Identification of sedative-hypnotic compounds shared by five medicinal Polyporales mushrooms using UPLC-Q-TOF-MS/MS-based untargeted metabolomics.

BACKGROUND: Five Polyporales mushrooms, namely Amauroderma rugosum, Ganoderma lucidum, G. resinaceum, G. sinense and Trametes versicolor, are commonly used in China for managing insomnia. However, their active components for this application are not fully understood, restricting their universal recognition. PURPOSE: In this study, we aimed to identify sedative-hypnotic compounds shared by these five Polyporales mushrooms. STUDY DESIGN AND METHODS: A UPLC-Q-TOF-MS/MS-based untargeted metabolomics, including OPLS-DA (orthogonal projection of potential structure discriminant analysis) and OPLS (orthogonal projections to latent structures) analysis together with mouse assays, were used to identify the main sedative-hypnotic compounds shared by the five Polyporales mushrooms. A pentobarbital sodium-induced sleeping model was used to investigate the sedative-hypnotic effects of the five mushrooms and their sedative-hypnotic compounds. RESULTS: Ninety-two shared compounds in the five mushrooms were identified. Mouse assays showed that these mushrooms exerted sedative-hypnotic effects, with different potencies. Six triterpenes [four ganoderic acids (B, C1, F and H) and two ganoderenic acids (A and D)] were found to be the main sedative-hypnotic compounds shared by the five mushrooms. CONCLUSION: We for the first time found that these six triterpenes contribute to the sedative-hypnotic ability of the five mushrooms. Our novel findings provide pharmacological and chemical justifications for the use of the five medicinal mushrooms in managing insomnia.

A tri-herb formulation protects against ethanol-induced mouse liver injury and downregulates mitogen-activated protein kinase phosphatase 1.

BACKGROUND: A tri-herb formulation comprising Ganoderma (the dried fruiting body of Ganoderma lucidum), Puerariae Thomsonii Radix (the dried root of Pueraria thomsonii) and Hoveniae Semen (the dried mature seed of Hovenia acerba) -GPH for short- has been using for treating liver injury; however, the pharmacological basis of this application of GPH is unknown. This study aimed to investigate the liver protective effects and mechanisms of action of an ethanolic extract of GPH (GPHE) in mice. METHODS: To control the quality of GPHE, the contents of ganodermanontriol, puerarin and kaempferol in the extract were quantified by ultra-performance liquid chromatography. An ethanol (6 ml/kg, i.g.)-induced liver injury ICR mouse model was employed to investigate the hepatoprotective effects of GPHE. RNA-sequencing analysis and bioassays were performed to reveal the mechanisms of action of GPHE. RESULTS: The contents of ganodermanontriol, puerarin and kaempferol in GPHE were 0.0632%, 3.627% and 0.0149%, respectively. Daily i.g. administration of 0.25, 0.5 or 1 g/kg of GPHE for 15 consecutive days suppressed ethanol (6 ml/kg, i.g., at day 15)-induced upregulation of serum AST and ALT levels and improved histological conditions in mouse livers, indicating that GPHE protects mice from ethanol-induced liver injury. Mechanistically, GPHE downregulated the mRNA level of Dusp1 (encoding MKP1 protein, an inhibitor of the mitogen-activated protein kinases JNK, p38 and ERK), and upregulated expression and phosphorylation of JNK, p38 and ERK, which are involved in cell survival in mouse liver tissues. Also, GPHE increased PCNA (a cell proliferation marker) expression and reduced TUNEL-positive (apoptotic) cells in mouse livers. CONCLUSION: GPHE protects against ethanol-induced liver injury, and this effect of GPHE is associated with regulation of the MKP1/MAPK pathway. This study provides pharmacological justifications for the use of GPH in treating liver injury, and suggests that GPHE has potential to be developed into a modern medication for managing liver injury.

Inhibition of STAT3 signaling contributes to the anti-melanoma effects of chrysoeriol.

