| 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 six 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. GZT can be used to treat biomedically defined disorders such as pyrexia, influenza, sore throat and inflammation. Recent research showed that it could affect the cellular signal transduction in the hypothalamus by regulating the level of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2) and other enzymes. In order to further analyze its antipyretic mechanism, an active fraction A (Fr.A) was extracted from GZT by a series of procedures, which contains more than 50 prematurely differentiated components such as benzoyl paeoniflorin, liquitigenin, cinnamic aldehyde, etc. The antipyretic effect of Fr.A was similar to that of the whole formula.The aim of the present study was to investigate the effects of phenylallyl compounds of Fr.A (etc. cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid) on a series of anabolizing and catabolizing enzymes of PGE2 which has cross-talk with COX. The effects of combinations of six phenylallyl compounds on catabolizing enzymes of PGE2 were also measured. For this purpose, rat cerebral microvascular endothelial cells (rCMEC) were cultured and incubated in medium containing IL-1β Then the combined effects of phenylallyl compounds on catabolizing enzymes of PGE2 were assayed by isoble. In the meanwhile, we compare the proteome of IL-1β stiumulated rCMEC with phenylallyl compounds and combination compound to identify proteins of interests. This investigation may contribute much to the study of the mechanism of formula.1. Effect of phenylallyl compounds of Fr.A on p-p 38 mitrogen-activated protein kinase pathway in rCMEC with IL-1β inducmentThe rCMEC were sub-cultured into six-well cell culture plates and maintained until sub-confluence. The medium was then replaced by a serum-free culture medium for 24 h prior to the addition of IL-1J3 or other reagents. To determine the time- and dose-dependent effect of IL-lp on p-p 38 mitrogen-activated protein kinase pathway (MAPK), cells were incubated with various concentrations of IL-ip for 4h, or 30ng /ml IL-ip for 0.5, 1, 2,4, 8, 12, 24h. Normalized value of p-p 38 MAPK was measured by western blot. Our results show that p-p 38 MAPK was increased significantly by IL-lp as early as 1 h, reached a maximum by 4 h of incubation and then declined thereafter, IL-ip also increased p-p 38 MAPK in a concentration dependent manner.rCMEC were incubated in the serum-free medium containing 30ng/ml IL-ip in the presence or absence of different concentrations of cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid for 4 h. Then normalized value of p-p 38 MAPK was measured by western blot. Our results show that cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid reduce p-p 38 MAPK with IC50 values of 154.82, 210.64, 67.60, 270.02, 596.94, 1003.29 ug/ml respectively.2. Effect of phenylallyl compounds of Fr.A on sPLA2 activity in rCMEC with IL-lp inducmentThe rCMEC were sub-cultured into 96-well cell culture plates and maintained until sub-confluence. The medium was then replaced by a serum-free culture medium for 24 h prior to the addition of IL-ip or other reagents. To determine the time- and dose-dependent effect of IL-ip on SPLA2 activity, cells were incubated with various concentrations of IL-ip for 4h, or 30ng /ml IL-ip for 0.5, 1, 2, 4, 8, 12, 24h. SPLA2 activity in the media was measured by enzyme-linked immunosorbent assay (ELISA). Our results show that SPLA2 activity was increased significantly by IL-ip as early as 1 h, reached a maximum by 4 h of incubation and then declined thereafter, IL-ip also increased sPLA2 activity in a concentration dependent manner.rCMEC were incubated in the serum-free medium containing 30ng/ml IL-ip in the presence or absence of different concentrations of cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid for 4 h. Then SPLA2 activity in media was measured by ELISA. Our results show that cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid reduce SPLA2 activity with IC50 values of 235.99, 123.35, 213.10, 425.31, 154.86, 468.65 ug/ml respectively.3. Effect of phenylallyl compounds of Fr.A on CPLA2 activity in rCMEC with IL-1JJ inducmentThe rCMEC were sub-cultured into six-well cell culture plates and maintained until sub-confluence. The medium was then replaced by a serum-free culture medium for 24 h prior to the addition of IL-lp or other reagents. To