Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

BACKGROUND AND AIM: Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

Dietary magnesium is able to influence the relationship between vitamin C and estimated glomerular filtration rate: A cross-sectional study.

The estimated glomerular filtration rate (eGFR) is a comprehensive index that is widely used to assess renal function. Although studies have confirmed a correlation between eGFR and dietary vitamin C, the impact of varying levels of vitamin C on eGFR remains unclear. Additionally, the interaction between dietary magnesium intake and vitamin C concentration on eGFR is not well understood. As such, the objective of this study was to investigate the relationship between dietary magnesium intake and vitamin C in relation to eGFR. This study analyzed the data of consecutive NHANES from 2005 to 2018. We included 17,633 participants aged 20 or older and used multiple linear regression analysis to evaluate the relationship between dietary vitamin C and eGFR. Dietary Mg intake from experimental data was dichotomized into a low dietary Mg intake group (≤254 mg/day) and a normal dietary Mg intake group (>254 mg/day). To evaluate the impact of dietary magnesium intake on eGFR, a multivariable linear regression was conducted utilizing an interaction test between dietary vitamin C and eGFR. We discovered a positive association between dietary vitamin C content and eGFR. The relationship between dietary vitamin C levels and eGFR differed between individuals with low Mg intake and those with normal Mg intake (ß: 2.96 95% CI:1.63 ~ 4.29 vs. ß: 1.05 95% CI: -0.15 to 2.25), and the positive association of high dietary vitamin C content with eGFR was stronger in the low Mg intake group. Furthermore, we observed that dietary magnesium intake significantly altered the positive association between dietary vitamin C and eGFR (interaction value of 0.020). Our experimental study revealed that the interaction between dietary magnesium and dietary vitamin C can significantly impact eGFR. This finding carries significant implications for the treatment of diseases resulting from abnormal eGFR, as well as the selection of clinically relevant drugs.

A traditional prescription comprising Astragali Radix and Schisandra chinensis Fructus induces apoptosis and protective autophagy in hepatocellular carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocellular carcinoma (HCC) poses a growing challenge to global health efforts. The 5-year survival rate of HCC patients is still dismal. A traditional prescription Qi-Wei-Wan (QWW) comprising Astragali Radix and Schisandra chinensis Fructus has traditionally been used for HCC treatment according to traditional Chinese medicine theory, but the pharmacological basis is not clear. AIM OF THE STUDY: This study aims to investigate the anti-HCC effects of an ethanolic extract of QWW (hereafter, QWWE) and the mechanism of action. MATERIALS AND METHODS: An UPLC-Q-TOF-MS/MS method was developed to control the quality of QWWE. Two human HCC cell lines (HCCLM3 and HepG2) and a HCCLM3 xenograft mouse model were employed to investigate the anti-HCC effects of QWWE. The anti-proliferative effect of QWWE in vitro was determined by MTT, colony formation and EdU staining assays. Apoptosis and protein levels were examined by flow cytometry and Western blotting, respectively. Nuclear presence of signal transducer and activator of transcription 3 (STAT3) was examined by immunostaining. Transient transfection of pEGFP-LC3 and STAT3C plasmids was performed to assess autophagy and determine the involvement of STAT3 signaling in QWWE's anti-HCC effects, respectively. RESULTS: We found that QWWE inhibited the proliferation of and triggered apoptosis in HCC cells. Mechanistically, QWWE inhibited the activation of SRC and STAT3 at Tyr416 and Tyr705, respectively; inhibited the nuclear translocation of STAT3; lowered Bcl-2 protein levels, while increased Bax protein levels in HCC cells. Over-activating STAT3 attenuated the cytotoxic and apoptotic effects of QWWE in HCC cells. Moreover, QWWE induced autophagy in HCC cells by inhibiting mTOR signaling. Blocking autophagy with autophagy inhibitors (3-methyladenine and chloroquine) enhanced the cytotoxicity, apoptotic effect and the inhibitory effect on STAT3 activation of QWWE. Intragastric administration of QWWE at 10 mg/kg and 20 mg/kg potently repressed tumor growth and inhibited STAT3 and mTOR signaling in tumor tissues, but did not significantly affect mouse body weight. CONCLUSION: QWWE exhibited potent anti-HCC effects. Inhibiting the STAT3 signaling pathway is involved in QWWE-mediated apoptosis, while blocking mTOR signaling contributes to QWWE-mediated autophagy induction. Blockade of autophagy enhanced the anti-HCC effects of QWWE, indicating that the combination of an autophagy inhibitor and QWWE might be a promising therapeutic strategy for HCC management. Our findings provide pharmacological justifications for the traditional use of QWW in treating HCC.

