p-ERK Protein Expression Research Articles

MiR-133b inhibits MPP+-induced apoptosis in Parkinson's disease model by inhibiting the ERK1/2 signaling pathway.

The aim of this study was to explore the effect of micro ribonucleic acid (miR)-133b on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in the Parkinson's disease (PD) model. PC12 cells were induced by different concentrations of MPP+ to establish the PD cell model. Subsequently, the survival rate of PC12 cells was detected using Cell Counting Kit-8 (CCK-8) assay. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of miR-133b in the PD model induced by different concentrations of MPP+. Next, PC12 cells were transfected with miR-133b mimic and miR-negative control (NC), and divided into MPP+ group, MPP+ + miR-NC group and MPP+ + miR-133b mimic group. Transfection efficiency was verified using qRT-PCR. The apoptosis of cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated (p)-ERK1/2 were determined using Western blotting. After MPP+ treatment, the survival rate of PC12 cells significantly declined (p<0.05). MPP+ exhibited toxicity against PC12 cells in a concentration-dependent manner. Meanwhile, cell survival rate decreased remarkably with the increase of MPP+ concentration (p<0.05). With increased concentration of MPP+, the expression of miR-133b in the PD cell model declined significantly (p<0.05). The apoptosis of PC12 cells was remarkably inhibited by overexpression of miR-133b in the PD cell model (p<0.05). In addition, the protein expression of p-ERK1/2 in PC12 cells was notably reduced after overexpression of miR-133b in the PD cell model (p<0.05). MiR-133b is lowly expressed in the PD cell model. Furthermore, overexpression of miR-133b inhibits cell apoptosis in the PD cell model by regulating the ERK1/2 signaling pathway.

Fibrillin 2 gene knockdown inhibits invasion and migration of lung cancer cells

To investigate the effect of Fibrillin 2 (FBN2) expression on the invasion and migration of lung cancer cells, and the underlying mechanism. Protein and mRNA expressions of FBN2 were assayed. The relationship between FBN2 protein expression and clinical characteristics of lung cancer patients was analyzed. Correlation between FBN2 expression level and patient survival time was analyzed. Moreover, the mRNA and protein expression of FBN2 in lung cancer cells and human normal lung epithelial cells were assayed. After constructing low-expressing FBN2 cells, the cell proliferation, clone formation, migration and invasion capabilities were tested. Lung cancer cells proliferation with low FBN2 expression in nude mice was measured with a nude mouse tumorigenic experiment. The mRNA and protein expressions of FBN2 in lung cancer tissues were significantly higher than those in para-cancerous tissues (p&lt;0.05). FBN2 protein expression was significantly correlated with TNM stage, lymph node metastasis and histological type (p&lt;0.05). Survival time was markedly reduced in patients with high FBN2 expression (p&lt;0.001). The expressions of FBN2 mRNA and protein were markedly higher in lung cancer cells than in human normal lung epithelial cells. The proliferation, migration and invasion of lung cancer cells were significantly inhibited by FBN2 knockdown. The FBN2 knockdown significantly inhibited the protein expressions of p-FAK, p-MEK and p-ERK. FBN2 is highly expressed in lung cancer tissues, and as an oncogene, it affects the pathogenesis of lung cancer. The knockdown of the expression of FBN2 significantly inhibits the proliferation, invasion and migration abilities of lung cancer cells.

4-Sulfonyloxy/alkoxy benzoxazolone derivatives with high anti-inflammatory activities: Synthesis, biological evaluation, and mechanims of action via p38/ERK-NF-κB/iNOS pathway.

In an effort to discover new agents with high anti-inflammatory activity, 22 new 4-sulfonyloxy/alkoxy benzoxazolone derivatives were synthesized, characterized, and evaluated for their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production and TNF-α expression in RAW 264.7 cells in vitro. Most of these compounds displayed greater inhibitory ability against NO production than the lead compound 4-o-methyl-benzenesulfonyl benzoxazolone, and the most active compound 2h exhibited the strongest inhibitory activity against NO, IL-1β, and IL-6 production with IC50 values 17.67, 20.07, and 8.61μΜ, respectively. The effects of 2h were comparable or stronger than those of the positive control celecoxib. Compound 2h also displayed higher activity in vivo than celecoxib in a mouse model of xylene-induced ear edema, based on their inhibitory rates of 42.69% and 30.87%, respectively. Further molecular analysis revealed that compound 2h significantly reduced the iNOS levels in cell supernatant and suppressed the protein expression of iNOS, p-p38, p-ERK, and nuclear NF-κB. The results indicated that the anti-inflammatory effect of 2h might be realized through the regulation of ERK- and p38-mediated mitogen-activated protein kinase (MAPK)-NF-κB/iNOS signaling, thereby reducing the excessive release of NO, IL-1β, and IL-6. Our findings demonstrated that compound 2h, a new benzoxazolone derivative, could inhibit activation of the MAPK-NF-κB/iNOS pathway, supporting its potential as a novel anti-inflammatory agent.

