 hours, extract six hours.2、Establish the mouse model infected by influenza virus and take the experiment materials for relevant testing:BALB/c mice of either sex were randomly divided into 5 groups, including Negative group, Influenza control group, Ribavirin group, Bing-Xiang Powder aerosol group, Bing-Xiang Powder intranasal group. All mice were intranasally challenged with 15LD50 (LD50=10-5’5) doses of FM1 except the negative control group. Mice were treated with Bing-Xiang Powder(in aerosol or intranasal route) or ribavirin (oral administration) for 7 days before virus infection, followed by additional 5 days. Take the lungs, spleen, trachea of the mice and wash away the blood then cryopreserve thoes materials in liquid nitrogen in 30 minutes.3、The detection of cytokines secreted by spleen lymphocytes using RT-PCR method:Extract the total RNA of spleen by guanidine thiocyanate one-step method and use the spectrophotometer method for quantitative. According to the cDNA sequence of IFN-γ、IL-4 and β-actin to design the upstream and downstream primers respectively. Take 2 ug total RNA joined with AMV and TaqDNA polymerase to process Reverse transcription polymerase chain reaction .The amplification parameter is 94 ℃ for 30 s,45 s 60 ℃,72 ℃ 60 s, loop 30 times.2% agarose gel electrophores was used to analyze the amplification products.4、Tracheal mucosa TLR2/TLR4, MYD88 mRNA Real-time PCR detection:Take 100mg of tracheal mucosa, trizol extracted total RNA, after quantitative UV spectrophotometer, nucleic acid protein UV analyzer RNA concentrations, require A260/A280 at 118-210 and adjust the concentration of RNA to 1 mg /L. Take lμg RNA, M-MuLV reverse transcriptase, reverse transcriptase synthesized cDNA. On the Step One QPCR instrument technology with fluorescent dye SYBR Green I line real-time quantitative PCR reactions. UDG 2min,95 ℃ denaturation for 2min, design cycle program:95 ℃ denaturation 15 s,62.5 ℃ annealing 30 s,72 ℃ extension 30s, a total of 40 cycles. Each sample was analyzed three times.5、The maximum non-toxic dose of the drug (TD0) determination Experiment has blank control, solvent control group, ribavirin injection group, patchouli volatile oil group, leaves volatile oil group, borneol group. Dose ribavirin injection were 10mg/embryo,5 mg/embryo,2.5 mg/embryo,1.25 mg/embryo, 0.625mg/embryo; patchouli volatile oil group drug doses were 20 mg/embryo, 15mg/embryo,10mg/embryo,5mg/embryo,2.5mg/embryo; dose leaves volatile oil group had 20mg/embryo,15mg/embryo, 10mg/embryo,5mg/embryo,2.5mg /embryo; administration borneol doses were 0.3 mg/embryo,0.25mg/embryo, 0.2mg/embryo,0.15mg/embryo,0.12mg/embryo; each drug at each dose group received 5 embryo, according to urine drug cavity inoculation method was inoculated into allantoic cavity,0.lml/embryo. The control group received the same volume of saline vehicle control group received the same volume of 0.5% aqueous polysorbate-80 fat. Injectable drugs, After sealing the mouth of the embryo into the 37 ℃, saturated humidity incubator and incubated 96h, turn the eggs twice a day, to observe the development of the situation chick embryos discarded within 24h to death to all surviving chick The maximum dose is the maximum nontoxic dose of drug.6、Influenza viruses infect half the amount of chicken embryos (EID50) Determination:Take virus seed, soaked with water rushing melt, the virus was diluted with saline to 10-1 to 10-8 times, according to the above method allantoic cavity inoculation virus inoculation, each dilution was inoculated six chick, 0. lml/embryo, after incubation at 37 ℃ incubator, humidity certain culture, daily observation of embryo survival, and death within 24h to abandon the rest of the embryo within 48h after incubation at 4 ℃ refrigerator overnight, the harvest of each embryo allantoic fluid, to HA test, a "+" agglutination positive for infection, calculated using the Reed-Muench influenza virus on chicken embryo EID50.7、The prevention and treatment function of each drug resist influenza virus:This experiment has two types of drug delivery,preventive and therapeutic administration, the intervals of giving the drug ang injecting virus is for one hour. this experiment are divided into six groups, including Negative group, Influenza control group, Ribavirin group, Pogostemon cablin group, Artemisi group, Borneol group. Each group is composed of five dose group except Negative group and Influenza control group. The dosage of Ribavirin group is 5mg/embryo; Patchouli essential oil dosage are 10 mg/embryo,5 mg/embryo,2.5 mg/embryo;the volatile oil of artemisi dosage are 5 mg/embryo respectively,2.5 mg/embryo,1.25 mg/embryo; Borneol dosage were 0.15 mg/embryo,0.12 mg/embryo,0.06 mg/embryo. Each dosage group has five chicken embryos, the dosing volume is 0.1 ml per embryo. The amount of injected virus is EID50. In the Prophylactic, Negative group and Influenza control group received 0. 1ml of 0.5% aqueous polysorbate fat-800. lml remaining drugs were given drugs, after 37 ℃ for 1h in addition to the control group was given 0.5%0. 1ml poly Yamanashi fat-80 solution outside other groups were inoculated 0.1ml influenza virus; therapeutic administration groups except the control group were given 0. 1ml of 0.5% polysorbate 80 Aqueous Solution fat outside the rest of the group were inoculated with 0. lml influenza virus,37 ℃ for 1h after each drug administration group received 0. 1ml Negative group, Influenza control group received 0. 1ml of 0.5% polysorbate 80 Aqueous Solution fat. Put all the chicken embryos in to the incubator, incubation conditions is 37 ℃ and a certain humidity. check and turn these embryo 1～2 times a day, throw away the embryos which are death in 24 hours. After 48 hours, put these embryos into 4 ℃ refrigerator Overnight, harvest each embryo allantoic fluid to carry out the HA experiment.ResultsThe photomicrograph of Influenza control group show large area of pneumonia with intense exudates in bronchial lumen.The mRNA expression of TLR2, TLR4, MYD88 in Influenza control group are markedly higher than that in Negative group (p<0.01), but the expression levels of IL-4, IL-5mRNA are declined (p<0.01).Bing-Xiang Powder(in aerosol or intranasal route)can significantly increase the expression levels of IL-4 mRNA、IL-5mRNA (P<0.01 or P<0.05), however, the expression level of IFN-γmRNA in Bing-Xiang Powder intranasal group is lower than that of Bing-Xiang Powder aerosol group (P<0.05).Bing-Xiang Powder intranasal group reduced the mRNA expression level of TLR2, TLR4, MYD88 most dramatically among all of the groups (P<0.01). Pogostemon cablin and Artemisi in the prescription of Bing-Xiang Powder has the effect of prevention and control the influenza on non-toxic and appropriate drug doses.ConclusionsBing-Xiang Powder can prevent the infection of influenza in some degree and regulating the mRNA expression level of cytokines and TLR2, TLR4, MYD88 may be the probable anti-virus mechanism. The main pharmacodynamics materials of Bing-Xiang Powder to anti-influenza virus need to be further discussed. |
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