BACKGROUND: Melanoma is an aggressive malignancy with a high mortality rate. Signal transducer and activator of transcription 3 (STAT3), an oncoprotein, is considered as an effective target for treating melanoma. Chrysoeriol is a flavonoid compound, and possesses anti-tumor activity in lung cancer, breast cancer and multiple myeloma; while whether it has anti-melanoma effects is still not known. Chrysoeriol has been shown to restrain STAT3 signaling in an inflammation mouse model. PURPOSE: In this study, the anti-melanoma effects of chrysoeriol and the involvement of STAT3 signaling in these effects were investigated. STUDY DESIGN AND METHODS: CCK8 assays, 5-ethynyl-2'-deoxyuridine (EdU) staining, Annexin V-FITC/PI staining, Western blot analyses of cleaved caspase-9 and wound healing assays were used to study the anti-melanoma effects of chrysoeriol in cell models. A B16F10 melanoma bearing mouse model was used to evaluate the in vivo anti-melanoma effects of chrysoeriol. Indicators of cell proliferation, cell apoptosis and angiogeneis in melanoma tissues were detected by immunohistochemistry (IHC) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Immune cells in melanoma tissues were analyzed by flow cytometry. STAT3-overactivated cell models were used to investigate the involvement of STAT3 signaling in the anti-melanoma effects of chrysoeriol. Molecular dynamics (MD) simulations and surface plasmon resonance (SPR) assays were conducted to determine whether chrysoeriol binds to Src, an upstream kinase of STAT3. RESULTS: The results of cell experiments showed that chrysoeriol dose-dependently inhibited viability, proliferation and migration of, and induced apoptosis in, A375 and B16F10 melanoma cells. Chrysoeriol inhibited the phosphorylation of STAT3, and downregulated the expression of STAT3-target genes involved in melanoma growth and metastasis. Mouse studies showed that chrysoeriol restrained melanoma growth and tumor-related angiogenesis, and altered compositions of immune cells in melanoma microenvironment. Chrysoeriol also inhibited STAT3 signaling in B16F10 allografts. Chrysoeriol's viability-inhibiting effects were attenuated by over-activating STAT3 in A375 cells. Furthermore, chrysoeriol bound to the protein kinase domain of Src, and suppressed Src phosphorylation in melanoma cells and tissues. CONCLUSION: This study, for the first time, demonstrates that chrysoeriol has anti-melanoma effects, and these effects are partially due to inhibiting STAT3 signaling. Our findings indicate that chrysoeriol has the potential to be developed into an anti-melanoma agent.

Network pharmacology and molecular docking-based prediction of active compounds and mechanisms of action of Cnidii Fructus in treating atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common inflammatory skin disease that compromises the skin's barrier function and capacity to retain moisture. Cnidii Fructus (CF), the dried fruits of Cnidium monnieri, has long been used to treat atopic dermatitis (AD) in China. However, the anti-AD compounds and mechanisms of CF are not fully understood. In this study, we evaluated the active compounds and molecular targets of CF in treating AD. METHODS: The Traditional Chinese Medicine Systems Pharmacology database was used to acquire information regarding the compounds that occur in the herb. Targets of these compounds were predicted using the SwissTargetPrediction website tool. AD-related genes were collected from the GeneCards database. Gene ontology (GO) enrichment analysis and KEGG pathway analysis of proteins that are targeted by active compounds of CF and encoded by AD-related genes were performed using Database for Annotation, Visualization, and Integrated Discovery Bioinformatics Resources. A "compound-target" network was constructed and analyzed using Cytoscape Software. Molecular docking was performed using BIOVIA Discovery Studio Visualizer and AutoDock Vina. RESULTS: We identified 19 active compounds in CF, 532 potential targets for these compounds, and 1540 genes related to AD. Results of GO enrichment indicated that CF affects biological processes and molecular functions, such as inflammatory response and steroid hormone receptor activity, which may be associated with its anti-AD effects. KEGG pathway analyses showed that PI3K-Akt signaling, calcium signaling, Rap1 signaling, and cAMP signaling pathways are the main pathways involved in the anti-AD effects of CF. Molecular docking analyses revealed that the key active compounds in CF, such as (E)-2,3-bis(2-keto-7-methoxy-chromen-8-yl)acrolein, ar-curcumene, and diosmetin, can bind the main therapeutic targets AKT1, SRC, MAPK3, EGFR, CASP3, and PTGS2. CONCLUSIONS: Results of the present study establish a foundation for further investigation of the anti-AD compounds and mechanisms of CF and provide a basis for developing modern anti-AD agents based on compounds that occur in CF.

Amelioration of TPA-induced skin inflammation by the leaf extract of Vernonia amygdalina involves ERK/STAT3 (Ser727) signaling inhibition.

