Experimental Research On The Cellular Signal Transduction Mechanism Of The Detoxification Of Liangge San

Background: With the intercommunion between traditional Chinese medicine and modern medicine,the occur,development,prevention and cure of disease have been known deeply. Traditional Chinese medicine consider "toxin" as substance foundation of many disease(especially "wenbing",seasonal febrile disease),as the determinal factor of development and transfer of disease. "Toxin" could invade into body from out,it could also be received by translation of "liuyin" in body.Disease due to "toxin" show characteristic of urgent-occur,severe-state,quick-tranfer. It is well known that "toxin" could make function disorder, "yinyang" maladjustment,disease transfer, and could induce blood stasis, bring on hot symptom. Today it is recognized that "toxin" is composed of microorganisms and their "toxin",antigen-antibody complex.Endotoxin is much important among these.It has been found that inflammation medium would be important factor of severy pathology damage induced by microorganisms and their toxin. Inflammation medium almost come into being from microphages,which make important effects in the immunoreaction,inflamationreaction and organization damage.It has two-ply effects: Recovery and damage effects.Suitable concentration of that would have recovery effect,but excessive medium would result in damage of organization.For example, hign concentration of TNF-α,IL-1, IL- 6, IL -8 would activate cruor and complement system,which would induce severe damage of organism.On the basis of these,we advance theory that:bacteria,virus,toxin which invade body from out are thought as "outer toxin",cytokine induced by "outer toxin" are thought as "inner toxin",the effect of "outer toxin" should depend on the "inner toxin"— "startup with "outer toxin" and transduction with "inner toxin"" is the process of a disease come on.So inhibiting the expression of inflammation medium is the key means of detoxification. Detoxification is basic way to treat disease due to "toxin" and is also important way of treating disease on TCM.Clearing away heat and toxins is mostimportant means of Detoxification on TCM.prior we study the effect of Liangge San,a famous Chinese herbs recipe of clearing away heat and toxins, which originated in ((Taipin Huimin Hejiju Fang)) in song dynasty.We found that Liangge San could decrease concentration of TNF- a in the blood plasma,inhibite the expression of TNF- a and IL-6 in lung on LPS injure-mice, alleviate the pathological damages and decrease mortality of LPS injure-mice. It has shown that Liangge San's inhibiting the excessive expression of cellkine would be main detoxification mechanism of Liangge San. With the deep investigation on the pathways of cell signal transduction,it has shown that the expression of inflammation medieum depend on a seris of pathways of cell signal transduction. Pathways of cell signal transduction on the expression of inflammation medieum induced by LPS have been shown as follow:first LPS combine with CD14 receptor with the help of LBP,then the signal transfer into the cell,activate a series of pathways of MAPK via phosphorus,then activate NF- k B, P50 and P65, submit of which,turn into neuceus of cell,combine with k B series on the target gene,startup and ruegulate expression of inflammation medieum.We had found that serum with Liangge San could inhibite expression of CD14mRNA and activation of p38 MAPK on the RAW264.7 cell induced by LPS .Liangge San could inhibite the activation of NF- k B on the lung of LPS injury-mice.lt has shown that detoxification of Liangge San rely on it's regulation on the pathway of cell signal transduction.Our study would further this mechanism.Source of Study: Natiaonal Nature Science Foundation of China. No.30171155 Objective:We study the cellular signal transduction mechanism of the detoxification of Liangge San in order to further mechanism investigation of Chinese herbs's detoxification,provide theoretical foundation of detoxification's "treating the different disease with the same methods", prove the theory of "startup with outer toxin,transduction with inner toxin"Methods: The experiment was composed of tow parts, in vivo and in vitro. In vivo:The model were established with intravenous injection of lipopolysaccharide(LPS) and different dose Liangge San were given, the expression of CD14 and Scaverger receptor were detected at the post injury 2nd,4th,8lh hour in immunohistochemistry and analyzed with computer image system,and the pathological changes of liver tissue were observed, the expression of p-p38MAPK was detected at the post injury 2nd hour in westernblot and analyzed with computer image system. In vitro:The macrophages from abdominal cavity of mouse were collected ^ incubated and Subsequently re-incubated with LPS. P65? the submit ofNF- k B, was detected by Immunofluorescence inO> 10 > 30 ^ 60 > 120 n 720minite after LPS being given and the Fluorescent intensity of P65 expression was measured by laser scaning confocal microscope(LSCM) and Leica QN software. Then we selected the time 60 minite after injecting LPS to perform our followed experiment: To prepare the serum with or without Liangge San. the macrophage from abdominal cavity of mouse was collected > incubated and subsequently re-incubated with LPS and different doses serum with or without Liangge San.Intranuclear P65 > the submit of NF- k B, was detected by Immunofluorescence 60 minite after injecting LPS and the Fluorescent intensity of p65 expression was measured by laser scaning confocal microscope(LSCM).Results: In vivo:After injecting lipopolysaccharide(LPS),the expression of CD14 and Scaverger receptor in macrophages of liver showed the tendency of up an down regulation respectively.Liangge San could inhibite the up-regulation of CD14 expression and down-regulation of Scaverger receptor expression in a dose-dependent manner wherever in three different times. Liver cells shew vacuole change. Changes of CD14 and SR expression were paralleled with the severity of liver damages of the mice, the express of p-p38MAPK of LPS-injury group rose significantly compared with that of the blank-control group, the express of p-p38MAPK of the different dose groups and drug-control group significantly decreased when compared with that of LPS-injury group.Those of drug-control group and high dose group were lowest and those of middle and low dose groups were lower compared with that of LPS injury group. In vitro:The Fluorescent intensity of Intranuclear p65 expression of incubated macrophages with LPS shew the regulation as follow:it began to reinforces in 10 minite, up to peak in 30 minite, began to decrease in 60 minite,decreased significantly in 120 minite, had no difference compared with that with no LPS (0 minite group) in 720 minite. The Fluorescent intensity of intranuclear p65 expression of incubated macrophages with LPS for 60 minite,significantly reinforced,compared with that with no LPS and serum of Liangge San. The Fluorescent intensities with inhibitor TLCK and different doses serum of Liangge San significantly decreased when compared with that with LPS. And the Fluorescent intensities with inhibitor TLCK and high dose serum of Liangge San were lowest and these of middle and low doses were lower compared with that of LPS.There were no difference between the Fluorescent intensity of Intranuclear P65 expression of incubated macrophages with LPS with that with serum without Liangge San.Conclusions: Liangge San shows the function of regulating the expression of CD14 and SR in vivo, inhibiting the activation of p38MAPK in vivo, restraining theactivity of NF- k B in vitro,which may be the signal transduction mechanism of Liangge San's detoxication.