determine the time- and dose-dependent effect of IL-ip on CPLA2 activity, cells were incubated with various concentrations of IL-ip for 12h, or 30ng /ml IL-lp for 0.5, 1, 2, 4, 8, 12, 24h. cPLA2 activity in rCMEC was measured by ELISA. Our results show that CPLA2 activity was increased significantly by IL-1J3 as early as 2 h, reached a maximum by 12 h of incubation and then declined thereafter, IL-ip also increased CPLA2 activity in a concentration dependent manner.rCMEC were incubated in the serum-free medium containing 30ng/ml IL-lp in the presence or absence of different concentrations of cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid for 12 h. Then CPLA2 activity was measured by ELISA. Our results show that cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid reduce cPLA2 activity with IC50 values of 36.44, 91.67, 65.27, 75.60, 699.80, 342.67 ug/ml respectively.4. Effect of phenylallyl compounds of Fr.A on COX mRNA expression in rCMEC with IL-lp* inducmentThe rCMEC were sub-cultured into six-well cell culture plates and maintained until sub-confluence. The medium was then replaced by a serum-free culture medium for 24 h prior to the addition of IL-ip or other reagents. To determine the time- and dose-dependent effect of IL-ip on COX mRNA expression, cells were incubated with various concentrations of IL-ip for 4h, or 30ng/ml IL-lp for 0.5, 1, 2, 4, 8, 12, 24h. Normalized value of COX mRNA was measured by real-time PCR. Our results show that COX-2 mRNA was detected by IL-ip as early as 1 h, reached a maximum by 4 h of incubation and then declined thereafter, IL-ip also increased COX-2 mRNA in a concentration dependent manner. In contrast, COX-1 mRNA was not significantly altered during the incubation condition.rCMEC were incubated in the serum-free medium containing 30ng/ml IL-ip in the presence or absence of different concentrations of cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid for 4 h. Then the COX-2 mRNA expression was measured by real-time PCR. Our results show that cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol,cinnamic acid and 2-methoxycinnamic acid reduce COX-2 mRNA expression with IC50 values of 22189.00, 545.34,434.79, 277.11, 373.71, 12181.88 ug/ml respectively.5. Effect of phenylallyl compounds of Fr.A on COX activity in rCMEC with IL-lp inducmentThe rCMEC were sub-cultured into six-well cell culture plates and maintained until sub-confluence. The medium was then replaced by a serum-free culture medium for 24 h prior to the addition of IL-ip or other reagents. To determine the time- and dose-dependent effect of IL-ip on COX activity, cells were incubated with various concentrations of IL-ip for 12h, or 30ng /ml IL-1J5 for 0.5, 1, 2, 4, 8, 12, 24h. COX activity was measured by ELISA. Our results show that COX-2 activity was increased significantly by IL-ip as early as 8 h, reached a maximum by 12 h of incubation and then declined thereafter, IL-ip also increased COX-2 activity in a concentration dependent manner. In contrast, COX-1 activity was not significantly altered during the incubation condition.rCMEC were incubated in the serum-free medium containing 30ng/ml IL-ip in the presence or absence of different concentrations of cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid for 12 h. Then COX-2 activity in rCMEC was measured by ELISA. Our results show that cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid reduce COX-2 activity with IC50 values of 77.88, 137.16,115.44, 95.70, 2387.82, 18886.55 ug/ml respectively.6. Effect of phenylallyl compounds of Fr.A on 15-PGDH activity in rCMEC with IL-ip inducmentThe rCMEC were sub-cultured into six-well cell culture plates and maintained until sub-confluence. The medium was then replaced by a serum-free culture medium for 24 h prior to the addition of IL-ip or other reagents. To determine the time- and dose-dependent effect of IL-ip on 15-PGDH activity, cells were incubated with various concentrations of IL-ip for 12h, or 30ng /ml IL-ip for 0.5, 1, 2, 4, 8, 12, 24h. 15-PGDH activity was measured by isotopic tracing assay. Our results show that 15-PGDH activity was increased significantly by IL-ip as early as 2 h, reached a maximum by 12 h of incubation and then declined thereafter, IL-ip also increased 15-PGDH activity in a concentration dependent manner.rCMEC were incubated in the serum-free medium containing 30ng/ml IL-ip in the presence or absence of different concentrations of cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid for12 h. Then 15-PGDH activity was measured by isotopic tracing assay. Our results show that cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid reduce 15-PGDH activity with IC50 values of 61.19, 250.87, 31.68, 2089.87, 149.20, 30.14 ug/ml respectively.7. Effect of phenylallyl compounds of Fr.A on PGE2 release of rCMEC with IL-ip inducmcntThe rCMEC were sub-cultured into 96-well cell culture plates and maintained until sub-confluence. The medium was then replaced by a serum-free culture medium for 24 h prior to the addition of IL-lf3 or other reagents. To determine the time- and dose-dependent effect of IL-ip on PGE2 production, cells were incubated with various concentrations of IL-1(3 for 4h, or 30ng /ml IL-lp for 0.5, 1, 2, 4, 8, 12, 24h. PGE2 release in rCMEC was measured by ELISA. Our results show that PGE2 production was increased significantly by IL-ip as early as 4 h, reached a maximum by 12 h of incubation and then declined thereafter, IL-ip also increased PGE2 production in a concentration dependent manner.rCMEC were incubated in the serum-free medium containing 30ng/ml IL-ip in the presence or absence of different concentrations of cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid for 12 h. Then PGE2 production was measured by ELISA. Our results show that cinnamaldehyde, 2-methoxycinnamaldehyde, cinnamyl alcohol, 2-methoxycinnamyl alcohol, cinnamic acid and 2-methoxycinnamic acid reduce PGE2 release with IC50 values of 50.22, 55.66, 58.77, 120.01, 300.81, 1431.54 ug/ml respectively.8. Combined effects of phenylallyl compounds on catabolizing enzymes of PGE2 in rCMEC with IL-ip inducmentThe effect of combination of phenylallyl compounds isolated from Fr.A on p-p 38 MAPK, sPLA2 activity, cPLA2 activity, COX-2 mRNA, COX -2 activity, 15-PGDH activity and PGE2 production were measured. Date show that combination of 6 compounds reduces p-p38 MAPK, sPLA2 activity, cPLA2 activity, COX-2 mRNA, COX-2 activity, 15-PGDH activity and PGE2 production with IC50 value of 100.32, 277.13, 72.57, 280.13, 76.52, 92.02, 48.79|xg/ml respectively. Then the combined effects of 6 compounds on p-p38 MAPK, sPLA2 activity, cPLA2 activity, COX-2 mRNA, COX-2 activity, 15-PGDH activity and PGE2 production were assessed by isobole. The q values were 0.87, 1.25, 1.68, 0.61, 0.87, 1.84 and 0.92 respectively. Our results show that the interactions of the 6 compounds to p-p 38 MAPK, COX mRNA expression, COX activity, 15- PGDH activity and PGE2 production were synergism, while theinteractions to SPLA2 and CPLA2 were antagonism.9. Changes in proteome of IL-lp* stimulated rCMEC with phenylallyl compounds of Fr.A and combination compoundPGE2 is an important medium that induces pyrexia. IL-ip, one of the endogenous pyrogen (EP) , could stimulate rCMEC to synthesis and release PGE2 to affect thermoregulatory center. By using 2D, 31 protein spots, about 3% of total valid spots in 2D gel, were found to express differently in control group and IL-1P stimulated group. 11 of 31 spots were changed after added with 6 compounds and the combination compound, which suggested that those protein spots may be targets of Fr.A.In conclusion, IL-ip induced PGE2 release in rCMEC, which shows that PGE2 in hypothalamus is mostly released from rCMEC. Our result partly supports the working hypothesis that EP (etc. IL-1) in blood induces PGE2 release in rCMEC, then PGE2 entrance into hypothalamus via paresecretion, and this is followed by PGE2-induced neuronal mechanisms to elevate the temperature set-point, resulting fever. Effects of 6 phenylallyl compounds isolated from Fr.A on a series of anabolizing and catabolizing enzymes of PGE2in rCMEC with IL-ip inducement were also investigated with molecular biology method. Results show that there are differences among 6 phenylallyl compounds on PGE2 metabolism, and their final effect on PGE2 release are also diffence. While the combined effect of 6 compounds on PGE2 release is synergism. Our data initially suggest the basic definition of substance and feature of traditional Chinese medicine and its prescriptions: "Traditional Chinese medicine and its prescriptions are composed of groups of active substances, which enter into compatibility and combination according to certain requirements and exert their functions on human body through multi-targets and various ways."...
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