The Mechanism Of Viruses Attachment To Host Cells And The Interference Effect Of Traditional Chinese Medicine On It

Human being has been threated seriously by viral diseases. At the 2th global conference which organized by International Academy of Medical Science on March 2006, 500 specialists indicated that we are in the danger of pandemic which induced by variant viruses. In developing country priority in controlling diseases, it is one in four challenges to human healthy that epidemic viral diseases will break out.With the application of molecular biology theories and techniques in virology, the mechanism of viruses infection have been identified to a certain extent. But up to now, there was no substantial advancement in the treatment. For this reason, it became important that we must find out an effective treatment to control viral deseases.It has long history of using the Chinese herbal medicine to prevent and cure viral diseases with remakable effect. It is able to inhibit the cycle of viral replication, prevent the cytopathic effect of viruses, improve clinical symptom. Moreover, traditional Chinese medicine is better than other anti-viral methods in rich natural resources, low price and hypotoxicity. For the past 20 years, the efficacy of traditional Chinese medicine has been verificated by a number of experiments in vivo and in vitro. Especially after SARS, scientists focused on screening for effective constituents to anti-viruses. But up to now, there was no substantial advancement in the anti-viral traditional Chinese medicine. The development of anti-viral traditional Chinese medicine is limited by the following reasons: 1.There is few train of thoughts which can fully manifest the feature of traditional Chinese medicine in anti-viral diseases. 2. There is few research means which can fully manifest the feature of traditional Chinese medicine in anti-viral diseases. So we should study the the anti-viral mechanism of traditional Chinese medicine not only under the direction of theory of traditional Chinese medicine anti-viruses but also on the base of advanced biology techniques and means. It has practical importance for traditional Chinese medicine to the world.The study is based on the screening for effective constituents of anti-viral traditionalChinese medicine and the preliminary discussion of mechanism of anti-viruses. We choose the extract ZG from Gardenia and total flavone of Scutellaria barbata as the object and 2³ strains of viruses as the samples to research the mechanism of viral attachment to host cells and the interference effect of traditional Chinese medincine on it. Nonspecific physiopathologic changes of host cells are studied post viral infection to find the definite target of traditional Chinese medicine. Our research was supported by national nature science fundation of China.1. The study on anti-viral effect of the extract ZG from Gardenia and total flavone of Scutellaria barbata in vitroHep-2 as host cells, Acyclovir as positive control to anti-HSV-1, we observe the effect of ZG and TFSB on 4 strains of viruses-induced cytopathic effect. The 0% toxicity concentration (TCo), 50% toxicity concentration (TC50), 50% inhibitor concentration (IC50), therapeutic index (TI) were determined by Reed-Muench method. The result demonstrated the TC0 and TC₅₀ of ZG were 5 mg.mL^-1 and 12.74 mg.mL^-1 respectively.The TC₀ and TC₅₀ of TFSB were 3.13mg.mL^-1 and 8.91 mg. mL^-1 respectively. The TCo and TC50 of Acyclovir were 0.2 mg.mL^-1 and l.Mmg.mL^-1 respectively. The cytopathic effect induced by herpes simplex virus type 1 (HSV-1) and Parainfiuenza virus type 1 (PIV-1) was inhibited by not only ZG but also TFSB significantly , while the cytopathic effect induced by respiratory syncytial virus (RSV) was inhibited only by TFSB. Coxsackie B virus type 4 (CoxB4) was inhibited by neither. The IC50 and TI of ZG anti-HSV-1 were 0.45 mg.mL^-1 and 28.31 respectively. The IC₅₀ and TI of ZG anti-PIV-1 were 1.00 mg.mL^-1 and 12.68 respectively. The IC₅₀ and TI of TFSB anti-HSV-1 were 1.28mg.mL^-1 and 6.96 respectively. The IC₅₀ and TI of TFSB anti-PIV-1 were 1.41mg.mL^-1 and 6.32 respectively. The IC₅₀ and TI of TFSB anti-RSV-1 were 0.68mg.mL^-1 and 13.10 respectively.The IC50 and TI of Acyclovir anti-HSV-1 were 0.05mg.mL^-1and 19.05 respectively.2. The effect of viruses