Synthesis and evaluation of ursolic acid-based 1,2,4-triazolo[1,5-a]pyrimidines derivatives as anti-inflammatory agents.

Here, two series of novel ursolic acid-based 1,2,4-triazolo[1,5-a]pyrimidines derivatives were synthesized and screened for their anti-inflammatory activity by evaluating their inhibition effect of using LPS-induced inflammatory response in RAW 264.7 macrophages in vitro; the effects of different concentrations of the compounds on the secretion of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6 were evaluated. Their toxicity was also assessed in vitro. Results showed that the most prominent compound 3 could significantly decrease production of the above inflammatory factors. Docking study was performed for the representative compounds 3, UA, and Celecoxib to explain their interaction with cyclooxygenase-2 (COX-2) receptor active site. In vitro enzyme study implied that compound 3 exerted its anti-inflammatory activity through COX-2 inhibition.

Circ_0000518 Promotes Macrophage/Microglia M1 Polarization via the FUS/CaMKKß/AMPK Pathway to Aggravate Multiple Sclerosis.

Evidence has shown that circ_0000518 is upregulated in peripheral of multiple sclerosis (MS) patients, suggesting that it may play an important role in the progression of MS. However, its specific mechanism in MS progression is unclear. In this study, the human microglial clone 3 (HMC3) cells were treated with 100 ng/mL of LPS for 24 h, then the short hairpin RNA against hsa_circ_0000518 (sh-hsa_circ_0000518) was transfected into cells and incubated for 48 h. We found increased circ_0000518 expressions, increased apoptosis and oxidative stress, increased M1 phenotype marker expression, and decreased M2 phenotype marker expression in cells, and that interfering with circ_0000518 expression reversed the effect of LPS on HMC3 cells. Online bioinformatics database analysis indicated that FUS is an RNA binding protein of circ_0000518. Next, we observed increased FUS expression in LPS treated HMC3 cells, and interfering with FUS expression reduced LPS triggered apoptosis and oxidative stress, decreased M1 phenotype marker expression, and promoted M2 phenotype marker expression. Mechanistic studies revealed that interfering with FUS promoted the polarization of HMC3 cells from the M1 phenotype to the M2 phenotype via activation of CaMKKß/AMPK-PGC-1α pathway, whereas this promoting effect was counteracted by STO-609. In an experimental autoimmune encephalomyelitis (EAE) mouse model, we observed that circ_0000518 knockdown reduced circ_0000518 and FUS expression in brain and spinal cord tissues, reduced neurological scores in mice, and alleviated inflammatory cell infiltration in the CNS. Summarily, our study identified that circ_0000518 promotes macrophage/microglial M1 polarization through the FUS/CaMKKß/AMPK pathway and aggravates MS.

Effect of Dihydroartemisinin on Plasmodium NADH-Dependent Glutamate Synthase: The Implication in Malaria Management.

Artemisinin and its analogues (ARTs) are currently the most effective anti-malarial drugs, but the precise mechanism of action is still highly controversial. Effects of ARTs on Plasmodium genes expression are studied in our Lab. The overexpression of an interesting amidotransferase, NADH-dependent glutamate synthase (NADH-GltS) was found in treated by dihydroartemisinin (DHA). The increased expression occurred not only from global transcriptomics analysis on the human malaria parasite Plasmodium falciparum (P. falciparum) 3D7 and gene expression screening on all of iron-sulphur cluster proteins from P.f. 3D7 in vitro but also from Plasmodium berghei (P. berghei) ANKA in mice. Influence of DHA on NADH-GltS was specifically at trophozoite stage of P. falciparum and in a dose-dependent manner below the effective doses. L-glutamine (Gln) and L-glutamate (Glu) are the substrate and product of NADH-GltS respectively. Azaserine (Aza) is specific inhibitor for NADH-GltS. Experimental data showed that Glu levels were significantly decreasing with DHA dose increasing but NADH-GltS enzyme activities were still remained at higher levels in parasites, and appropriate amount of exogenous Glu could significantly reduce anti-malarial action of DHA but excessive amount lost the above effect. Aza alone could inhibit proliferation of P. falciparum and had an additive effect in combination with DHA. Those results could suggest that: Glutamate depletion is one of the anti-malarial actions of DHA; overexpression of NADH-GltS would be a feedback pattern of parasite itself due to glutamate depletion, but not a direct action of DHA; the "feedback pattern" is one of protective strategies of Plasmodium to interfere with the anti-malarial actions of DHA; and specific inhibitor for NADH-GltS as a new type of anti-malarial agents or new partner in ACT might provide a potential.

