Ratio Of p-ERK1 Research Articles

Determination of the signaling proteins responsible for the antioxidant potential of the Yishenhuoxue formula for treating asthenospermia in rats.

Asthenospermia is a predominant cause of male infertility, and antioxidant supplements can be effective in treating asthenospermia. We demonstrate the antioxidant potential of traditional Chinese medicine, the Yishenhuoxue (YSHX) formula, in treating polyglycosides of Tripterygium wilfordii (GTW)-induced asthenospermia in rats. Fifty male rats were randomly divided into the normal, model, and treatment groups. HE staining was used to evaluate the improvement of spermatogenic function of rats, and TBA reaction, qRT-PCR, Western Blot and other methods were used to determine the changes of oxidative stress indicators and to evaluate the improvement of antioxidant capacity of rats by YSHX. Comparison with the model group showed significant improvement in pathological damage caused by GTW to seminiferous tubules. MDA and NO content in rat testes decreased, especially in middle- and high-dosage groups. No significant changes were observed in SOD and CAT activity or mRNA expression. GSH-Px activity and GSH mRNA expression were significantly higher in the low-dosage group than in the model group. Compared to the model group, GR activity was significantly lower in the middle and high dosage groups, while the mRNA expression was higher. The PKC-beta level increased, while p-ERK1/2, NF-κB, and the ratio of p-ERK1/2*(ERK1/2)-1 decreased significantly in the treatment groups. Therefore, YSHX can alleviate GTW-induced testicular damage, enhance GSH-Px activity, regulate GSH redox cycling, and mitigate oxidative stress injury. Furthermore, YSHX can promote PKC-beta expression and inhibit the phosphorylation of ERK1/2 and NF-κB. Using YSHX may be an effective way to increase sperm motility via the PKC-ERK1/2-NF-ĸB axis.

Integrated network pharmacology and experimental verification to explore the mechanism of Sangqi Qingxuan formula against hypertensive vascular remodeling

To investigate the bioactive components of Sangqi Qingxuan formula (SQQX), predict the pharmacological targets, and explore the mechanism of hypertensive vascular remodeling (HVR). Network pharmacology was adopted to predict how SQQX acts in HVR. The effectiveness was assessed by blood pressure measurements and pathological morphology observation based on a spontaneously hypertensive rat model, while the mechanism of SQQX on HVR was validated by immunohistochemistry (IHC) and western blot (WB) according to the results of network pharmacology. There were 130 bioactive components of SQQX and 231 drug targets predicted by the Traditional Chinese Medicine Systems Pharmacology Database. Subsequently, 181 common targets were identified for SQQX against HVR, with TP53, MAPK1, and AKT1 as the core targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses was employed to identify the top 20 enriched functions and the top 20 pathways ( P < .01). Finally, the key role of the ERK/MAPK signaling pathway in HVR was determined. The in vivo results suggested that SQQX reduced systolic blood pressure and increased the ratio of thoracic aortic wall thickness to lumen diameter. Additionally, compared with the model group, SQQX increased the expression of smooth muscle 22 alpha (IHC: P < .001; WB: P < .05) and decreased the expression of osteopontin (IHC: P < .001; WB: P < .05), ERK1/2 (IHC: P < .001; WB: ERK1 & ERK2, all P < .05), p-ERK1/2 (IHC: P < .001; WB: ERK1 & ERK2, all P < .05), and the ratio of p-ERK1/2 to ERK1/2 protein (IHC: P < .001). SQQX, which has multiple bioactive ingredients and potential targets, is an effective treatment for HVR. The mechanism of antihypertensive and vascular protection may be related to the inhibition of phenotypic transformation of vascular smooth muscle cells and the ERK/MAPK signaling pathway.

Effects and molecular mechanism of histone deacetylase 6 inhibitor Tubastatin A on the prolifera- tion and movement of human skin fibroblasts