BACKGROUND: Uncontrolled inflammation causes health problems. Extracellular signal-regulated kinase (ERK) phosphorylates signal transducer and activator of transcription 3 (STAT3) at Ser727, resulting in inflammation. The leaf of Vernonia amygdalina (VA) is a medicinal herb for managing inflammation-associated diseases. Oral administration or topical application of VA leaf extract exerts anti-inflammatory effects in rat models. However, the anti-inflammatory mechanisms of the herb are not fully understood. PURPOSE: In this study, we aimed to investigate the involvement of ERK/STAT3 (Ser727) signaling in the anti-inflammatory effects of an ethanolic extract of VA leaves. STUDY DESIGN AND METHODS: Extracts of VA leaves were prepared with different concentrations of ethanol. A LPS-stimulated RAW264.7 cell model was used for in vitro assays, and a TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model was employed for in vivo assays. The 95% ethanol extract of VA leaves (VAE) exerted the strongest inhibitory effect on nitric oxide (NO) production in LPS-stimulated macrophages; thus it was selected for use in this study. Hematoxylin and eosin (H&E) staining was used to examine pathological conditions of mouse ear tissues. Griess reagent was employed to examine NO generation in cell cultures. Immunoblotting and ELISA were used to examine protein levels, and RT-qPCR was employed to examine mRNA levels. RESULTS: Topical application of VAE ameliorated mouse ear edema induced by TPA. VAE suppressed the phosphorylation of ERK (Thr202/Tyr204) and STAT3 (Ser727); and decreased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, IL-1ß and tumor necrosis factor-α (TNF-α) in the mouse ear tissues and in LPS-stimulated RAW 264.7 cells. VAE also inhibited NO production, and lowered mRNA levels of IL-6, IL-1ß and TNF-α in the macrophages. CONCLUSIONS: VAE ameliorates TPA-induced mouse ear edema. Suppression of ERK/STAT3 (Ser727) signaling is involved in VAE's anti-inflammatory effects. These novel data provide further pharmacological justifications for the medicinal use of VA in treating inflammation-associated diseases, and lay the groundwork for developing VAE into a new anti-inflammatory agent.

Inhibition of the Akt/NF-κB pathway is involved in the anti-gastritis effects of an ethanolic extract of the rhizome of Atractylodes macrocephala.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastritis can lead to ulcers and the development of gastric cancer. The rhizome of Atractylodes macrocephala Koidz. (Asteraceae), a traditional Chinese medicinal herb, is prescribed for the treatment of gastric disorders, hepatitis and rheumatism. Its bio-active compounds are considered to be particularly effective in this regard. However, the molecular processes of the herb's anti-inflammatory activity remain obscure. This study elucidates a mechanism upon which an ethanolic extract of this herb (Am-EE) exerts anti-inflammation effects in RAW264.7 macrophage cells (RAW cells) stimulated by lipopolysaccharide (LPS) treatment and HCl Ethanol-stimulated gastritis rats. AIM OF THE STUDY: To investigate the anti-gastritis activities of Am-EE and explore the mode of action. MATERIALS AND METHODS: Ethanol (95%) was used to prepare Am-EE. The quality of the extract was monitored by HPLC analysis. The in vivo effects of this extract were examined in an HCl Ethanol-stimulated gastritis rat model, while LPS-stimulated RAW cells were used for in vitro assays. Cell viability and nitric oxide (NO) production were observed by MTT and Griess assays. Real-time PCR was used to examine mRNA expression. The PGE2 ELISA kit was employed to detect prostaglandin E2 (PGE2). Enzyme activities and protein contents were examined by immunoblotting. Luciferase reporter gene assays (LRA) were employed to observe nuclear transcription factor (NF)-κB activity. The SPSS (SPSS Inc., Chicago, Illinois, United States) application was used for statistical examination. RESULTS: HPLC analysis indicates that Am-EE contains atractylenolide-1 (AT-1, 1.33%, w/w) and atractylenolide-2 (AT-2, 1.25%, w/w) (Additional Figure. A1). Gastric tissue damage (induced by HCl Ethanol) was significantly decreased in SD rats following intra-gastric application of 35 mg/kg Am-EE. Indistinguishable to the anti-inflammation effects of 35 mg/kg ranitidine (gastric medication). Am-EE treatment also reduced LPS-mediated nitric oxide (NO) and prostaglandin E2 (PGE2) production. The mRNA and protein synthesis of inducible cyclooxygenase (COX)-2 and NO synthase (iNOS) was down-regulated following treatment in RAW cells. Am-EE decreased NF-κB (p50) nuclear protein levels and inhibited NF-κB-stimulated LRA activity in RAW cells. Lastly, Am-EE decreased the up-regulated levels of phosphorylated IκBα and Akt proteins in rat stomach lysates and in LPS challenged RAW cell samples. CONCLUSION: Our study illustrates that Am-EE suppresses the Akt/IκBα/NF-κB pathway and exerts an anti-inflammatory effect. These novel conclusions provide a pharmacological basis for the clinical use of the A. macrocephala rhizome in the treatment and prevention of gastritis and gastric cancer.

