Lactobacillus plantarum L67 glycoprotein protects against cadmium chloride toxicity in RAW 264.7 cells.

The food and water we consume may be contaminated with a range of chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and mercury by accumulation through the food chain. Cadmium is known to be one of the major components in cigarette smoke and can cause lesions in many organs. Some lactobacilli can bind and remove heavy metals such as cadmium, lead, and copper. However, the mechanisms of cadmium toxicity and inhibition by probiotics are not clear. In this study, we demonstrated that glycoprotein (18 kDa) isolated from Lactobacillus plantarum L67 protected RAW 264.7 cells from expression of inflammation-related factors stimulated by cadmium chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium using the MTT assay and intracellular Ca(2+) using fluorescence, and assessed activities of activator protein kinase C (PKC-α), inducible nitric oxide synthase, activator protein (AP)-1, and mitogen-activated protein kinases using immunoblot. Our results indicated that glycoprotein isolated from L. plantarum L67 inhibited intracellular Ca(2+) mobilization. It also significantly suppressed inflammatory factors such as AP-1 (c-Jun and c-Fos), mitogen-activated protein kinases (ERK, JNK, and p38), and inducible nitric oxide synthase. Our findings suggest that the 24-kDa glycoprotein isolated from L. plantarum L67 might be used as a food component for protection of inflammation caused by cadmium ion.

The anti-allergic activity of Lactobacillus plantarum L67 and its application to yogurt.

Recently, interest in the beneficial role of probiotics in the protection and management of allergic diseases caused by immune disorders has been increasing. This study investigated the inhibitory effect of Lactobacillus plantarum L67 on induced allergic inflammatory response in bisphenol A-treated rat basophilic leukemia 2H3 (RBL-2H3) cells and mouse splenocytes. We also evaluated the applicability of L. plantarum L67 as a yogurt starter culture. We measured the ability of Lactobacillus strains to induce the production of IL-12 and IFN- γ in cultured splenocytes by ELISA. Bisphenol A (50µM)-treated RBL-2H3 cells were cotreated with a glycoprotein (18kDa) isolated from L. plantarum L67 (5-100µg/mL) for 30min. We measured the expression of mitogen-activated protein kinase (ERK and p38), AP-1 (c-Fos and c-Jun), T-bet, and GATA-binding protein 3 (GATA-3) using Western blotting to examine the differentiation of T helper cells. Furthermore, we evaluated the gene expression of IL-1ß, IL-6, and IL-10 using real-time quantitative PCR. Finally, we evaluated the applicability of L. plantarum L67 as a yogurt starter by measuring pH, enumeration of bacteria, and sensory scores. Our results showed that L67 protein inhibited the phosphorylation of ERK and p38 mitogen-activated protein kinase through the transcriptional activation of AP-1 in bisphenol A-treated RBL-2H3 cells. During differentiation of T helper cells, the expression of transcription factor GATA-3 was significantly suppressed by L67 protein (100µg/mL) treatment, whereas expression of transcription factor T-bet was increased. In addition, the L67 protein significantly attenuated the expression of T helper 2-linked cytokines IL-1ß, IL-6, and IL-10. These results indicate that L. plantarum L67, made available as yogurt starters and dietary supplements, has the potential to prevent allergy-related immune disorders.

ZPDC glycoprotein (24 kDa) induces apoptosis and enhances activity of NK cells in N-nitrosodiethylamine-injected Balb/c.

Natural killer (NK) cells have anti-tumor activity in hepatocellular carcinoma (HCC) using secreting granules and cytotoxic ability. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC) has anti-oxidant effect and anti-cancer effect. The objective of this study was to determine whether ZPDC glycoprotein enhances activity of NK cells and induces apoptosis of liver cancer cells in diethylnitrosamine (DEN)-treated Balb/c mice. This study evaluated the secreting of perforin and granzyme B and cytotoxicity of NK cells, interleukin (IL)-2 and IL-12, apoptosis-related factors (bid, cytochrome c, and caspase-3) in liver tissue using Immunoblot and ELISA. The results demonstrated that ZPDC glycoprotein (20mg/kg, BW) induces secretion of perforin and granzyme B and NK cells activity. Also, it induces expression of apoptosis-related factors (bid, cytochrome c, and caspase-3) in liver tissues. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatocarcinogenesis without immunosuppression.

