Gui Zhi Tang "square Card Corresponding Experimental Studies

Toll-like receptors(TLRs) owe their name to a closely related receptor called Toll, first identified in Drosophila.TLRs are typeⅠtransmembrane receptors,at least 11 TLRs have been identified in humans and 13 TLRs in mice.As a member of pattern-recognition receptors(PPRs),TLRs mainly recognize pathogen associated molecule pattern(PAMP). Certain TLRs seem to recognize specifically endogenous ligands.When TLRs recognize and specific bind with their ligands,they initiate two major intracellular signaling pathways,such as Myeloid differentiation factor(MyD88)-dependent and MyD88-independent pathways,and activate a series of protein cascade reactions of the signaling downstream,including transmiting signals into nucleus,leading to the activation of nuclear factor-kappa B(NF-κB),activating protein-1 and interferon regulatory factors,induce the synthesis and release of corresponding cytokines of interleukin(IL)-1,IL-2,tumor necrosis factor-α(TNF-α) and interferon a full stop.Finaly they stimulate a series of immune event according to different pathogenic microorganism.Researches certified that many kinds of immune defense reaction needed TLRs.They not only provide the first line of host defense response against microbes and initiate early phage of innate immunity,but also shape the adaptive immunity and play the "bridge" action between innate and adaptive immunity.Fangji(Formula) is a way to be used for the treatment of the "disease-complex" (zheng) under the direction of therapeutic methods of traditional Chinese medicine(TCM), where the Chinese herbs are selected and used alone or in combination according to the natures and functions of the herbs as well as the reciprocal action relationships among them.The theory of "gearing of formula to disease-complex" in TCM means therapeutic effect of a specific formula composed of corresponding herbs is closely consistent with the pathogenic mechanisms and disease-complex it targets for,where the internal links between the herbs in the formula and the disease-complex are highlighted.In the theory of TCM,selection of formulas as well as the herbs in it must depend on the pathogenic mechanisms of the specific disease-complex and the compatible relationships among the herbs in the formula are always organized to regulate or reverse the pathogenic mechanism of the disease-complex.Only through interaction between the herbs in the formula and the disease-complex,the functions of the formula can be realized,and this is a key feature of the therapeutic system based on the disease-complex in TCM.The research of theory of "gearing of formula to disease-complex" in TCM is an essential part for uncovering the scientific nature of functions and main therapeutic actions for the formulas of TCM.The comparative studies on the differences of functions among the representative formulas mainly for treating the exterior syndrome of various febrile diseases is thought to be effective way to start the experimental research on the theory of "gearing of formula to disease-complex".In the theory of TCM,all the formulas mainly consisted of herbs relieving exterior syndrome,which have the actions of expelling the exterior evil,inducing sweating,letting out eruptions and eliminating toxic materials belong to the territory of agents for relieving exterior evils,which are mainly for the treatment of exterior syndromes as well as measles at early stages along with manifestations as fever,aversion to cold,headache,pain over the body and floating pulse. The exterior-expelling formulas can be subclassified into exterior-expelling formulas with the nature of pungent coolness and exterior-expelling formulas with the nature of pungent warmth.The pharmacological studies have shown better therapeutic effects could be achieved on the virus-infected diseases for the exterior-expelling formulas.In this experiment,we first got serum samples from the patients suffering from both upper respiratory tract infection and Guizhi Tang syndrome identified by the doctors of TCM in clinics.Four formulas with therapeutic actions of pungent warmth or pungent coolness for superficial syndromes were chosen and rat serum samples containing the above herbal formulas were collected,then the