Chrysoeriol suppresses hyperproliferation of rheumatoid arthritis fibroblast-like synoviocytes and inhibits JAK2/STAT3 signaling.

BACKGROUND: Fibroblast-like synoviocytes (FLS) have cancer cell-like characteristics, such as abnormal proliferation and resistance to apoptosis, and play a pathogenic role in rheumatoid arthritis (RA). Hyperproliferation of RA-FLS that can be triggered by the activation of interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling destructs cartilage and bone in RA patients. Chrysoeriol is a flavone found in medicinal herbs such as Chrysanthemi Indici Flos (the dried capitulum of Chrysanthemum indicum L.). These herbs are commonly used in treating RA. Chrysoeriol has been shown to exert anti-inflammatory effects and inhibit STAT3 signaling in our previous studies. This study aimed to determine whether chrysoeriol inhibits hyperproliferation of RA-FLS, and whether inhibiting STAT3 signaling is one of the underlying mechanisms. METHODS: IL-6/soluble IL-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS were used to evaluate the effects of chrysoeriol. CCK-8 assay and crystal violet staining were used to examine cell proliferation. Annexin V-FITC/PI double staining was used to detect cell apoptosis. Western blotting was employed to determine protein levels. RESULTS: Chrysoeriol suppressed hyperproliferation of, and evoked apoptosis in, IL-6/sIL-6R-stimulated RA-FLS. The apoptotic effect of chrysoeriol was verified by its ability to cleave caspase-3 and caspase-9. Mechanistic studies revealed that chrysoeriol inhibited activation/phosphorylation of Janus kinase 2 (JAK2, Tyr1007/1008) and STAT3 (Tyr705); decreased STAT3 nuclear level and down-regulated protein levels of Bcl-2 and Mcl-1 that are transcriptionally regulated by STAT3. Over-activation of STAT3 significantly diminished anti-proliferative effects of chrysoeriol in IL-6/sIL-6R-stimulated RA-FLS. CONCLUSIONS: We for the first time demonstrated that chrysoeriol suppresses hyperproliferation of RA-FLS, and suppression of JAK2/STAT3 signaling contributes to the underlying mechanisms. This study provides pharmacological and chemical justifications for the traditional use of chrysoeriol-containing herbs in treating RA, and provides a pharmacological basis for developing chrysoeriol into a novel anti-RA agent.

Tea and tea drinking: China's outstanding contributions to the mankind.

BACKGROUND: Tea trees originated in southwest China 60 million or 70 million years ago. Written records show that Chinese ancestors had begun drinking tea over 3000 years ago. Nowadays, with the aging of populations worldwide and more people suffering from non-communicable diseases or poor health, tea beverages have become an inexpensive and fine complementary and alternative medicine (CAM) therapy. At present, there are 3 billion people who like to drink tea in the world, but few of them actually understand tea, especially on its development process and the spiritual and cultural connotations. METHODS: We searched PubMed, Google Scholar, Web of Science, CNKI, and other relevant platforms with the key word "tea", and reviewed and analyzed tea-related literatures and pictures in the past 40 years about tea's history, culture, customs, experimental studies, and markets. RESULTS: China is the hometown of tea, tea trees, tea drinking, and tea culture. China has the oldest wild and planted tea trees in the world, fossil of a tea leaf from 35,400,000 years ago, and abundant tea-related literatures and art works. Moreover, tea may be the first Chinese herbal medicine (CHM) used by Chinese people in ancient times. Tea drinking has many benefits to our physical health via its antioxidant, anti-inflammatory, immuno-regulatory, anticancer, cardiovascular-protective, anti-diabetic, and anti-obesity activities. At the moment, COVID-19 is wreaking havoc across the globe and causing severe damages to people's health and lives. Tea has anti-COVID-19 functions via the enhancement of the innate immune response and inhibition of viral growth. Besides, drinking tea can allow people to acquire a peaceful, relaxed, refreshed and cheerful enjoyment, and even longevity. According to the meridian theory of traditional Chinese medicine, different kinds of tea can activate different meridian systems in the human body. At present, black tea (fermented tea) and green tea (non-fermented tea) are the most popular in the world. Black tea accounts for over 90% of all teas sold in western countries. The world's top-grade black teas include Qi Men black in China, Darjeeling and Assam black tea in India, and Uva black tea in Sri Lanka. However, all top ten famous green teas in the world are produced in China, and Xi Hu Long Jing tea is the most famous among all green teas. More than 700 different kinds of components and 27 mineral elements can be found in tea. Tea polyphenols and theaflavin/thearubigins are considered to be the major bioactive components of black tea and green tea, respectively. Overly strong or overheated tea liquid should be avoided when drinking tea. CONCLUSIONS: Today, CAM provides an array of treatment modalities for the health promotion in both developed and developing countries all over the world. Tea drinking, a simple herb-based CAM therapy, has become a popular man-made non-alcoholic beverage widely consumed worldwide, and it can improve the growth of economy as well. Tea can improve our physical and mental health and promote the harmonious development of society through its chemical and cultural elements.

