Jine Yi

Immunomodulatory effects of betulinic acid from the bark of white birch on mice.

The objective of this study was to explore the immunomodulatory effects of betulinic acid (BA) extracted from the bark of white birch on mice. Female mice were orally administered BA for 14 days in doses of 0, 0.25, 0.5, and 1 mg/kg body weight. We found that BA significantly enhanced the thymus and spleen indices, and stimulated lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide as shown by MTT assay. Flow cytometry revealed that BA increased the percentage of CD4+ cells in thymus as well as the percentage of CD19+ and the ratios of CD4+/CD8+ in spleen. BA increased the number of plaque-forming cell and macrophage phagocytic activity as indicated by a neutral red dye uptake assay, and the peritoneal macrophages levels of TNF-α were also increased. In contrast, serum levels of IgG and IgM and serum concentrations of IL-2 and IL-6 were significantly decreased in BA-treated mice compared to the control as assayed by haemagglutination tests and ELISA, respectively. Taken together, these results suggest that BA enhances mouse cellular immunity, humoral immunity, and activity of macrophages. Thus, BA is a potential immune stimulator and may strengthen the immune response of its host.

Gossypol acetic acid induces apoptosis in RAW264.7 cells via a caspase-dependent mitochondrial signaling pathway

To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 µmol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (ΔΨm) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of ΔΨm in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.

Gynostemma pentaphyllum protects mouse male germ cells against apoptosis caused by zearalenone via Bax and Bcl-2 regulation.

The objective of this study was to explore the effects of Gynostemma pentaphyllum on Zearalenone-induced apoptosis in mouse male germ cells. Fifty Kunming male mice at 25-days-old were classified into five groups: group A was the control (10% ethanol, 0.5 ml/day); group B with 10 µg Zearalenone/day; group C with 10 µg Zearalenone and 50 mg/kg/day Gynostemma pentaphyllum; group D with 10 µg Zearalenone and 100 mg/kg/day Gynostemma pentaphyllum; and group E with 10 µg Zearalenone and 200 mg/kg/day Gynostemma pentaphyllum. It was found that Gynostemma pentaphyllum has a marked effect on protecting male germ cells against Zearalenone-induced apoptosis, as evidenced by a reduced apoptosis rate of male germ cells and Bax expression as well as an enhancement of Bcl-2 expression in Gynostemma pentaphyllum-treated groups compared to the control. In addition, Gynostemma pentaphyllum remarkably improved pathologic changes of testicular tissue, reduced the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD) caused by Zearalenone. Taken together, these results suggest that Gynostemma pentaphyllum protects against toxicity caused by Zearalenone through anti-oxidation and anti-apoptosis via the regulation of Bax and Bcl-2 expression.

T-2 toxin-induced cytotoxicity and damage on TM3 Leydig cells

T-2 toxin is a highly toxic mycotoxin produced by various Fusarium species, mainly, Fusarium sporotrichoides, and has been reported to have toxic effects on reproductive system of adult male animals. This study investigated the dose-dependent cytotoxicity of T-2 toxin on reproductive cells using TM3 Leydig cells. Specifically, the cytotoxic effect of T-2 toxin was assessed by measuring cell viability; lactate dehydrogenase (LDH); malondialdehyde (MDA); antioxidant activity by measuring superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and DNA damage; and cell apoptosis. Results showed that T-2 toxin is highly cytotoxic on TM3 Leydig cells. However, Trolox-treated TM3 Leydig cells showed significantly reduced oxidative damage, DNA damage, and apoptosis induced by T-2 toxin. This study proves that T-2 toxin can damage the testes and thus affects the reproductive capacity of animals and humans. Furthermore, oxidative stress plays an important role in the cytotoxic effect of T-2 toxin.

