Activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrogenesis. armepavine (Arm, C 19 H 23 O 3 N), an active compound from Nelumbo nucifera, has been shown to exert immunosuppressive effects on T lymphocytes and on lupus nephritic mice. The aim of this study was to investigate whether Arm could exert anti-hepatic fibrogenic effects in vitro and in vivo. A cell line of rat HSCs (HSC-T6) was stimulated with tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) to evaluate the inhibitory effects of Arm. An in vivo therapeutic study was conducted in bile duct-ligated (BDL) rats. BDL rats were given Arm (3 or 10 mg/kg) by gavage twice daily for 3 weeks starting from the onset of BDL. Liver sections were taken for fibrosis scoring, immuno-fluorescence staining and quantitative real-time mRNA measurements. In vitro, Arm (1-10 μM) concentration-dependently attenuated TNF-α-and LPS-stimulated α-SMA protein expression and AP-1 activation by HSC-T6 cells without adverse cytotoxicity. Arm also suppressed TNF-α-induced collagen collagen deposition, NFκB activation and MAPK (p38, ERK1/2, and JNK) phosphorylations. In vivo, Arm treatment significantly reduced plasma AST and ALT levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of BDL rats as compared with vehicle treatment. Moreover, Arm attenuated the mRNA expression levels of col 1α2, TGF-β1, TIMP-1, ICAM-1, iNOS, and IL-6 genes, but up-regulated metallothionein genes. Our study results showed that Arm exerted both in vitro and in vivo antifibrotic effects in rats, possibly through anti-NF-κB activation pathways.

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Lung cancer is the leading cause of cancer death in the world, and non-small cell lung carcinoma (NSCLC) accounts for approximately 75-85% of these cancers. Non-small cell lung cancers commonly develop resistance to radiation and chemotherapy, and they often present at stages beyond surgical remedy. Since current treatment modalities are inadequate, novel therapies are necessary to reduce the effects of the increasing incidence in pulmonary neoplasm. 1,2)Bupleuri Radix (Chai-hu in Chinese and Saiko in Japanese) is one of the most important traditional Chinese crude drugs for treating hepatitis malaria and intermittent fever. It has been used in many Chinese medicine prescriptions to treat deafness, dizziness, irregular menstruation, lung disease, jaundice, liver disease and amenorrhea. Among them, B. kaoi is one of the Bupleurum spp. families locally found in Taiwan. 3) Previous studies have shown that Bupleuri Radix possesses a wide range of immunopharmacologic functions, such as anti-inflammatory, mitogen-induced lymphocyte transformation, or antiviral activities. [4][5][6] A recent study reported that the acetone extract of Bupleurum scorzonerifolium could inhibit proliferation of A549 human lung cancer cells by inducing apoptosis and suppressing telomerase activity. 1)Apoptosis has been characterized as a fundamental cellular activity to maintain the physiological balance of the organism. It is also involved in immune defense machinery and plays a necessary role as a protective mechanism against carcinogenesis by eliminating damaged cells or abnormal excess cells proliferated owing to various chemical agents' induction. 7) In this study, we determined the antiproliferative activity of SEF, and examined its effect on apoptosis in the human lung cancer cell line, A549. Furthermore, to establish the anticancer mechanism of SEF, we assayed the levels of Fas/ APO-1 receptor and Fas ligand which are strongly associated with the signal transduction pathway of apoptosis and affect the chemosensitivity of tumor cells to anticancer agents. MATERIALS AND METHODS MaterialsFetal bovine serum (FBS), penicillin G, streptomycin, and amphotericin B were obtained from GIBCO BRL (Gaithersburg, MD, U.S.A.). Dimethyl sulfoxide (DMSO) and RPMI-1640 were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). Sodium 3Ј-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfoic acid hydrate (XTT) kit was obtained from Roche Diagnostics GmbH (Germany). Nucleosome ELISA, Fas Ligand, and Fas/APO-1 ELISA kits were purchased from Calbiochem (Cambridge, MA, U.S.A.).Preparation of B. kaoi Extracts Saponin-enriched fraction (SEF) was extracted from the roots of B. kaoi using the methods of Lin et al.3) SEF was dissolved in dimethyl sulfoxide (DMSO) and stored at Ϫ20°C. For all experiments, final concentrations of the tested compound were prepared by diluting the stock with RPMI-1640. Control cultures received the carrier solvent (0.1% DMSO).Cell Culture A549 (American Type Culture Collection [ATCC] CCL185) was maintaine...

The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5-20 mM), cortiosterone (10 -10 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side-chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate-stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135-2144, 2017. © 2016 Wiley Periodicals, Inc.

The aim of this study was to investigate if armepavine (Arm, C₁₉H₂₃O₃N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1-10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H₂O₂ production; and (iii) NF-κB, JunD and C/EBPß (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-ß (EBPß)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-ß1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPß pathways.