Ethyl acetate extract of the Musa nana flower inhibits osteoclastogenesis and suppresses NF-κB and MAPK pathways.

Banana flowers are consumed as a vegetable and traditionally used for managing several health problems including joint pain, a symptom of bone loss. Osteoclasts are key effector cells responsible for bone loss. Some flavonoids in banana flowers, such as quercetin and quercitrin, have been shown to be able to inhibit osteoclastogenesis. Whether banana flowers can inhibit osteoclast formation is unknown. In this study, we prepared the ethyl acetate fraction (FFE-EA) of an ethanolic extract of fresh flowers of Musa nana. Using UPLC-MS/MS analyses, 76 polyphenols were identified in FFE-EA. In RANKL-stimulated RAW264.7 macrophages, FFE-EA inhibited osteoclastogenesis and osteoclastic bone resorption. Mechanistic studies revealed that FFE-EA suppressed NF-κB and MAPK pathways, and lowered mRNA levels of osteoclast formation/function-related genes. These findings suggest that flowers of M. nana could be a source for formulating functional food that benefits bone health.

A proprietary herbal drug Young Yum Pill ameliorates chronic fatigue syndrome in mice.

BACKGROUND: Chronic fatigue syndrome (CFS) is a complex disease with few effective and safe therapies. Young Yum Pill (YYP), a proprietary herbal drug, has been used to relieve CFS-like symptoms. The pharmacological basis of this application of YYP is unknown. PURPOSE: This study aimed to investigate the pharmacological effects and mechanisms of action of YYP in a mouse model of CFS. STUDY DESIGN AND METHODS: A food restriction and exhaustive swimming-induced mouse CFS model was used to evaluate the effects of YYP. Lymphocyte proliferation was assessed by MTT assays. T-lymphocyte subsets were analyzed by flow cytometry. Serum biochemical parameters were determined using commercial kits. Protein levels were measured by immunoblotting. RESULTS: Intragastric administration of YYP (2.85, 5.70, 11.40 g/kg) daily for 21 consecutive days significantly prolonged swimming time and diminished body weight loss of CFS mice. Mechanistic investigations revealed that YYP increased thymus and spleen indices of CFS mice, enhanced proliferation of lipopolysaccharide- or concanavalin A-stimulated spleen lymphocytes, and increased CD3+CD4+ and CD3+CD8+ T-cells in the spleen. YYP increased glycogen content in gastrocnemius muscle and liver, and lowered levels of triglyceride, lactic acid and urea nitrogen in sera of CFS mice. YYP suppressed the elevation of serum level of malondialdehyde, the increase of activities of lactic dehydrogenase and creatine phosphokinase, and the decrease of activity of the serum antioxidant enzyme superoxide dismutase in CFS mice. Moreover, YYP upregulated protein level of activated AMPK in gastrocnemius muscle and liver of CFS mice. CONCLUSIONS: YYP ameliorates CFS by reversing metabolic changes, reducing oxidative damage, and improving some immune function parameters in mice. This study provides pharmacological justifications for the use of YYP in treating fatigue, including CFS.

