Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages.

The white-rot fungi Ceriporia lacerata is used in bioremediation, such as lignocellulose degradation, in nature. Submerged cultures and extracts of C. lacerata mycelia (CLM) have been reported to contain various active ingredients, including ß-glucan and extracellular polysaccharides, and to exert anti-diabetogenic properties in mice and cell lines. However, the immunostimulatory effects have not yet been reported. This study aimed to identify the immunomodulatory effects, and underlying mechanisms thereof, of submerged cultures of CLM using RAW264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression in mice. Compared to CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice orally administered CLM were significantly increased; body weight loss was alleviated; and natural killer (NK) cytotoxicity, lymphocyte proliferation, and cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, and interleukin [IL]-2) production were elevated in the serum. In RAW264.7 macrophages, treatment with CLM induced phagocytic activity, increased the production of nitric oxide (NO), and promoted mRNA expression of the immunomodulatory cytokines TNF-α, IFN-γ, IL-1ß, IL-6, IL-10, and IL-12. In addition, CLM increased the inducible NO synthase (iNOS) concentration in macrophages, similar to lipopolysaccharide (LPS) stimulation. Mechanistic studies showed that CLM induced the activation of the NF-κB, PI3k/Akt, ERK1/2, and JNK1/2 pathways. Moreover, the phosphorylation of NF-κB and IκB induced by CLM in RAW264.7 cells was suppressed by specific MAPKs and PI3K inhibitors. Further experiments with a TLR4 inhibitor demonstrated that the production of TNF-α, IL-1ß, and IL-6 induced by CLM was decreased after TLR4 was blocked. Overall, CLM protected against CTX-induced adverse reactions by enhancing humoral and cellular immune functions, and has potential as an immunomodulatory agent.

Effect of Porcine Whole Blood Protein Hydrolysate on Slow-Twitch Muscle Fiber Expression and Mitochondrial Biogenesis via the AMPK/SIRT1 Pathway.

Skeletal muscle is a heterogeneous tissue composed of a variety of functionally different fiber types. Slow-twitch type I muscle fibers are rich with mitochondria, and mitochondrial biogenesis promotes a shift towards more slow fibers. Leucine, a branched-chain amino acid (BCAA), regulates slow-twitch muscle fiber expression and mitochondrial function. The BCAA content is increased in porcine whole-blood protein hydrolysates (PWBPH) but the effect of PWBPH on muscle fiber type conversion is unknown. Supplementation with PWBPH (250 and 500 mg/kg for 5 weeks) increased time to exhaustion in the forced swimming test and the mass of the quadriceps femoris muscle but decreased the levels of blood markers of exercise-induced fatigue. PWBPH also promoted fast-twitch to slow-twitch muscle fiber conversion, elevated the levels of mitochondrial biogenesis markers (SIRT1, p-AMPK, PGC-1α, NRF1 and TFAM) and increased succinate dehydrogenase and malate dehydrogenase activities in ICR mice. Similarly, PWBPH induced markers of slow-twitch muscle fibers and mitochondrial biogenesis in C2C12 myotubes. Moreover, AMPK and SIRT1 inhibition blocked the PWBPH-induced muscle fiber type conversion in C2C12 myotubes. These results indicate that PWBPH enhances exercise performance by promoting slow-twitch muscle fiber expression and mitochondrial function via the AMPK/SIRT1 signaling pathway.

Influence of o,p'-DDT on MUC5AC expression via regulation of NF-κB/AP-1 activation in human lung epithelial cells.

o,p'-Dichlorodiphenyltrichloroethane (o,p'-DDT) is a representative endocrine disruptor, and exposure to o,p'-DDT may produce immune disorders and inflammation, leading to various diseases such as cancer. Chronic airway inflammation is characterized by excessive mucus secretion resulting in chronic obstructive pulmonary disease (COPD). Mucin 5AC  (MUC5AC), one of the mucus genes, plays an important role in mucus secretion and inflammation in the airways. The aim of this study was to examine the effects of o,p'-DDT on the regulation of MUC5AC expression in human lung epithelial A549 cell line. o,p'-DDT increased mRNA levels and the promoter activity of MUC5AC. Transient transfection with mutation promoter constructs of MUC5AC demonstrated that nuclear factor kappa-b (NF-κB) and activator protein 1(AP-1) response elements were essential for the consequences of o,p'-DDT on MUC5AC expression. In addition, o,p'-DDT induced phosphorylation of ERK, JNK, p38, and Akt, which are involved in the regulation of MUC5AC expression. It is noteworthy that inhibitors of NF-κB, AP-1, Akt, and MAPKs blocked enhanced o,p'-DDT-induced MUC5AC mRNA expression. Data indicate that o,p'-DDT increase in NF-κB, and AP-1 transcriptional activation-dependent MUC5AC expression is associated with stimulation of Akt and MAPK signaling pathways in A549 cells.

