Zhi-Yi Yuan

PI3K p55γ promoter activity enhancement is involved in the anti-apoptotic effect of berberine against cerebral ischemia-reperfusion.

Berberine is a candidate clinical neuroprotective agent against ischemic stroke. In the present study, we examined the influence of the PI3K/Akt pathway in mediating the anti-apoptotic effects of berberine. Oxygen-glucose deprivation and reoxygenation of nerve growth factor-differentiated PC12 cells and primary neurons, and bilateral common carotid artery occlusion in mice were used as in vitro and in vivo ischemia models. We found that the anti-apoptotic effects of berberine against ischemia were indeed mediated by the increased phosphor-activation of Akt (higher p-Akt to total Akt), leading to the intensified phosphorylation of Bad and the decreased cleavage of the pro-apoptotic protease caspase-3. Berberine action is specific for PI3K, rather than the upstream receptor tyrosine kinase. The anti-apoptotic effect is maintained in the presence of tyrosine kinase inhibitor genistein and the epidermal growth factor receptor inhibitor PD153035, but is suppressed by the PI3K inhibitor Ly294002 and the Akt inhibitor Akti-1/2.The unique PI3K regulatory subunit p55γ was upregulated by berberine during ischemia-reperfusion and was not blocked by these inhibitors. We constructed a reporter plasmid to detect PI3K p55γ promoter activity and found that berberine enhanced PI3K p55γ promoter activity during cerebral ischemia-reperfusion.

Comprehensive study of baicalin down-regulating NOD2 receptor expression of neurons with oxygen-glucose deprivation in vitro and cerebral ischemia-reperfusion in vivo

Cerebral ischemia-reperfusion can activate several transcription factors and lead to inflammatory reactions, which related to pattern recognition receptors with immune activating functions. NOD2 (nucleotide-binding oligomerization domain protein 2) is one of the receptors involved in innate immune response and is genetically associated with several inflammatory reactions. Since baicalin has the pharmacological effects of anti-inflammation and protection of brain from cerebral ischemia-reperfusion, we studied baicalins effect on NOD2/TNFα in the cell of oxygen-glucose deprivation (OGD) in vitro and the mice of cerebral ischemia-reperfusion in vivo. The results showed that NOD2 and TNFα were up regulated in the cells with oxygen-glucose deprivation, not only in BV2 cells, but also in both of PC12 cells and primary neuron cells, which suggested NOD2 could express directly in neuron while OGD treatment. Baicalin (10 μg/ml) could effectively down regulate the expression of NOD2 and TNFα in both mRNA and protein levels. Meanwhile, baicalin (50 mg/kg, i.p.) could also down regulate the expression of NOD2 and TNFα in protein levels significantly, in which agreed with its effect in vitro study. These data demonstrated that targeting on NOD2 especially in neurons directly was possibly attributed to the neural-protective effect of baicalin in the injury of cerebral ischemia-reperfusion.

Comparative study of the effect of baicalin and its natural analogs on neurons with oxygen and glucose deprivation involving innate immune reaction of TLR2/TNFα.

This work is to study the baicalin and its three analogs, baicalin, wogonoside, and wogonin, on the protective effect of neuron from oxygen-glucose deprivation (OGD) and toll-like receptor 2 (TLR2) expression in OGD damage. The results showed that baicalin and its three analogs did protect neurons from OGD damage and downregulated protein level of TLR2. D-Glucopyranosiduronic acid on site 7 in the structure played a core of cytotoxicity of these flavonoid analogs. The methoxyl group on carbon 8 of the structure had the relation with TLR2 protein expression, as well as the anti-inflammation. In addition, we detected caspase3 and antioxidation capability, to investigate the effect of four analogs on cell apoptosis and total antioxidation competence in OGD model.

Purgative components in rhubarbs: Adrenergic receptor inhibitors linked with glucose carriers

Rhubarbs and their extractives have been used as cathartic for many years. There have been numerous breakthroughs in the pharmacological research of the drug. However, as the key point of the mechanism, the targets of the effective components still remain unclear. In this paper, with an in vitro system of isolated intestine, we found that both the rhubarb extractives and the anthraquinone derivatives can antagonize the adrenaline effectively. Furthermore, computer based docking provided the binding model of the anthraquinone derivatives and adrenergic receptor. Then, based on the results of the small intestinal promotion and purgative effect experiments in vivo, we built an inhibitor-carrier hypothesis to elucidate the mechanism of rhubarb. This work provided key massages for the pharmacological research of rhubarb, such a common and active medicinal plant, and might be of help for the development of new purgative drugs.

Mitochondria play an important role in the cell proliferation suppressing activity of berberine

After being studied for approximately a century, berberine (BBR) has been found to act on various targets and pathways. A great challenge in the pharmacological analysis of BBR at present is to identify which target(s) plays a decisive role. In the study described herein, a rescue experiment was designed to show the important role of mitochondria in BBR activity. A toxic dose of BBR was applied to inhibit cell proliferation and mitochondrial activity, then α-ketobutyrate (AKB), an analogue of pyruvate that serves only as an electron receptor of NADH, was proven to partially restore cell proliferation. However, mitochondrial morphology damage and TCA cycle suppression were not recovered by AKB. As the AKB just help to regenerate NAD+, which is make up for part function of mitochondrial, the recovered cell proliferation stands for the contribution of mitochondria to the activity of BBR. Our results also indicate that BBR suppresses tumour growth and reduces energy charge and mitochondrial DNA (mtDNA) copy number in a HepG2 xenograft model. In summary, our study suggests that mitochondria play an important role in BBR activity regarding tumour cell proliferation and metabolism.

