Studies Of Phenylallyl Compounds Of Guizhi-Tang On Structure-activity Relationship And PGE₂ Release With TRPV4 Expression

Chinese formula has more than a thousand years' history. Guizhi-Tang (GZT) is one of the most famous formula in the classical works Shang-han-lun, which contains five herbal ingredients: Ramulus of cinnamomum cassia Presl, Radix of paeonia lactiflora Pall., Rhizoma of zingiber officinale Rosc, Fructus of ziziphus jujuba Mill, and Glycyrrhiza uralensis Fisch. In tradititonal Chinese medicine (TCM) theory, GZT expels pathogenic factors from muscles and skin, and regulates Ying and Wei, which increases patient's ability to protect from invasion by external pathogenic influence. Since 1985, many kinds of experiments have been carried out in our lab focusing on the pharmacological effects of the GZT. It was demonstrated that the active fration A (Fr.A), extracted from GZT, has the obvious antipyretic effect and could decrese the contents of PGE₂ in the in the hypothalamus of the febrile rats. Using MS, GC-MS and chemical separation, it confirmed that Fr.A contains more than 10 of phenylallyl compounds, which is the main ingredient of Fr.A.In recent years, several genes related to temperature of biological molecules had been cloned, which is encoded by transient receptor potential (TRP) family. It had be found that TRP vanilloid 4 (TRPV4) felt thermal stimulation within physiological range (>27℃) and it expressed in the hypothalamus, which suggested that TPRV4 might be a thermal transfer device of hypothalamus region. TCM active component on the role of TRP involved in the regulation of body temperature is worth exploring. We hope to further clarify the role of phenylallyl compounds antipyretic mechanism, so as to reveal "Chinese medicine and its active group take action through the multi-targets and multi-channels".Regarding the critical role of cerebral microvascular endothelial cells and PGE2 in the pyrexia mechanism, mouse cerebral microvascular endothelial cells (b.End3) were adopted in our experiments The aim of the study was to observe the effects of phenylallyl compounds on PGE2 release in b.End3 cells stimulated by IL-1β, and to analyze their structure-activity relationships. On the basis of the above results, cinnamaldehyde as the best inhibitory compound was adopted for the following experiments in the present study, the antipyretic-analgesic and anti-inflammatory effects of cinnamaldehyde were observed, and the relationship of PGE2 release and TRPV4 expression was analyzed by using Real-time RT-PCR technique, Western blot, fluorescence immunocytochemistry and ELISA method, respectively.1. Structure-activity relationships of phenylallyl compounds PGE₂ release in mouse cerebral microvascular endothelial cells induced by IL-1βOn the basis of the studies of GZT or Fr.A, we collected or synthesized a total of 33 phenylallyl compounds. The aim of the study was to observe the effects of phenylallyl compounds on PGE2 release in b.End3 cells stimulated by IL-1β, and to analyze their structure-activity relationships. Releasing PGE2 form b.End3 cells induced by IL-1β, different concentrations of phenylallyl compounds were added respectively and production of PGE₂ in the conditioned media was measured by ELISA, and calculated the 50% inhibitory concentration (IC₅₀) of PGE₂. Phenylallyl compounds exhibited inhibitory activities at different extent to PGE₂ release in b.End3 cells stimulated by IL-1β. Close relationships were shown between the inhibitory activities and the location/ number of the side chain substituent groups.2. Experimental studies on the antipyretic, analgesic and anti-inflammatory actions and antipyretic mechanism of cinnamaldehydeIn this study, cinnamaldehyde as representative of phenylallyl compounds and the aim was to observe the antipyretic-analgesic and anti-inflammatory effects of cinnamaldehyde. The rats febrile models were induced by injecting fresh yeast subcutaneously. The pain models were established by acetic acid and hot plate in mice. The mice inflammatory models were caused with dimethylbenzene (auricle smearmethod) and HAc-induced peritoneal capillary permeability, respectively. Results showed that cinnamaldehyde depressed the fervescence by yeast effectively in rats and remarkable decreased the contents of PGE₂ in the hypothalamus of febrile rats. It could inhibit the pain caused by acetic acid and hot plate evidently in mice. At the same time, it was found that the mouse's ear edema induced by dimethylbenzene and the capillary permeability were significantly suppressed by cinnamaldehyde. In conclusion, cinnamaldehyde has remarkable antipyretic-analgesic and anti-inflammatory effects and antipyretic effect might be related with the interferencing the content of PGE₂.3. Study on the antipyretic mechanism of cinnamaldehyde—the relationship of PGE₂ release and the expression of TRPV4In the present study, b.Ende3 cells were used for investigate object. Ruthenium red (RR), a known TRPV4 inhibitor, was used as a tool to block TRPV4. The relative expression of TRPV4 mRNA were determined by Realtime RT-PCR, the expression of TRPV4 protein were determined by Western blot and fluorescence immunocytochemistry, and the release levels of PGE₂ were determed by ELISA. The results suggested that IL-1βsignificantly increased production of PGE₂ and cinnamaldehyde evidently decreased IL-1β-induced PGE₂ production, while RR showed no inhibitory effect on that. Moreover, we identified TRPV4 that was expressed at the mRNA and protein levels in b.End3 cells. IL-1βcould up-regulation the expression of TRPV4, RR and cinnamaldehyde could down-regulation the high expression of mRNA and protein of TRPV4 by IL-1βinduced in b.End3 cells. In conclusion, PGE₂ release in End.3 cells induced by IL-1βis sensitive to inhibition by cinnamaldehyde, which indicate that TRPV4 wasn't directly involved PGE₂ release and the effect of body temperature regulation of TRPV4 might be achieved through other pathways.