[Mechanism of total saponins of Panax japonicus against liver injury induced by acetaminophen in mice based on ERK/NF-κB/COX-2 signaling pathway].

This study investigated the effects and mechanisms of total saponins of Panax japonicus(TSPJ) against liver injury induced by acetaminophen(APAP). Male Kunming mice were randomly divided into a blank control group, TSPJ group(200 mg·kg~(-1), ig), model group, APAP+ TSPJ low-dose group(50 mg·kg~(-1), ig), APAP+ TSPJ medium-dose group(100 mg·kg~(-1), ig), APAP+ TSPJ high-dose group(200 mg·kg~(-1), ig), and APAP+ N-acetyl-L-cysteine group(200 mg·kg~(-1), ip). The administration group received the corresponding medications via ig or ip once a day for 14 consecutive days. After the last administration for one hour, except for the blank control group and TSPJ group, all groups of mice were given 500 mg·kg~(-1) APAP by gavage. After 24 hours, mouse serum and liver tissue were collected for serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), reactive oxygen species(ROS), tumor necrosis factor alpha(TNF-α), interleukin-1 beta(IL-1ß), cyclooxygenase-2(COX-2), IL-6, IL-4, IL-10, as well as lactate dehydrogenase(LDH), glutathione(GSH), superoxide dismutase(SOD), catalase(CAT), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and myeloperoxidase(MPO) liver tissue. Hematoxylin-eosin staining was used to observe the morphological changes of liver tissue. The mRNA expression levels of lymphocyte antigen 6G(Ly6G), galectin 3(Mac-2), TNF-α, IL-1ß, COX-2, IL-6, IL-4, and IL-10 in liver tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expression levels of Ly6G, Mac-2, extracellular regulated protein kinases(ERK), phosphorylated extracellular regulated protein kinases(p-ERK), COX-2, inhibitor of nuclear factor κB protein α(IκBα), phosphorylated inhibitor of nuclear factor κB protein α(p-IκBα), and nuclear factor-κB subunit p65(NF-κB p65) in cytosol and nucleus in liver tissue. The results manifested that TSPJ dramatically reduced liver coefficient, serum ALT, AST, ROS, TNF-α, IL-1ß, IL-6, and COX-2 levels, LDH, MPO, and MDA contents in liver tissue, and mRNA expressions of TNF-α, IL-1ß, and IL-6 in APAP-induced liver injury mice. It prominently elevated serum IL-4 and IL-10 levels, GSH, CAT, SOD, and T-AOC contents, and mRNA expressions of IL-4 and IL-10 in liver tissue, improved the degree of liver pathological damage, and suppressed neutrophil infiltration and macrophage recruitment in liver tissue. In addition, TSPJ lessened the mRNA and protein expressions of neutrophil marker Ly6G, macrophage marker Mac-2, and COX-2 in liver tissue, protein expressions of p-ERK, p-IκBα, and NF-κB p65 in nuclear, and p-ERK/ERK and p-IκBα/p-IκBα ratios and hoisted protein expression of NF-κB p65 in cytosol. These results suggest that TSPJ has a significant protective effect on APAP-induced liver injury in mice, and it can alleviate APAP-induced oxidative damage and inflammatory response. Its mechanism may be related to suppressing ERK/NF-κB/COX-2 signaling pathway activation, thus inhibiting inflammatory cell infiltration, cytokine production, and liver cell damage.

Puerarin alleviates depressive-like behaviors in high-fat diet-induced diabetic mice via modulating hippocampal GLP-1R/BDNF/TrkB signaling.

OBJECTIVE: Depression is one of the most common complications in patients with diabetes. Our previous study demonstrated puerarin, a dietary isoflavone, improved glucose homeostasis and ß-cell regeneration in high-fat diet (HFD)-induced diabetic mice. Here, we aim to evaluate the potential effect of puerarin on diabetes-induced depression. METHODS: The co-occurrence of diabetes and depression with related biochemical alterations were confirmed in HFD mice and db/db mice, respectively using behavioral analysis, ELISA and western blotting assay. Furthermore, impacts of puerarin on depression-related symptoms and pathological changes were investigated in HFD mice. RESULTS: The results showed that puerarin effectively alleviated the depression-like behaviors of HFD mice, down-regulated serum levels of corticosterone and IL-1ß, while up-regulated the content of 5-hydroxytryptamine. Simultaneously, puerarin increased the number of hippocampal neurons in HFD mice, and suppressed the apoptosis of neurons to protect the hippocampal neuroplasticity. GLP-1R expression in hippocampus of HFD mice was enhanced by puerarin, which subsequently activated AMPK, CREB and BDNF/TrkB signaling to improve neuroplasticity. Importantly, our data indicated that puerarin had an advantage over fluoxetine or metformin in treating diabetes-induced depression. CONCLUSION: Taken together, puerarin exerts anti-depressant-like effects on HFD diabetic mice, specifically by improving hippocampal neuroplasticity via GLP-1R/BDNF/TrkB signaling. Puerarin as a dietary supplement might be a potential candidate in intervention of diabetes with comorbid depression.