Objective: To explore the effects and possible molecular mechanism of histone deacetylase 6 (HDAC6) inhibitor Tubastatin A on the proliferation and movement of human skin fibroblasts (HSFs). Methods: The experimental research method was used. HSFs in logarithmic growth phase were taken and divided into negative control group, 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group according to the random number table. The HSFs in negative control group were added with Dulbecco's modified eagle medium with the final volume fraction of 0.1% dimethyl sulfoxide (hereinafter referred to as the complete medium), and the other three groups were added with the complete medium with the corresponding final molarity of Tubastatin A. After 24 h of conventional culture, the cell proliferation activity was detected using cell counting kit 8 (CCK-8) method and 5-ethynyl-2'-deoxyuridine (EdU) staining; the range of motion of cells within 3 h was observed under the living cell workstation, and the curve movement velocity of the cells was calculated. The protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) were detected by Western blotting, and the ratio of p-ERK1/2 to ERK1/2 was calculated to represent the activity of ERK1/2. The sample number in cell proliferation activity detection with CCK-8 method was 6, while the sample numbers in other experiments were 3. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: After 24 h of culture, CCK-8 method and EdU staining showed that compared with negative control group, the cell proliferation activities in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were significantly decreased (P<0.01). After 24 h of culture, CCK-8 method showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activity in 10 μmol/L Tubastatin A group was significantly decreased (P<0.05); EdU staining showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activities in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were significantly decreased (P<0.05 or P<0.01). Within 3 h of observation, the ranges of cell motion in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were obviously reduced compared with that in negative control group. Within 3 h of observation, the curve movement velocity of cells in negative control group was (0.780±0.028) μm/min, which was obviously faster than (0.594±0.023), (0.469±0.028), and (0.391±0.021) μm/min of 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 1 μmol/L Tubastatin A group was obviously faster than those in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 5 μmol/L Tubastatin A group was obviously faster than that in 10 μmol/L Tubastatin A group (P<0.05). After 24 h of culture, compared with negative control group, the activities of ERK1/2 of cells in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 1 μmol/L Tubastatin A group, the activities of ERK1/2 of cells in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 5 μmol/L Tubastatin A group, the activity of ERK1/2 of cells in 10 μmol/L Tubastatin A group was decreased significantly (P<0.05). Conclusions: HDAC6 inhibitor Tubastatin A may mediate the inhibitory effect on proliferation and movement of HSFs by inhibiting the activity of ERK1/2.

Silencing Aurora-kinase-A (AURKA) reinforced the sensitivity of diffuse large B-cell lymphoma cells to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) via suppressing β-Catenin and RAS-extracellular signal-regulated protein kinase (ERK1/2) pathway

ABSTRACT The therapeutic effects of standard cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) therapy for prevalent lymphoma diffuse large B-cell lymphoma (DLBC, DLBCL) still require improvement. Cancer-related aurora-kinase-A (AURKA) may work as a target for DLBCL treatment and its effect on CHOP therapy was investigated in the present study. The Gene Expression Profiling Interactive Analysis 2 was applied to analyze AURKA expression in DLBC tumor tissues and normal lymphoid tissues. The DLBCL tissues and normal lymphoid tissues were obtained from the DLBCL patients and healthy volunteers. Clinic data of patients were recorded, and AURKA expression in tissues and cells was detected and analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. After AURKA in DLBCL cells was silenced or overexpressed and treated with CHOP, viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Expressions of AURKA, β-Catenin, phosphorylated (p)-β-Catenin, extracellular signal-regulated protein kinase (ERK1/2), p-ERK1/2 and RAS were detected using qRT-PCR and Western blot. AURKA was highly expressed in DLBCL tissues and cells. Silencing AURKA inhibited AURKA expression and viability, but promoted apoptosis of DLBCL cells. CHOP had no obvious effects on AURKA expression while reducing viability and promoting apoptosis of DLBCL cells. Silencing AURKA enhanced the effects of CHOP on cell apoptosis of DLBCL cells by inhibiting the expressions of RAS and β-Catenin as well as the ratio of p-ERK1/2/ERK1/2 and promoting the ratio of p-β-Catenin/β-Catenin. Silencing AURKA reinforced the therapeutic effects of CHOP on reducing viability and promoting apoptosis of DLBCL cell via repressing β-Catenin and RAS-ERK1/2 pathway.

The effects of YKL-40 on angiogenic potential of HUVECs are partly mediated by syndecan-4.

Background: YKL-40, a secreted glycoprotein, has a role in promoting tumor angiogenesis through syndecan-1 receptor. Syndecan-4 is a member of syndecan family. However, the effects of YKL-40 on migration and tube formation of human umbilical vein cells (HUVECs) mediated by syndecan-4 receptor are unknown.Materials and methods: HUVECs were transfected with lentivirus encoding syndecan-4 short hairpin (sh) RNAs (lenti-synd4 shRNAs) and the efficiency of transfection was measured using qRT-PCR and western blotting. The effects of recombinant protein of YKL-40 on migration and angiogenesis of HUVECs adjusted by syndecan-4 were determined by wound healing and tube formation assay. The expressions of protein kinase Cα (PKCα) and extracellular signal regulated kinases (ERKs) 1 and 2 (ERK1/2) in HUVECs were measured using western blotting.Results: The mRNA and protein expression of syndecan-4 were significantly decreased in HUVECs successfully transfected with lenti-synd4 shRNAs. Lenti-synd4 shRNAs remarkably inhibited the migration and tube formation of HUVECs stimulated by recombinant protein of YKL-40. The levels of PKCα and ratio of p-ERK1/2 to ERK1/2 in HUVECs were also decreased by down-regulating syndecan-4.Conclusion: The effects of YKL-40 on migration and tube formation of HUVECs are partly inhibited by knock-downing syndecan-4 through suppressing PKCα and ERK1/2 signaling pathways.