Non-destructive assessment of equivalent umami concentrations in salmon using hyperspectral imaging technology combined with multivariate algorithms.

This study utilized equivalent umami concentrations (EUC) to characterize umami intensity in salmon with different freeze-thaw times. A rapid and non-destructive method was established to determine EUC values in salmon which is based on hyperspectral imaging (HSI) system combined with multiple characteristic variable screening methods. The established CARS-PLS model showed greater advantages in correlating the reference values of spectral data with EUC in salmon with Rc of 0.9012, Rp of 0.9009, RMSECV of 0.82, and RMSEP of 0.88. The model was employed pixel-wise to visualize the distribution of EUC with different freeze-thaw times, which demonstrated the reduction of EUC value with the increasing of freeze-thaw times. Therefore, this reseearch showed hyperspectral imaging (HSI) system combined with chemometrics possesses a substantial capability to predict and visualize the EUC of salmon, which would provide an intuitive understanding of salmon quality prediction and detection.

Gracillin exerts anti-melanoma effects in vitro and in vivo: role of DNA damage, apoptosis and autophagy.

BACKGROUND: Melanoma is an aggressive cancer. Gracillin has been reported to treat various types of cancer, such as colorectal and lung cancer. However, there is a paucity of research on the anti-melanoma effects of gracillin. PURPOSE: The aim of this study was to assess the anti-melanoma effects and mechanisms of action of gracillin in vitro and in vivo. METHODS: Cell viability was detected using MTT and crystal violet staining assays. Cell proliferation was examined by EdU staining assays. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Autophagic flux was monitored under a confocal microscope. Protein levels were determined by immunoblotting. LY294002 and rapamycin (Rapa) were used to determine the involvement of PI3K/AKT/mTOR signaling in gracillin-mediated autophagy. Signal transducer and activator of transcription 3 (STAT3) was overactivated to explore the contribution of the STAT3 signaling pathway in the anti-melanoma effects of gracillin. A B16F10 allograft mouse model was developed to evaluate the anti-melanoma effects of gracillin in vivo. RESULTS: We demonstrated that in melanoma cells, gracillin inhibited proliferation, induced G0/G1 phase cell cycle arrest, evoked apoptosis, and triggered autophagic cell death. Gracillin induced DNA damage in melanoma cells. Moreover, it suppressed the phosphorylation/activation of PI3K, AKT, mTOR, and 4E-BP1 in melanoma cells. Inhibiting PI3K/AKT and mTOR activity using LY294002 and Rapa, respectively, increased the protein level of LC3B-II in gracillin-treated melanoma cells. Furthermore, gracillin downregulated the protein levels of p-JAK2 (Tyr1007/1008), p-Src (Tyr416), and p-STAT3 (Tyr705) in melanoma cells. Over-expression of STAT3 in A375 cells significantly mitigated the cytotoxic and apoptotic effects of gracillin. In vivo studies showed that gracillin (1 mg/kg or 8 mg/kg, administered intraperitoneally for 16 consecutive days) suppressed B16F10 tumor growth and Src/STAT3 and AKT/mTOR signaling in tumors. No overt toxicity was observed in mice. CONCLUSION: Induction of DNA damage, inhibition of PI3K/AKT/mTOR signaling and suppression of STAT3 signaling are involved in gracillin-mediated cell cycle arrest, autophagic cell death and apoptosis, respectively, in melanoma cells. These findings provide novel insights into the anti-melanoma molecular mechanisms of gracillin, and suggest a potential role of gracillin in melanoma management.

Exploring the Effective Components and Mechanism of Action of Japanese Ardisia in the Treatment of Autoimmune Hepatitis Based on Network Pharmacology and Experimental Verification.