attachment on membrane potential and the interference effect of traditional Chinese medicine on itWe took acetylcholine as positive control to observe the changes of membrane potential on the process HSV-1, PIV-1 and Ad3 (adenovirus type 3) attachment to host cells and the effect of ZG and TFSB on it. It can provide theoretical foundation for the research of viruses attachment to host cells and the interference effect of traditional Chinese medicine on it .The Hep-2 cells membrane petential was fluorescent labeled with DiBAC4(3) and the changes of membrane potential were measured by flow cytometer .The result demonstrated that the resting potential of Hep-2 cells seldom fluctuate,but post viuses infection for 10³0min, membrane potential decreased significantly and membrane was in the state of hyperpolarization. There was no apparent effect of ZG and TFSB on the membrane potential of normal cells, so was they on the hyperpolarization of membrane which had infected by HSV-1 and PIV-1. It may be one mechanism of viruses attachment to host cells that membrane potential decreased and membrane hyperpolarized continuously. Acetylcholine could improve the state of hyperpolarization, while ZG and TFSB couldn't. It indicated that the anti-viral mechanism of ZG and TFSB had nothing to do with the changes of membrane potential on the process of attachment. Further investigations are needed for the mechanism.3. The effect of viruses attachment on the Na+-K+-ATPase activity of host cells membrane and the interference effect of traditional Chinese medicine on itWe made use of phosphorus determination method and spectro photometer to measure the Na+-K+-ATPase activity of Hep-2 cells membrane post HSV-1 and PIV-1 attachment so as to discuss the mechanism of viruses attachment. Heparin sodium as positive control, we observed the interference effect of ZG and TFSB in different methods on Na+-K+-ATPase activity of Hep-2 cells post HSV-1 and PIV-1 infection. The result demonstrated that Na+-K+-ATPase activity increased significantlly post both HSV-1 and PIV-1 attachment (P < 0.01 and 0.05 respectively). While there was no apparente interference effect of ZG and TFSB on the changes of Na+-K+-ATPase activity post viruses attachment. Compared with viruses control groups, there was no significant difference. Neither did heparin sodium. During HSV-1 and PIV-1 attachment to host cells, Na+-K+-ATPase activity increased significantly which indicated that during viruses attachment, energy metabolism increased and viruses drew assistance fromthe energy producted by ATP to infection. There was no apparente interference effect of ZG, TFSB and heparin sodium on the changes of Na+-K+-ATPase activity post viruses attachment. So it is possible that the mechanisms of traditional Chinese medicine and heparin sodium have nothing to do with it.4.The effect of viruses attachment on the host cells membrane fluidity in and the interference effect of traditional Chinese medicine on itIn order to research into the common effect of viruses on attachment to host cells and provide theoretical foundation for making clear the target and mechanism of traditional Chinese medicine anti-viruses. Confocal scanning laser microscope was adopted to study the changes of Hep-2 membrane fluidity which infected by HSV-1 or PIV-1 and ZG and TFSB on it. The result demonstrated that the membrane fluidity recovery of normal Hep-2 cell was 72.66% .The fluidity of Hep-2 membrane decreased significantlly post HSV-1 and PIV-1 infection and the recovery decreased to 26.73% and 19.85% respectively. In three different treating methods, the Extract ZG from Gardenia could improve the membrane fluidity post viruses attachment. Both in the group of treating with TFSB ahead and simultaneously to anti-HSV-1, the membrane fluidity increased significantly which compared with HSV-1 control group. But in the group of treating with TFSB behind and treating with TFSB to anti-PIV-1 in three different methods, diffusion coefficients had no significant difference which compared with viruses control groups;While recovery rate increased significantly which indicated that membrane potential improved to a certain extent. That traditional Chinese medicine could improve membrane fluidity and interfere viruses attachment was one possible mechanism of anti-viruses activity.