Extraction of polysaccharides from Fomes officinalis Ames and their antitumor activity.

The aim of this study was to optimize the extraction parameters of Fomes officinalis Ames polysaccharides (FOAPs) and evaluate their antitumor activity. FOAPs were extracted using the hot water extraction, acid extraction and alkali extraction methods, respectively. Alcohol precipitation and acetone washes were conducted to separate and purify the FOAPs. The FOAP content was determined using the phenol-sulfuric acid method. The effects of raw material particle size, extraction time and material-liquid ratio on the yield of FOAPs were investigated, and the effects of FOAPs on the immune function of S180 tumor-bearing mice and their antitumor activity were evaluated. The yield of FOAPs obtained with the hot water extraction method was higher compared with the yields of the other methods. The optimum extraction conditions were as follows: a raw material particle size of 24 mesh; an extraction time of 2.5 h; and a material-liquid ratio of 1 g:12 ml. Under these conditions, the yield of FOAPs was 1.13%. FOAPs significantly inhibited tumor growth and enhanced the immune function in S180 tumor-bearing mice. FOAPs extracted using the hot water extraction method have antitumor activity.

Reciprocal effects of Guizhi decoction to the Guizhi decoction syndrome by toll-like receptor mRNA expression and cytokines secretion.

OBJECTIVE: To explore the pathological mechanisms of Guizhi Decoction () syndrome and the therapeutic molecular mechanisms of the Guizhi Decoction, Mahuang Decoction (), Sangju Decoction ( ) and Yinqiao Powder (), as well as the potentially biological basis that Guizhi Decoction is most effective only for the patients with Guizhi Decoction syndrome in clinical practice. METHODS: We first got serum samples from the patients suffering from both upper respiratory tract infection and Guizhi Decoction syndrome identified by the doctors of Chinese medicine (CM) in the clinic. Four formulas with therapeutic actions of pungent warmth or pungent coolness for superficial syndromes were chosen and four kinds of rat serum samples each containing one of the above-mentioned herbal formulas were collected, then the effects of Guizhi Decoction syndromes' patient serum as well as the effects of sera containing the formulas after being stimulated by the patient serum samples on both the mRNA expression of certain toll-like receptor (TLR) subtypes and the release of some inflammatory cytokines in RAW264.7 cells were tested and analyzed in vitro. RESULTS: The expression of TLR-3, TLR-4 and TLR-9 mRNA among the 9 tested TLR subforms were up-regulated in the macrophages stimulated by the sera from untreated upper respiratory infection patients with the Guizhi Decoction syndrome (symptomcomplex). The products such as interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-ß from stimulated macrophages through TLR signaling pathways were also increased correspondingly. Interestingly, the changes induced by the Guizhi Decoction syndrome patients' sera were masked significantly after the macrophages were incubated with the sera from donors treated with Guizhi Decoction. Similarly, the three other exterior-releasing formulas were all effective in reversing the up-regulated changes of certain TLR subforms to different degrees, but both the number of targeted TLRs and efficacy of them seemed to be inferior to that of Guizhi Decoction. CONCLUSION: Evidence from these experiments might contribute to the scientific explanation of both the pharmacological mechanisms of Guizhi Decoction and also the CM theory that Guizhi Decoction is specifically prescribed for the treatment of Guizhi Decoction syndrome (The gearing formula to the symptom-complex).

The role of cytochrome c on apoptosis induced by Anagrapha falcifera multiple nuclear polyhedrosis virus in insect Spodoptera litura cells.

There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell-free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.

[Cinnamaldehyde decreases interleukin-1beta induced PGE2 production by down-regulation of mPGES-1 and COX-2 expression in mouse macrophage RAW264.7 cells].