effects of Guizhi Tang syndrome patient serum as well as the effects of serum containing the four formulas after being stimulated by the patient serum samples on both the mRNA expression of certain TLR subtypes and the release of some inflammatory cytokines in RAW264.7 cells were tested and analyzed in vitro,with an aim to explore the pathological mechanisms of Guizhi Tang syndrome and therapeutically molecular mechanisms of the four formulas,as well as the potentially biologic basis that Guizhi Tang is most effective only for the patients with Guizhi Tang syndrome in clinics.The results are as follows.1 Effects of serum from healthy volunteers and patients with Guizhi Tang syndrome on the viability of RAW 264.7 cellsResults showed that serum from healthy volunteers had no significant influence on the viability of RAW264.7 cells,but serum from Guizhi Tang syndrome patients could reduce the viability of the cells evidently when the concentration of the serum increased to 2.5%. To be sure the cells was in a normal state,all the concentrations of serum from Guizhi Tang syndrome patients selected for subsequent tests were kept below2.5%.2 Effect of serum from healthy volunteers and serum from patients with Guizhi Tang syndrome on the mRNA expression of TLRs in RAW 264.7 cellsThe final serum concentrations of 10%,6%,3.5%,2%and 1.3%from healthy volunteers or patients with Guizhi Tang syndrome were chosen and the influence of those samples on the mRNA expression of TLR1～9 in RAW264.7 cells was tested,respectively. The results showed that the serum of varing concentrations from healthy volunteers had no significant effects on the mRNA expression of all the subforms of TLR.In contrast,the mRNA expression of TLR-3,TLR-4 and TLR-9 in RAW264.7 cells was markedly up-regulated after bing stimulated by serum from the Guizhi-Tang-syndrome patients with the highest expression at the serum concentration of 2%(p＜0.01).The up-regulated mRNA levels of TLR-3,TLR-4 and TLR-9 in RAW264.7 cells evoked by the patient serum reached 1.72,1.77,and 1.77 times that of the untreated ones,respectively and no significant changes of the mRNA expression of other TLR subforms induced by the patient serum were found.From the data in the results,it seems to be concluded that the up-regulation of the specific TLRs might constitutes one of the pathogenic basis for the formation of Guizhi Tang syndrome in terms of immunological regulation.3 Effects of serum from healthy volunteers and serum from patients with Guizhi Tang syndrome on the secretion of inflammatory cytokines in RAW 264.7 cellsThe final serum concentrations of 10%,6%,3.5%,2%and 1.3%from healthy volunteers or patients with Guizhi Tang syndrome were chosen and the influence of those samples on the secretion of IL-1,IL-6,TNF-αand IFN-βin RAW264.7 cells was tested, by ELISA,respectively.The results showed that the serum of varing concentrations from healthy volunteers had no promotional effects on the secretion of IL-1β,IL-6,TNF-αand IFN-β.In contrast,IL-1,IL-6,TNF-α,IFN-βin RAW264.7 cells was markedly increased after stimulated with serum from the Guizhi-Tang-syndrome patients and the highest secretion appeared when the serum concentration of 2%was used(p＜0.05 or p＜0.01) except for IFN-β,IL-1,IL-6 and TNF-α.From the data in the results,it appears that the up-regulated secretion of the specific cytokines might constitute another important pathogenic basis for the formation of Guizhi Tang syndrome in terms of immunological regulation.4 Effect of serum containing the four formulas on the mRNA expression of TLRs in the patient serum-incubated RAW 264.7 cellsIn this part,influence of the serum containing the four formulas individually on the viability of RAW264.7 cells was studied.The serum containing the four formulas of various concentrations was given to the RAW264.7 cells along with the addition of Guizhi tang-syndrome patient serum.The concrete concentrations that were selected for each formula are as follows.Mahuang Tang,2.4,1.4,0.8,0.5 and 0.3 g·kg^-1·U^-1;Guizhi Tang, 3.3,2.0,1.2,0.7 and 0.4 g·kg^-1·U^-1;Yinqiao San,6.3,3.8,2.2,1.3 and 0.8 g·kg^-1·U^-1; Shangju Yin,3.9,2.3,1.3,0.8 and 0.5 g·kg^-1·U^-1.After 24 h,the RAW264.7 cells were detected for their viability by the method of MTT.The results showed that