Preventive effects of ZPDC glycoprotein (24 kDa) on hepatotoxicity induced by mercury chloride in vitro and in vivo.

Mercury is a potent environmental contaminant that exerts toxic effect on various vital organs in the human body. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC), which has antioxidant and anticancer effects. In the present study, we determined the preventive effects of ZPDC glycoprotein on hepatic damage induced by mercury chloride (HgCl2 ). We evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), cyclo-oxygenase (COX-2), inducible nitric oxide synthetase (iNOS), and activator protein (AP-1) and the quantitative expressions of nuclear factor E2-related factor (Nrf2), heme oxygenase (HO-1), metallothionein (MT) and reduced glutathione (GSH) in mercury-chloride-exposed (50 µM and 10 mg/kg body weight) primary cultured hepatocytes and ICR mice, using biochemical assays, radioactivity and immunoblot analysis. The results demonstrated that ZPDC glycoprotein decreased the levels of LDH, ALT, HO-1 and MT, whereas it increased the activities of hepatic antioxidant enzymes (SOD, CAT and GPx) and reduced GSH in mercury-chloride-exposed primary cultured hepatocytes. Also, it suppressed arachidonic acid release and expression of ERK, p38 MAPK, COX-2, iNOS, AP-1 and Nrf-2 in primary cultured hepatocytes and ICR mice exposed to mercury chloride. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatotoxicity induced by mercury chloride.

Normalizing effect of SJSZ glycoprotein (38 kDa) on proliferating cell nuclear antigen and interferon-γ in diethylnitrosamine-induced mice splenocytes.

One of the immunosuppressive responses when hepatocellular carcinoma (HCC) develops in mammals is defective proliferation in the spleen. The objective of this study was to investigate the protective effect of the Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on the proliferation of splenocytes induced by diethlynitrosamine (DEN). To assess whether the SJSZ glycoprotein modulates splenocyte proliferation, Balb/c mice were injected intraperitoneally with DEN (50 mg/kg, BW) for 7 weeks. After 7 weeks, the mice were sacrificed, and spleens were isolated. We evaluated [(3) H]-thymidine incorporation, extracellular signal-regulated kinase (ERK), cell cycle-related factors [p53, p21, p27, cyclin D1/cyclin dependent kinase (CDK) 4], proliferating cell nuclear antigen and interferon (IFN)-γ using radiation activity, immunoblot analysis, and the reverse transcription-polymerase chain reaction. The results revealed that the SJSZ glycoprotein (10 mg/kg, BW) increased [(3) H]-thymidine incorporation, ERK phosphorylation, expression levels of cyclin D1/cyclin dependent kinase 4, and IFN-γ. However, the SJSZ glycoprotein decreased levels of p53, p21, and p27. Taken together, these results suggest that the SJSZ glycoprotein inhibited defective splenocyte proliferation induced by DEN.

SJSZ glycoprotein (38 kDa) modulates macrophage type 1/2-related factors at hepatocarcinogenic stage in N-nitrosodiethylamine-treated Balb/c.

Macrophage plays critical role for tumor progression: Type 1 (M1) for tumor prevention and type 2 (M2) for promotion in hepatocellular carcinoma. In order to study the chemopreventive effects of the SJSZ glycoprotein (38 kDa) on M1- or M2-related factors, Balb/c was injected intraperitoneally with N-nitrosodiethylamine (DEN; 50 mg/kg, BW) for 7 weeks. After 7 weeks, the mice were sacrificed. After that, peritoneal macrophages were isolated. We evaluated the production of reactive oxygen species (ROS) and nitric oxide (NO), hepatocarcinogenic signals [activities of mitogen-activated associated kinase (MAPKs), inducible nitric oxide synthase (iNOS), nuclear factor (NF)-κB, and signal transducer and activator of transcription (STAT) 6,], cytokines [interleukin (IL)-10, IL-4, IL-12, and interferon (IFN)-γ], and CD163-positive macrophages (M2 polarization) using biochemical methods, immunoblot analysis, qRT-PCR, ELISA, and flow cytometry. The results revealed that the SJSZ glycoprotein (10 mg/kg, BW) inhibits the phosphorylation of MAPKs and expression of NF-κB, pSTAT6, IL-10, and IL-4; and normalizes production of ROS and NO, and expression of iNOS, IL-12, and IFN-γ. Especially, it inhibited CD163-positive macrophages. In conclusion, these results indicated that SJSZ glycoprotein modulates polarization of macrophage type 1 and type 2 at hepatocarcinogenic initial stage in DEN-treated Balb/c. Thus, SJSZ glycoprotein may be useful as one of immunomodulating agents which have to regulate M1- and M2-related factors to prevent tumor progression.