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKß/AMPK Signaling Pathway in Human Endothelial Cells.

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca2+ concentration and phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), and Ca2+/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca2+ concentration and phosphorylation of CaMKII, CaMKKß, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKß inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca2+/CaMKII and CaM/CaMKKß/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.

Impressic Acid Ameliorates Atopic Dermatitis-Like Skin Lesions by Inhibiting ERK1/2-Mediated Phosphorylation of NF-κB and STAT1.

Impressic acid (IPA), a lupane-type triterpenoid from Acanthopanax koreanum, has many pharmacological activities, including the attenuation of vascular endothelium dysfunction, cartilage destruction, and inflammatory diseases, but its influence on atopic dermatitis (AD)-like skin lesions is unknown. Therefore, we investigated the suppressive effect of IPA on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin symptoms in mice and the underlying mechanisms in cells. IPA attenuated the DNCB-induced increase in the serum concentrations of IgE and thymic stromal lymphopoietin (TSLP), and in the mRNA levels of thymus and activation regulated chemokine(TARC), macrophage derived chemokine (MDC), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) in mice. Histopathological analysis showed that IPA reduced the epidermal/dermal thickness and inflammatory and mast cell infiltration of ear tissue. In addition, IPA attenuated the phosphorylation of NF-κB and IκBα, and the degradation of IκBα in ear lesions. Furthermore, IPA treatment suppressed TNF-α/IFN-γ-induced TARC expression by inhibiting the NF-κB activation in cells. Phosphorylation of extracellular signalregulated protein kinase (ERK1/2) and the signal transducer and activator of transcription 1 (STAT1), the upstream signaling proteins, was reduced by IPA treatment in HaCaT cells. In conclusion, IPA ameliorated AD-like skin symptoms by regulating cytokine and chemokine production and so has therapeutic potential for AD-like skin lesions.

WY-14643 Regulates CYP1B1 Expression through Peroxisome Proliferator-Activated Receptor α-Mediated Signaling in Human Breast Cancer Cells.

Human cytochrome P450 1B1 (CYP1B1)-mediated biotransformation of endobiotics and xenobiotics plays an important role in the progression of human breast cancer. In this study, we investigated the effects of WY-14643, a peroxisome proliferator-activated receptor α (PPARα) agonist, on CYP1B1 expression and the related mechanism in MCF7 breast cancer cells. We performed quantitative reverse transcription-polymerase chain reaction, transient transfection, and chromatin immunoprecipitation to evaluate the effects of PPARα on peroxisome proliferator response element (PPRE)-mediated transcription. WY-14643 increased the protein and mRNA levels of CYP1B1, as well as promoter activity, in MCF-7 cells. Moreover, WY-14643 plus GW6471, a PPARα antagonist, significantly inhibited the WY-14643-mediated increase in CYP1B1 expression. PPARα knockdown by a small interfering RNA markedly suppressed the induction of CYP1B1 expression by WY-14643, suggesting that WY-14643 induces CYP1B1 expression via a PPARα-dependent mechanism. Bioinformatics analysis identified putative PPREs (-833/-813) within the promoter region of the CYP1B1 gene. Inactivation of these putative PPREs by deletion mutagenesis suppressed the WY-14643-mediated induction of CYP1B1 promoter activation. Furthermore, WY-14643 induced PPARα to assume a form capable of binding specifically to the PPRE-binding site in the CYP1B1 promoter. Our findings suggest that WY-14643 induces the expression of CYP1B1 through activation of PPARα.

Molecular Discrimination of Cynanchum wilfordii and Cynanchum auriculatum by InDel Markers of Chloroplast DNA.

