Anti-virus Effects And Mechanisms Of Gardenia Extracts T9 And TH Against Herpes Simplex Virus Type-1

Viral diseases are becoming serious to human being in these days. It is an huge challenge to the medical fields, because the originally viral diseases resurgence and many new viral diseases appear unceasingly. Vaccine has produce a galactic marked effect in controling viral diseases, but it is confined to finding new specifically good effective vaccine for many reasons. Curative effect of chemosynthesis medicine in existence is not affirmative ,it can't reach the broad-spectrum and large effect,it also easy to bring into drug-fast effect.Those medincine has distrub the metablize of normal cells seriouly,so it show the side-effect and toxicity ,it is limited in used for the clinic and developing very slowly.In a word, to find more safe and effective anti-viral mentods is imminent.It has a long history of using the Chinese medicine to cure viral diseases .The Chinese medicine has the characteristics of large-effect,broad-spectrum,low-toxicity and relative steady-effect. The nature resouce of the Chinese medicine is abundant and the kinds of the Chinese medicine is various,but the anti-viral mechanism of the Chinese medicine is not recognized truly.So the development and application of the Chinses medicine is limited .Along with the development of modern biology and new model of experiments provid large foreground for the reseach of anti-viral medicine.It offer the good conditions for searching for good-effectvie anti-viral medicine from the Chinese medicine.Based on original reseaches, in our studies we picked out Gardenia extracts——T9, TH, which are confirmed the obvious activity of anti-HSV-1.We aim toinvestigate their effective targets of anti-HSV and reveal the anti-viral mechanism of traditional Chinese medicines. These findings played a foundation for development of new traditional Chinese medicines for HSV.Part I Gardenia extracts——T9, TH effect on HSV-VP16VP16 is a constitutive protein of HSV-1.It participates in viral particles' assemble and install. It is also a function protein which plays an important part in transcription regulation during the nonage of viral infection. Expression of VP16 gene can be considered as a marker of on going lytic HSV infection.Because of the VP16's important effect during HSV-1 infection, our experiment uses BALB/C mouse which are susceptible to HSV to study and research the expression level of HSV-VP16 by RT-PCR.We compare with the expression of HSV-VP16 between each group. RT-PCR can produce semiquantitive results by 'VP16 optical density value/β-actin optical density value'.We hope to investigate the mechanism of action that Gardeniaextracts——T9, TH resist HSV-1 by this method. The result demonstrains that thegroup of viral contrast can be detected the expression of VP16 mRNA on the 4^th day after infection, and it rises along with time. It reaches the peak on the 10^th day, and then falls-off. The two dosages and administration routes of T9,TH can degrade the expression of VP16 mRNA in mice's brains.Their overall tendency display that the groups of large doses surpass the groups of low doses in the two administration route, the groups of oral application surpass the groups of intraperitoneal injection,T9surpass TH. That shows Gardenia extracts——T9, TH can degrade the expression ofVP16 mRNA in HSV infected mice's brains.It may be one of their anti-HSV mechanisms of action. It is not reported that any drug is effect on this target at present.Part II Gardenia extracts——T9,TH effect on IFN-γin mice's brainsIFN-γis an essential immune factor for clearance of HSV. IFN-γis a cytokine secreted by activiated T cells and NK cells after viral infection. IFN-γhas been reported to irritate viral infection cells to express MHC I ,promote CTL to kill viral infection cells and enhance NK cells' activity of killing viral infected cells.However IFN-γplays a very important part in both innate immunity and adaptive immunity. We observe time-effect of IFN-γin brains of HSV-1 infected mice by RT-PCR, aimto investigate that Gardenia extracts——T9,TH resist anti-HSV-1's mechanism ofaction. The result demonstrains that the group of viral contrast can be detected the expression of IFN-γmRNA on the 4^th day after infection. From the 4^th day to the 10^th day after infected it shows a steady expression.The relative expression of IFN-γmRNA is going to descend on the 10^th day.After 10 days infected compared with the initial stage infected it shows a lower steady expression. The two dosages and administration routes of T9,TH can up-regulate the expression of IFN-γmRNA in mice's brains obviously.Except T9 low dose, others of T9 medication administration team reach peak on the 7^th day after infected,and after 21 days still show a steady expression. T9 medication administration reaches its peak on the 14^th day,and the expression quantity is higher than others of T9 medication administration team. Among TH medication administration team, the oral administration groups reach peak on the 10^th day and the injection groups reach peak on the 14^th day. That showsGardenia extracts——T9,TH can up-regulate the expression of IFN-γmRNA in HSVinfected mice's brains through some kind of mechanisms.It may be one of their anti-HSV mechanisms of action.Part III Gardenia extracts——T9,TH effect on the receptor movement ofHSV-1To utilize single particle tracking to study viral infection and surface receptor of membrane before and after adding drugs can explain the architecture and function of membrane and the effective target of drugs very well. Our experiment use HEP-2 cell which are easily cultivated to research kinestate of different groups of infected cells, so as to reveal cellular membrane's characters and pharmacal mechanism of action. The ligand is the complex of F-HSV, HSV which are labeled combines surface HSV receptor of cellular membrane, use fluorescence microscopy to observe and cCCD to real-time record. Single particle tracking that our develop a systems software to micro-observe cell traces single particle,analyze its trajectory and data .Meanwhile we manipulate fluorescence intensity of every group in the same time by Aquacomos and calculate every group's adsorption rate and inhibition ratio. The result demonstracts that ,because of FITC fast quenching and the short time of the receptor's movement,the mobile offset of receptor is not enough in viral contrast, the rest medication administration teams can not detect their receptors' movement.Now we are still working on searching for some fluorescent probe suiting for the observation of receptors' motion HSV-1.It may possess the characteristic of conjugated with virus easily,and diminish the quenching time to ensure scanning flame emission. The experiments related are going on. Added Gardeniaextracts——T9, TH using different methods can prevent the HSV-1 attach on theHEP-2 surface,reduce the quantity of viral binding.That shows Gardeniaextracts——T9,TH can also occupy receptor bindig sites,block out receporor change receptor,or contest with VAP to prevent the viral binding and infection.