T-2 toxin regulates steroid hormone secretion of rat ovarian granulosa cells through cAMP-PKA pathway

T-2 toxin is a secondary metabolite produced by Fusarium genus and is a common contaminant in food and feedstuffs of cereal origin. In porcine granulosa cells(GC), T-2 toxin has been shown to inhibit the steroidogenesis; however, the mechanism has not been well understood. Gonadotropin-stimulated steroidogenesis is regulated by the cAMP-PKA pathway. In this study, we investigated potential mechanisms for T-2 toxin-induced reproductive toxicity focusing on the critical steps of the cAMP-PKA pathway affected by T-2 toxin. We first analyzed the effects of T-2 toxin on progesterone and estrogen production in rat granulosa cells. For this purpose the granulosa cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 24h in serum-free medium containing FSH (10 ng/ml) and androstenedione (3 ng/ml), both are required for normal steroidogenesis. Treatment of these cells with T-2 toxin dose-dependently inhibited the growth of cells and the steroid hormone production. Cellular cyclic AMP levels were dose-dependently inhibited by T-2 toxin (0, 1, 10 and 100 nM, 24 h). Furthermore, we found that although the induction of progesterone by 8-Br-cAMP (a FSH mimetic) and 22R-HC (substrate for progesterone) could both be inhibited by T-2 toxin treatment, the T-2-imposed inhibitory effects could be reversed by increasing doses of 22R-HC, while increasing 8-Br-cAMP had no effects, suggesting that T2 toxin targeted at distinct mechanisms. cAMP-stimulated steroidogenic acute regulatory protein (StAR) is a rate limiting protein in progesterone synthesis. Exposure to T2 toxin caused significant suppression of StAR expression as determined by Western blotting and semi-quantitative RT-PCR suggesting StAR is a sensitive target for T-2 toxin. Taken together, our results strongly suggest that T2 toxin inhibits steroidogenesis by suppressing cAMP-PKA pathway and StAR is a target for T-2-toxin. The antisteroidogenesis effects were observable at low T-2 dose (1 ng/ml) suggesting T-2 toxin has an endocrine disruptive effect.

Arsanilic acid causes apoptosis and oxidative stress in rat kidney epithelial cells (NRK-52e cells) by the activation of the caspase-9 and -3 signaling pathway

Abstract Arsenic exists widely in rock, water and air, and arsanilic acid (also known as aminophenyl arsenic acid) is an organoarsenic compound and has been used as feed additives. Organoarsenic compounds in foodstuff cause adverse effects, including acute and chronic toxicity, in animals and humans. However, little is known about the cellular toxicity and mechanisms of organic arsenic on the kidney. In this study, we explored the toxicity and molecular mechanisms of arsanilic acid on rat kidney epithelial cells (NRK-52e cells). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that arsanilic acid inhibited the proliferation of rat NRK-52e cells in a dose-dependent manner, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and flow cytometry revealed that arsanilic acid induced cellular apoptosis in NRK-52e cells. Fluorescence spectrophotometer displayed that arsanilic acid caused a loss of mitochondrial transmembrane potential (MMP) of NRK-52e cells, but enhanced reactive oxygen species level of these cells. Notably, trolox, a water-soluble derivative of vitamin E, protected NRK-52e cells against MMP loss and apoptosis caused by arsanilic acid. Western blots with caspase inhibitors further indicated that arsanilic acid increased expression of active caspase-3 and -9 in NRK-52e cells. Collectively, these results suggest that arsanilic acid causes apoptosis and oxidative stress in rat kidney epithelial cells through activation of the caspase-9 and -3 signaling pathway. This study thus provides a novel insight into molecular mechanisms by which arsanilic acid has adverse cytotoxicity on renal tubular epithelial cells.

Lead induces apoptosis in mouse TM3 Leydig cells through the Fas/FasL death receptor pathway

The study was aimed to investigate the effect of Pb toxicity on mouse Leydig cells and its molecular mechanism. The TM3 cells were cultured in vitro and exposed to Pb at different concentrations for 24h. The effects of Pb on cell proliferation and apoptosis were analyzed with MTT and Annexin V-FITC/PI via flow cytometry, respectively. Expression levels of Fas, Fas-L and caspase-8 in TM3 cells were determined by western blot. As well as the inhibitory effect of the caspase-8 inhibitor Z-IETD-FMK on cell apoptosis. We found that Pb treatment significantly decreased the cellar viability (P<0.05), increased the apoptosis (P<0.01) and the Fas, FasL, and caspase-8 expression levels in Pb-treated cells as compared to the control cells (P<0.05 or P<0.01). Furthermore, the caspase-8 inhibitor effectively block the Pb-induced cell apoptosis. Taken together, our data suggest that Pb-induced TM3 cell toxic effect may involve in the Fas/FasL death receptor signaling pathway.

