Extract of Fructus Schisandrae chinensis Inhibits Neuroinflammation Mediator Production from Microglia via NF-κ B and MAPK Pathways.

OBJECTIVE: To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis (EFSC) on lipopolysaccharide (LPS)-induced BV-2 cells and the possible involved mechanisms. METHODS: Primary cortical neurons were isolated from embryonic (E17-18) cortices of Institute of Cancer Research (ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group (treated with 1 µg/mL LPS only) and EFSC groups (treated with 1 µg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide (MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide (NO) and interleukin-6 (IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence. RESULTS: EFSC (200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression in LPS-induced BV-2 cells (P<0.01 or P<0.05). EFSC (200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia (P<0.01). In addition, EFSC alleviated cell apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium (P<0.01). The mechanistic studies indicated EFSC could suppress nuclear factor (NF)-?B phosphorylation and its nuclear translocation (P<0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase (MAPK) pathway (P<0.01 or P<0.05). CONCLUSION: EFSC acted as an anti-inflammatory agent in LPS-induced glia cells. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.

Relationship between Tissue Distributions of Modified Wuzi Yanzong Prescription () in Rats and Meridian Tropism Theory.

OBJECTIVE: To investigate the relationship between tissue distributions of modified Wuzi Yanzong prescription (, MWP) in rats and meridian tropism theory. METHODS: A high-performance liquid chromatography with Fourier transform-mass spectrometry (HPLC-FT) method was used to identify the metabolites of MWP in different tissues of rats after continued oral administration of MWP for 7 days. The relationship between MWP and meridian tropism theory was studied according to the tissue distributions of the metabolites of MWP in rats and the relevant literature. RESULTS: Nineteen metabolites, mainly flavanoid compounds, were detected in the different rat tissues and classified to each herb in MWP. Further, it was able to establish that the tissue distributions of the metabolites of MWP were consistent with the descriptions of meridian tropism of MWP available in literature, this result might be useful in clarifying the mechanism of MWP on meridian tropism. In the long run, these data might provide scientific evidence of the meridian tropism theory to further promote the reasonable, effective utilization, and modernization of Chinese medicine. CONCLUSION: The tissue distributions of MWP in vivo were consistent with the descriptions of meridian tropism of MWP.

Antineuroinflammatory Effects of Modified Wu-Zi-Yan-Zong Prescription in ß-Amyloid-Stimulated BV2 Microglia via the NF-κB and ERK/p38 MAPK Signaling Pathways.

Modified Wu-Zi-Yan-Zong prescription (MWP), a traditional Chinese medicinal decoction, has possessed the neuroprotective and anti-inflammatory properties. The mechanisms associated with these properties, however, are not completely understood. We designed the experiments to elucidate the antineuroinflammatory property of MWP in BV2 microglia activated by ß-amyloid (Aß), which is a characteristic feature of Alzheimer's disease (AD). The composition of MWP was studied using HPLC. BV2 microglia cells were then treated with Aß in the presence or absence of MWP. The effects of MWP treatment on Aß-activated neuroinflammation were determined using PCR, western blotting, and immunofluorescence staining. MWP significantly inhibited the mRNA expression of inflammatory mediators such as IL-1ß, IL-6, TNF-α, and MCP-1, as well as the expression of inducible nitric oxide synthase (iNOS) in Aß-activated BV2 microglia. MWP also inhibited the nuclear translocation and signaling pathway of nuclear factor kappa B (NF-κB) by suppressing inhibitor of nuclear factor-κB (IκB) degradation and downregulating IκB kinase ß (IKKß) phosphorylation. Moreover, MWP decreased extracellular regulated protein kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) phosphorylation, which is an important signaling pathway for proinflammatory gene expression. We concluded that MWP could suppress neuroinflammatory responses in Aß-activated BV2 microglia via the NF-κB and ERK/p38 MAPK signaling cascades and could prove an effective therapeutic agent for the prevention and treatment of neuroinflammatory diseases such as AD.

Caruifolin D from artemisia absinthium L. inhibits neuroinflammation via reactive oxygen species-dependent c-jun N-terminal kinase and protein kinase c/NF-κB signaling pathways.

This work aims to evaluate the anti-neuroinflammatory effects of natural sesquiterpene dimer caruifolin D from Artemisia absinthium L., which is an edible vegetable or traditional medicinal food in East Asia due to its sedation, anti-asthma and antipruritic effects. In this study, we reported that caruifolin D significantly inhibited the productions of various neuroinflammatory mediators from microglia in response to bacterial lipopolysaccharide stimulation. Moreover, anti-inflammatory mechanism study showed that caruifolin D markedly suppressed the production of intracellular reactive oxygen species, which was an important player involved in neuroinflammation, leading to inhibitory effects on the activations of protein kinase C (PKC) and c-Jun N-terminal kinase (JNK), which were two major neuroinflammatory signaling pathways in the brains. Furthermore, caruifolin D protected neurons against microglia-mediated neuronal inflammatory damages by up-regulating neuronal viability and maintaining healthy neuronal morphology. Taken together, these results expanded our knowledge about the anti-neuroinflammatory and neuroprotective mechanism of Artemisia absinthium L., and also suggested that caruifolin D was a major anti-inflammatory component from Artemisia absinthium L., which might be developed as a drug candidate for neuroinflammation-related diseases.