Cinnamaldehyde was shown to have significant anti-inflammatory and anti-pyretic actions in studies from both others' and our lab. Prostaglandin E2 (PGE2) plays a key role in generation of these pathological states, while PGE, synthase-1 (mPGES-1) is one of crucial biological elements in the process of PGE2 production. And as a downstream inducible terminal prostaglandin synthase of COX-2, mPGES-1 is now regarded as a more promising novel drug target than COX-2 and is attracting more and more attention from both academia and pharmaceutical industry. The purpose of present study was to further investigate the anti-inflammatory and antipyretic molecular mechanisms of cinnamaldehyde based on the mouse macrophage cell line RAW264. 7 in vitro. The PGE2 was identified by using the method of enzyme-linked immunosorbent assay (ELISA) and the expression of COX-2 and mPGES-1 at mRNA and protein levels was detected by the Real-time PCR and Western blotting methods respectively. The experimental results suggested that cinnamaldehyde could evidently reverse the increased production of PGE2induced by IL-1beta. Moreover, the up-regulated expression levels of mPGES-1 and COX-2 were significatly decreased. Together, these results provide compelling evidence that the down-regulated actions to both the production of PGE2 as well as the expression of mPGES-I might account for, at least in part, the anti-inflammatory and anti-pyretic effects of cinnamaldehyde.

Simultaneous determination of trace arsenic and antimony in fomes officinalis ames with hydride generation atomic fluorescence spectrometry.

A robust, accurate and sensitive analytical procedure was developed for the simultaneous determination of trace arsenic and antimony in fomes officinalis ames by hydride generation atomic fluorescence spectrometry. The parameters were studied systematically, such as acid concentration of the reaction medium, flow rate of the carrier gas and shield gas, the atomizer of height, etc. Ascorbic acid, potassium iodide and thiourea were used as reducer or masking agents to enhance the generation efficiency of the volatile species of As and Sb. In the presence of thiourea, potassium iodide and ascorbic acid, the influences of some coexisting elements on the determination of arsenic and antimony were investigated.

Preparative isolation of three anthraquinones from Rumex japonicus by high-speed counter-current chromatography.

Three anthraquinones--emodin, chrysophanol, and physcion--were successfully purified from the dichloromethane extract of the Chinese medicinal herb Rumex japonicus by high-speed counter-current chromatography (HSCCC). The extract was separated with n-hexane-ethanol-water (18:22:3, v/v/v) as the two-phase solvent system and yielded 3.4 mg of emodin, 24.1 mg of chrysophanol, and 2.0 mg of physcion from 500 mg of sample with purities of 99.2 %, 98.8% and 98.2%, respectively. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the chemical structures of the three anthraquinones were confirmed by ¹H-NMR and ¹³C-NMR analysis. This is the first time these anthraquinones have been obtained from R. japonicus by HSCCC.

[Effects of ingredients from Chinese herbs with nature of cold or hot on expression of TRPV1 and TRPM8].

OBJECTIVE: To study the effects of the ingredients from Chinese herbs with the nature of cold or hot on the expression of TRPV1 and TRPM8. METHOD: The effects of ingredients from herbs on primary culture DRG neurons are observed in vitro. The expression quantity of gene is detected by the method of real time PCR. the 2 (-deltadeltaCT) method is applied to analyze the data. RESULT: Ingredients from herbs with the nature of cold up-regulate the expression level of TRPV1 and down-regulate that of TRPM8, especially under the temperature condition of 39 degrees C; while ingredients from herbs with the nature of hot up-regulate the expression level of TRPM8 and down-regulated that of TRPV1, which is more significant under the temperature condition of 19 degrees C. CONCLUSION: The regulatory changes of TRPV1 and TRPM8 mRNA expression induced by the chemical ingredients might be related to the cold and hot natures of the herbs from which the ingredients are extracted. And this could be one of the therapeutic mechanisms for the treatment of Chinese herbal medicines to cold- and heat-related diseases.

[Expression of hTERT mRNA in Anip973 and Anip973/NVB cell detected by quantitative real-time PCR].

BACKGROUND AND OBJECTIVE: Human telomerase reverse transcriptase is the catalytic subunit of telomerase, and its activity is correlated with cell's sensitivity to chemotherapy. The aim of this study is to investigate the differential expression of human telomerase reverse transcriptase (hTERT) mRNA in human lung adenocarcinoma cell line Anip973 and Anip973/NVB, and to observe the correlation between hTERT mRNA and drug-resistance. METHODS: The real-time fluorescence quantitative RT-PCR was used to detect the change of hTERT mRNA in human lung adenocarcinoma drug-resistant cell Anip973/NVB and parental cell Anip973 treated by NVB. RESULTS: In the control group, the expression of hTERT mRNA showed no significant difference between drug-resistant cell Anip973/NVB and parental cell Anip973. After been treated by NVB, the expression of hTERT mRNA in parental cell was significantly decreased (P < 0.01), and drug-resistant cell Anip973/ NVB had no evidently variant (P > 0.05). The down-regulated hTERT mRNA in Anip973 cell was higher than that in Anip973/ NVB cell. CONCLUSION: Telomerase correlates with the drug-resistant cell A973/NVB, and telomerase may be a new target for multi-drug resistant inversion.