[Y-box-binding protein 1 mediates sorafenib resistance via the extracellular signal regulated-protein kinase pathway in hepatoma cells].

Objective: To investigate the effect and possible mechanism of Y-box-binding protein 1 (YB-1) on sorafenib resistance in hepatoma cells. Methods: Lentiviral vectors with YB-1 overexpression and knockdown were constructed, respectively, to stimulate human hepatoma cell lines (HepG2 and Huh7) alone or in combination with sorafenib.The overexpression part of the experiment was divided into four groups: overexpression control group (Lv-NC), YB-1 overexpression group (Lv-YB-1), overexpression control combined with sorafenib resistance group (Lv-NC+sorafenib), YB-1 overexpression combined with sorafenib resistance group (Lv-YB-1 + sorafenib). The knockdown part of the experiment was also divided into four groups: knockdown control group (Lv-shNC), YB-1 knockdown group (Lv-shYB-1), knockdown control combined with sorafenib resistance group (Lv-shNC + sorafenib), YB-1 knockdown combined with sorafenib resistance group (Lv-shYB-1 + sorafenib). The occurrence of cell apoptosis was detected by TUNEL. The protein expression levels of phosphorylated (p)-ERK and ERK, key proteins in the extracellular regulatory protein kinase (ERK) signaling pathway, were detected by Western blot and quantified by ImageJ software. Subcutaneous tumorigenesis experiments were performed in nude mice. The effect of YB-1 on the efficacy of sorafenib was verified in vivo. The comparison between the two sets of data was carried out by an independent sample t-test. One-way ANOVA was used for comparisons between the three groups of data above. Results: Sorafenib had accelerated the occurrence of apoptosis in hepatoma cells, while YB-1 overexpression had inhibited cell apoptosis, and at the same time also inhibited the apoptosis-accelerating impact of sorafenib. On the contrary, YB-1 knockdown accelerated cell apoptosis and amplified the induction effect of sorafenib on apoptosis. Furthermore, sorafenib resistance had down-regulated p-ERK levels (HepG2: Lv-NC 0.685 ± 0.143, Lv-NC + sorafenib 0.315 ± 0.168, P < 0.05; Huh7: Lv-NC 0.576 ± 0.078, Lv-NC + sorafenib 0.150 ± 0.131, P < 0.01), whereas YB-1 overexpression had inhibited sorafenib resistance p-ERK reduction (HepG2: Lv-NC + sorafenib 0.315 ± 0.168, Lv-YB-1 + sorafenib 0.688 ± 0.042, P < 0.05; Huh7: Lv-NC + sorafenib 0.150 ± 0.131, Lv-YB-1 + sorafenib 0.553 ± 0.041, P < 0.05). YB-1 knockdown further increased sorafenib-induced p-ERK downregulation (HepG2: Lv-shNC + sorafenib 0.911 ± 0.252, Lv-shYB-1 + sorafenib 0.500 ± 0.201, P < 0.05; Huh7: Lv-shNC + sorafenib 0.577 ± 0.082, Lv-shYB-1 + sorafenib 0.350 ± 0.143, P < 0.05), which was further verified in naked mice (Lv-shNC + sorafenib 0.812 ± 0.279, Lv-shYB-1 + sorafenib 0.352 ± 0.109, P < 0.05). Conclusion: YB-1 mediates the occurrence of sorafenib resistance via the ERK signaling pathway in hepatoma cells.

High expression level of CXCL1/GROα is linked to advanced stage and worse survival in uterine cervical cancer and facilitates tumor cell malignant processes.

BACKGROUND: CXCL1 belongs to a member of the ELR + CXC chemokine subgroups that also known as GRO-alpha. It has been recognized that several types of human cancers constitutively express CXCL1, which may serve as a crucial mediator involved in cancer development and metastasis via an autocrine and/or paracrine fashion. However, the expression pattern and clinical significance of CXCL1 in human uterine cervix cancer (UCC), as well as its roles and mechanisms in UCC tumor biology remains entirely unclear. METHODS: The expression and clinical significance of CXCL1 in UCC tissues was explored using immunohistochemistry and bioinformatics analyses. The expression and effects of CXCL1 in HeLa UCC cells were assessed using ELISA, CCK-8 and transwell assays. Western blotting experiments were performed to evaluate the potential mechanism of CXCL1 on malignant behaviors of HeLa UCC cells. RESULTS: The current study demonstrated that CXCL1 was expressed in HeLa UCC cells, PHM1-41 human immortalized cervical stromal cells, as well as cervical tissues, with UCC tissues having an evidently high level of CXCL1. This high level of CXCL1 in cancer tissues was notably related to poor clinical stages and worse survival probability, rather than tumor infiltration and patient age. In addition, CXCL1 expression was extremely correlated with CCL20, CXCL8 and CXCL3 cancer-associated chemokines expression. In vitro, the growth and migration abilities of HeLa cells were significantly enhanced in the presence of exogenous CXCL1. Gain-function assay revealed that CXCL1 overexpression significantly promoted growth and migration response in HeLa cells in both autocrine and paracrine manners. Finally, we found that CXCL1 overexpression in HeLa cells influenced the expression of ERK signal-related genes, and HeLa cell malignant behaviors derived from CXCL1 overexpression were further interrupted in the presence of the ERK1/2 blocker. CONCLUSION: Our findings demonstrate the potential roles of CXCL1 as a promoter and a novel understanding of the functional relationship between CXCL1 and the ERK signaling pathway in UCC.

