Effects Of Diaphoretic Recipe, Heat-clearing Recipe And Their Related Components On TLRs And Downstream Signaling Transduction Pathway

Toll-like receptors (TLRs) owe their name to a closely related receptor called Toll,first identified in Drosophila. TLRs are typeâ… transmembrane receptors, at least 11 TLRshave been identified in humans and 13 TLRs in mice. As a member of pattern-recognitionreceptors (PPRs), TLRs mainly recognize pathogen associated molecule pattern (PAMP).certain TLRs seem to recognize specific endogenous ligands, for example, TLR4 mainlyrecognizes lipopolysaccharide (LPS), a major gram-negative bacterial cell-wall component,and derivants of lipoid A, TLR3 mainly recognize double-stranded RNA (dsRNA).WhenTLRs recognize and specific bind with their ligands, they initiate two major intracellularsignaling pathways, Myeloid differentiation factor (MyD88) -dependent and MyD88-independent pathways, activate a series of protein cascade reactions of the signalingdownstream, transmit signals into nucleus, lead to the activation of nuclear factor-kappa B(NF-?B), activate protein-1, interferon regulatory factors, induce the synthesis and releaseof corresponding cytokines such as interleukin (IL)-1, IL-2, tumor necrosis factor-α(TNF-α) and interferon, as a result, stimulate a series of immune event according todifferent pathogenic microorganism. Researches certified that many kinds of immunedefense reaction needed TLRs, TLRs not only provide the first line of host defenseresponse against microbes, initiate early phage of innate immunity, but also shape theadaptive immunity, play the "bridge" action between innate and adaptive immunity.The immune response of organism plays the "double-edged sword" action, that is tosay, when the immune response is weak, the organism is easily to be infected with bacteriaor virus; but over activated responses of immune action is dangerous to the host, whichlead to the aggravation of pathogenetic condition as exemplified by sepsis. Immuneresponse regulation has been one of the most important methods on clinic to prevent andcure diseases. Studies showed that some Chinese medicine strengthened the immuneresponsibility of the organism, but some Chinese medicine inhibited the over activatedimmune action to cure diseases. As main receptors of PAMP recognization, TLRs is veryimportant to the defence reaction of anti-infection of the organism. As a matter of course,researchers paid close attention to the relationship between Chinese medicine and TLRs.The prescriptions for relieving exterior syndrome refer to those that are chieflycomposed of traditional Chinese drugs having the therapeutic effects of inducingdiaphoresis, expelling pathogenic factors from the muscles and skin as well as promotingeruption. Modern research showed that can be used to treat viral infectious diseases. Heat-clearing prescriptions refer to those that are chiefly composed of heat-clearingdrugs for the treatment of interior purging, blood-cooling and toxin-removing. Now it iswidely used in clinics for fever and inflammation caused by bacterial infection.In this study, we chose the drug sera of Guizhi Tang (GZT), the pungent and warmrecipe, Shensu Yin (SSY), the tonic recipes for relieving exterior syndrome,Huanglianjiedu Tang (HLJD) and Qingkailing (QKL), which are prescriptions for cleaningaway heat and toxic material, as the experimental drugs, chose murine pulmonarymacrophage cell line RAW264．