Emodin down-regulates expression of TRPV1 mRNA and its function in DRG neurons in vitro.

Emodin is a principle ingredient isolated from rhubarb rhizome, which is commonly used for constipation or pain-related diseases in traditional Chinese medicine (TCM) practice. The transient receptor potential vanilloid 1 ion channel proteins (TRPV1) are abundantly expressed in the peripheral sensory neurons and are assumed to act as a kind of nociceptor involved in the perception of pain and development of hyperalgesia. The aim of this study was to further unravel the analgesic mechanisms of rhubarb through investigating the effects of its main constitutive ingredient emodin on the expression of TRPV1 mRNA as well as on its calcium- mediating functions in vitro. The primary DRG neurons with a high purity and viability were obtained, and the TRPV1 mRNA expression levels were examined by using real-time RT-PCR and the elevated amplitudes of intracellular [Ca(2+)]i in the DRG neurons evoked by TRPV1 agonist capsaicin were examined by confocal microscopy. The results showed that emodin could significantly down-regulate both the mRNA expression of TRPV1 and the capsaicin-evoked intracellular fluorescent intensity in the DRG neurons under both 37 degrees C and 39 degrees C in vitro. Concomitantly, all of the changes induced by emodin could not be blocked by pretreatment of the primary neurons with capsazepine, an antagonist of TRPV1. In conclusion, we established that the mRNA expression level of TRPV1 and its calcium-mediating function in naive DRG neurons could be down-regulated by emodin through perhaps the non-TRPV1 channel pathways, and this might be the molecular mechanisms for rhubarb to inhibit hyperalgesia induced by inflammatory stimuli.

Anti-nociceptive mechanism of baicalin involved in intervention of TRPV1 in DRG neurons in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis Georgi (Lamiaceae) is often included as an ingredient in traditional Chinese compound prescriptions for the treatment of fever-related or inflammatory conditions. AIM OF THE STUDY: The present work was to further uncover the analgesic mechanisms of baicalin (a known principal constituent of Scutellaria baicalensis) by investigating its effects on the expression of TRPV1 mRNA as well as on its functions as mediators of calcium entrance into the cytoplasm of dorsal root ganglion (DRG) neurons in vitro. MATERIALS AND METHODS: By using CPT as an agent to eliminate the non-neuronal cells and using serum-free neurobasal as culture medium, primary cultures of rat DRG neurons with high purity and viability were established. On this basis, effects of baicalin on both the expression of TRPV1 mRNA and on the function of TRPV1 in vitro under two various temperature conditions were studied. The TRPV1 mRNA expression levels were examined by using qRT-PCR and analyzed by the method of 2(-DeltaDeltaCT). The elevation amplitudes of intracellular [Ca(2+)]i evoked by TRPV1 agonist capsaicin in DRG neurons were examined by the calcium fluorescence imaging method under confocal microscopy. RESULTS: Baicalin was shown to down-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and under the latter temperature, the intracellular fluorescent intensity evoked by capsaicin was significantly decreased following incubation with baicalin in vitro. We also demonstrated that the actions of baicalin to TRPV1 were not achieved through pathways of TRPA1 or TRPV subfamily members. CONCLUSIONS: Collectively, these results provide compelling evidence that the down-regulated actions of baicalin to TRPV1 in DRG neurons might account for part of the anti-nociceptive mechanism of baicalin.

Cinnamaldehyde up-regulates the mRNA expression level of TRPV1 receptor potential ion channel protein and its function in primary rat DRG neurons in vitro.