The role and mechanism of miR-557 in inhibiting the differentiation and maturation of megakaryocytes in immune thrombocytopenia.

Specific miRNA in immune thrombocytopenia (ITP) was screened to explore its intervention effects and mechanisms in ITP. MTT assay and CFSE staining were used to detect the effects of gradient concentrations of thrombopoietin (TPO) on cell proliferation. Expressions of differentially expressed miRNAs were analysed via qRT-PCR in TPO-induced megakaryocytes and ITP plasma. Effects of miR-557 on cell physiological functions were examined by MTT and flow cytometry. Expressions of miR-557, apoptosis-associated genes and Akt/ERK pathways were detected by qRT-PCR and Western blot as needed. Multinucleation of TPO-induced megakaryocytes was determined by megakaryocyte colonies. The toe skin and intestinal bleeding of the ITP rat model were observed and evaluated. Effects of miR-557 on the numbers of platelets, megakaryocytes, and peripheral blood platelets and the expressions of CD4+ T cells, Treg cells, TGF-ß, IL-6 and miR-557 in the ITP rats were detected by Giemsa staining, flow cytometry, ELISA and qRT-PCR. MiR-557 was identified as an specific miRNA associated with both ITP and TPO treatment. MiR-557 inhibitor enhanced the physiological functions of TPO-induced megakaryocytes, while miR-557 mimic had the opposite effect. At the molecular level, the expressions of miR-557, cleaved Caspase-3 and Bax were further silenced by inhibitor, on the contrary, the expressions of bcl-2, p-Akt and p-ERK were upregulated. Animal experiments showed that, miR-557 inhibitor increased the numbers of platelets and megakaryocytes, and improved the symptoms of ITP model rats. Our results indicated that miR-557 inhibitor improved ITP by regulating apoptosis-related genes and cellular immunity and activating the Akt/ERK pathway.

Nimodipine attenuates dibutyl phthalate-induced learning and memory impairment in kun ming mice: An in vivo study based on bioinformatics analysis.

Dibutyl phthalate (DBP), a typical representative of phthalate esters (PAEs), is used as a plasticizer in various industrial applications and has been reported to be responsible for neurobehavioral changes. Despite mounting evidence showing that nimodipine (Nim) palys a neuropharmacological and psychopharmacological role in neurons, the attenuating effects of Nim on learning and memory impairment induced by DBP exposure remain unknown. Based on bioinformatics analysis we found that the biological processes affected by both DBP and Nim may involve the calcium signaling pathway, the MAPK signaling pathway and the apoptosis pathway. The results of an in vivo study confirmed that DBP affects the levels of Ca2+ -related proteins, up-regulates phosphorylated -ERK1/2 expression and results in hippocampal neuronal damage and apoptosis, whereas Nim as a Ca2+ antagonist, has a certain neuroprotective role to avoid these adverse effects. Our data suggest that Nim could be used to attenuate the learning and memory impairment in DBP-exposed mice, to down-regulate intracellular Ca2+ levels, subordinate the ERK1/2 pathway and attenuate apoptosis in hippocampal tissue.

[Fat1 inhibits cell proliferation via ERK signaling pathway in esophageal squamous cell carcinoma].

