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.

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.

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.

Chrysoeriol ameliorates TPA-induced acute skin inflammation in mice and inhibits NF-κB and STAT3 pathways.

BACKGROUND: Chrysoeriol is a flavone found in diverse dietary and medicinal herbs such as Lonicerae Japonicae Flos (the dried flower bud or newly bloomed flower of Lonicera japonica Thunb.). These herbs are commonly used for treating inflammatory diseases. Herbal extracts containing chrysoeriol have been shown to have anti-inflammatory effects and inhibit nuclear factor-kappa B (NF-κB) signaling. Some of these extracts can inhibit signal transducers and activators of transcription 3 (STAT3) signaling in cancer cells. PURPOSE: This study aimed to determine whether chrysoeriol has anti-inflammatory effects and whether NF-κB and STAT3 pathways are involved in the effects. STUDY DESIGN AND METHODS: A TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model and LPS-stimulated RAW264.7 cells were used to evaluate the effects of chrysoeriol. Griess reagent was used to measure the production of nitric oxide (NO). Western blot and enzyme-linked immunosorbent assays were employed to detect protein levels. RT-qPCR analyses were used to detect mRNA levels. Haematoxylin and eosin (H&E) staining was employed to examine the pathological conditions in animal tissues. RESULTS: In the mouse model, chrysoeriol ameliorated acute skin inflammation, evidenced by reduced ear thickness, ear weight and number of inflammatory cells in inflamed ear tissues. The compound lowered protein levels of phospho-p65 (Ser536), phospho-STAT3 (Tyr705), inducible nitric oxide synthases (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), IL-1ß and tumor necrosis factor α (TNF-α) in mouse swollen ears. In LPS-stimulated RAW264.7 cells, chrysoeriol also lowered levels of these proteins. In addition, chrysoeriol decreased the production of NO and prostaglandin E2; inhibited the phosphorylation of inhibitor of κB (Ser32), p65 (Ser536) and Janus kinase 2 (Tyr1007/1008); decreased nuclear localization of p50, p65 and STAT3; and down-regulated mRNA levels of pro-inflammatory cytokines IL-6, IL-1ß and TNF-α that are transcriptionally regulated by NF-κB and STAT3 in the cell model. CONCLUSION: We for the first time demonstrated that chrysoeriol ameliorates TPA-induced ear edema in mice, and that inhibition of JAK2/STAT3 and IκB/p65 NF-κB pathways are involved in the anti-inflammatory effects of chrysoeriol. This study provides chemical and pharmacological justifications for the use of chrysoeriol-containing herbs in treating inflammatory diseases, and provides pharmacological groundwork for developing chrysoeriol as a novel anti-inflammatory agent.

MiR-let-7a/f-CCR7 signaling is involved in the anti-metastatic effects of an herbal formula comprising Sophorae Flos and Lonicerae Japonicae Flos in melanoma.

BACKGROUND: Metastasized melanoma is extremely difficult to treat. Activation of C-C chemokine receptor type 7 (CCR7) has been linked to melanoma metastasis. CCR7 can be directly regulated by miR-let-7. We have previously shown that an ethanolic extract of an herbal formula comprising Sophorae Flos and Lonicerae Japonicae Flos (SLE) inhibits melanoma cell migration and invasion. PURPOSE: In this study, we determined whether SLE suppresses melanoma metastasis, and whether regulation of miR-let-7a/f-CCR7 signaling is involved in the effect. STUDY DESIGN AND METHODS: Small RNA sequencing was conducted to compare miRNA expression profiles of B16F10 tumors dissected from SLE-treated or untreated mice. Western blot and RT-qPCR analyses were employed to examine protein and miRNA levels, respectively. A B16F10 melanoma lung metastasis mouse model was used to evaluate the effects of SLE on melanoma metastasis. MiR-let-7a/f-knockdown and CCR7-overexpression cell models were used to investigate the involvement of miR-let-7a/f-CCR7 signaling in the anti-metastatic effects of SLE. RESULTS: It was found that SLE upregulated levels of miR-let-7a/f in B16F10 melanoma tissues. SLE significantly elevated levels of miR-let-7a/f, lowered the protein level of CCR7, inhibited the phosphorylation of CCR7 downstream molecules p38 and JNK in B16F10 and A375 melanoma cells. SLE inhibited B16F10 melanoma lung metastasis in mice. SLE upregulated levels of miR-let-7a/f, and lowered protein levels of CCR7, MMP-2, MMP-9, phospho-p38 (Thr180/Tyr182) and phospho-JNK (Thr183/Tyr185) in melanoma-invaded lung tissues. Knockdown of miR-let-7a/f diminished the effects of SLE on CCR7 signaling in, and invasion of, melanoma cells. Overexpression of CCR7 lessened the effects of SLE in inhibiting the phosphorylation of p38 and JNK in, and the invasive capability of, melanoma cells. CONCLUSION: We for the first time demonstrated that SLE inhibits melanoma metastasis in mice, and that regulation of the miR-let-7a/f-CCR7 pathway contributes to the anti-metastatic mechanisms of SLE. These findings provide a pharmacological basis for developing SLE as a modern agent for treating metastatic melanoma. Additionally and importantly, this study suggests that regulating the miR-let-7a/f-CCR7 pathway is a novel strategy for controlling melanoma metastasis.

A TCM formula comprising Sophorae Flos and Lonicerae Japonicae Flos alters compositions of immune cells and molecules of the STAT3 pathway in melanoma microenvironment.

A traditional Chinese medicine (TCM) formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos was used for treating melanoma in ancient China. We have previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects and suppresses STAT3 signaling in vitro and in vivo. STAT3 has been linked to the development of melanoma immunosuppressive microenvironment. In this work, we investigated whether SLE inhibits melanoma growth by reprogramming the tumor microenvironment in mouse and co-culture cell models. In B16F10 melanoma-bearing mice, we found that intragastric administration of SLE (1.2 g/kg) dramatically inhibited tumor growth. This observation was associated with the downregulation of protein levels of phospho-STAT3 (Tyr 705) and STAT3-regulated immunosuppressive cytokines, and mRNA levels of STAT3-targeted genes involved in tumor growth and immune evasion. We also observed increased Th, Tc and dendritic cells in the melanomas and spleens in SLE-treated mice compared to that in control mice. In a co-culture system composed of B16F10 cells and mouse primary splenic lymphocytes, it was found that SLE not only inhibited STAT3 activation in B16F10 cells, but also downregulated mRNA levels of STAT3-targeted genes in the splenic lymphocytes. In this co-culture setting, SLE decreased the levels of STAT3-regulated immunosuppressive cytokines, increased the percentages of Th, Tc and dendritic cells as well. Furthermore, effects of SLE on STAT3 phosphorylation, cytokine levels and immune cell subtype percentages were significantly weaker in the B16STAT3C cells (stable cells harboring a constitutively active STAT3 variant STAT3C)/splenic lymphocytes co-culture system than in the B16V cells (cells stably transfected with the empty vector)/splenic lymphocytes co-culture system, indicating that STAT3 over-activation diminishes SLE's effects. In summary, our findings indicate that reprograming the immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. This study provides further pharmacological groundwork for developing SLE as a modern agent for melanoma prevention/treatment, and supports the notion that reprograming immunosuppressive microenvironment is a viable anti-melanoma strategy.