The tuber of Cynanchum wilfordii (Baekshuoh Radix in Korean) is an important medicinal herb in Korea and China; however, it is difficult to differentiate C. wilfordii from a related medicinal herb, C. auriculatum (Baishouwu Radix in Chinese). We sought to develop a molecular method that could be used to distinguish between the tubers of C. wilfordii and C. auriculatum. We aligned the chloroplast genome sequences (available in the NCBI database) of the two species and identified three species-specific insertion and deletion (InDel) sites in the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 intergenic spacer (IGS) regions. To confirm the presence of these three InDels and validate their use as markers, we designed three primer pairs to amplify the trnQ-psbK, rps2-rpoC2, and psaJ-rpl33 IGS regions. Polymerase chain reaction (PCR) amplification of the trnQ-psbK IGS region yielded a 249 bp fragment for C. wilfordii, and 419 bp fragment for C. auriculatum, whereas the rps2-rpoC2 IGS primers produced a 629 bp fragment from C. wilfordii and a 282 bp fragment from C. auriculatum. In the psaJ-rpl33 IGS region, allele fragments of 342 and 360 bp in length were amplified from C. wilfordii, whereas 249 and 250 bp fragment were amplified from C. auriculatum. We propose these three InDel markers as a valuable, simple, and efficient tool for identifying these medicinal herbs and will thus reduce adulteration of these herbal materials in commercial markets.

Effects of isometric exercise using biofeedback on maximum voluntary isometric contraction, pain, and muscle thickness in patients with knee osteoarthritis.

[Purpose] The purpose of our study was to investigate the effects of isometric exercises using electromyographic biofeedback (EMGBF) and ultrasound biofeedback (USBF) on maximum voluntary isometric contraction (MVIC), pain assessed by the Visual Analogue Scale (VAS), and vastus medialis oblique (VMO) thickness in patients with knee osteoarthritis (OA). [Subjects and Methods] Thirty females over 65 years of age who had been diagnosed with knee osteoarthritis were recruited and randomly assigned to three groups, each comprising of 10 subjects. The Subjects in the EMGBF training and USBF training groups were trained with the corresponding physical training exercise program targeting the vastus medialis oblique, whereas the subjects in the control group were treated with conventional physical therapies, such as a hot pack, ultrasound, and transcutaneous electrical nerve stimulation. Subjects in each group were trained or treated for 20 min, 3 times a week for 8 weeks. [Results] The MVIC in the EMGBF and USBF training groups was significantly increased compared with that in the control group, and the VAS score (for measurement of pain) in the EMGBF and USBF training groups was significantly decreased compared with that in the control group. Only the EMGBF training group showed a significantly increased VMO thickness compared with before training. [Conclusion] These results suggest that USBF training is similar to EMGBF training in terms of its effectiveness and is helpful for treating patients with knee OA.

Saponins from the roots of Platycodon grandiflorum suppresses TGFß1-induced epithelial-mesenchymal transition via repression of PI3K/Akt, ERK1/2 and Smad2/3 pathway in human lung carcinoma A549 cells.

Transforming growth factor ß (TGFß) is a multifunctional cytokine that induces growth arrest, tissue fibrosis, and epithelial-mesenchymal transition (EMT) through activation of Smad and non-Smad signaling pathways. EMT is the differentiation switch by which polarized epithelial cells differentiate into contractile and motile mesenchymal cells. Our previous studies have shown that saponins from the roots of Platycodon grandiflorum (CKS) have antiinflammatory, antioxidant, antimetastatic, and hepatoprotective effects. In this study, we investigated the inhibitory effect of CKS on TGFß1-induced alterations characteristic of EMT in human lung carcinoma A549 cells. We found that CKS-treated cells displayed inhibited TGFß1-mediated E-cadherin downregulation and Vimentin upregulation and also retained epithelial morphology. Furthermore, TGFß1-increased Snail expression, a repressor of E-cadherin and an inducer of the EMT, was reduced by CKS. CKS inhibited TGFß1-induced phosphorylation of Akt, ERK1/2, and glycogen synthase kinase-3ß (GSK-3ß). Inhibition of PI3K/Akt and ERK1/2 also blocked TGFß1-induced GSK-3ß phosphorylation and Snail activation. Furthermore, TGFß1-increased Snail expression was reduced by selective inhibitors of Akt and ERK1/2. Moreover, CKS treatment attenuated TGFß1-induced Smad2/3 phosphorylation and upregulated Smad7 expression. These results indicate that pretreatment with the CKS inhibits the TGFß1-induced EMT through PI3K/Akt, ERK1/2, GSK-3ß and Smad2/3 in human lung carcinoma cells.

