Anti-tumor and Phenotypic Regulation Effect of Matrine on Dendritic Cells through Regulating TLRs Pathway.

OBJECTIVE: To investigate the effects of matrine on antigen presentation of dendritic cells (DCs), and to explore the pharmacological mechanism of matrine on anti-tumor effect. METHODS: Different concentrations (0, 1, 2, 4, 8 and 16 µ g/mL) of matrine were co-cultured with DCs, the harvested DCs were co-cultured with antigens of Lewis lung cancer (LLC) cells, and then DCs and T cells were co-cultured to produce DCs-activated killer (DAK) cells, which have significant tumor-killing activity. The expression of cytokines, mRNA and protein of toll-like receptors (TLRs) in DCs were detected by enzyme linked immunosobent assay, polymerase chain reaction and Western blot, respectively. And the killing effect of DAK were measured by MTT assay. RESULTS: Matrine significantly increased the mRNA expression of TLR7, TLR8, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF-6) and I κ B kinase (IKK), as well as the protein expression of TLR7 and TLR8, and up-regulated the levels of interleukin-12 (IL-12), IL-6 and tumor necrosis factor-α (TNF-α), meanwhile, it also increased the expressions of MHC-II, CD54, CD80 and CD86 in DCs. DCs-activated effector T cells had significant tumor-killing activity. When the concentration of matrine was more than 4 µg/mL, all indices had significant difference (P<0.01 or P<0.05). CONCLUSION: Matrine plays an anti-tumor role by regulating TLRs signal transduction pathway, promoting the secretion of inflammatory cytokines and enhancing immune function.

In vitro screening and toxic mechanism exploring of leading components with potential hepatotoxicity of Herba Epimedii extracts.

Herba Epimedii is a famous Chinese edible herb, and due to its potential hepatotoxic effects, the safety associated with this herb has attracted a great deal of attention. In this study, the components of four types of the Herba Epimedii extracts were identified by HPLC-MS/MS. Among these components, 11 components that were present in all four extracts and could be obtained as reference substances were evaluated for their ability of cytotoxicity in HL-7702 and HepG2 cells, resulting in the identification of icarisid I and sagittatoside A as the most relevant with respect to the toxicity of the extracts. The targeted toxicological effects were further investigated using a series of correlated biological indicators to elucidate potentially hepatotoxic mechanisms. The results showed that the extracts and the selected compounds had varying degrees of influence on the leakage of ALT, AST and LDH; the activity of SOD, GSH and MDA; the increase in intercellular ROS; and the decrease in MMP. Among the tested substances, the ethanol extracts exhibited stronger hepatotoxicity, with icarisid I and sagittatoside A correlating with this toxic effect, and the hepatoxic mechanisms of which may be associated with damaged cell structure, increased oxidative stress and induction of apoptosis.

Effect of 2″-O-Rhamnosyl Icariside II, Baohuoside I and Baohuoside II in Herba Epimedii on Cytotoxicity Indices in HL-7702 and HepG2 Cells.

Herba Epimedii, a commonly used Chinese medicine, has attracted much attention recently because of its potential hepatotoxic effects. 2″-O-Rhamnosyl icariside II, baohuoside I and baohuoside II are the main components of Herba Epimedii, and previous research indicates that these three compounds are related to the hepatotoxicity of Herba Epimedii. To test this idea, in this study, HL-7702 and HepG2 cells were chosen as the in vitro models and the influences of these three compounds on a series of cytotoxicity indices, including ALT, AST, LDH, SOD, GSH, MDA, ROS and MMP, were determined. The results showed that at certain concentrations, the three compounds had different effects on the indices. Among them, baohuoside I at high concentration (32 µg/mL) displayed more significant cytotoxicity than the other two compounds; therefore, it was inferred to be more closely correlated with the liver injury induced by Herba Epimedii combined with the previous study, and its toxic mechanisms may be involved in increasing oxidative stress and inducing apoptosis. The findings of this study may provide evidence of the toxic composition of Herba Epimedii to preliminarily discuss the toxic mechanisms and provide improved guidance for its clinical safety.

[Rapid characterization of constituents absorbed into blood after oral administration of steroidal saponins from Radix Ophiopogonis using UHPLC-LTQ-Orbitrap MS coupled with higher energy collision induced dissociation].

The method of UHPLC-LTQ-Orbitrap mass spectrometry coupled with higher energy collision dissociation (HCD) was established to rapidly analyze the constituents absorbed into blood after oral administration of steroidal saponins from Radix Ophiopogonis. A total of 31 constituents, including 13 furostanol steroidal saponins and 18 spirostanol steroidal saponins, were characterized based on the accurate mass measurements, fragmentation patterns, chromatographic retention times, and diagnostic product ions. Among them, 8 compounds were unambiguously identified by comparison with their corresponding standards. The results provide comprehensive insights and guidance for elucidation of material basis of Radix Ophiopogonis activity.