Objective: To clarify the mechanism of Fat1 on the proliferation of esophageal squamous cell carcinoma (ESCC). Methods: KYSE450 cells were transfected with Plko.1-puro-GFP-shRNA-Fat1 plasmid and real time polymerase chain reaction (PCR) was used to verify the efficiency of Fat1 knockdown. The effects of Fat1 and extracellular regulated protein kinase (ERK) pathway inhibitor U0126 on the proliferation of ESCC cells were detected by methyl thiazolyl tetrazolium (MTT). Colony formation assay was used to detect the colony formation ability. Cell cycle was detected by live cell imaging. Western blot was used to observe the level of target protein. Mouse xenograft assay was applied to detect the effect of Fat1 knockdown on KYSE450 cell tumor growth. Immunohistochemistry was used to detect the expressions of related proteins in tumor sections. Results: The efficiency of Fat1 knockdown was (77.1±6.9)% in Fat1 sh1 group and (77.7±7.1)% in Fat1sh2 group. Compared with the control group, the cell proliferation and the expression of p-ERK1/2 were significantly increased in Fat1 sh1 and Fat1sh2 group (P<0.05). After U0126 treatment, the effect of Fat1 knockdown on the proliferation of KYSE450 cells disappeared, and the expression of p-ERK1/2 in KYSE450 cells decreased to a level similar to that in the control group. The number of cell clones in the control group was (72±8), lower than (155±28) and (193±9) in the Fat1sh1 and Fat1sh2 groups, respectively (P<0.05). In KYSE450 cell, division time was shortened from 1 622±32 min in control group to 1 408±29 min in Fat1 sh1 group, the difference was statistically significant (P<0.05). Compared with the control group, the tumor volume of Fat1 knockdown group increased significantly. The tumor weight of control group and Fat1 knockdown group were (0.224±0.028) g and (1.532±0.196) g, respectively, at 4 weeks after inoculation, and the difference was statistically significant (P<0.05). Conclusion: Fat1 inhibits cell proliferation via ERK signaling in ESCC.

Long non coding RNAs involved in MAPK pathway mechanism mediates diabetic neuropathic pain.

Diabetes is the largest global epidemic of the 21st century, and the cost of diabetes and its complications comprise about 12% of global health expenditure. Diabetic neuropathy is the most common complication of diabetes, affecting up to 50% of patients over the course of their disease. Among them, 30%-50% develop neuropathic pain, which has typical symptoms that originate from the toes and progress to foot ulcers and seriously influence quality of life. The pathogenesis of diabetic neuropathic pain (DNP) is complicated and incompletely understood and there is no effective treatment except supportive treatment. Long noncoding RNAs (lncRNAs), a class of noncoding RNAs exceeding 200 nucleotides in length, have been shown to play key roles in fundamental cellular processes, and are considered to be potential targets for treatment. Recent research indicates that lncRNA is involved in the pathogenesis of DNP. Certain overexpressed lncRNAs can enhance the purinergic receptor-mediated neuropathic pain in peripheral ganglia and inflammatory cytokines are released due to receptors activated by adenosine triphosphate. In recent years, our laboratory also has been exploring the relationship and pathogenesis between lncRNAs and DNP. In this review, we focus on the recent progress in functional lncRNAs associated with DNP and investigate their roles related to respective receptors.

Dendrobium candidum aqueous extract attenuates isoproterenol-induced cardiac hypertrophy through the ERK signalling pathway.

CONTEXT: The pharmacological functions of Dendrobium candidum Wall. ex Lindl. (Orchidaceae) in cardiac hypertrophy remains unclear. OBJECTIVE: To evaluate whether D. candidum aqueous extract (DCAE) can attenuate experimental cardiac hypertrophy. MATERIALS AND METHODS: Cardiac hypertrophy in SD rats was induced by subcutaneously injection of isoproterenol (2 mg/kg), once a day for ten days. Rats were gavaged with DCAE (0.13 and 0.78 g/kg) daily for one month. At the end of treatment, measurement of left ventricular systolic pressure (LVSP), heart-to-body weight ratio (HW/BW), left ventricular/tibia length (LV/TL), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) levels, haematoxylin-eosin staining, and Masson's trichrome staining were conducted. In cultured H9c2 cells, DCAE (2 mg/mL) and U0126 (10 µM) were added 2 h before the isoproterenol (10 µM) stimulus. Phalloidin staining was used to evaluate cellular hypertrophy. The mRNA expression of ANP and BNP was measured by qRT-PCR. The expression of p-ERK was determined by immunoblotting. RESULTS: DCAE treatment significantly reduced the following indicators in vivo: (1) the LVSP (16%); (2) HW/BW (13%); (3) LV/TL (6%); (4) ANP (39%); (5) BNP (32%). In cultured H9c2 cells, phalloidin staining showed that DCAE relieved cellular hypertrophy (53% reduction). Furthermore, immunoblotting showed that DCAE can significantly inhibit p-ERK protein expression in vivo and in vitro (39% and 27% reduction, respectively). DISCUSSION AND CONCLUSIONS: DCAE prevents cardiac hypertrophy via ERK signalling pathway and has the potential for treatment of cardiac hypertrophy.

Blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes.

Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant widely used in various industrial fields. It is well known that mitochondria are the most vulnerable targets of heavy metals, but the key molecule/event that directly mediated mitochondrial dysfunction after Cr(VI) exposure is still unclear. The present study was aimed to explore whether Cr(VI) exposure could affect the mitochondrial fission/fusion process, and whether the related abnormal mitochondrial dynamics have been implicated in Cr(VI)-induced mitochondrial dysfunction. We found that the mitochondrial dysfunction caused by Cr(VI) exposure was characterized by decreased mitochondrial respiratory chain complex (MRCC) I/II activities and levels, collapsed mitochondrial membrane potential (MMP), depleted ATP, and increased reactive oxygen species (ROS) level. Cr(VI) induced abnormal mitochondrial fission/fusion events, the antioxidant Nacetyl-L-cysteine (NAC) restored the abnormal mitochondrial function as well as the fission/fusion dynamics. ROS was the up-stream regulator of extracellular regulated protein kinases (ERK) signaling, and the application of a specific ERK1/2 inhibitor PD98059 confirmed that activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction. We also demonstrated that treatment with dynamic-like protein 1 (DLP1)-siRNA rescued mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. We reached the conclusion that blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes, which may provide the new avenue for developing effective strategies to protect against Cr(VI)-induced hepatotoxicity.

Mel1c Mediated Monochromatic Light-Stimulated IGF-I Synthesis through the Intracellular Gαq/PKC/ERK Signaling Pathway.

Previous studies have demonstrated that monochromatic light affects plasma melatonin (MEL) levels, which in turn regulates hepatic insulin-like growth factor I (IGF-I) secretion via the Mel1c receptor. However, the intracellular signaling pathway initiated by Mel1c remains unclear. In this study, newly hatched broilers, including intact, sham operation, and pinealectomy groups, were exposed to either white (WL), red (RL), green (GL), or blue (BL) light for 14 days. Experiments in vivo showed that GL significantly promoted plasma MEL formation, which was accompanied by an increase in the MEL receptor, Mel1c, as well as phosphorylated extracellular regulated protein kinases (p-ERK1/2), and IGF-I expression in the liver, compared to the other light-treated groups. In contrast, this GL stimulation was attenuated by pinealectomy. Exogenous MEL elevated the hepatocellular IGF-I level, which is consistent with increases in cyclic adenosine monophosphate (cAMP), Gαq, phosphorylated protein kinase C (p-PKC), and p-ERK1/2 expression. However, the Mel1c selective antagonist prazosin suppressed the MEL-induced expression of IGF-I, Gαq, p-PKC, and p-ERK1/2, while the cAMP concentration was barely affected. In addition, pretreatment with Ym254890 (a Gαq inhibitor), Go9863 (a PKC inhibitor), and PD98059 (an ERK1/2 inhibitor) markedly attenuated MEL-stimulated IGF-I expression and p-ERK1/2 activity. These results indicate that Mel1c mediates monochromatic GL-stimulated IGF-I synthesis through intracellular Gαq/PKC/ERK signaling.

Cyclic hydrostatic pressure promotes uroplakin expression in human urothelial cells through activation of ERK1/2 signaling.

OBJECTIVES: To investigate the effect of cyclic hydrostatic pressure on the expression of uroplakins and the role of extracellular regulated protein kinases 1/2 (ERK1/2) in the hydrostatic pressure-induced uroplakin expression of human urothelial cells (UCs). METHODS: Human UCs were seeded into a cell culture flask and subjected to cyclic hydrodynamic pressures for 24 h. Pressure parameters were set as follows: static, 100 cm H2O, 200 cm H2O and 300 cm H2O pressure. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect the expression of uroplakins. The role of the ERK1/2 was investigated using ERK1/2 inhibitor. RESULTS: Compared with the 0 cm H2O control group, 200 cm H2O hydrostatic pressure significantly increased the expression of uroplakins, however, 100 cm and 300 cm pressures could not promote uroplakin expression. Hence, ERK1/2 expression was also detected under 200 cm H2O hydrostatic pressure. Western blot showed that 200 cm H2O pressure promoted the expression of ERK1/2. ERK1/2 inhibitor decreased the pressure-induced ERK1/2 activivation and uroplakin expression. CONCLUSIONS: Cyclic hydrostatic pressure increases the expression of uroplakins via activating ERK1/2 signaling pathway in human UCs, and 200 cm H2O pressure may be an optimal stress parameter to promote the uroplakin expression.

[Effects of glycyrrhizic acid on ERK1/2 and p38 MAPK signaling pathway in a murine model of asthma].