Natural small molecule FMHM inhibits lipopolysaccharide-induced inflammatory response by promoting TRAF6 degradation via K48-linked polyubiquitination.

TNF receptor-associated factor 6 (TRAF6) is a key hub protein involved in Toll-like receptor-dependent inflammatory signaling pathway, and it recruits additional proteins to form multiprotein complexes capable of activating downstream NF-κB inflammatory signaling pathway. Ubiquitin-proteasome system (UPS) plays a crucial role in various protein degradations, such as TRAF6, leading to inhibitory effects on inflammatory response and immunologic function. However, whether ubiquitination-dependent TRAF6 degradation can be used as a novel anti-inflammatory drug target still remains to be explored. FMHM, a bioactive natural small molecule compound extracted from Chinese herbal medicine Radix Polygalae, suppressed acute inflammatory response by targeting ubiquitin protein and inducing UPS-dependent TRAF6 degradation mechanism. It was found that FMHM targeted ubiquitin protein via Lys48 site directly induced Lys48 residue-linked polyubiquitination. This promoted Lys48 residue-linked polyubiquitin chain formation on TRAF6, resulting in increased TRAF6 degradation via UPS and inactivation of downstream NF-κB inflammatory pathway. Consequently, FMHM down-regulated inflammatory mediator levels in circulation, protected multiple organs against inflammatory injury in vivo, and prolong the survival of endotoxemia mouse models. Therefore, FMHM can serve as a novel lead compound for the development of TRAF6 scavenging agent via ubiquitination-dependent mode, which represents a promising strategy for treating inflammatory diseases.

[Study of effect of Humifuse Euphorbia Herb on alleviating insulin resistance in type 2 diabetic model KK-Ay mice].

[To explore the effect of Humifuse Euphorbia Herb ( HEH) on alleviating insulin resistance in type 2 diabetic KK-Ay mice. Totally 40 KK-Ay mice fed with high-fat diet were divided into four groups: the metformin group, the model group, the HEH low-dose group and the HEH high-dose group, and orally administrated with metformin hydrochloride (250 mg x kg(-1)), distilled water, humifuse euphorbia herb 1 g x kg(-1) and 2 g x kg(-1). Besides, C57BL/6J mice with ordinary feed were taken as the normal control group and orally administrated with equal distilled water. The oral administration for the five groups lasted for eight weeks. Before and after the experiment, weight, fasting glucose and insulin tolerance were determined. The morphological changes in pancreas were observed through hematoxylin-eosin (HE) staining on pancreatic tissue sections. The serum insulin, TNF-α, IL-6, adiponectin (ADPN) and leptin (LEP) were detected by ELISA. The results showed that HEH could reduce weight and fasting glucose in KK-Ay mice, alleviate hyperinsulinemia, reduce blood glucose-time AUC, increase 30-min blood glucose decline rate, relieve insulin resistance, significantly ameliorate the pathomorphological changes in pancreas in each group, decrease serum TNF-α, IL-6 and leptin levels in KK-Ay mice and rise serum ADPN level. This study proved that humifuse euphorbia herb can ameliorate the insulin resistance in KK-Ay mice, and its mechanism may be related to the effect on inflammatory factors and adipocytokines.

Anti-Neuroinflammatory Effect of MC13, a Novel Coumarin Compound From Condiment Murraya, Through Inhibiting Lipopolysaccharide-Induced TRAF6-TAK1-NF-κB, P38/ERK MAPKS and Jak2-Stat1/Stat3 Pathways.

MC13 is a novel coumarin compound found in Murraya, an economic crop whose leaves are widely used as condiment (curry) in cuisine. The aims of the present study were to investigate the neuroprotective effects of MC13 on microglia-mediated inflammatory injury model as well as potential molecular mechanism. Cell viability and apoptosis assay demonstrated that MC13 was not toxic to neurons and significantly protected neurons from microglia-mediated inflammatory injury upon lipopolysaccharide (LPS) stimulation. Results showed that MC13 markedly inhibited LPS-induced production of various inflammatory mediators, including nitrite oxide (Griess method), TNF-α and IL-6 (ELISA assay) in a concentration-dependent manner. Mechanism study showed that MC13 could suppress the activation of NF-κB, which was the central regulator for inflammatory response, and also decreased the interaction of TGF-ß-activated kinase 1 (TAK1)-binding protein (TAB2) with TAK1 and TNF receptor associated factor (TRAF6), leading to the decreased phosphorylation levels of NF-κB upstream regulators such as IκB and IκB kinase (IKK). MC13 also significantly down-regulated the phosphorylation levels of ERK and p38 MAPKs, which played key roles in microglia-mediated inflammatory response. Furthermore, MC13 inhibited Jak2-dependent Stat1/3 signaling pathway activation by blocking Jak2 phosphorylation, Stat1/3 phosphorylation, and nuclear translocation. Taken together, our results demonstrated that MC13 protected neurons from microglia-mediated neuroinflammatory injury by inhibiting TRAF6-TAK1-NF-κB, p38/ERK MAPKs, and Jak2-Stat1/3 pathways. Finally, MC13 might interact with LPS and interfere LPS-binding to cell membrane surface. These findings suggested that coumarin might act as a potential medicinal agent for treating neuroinflammation as well as inflammation-related neurodegenerative diseases.