Circadian Rhythm Protein Bmal1 Modulates Cartilage Gene Expression in Temporomandibular Joint Osteoarthritis via the MAPK/ERK Pathway.

The purpose of this study was to elucidate the role of the circadian gene Bmal1 in human cartilage and its crosstalk with the MAPK/ERK signaling pathway in temporomandibular joint osteoarthritis (TMJ-OA). We verified the periodical variation of the circadian gene Bmal1 and then established a modified multiple platform method (MMPM) to induce circadian rhythm disturbance leading to TMJ-OA. IL-6, p-ERK, and Bmal1 mRNA and protein expression levels were assessed by real-time RT-PCR and immunohistochemistry. Chondrocytes were treated with an ERK inhibitor (U0126), siRNA and plasmid targeting Bmal1 under IL-6 simulation; then, the cells were subjected to Western blotting to analyze the relationship between Bmal1 and the MAPK/ERK pathway. We found that sleep rhythm disturbance can downregulate the circadian gene BMAL-1 and improve phosphorylated ERK (p-ERK) and IL-6 levels. Furthermore, Bmal1 siRNA transfection was sufficient to improve the p-ERK level and aggravate OA-like gene expression changes under IL-6 stimulation. Bmal1 overexpression relieved the alterations induced by IL-6, which was consistent with the effect of U0126 (an ERK inhibitor). However, we also found that BMAL1 upregulation can decrease ERK phosphorylation, whereas ERK downregulation did not change BMAL1 expression. Collectively, this study provides new insight into the regulatory mechanism that links chondrocyte BMAL1 to cartilage maintenance and repair in TMJ-OA via the MAPK/ERK pathway and suggests that circadian rhythm disruption is a risk factor for TMJ-OA.

Leptin promotes proliferation and inhibits apoptosis of prostate cancer cells by regulating ERK1/2 signaling pathway.

The aim of this study was to investigate the effect of leptin (Lep) on the proliferation, invasion and apoptosis of prostate cancer cells through the extracellular regulated protein kinase 1/2 (ERK1/2) signaling pathway. Prostate cancer DU145 cells in the logarithmic growth phase were randomly divided into Lep (10, 20, 40, 80, 160 and 320 ng/mL) groups and blank control (Con) group. After culture, the cells were treated for 6 h, 12 h and 24 h, respectively. The effects of Lep on the proliferation and invasion of DU145 cells were detected via methyl thiazolyl tetrazolium (MTT) assay and transwell chamber assay, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to examine the messenger ribonucleic acid (mRNA) expressions of ERK1/2, b-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in DU145 cells after Lep treatment for 24 h. Thereafter, immunofluorescence assay was performed to detect the localization of ERK1/2 protein in prostate cancer DU145 cells. In addition, the expressions of phosphorylated (p)-ERK, ERK1/2 and apoptosis-related proteins, Bcl-2, Bax and cleaved cysteinyl aspartate specific proteinase (c-Caspase 3) in prostate cancer DU145 cells after treatment with different concentrations of Lep for 24 h were examined by Western blotting. MTT assay results showed that the proliferation rate of DU145 cells increased significantly at 6 h, 12 h and 24 h after 5-320 ng/mL of Lep treatment (p<0.05). Transwell assay manifested that the number of invasive cells was significantly raised after Lep treatment for 24 h (p<0.05). Meanwhile, the invasion ability of cells increased gradually with the elevation of Lep concentration. Subsequent qRT-PCR results demonstrated that after treatment with different concentrations of Lep, the mRNA expressions of ERK1/2 and Bcl-2 rose markedly (p<0.05). However, the mRNA expression of Bax was remarkably down-regulated (p<0.05) with the increase of Lep concentration in a concentration-dependent manner. According to the detection using a laser scanning confocal microscope, ERK1/2 red fluorescence showed punctiform aggregation, which was gradually raised with the increase of Lep concentration for 24 h. Moreover, Western blotting results denoted that with the increase of Lep concentration, the protein expressions of p-ERK, ERK1/2 and Bcl-2 were notably elevated (p<0.05), while those of Bax and c-Caspase 3 were distinctly reduced (p<0.05). Lep activation induces the proliferation, promotes the invasion and inhibits the apoptosis of prostate cancer cells through the ERK1/2 signaling pathway.

Attenuation of Lipopolysaccharide-Induced Acute Lung Injury by Hispolon in Mice, Through Regulating the TLR4/PI3K/Akt/mTOR and Keap1/Nrf2/HO-1 Pathways, and Suppressing Oxidative Stress-Mediated ER Stress-Induced Apoptosis and Autophagy.