7 cells as the study object, analyzed the influences of thesefour formulas to TLR3, TLR4 and the downstream signaling components such as MyD88,Tumor necrosis factor receptor-associated factor-6 (TRAF-6) , TIR domain-containingadaptor inducing IFN-β(TRIF), Toll-like receptor-associated molecule (TRAM) andrelated gene-induced productions. This study also observed the influence ofCinnamaldehyde, Panaxsaponin Rg1, Berberine and Baicalin, the principal activeconstituent of the formulas above, to TLR3, TLR4 and their downstream signalingcomponents, discussed the immunological basis of these formulas and their chief activecomponents in interfering infectious diseases. The experimental results are as follow:1 The influence of diaphoretic recipe and heat-clearing recipe to the secretion ofTNF-αand IFN-βinduced by LPS or Poly (I:C) on RAW264．7 cells.RAW264．7 cells were stimulated with LPS (5μg·mL^-1 , same as follow) or Poly (I:C)(50μg·mL^-1 , same as follow), then interfered with drug sera of the indicated formulas,measured the contents of TNF-αand IFN-βin the cell culture supernate, calculated the 50 %inhibiting concentration (IC₅₀ ) of every formula with Logit method. According to theresults of the experiments, we chose the dose as follow to do next experiments: when LPSstimulated: GZT: 4．2 g·kg^-1·U^-1 ; SSY: 11．2 and 2．5 g·kg^-1·U^-1 ; HJJD: 2．4 g·kg^-1·U^-1 ;QKL: 1．9 and 0．6 g. kg^-1·U^-1 ; when Poly (I:C) stimulated: GZT: 4．2 g·kg^-1·U^-1 ; SSY: 4．8g·kg^-1·U^-1 ; HLJD: 6．0 and 2．9 g·kg^-1·U^-1 ; QKL: 3．2 and 1．5 g·kg^-1·U^-1 respectively.2 The influence of diaphoretic recipe and heat-clearing recipe to the expression ofTLR3 and TLR4 on RAW264．7 cells.RAW264．7 cells were stimulated with LPS or Poly (I:C), then interfered with drug seraof GZT, SSY, HLJD or QKL, analyzed the mRNA and protein expression of TLR3 and TLR4．The results showed that Poly (I:C) up-regulated the expression of TLR3 mRNA, LPSup-regulated the expression TLR4 mRNA respectively. When cells were induced by LPSor Poly (I:C), the proteins of TLR3 and TLR4 also increased too. This certified therecognition that Poly (I:C) is the special ligand of TLR3, and LPS is the special ligand ofTLR4．Drug sera of GZT and SSY degraded the over expression of TLR3 mRNA (P<0．01) ,but they had no effect on the mRNA expression of TLR4; In contrast, HLJD and QKL drug sera had no influence on the mRNA expression of TLR3, but inhibited the mRNAexpression of TLR4 markedly.The effects of these formulas on the TLR3 and TLR4 proteins were similar to that ofmRNA, but the inhibition ratios were much higher. For example, the inhibition ratios ofGZT and SSY drug sera to TLR3 proteins were 74．2 % and 55．3 %, which were muchhigher than that of mRNA, seemed to indicate that these formulas had different extent, buttransparent inhibiting effect on TLRs genetic transcription activity and correspondingproteins.3 The influence of diaphoretic recipe and heat-clearing prescriptions to the expressionof MyD88 and TRAF-6 on RAW264．7 cells.As we all know, MyD88 and TRAF-6 are essential adaptor proteins ofMyD88-dependent signaling transduction pathway. Both LPS and Poly (I:C) induced overexpression of MyD88 mRNA and protein (p<0．01) . LPS also induced the over expressionof TRAF-6 mRNA and protein.GZT drug serum significantly down-regulated the over expression of MyD88 mRNAand protein (P<0．01) ; both GZT and SSY drug sera decreased the Poly (I;C)-inducedMyD88 expression (P<0．05 or P<0．01) , these indicated that GZT and SSY influenced theMyD88-dependent pathway.Both HHJD and QKL drug sera markedly degraded the expression of LPS-inducedMyD88 and TRAF-6 (P<0．01) , QKL drug serum also cut down the over expression of Poly(I:C)-induced MyD88．4 The influence of diaphoretic recipe and heat-clearing prescriptions to the expressionof TRAM and TRIF on RAW264．