Mollugin inhibits proliferation and induces apoptosis by suppressing fatty acid synthase in HER2-overexpressing cancer cells.

Mollugin is a naphthohydroquine found in the roots of Rubia cordifolia, and has been reported to have a variety of biological activities, including anti-inflammatory and apoptotic effects. In the present study, we investigated the molecular mechanisms by which mollugin exerts anti-tumor effect in HER2-overexpressing cancer cells. Our results showed that mollugin exhibited potent inhibitory effects on cancer cell proliferation, especially in HER2-overexpressing SK-BR-3 human breast cancer cells and SK-OV-3 human ovarian cancer cells in a dose- and time-dependent manner without affecting immortalized normal mammary epithelial cell line MCF-10A. Furthermore, we found that a blockade of Akt/SREBP-1c signaling through mollugin treatment significantly reduced FAS expression and subsequently suppressed cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Mollugin treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level, potentially in part through suppression of NF-κB activation. The combination of mollugin with a MEK1/2 inhibitor may be required in order to achieve optimal efficacy in HER2-overexpressing cancers. These data provide evidence that mollugin inhibits proliferation and induces apoptosis in HER2-overexpressing cancer cells by blocking expression of the FAS gene through modulation of a HER2/Akt/SREBP-1c signaling pathway. Our findings suggest that mollugin is a novel modulator of the HER2 pathway in HER2-overexpressing cancer cells with a potential role in the treatment and prevention of human breast and ovarian cancer with HER2 overexpression.

Saponins, especially platycodin D, from Platycodon grandiflorum modulate hepatic lipogenesis in high-fat diet-fed rats and high glucose-exposed HepG2 cells.

AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP-1c) pathway. Saponins, particularly platycodin D, from the roots of Platycodon grandiflorum (Changkil saponins, CKS) have a variety of pharmacological properties, including antioxidant and hepatoprotective properties. The aim of this study was to investigate the effects of CKS on hepatic lipogenesis and on the expression of genes involved in lipogenesis, and the mechanisms involved. CKS attenuated fat accumulation and the induction of the lipogenic genes encoding SREBP-1c and fatty acid synthase in the livers of HFD-fed rats and in steatotic HepG2 cells. Blood biochemical analyses and histopathological examinations showed that CKS prevented liver injury. CKS and platycodin D each increased the phosphorylation of AMPK and acetyl-CoA carboxylase in HFD-fed rats and HepG2 cells. The use of specific inhibitors showed that platycodin D activated AMPK via SIRT1/CaMKKß in HepG2 cells. This study demonstrates that CKS or platycodin D alone can regulate hepatic lipogenesis via an AMPK-dependent signalling pathway.

Effect of 3-caffeoyl, 4-dihydrocaffeoylquinic acid from Salicornia herbacea on endothelial nitric oxide synthase activation via calcium signaling pathway.

3-Caffeoyl-4-dicaffeoylquinic acid (CDCQ) is a natural chlorogenic acid isolated from Salicornia herbacea that protects against oxidative stress, inflammation, and cancer. Nitric oxide (NO) plays a physiologically beneficial role in the cardiovascular system, including vasodilation, protection of endothelial cell function, and anti-inflammation. However, the effect of CDCQ on NO production and eNOS phosphorylation in endothelial cells is unclear. We investigated the effect of CDCQ on eNOS phosphorylation and NO production in human endothelial cells, and the underlying signaling pathway. CDCQ significantly increased NO production and the phosphorylation of eNOS at Ser1177. Additionally, CDCQ induced phosphorylation of PKA, CaMKII, CaMKKß, and AMPK. Interestingly, CDCQ increased the intracellular Ca2+ level, and L-type Ca2+ channel (LTCC) blockade significantly attenuated CDCQ-induced eNOS activity and NO production by inhibiting PKA, CaMKII, CaMKKß, and AMPK phosphorylation. These results suggest that CDCQ increased eNOS phosphorylation and NO production by Ca2+-dependent phosphorylation of PKA, CaMKII, CaMKKß, and AMPK. Our findings provide evidence that CDCQ plays a pivotal role in the activity of eNOS and NO production, which is involved in the protection of endothelial dysfunction.

