The ethanolic extract of peanut shell attenuates the depressive-like behaviors of mice through modulation of inflammation and gut microbiota.

Peanut shell is an agricultural byproduct being wasted on a large scale, which is in urgent need to be recycled. To fully utilize its pharmacological ingredients, e.g. luteolin, eriodyctiol, and 5,7-dihydroxychromone, we evaluated the curative effect of ethanol extract deriving from peanut shell (PSE) in treating chronic unpredictable mild stress (CUMS)-induced depressive mice. The chronic stress lasted for 10 weeks, and PSE at 100-900 mg/kg/day was gavaged to mice in the last 2 weeks of modeling. The depressive behaviors were assessed by analyses of sucrose preference, tail suspension, and forced swimming. The brain injury was demonstrated by Hematoxylin and Eosin (H&E), Nissl body, and TdT-mediated dUTP nick end labeling (TUNEL) stainings in the mouse hippocampus. Biochemical indicators were analyzed, including levels of neurotrophic factors, neurotransmitters, stress hormones, and inflammatory mediators. The feces were collected for the 16S rDNA sequencing of gut microbiome. Administration of PSE improved the sucrose water consumption of depressive mice, while it decreased the immobile time in tail suspension and forced swimming tests. Meanwhile, the anti-depressive effect of PSE was supported by ameliorated histochemical staining, increased levels of neurotrophic factors and neurotransmitters, as well as down-regulated stress hormones. Furthermore, the treatment of PSE was able to mitigate the levels of inflammatory cytokines in brain, serum, and small intestine. Besides, the tight junction proteins, e.g., occludin and ZO-1, of gut showed elevated expressions, which coincided with the elevated abundance and diversity of gut microbiota upon PSE treatment. This study validated the therapeutic efficacy of PSE in fighting against depression, as well as its modulatory action on inflammation and gut microbiota, which promoted the recycling of this agricultural waste to be health supplements of added value.

The inclusion of extract from aerial part of Scutellaria baicalensis in feeding of pearl gentian grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceo-latus♂) promotes growth and immunity.

The root of Scutellaria baicalensis (Scutellaria Radix) has been used as herbal medicine for years in China; however, its stem and leaf (aerial part) are considered as waste. The water extract of aerial part of S. baicalensis, named as SBA, having anti-microbial property has been applied in fish aquaculture. To extend the usage of SBA in fish feeding, SBA was employed to feed pearl gentian grouper (a hybrid of Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂), and subsequently the total fish output, the levels of digestive enzymes and inflammatory cytokines were determined. Feeding the fish with different doses of SBA for two months, the body length and weight were significantly increased by 5%-10%. In parallel, the expressions of alkaline phosphatase and growth-related factors in bone, liver and muscle of SBA-fed fish were doubled, which could account the growth promoting effect of SBA. Besides, the activity of digestive enzyme, lipase, and the expressions of anti-inflammatory cytokines were markedly stimulated by 2-3 times under the feeding of 3% SBA-containing diet. The results indicated the growth promoting activity of SBA in culture of pearl gentian grouper, as well as the effect of SBA in strengthening the immunity. These beneficial effects of SBA feeding can increase the total yield of pearl gentian grouper in aquaculture. Thus, the re-cycle of waste products during the farming of S. baicalensis herb in serving as fish feeding should be encouraged.

Feeding containing the aerial part of Scutellaria baicalensis promotes the growth and nutritive value of rabbit fish Siganus fuscescens.

The root of Scutellaria baicalensis (Scutellaria Radix) has been used as herbal medicine for years, while its stem and leaf (aerial part) are considered as waste. The water extract from the aerial part of S. baicalensis (named as SBA) being included in the feeding of Siganus fuscescens (grey rabbit fish) has been shown to replace antibiotics in aquaculture with excellent outcome. To strengthen the usage of SBA in fish feeding, the total fish output and its nutritive value were determined here. Feeding the fishes with different doses of SBA for a month, the body length and weight were significantly increased after intake of standard feed containing 1% SBA. In parallel, the expressions of alkaline phosphatase and growth-related factors in bone, liver, and muscle of 1% SBA-fed fishes were markedly increased, suggesting the beneficial effects of SBA. The composition of amino acid and fatty acid in fish muscle, after intaking 1% SBA-containing feed, was altered. In SBA-fed fish muscle, the amounts of threonine and methionine were increased, while the amount of leucine was decreased, as compared with control group. The amounts of fatty acids, including docosahexaenoic acid, phosphatidylcholine, and phosphatidylethanolamine, were increased in the 1% SBA-fed fish, while the amounts of triglycerides were decreased. The results indicated the growth-promoting activity of SBA in an in vivo culture of S. fuscescens, as well as to increase the nutritive values of the muscle. Thus, the re-cycle of waste products during the farming of S. baicalensis herb in serving as fish feeding should be encouraged.