Japanese Ardisia is widely used as a hepatoprotective and anti-inflammatory agent in China. However, the active ingredients in Japanese Ardisia and their potential mechanisms of action in the treatment of autoimmune hepatitis (AIH) are unknown. The pharmacodynamic substance and mechanism of action of Japanese Ardisia in the treatment of AIH were investigated using network pharmacology and molecular docking technology in this study. Following that, the effects of Japanese Ardisia were evaluated using the concanavalin A (Con A)-induced acute liver injury rat model. The active ingredients and targets of Japanese Ardisia were searched using the Traditional Chinese Medicine Systems Pharmacology database, and hepatitis-related therapeutic targets were identified through GeneCards and Online Mendelian Inheritance in Man databases. A compound-target network was then constructed using Cytoscape software, and enrichment analysis was performed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Molecular docking technology was used to simulate the docking of key targets, and the AIH rat model was used to validate the expression of key targets. Nineteen active chemical components and 143 key target genes were identified. GO enrichment analysis revealed that the treatment of AIH with Japanese Ardisia mainly involved DNA-binding transcription factor binding, RNA polymerase II-specific DNA transcription factor binding, cytokine receptor binding, receptor-ligand activity, ubiquitin-like protein ligase binding, and cytokine activity. In the KEGG enrichment analysis, 165 pathways were identified, including the lipid and atherosclerotic pathway, IL-17 signaling pathway, TNF signaling pathway, hepatitis B pathway, and the AGE-RAGE signaling pathway in diabetic complications. These pathways may be the key to effective AIH treatment with Japanese Ardisia. Molecular docking showed that quercetin and kaempferol have good binding to AKT1, IL6, VEGFA, and CASP3. Animal experiments demonstrated that Japanese Ardisia could increase the expression of AKT1 and decrease the expression of CASP3 protein, as well as IL-6, in rat liver tissues. This study identified multiple molecular targets and pathways for Japanese Ardisia in the treatment of AIH. At the same time, the effectiveness of Japanese Ardisia in treating AIH was verified by animal experiments.

Polyphyllin II Induces Protective Autophagy and Apoptosis via Inhibiting PI3K/AKT/mTOR and STAT3 Signaling in Colorectal Cancer Cells.

Polyphyllin II (PPII) is a natural steroidal saponin occurring in Rhizoma Paridis. It has been demonstrated to exhibit anti-cancer activity against a variety of cancer cells. However, the anti-colorectal cancer (CRC) effects and mechanism of action of PPII are rarely reported. In the present study, we showed that PPII inhibited the proliferation of HCT116 and SW620 cells. Moreover, PPII induced G2/M-phase cell cycle arrest and apoptosis, as well as protective autophagy, in CRC cells. We found that PPII-induced autophagy was associated with the inhibition of PI3K/AKT/mTOR signaling. Western blotting results further revealed that PPII lowered the protein levels of phospho-Src (Tyr416), phospho-JAK2 (Tyr1007/1008), phospho-STAT3 (Tyr705), and STAT3-targeted molecules in CRC cells. The overactivation of STAT3 attenuated the cytotoxicity of PPII against HCT116 cells, indicating the involvement of STAT3 inhibition in the anti-CRC effects of PPII. PPII (0.5 mg/kg or 1 mg/kg, i.p. once every 3 days) suppressed HCT116 tumor growth in nude mice. In alignment with the in vitro results, PPII inhibited proliferation, induced apoptosis, and lowered the protein levels of phospho-STAT3, phospho-AKT, and phospho-mTOR in xenografts. These data suggest that PPII could be a potent therapeutic agent for the treatment of CRC.

Isoalantolactone Induces Cell Cycle Arrest, Apoptosis and Autophagy in Colorectal Cancer Cells.

Colorectal cancer (CRC) is an aggressive cancer. Isoalantolactone (IATL) has been reported to exert cytotoxicity against various cancer cells, but not CRC. In this study, we explored the anti-CRC effects and mechanism of action of IATL in vitro and in vivo. Our results demonstrated that IATL inhibited proliferation by inducing G0/G1 phase cell cycle arrest, apoptosis and autophagy in CRC cells. Repression of autophagy with autophagy inhibitors chloroquine (CQ) and Bafilomycin A1 (Baf-A1) enhanced the anti-CRC effects of IATL, suggesting that IATL induces cytoprotective autophagy in CRC cells. Mechanistic studies revealed that IATL lowered protein levels of phospho-AKT (Ser473), phospho-mTOR (Ser2448), phospho-70S6K (Thr421/Ser424) in CRC cells. Inhibition of AKT and mTOR activities using LY294002 and rapamycin, respectively, potentiated the inductive effects of IATL on autophagy and cell death. In vivo studies showed that IATL suppressed HCT116 tumor growth without affecting the body weight of mice. In consistent with the in vitro results, IATL lowered protein levels of Bcl-2, Bcl-XL, phospho-AKT (Ser473), phospho-mTOR (Ser2448), and phsopho-70S6K (Thr421/Ser424), whereas upregulated protein levels of cleaved-PARP and LC3B-II in HCT116 tumors. Collectively, our results demonstrated that in addition to inhibiting proliferation, inducing G0/G1-phase cell cycle arrest and apoptosis, IATL initiates cytoprotective autophagy in CRC cells by inhibiting the AKT/mTOR signaling pathway. These findings provide an experimental basis for the evaluation of IATL as a novel medication for CRC treatment.