Objective: To explore the effects of glycyrrhizic acid (GA) on Extracellular regulated protein kinases (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) signaling pathways in a murine model of asthma. Methods: Sixty female BALB/c mice were randomly divided into 6 groups (n=10 each): a control group, an asthmatic group, two treatment groups with low and high doses of GA, U0126 group and SB203580 group. Within 24 hours after the last OVA challenge, histological studies of lung were conducted with the hematoxylin and eosin staining (HE) and alcian blue-periodic acid-Schiff (AB-PAS), the relative protein expression of ERK1/2 and p38 MAPK were detected by immunohistochemistry and Western blotting in vivo. CD4(+) T cells were purified from spleens of OVA-sensitized and challenged mice by using the Mouse CD4 Cell Positive Isolation Kit and incubated with anti-CD3 mAb (1 µg/ml) in the presence of various concentrations of GA (10 and 100 µg/ml), U0126 (10 µmol/L) or SB203580(10 µmol/L). After 72 h of incubation, the relative protein expression of ERK1/2 and p38 MAPK of CD4(+) T cells were detected by Western blotting in vitro. Results: The asthmatic mice induced infiltration of inflammatory cells around airways and blood vessels, airway goblet cell hyperplasia and mucus production. Administration of GA at a dose of 100 mg/kg, U0126 or SB203580 significantly reduced the infiltration of inflammatory cells in the peribronchial areas and goblet cell hyperplasia compared with the asthmatic mice. The protein expressions of p-ERK1/2 were lower in GA at a dose of 100 mg/kg (0.090±0.022) and U0126 group (0.072±0.017) than those in asthmatic group (0.143±0.022) (all P<0.05). The protein expressions of p-p38 MAPK were lower in GA at a dose of 100 mg/kg (0.072±0.019) and SB203580 group (0.061±0.015) than those in asthmatic group (0.121±0.022) (all P<0.05) by immunohistochemistry. Compared with asthmatic group (0.783±0.133, 0.649±0.095), the protein expressions of p-ERK1/2 and p-p38 MAPK in GA at a high dose group (0.385±0.186, 0.275±0.089) and in U0126 group (0.117±0.051) or in SB203580 group (0.108±0.043) were decreased by Western blotting (all P<0.05). The expressions of p-ERK1/2 in CD4(+) T cells after 72 h incubation were lower in 100 µg/ml concentrations of GA (0.579±0.184) and group U0126 (0.249±0.082) and the expressions of p-p38 MAPK were much lower in 100 µg/ml concentrations of GA (0.445±0.081) and group SB203580 (0.249±0.082) compared with those in group CD3 (1.028±0.147, 0.902±0.107) (all P<0.05). Conclusion: ERK1/2 and p38 MAPK signaling pathways are activated in asthmatic mice and GA may negative regulate this activation.

Simulated physiological stretch increases expression of extracellular matrix proteins in human bladder smooth muscle cells via integrin α4/αv-FAK-ERK1/2 signaling pathway.

OBJECTIVES: To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). METHODS: HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. RESULTS: The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. CONCLUSION: Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.

Tetramethylpyrazine Protects Bone Marrow-Derived Mesenchymal Stem Cells against Hydrogen Peroxide-Induced Apoptosis through PI3K/Akt and ERK1/2 Pathways.

Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation is one of the new therapeutic strategies for treating ischemic stroke. However, the poor survival rate of transplanted BMSCs in ischemic tissue limits the therapeutic efficacy of this approach. Oxidative stress is a major mechanism underlying the pathogenesis of brain ischemia and has a negative impact on the survival of transplanted BMSCs. Tetramethylpyrazine (TMP) has been reported to possess potent antioxidant activity. In the present study, we aimed to investigate the protective effects of TMP pretreatment on BMSCs survival of hydrogen peroxide (H2O2)-induced apoptosis in vitro and to elucidate the potential antiapoptotic mechanisms of TMP pretreatment on BMSCs. BMSCs were pretreated with TMP (10, 25, 50, 100, and 200 µmol/L) for 24 h and then exposed to 500 µmol/L of H2O2 for 24 h. We found that TMP pretreatment significantly increased cell viability and decreased cell apoptosis and intracellular reactive oxygen species (ROS) generation. Furthermore, the protective effects of TMP were related to increased Bcl-2 expression, attenuated Bax expression, and enhanced levels of phosphorylated Akt (p-Akt) and extracellular regulated protein kinases1/2 (p-ERK1/2). Further studies found that these beneficial effects of TMP were significantly blocked by wortmannin (an inhibitor of phosphoinositide-3 kinase (PI3K)) or PD98059 (an inhibitor of ERK1/2). In conclusion, our results confirm that TMP protects BMSCs against H2O2-induced apoptosis by regulating the PI3K/Akt and ERK1/2 signaling pathways, suggesting that TMP may be used in combination with BMSCs to improve cell survival for the treatment of ischemic stroke.

Diallyl disulphide inhibits apolipoprotein(a) expression in HepG2 cells through the MEK1-ERK1/2-ELK-1 pathway.