Betulinic acid attenuates dexamethasone-induced oxidative damage through the JNK-P38 MAPK signaling pathway in mice

Dexamethasone (Dex), a potent anti-inflammatory/immunosuppressive agent, has been shown to induce oxidative stress. Betulinic acid (BA) is a pentacyclic lupane triterpene with a potent antioxidant activity. The aim of this study was to investigate the ameliorative effect and underlying mechanisms of BA on Dex-induced oxidative damage. Mice were pretreated with BA orally (0, 0.25, 0.5, and 1.0 mg/kg) daily for 14 days, and then a single dose of Dex (25 mg/kg body weight) was administered intraperitoneally 8 h after the last administration of BA to induce oxidative stress. BA pretreatment significantly alleviated Dex-induced changes of blood biochemical indices, increased the total antioxidant capacity (T-AOC), the activity of superoxide dismutase (SOD), and the ability of inhibiting hydroxyl radical (AIHR), reduced the level of malondialdehyde (MDA) in serum. Moreover, BA pretreatment enhanced the T-AOC, AIHR and the activity of peroxidase (POD) in liver, spleen and thymus. Concomitant with these biochemical parameters, BA pretreatment significantly reduced gene and protein expressions of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) in the lymphatic organs of Dex-treated mice. BA was found to effectively attenuate Dex-induced oxidative damage. These protective effects may be mediated in part through the JNK-P38 MAPK signaling transduction pathway and BA may be a potential therapeutic agent due to its anti-oxidative properties.

Anti-obesity effect of a traditional Chinese dietary habit—blending lard with vegetable oil while cooking

Obesity, which is associated with dietary habits, has become a global social problem and causes many metabolic diseases. In China, both percentages of adult obesity and overweight are far lower compared to western countries. It was designed to increase the two levels of daily intake in human, namely 3.8% and 6.5%, which are recommendatory intake (25 g/d) and Chinese citizens’ practical intake (41.4 g/d), respectively. The mice were respectively fed with feeds added with soybean oil, lard or the oil blended by both for 12 weeks. In the mice fed with diet containing 3.8% of the three oils or 6.5% blended oil, their body weight, body fat rate, cross-sectional area of adipocytes, adipogenesis and lipogenesis in adipose were decreased, whereas hydrolysis of triglyserides in adipose was increased. This study demonstrated that the oil mixture containing lard and soybean oil had a remarkable anti-obesity effect. It suggests that the traditional Chinese dietary habits using oils blended with lard and soybean oil, might be one of the factors of lower percentages of overweight and obesity in China, and that the increasing of dietary oil intake and the changing of its component resulted in the increasing of obesity rate in China over the past decades.

Antitumor Activity of Betulinic Acid and Betulin in Canine Cancer Cell Lines

Background/Aim: Betulinic acid (BA) and betulin (BT) exhibit a variety of pharmacological properties including anti-cancer, anti-inflammatory and anti-oxidant ones. Canine lymphoma and osteosarcoma have a high mortality rate and need more effective therapeutic approaches. In this study, the anti-proliferative and pro-apoptotic effects of BA and BT were investigated in canine T-cell lymphoma (CL-1), canine B-cell lymphoma (CLBL-1) and canine osteosarcoma (D-17) cell lines. Materials and Methods: The cultured cells were treated with several concentrations of BA or BT for 24, 48 and 72 h, and cell proliferation was assessed by the MTT assay. Cell apoptotic rate and cell cycle were analyzed using flow cytometry. Results: Anti-proliferative effect of BT and BA was concentration- and time-dependent. Moreover, BA and BT arrested cell cycle in S phase in CL-1 and D-17 cells, and in G0/G1 phase in CLBL-1 cells. Conclusion: Both compounds showed an antitumor activity, and the effects of BA were stronger than that of BT.