Inductive effect of phytoglycoprotein (38 kDa) on G0/G1 arrest and apoptosis in diethylnitrosamine-treated ICR mice.

Promotion in the carcinogenic stage is closely related to the growth and proliferation of liver cancer cells. The purpose of this study was whether Styrax japonicum Siebold & Zuccarini (SJSZ) glycoprotein has preventive effect on the growth of hepatocarcinoma cells in diethylnitrosamine (DEN)-induced ICR mice. The study evaluated cell cycle and the activities of cell cycle-related factors [cyclin D1/cyclin dependent kinase (CDK) 4], cell cycle inhibitors (CKIs; p53, p21, and p27), proliferating cell nuclear antigen (PCNA), cytochrome c, Bid, caspase-3, and caspase-9 in DEN-induced ICR mice by flow cytometry, immunoblot analysis and qRT-PCR. The results showed that SJSZ glycoprotein (10 mg/kg, BW) arrested G(0)/G(1) phase and the activity of cyclin D1/CDK4, PCNA, and Bid. However, it induced activities of CKIs, cytochrome c, caspase-3, and caspase-9. Taken together, this present study suggested that SJSZ glycoprotein might be a potent inhibition of hepatic tumor promotion.

Protection against cyclophosphamide-induced myelosuppression by ZPDC glycoprotein (24 kDa).

Immunomodulatory agents are often used to reduce myelosuppression and enhance immune response for cancer treatment. Cyclophosphamide (CTX) can induce oxidative stress in bone marrow resulting in suppression of anti-oxdiantive enzymes and causes myelosuppression. We isolated glycoprotein from Zanthoxylum piperitum DC fruit (ZPDC), and it consists of a carbohydrate (18%) and a protein (82%). The objective of this study was to investigate its protective activity against CTX-induced myelosuppression in Balb/c (n=6/group). The mice were orally administrated by ZPDC glycoprotein (10 and 20 mg/kg, BW) for 1 week in the presence or absence of CTX. Intracellular reactive oxygen species (ROS), anti-oxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)], cyclin kinase inhibitors (CKIs: p53, p21 and p27), cyclin D1/ cyclin dependent kinase (CDK) 4, PCNA and cytokines [interleukin (IL)-3, and granulocyte/ macrophage-colony-stimulating factor (GM-CSF)] were evaluated using biochemical activity, Western blot analysis, and ELISA. The results obtained from this study showed that CTX decreased spleen and thymic indices, bone marrow cellularity and expression of cyclin D1/CDK4 and PCNA, but it increased CKIs, whereas ZPDC glycoprotein (20 mg/kg, BW) resulted in vice versa in CTX-induced Balb/c. Expression of IL-3 and GM-CSF were normalized by ZPDC glycoprotein. Thus, this study suggested that ZPDC glycoprotein prevents oxidative stress and myelosuppression in CTX-induced mice and might be a potential immunomodulatory agent.

Activity of tumor necrosis factor-α blocked by phytoglycoprotein (38 kDa) at initiation stage in N-nitrosodiethylamine-induced ICR mice.