Effects Of Hypoxic Training On Gdnf In The Cortex Of Obese Rats

Glial cell-derived neurotrophic factor (GDNF) is an important indicator that represent the function of nervous system. Exercise and hypoxic environment both can reduce weight and improve nervous system function in obese rats. But the mechanism by which hypoxic training affects the nervous system is unclear. PURPOSE: To determine the potential mechanisms of hypoxic training on GDNF in the cortex of obese rats. METHODS: Forty SD obese rats were assigned into one of the following groups (n=10 each): normal oxygen control group (NC), normal oxygen training group (NT), hypoxic control group (HC) and hypoxic training group (HT). Rats were kept continuous exercise 1 hour per day, 6 days per week for 4 weeks on a rat treadmill at a speed of 25 m/min in normoxic conditions or 20 m/min in hypoxic conditions (13.6% O2). After 4 weeks, the expressions of GDNF in the cortex were detected by quantitative fluorescent PCR and Western blotting. The level of inflammatory cytokine interleukin 1β (IL-1β) and interleukin 6 (IL-6) in the cortex were measured by ELISA. The phosphorylation of ERK1/2 and JNK of cortex were detected by Western blotting. RESULTS: 1) The results of qPCR showed that the expression of GDNF in the cortex of HT were higher than NT and HC (2.75±0.69 vs. 1.66±0.88 and 1.06±0.45, p<0.05). 2) The results of Western blotting showed that the expression of GDNF in the cortex of HT were higher than NT and HC (489±23.58 vs. 337±46.22 and 387±36.98, p<0.05). 3) The results of phosphorylation of ERK1/2 and JNK showed that the ratio of ERK1/2 and JNK to β-actin integral optical density in the cortex showed no significant difference in all groups(p>0.05), while compared with NC, the ratio of p-ERK1/2 to ERK1/2 and p-JNK to JNK integral optical density in HC, NT and HT were significantly increased (P < 0.05, P < 0.05, P < 0.01). 4) The results of ELISA showed that IL-1β and IL-6 serum levels in HC, NT and HT were decreased compared with NC (19.28±1.41,19.99±1.26 and 17.8±1.5 vs. 27.15±2.27, p<0.01; 53.82±2.54,57.53±1.96 and 52.22±1.4 vs. 66.87±2.73, p<0.01). CONCLUSION: Hypoxic training can increase the expression of GDNF in the cortex of obese rats and improve the function of the central nervous system. Its mechanisms may relate to the activation of ERK1/2 and JNK signaling pathways and inhibit inflammatory cytokine IL-1β and IL-6 level in the cortex.

The mechanism of irbesartan against diabetes induced myocardial fibrosis in rat model

To observe the protective effect of irbesartan on myocardial fibrosis in diabetic rats, and analyze the role of extracellular signal-regulated kinase (ERK) pathway in this protection. Thirty two male SD rats were randomly divided into two groups:normal control group (CON, n=10), experimental group (n=22). Twenty diabetic rats which had modelled successfully were randomly divided into two groups:diabetic group (DM, n=10), irbesartan + DM group (Ir+DM, n=10). After 8 weeks, fasting blood glucose (FBG) level, body weight (BW), the ratio of heart weight/body weight (H/B) and left ventricular weight index (LVWI) were measured. The myocardial morphological and fibrotic changes were observed by Masson staining. Col I and col Ⅲ contents were evaluated using ELISA method, and the protein expressions of ERK1/2, p-ERK1/2 in heart tissue were tested by Western blot. Compared with CON group, in diabetic rats, the levels of FBG, H/B and LVWI were increased while BW was decreased. The contents of col I and col Ⅲ were increased as well as the protein expression of p-ERK1/2 and the ratio of p-ERK1/2/ERK1/2(P<0.05,P<0.01), which had the statistic differences, while ERK1/2 protein expression was not changed. Masson staining showed myocardial collagen was increased, arranged in disorder and uneven distribution. However, in Ir+ DM group, BW was increased obviously, H/B, LVWI, the contents of col I and col Ⅲ were decreased significantly (P<0.05,P<0.01), p-ERK1/2 protein expression and the ratio of p-ERK1/2/ERK1/2 were decreased (P<0.01), which had the statistic differences. Meanwhile myocardial morphology was improved significantly. Diabetes can induce the happening of myocardial fibrosis, and irbesartan can induce the damage of myocardial fibrosis through inhibitting the activation of ERK.