Effects of Angelica dahurica on obesity and fatty liver in mice

Angelica dahurica (A. dahurica) is a traditional Chinese medicinal plant being used in clinical practice. The present study demonstrated that A. dahurica could reduce white-fat weight in high-fat-diet hyperlipidemic mice, decrease total cholesterol and triglyceride concentrations in the livers of both high-fat-diet and Triton WR1339 induced hyperlipidemic mice, and enhance the total hepatic lipase activities of them. These findings were further supported by the results derived from the experiments with HepG2 cells in vitro. In addition, the proteins related to lipids metabolism were investigated using LC-MS/MS, indicating that genes of lipid metabolism and lipid transport were regulated by A. dhurica. The results from LC-MS/MS were further conformed by Western blot and real time PCR assays. A. dahurica could down-regulate the expression of catalase (CAT) and sterol carrier protein2 (SCP2) and up-regulate the expression of lipid metabolism related genes-lipase member C (LIPC) and peroxisome proliferator-activated receptor gamma (PPARγ). In the Triton WR1339 mouse liver and HepG2 cells in vitro, A. dahurica was able to increase the expression of LIPC and PPARγ, confirming the results from in vivo experiments. Imperatorin showed the same activity as A. dahurica, suggesting it was one of the major active ingredients of the herb. In conclusion, our work represented a first investigation demonstrating that A. dahurica was able to regulate lipid metabolism and could be developed as a novel approach to fighting against fatty liver and obesity.

TRPM8 in the negative regulation of TNFα expression during cold stress

Transient Receptor Potential Melastatin-8 (TRPM8) reportedly plays a fundamental role in a variety of processes including cold sensation, thermoregulation, pain transduction and tumorigenesis. However, the role of TRPM8 in inflammation under cold conditions is not well known. Since cooling allows the convergence of primary injury and injury-induced inflammation, we hypothesized that the mechanism of the protective effects of cooling might be related to TRPM8. We therefore investigated the involvement of TRPM8 activation in the regulation of inflammatory cytokines. The results showed that TRPM8 expression in the mouse hypothalamus was upregulated when the ambient temperature decreased; simultaneously, tumor necrosis factor-alpha (TNFα) was downregulated. The inhibitory effect of TRPM8 on TNFα was mediated by nuclear factor kappa B (NFκB). Specifically, cold stress stimulated the expression of TRPM8, which promoted the interaction of TRPM8 and NFκB, thereby suppressing NFκB nuclear localization. This suppression consequently led to the inhibition of TNFα gene transcription. The present data suggest a possible theoretical foundation for the anti-inflammatory role of TRPM8 activation, providing an experimental basis that could contribute to the advancement of cooling therapy for trauma patients.

Comprehensive study of the intestinal absorption of four phenolic compounds after oral administration of Ananas comosus leaf extract in vivo and in vitro

The extract ofxa0Ananas comosusxa0leaf (EAL) was proved to be antihyperlipidemia and antihyperglycemia consisting of rich phenolic acids. Pharmacokinetic study showed p-coumaric acid was a rich compound detected in the mouse plasma after EAL oral administration. The aim of this study was to explore the alteration of the absorption of four principle phenolic compounds through the intestines by oral administration. Liquid chromatography-mass spectrometry (LC-MS-MS) was used to detect the phenolic compounds in EAL. The assay for the absorption of the small intestines of mice was employedxa0in vivoxa0andxa0in vitro. According to our results, 1-O-p-coumaroylglycerol and 1-O-caffeoylglycerol would be converted into p-coumaric acid and caffeic acid by passing though the intestines. Caffeic acid might be transformed into a new compound with relative molecular mass of 359 in liver. The phenolic components of EAL were absorbed mainly in the form of p-coumaric acid while caffeic acid were absorbed through the intestines. p-Coumaric acid and caffeic acid are considered the active components of EAL in the body. n n xa0 n n Key words:xa0Ananas comosus, phenolic acid, absorption, mouse, metabolism.

Brazilein inhibits neuronal inflammation induced by cerebral ischemia and oxygen-glucose deprivation through targeting NOD2 expression

Brazilein is reported to have immunosuppressive effect on cardiovascular and cerebral-vascular diseases. The essential roles of innate immunity in cerebral ischemia are increasingly identified, but no studies concerning the influence of brazilein on the innate immunity receptors have been reported. The present study was designed to investigate the regulation of NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) by brazilein for its protection of neuron in cerebral ischemia in vivo and oxygen-glucose deprivation in vitro. The results showed that brazilein could reverse the elevated expression of NOD2 and TNFα (tumor necrosis factor alpha) elicited by cerebral ischemia and reperfusion. This reduction could also be detected in normal mice and C17.2 cells, indicating that this suppressive effect of brazilein was correlated with NOD2. The results from GFP reporter plasmid assay suggested brazilein inhibited NOD2 gene transcription. In conclusion, brazilein could attenuate NOD2 and TNFα expression in cerebral ischemia and NOD2 may be one possible target of brazilein for its immune suppressive effect in neuro-inflammation.

Mechanism underlying berberine's effects on HSP70/TNFα under heat stress: Correlation with the TATA boxes

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.