Hepatocellular carcinoma is becoming one of the most prominent types of cancer in the world. Recently, from Styrax japonica Siebold et al. Zuccarini (SJSZ), we isolated a glycoprotein which consists of carbohydrate moiety (52.64%) and protein moiety (42.35%). We evaluated whether SJSZ glycoprotein prevents hepatocarcinogenesis induced by N-nitrosodiethylamine (DEN). The purpose of this study was to evaluate the effect of SJSZ glycoprotein in DEN-induced hepatocarcinogenesis in ICR mice. To know chemopreventive effect of SJSZ glycoprotein on hepatocarcinogenesis, ICR mice were intraperitoneally injected with N-nitrosodiethylamine (DEN, 10 mg/kg) for 7 weeks. After sacrifice, we evaluated indicators of liver tissue damage [the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid reactive substances (TBARS)], antioxidative enzymes [activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], and initiating hepatocarcinogenic indicator [heat shock protein (HSP) 27 and 70] and hepatocarcinogenic signals [protein kinase C (PKC), extracellular signal-regulating kinase (ERK) 1/2, nuclear factor (NF)-κB (p50 and p65) and tumor necrosis factor-α (TNF-α)] using biochemical methods, immunoblot analysis, and RT-PCR. The results obtained from this study revealed that SJSZ glycoprotein (10 mg/kg, BW) decreased the levels of LDH, ALT, and TBARS, whereas the activities of SOD, GPx, and CAT increased in the DEN-induced ICR mice. With respect to the hepatocarcinogenic indicator and hepatocarcinogenic signals, HSP27, HSP70, PKC, ERK1/2, NF-κB (p50 and p65), and TNF-α, activity decreased. Hence, SJSZ glycoprotein might prevent expression of HSP27 and HSP70 by DEN.

Inhibitory effect of SJSZ glycoprotein (38 kDa) on expression of heat shock protein 27 and 70 in chromium (VI)-treated hepatocytes.

Chromium (VI) is as an extremely toxic chemical substance, and is also an internationally recognized human carcinogen. The principal objective of this study was to determine whether or not Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents hepatocarcinogenesis in chromium-treated BNL CL.2 cells and ICR mice. Firstly, it was evaluated that SJSZ glycoprotein has strong antioxidant character and scavenges radicals. In an effort to assess the chemopreventive effects of SJSZ glycoprotein on hepatocarcinogenesis, ICR mice were intraperitoneally injected with chromium (10 mg/kg, BW) for 8 weeks. After sacrifice, we evaluated indicators of liver tissue damage [the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS)], antioxidative enzymes [activities of superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (GPx)], and initiating hepatocarcinogenic indicator [heat shock protein (HSP) 27 and 70] and protein kinase C (PKC), p38 MAPK and PCNA via biochemical methods and immunoblot analysis. The results obtained from this study demonstrated that the SJSZ glycoprotein (50 µg/ml) inhibited the production of intracellular ROS in BNL CL.2 cells. In addition, the SJSZ glycoprotein (10 mg/kg, BW) attenuated the levels of LDH, ALT, and TBARS, whereas it increased antioxidative enzymes in mouse serum. SJSZ glycoprotein attenuated the activity of HSP27, HSP70, PKC, MAPKs, and PCNA in BNL CL.2 cells and liver tissue. Taken together, our results indicate that SJSZ glycoprotein might be have a potent preventive effect against hepatocarcinogenesis induced by oxidative stress.

Inhibitory effect of Styrax Japonica Siebold et al. Zuccarini glycoprotein (38 kDa) on interleukin-1ß and induction proteins in chromium(VI)-treated BNL CL.2 cells.

Chromium(VI) [Cr(VI)] induces chronic inflammation in hepatocytes. Inflammation has been shown to play an important role in tumorigenesis, tumor progression, and metastasis. To examine the effects of the Styrax Japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on inflammation in BNL CL.2 cells, we evaluated the activities of c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), nuclear factor (NF)-κB (p50 and p65), and inflammation-related factors [cyclooxygenase (COX)-2, inducible nitric oxide syntheses (iNOS) and interleukin (IL)-1ß] in Cr-induced BNL CL.2 cells using immunoblot analysis and RT-PCR. We also used two-dimensional gel electrophoresis (2-DE) to compare between treatments. To determine which proteins are induced by Cr(VI), we evaluated total protein lysates using 2-DE. After Cr(VI) treatment, total protein lysates were prepared and resolved by 2-DE. The results obtained from this study demonstrated that the SJSZ glycoprotein (50 µg/ml) inhibits expression of JNK, ERK, NF-κB, and the expression of COX-2, iNOS, and IL-1ß. Moreover, the results obtained from 2DE showed that four proteins out of nine proteins were relatively expressed strongly, while the rest of them were relatively appeared weakly on the gel. Taken together, these data indicate that the SJSZ glycoprotein prevents expression of COX-2, iNOS, and IL-1ß by blocking NF-κB and MAPKs in Cr(VI)-induced BNL CL.2 cells.