7 cells.TRAM and TRIF are essential adaptor proteins of MyD88-independent signalingtransduction pathway. When cells were stimulated by LPS or Poly (I:C), the mRNAexpression of TRAM and TRIF increased markedly.GZT and SSY drug sera obviously inhibited the LPS-induced TRAM and TRIFmRNA expression (P<0．05 or P<0．01) . When cells were induced by Poly (I:C), GZT andSSY drug sera only inhibited the expression of TRAM mRNA, but showed no influence onthe expression of TRIF mRNA.HLJD and QKL drug sera obviously inhibited the mRNA expression of LPS-and Poly(I:C)-induced TRAM (P<0．05 or P<0．01) , but the inhibiting effect of both drug sera was alittle lower, QKL had no effect on the LPS-induced TRIF.The results of above indicated that diaphoretic recipe played TLR3-antagonist-likeaction, and heat-clearing recipe played TLR4-antagonist-like action, this maybe thedifferent immune basis for diaphoretic recipe and heat-clearing recipe, in dealing withinfectious diseases. 5 The influence of Cinnamaldehyde, Panaxsaponin Rg1, Berberine and Baicalin tothe secretion of inflammatory factors on RAW264．7 cells.In this part, we observed the damage of Cinnamaldehyde, Panaxsaponin Rg1,Berberine and Baicalin to RAW264．7 cells. The cells were stimulated with LPS or Poly(I:C) respectively, and added monomers above with different concentrations:Cinnamaldehyde: 302．66, 181．60, 108．96 and 65．07μmol·L^-1 ; Panaxsaponin Rg1: 3．61,2．17, 1．33 and 0．84μmol·L^-1 ; Berberine: 11．89, 7．14, 4．16 and 2．68μmol·L^-1 ; Baicalin:49．36, 29．61, 17．81 and 10．73μmol·L^-1 . 24 h later, observed the cells damage extent withMTT method. The result indicated that all of these four monomers had no influence on thecells activity.Using the data of above, we calculated the IC₅₀ of these four monomers to TNF-αandIFN-β. When cells were stimulated by LPS or Poly (I:C), the IC₅₀ to TNF-αof everymonomers were: Cinnamaldehyde: 98．67 and 82．48μmol·L^-1 ; Baicalin: 17．38 and 4．64μmol·L^-1 ; Panaxsaponin Rg1: 0．98 and 1．05μmol·L^-1 ; Berberine: 2．53 and 2．74μmol·L^-1 ;IC₅₀ to IFN-βwere: Cinnamaldehyde: 74．30 and 74．91μmol·L^-1 ; Baicalin: 9．14 and 16．46μmol·L^-1 ; Panaxsaponin Rg1: 0．82 and 0．87μmol·L^-1 ; Berberine: 2．35 and 2．50μmol·L^-1 .In this paper, we determined the interfere drug dosage as follow: Cinnamaldehyde: 75．67μmol·L^-1 ; Baicalin: 17．17μmol·L^-1 and 8．58μmol·L^-1 ; Panaxsaponin Rg1: 1．08μmol·L^-1 ;Berberine: 2．68μmol·L^-1 .6 The influence of Cinnamaldehyde, Panaxsaponin Rg1, Berberine and Baicalin tothe expression of TLR3 and TLR4 on RAW264．7 cells.Similar to that of part one, LPS specially induced the expression of TLR4, and Poly(I:C) specially induced the expression of TLR3．Cinnamaldehyde and Baicalin inhibited the expression of TLR3 and TLR4 (p<0．05 orp<0．01) , Panaxsaponin Rg1 had no influence on the expression of TLR3 and TLR4,Berberine only blocked the expression of TLR4．7 The influence of Cinnamaldehyde, Panaxsaponin Rg1, Berberine and Baicalin tothe expression of MyD88 and TRAΓ-6 on RAW264．7 cells.Cinnamylaldehyde had obvious inhibitory action on MyD88 and TRAF-6's overexpression induced by LPS or Poly (I:C) respectively; Ginsenoside Rg1 and Baicalin onlyblocked the expression of Poly (I:C)-induced MyD88(p<0．05) , but they had no effect onthe expression of LPS-induced MyD88 and TRAF-6．On the contrary, Berberine notablyinhibited the over expression of LPS-induced MyD88 and TRAF-6, but have no effect onPoly (I:C)-induced MyD88． 8 The influence of Cinnamaldehyde, Panaxsaponin Rg1, Berberine and Baicalin tothe expression of TRAM and TRIF on RAW264．