BACKGROUND: Lipoprotein(a) [LP(a)] is implicated as a common and independent risk factor for cardiovascular diseases. The therapeutic options currently available for reducing plasma LP(a) concentrations are limited. Diallyl disulphide (DADS), the main component of garlic, regulates lipid metabolism in hepatocytes and adipocytes through ERK1/2 signalling. This study aimed to assess the effect of DADS on apolipoprotein(a) [apo(a)] in HepG2 cells. We also determined the effects of DADS on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms. METHODS: We examined the role of DADS on apo(a) expression in HepG2 cells by treating cell with different concentrations of DADS (10, 20, 40 and 80 µg/mL) for 24 h or treating cells with 40 µg/mL DADS for 0, 6, 12, 24 and 48 h. Then we used quantitative real-time PCR to analysis apo(a) mRNA levels, used Western blot to analysis apo(a) protein levels and used enzyme-linked immunosorbent assay to test apo(a) secreted levels. To farther determined the role of DADS, we applied Transfection of small interfering RNA to knockdown ELK-1levels and applied PD98059, a specific inhibitor of ERK1/2, to block ERK1/2 signal. RESULTS: The results show DADS inhibited apo(a) at both the mRNA and protein levels in HepG2 cells in a dose-dependent manner. DADS-mediated inhibition of apoa(a) expression in HepG2 cells was attenuated when the cells were cultured in medium containing PD98059 (ERK1/2 inhibitor) or were transfected with siRNAs against MEK1 or ELK-1. Overexpression of apo(a) yielded similar results. CONCLUSIONS: This study reveals that DADS can downregulate apo(a) expression in a dose-dependent manner via the MEK-ERK12-ELK-1 pathway.

[Effects of overexpression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells and its mechanism].

Objective: To explore the effect and mechanism of over-expression of miR-17-92 gene cluster on the biological characteristics of prostate cancer cells. Methods: DU145 cells were transfected with miR-17-92 gene expression plasmid and clones with stable ectopic miR-17-92 overexpression were established. The cell viabilities of DU145-17-92 and DU145-control cells were monitored by xCELLigence system. Cell proliferation and apoptosis were analyzed by Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Cell cycle was detected by flow cytometry. Expression levels of proteins involved in apoptosis and Akt pathway were determined by western blotting. Results: xCELLigence RTCA array data showed that the growth rate of DU145-17-92 cells was significantly higher than that of DU145-control cells after 24 h of seeding (P<0.01). The Ki-67-positive rates of the DU145-control group at 24, 48 and 72 hours were (56.57±1.68)%, (85.48±0.26)% and (90.85±2.08)%, respectively. While the Ki-67 positive rates of the DU145-17-92 group at the desired time points were (73.64±0.68)%, (93.43±1.23)% and (97.36±0.86)%, respectively, with a statistically significant difference at 24 hours (P<0.01). The percentages of apoptotic cells of the DU145-control group at 24, 48 and 72 hours were (6.76±0.09)%, (14.51±0.86)% and (20.73±1.64)%, respectively, while the apoptotic percentages of the DU145-17-92 group were (1.86±0.15)%, (7.90±0.40)% and (4.92±0.48)%, respectively. The percentages of apoptotic cells of the DU145-control group at different time were significantly higher than those of DU145-17-92 group (P<0.01 for all). The result of western blotting showed that the protein expression levels of Bcl-2 interacting mediator of cell death (BIM) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in DU145-control cells were 0.83±0.00 and 0.91±0.00, respectively, significantly higher than 0.16±0.00 and 0.13±0.00 of DU145-17-92 cells (both P<0.01). Overexpression of miR17-92 induced the phosphorylation of protein kinase B (Akt) at Ser473 while no appreciable effect on the phosphorylation of Akt at Thr308. The phosphorylated level of extracellular regulated protein kinases (ERK) in DU145-control cells was 0.21±0.01, significantly lower than 0.72±0.01 of DU145-17-92 cells (P<0.01). Conclusions: Overexpression of miR-17-92 gene plays a pivotal role in growth, proliferation, apoptosis and cell cycle of DU145 cells through down-regulation of apoptotic protein BIM and tumor suppressor PTEN and activation of Akt and ERK signaling pathway.

[Glabridin attenuates MPTP-induced parkinson disease by inhibiting extracellular regulated protein kinases signaling pathway].