SJSZ glycoprotein (38 kDa) modulates expression of IL-2, IL-12, and IFN-γ in cyclophosphamide-induced Balb/c.

OBJECTIVE: Cyclophosphamide (CTX) often results in immunosuppression and cytotoxic effects. The object of this study was to understand whether Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents immunosuppression in CTX-induced Balb/c. METHODS: The mice were injected intraperitoneally with CTX (80 mg/kg, BW) for 1 week in the presence or absence of the SJSZ glycoprotein, and divided into five groups. Weights of the spleen and thymus, phagocytic macrophages, proliferation of splenocytes and thymocytes ([(3)H]-thymidine incorporation and expression of PCNA), natural killer (NK) cytotoxicity [MTT assay, perforin, and granzyme B], and cytokines [interleukin (IL)-2, IL-12, and interferon (IFN)-γ] were evaluated using radioactivity, biochemical reactions, immunoblot analysis, and qRT-PCR. Activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione dismutase (GPx) and catalase (CAT)] was also assessed. RESULTS: The results revealed that while the parameters assessed decreased with treatment with CTX alone, SJSZ glycoprotein (10 mg/kg, BW) in the presence of CTX significantly normalized the weights of spleen and thymus, the phagocytic effect of peritoneal macrophages, the activity of antioxidant enzymes, proliferation (splenocytes and thymocytes), NK cell cytotoxicity, and expression of IL-2, IL-12, and IFN-γ. CONCLUSION: SJSZ glycoprotein can normalize activity of anti-oxidative enzymes and immune-related factors.

Preventive effect of phytoglycoprotein (38 kDa) on expression of alpha-fetoprotein and matrix metalloproteinase-9 in diethylnitrosamine-treated ICR mice.

Metastasis is one of the major causes of cancer-associated mortality. Aberrant expression of matrix metalloproteinase-9 (MMP-9) has been implicated in the metastasis of various cancer cells. The aim of this study was to investigate the inhibitory effect of a glycoprotein (38 kDa) isolated from Styrax japonica Siebold et al Zuccarini (SJSZ) on metastasis of hepatocellular carcinoma (HCC) in diethylnitrosamine (DEN)-treated imprinting control region (ICR) mice. To study the chemopreventive effect of SJSZ glycoprotein on the metastasis of HCC, ICR mice were injected intraperitoneally with DEN (75 mg/kg) for 11 weeks. Subsequently, we evaluated nitric oxide (NO), alpha-fetoprotein (AFP), activator protein (AP)-1, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-9, and interleukin (IL)-6 using biochemical reaction, immunoblot analysis, and reverse-transcription polymerase chain reaction. Here, the results showed that SJSZ glycoprotein (10 mg/kg body weight) reduced the production of NO in DEN (75 mg/kg)-treated ICR mice. Also, it suppressed the activity of AFP, AP-1 (c-Jun and c-Fos), COX-2, iNOS, and MMP-9. Taken together, SJSZ glycoprotein inhibits the activity of MMP-9 as a metastasis factor.

Phytoglycoprotein (38 kDa) induces cell cycle (G0/G1) arrest and apoptosis in HepG2 cells.