The anti-inflammatory effects of an ethanolic extract of the rhizome of Atractylodes lancea, involves Akt/NF-κB signaling pathway inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried rhizome of Atractylodes lancea (Thumb.) DC. (Compositae) has been prescribed in folk medicine for the management of various inflammatory conditions such as rheumatic diseases, gastritis and hepatitis. However, the molecular mechanisms underlying the beneficial properties of this herb remain elusive. AIM OF THE STUDY: In this study, we investigated the anti-gastritis activities of Al-EE (an ethanolic extract of the herb) and explored the mechanism of action. MATERIALS AND METHODS: An ethanolic extract of the Atractylodes lancea (Thumb.) DC. (Compositae) rhizome, Al-EE, was prepared with ethanol (95%) and quality controlled using HPLC analysis. To determine the in vivo effects of this extract, we utilised a HCl/EtOH-induced gastritis rat model. In vitro assays were carried out using a lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cell model. MTT assays were used to examine cell viability, while Griess assays were carried out to measure nitric oxide (NO) production. Messenger RNA expression was examined by real-time PCR. Prostaglandin E2 (PGE2) production was examined using ELISA assays. To examine protein expression and enzymatic activities, we employed western blot analysis. Nuclear transcription factor (NF)-κB activity was determined by Luciferase reporter assays. RESULTS: The content of atractylenolide (AT)-1 and AT-2 in Al-EE was 0.45% and 5.07% (w/w), respectively (Supplementary Fig. 1). Al-EE treatment suppressed the production of NO and PGE2, reduced the mRNA expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-α, while also reducing the protein levels of iNOS and COX-2 in RAW264.7 macrophage cells. Furthermore, Al-EE inhibited the nuclear protein levels of NF-κB (p65) and NF-κB-driven luciferase reporter gene activity in RAW264.7 macrophage cells. Critically, intra-gastric injection of Al-EE (25 mg/kg) attenuated HCl/EtOH-induced gastric damage in SD rats, while the phosphorylation of Akt and IκBα was suppressed by Al-EE in vitro and in vivo. CONCLUSION: In summary, Al-EE has significant anti-gastritis effects in vivo and in vitro, which can be associated with the inhibition of the Akt/IκBα/NF-κB signalling pathway. This mechanistic finding provides a pharmacological basis for the use of the A. lancea rhizome in the clinical treatment of various inflammatory conditions.

Evaluation of Beneficial and Adverse Effects of a Diet Supplemented with Schisandrae Fructus Seed Ethanol Extract on Lipid and Glucose Metabolism in Normal and Hypercholesterolemic/Hyperglycemic Mice.

Schisandrae Fructus (SF), the fruit of Schisandra chinensis (Turcz.) Baillon, has been used for the treatment of liver injury and metabolism-related disorders in China. The objective of this study was to investigate the effects of supplementation with ethanol extract of SF seed (EtSF-S) on serum/hepatic lipid and glucose levels as well as fecal total cholesterol (TC) contents in mice fed a normal diet (ND) or high-fat/fructose diet (HFFD) containing 15% lard oil and 15% fructose. Female ICR mice (18-20 g in body weight) were fed with ND or HFFD for 3 months, and then EtSF-S was added to both chow diets at increasing concentrations of 1, 5, and 10% (w/w). Thirty days later, serum and hepatic lipids, including TC, triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and glucose, were measured. Dietary supplementation with EtSF-S reduced hepatic TC (36 and 18%) and TG levels (38 and 28%) and increased serum HDL/LDL ratio (16 and 26%) in both ND- and HFFD-fed mice, respectively. Moreover, supplementation with EtSF-S elevated serum HDL (31%) in HFFD-fed mice and reduced serum LDL (27%) in ND-fed mice. EtSF-S treatment reduced fat mass (40%) in ND-fed mice and increased fecal TC contents (33%) in HFFD-fed mice. EtSF-S supplementation decreased hepatic glucose contents (29%) in both ND- and HFFD-fed mice. However, diet supplemented with EtSF-S elevated serum TG levels (up to 123%) and hepatic size (28%), but more importantly, suppressed the body weight gain (approximately 130%) in mice fed with HFFD. These findings suggested that dietary supplementation with EtSF-S as natural herbal function food may be a useful strategy for the treatment of patients with fatty liver disease or overweight without a high intake of sugar and fat.

A two-herb formula inhibits hyperproliferation of rheumatoid arthritis fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) play a pathogenic role in rheumatoid arthritis (RA). STAT3 signaling is activated in FLS of RA patients (RA-FLS), which in turn causes RA-FLS hyperproliferation. RL is a traditional remedy for treating inflammatory diseases in China. It comprises Rosae Multiflorae Fructus and Lonicerae Japonicae Flos. A standardized ethanolic extract of RL (RLE) has been shown to exert anti-arthritic effects in collagen-induced arthritis (CIA) rats. Some constituents of RLE were reported to inhibit JAK2/STAT3 signaling in rat FLS. Here, we determined whether RLE inhibits FLS hyperproliferation, and explored the involvement of STAT3 signaling in this inhibition. In joints of CIA rats, RLE increased apoptotic FLS. In IL-6/sIL-6R-stimulated RA-FLS, RLE reduced cell viability and evoked cell apoptosis. In synovial tissues of CIA rats, RLE lowered the protein level of phospho-STAT3. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited activation/phosphorylation of STAT3 and JAK2, decreased the nuclear localization of STAT3, and downregulated protein levels of Bcl-2 and Mcl-1. Over-activation of STAT3 diminished RLE's anti-proliferative effects in IL-6/sIL-6R-stimulated RA-FLS. In summary, RLE inhibits hyperproliferation of FLS in rat and cell models, and suppression of STAT3 signaling contributes to the underlying mechanisms. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug.