Mass spectrometry-based multi-omics analysis reveals the thermogenetic regulation of herbal medicine in rat model of yeast-induced fever.

ETHNOPHARMACOLOGICAL RELEVANCE: In the principle of traditional Chinese medicine (TCM), clinical usage is based on drug attributes of the herbal medicine. The cold and hot properties of TCM are classified accordingly to their pharmacological effects, such as temperature change. Herbal medicine has been used as food supplements in our daily life, and the thermogenetic regulation is one of their primary applications. However, the underlying mechanism of "cold" or "hot" stimulating effect of herbal medicine has not been fully identified. AIM OF THE STUDY: Thermogenetic regulation and classification of herbal medicine of hot/cold herbs were determined by rat model of yeast-induced fever. MATERIALS AND METHODS: Here, a novel method in classifying and characterizing cold- and hot-herbal medicines was established by analyses of mass spectrometry (MS)-based metabolomics and lipidomics from the serum of herbal extract-treated rats. The yeast-induced inflammatory rats were used as the model system, which were subjected to the treatments of cold- or hot-herbal medicine. RESULTS: The multi-omics approach identified the clustering of metabolites from cold and hot herb-treated rat serum by using partial least squares discriminant analysis (PLS-DA), and which subsequently identified that the 8-h treatment was the metabolic perturbation point of herb-mediated thermogenesis. Meanwhile, the levels of identified metabolites in the serum, i.e. lysoPE, lysoPC and carnitine, showed a positive relationship with the regulation of body temperature; while the levels of amino acid, fatty acid and bile acid were contrary correlated with the temperature change. In addition, the differential expressed metabolites were subjected to pathway enrichment and network analyses in revealing the possible action mechanism of herbal medicines in relating to thermogenetic regulation. CONCLUSION: The developed MS-based omics provides a new insight in characterizing the properties of cold/hot herbal medicine.

Nuclear Factor-κB Signaling Mediates Antimony-induced Astrocyte Activation.

OBJECTIVE: Antimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation. METHODS: Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Antimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation in vivo and in vitro. Antimony exposure consistently upregulated the expression of inflammatory factors. Moreover, it induced the NF-κB signaling, indicated by increased p65 phosphorylation and translocation to the nucleus. NF-κB inhibition effectively attenuated antimony-induced astrocyte activation. Furthermore, antimony phosphorylated TGF-ß-activated kinase 1 (TAK1), while TAK1 inhibition alleviated antimony-induced p65 phosphorylation and subsequent astrocyte activation. CONCLUSION: Antimony activated astrocytes by activating the NF-κB signaling pathway.

Characterization of a macrophagic-like cell line derived from rabbit fish (Siganus fuscescens): An illustration of anti-inflammatory responses of the herbal extract of Scutellaria baicalensis.

A new cell line was isolated and characterized from the head kidney of Siganus fuscescens (rabbit fish). The new macrophagic-like cell line was named as rabbit fish macrophage (RFM), and which could be sub-cultured for over 50 cycles since the development. RFM cell line was tested for growth in different temperatures and serum concentrations: the best growing condition was optimized at 20% serum under 28 °C. In cultured RFM cells, sequencing of 18S rRNA, as well as immunostaining of cytokeratin and CD 68, confirmed the identity as macrophagic cell of S. fuscescens. Cultured RFM cells were exposed to challenge of inflammation, as triggered by LPS, showing highly sensitive responses to inflammation, including release of nitric oxide, expression of cytokine, and activation of phagocytosis. The water extract of aerial part of Scutellaria baicalensis, named as SBA, has been shown anti-inflammatory property in S. fuscescens fish. In order to extend the application of SBA in aquaculture, the extract and its effective flavonoids, i.e. baicalin and scutellarin, were applied in LPS-treated RFM cells. Application of SBA extract, baicalin or scutellarin, inhibited the expressions of LPS-induced inflammatory cytokines, i.e. IL-1ß, TNF-α, as well as the signaling of transcription factor NF-κB. The results support the established RFM cell line could be an ideal in vitro model in drug screening relating to inflammation. Additionally, the notion of SBA herbal extract in fish aquaculture is supported by its efficacy against inflammation.

The herbal extract deriving from aerial parts of Scutellaria baicalensis shows anti-inflammation and anti-hypoxia responses in cultured fin cells from rabbit fish.

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1ß, IL-6, as well as the signaling of NF-κB. The application of CoCl2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

Synergistic Inhibition of Acetylcholinesterase by Alkaloids Derived from Stephaniae Tetrandrae Radix, Coptidis Rhizoma and Phellodendri Chinensis Cortex.