Styrax japonica Siebold et al Zuccarini (SJSZ) has been used to heal inflammation and bronchitis as folk medicine in Korea. Firstly, glycoprotein isolated from SJSZ (SJSZ glycoprotein) has a molecular weight with 38 kDa and consists of carbohydrate (57.64%) and protein (42.35%). In the composition of SJSZ glycoprotein, carbohydrate mostly consists of glucose (28.17%), galactose (21.85%), and mannose (2.62%) out of 52.64%, respectively. The protein consists of Trp (W, 7.01%), Pro (P, 6.72%), and Ile (I, 5.42%) out of 42.35% as three major amino acids, while total amount of other amino acids is 23.20%. The purpose of this study is to know whether the SJSZ glycoprotein (38 kDa) induces the cell cycle arrest and apoptosis in HepG2 cells. Cytotoxicity was evaluated using MTT and lactate dehydrogenase assay and amount of intracellular reactive oxygen species (iROS) and nitric oxide (NO) was measured using fluorescence microplate reader. Activities of cell cycle-related proteins [p53, p21, p27, Cyclin D1, and cyclin-dependent kinase (CDK)4] and apoptosis-related factors [iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and poly-(ADP-ribose) polymerase (PARP)] were assessed by Western blot and fluorescence-activated cell sorter (FACS) analysis. In the cell cycle-related proteins, SJSZ glycoprotein (50 µg/ml) significantly enhances the expression of p53, p21, and p27, whereas it suppressed the activity of cyclin D1/CDK4. In the apoptosis-related factors, SJSZ glycoprotein (50 µg/ml) stimulates to increase iROS, and NO, to activate iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and PARP. SJSZ glycoprotein (50 µg/ml) has potent effect to arrest cell cycle from G(0) /G(1) to S and to induce apoptosis in HepG2 cells.

Preventive effect of phytoglycoprotein (27 kDa) on inflammatory factors at liver injury in cadmium chloride-exposed ICR mice.

Cadmium is one of the inflammation-related xenobiotics and has been regarded as a potent carcinogen. Gardenia jasminoides Ellis (GJE) has been used to cure inflammation in Korean folk medicine for a long time. The purpose of present study is the inhibitory effect of glycoprotein isolated from GJE (27 kDa) on inflammation mechanism in cadmium chloride-exposed ICR mice. We evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS), activities of anti-oxidative enzymes [superoxide dismutase (SOD) and gluthathione peroxidase (GPx)], activities of c-Jun N-terminal protein kinase (JNK), heat shock protein 27 (Hsp27), activator protein (AP)-1, nuclear factor (NF)-κB and expression of inflammation-related mediators including tumor necrosis factor (TNF)-α and interleukin (IL)-6 in cadmium chloride-exposed ICR mice using immunoblot analysis, EMSA and RT-PCR. It notes that mice plasma was used to measure ALT, LDH, and TBARS after treatment with cadmium chloride alone or cadmium chloride under the pretreatment with GJE glycoprotein. Liver tissues were used to assess activities of anti-oxidant enzymes, SAPK/JNK, Hsp27, AP-1, NF-κB, TNF-α, and IL-6 in this study. The results obtained from this study revealed that GJE glycoprotein (10 mg/kg) decreased the levels of LDH, ALT and TBARS, whereas increased the activity of hepatic anti-oxidant enzymes (SOD and GPx) in cadmium chloride-exposed ICR mice. Moreover, it decreased the activity of JNK/AP-1, NF-κB, Hsp27, and pro-inflammatory cytokines (TNF-α and IL-6). Taken together, the results in this study suggest that GJE glycoprotein inhibits the expression of inflammation-related cytokines (TNF-α and IL-6) in cadmium chloride-exposed ICR mice.

Anti-inflammatory effect of glycoprotein isolated from Cudrania tricuspidata Bureau: involvement of MAPK/NF-κB signaling.

The aim of this study was to evaluate inhibitory effect of glycoprotein isolated from Cudrania tricuspidata Bureau (CTB glycoprotein) on allergic responses. We evaluated the activities of mitogen-activated protein kinase (MAPK), transcriptional factor, and production of immunoglobulin (Ig)E and interleukin (IL)-4 in RBL-2H3 cells and BALB/c mice. Our results showed that CTB glycoprotein inhibited the production of IgE and IL-4 in serum from ovalbumin (OVA)-treated BALB/c mice. We also found that CTB glycoprotein inhibited the phosphorylation of p38 MAPK, and transcriptional activation of nuclear factor (NF)-κB in RBL-2H3 cells. The activation of NF-κB was effectively blocked by treatment with p38 MAPK inhibitor (SKF86002). The results from these experiments indicate that the CTB glycoprotein inhibits release of ß-hexosaminidase, and production of IgE and IL-4 via down regulation of MAPK/ NF-κB on the stage of mast cell degranulation. In conclusion, we suggest that the CTB glycoprotein might be a potent preventive agent in allergic responses.