Role of metabolism by the human intestinal microflora in arbutin-induced cytotoxicity in HepG2 cell cultures.

A possible role for metabolism by the human intestinal microflora in arbutin-induced cytotoxicity was investigated using human hepatoma HepG2 cells. When the cytotoxic effects of arbutin and hydroquinone (HQ), a deglycosylated metabolite of arbutin, were compared, HQ was more toxic than arbutin. Incubation of arbutin with a human fecal preparation could produce HQ. Following incubation of arbutin with a human fecal preparation for metabolic activation, the reaction mixture was filter-sterilized to test its toxic effects on HepG2 cells. The mixture induced cytotoxicity in HepG2 cells in a concentration-dependent manner. In addition, the mixture considerably inhibited expression of Bcl-2 together with an increase in Bax expression. Likewise, activation stimulated cleavage of caspase-3 and production of reactive oxygen species in HepG2 cell cultures. Furthermore, induction of apoptosis by the intestinal microflora reaction mixture was confirmed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling assay. Taken together, these findings suggest that the human intestinal microflora is capable of metabolizing arbutin to HQ, which can induce apoptosis in mammalian cells.

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase.

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with N(G)-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease.

Novel role of Pin1 induction in type II collagen-mediated rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints and subsequent destruction of cartilage and bone. Inflammatory mediators such as PGs and proinflammatory cytokines contribute to RA progress. Pin1, a peptidyl prolyl isomerase, plays important pathophysiological roles in several diseases, including cancer and neurodegeneration. We found that both Pin1 and cyclooxygenase-2 (COX-2) were highly expressed in ankle tissues of type II collagen-induced RA mice. HTB-94 cells overexpressing Pin1 and primary cultured human chondrocytes showed increased basal expression of proinflammatory proteins (COX-2, inducible NO synthase, TNF-alpha, and IL-1beta). Site-directed mutagenesis revealed that Pin1-mediated transcriptional activation of COX-2 was coordinately regulated by NF-kappaB, CREB, and C/EBP. Gel shift, reporter gene, and Western blot analyses confirmed that NF-kappaB, CREB, and C/EBP were consistently activated in chondrocytes overexpressing Pin1. Treatment of RA mice with juglone, a chemical inhibitor of Pin1, significantly reduced RA progress and COX-2 expression in the ankle tissues. Moreover, juglone dose dependently decreased the basal COX-2 expression in primary cultured chondrocytes from RA patients. These results demonstrate that Pin1 induction during RA progress stimulates proinflammatory protein expression by activating NF-kappaB, CREB, and C/EBP, and suggest that Pin1 is a potential therapeutic target of RA.

Rutaecarpine Protects against Acetaminophen-Induced Acute Liver Injury in Mice by Activating Antioxidant Enzymes.

Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, is used to treat hypertension, postpartum hemorrhage, dysentery, and amenorrhea as a traditional medicine in Asia. We investigated the effect of rutaecarpine on acetaminophen-induced hepatotoxicity in mice. Rutaecarpine was administered orally daily for seven consecutive days, followed by intraperitoneal injection of acetaminophen in mice on day seven to induce hepatotoxicity. Rutaecarpine pretreatment significantly decreased acetaminophen-induced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) activities and hepatic malondialdehyde content and prevented acetaminophen-induced hepatic glutathione depletion. Furthermore, CYP2E1 expression was decreased by rutaecarpine pretreatment in a dose-dependent manner. Rutaecarpine pretreatment inhibited acetaminophen-induced expression of inflammatory cytokines by inhibiting NF-κB activation by JNK1/2. Also, rutaecarpine pretreatment promoted Nrf2-mediated activation of the antioxidant enzymes GCLC, HO-1, and NQO1. This indicates that the protective effect of rutaecarpine during acetaminophen-induced acute liver injury is mediated by the activation of antioxidant enzymes. Therefore, rutaecarpine has a protective effect of APAP-induced liver damage.

G protein-coupled estrogen receptor regulates the KLF2-dependent eNOS expression by activating of Ca2+ and EGFR signaling pathway in human endothelial cells.