the four formulas of varing concentrations had promotional effects on the viability of the cells within the concentrations chosen for assay.On this basis,influence of the serum containing the four formulas on the mRNA expression of TLR1～9 in the patient serum-treated RAW264.7 cells was further studied.The data from the results showed that the serum containing various concentrations of Mahuang Tang had significant inhibition on the up-regulated mRNA expression evoked by the patient serum in a dose-dependent manner(p＜0.05 or p＜0.01) except the lowest concentration had no marked influence on the mRNA expression of TLR-4 and TLR-9.For the groups of Mahuang Tang,apart from the group of lowest concentration,significant inhibition on the up-regulated mRNA expression of TLR-3 and TLR-4 was found in other groups in a dose-dependent manner(p＜0.05 or p＜0.01).For the groups of Yinqiao San and Shangju Yin,apart from the groups of lowest concentrations,significant inhibition on the up-regulated mRNA expression of TLR-4 was found in other groups in a dose-dependent manner(p＜0.05 or p＜0.01).The 50%inhibition concentrations(IC₅₀) on the up-regulated mRNA expression of TLR-3,TLR-4 and TLR-9 were calculated for all the groups of serum containing the four formulas by using the method of Logit.The data from the results showed that for the groups of Guizhi Tang,the value of IC₅₀ on TLR-3,TLR-4,TLR-9 were 5.1%(1.7 g/kg/U), 2.7%(0.9g/kg/U) and 4.1%(1.4g/kg/U) respectively,which were all lowest among all the four formulas.The values of IC₅₀ on TLR-3 and TLR-4 were 8.0%(1.9 g/kg/U) and 7.1% (1.7g/kg/U) respectively for the groups of Mahuang Tang.The value of IC₅₀ on TLR-4 was 5.4%(3.4g/kg/U) and 6.9%(2.6g/kg/U) for the groups of Yinqian San and Shangju Yin, respectively,while the values of IC₅₀ on TLR-3 and TLR-9 could be figured out only by exterior inference.5 Effects of serum containing the four formulas on the secretion of inflammatory cytokines in the patient serum-incubated RAW 264.7 cellsInfluence of the serum containing the four formulas on the up-regulated secretion of cytokines in the patient serum(2%)-treated RAW264.7 cells was further studied by ELISA. The 50%inhibition concentrations(IC₅₀) on the up-regulated secretion of IL-Iβ,IL-6, TNF-αand IFN-βwere close and were lowest among groups of the four formulas.For the groups of Mahuang Tang,the values of IC₅₀ on the secretion of IL-1βand IFN-βwere 8.2%(1.9 g/kg/U) and 7.4%(1.7 g/kg/U) respectively,while the values of IC₅₀ on IL-6 and TNF-αcould be calculated only through exterior inference,which were the same as that on the secretion of IL-1βand IL-6 for the groups of Yinqiao San.The values of IC₅₀ on the TNF-αand IFN-βcould not be calculated for lack of the dose-activity relationships for the Yinqiao San groups.For the groups of Shangju Yin,the IC₅₀ on the secretion of IL-1βwas 4.3%(1.7g/kg/U),while the values of IC₅₀ on the secretion of IL-6 and IFN-βcould be figured out only by exterior inference and the IC₅₀ on TNF-αcould not calculated even through exterior inference for lack of the dose-activity relationships.In general,no significant influence on both the mRNA expression of TLR1-9 and possibly TLR-mediated secretion of cytokines was found in group of healthy volunteer serum.In contrast,the serum from untreated patients with both upper tract infection and Guizhi Tang syndrome could induce up-regulation on both the mRNA expression of TLR-3, TLR-4 and TLR-9 and the secretion of IL-1β,IL-6,TNF-αand IFN-β,while the serum containing the four formulas had significant inhibition on the up-regulated mRNA expression evoked by the patient serum.At the same concentrations as Guizhi Tang,the serum containing Mahuang Tang could only decrease the up-regulated mRNA expression of TLR-3 and TLR-4 and secretion of IL-1βand IL-6,the serum containing Yinqiao San could only decrease the up-regulated mRNA expression of TLR-4 and secretion of IL-1βand the serum containing Shangju Yin could only decrease the up-regulated mRNA expression of TLR-4 and secretion of IL-1βand IL-6.The data from the results reflect and provide experimental basis for the scientific value and rationality of the theories "gearing of formula to disease-complex" and "treatment according to the disease-complex" on the molecular level in TCM.