Cudrania tricupidata bureau (CTB) glycoprotein inhibits proliferation by Di(2-ethylhexyl) phthalate in primary splenocytes: responses in cell proliferation signaling.

The aim of the present study was to evaluate inhibitory effect of CTB glycoprotein isolated from Cudrania tricuspidata Bureau on DEHP-induced cell proliferation in lymphocytes. Our results revealed that DEHP increased lymphocyte proliferation as confirmed by increasing [(3)H]thymidine incorporation, and proliferating cell nuclear antigen (PCNA), cyclin D1, and cyclin-dependent kinase (CDK)-4 expression. This was accompanied by induced intracellular Ca(2+) level, protein kinase C (PKC) translocation from cytosol to membrane, ERK1/2 phosphorylation, and nuclear factor (NF)-κB transcriptional activation in DEHP-treated cells. However, CTB glycoprotein (100 µg/ml) reduced labeled thymidine incorporation and PCNA expression in DEHP-treated cells. Additionally CTB glycoprotein reduced Ca(2+) level, PKC translocation, ERK1/2 phosphorylation, NF-κB transcriptional activation, cell cycle proteins (cyclin D1 and CDK4) expression in cells. The activation of NF-κB was collectively blocked by pretreatment with PKC inhibitor (staurosporine) and ERK1/2 inhibitor (PD98059), respectively. The results from these experiments indicate that CTB glycoprotein inhibits cell proliferation via down regulations of Ca(2+)/PKC, ERK1/2, and cell cycle proteins induced by DEHP. Therefore, we suggest that the CTB glycoprotein might be one component for prevention of cell proliferation-related immune diseases.

Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate.

Di(2-ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP-induced BNL CL. 2 cells. [³H]-thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²âº mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)], activator protein (AP)-1 (c-Jun and c-Fos), proliferating cell nuclear antigen (PCNA) and cell cycle-related factors (cyclin D1/cyclin-dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]-thymidine incorporation, intracellular ROS, intracellular Ca²âº mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP-induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP-1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate-induced BNL CL. 2 cells.

Allergy-related cytokines (IL-4 and TNF-α) are induced by Di(2-ethylhexyl) phthalate and attenuated by plant-originated glycoprotein (75 kDa) in HMC-1 cells.

Phthalate esters as plasticizers have been widespread in the environment and may be associated with development of allergic diseases such as asthma and atopic dermatitis. In this study, we demonstrated that the CTB glycoprotein attenuates allergic reactions caused by di(2-ethylhexyl) phthalate (DEHP) in human mast cells (HMC-1). This experiment evaluated degranulation of histamine and ß-hexosaminidase as well as activities of protein kinase C (PKC), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), activator protein (AP)-1 and interleukin (IL)-4 and tumor necrosis factor (TNF)-α using immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Our results revealed that the CTB glycoprotein in the presence of DEHP inhibits degranulation of mast cell, translocation of PKC from cytosol to membrane, and phosphorylation of SAPK/JNK in HMC -1 cells. We also found that the CTB glycoprotein (100 µg mL(-1) ) has suppressive effects on transcriptional activation of AP-1, and on the expression of IL-4 and TNF-α in DEHP-treated HMC-1 cells. We suggest that the CTB glycoprotein inhibits degranulation of mast cells and expressions of cytokines in HMC-1 cells.

A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium.

Cudrania tricuspidata Bureau (CTB), a species of the Moraceae plant, has been used as a bruise recovery treatment. This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease (IBD). We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C. CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB, which are responsible for the expression of cell-cycle-related proteins (CDK2, CDK4, cyclin D1 and cyclin E) during its promotion of cell proliferation. Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4% (W/V) for seven days. We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score, which was composed of body weight change, diarrhea, and hematochezia in ICR mice treated with dextran sulfate sodium. Hence, we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC, JNK and NF-κB in colon epithelial cells.