G protein-coupled estrogen receptor (GPER) is important for maintaining normal blood vessel function by preventing endothelial cell dysfunction. It has been reported that G-1, an agonist of GPER, increases nitric oxide (NO) production through the phosphorylation of endothelial nitric oxide synthase (eNOS). However, the effect of GPER activation on eNOS expression has not been studied. Our results show that G-1 significantly increased the expression of eNOS and Kruppel-like factor 2 (KLF2) in human endothelial EA.hy926 cells. The individual silences of KLF2 and GPER attenuated G-1-induced eNOS expression. In addition, inhibition of the Gαq and Gßγ suppressed G-1-induced the expression of eNOS and KLF2 in EA.hy926 cells. Interestingly, these effects were similar in HUVECs. Furthermore, we found that GPER-mediated Ca2+ signaling increased the phosphorylation of CaMKKß, AMPK, and CaMKIIα in the cells. The phosphorylation of histone deacetylase 5 (HDAC5) by activation of AMPK and CaMKIIα increased the expression of eNOS via transcriptional activity of KLF2. We further demonstrate that GPER activation increased the phosphorylation of Src, EGFR, ERK5, and MEF2C and consequently induced the expression of eNOS and KLF2. Meanwhile, inhibition of ERK5 and HDAC5 suppressed the expression of eNOS and KLF2 induced by G-1 in the cells. These findings suggest that GPER provides a novel mechanism for understanding the regulation of eNOS expression and is an essential therapeutic target in preventing cardiovascular-related endothelial dysfunction.

Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells.

Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gbeta1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation, which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gbeta1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.

Upregulation of cyclooxygenase-2 by 4-nonylphenol is mediated through the cyclic amp response element activation pathway.

The organic compound nonylphenol (NP) belongs to the family of alkylphenols and is a product of industrial synthesis formed during phenol alkylation. Nonylphenol is considered to be an endocrine disruptor due to weak ability to mimic estrogen and subsequently to disrupt the natural balance of hormones in a given organism. Since the endocrine and immune systems share portions of common signaling pathways, it is conceivable that NP may also affect immune system functions. However, the influence of NP on inflammation and macrophages responsiveness to NP is unclear. Thus, the effects of NP were investigated on cyclooxygenase (COX)-2 expression in cultured macrophages. NP induced COX-2 protein and gene expression in murine macrophage RAW264.7 cells and enhanced COX-2 promoter activity and prostaglandin E(2) production. Transfection of RAW264.7 cells with hCOX-2 or various deletion and mutation promoter constructs revealed that the cyclic AMP response element (CRE) was the predominant mediator responsive to NP-induced effects. Moreover, transfection with pCRE-Luc plasmid followed by immunoblotting demonstrated that NP activated CRE sites and CRE binding protein (CREB) phosphorylation. NP also increased nuclear CREB accumulation and CREB binding to the COX-2 promoter. Phosphatidylinositol 3 (PI3)-kinase, Akt, and the mitogen-activated protein kinases (MAP kinases) p38 and JNK were also significantly activated by NP. Our data demonstrate that NP induces COX-2 expression through the PI3-kinase/Akt/MAP kinases/CRE pathway. These findings provide insight into the signal transduction pathways involved in the inflammatory responses induced by NP in macrophages.

Hepatoprotective Effects of Streptococcus thermophilus LM1012 in Mice Exposed to Air Pollutants.

In this study, we explored whether the use of Streptococcus thermophilus LM1012 (TL-LM1012) as a safe probiotic exerts hepatoprotective effects by suppressing oxidative stress and inflammation in vitro and alleviating aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) production in vivo. In a series of safety tests, TL-LM1012 was found to have a negative response to hemolysis and biogenic amines, as well as susceptibility to antibiotics. TL-LM1012 protected cell viability and suppressed cytotoxicity by inhibiting oxidative stress and induced heme oxygenase-1 and superoxide dismutase activity in a dose-dependent manner in diesel exhaust particulate matter (DEPM)-treated HepG2 cells. Moreover, proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß, were suppressed in DEPM-treated splenocytes. In DEPM-treated mice, oral administration of TL-LM1012 regulated AST, ALT, and LDH production in the serum after 14 days of treatment. These findings indicate that TL-LM1012, a safe probiotic, provides a potent preventive or therapeutic effect against liver disease caused by air pollution.