Cinnamaldehyde (1) is a pharmacologically active ingredient isolated from cassia twig (Ramulus Cinnamomi), which is commonly used in herbal remedies to treat fever-related diseases. Both TRPV1 and TRPM8 ion channel proteins are abundantly expressed in sensory neurons, and are assumed to act as a thermosensor, with the former mediating the feeling of warmth and the latter the feeling of cold in the body. Both of them have recently been reported to be involved in thermoregulation. The purpose of this paper is to further uncover the antipyretic mechanisms of 1 by investigating its effects on the mRNA expression levels and functions of both TRPV1 and TRPM8. The results showed that 1 could up-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and its calcium-mediating function was significantly increased at 39 degrees C, all of which could not be blocked by pretreatment of the neuronal cells with ruthenium red, a general transient receptor potential (TRP) blocker, indicating that the action of 1 was achieved through a non-TRPA1 channel pathway. In conclusion, the findings in our in vitro studies might account for part of the peripheral molecular mechanisms for the antipyretic action of 1.

Sini tang prevents depression-like behavior in rats exposed to chronic unpredictable stress.

Sini Tang, a Chinese traditional prescription containing three herbs, has been widely used for Yang-deficiency. Recent clinical studies have shown that Sini Tang could treat and improve depression symptoms, but the mechanisms underlying the antidepressant effect of Sini Tang remains unknown. In rats with chronic unpredictable stress (CUS), we examined the effects of Sini Tang on sucrose preference and open field exploratory behavior. The levels of corticosterone level in plasma and corticotropin-releasing hormone (CRH) mRNA expression in hypothalamus were also measured by enzyme-linked immunosorbent assays (ELISA) and real-time reverse transcription PCR (RT-PCR), respectively. Rats subjected to CUS exhibited decreases in sucrose preference and ambulation in the open field test. These were all attenuated by Sini Tang in a dose-dependent manner. Biochemically, Sini Tang also reversed CUS-induced increases in corticosterone in plasma and CRH mRNA in the hypothalamus. The behavioral effects of the Sini Tang were correlated to the biochemical actions. These results suggest that Sini Tang produces an antidepressant-like effect, which appears to involve CRH in the brain.

Effects of cinnamaldehyde on PGE2 release and TRPV4 expression in mouse cerebral microvascular endothelial cells induced by interleukin-1beta.

Cinnamaldehyde is a principle compound isolated from Guizhi-Tang (GZT), which is a famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions. Transient receptor potential vanilloid subtype 4 (TRPV4) is expressed in the anterior hypothalamus and may act as thermosensor. The purpose of the present study was to investigate the effects of cinnamaldehyde on the production of prostaglandin E2 (PGE2) and the expression of TRPV4 in mouse cerebral microvascular endothelial cell strain (b.End3). In the research work, the b.End3 cells were cultured in DMEM medium containing interleukin-1beta (IL-1beta) in the presence or absence of ruthenium red (RR), a kind of known TRPV4 inhibitor, or different concentrations of cinnamaldehyde. The results suggested that IL-1beta significantly increase production of PGE2 and cinnamaldehyde evidently decrease IL-1beta-induced PGE2 production, while RR showed no inhibitory effect on PGE2 production. Moreover, it was identified that TRPV4 was expressed at the mRNA and protein levels in b.End3 cells. IL-1beta could up-regulate the expression of TRPV4, RR and cinnamaldehyde could down-regulate the high expression of mRNA and protein of TRPV4 by IL-1beta induced in b.End3 cells. In conclusion, cinnamaldehyde decreased the production of PGE2 and the expression of TRPV4 in b.End3 cells induced by IL-1beta.

Effects of 3-phenyl-propenal on the expression of toll-like receptors and downstream signaling components on raw264.7 murine macrophages.

3-phenyl-propenal is one of the principle compounds isolated from Guizhi (Ramulus Cinnamomi), the principal drug in Guizhi-Tang (GZT), a famous traditional Chinese medical formula. The aim of the present study was to investigate the effects of 3-phenyl-propenal on the expression of toll-like receptor 3 (TLR3), TLR4 and the downstream signaling components on Raw264.7 murine microphages. Raw264.7 cells were cultured in RPMI-1640 medium containing LPS (lipopolysaccharide) or poly (I:C) in the presence or absence of 3-phenyl-propenal. After 24-hour incubation, the medium was collected and the amount of TNF-alpha and IFN-beta was measured by ELISA. mRNA expression of TLR3, TLR4, myeloid differentiation factor (MyD88), TRAF-6 (tumor necrosis factor receptor-associated), TRAM (toll-like receptor-associated molecule) and TRIF (TIR domain-containing adaptor inducing IFN-beta) were analyzed by real-time PCR with SYBR green dye. Protein expression of TLR3 and TLR4 was analyzed by Western blotting and that of MyD88 and TRAF-6 was analyzed by immunofluorescence assay. The results indicate that LPS increased the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF, but had no influence on TLR3, while poly (I:C) up-regulated the expression of TLR3, MyD88, TRAM and TRIF. 3-phenyl-propenal significantly decreased the expression of LPS-induced TLR4, MyD88, TRAF-6, while possessing no effect on LPS-induced TRAM and TRIF expression in Raw264.7 cells. When cells were stimulated by poly (I:C), 3-phenyl-propenal significantly decreased TLR3 and MyD88 expression. In conclusion, 3-phenyl-propenal blocked the over-expression of TLR3, TLR4, their downstream signaling components MyD88 and TRAF-6, which indicate that it had an antagonistic effect on TLR3 and TLR4.