Alkaloids having acetylcholinesterase (AChE) inhibitory activity are commonly found in traditional Chinese medicine (TCM); for example, berberine from Coptis chinensis, galantamine from Lycoris radiata, and huperzine A from Huperzia serrata. In practice of TCM, Stephaniae Tetrandrae Radix (STR) is often combined with Coptidis Rhizoma (CR) or Phellodendri Chinensis Cortex (PCC) as paired herbs during clinical application. Fangchinoline from STR and coptisine and/or berberine from CR and/or PCC are active alkaloids in inhibiting AChE. The traditional usage of paired herbs suggests the synergistic effect of fangchinoline-coptisine or fangchinoline-berberine pairing in AChE inhibition. HPLC was applied to identify the main components in herbal extracts of STR, CR, and PCC, and the AChE inhibition of their main components was determined by Ellman assay. The synergism of herb combination and active component combination was calculated by median-effect principle. Molecular docking was applied to investigate the underlying binding mechanisms of the active components with the AChE protein. It was found that fangchinoline showed AChE inhibitory potency; furthermore, fangchinoline-coptisine/berberine pairs (at ratios of 1:5, 1:2, 1:1, and 2:1) synergistically inhibited AChE; the combination index (CI) at different ratios was less than one when Fa = 0.5, suggesting synergistic inhibition of AChE. Furthermore, the molecular docking simulation supported this enzymatic inhibition. Therefore, fangchinoline-coptisine/berberine pairs, or their parental herbal mixtures, may potentially be developed as a possible therapeutic strategy for Alzheimer's patients.

Asarones from Acori Tatarinowii Rhizoma stimulate expression and secretion of neurotrophic factors in cultured astrocytes.

Acori Tatarinowii Rhizoma (ATR, the dried rhizome of Acorus tatarinowii Schott.) is a traditional Chinese medicine widely used to treat brain diseases, e.g. depression, forgetfulness, anxiety and epilepsy. Several lines of evidence support that ATR has neuronal beneficial functions in animal models, but its action mechanism in cellular level is unknown. Here, we identified α-asarone and ß-asarone could be the major active ingredients of ATR, which, when applied onto cultured rat astrocytes, significantly stimulated the expression and secretion of neurotrophic factors, i.e. nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF), in dose-dependent manners. These results suggested that the neuronal action of ATR, triggered by asarone, might be mediated by an increase of expression of neurotrophic factors in astrocytes, which therefore could support the clinical usage of ATR. In addition, application of PKA inhibitor, H89, in cultured astrocytes partially blocked the asarone-induced neurotrophic factor expression, suggesting the involvement of PKA signaling. The results proposed that α-asarone and ß-asarone from ATR could serve as potential candidates for drug development in neurodegenerative diseases.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Promotes Proliferation of Astrocyte Cells via the Akt/STAT3/Cyclin D1 Pathway.

OBJECTIVE: The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent organic pollutant, is harmful to the nervous system, but its effects on the brain are still unclear. This study aimed to investigate the effects of TCDD on astrocytes proliferation and underlying molecular mechanism. METHODS: The cell proliferation was measured by EdU-based proliferation assay and PI staining by flow cytometry. Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of signal transducer and activator of transcription 3 (STAT3). RESULTS: C6 cells treated with 10 and 50 nmol/L TCDD for 24 h showed significant promotion of the proliferation of. The exposure to TCDD resulted in the upregulation in the expression levels of phosphorylated protein kinase B (p-Akt), phosphorylated STAT3, and cyclin D1 in a dose- and time-dependent manner. The inhibition of Akt expression with LY294002 or STAT3 expression with AG490 abolished the TCDD-induced cyclin D1 upregulation and cell proliferation. Furthermore, LY294002 suppressed the activation of STAT3. Finally, TCDD promoted the translocation of STAT3 from the cytoplasm to the nucleus, and LY294002 treatment blocked this effect. CONCLUSION: TCDD exposure promotes the proliferation of astrocyte cells via the Akt/STAT3/cyclin D1 pathway, leading to astrogliosis.

Isoorientin derived from Gentiana veitchiorum Hemsl. flowers inhibits melanogenesis by down-regulating MITF-induced tyrosinase expression.