Protosappanin B protects PC12 cells against oxygen-glucose deprivation-induced neuronal death by maintaining mitochondrial homeostasis via induction of ubiquitin-dependent p53 protein degradation.

Protosappanin B (PTB) is a bioactive dibenzoxocin derivative isolated from Caesalpinia sappan L. Here, we investigated the neuroprotective effects and the potential mechanisms of PTB on oxygen-glucose deprivation (OGD)-injured PC12 cells. Results showed that PTB significantly increased cell viability, inhibited cell apoptosis and up-regulated the expression of growth-associated protein 43 (a marker of neural outgrowth). Moreover, our study revealed that PTB effectively maintained mitochondrial homeostasis by up-regulation of mitochondrial membrane potential (MMP), inhibition of cytochrome c release from mitochondria and inactivation of mitochondrial caspase-9/3 apoptosis pathway. Further study showed that PTB significantly promoted cytoplasmic component degradation of p53 protein, a key negative regulator for mitochondrial function, resulting in a release of Bcl-2 from p53-Bcl-2 complex and an enhancing translocation of Bcl-2 to mitochondrial outer membrane. Finally, we found the degradation of p53 protein was induced by PTB via activation of a MDM2-dependent ubiquitination process. Taken together, our findings provided a new viewpoint of neuronal protection strategy for anoxia and ischemic injury with natural small molecular dibenzoxocin derivative by activating ubiquitin-dependent p53 protein degradation as well as increasing mitochondrial function.

Deoxysappanone B, a homoisoflavone from the Chinese medicinal plant Caesalpinia sappan L., protects neurons from microglia-mediated inflammatory injuries via inhibition of IκB kinase (IKK)-NF-κB and p38/ERK MAPK pathways.

Caesalpinia sappan L. (Lignum Sappan) is a Chinese medicinal plant for treating ischemic cerebral apoplexy. Deoxysappanone B (DSB), a homoisoflavone compound isolated from C. sappan L. (Lignum Sappan), was studied for anti-neuroinflammatory and neuroprotective properties using lipopolysaccharide (LPS)-induced BV-2 microglia neuroinflammation model and LPS-induced microglia-neuron co-culture system. Our findings showed that DSB effectively inhibited BV-2 microglia-mediated neuroinflammatory mediators׳ release including NO, PGE2, TNF-α, IL-6 and reactive oxygen species. Moreover, DSB markedly protected neurons against inflammatory microglia-mediated neurotoxicity in a microglia-neuron co-culture system. Mechanism study revealed that DSB blocked two major neuroinflammation-related signaling pathways including IKK-IκB-nuclear factor kappaB (NF-κB) and p38/ERK mitogen-activated protein kinase (MAPK) cascades, further leading to the inhibition of neuroinflammatory mediators׳ production. The present study provides evidence that the anti-neuroinflammatory and neuroprotective effect of DSB are due to the suppression of neuroinflammatory mediators׳ production as well as inflammation-induced neurotoxicity through regulation of multi-targets. Therefore, DSB may serve as a neuroprotective agent for the treatment of neuroinflammatory disorders and inflammation-related neuronal injury.

ASC, a bioactive steroidal saponin from Ophitopogin japonicas, inhibits angiogenesis through interruption of Src tyrosine kinase-dependent matrix metalloproteinase pathway.

As angiogenesis is an important target for antitumour drugs, the agents that inhibit angiogenesis may help reduce the use of chemotherapy by blocking tumour blood supply. In this study, we investigated a potent angiogenesis inhibitor, ASC, a steroidal saponin compound, which has been purified from Ophitopogin japonicus (L.f) Ker.-Gawl. Our observations showed that ASC significantly suppressed human umbilical vein endothelial cell (HUVECs) growth both in vitro and in vivo. This may be resulted from the G2/M cell cycle arrest effects of ASC. Moreover, ASC inhibited HUVECs invasion and tube formation processes, which were associated with endothelial cells remodelling. A mechanism study indicated that ASC down-regulated the expression of Src tyrosine kinase, further leading to the blockage of Akt-dependent matrix metalloproteinases (mainly for MMP-9) signalling pathway, which was functionally associated with angiogenic blood vessels. Finally, ASC significantly inhibited angiogenesis and MMPs/VEGF expression in the subcutaneously injected matrigel in C57/BL mice. These findings suggest that ASC might be a potential drug candidate in anti-angiogenesis and anticancer therapies.

Induction of hepatoma carcinoma cell apoptosis through activation of the JNK-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS self-driven death signal circuit.

As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase-ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK-NADPH oxidase-ROS-dependent self-driven signal circuit pathway.