[Effect of Shensu Yin on the expression of toll-like receptors and the downstream signaling components on RAW 264.7 cells].

OBJECTIVE: To investigate the influences of Shensu Yin to RAW 264.7 on the expression of TLR3, TLR4 and the factors of the downstream in RAW 264. 7 cells. METHOD: RAW 264.7 cell line was stimulated with Lipopolysaccharide and POLY I: C, respectively, and treated with the drug serum of Shensuyin simultaneously. 24 hours later, collected the supernatant and measured the inflammatory factors TNF-alpha and IFN-beta, extracted mRNA and measured the expression of TLR3, TLR4 and other correlated indexes of the downstream, analyzed and evaluated Shensu Yin's substance basis of pharmacodynamic actions. RESULT: Shensu Yin drug serum depressed the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF mRNA, as a result, it decreased the amount of TNF-alpha and IFN-beta. CONCLUSION: Depressing the expression of TLR3, MyD88, TRAM and TRIF mRNA may be the elementary basis of Shensu Yin to play heat-clearing and detoxicating effect.

2-methoxycinnamaldehyde reduces IL-1beta-induced prostaglandin production in rat cerebral endothelial cells.

Prostaglandin E2 (PGE2) works as a common final mediator of the febrile. Guizhi-Tang, one of the most famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions, was previously reported to reduce the production of PGE 2 in rats. 2-Methoxycinnamaldehyde is a principle compound isolated from Guizhi-Tang. The aim of the present study was to investigate the effects of 2-methoxycinnamaldehyde on PGE2 production of rat cerebral endothelial cells (CECs). 2-Methoxycinnamaldehyde dose-dependently inhibited interleukin (IL)-1beta-induced PGE2 production in CECs with IC50 values of 174 microM. IL-1beta stimulation increased the protein, activity and mRNA expression of cyclooxygenase (COX)-2 but not COX-1. 2-Methoxycinnamaldehyde reduced IL-1beta-induced protein and activity of COX-2, but did not influence the COX-2 mRNA expression. Our results show that prostaglandin production in CECs during stimulated conditions is sensitive to inhibition by 2-methoxycinnamaldehyde and suggest that 2-methoxycinnamaldehyde may reduce COX-2 protein level and activity but not COX-2 mRNA.

[Effect of 2-methoxycinnamaldehyde on activity of COX and PGE2 release in cerebral microvascular endothelial cells stimulated by IL-1].

OBJECTIVE: To observe the effect of 2-methoxycinnamaldehyde (isolated from fraction A of Guizhi Tang) on activity of COX and PGE2 release in rat cerebral microvascular endothelial cells (rCMEC) stimulated by IL-1. METHOD: rCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor (VIII factor, a marker for all endothelial cells) in cytoplasm of the cells. Different concentrations of 2-methoxycinnamaldehyde were added respectively and incubated for 3 hours, then stimulated for another 12 hours by IL-1. Activities of COX-1 and COX-2 in rCMEC, and production of PGE2 in the conditioned media were measured by ELISA. RESULT: Positive immunostaining for VIII factor was present diffusely in the cytoplasm of > 90% rCMEC. After being exposed to 30 ng x mL(-1) IL, the activity of COX-2 in rCMEC and the production of PGE2 in conditioned media were higher than those of control group, while there was no difference on activity of COX-1 in the two groups. 2-methoxycinnamaldehyde could down-regulate them in concentration-dependently, and significant differences on the activity of COX-2 and amount of PGE2 were showed in 200 microg x mL(-1) concentration. CONCLUSION: 2-methoxycinnamaldehyde can affect the PGE2 release in rCMEC induced by IL-1, which might be related with its inhibition on the activity of COX-2.