BACKGROUND: Gentiana is a genus of flowering plants in Gentianaceae family, which comprises of 1,600 species. The roots of few species of Gentiana, also known as Long Dan Cao in Chinese, are traditionally used in herbal remedies for a wide variety of liver-associated diseases. The medicinal part of Gentiana is root; however, the trumpet-shaped flowers are seldom being used. PURPOSE: We investigated the anti-melanogenesis effect of water extract of Gentiana veitchiorum Hemsl. flowers, and isoorientin was identified to be the active compound. STUDY DESIGN: We tested the anti-melanogenesis effects of extracts deriving from different parts of G. veitchiorum, followed by identification of active ingredients within the extracts. The mechanism of inhibitory effect on melanogenesis, triggered by isoorientin, was elucidated by in vitro analyses. METHODS: HPLC was applied to identify the components in water extracts from different parts of G. veitchiorum. The cytotoxicity of extracts and pure compounds in cultured B16F10 murine melanoma cells was determined by MTT and trypan blue assays. Melanin assay, tyrosinase assay, RT-PCR, luciferase assay and western blot were used to analyze the effect of isoorientin in melanin content, tyrosinase activity, as well as the expressions of those related genes and proteins. RESULTS: We identified an inhibitory effect on melanogenesis from water extract of G. veitchiorum flowers in B16F10 cells. Isoorientin, a major flavone in the extract, was identified to be an active ingredient causing reduction in melanin content in a dose-dependent manner. Such reduction was suggested to be a result of suppressed expression of tyrosinase (TYR), tyrosinase related protein-1 (TRP1) and DOPA-chrome tautomerase (DCT). Isoorientin also suppressed the expression of microphthalmia- associated transcription factor (MITF) through the phosphorylation of cAMP response element-binding protein (CREB). CONCLUSION: These findings indicate that isoorientin derived from G. veitchiorum flowers may be a potential skin lightening agent for the treatment of skin pigmentary disorders.

The extract of Polygoni Cuspidati Rhizoma et Radix suppresses the vascular endothelial growth factor-induced angiogenesis.

BACKGROUND: Polygoni Cuspidati Rhizoma et Radix (PCRR; the root and rhizome of Polygonum cuspidatum Sieb. et Zucc) is a traditional Chinese medicine for the treatment of inflammation, hyperlipemia, favus, jaundice and scald. HYPOTHESIS/PURPOSE: The extract of PCRR inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis. The hypothesis is supported by analysis of PCRR extract and investigation of pharmacological role and signaling mechanism of PCRR extract in regulating angiogenic responses. STUDY DESIGN: The PCRR ethanolic extract was examined for its inhibitory effects on angiogenesis based on VEGF-treated human umbilical vein endothelial cells and in zebrafish model METHODS: The effects and signaling mechanism of a standardized ethanolic extract of PCRR were tested on cell proliferation, migration and tube formation in VEGF-treated human umbilical vein endothelial cells, and which was further validated in zebrafish embryo model. RESULTS: The treatment of PCRR extract in cultured endothelial cells inhibited VEGF-induced cell proliferation, cell migration and tube formation in a dose-dependent manner and also suppressed the formation of sub-intestinal vessels in zebrafish embryos. Moreover, the applied PCRR extract suppressed VEGF-induced phosphorylations of VEGF receptor 2 (VEGFR2) and JNK. Thus, the site of effect triggered by PCRR was proposed to be mediated by VEGFR2. To further support this notion, the phosphorylations of Erk, Akt and eNOS, induced by VEGF, were markedly reduced under the challenge of PCRR extract: the reductions were subsequently further decreased in the present of inhibitors of Erk, Akt and eNOS. In parallel, the formation of ROS induced by VEGF in cultured endothelial cells was markedly reduced in the present of PCRR extract. CONCLUSION: Collectively, our studies demonstrated the pharmacological role and signaling mechanism of PCRR in regulation of angiogenic responses, which supported further evaluation and development of PCRR as a potential therapeutic agent for the treatment and prevention of diseases related with angiogenesis.

Yu Ping Feng San reverses cisplatin-induced multi-drug resistance in lung cancer cells via regulating drug transporters and p62/TRAF6 signalling.

Yu Ping Feng San (YPFS), an ancient Chinese herbal decoction composed of Astragali Radix, Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix, has been used in the clinic for treating immune deficiency. In cancer therapy, YPFS is being combined with chemotherapy drugs to achieve improved efficacy; however, scientific evidence to illustrate this combination effect is lacking. The present study aims to demonstrate the anti-drug resistance of YPFS in cisplatin (DDP)-resistant non-small cell lung cancer cells (A549/DDP). The application of YPFS exhibited a synergistic enhancement of DDP-induced cytotoxicity as well as of the apoptotic signalling molecules. DDP-induced expression of the multi-drug-resistance efflux transporters was markedly reduced in the presence of YPFS, resulting in a higher intracellular concentration of DDP. In addition, the application of YPFS increased DDP-induced ROS accumulation and MMP depletion, decreased p62/TRAF6 signalling in DDP-treated A549/DDP cells. The co-treatment of DDP and YPFS in tumour-bearing mice reduced the tumour size robustly (by more than 80%), which was much better than the effect of DDP alone. These results indicate that YPFS can notably improve the DDP-suppressed cancer effect, which may be a consequence of the elevation of intracellular DDP via the drug transporters as well as the down regulation of p62/TRAF6 signalling.