[Combination analysis of new drug discovery with "Xiaohe Silian" method and traditional Chinese medicine clinical pharmacy].

With the kernel of efficacy, "Xiaohe Silian" was a pattern and method for new drug discovery which was constituted with "metabolism-efficacy, toxicity-efficacy, quality-efficacy and structure-efficacy". Its connotation was in keeping with traditional Chinese medicine (TCM) clinical pharmacy. This paper systematically summarized the research method of new drug discovery practice process for TCM. To avoid western drug like in TCM new drug discovery, we carried out combination analysis with TCM clinical pharmacy. The correlation analysis between basic elements of "Xiaohe Silian(n) and TCM clinical pharmacy was studied to guarantee this method could integrate closely with TCM clinic from all angles. Hence, this method aimed to provide a new method for TCM new drug discovery on the basis of TCM clinical pharmacy with insisting on holistic view of multicomponent study, kinetic view of metabolic process when the curative effect occurred and molecular material view of quality control and structure-activity exposition.

Effects of ginsenoside Rg1 on the expression of toll-like receptor 3, 4 and their signalling transduction factors in the NG108-15 murine neuroglial cell line.

As one of the most important components of Panax ginseng, ginsenoside Rg1 has certain anti-aging effects, improving the activity of learning and memory. Studies have showed that ginsenoside Rg1 improves the memory impairment associated with Alzheimer's disease (AD). In this study, the effects of ginsenoside Rg1 were investigated through the activity of toll-like receptor (TLR) 3, TLR4 and their signaling transduction pathways in amyloid ß peptide 25-35 (Aß25-35) induced AD cell model. Thus we investigated several critical components of the TLR pathway. The neuroglial cell line NG108-15 was stimulated with or without Aß25-35, while different concentrations of ginsenoside Rg1 were administered. After 24 h, tumor necrosis factor-α (TNF-α), interferon-ß (IFN-ß) in cell supernatant and inducible nitric oxide synthase (iNOS) in cell lysate supernatant were measured with enzyme-linked immunosorbent assays (ELISAs). The mRNA and protein expression of TLR3, TLR4, nuclear factor kappa B (NF-κB) and tumor necrosis factor receptor-associated factor-6 (TRAF-6) were detected by real-time PCR and western blot methods, respectively. The experimental results showed that Aß25-35 could markedly raise the level of TNF-α, IFN-ß and iNOS, and increase the expressions of mRNA and TLR3, TLR4, NF-κB and TRAF-6 protein in the NG108-15 cells. At the same time, the ginsenoside Rg1 significantly reduced the expressions of proteins and mRNA of TLR3, TLR4, NF-κB and TRAF-6, and down-regulated the levels of TNF-α, IFN-ß of cell supernatant and iNOS of cell lysate supernatant in a concentration-dependent manner. In conclusion, ginsenoside Rg1 has good activity for suppressing the signaling transduction pathway of TLR3 and TLR4, and decreasing the inflammation factors induced by Aß25-35 in NG108-15 cells, and this may be the mechanism of ginsenoside Rg1 action in AD treatment, but more studies are needed to identify its specificity.

[Exploration of property theory of Tibetan medicine].

Tibetan Herbal medicine has its own complete theory based on five sources doctrine. And the theories of "Liuwei", "Baxing" and "Shiqi Gongxiao" formed the basic core components of the property theory of Tibetan medicine. However, books and literature of Tibetan medicine have never been systematically expounded and discussed about it specially which thus will limit the further development of Tibetan medicine theory. In this thesis, we firstly introduced three basic core components of the property theory-the "Liu Wei", "Baxing", and "Shiqi Gongxiao" and their interactions as well. At the same time, the links and similarities between the theory of Tibetan medicine and Chinese medicine theory were compared. The job of the thesis done above is to lay the foundation for further systematic reveal and development of Tibetan medicine theory.

[Analysis of multicomponent drug metabolism used in clinical pharmacy research of traditional Chinese medicine].

Multicomponent drug metabolism can be defined as a research area that, rather than pharmacokinetics and pharmacodynamics, is a concerted dynamic metabolic variation of one component in several other compounds circumstance with the interaction of transport protein and drug metabolizing enzymes, and the study of the dynamic course of multiple components must be simultaneously determined. By the use of multicomponent drug metabolism in the clinical pharmacy research of traditional Chinese medicine (TCM), it can become a useful tool with the integration of the overall dialectical method and the concrete molecular approach.

Evaluation of anxiolytic activity of compound Valeriana jatamansi Jones in mice.