A two-herb formula inhibits STAT3 signaling and exerts anti-melanoma effects in cell and animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: Malignant melanoma is a fatal cancer. Signal transducer and activator of transcription 3 (STAT3) has been proposed as a therapeutic target of melanoma. An herbal formula Huai-Hua-San (HHS) comprising Sophorae Flos (SF) and Gardeniae Fructus (GF) is traditionally used for treating cancers including melanoma, but the pharmacological basis is unknown. AIMS OF THIS STUDY: This study aimed to investigate the anti-melanoma effects of an ethanolic extract of HHS (HHSE), and explore the involvement of STAT3 signaling in the effects. MATERIALS AND METHODS: An UPLC-TOF/MS method was developed to control the quality of HHSE. A B16F10 allograft mouse model and three melanoma cell lines (B16F10, A375 and A2058) were used to determine the anti-melanoma effects of HHSE. Dacarbazine (DTIC) and Stattic were used as positive controls. Cell viability was detected using MTT and crystal violet staining assays. Cell apoptosis was analyzed by flow cytometry after the cells were stained with Annexin-V/PI. Cell invasive ability was examined using the transwell assay. Protein levels were determined by Western blotting. RESULTS: The contents of crocin I, crocin II, quercetin and kaempferol in HHSE were 0.59%, 0.98%, 4.66% and 1.15%, respectively. A clinically relevant dose of HHSE (0.1 g/kg/day, i.g. for 15 consecutive days) significantly suppressed B16F10 tumor growth in mice. HHSE dose-dependently reduced cell viability and dampened invasion of, and induced apoptosis in, melanoma cells. Mechanistic studies revealed that HHSE inhibited the phosphorylation/activation of STAT3 in B16F10 allografts and in cultured melanoma cells. In cell models, HHSE also inhibited the phosphorylation of STAT3 upstream kinases, JAK2 (Tyr1007/1008) and Src (Tyr416), lowered STAT3 nuclear levels, and down-regulated the protein levels of STAT3-targeted molecules. Over-activation of STAT3 in A375 cells significantly attenuated the cytotoxic effects of HHSE. CONCLUSIONS: HHSE exhibits anti-melanoma effects in cell and mouse models. Inhibition of STAT3 signaling contributes to the anti-melanoma mechanisms of HHSE. Our findings lay a groundwork for developing HHSE as a modern agent for melanoma management, and provide pharmacological justifications for the traditional use of HHS in treating melanoma.

Twenty-four-week oral dosing toxicities of Herba Siegesbeckiae in rats.

BACKGROUND: Herba Siegesbeckiae (HS), the dried aerial parts of Siegesbeckia orientalis L., S. pubescens Makino, or S. glabrescens Makino, is traditionally used for treating chronic diseases in China. However, there is no information about the chronic toxicity of HS. The objective of this study is to evaluate the 24-week oral dosing toxicities of HS aqueous extract (HSE) in rats. METHODS: S. orientalis-originated HS was reflux-extracted with distilled water. Sprague-Dawley rats were randomly divided into four groups, with 10 males and 10 females in each group. The rats were intragastrically administered with HSE at 5, 1.67 and 0.56 g/kg (experimental groups) or an equal volume of distilled water (control group), 6 days a week, for 24 weeks. The high dose of HSE (5 g/kg) was its maximum tolerated dose. Body weight was recorded every 2 days during the experimental period. Chemical, hematological and histopathological parameters, as well as organ weights, were measured at the end of the experiment. RESULTS: Decreased body weight gain; increased liver and lung relative weights; histopathological alterations in liver and lung tissues; elevated serum levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase were found after HSE treatments. In liver tissues, HSE treatment upregulated levels of three pro-inflammatory cytokines: IL-6, IL-1ß and TNF-α. In lung tissues, HSE treatment caused oxidative stress and activated mitogen-activated protein kinases (MAPKs). CONCLUSION: Long-term oral administration of HSE caused toxicities in rats evidenced by decreased body weight gain, as well as liver and lung damage. Treatment-induced oxidative stress, inflammation and MAPK activation are involved in HSE's toxicities. Caution should be taken when using HS to treat chronic diseases.