Luteolin binds Src, promotes STAT3 protein ubiquitination and exerts anti-melanoma effects in cell and mouse models.

Signal transducer and activator of transcription 3 (STAT3) has been proposed as a target for melanoma prevention. Luteolin, a bioactive flavonoid abundant inmedicinal herbs, has been reported to have anti-melanoma activity in vitro. However, its in vivo anti-melanoma effects and underlying mechanisms have not been fully elucidated. In this study, ten cell lines and two mouse models (B16F10 allograft and A375 xenograft models) were used for assessing the in vitro and in vivo anti-melanoma effects of luteolin. A STAT3 over-activated stable A375 cell line was used to determine the contribution of STAT3 signaling in luteolin's anti-melanoma effects. Results showed that luteolin dose-dependently reduced viability of melanoma cells. Luteolin also induced apoptosis in, and suppressed migration and invasion of, A375 and B16F10 melanoma cells. Mechanistically, luteolin inhibited phosphorylation of STAT3 and Src (an upstream kinase of STAT3), accelerated ubiquitin-proteasome pathway-mediated STAT3 degradation, and downregulated the expression of STAT3-targeted genes involved in cell survival and invasion in melanoma cells. Molecular modelling and surface plasmon resonance imaging showed that luteolin stably bound to the protein kinase domain of Src. Animal studies demonstrated that prophylactic administration of luteolin restrained melanoma growth and Src/STAT3 signaling in both A375 and B16F10 melanoma-bearing mice. Moreover, luteolin's anti-melanoma effects were diminished by STAT3 over-activation in A375 cells. Our findings indicate that luteolin inhibits STAT3 signaling by suppressing STAT3 activation and promoting STAT3 protein degradation in melanoma cells, thereby exhibiting anti-melanoma effects. This study provides further pharmacological groundwork for developing luteolin as a chemopreventive agent against melanoma.

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.

Inhibition of Src/STAT3 signaling-mediated angiogenesis is involved in the anti-melanoma effects of dioscin.

Angiogenesis plays an important role in the growth and metastasis of solid tumors including melanoma. Inhibiting tumor-associated angiogenesis is a tactic in treating melanoma. Dioscin restrains angiogenesis in colon tumor and has anti-melanoma effects in cell and animal models. In a previous study, we found that dioscin inhibits Src/STAT3 signaling in melanoma cells. Activation of the Src/STAT3 pathway has been shown to promote tumor angiogenesis. This study aimed to determine whether dioscin's anti-melanoma effects is related to inhibiting Src/STAT3 signaling-mediated angiogenesis. In a B16F10 allograft mouse model, we found that dioscin inhibited melanoma growth and angiogenesis. To exclude the impact of tumor growth on angiogenesis, a chicken chorioallantoic membrane (CAM) model was used to verify the anti-angiogenic effect of dioscin. Results showed that dioscin suppressed vessel formation in CAM. To determine if tumor secreted pro-angiogenic cytokines are involved in the anti-angiogenic effect of dioscin, conditioned media from dioscin-treated A375 melanoma cells were used to culture human umbilical vein endothelial cells (HUVECs), and tube formation was monitored. It was observed that the tube formation of HUVECs was inhibited. Mechanistic studies revealed that dioscin inhibited the activation of Src and STAT3, and lowered mRNA and protein levels of STAT3 transcriptionally-regulated genes, in B16F10 melanomas. ELISA assays showed that dioscin decreased the secretion of MMP-2, MMP-9 and VEGF from A375 cells. Over-activation of STAT3 lessened the effects of dioscin in decreasing the secretion of pro-angiogenic cytokines from melanoma cells, and in inhibiting tube formation of HUVECs cultured with conditioned media from melanoma cell cultures. In summary, we for the first time demonstrated that inhibiting Src/STAT3 signaling-mediated angiogenesis is involved in the anti-melanoma effects of dioscin. This study provides further pharmacological groundwork for developing dioscin as an anti-melanoma agent.

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.

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.

Gomisin N Exerts Anti-liver Cancer Effects and Regulates PI3K-Akt and mTOR-ULK1 Pathways in Vitro.