BACKGROUND: Compound Valeriana jatamansi Jones is a formula for treating anxiety-related diseases in the clinic, which is composed of Valeriana jatamansi Rhizoma et Radix, Ziziphi Spinosae Semen, Albiziae Cortex and Junci Medulla. The purpose of this study was to explore the anxiolytic properties of this compound in mice. METHODS: Male ICR mice were treated with compound Valerianae Jatamansi Jones (1.2 g/kg, 2.4 g/kg, 4.8 g/kg), saline, diazepam (2 mg/kg) orally for 10 days and then exposed to elevated maze-plus (EPM) and light-dark box (LDB). The effects of the compound on spontaneous activity were evaluated by locomotor activity test. We further investigated the mechanism of action underlying the anxiolytic-like effect of compound by pre-treating animals with antagonists of benzodiazepine (flumazenil, 3mg/kg) prior to evaluation using EPM and LDB. RESULTS: Compound Valerianae Jatamansi Jones (2.4, 4.8 g/kg, p.o.) significantly increased entries (P<0.05) into and time spent (P<0.05) on the open arms of the EPM, and number of transitions (P<0.05) and time spent (P<0.05) in the light compartment of the LDB. However, the anxiolytic-like effects of compound were significantly reduced by pre-treatment with flumazenil (P>0.05). In addition, compound Valerianae Jatamansi Jones treatment didn't affect the spontaneous activity in mice (P> 0.05). CONCLUSIONS: The present study supports the hypothesis that compound Valeriana jatamansi Jones exert anxiolytic action but no sedative effects in mice and that this effect might be mediated by benzodiazepine receptors.

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.

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.

Effect of 3-Phenyl-2-Propene-1-ol on PGE2 release from rat cerebral microvascular endothelial cells stimulated by IL-1beta.

Fever, an elevation in body temperature, is thought to be terminally mediated by prostaglandin E(2) (PGE(2)). Both Guizhi Tang (GZT) and its active fraction A (Fr.A) showed an antipyretic effect in rats. 3-Phenyl-2-propene-1-ol was one of the active compounds isolated from Fr.A. In the present study, we examined the influence of interleukin-1beta (IL-1beta) on prostaglandin E(2) (PGE(2)) release, and the effect of 3-phenyl-2-propene-1-ol on IL-1beta-induced PGE(2) release from rat cerebral endothelial cells (rCMEC). Cultured rCMEC were used in the study. In vitro, cells express typical phenotypic markers of brain endothelium. Using a monoclonal antibody against von Willebrand factor, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured cells. rCMEC were incubated in M199 medium containing IL-1beta in the presence or absence of 3-phenyl-2-propene-1-ol. After incubation, the conditioned media were collected and the amount of PGE(2) was measured by enzyme-linked immunosorbent assay (ELISA). IL-1beta increased the production of PGE(2) in a dose- and time-dependent manner. 3-Phenyl-2-propene-1-ol significantly decreased IL-1beta-induced PGE(2) release in a dose-dependent manner. Our results indicate that 3-phenyl-2-propene-1-ol inhibits the PGE(2) release from rCMEC stimulated by IL-1beta, and may have an antipyretic effect.

Cinnamaldehyde reduces IL-1beta-induced cyclooxygenase-2 activity in rat cerebral microvascular endothelial cells.

Cinnamaldehyde is a principle compound isolated from Guizhi-Tang, which is a famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions. The aim of the present study was to investigate the effects of cinnamaldehyde on expression and activity of cyclooxygenase (COX) and prostaglandin E(2) (PGE(2)) in rat cerebral microvascular endothelial cells (RCMEC). RCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor in cytoplasm of the cells. Then cells were incubated in M199 medium containing interleukin (IL)-1beta in the presence or absence of cinnamaldehyde. After incubation, the medium was collected and the amount of PGE(2) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, mRNA expression and activity of COX were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) with SYBR Green dye and ELISA respectively. Positive immunostaining for von Willebrand factor was present diffusely in the cytoplasm of >95% RCMEC. IL-1beta increased the mRNA expression and activity of COX-2, and production of PGE(2) in a dose- and time-dependent manner in RCMEC, while mRNA and activity of COX-1 were not significantly altered. Cinnamaldehyde significantly decreased IL-1beta-induced COX-2 activity and PGE(2) production in a dose-dependent manner, while it showed no inhibitory effect on IL-1beta-induced COX-2 mRNA expression in cultured RCMEC. In conclusion, cinnamaldehyde reduces IL-1beta-induced COX-2 activity, but not IL-1beta-induced COX-2 mRNA expression, and consequently inhibits production of PGE(2) in cultured RCMEC.