An herbal formula inhibits STAT3 signaling and attenuates bone erosion in collagen-induced arthritis rats.

BACKGROUND: Receptor activator of NF-κB ligand (RANKL) facilitates differentiation of osteoclast precursors into osteoclasts, resulting in bone erosion in rheumatoid arthritis (RA) patients. Fibroblast-like synoviocytes (FLS) are the main cells for producing RANKL. Signal transducer and activator of transcription 3 (STAT3) signaling is activated in FLS of RA patients (RA-FLS), which has been linked to RANKL production. A two-herb formula (RL) comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos is traditionally used for treating RA in China. We have found that a standardized ethanolic extract of RL (RLE for short) alleviates bone erosion in collagen-induced arthritis (CIA) rats. PURPOSE: This study aimed to determine whether RLE inhibits RANKL production and osteoclastogenesis in cell and rat models, and to explore the involvement of the STAT3 pathway in this inhibition. STUDY DESIGN AND METHODS: A CIA rat model, interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-stimulated RA-FLS and a co-culture system (IL-6/sIL-6R-stimulated RA-FLS/peripheral blood mononuclear cells) were used to evaluate the effects of RLE. Micro-computed tomography analysis was used to observe bone erosion in CIA rats. Tartrate-resistant acid phosphatase staining was used to evaluate osteoclastogenesis. Western blotting and ELISA assays were employed to examine protein levels. RT-qPCR was used to detect mRNA levels. STAT3-over-activated RA-FLS were used to investigate the involvement of STAT3 signaling in the anti-osteoclastogenic effects of RLE. RESULTS: RLE alleviated bone erosion in joints of CIA rats. In both synovial tissues of CIA rats and IL-6/sIL-6R-stimulated RA-FLS, RLE downregulated the protein level of RANKL. In the co-culture system, RLE significantly and dose-dependently inhibited IL-6/sIL-6R-induced osteoclastogenesis. Mechanistic studies revealed that RLE lowered the protein level of phospho-STAT3 (Tyr705) in synovial tissues of CIA rats. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited the activation/phosphorylation of a STAT3 upstream kinase Janus kinase 2 (Tyr1007/1008) and STAT3 (Tyr705), decreased the nuclear localization of STAT3, lowered mRNA levels of STAT3-transcriptionally regulated genes IL-1ß and TNF-α. RLE's inhibitory effects on RANKL production in RA-FLS gradually decreased when IL-6/sIL-6R doses increased. Over-activation of STAT3 diminished the inhibitory effects of RLE on RANKL production in IL-6/sIL-6R-stimulated RA-FLS, and attenuated the anti-osteoclastogenic effects of RLE in the co-culture system. CONCLUSION: We, for the first time, demonstrated that suppressing STAT3 signaling contributes to the inhibition of RANKL production and osteoclastogenesis, and thereby supports the mechanisms responsible for the reduction in bone erosion in RLE-treated CIA rats. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug, and supports the notion that targeting STAT3 signaling is a viable strategy for managing bone erosion.

A traditional Chinese medicine formula inhibits tumor growth in mice and regulates the miR-34b/c-Met/ß-catenin pathway.

ETHNOPHARMACOLOGY RELEVANCE: Si-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. AIM OF THE STUDY: To investigate the anti-melanoma effects and mechanism of action of an ethanolic extract of SJZT. MATERIALS AND METHODS: SJZT was extracted using 50% ethanol. A murine B16 melanoma-bearing mouse model was employed to investigate the anti-melanoma effects of SJZT. microRNA (miRNA) and mRNA levels were examined by RT-qPCR, and protein levels were measured by Western blotting. RESULTS: SJZT significantly inhibited B16 tumor growth in mice. Mechanistic investigations revealed that SJZT elevated miR-34b (a tumor suppressing miRNA), and lowered c-Met (a miR-34b target gene) and ß-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. CONCLUSIONS: In this study we found, for the first time, that SJZT exerts anti-melanoma effects and regulates the miR-34b/c-Met/ß-catenin pathway in a melanoma mouse model. Our findings provide pharmacological justifications for the clinical use of SJZT in treating melanoma.