7 cells.Cinnamylaldehyde had no influence on the over expression of TRAM and TRIFwhich were induced by LPS or Poly (I:C); Ginsenoside Rg1 significantly inhibited the overexpression of TRAM and TRIF which were induced by LPS or Poly (I.C) (p<0．05 orp<0．01) ; Berberine had no influence on the over expression of LPS-induced MyD88 andTRAF-6, but it did block the expression of Poly (I. C)-induced TRAM and TRIF; Differentwith that of Berberine, Baicalin inhibited the over expression of TRAM and TRIF mRNAinduced by LPS (p<0．05 or p<0．01) , but had no effect on the mRNA expression of Poly(I:C)-induced TRAM and TRIF.According to the effects of Cinnamylaldehyde on MyD88 and TRAF-6, it appearedthat we might draw a conclusion as follow: Cinnamylaldehyde played its pharmacody-namic action through inhibiting TLR3, TLR4 and the signaling components on MyD88-dependent transduction pathway; Panaxsaponin Rg1 didn't inhibit the over expression ofTLR3 and TLR4 directly, but markedly blocked the expression of TRAM and TRIF, it alsoinhibited the expression of Poly (I:C)-induced MyD88．These indicated that PanaxsaponinRg1 played its pharmacological action by regulating MyD88-independent signalingtransduction pathway; Berberine not only inhibited the expression of TLR4, but alsoinhibited MyD88-dependent signaling transduction pathway when LPS stimulated. WhenPoly (I:C) stimulated the cells, Berberine played its pharmacodynamic action mainly byregulating the MyD88-independent signaling transduction pathway. Baicalin inhibited theexpression of TLR3 and TLR4, as to the effect on the downstream signaling transductionpathway, it depends on the exogenous stimulating factors. When the cells were stimulatedby LPS, Baicalin obviously influenced MyD88-dependent and MyD88-independentsignaling transduction pathway. When the cells were stimulated by Poly (I:C), Baicalinmainly effected the MyD88-dependent signaling transduction pathway.In one word, Cinnamylaldehyde remarkably influenced MyD88-dependent signalingtransduction pathway, played the TLR3-and TLR4-antagonist-like action; GinsenosideRg1 mainly influenced the MyD88-independent pathway. Berberine's different influenceto TLR signaling transduction components depended on different stimulating factors.When cells were stimulated by LPS, Berberine mainly effected MyD88-dependentpathway. But when cells were stimulated by Poly (I:C), Berberine influenced the processof MyD88-independent signaling transduction pathway, played TLR4-antagonist-likeaction; When LPS stimulated the cells, Baicalin mainly effected MyD88-independentpathway; But when Poly (I:C) stimulated, it influenced the MyD88-dependent pathway.Similar to that of Cinnamylaldehyde, Baicalin also played the TLR3-and TLR4-antagonist-like action.In addition, comparing the influences of Chinese medicinal formulas with that of corresponding chemical monomers to TLRs, we found there were difference between theformulas and monomers. For example, drug serum of GZT inhibited the expression ofTLR3 directly, blocked the expression of MyD88-independent signaling transductioncomponents, played TLR3-antagonist-like action, Cinnamaldehyde inhibited theexpression of both TLR3 and TLR4, influenced the MyD88-dependent signalingtransduction pathway. This result seemed to indicated that monomer couldn't substituteChinese formulas, the effect of Chinese prescriptions is more or lesser than that ofmonomer, perhaps it is because of the integration effect of formula's multicomponents andmulticomponents. In this integrating process, part of formula's effects may be weakened,even vanished. In contrary, new pharmacological effect may be emerge in this process.