Objective: To investigate whether Glabridin had a beneficial effect on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine(MPTP) induced parkinson disease (PD) in mice, and explore the possible underlying mechanisms. Methods: Forty C57BL/6N mice were randomly assigned into control group, MPTP group, Glabridin therapy(MPTP+ GLA)group, Levodopa therapy(MPTP+ LD)group, with 10 in each group. PD model was induced by intraperitoneal administration of MPTP(20 mg/kg). The mice in MPTP+ GLA group, MPTP+ LD group and control group were gavaged by glabridin (50 mg/kg), levodopa (40 mg/kg), and equal volume of normal saline, respectively. The behavioral changes of each group were observed and Y-type electric maze test was performed. The levels of tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18) were assayed by enzyme-linked immunosorbent assay (ELISA). Malondialdehyde (MDA), and superoxide dismutase (SOD) were analyzed. The protein expression of tyrosine hydroxylase (TH) and extracellular signal regulated protein kinase (ERK) protein expression of hippocampus tissue was detected using immunohistochemical techniques. TH and pERK protein expression of hippocampus tissue were detected by Western blotting. Results: Mice in MPTP group showed typical behavior of PD, and the ability of learning and memory was significantly lower than those in the control group. Compared with MPTP group, the expressions of TNF-α and IL-18 were suppressed by GLA in hippocampus[TNF-α(µg/L): 84.04±18.66 vs 106.53±28.54; IL-18(µg/L): 42.34±6.01 vs 58.42±8.39]. The levels of MDA in hippocampus were down-regulated significantly in groups administrated with GLA[MDA(nmol/mgprot): 2.64±0.52vs 3.78±0.31], while the SOD level increased after GLA administration[SOD(U/mgprot): 93.45±9.59 vs 77.83±8.98]. The results of immunohistochemistry showed the expression of TH protein in MPTP group was significantly decreased compared with that in control group, while the p-ERK protein in MPTP group was significantly increased. In MPTP+ GLA group and MPTP+ LD group, the expression of TH protein was significantly higher than that in MPTP group, and the expression of p-ERK protein was significantly lower than that in MPTP group. Western blot results showed that compared with control group, the expression of TH was significantly decreased, and p-ERK protein in hippocampus was significantly higherin MPTP group. The expression of TH protein was significantly higher in MPTP+ GLA group than that in MPTP group, while the expression of p-ERK protein were inhibited by GLA in MPTP-induced PD mice. Conclusion: Traditional Chinese medicine glabridin can protect the learning and memory ability of PD mice induced by MPTP by inhibition of the ERK signal pathway, antioxidation and reduction of inflammation.

Exposure to bisphenol-A affects fear memory and histone acetylation of the hippocampus in adult mice.

Bisphenol-A (BPA), an environmental endocrine disruptor, has been reported to possess weak estrogenic, anti-estrogenic, and anti-androgen properties. Previous evidence indicates that perinatal exposure to low levels of BPA affects anxiety-like and cognitive behaviors in adult rodents. The present study aims to investigate the effect of BPA on emotional memory using the contextual fear conditioning of male mice in adulthood exposed to BPA for 90days. The results indicated that exposure to BPA increased the freezing time 1h and 24h after fear conditioning training. Furthermore, western blot analyses showed that BPA exposure decreased the level of N-methyl-d-aspartic acid (NMDA) receptor subunit NR1 and increased the expression of histone deacetylase 2 (HDAC2) before fear conditioning training in the hippocampus of male mice. One and twenty-four hours after fear conditioning training, BPA enhanced the changes of the expressions of NR1, phosphorylated extracellular regulated protein kinases (ERK1/2), and histone acetylation induced by contextual fear conditioning in the hippocampus. These results suggest that long term exposure to BPA enhanced fear memory by the concomitant increased level of NMDA receptor and/or the enhanced histone acetylation in the hippocampus, which may be associated with activation of ERK1/2 signaling pathway.

Optimized new Shengmai powder inhibits myocardial fibrosis in heart failure by regulating the rat sarcoma/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase kinase/extracellular regulated protein kinases signaling pathway.

OBJECTIVE: Exploring the effect of Optimized New Shengmai powder (, ONSMP) on myocardial fibrosis in heart failure (HF) based on rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway. METHODS: Randomized 70 Sprague-Dawley rats into sham (n = 10) and operation (n = 60) groups, then established the HF rat by ligating the left anterior descending branch of the coronary artery. We randomly divided the operation group rats into the model, ONSMP [including low (L), medium (M), and high (H) dose], and enalapril groups. After the 4-week drug intervention, echocardiography examines the cardiac function and calculates the ratios of the whole/left heart to the rat's body weight. Finally, we observed the degree of myocardial fibrosis by pathological sections, determined myocardium collagen (COL) I and COL Ⅲ content by enzyme-linked immunosorbent assay, detected the mRNA levels of COL I, COL Ⅲ, α-smooth muscle actin (α-SMA), and c-Fos proto-oncogene (c-Fos) by universal real-time, and detected the protein expression of p-RAS, p-RAF, p-MEK1/2, p-ERK1/2, p-ETS-like-1 transcription factor (p-ELK1), p-c-Fos, α-SMA, COL I, and COL Ⅲ by Western blot. RESULTS: ONSMP can effectively improve HF rat's cardiac function, decrease cardiac organ coefficient, COL volume fraction, and COL I/Ⅲ content, down-regulate the mRNA of COL I/Ⅲ, α-SMA and c-Fos, and the protein of p-RAS, p-RAF, p-MEK1/ 2, p-ERK1/2, p-ELK1, c-Fos, COL Ⅰ/Ⅲ, and α-SMA. CONCLUSIONS: ONSMP can effectively reduce myocardial fibrosis in HF rats, and the mechanism may be related to the inhibition of the RAS/RAF/MEK/ERK signaling pathway.