Antidepressant-like activity of Chaihu-Shugan-San aqueous extract in rats and its possible mechanism.

Background:Chaihu-Shugan-San (CHSGS), a traditional Chinese medicinal formula, is commonly used for the treatment of depression in China. However, the molecular mechanism underlying its antidepressant action is unknown.Objective:The objective of this study is to evaluate the antidepressant-like effects of CHSGS and further explore the possible molecular mechanism implicated in its actions.Materials and Methods:The rats were randomly divided into four groups: The normal control group, the model control group, the CHSGS group and the fluoxetine control group. The antidepressant-like effects of CHSGS aqueous extract were assessed in rats exposed to chronic mild stress (CMS) using the open-field test and sucrose water consumption test, its underlying mechanism of anti-depression was explored by determining the effect of CHSGS on the extracellular signal-regulated kinase (ERK) and phospho-ERK (P-ERK) in the hippocampus using western blot. The aqueous extract of CHSGS at a dose of standard (5.9 g/kg·d) was administered intragastrically for 14 days during the CMS model while the fluoxetine control group was given at the same time using fluoxetine hydrochloride (1.8 mg/kg·d).Results:The stressed rats demonstrated decreased locomotor activity in open field test and reduction in sucrose consumption and decreased levels of P-ERK1/2 and the ratio of P-ERK1/2 to total ERK1/2 in the hippocampus. CHSGS alleviated the depressive-like behaviors and increased levels of P-ERK1/2 and the ratio of P-ERK1/2 to total ERK1/2 in stressed rats as well as fluoxetine.Conclusion:In summary, these results suggest that CHSGS aqueous extract possesses an antidepressant-like activity in CMS induced depression model rats, which might be mediated, at least in part, by reversing the stress-induced disruption of ERK activity.

Mechanisms of Periodic Acceleration Induced Endothelial Nitric Oxide Synthase (eNOS) Expression and Upregulation Using an In Vitro Human Aortic Endothelial Cell Model

Following severe injury in cases like ischemia reperfusion, recovery and tissue repair are compromised due to microvascular failure. Nitric oxide (NO) has been shown to have both vasodilatory and anti-inflammatory effects, and to enhance recovery and tissue repair by increasing microcirculation. Therefore, techniques that induce endothelial derived nitric oxide (eNO) may provide a suitable supplemental treatment following injury. Periodic acceleration (pGz) applied to the supine positioned body in a repetitive head-foot direction increases pulsatile shear stress to the vascular endothelium. pGz provides cytoprotection in part because it increases expression of eNOS and eNOS phosphorylation (p-eNOS) in the heart and vasculature in models of whole body and focal ischemia reperfusion injuries in vivo. To confirm that endothelial cells are indeed the central mediator of NO production and to understand the signal transduction pathway through which pGz upregulates eNOS, we used an in vitro model of human aortic endothelial cells coated tubing, mimicking realistic conditions of blood vessels in vivo that are exposed to linear pulsatile flow (PF) or PF plus pGz (PF + pGz). PF + pGz produced significantly higher values of total eNOS and p-eNOS content than PF alone. Periodic acceleration also increased the ratio of phosphorylated Akt (p-Akt) to total Akt, as well as the ratio of p-ERK1/2 to total ERK1/2. Further, Wortmannin (PI3K inhibitor) inhibited phosphorylation of Akt and eNOS but not eNOS upregulation, while PD98059 (MEK inhibitor) inhibited phosphorylation of ERK1/2 and eNOS upregulation, but not eNOS phosphorylation. Therefore, this investigation shows that, pGz increases eNO and p-eNOS through the PI3K–Akt pathway and upregulated eNOS expression through the MEK-ERK1/2 pathway in cultured endothelial cells.

Role of extracellular signal-regulated kinase 1/2 in cigarette smoke-induced mucus hypersecretion in a rat model.

Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease, which is closely associated with cigarette smoking. However, the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known. This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats. A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks.Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126, a specific MEK1 kinase inhibitor, at doses of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 14 days. Expression of MUC5AC mRNA and protein, ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR, immunohistochemistry and Western blotting. Cigarette smoke significantly increased airway goblet cells metaplasia, induced the overexpression of MUC5AC mRNA and protein in bronchial epithelia, and increased the ratio of p-ERK 1/2 and ERK 1/2. U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P < 0.05). Moreover, there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P < 0.05). Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein. ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.