Primary liver cancer is a lethal cancer. The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway has been implicated in the pathogenesis of liver cancer. Gomisin N (GN), a lignan isolated from the dried fruits of Schisandra chinensis (Turca.) Baill., has been reported to reduce viability of, and induce apoptosis in, HepG2 liver cancer cells. In preadipocytes, GN was found to inhibit Akt activity. In the present study, Akt signaling-related anti-liver cancer mechanisms of GN were investigated. We confirmed that GN reduces cell viability of, and triggers apoptosis in, more liver cancer cell lines. Mechanistic studies revealed that GN lowers protein levels of phospho-PI3K (p85 tyrosine (Tyr)458), phospho-Akt (serine (Ser)473), and Akt downstream molecules Mcl-1 in HepG2 and HCCLM3 cells. Meanwhile, GN activates mTOR and inhibits ULK1 (a negative downstream effector of mTOR) activities. Activation of mTOR has been reported to suppress ULK1 activity and repress autophagy. Indeed, we observed that GN inhibits autophagy in liver cancer cells. In summary, we for the first time demonstrated that GN inhibits the PI3K-Akt pathway and regulates the mTOR-ULK1 pathway in liver cancer cells.

Activation of STAT3 is a key event in TLR4 signaling-mediated melanoma progression.

Malignant melanoma is aggressive and has a high mortality rate. Toll-like receptor 4 (TLR4) has been linked to melanoma growth, angiogenesis and metastasis. However, signal transduction mediated by TLR4 for driving melanoma progression is not fully understood. Signal transducer and activator of transcription 3 (STAT3) has been identified as a major oncogene in melanoma progression. We found: that TLR4 expression positively correlates with activation/phosphorylation of STAT3 in human melanoma samples; that TLR4 ligands activate STAT3 through MYD88 and TRIF in melanoma cells; and that intratumoral activation of TLR4 increases STAT3 activation in the tumor and promotes tumor growth, angiogenesis, epithelial-mesenchymal transition (EMT) and the formation of an immunosuppressive tumor microenvironment in mice. Further, we found that the effects mediated by activating TLR4 are weakened by suppressing STAT3 function with a dominant negative STAT3 variant in melanoma. Collectively, our work identifies STAT3 activation as a key event in TLR4 signaling-mediated melanoma progression, shedding new light on the pathophysiology of melanoma.

Inhibiting the Src/STAT3 signaling pathway contributes to the anti-melanoma mechanisms of dioscin.

Late stage melanoma is associated with a high mortality rate. Signal transducer and activator of transcription 3 (STAT3) is currently a target for melanoma treatment as it is constitutively activated with high frequency in melanoma. Dioscin is a natural steroid saponin that is present in several medical herbs. A previous study demonstrated that dioscin inhibits STAT3 signaling in a cerebral ischemia-reperfusion injury rat model. Furthermore, dioscin has been reported to exert anti-melanoma effects in B16 melanoma cells and a B16 allograft mouse model. The present study investigated whether inhibition of STAT3 signaling is involved in the anti-melanoma effects of dioscin. The results of the present study demonstrated that dioscin significantly decreased viability, induced apoptosis and suppressed migration of human A375 melanoma cells and murine B16F10 melanoma cells. Furthermore, dioscin inhibited the phosphorylation of STAT3 and Src (an upstream kinase of STAT3), and downregulated mRNA levels of STAT3-targeted genes, including B-cell lymphoma-2, cyclin D1 and matrix metalloproteinase-2. In addition, overexpression of STAT3 decreased the anti-proliferative effects of dioscin. Overall, the results of the present study indicate that inhibiting the Src/STAT3 signaling pathway contributes to the anti-melanoma molecular mechanisms of dioscin. These results provide further pharmacological groundwork for developing dioscin as a novel anti-melanoma agent.

Rapid detection of Atlantic salmon multi-quality based on impedance properties.

To establish a rapid, convenient, and low-cost method to assess the quality of Atlantic salmon, we analyzed the impedance between 10-1 and 105 Hz for Atlantic salmon/rainbow trout, chilled/frozen-thawed salmon, and fresh/stale salmon. We combined chemometrics with impedance properties to create a multi-quality index for Atlantic salmon. The accuracy of all three models established can reach 100% in distinguishing Atlantic salmon from rainbow trout and distinguishing chilled salmon from frozen-thawed salmon. We applied a partial least squares method to create a quantitative prediction model of bioimpedance spectroscopy and the value of total volatile basic nitrogen. The correlation coefficients of the training and test sets were 0.9447 and 0.9387. Our results showed that the combination of impedance properties and chemometrics was a simple and effective application to evaluate Atlantic salmon quality.