The anti-inflammatory effect of hispolon has identified it as one of the most important compounds from Sanghuangporus sanghuang. The research objectives were to study this compound using an animal model by lipopolysaccharide (LPS)-induced acute lung injury. Hispolon treatment reduced the production of the pro-inflammatory mediator NO, TNF-α, IL-1β, and IL-6 induced by LPS challenge in the lung tissues, as well as decreasing their histological alterations and protein content. Total cell number was also reduced in the bronchoalveolar lavage fluid (BALF). Moreover, hispolon inhibited iNOS, COX-2 and IκB-α and phosphorylated IKK and MAPK, while increasing catalase, SOD, GPx, TLR4, AKT, HO-1, Nrf-2, Keap1 and PPARγ expression, after LPS challenge. It also regulated apoptosis, ER stress and the autophagy signal transduction pathway. The results of this study show that hispolon regulates LPS-induced ER stress (increasing CHOP, PERK, IRE1, ATF6 and GRP78 protein expression), apoptosis (decreasing caspase-3 and Bax and increasing Bcl-2 expression) and autophagy (reducing LC3 I/II and Beclin-1 expression). This in vivo experimental study suggests that hispolon suppresses the LPS-induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy and has the potential to be used therapeutically in major anterior segment lung diseases.

ALA/LA Protect Against DN db/db mice Oxidative Stress and Fibrosis Through the ROS/p38/TGF-β1 Pathway

Abstract Objectives Both oxidative stress (OS) and fibrosis play a significant role in the pathobiology of diabetic nephropathy (DN). Hence, we observed the effects of a-linolenic acids/linoleic acid (ALA/LA) on renal oxidative stress and tubule fibrosis on DN db/db mice and clarified its possible mechanisms in vivo. Methods db/db and db/m mice were divided into four groups, including normal control group(db/m mice), DN model group(DN db/db mice), low-dose group (DN db/db mice intervened by 250 mg/kg.d mixture of ALA and LA, with the ratio of 1:4), high-dose group (DN db/db mice intervened by500mg/kg.d ALA/LA, with the ratio of 1:4). The effect of ALA/LA on DN db/db mice analyzed by ELISA, realtime PCR, Westen Blot and pathophysiology methods. Results Compared to the DN model group, after ALA/LA intervention, fasting plasma glucose, renal function, inflammation factors, the level of oxidative stress and the degree renal pathological morphology damage of db/db mice were lower in the intervention group(P &amp;lt; 0.05). The SOD, CAT, MDA level (nmol/mgprot) in intervention group was 11.97 ± 1.95, 20.13 ± 1.67, 0.81 ± 0.12, and that in the model group was5.87 ± 1.87, 12.37 ± 1.50, 1.07 ± 0.10. BUN, SCr, UA in intervention group was 19.21 ± 6.16 mmol/L, 30.86 ± 5.37μmol/L, 230.14 ± 56.44μmol/L; and that in the model group was 9.74 ± 3.77 mmol/L, 22.22 ± 3.9μmol/L, 172.56 ± 27.32μmol/L.Intervention of ALA/LA decreased the protein and mRNA expression of TGF-β1, p-p38 (p38), p-ERK (ERK) and COLⅣ in kidney of db/db mice (P &amp;lt; 0.05). The relative mRNA level of TGF-β1, p38, ERK and COLⅣ in the intervention group was 0.64 ± 0.14, 2.71 ± 0.15, 2.48 ± 0.47, 4.37 ± 0.71, while that in the model group was17.63 ± 4.84, 127.43 ± 4.84, 15.91 ± 3.55, 32.05 ± 4.13.The relative protein level of TGF-β1, p-p38, p-ERK and COL-Ⅳ in the intervention group was 0.42 ± 0.07, 0.13 ± 0.04, 0.35 ± 0.04, and 0.46 ± 0.03, while that in the model group was 0.74 ± 0.05, 0.90 ± 0.03, 0.98 ± 0.02, 1.06 ± 0.10. Conclusions ALA/LA(1:4, 250 mg/kg/d) has good antioxidant capacity in vivo. It inhibited the transduction action of p38 and ERK, reduced the activity of TGF-β1, COL-Ⅳand ameliorate kidney fibrosis of DN db/db mice. The study on ALA/LA might provide research evidence and possible clinical treatment for DN. Funding Sources This work was financially supported by the National Natural Science Foundation of China (No. 81,372,986).

Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated

BackgroundMost mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.MethodsDNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.ResultsThree cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.ConclusionCell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.

MKP1 in the medial prefrontal cortex modulates chronic neuropathic pain via regulation of p38 and JNK1/2

Aim: The medial prefrontal context (mPFC) plays pivotal roles in initiation, development, and maintenance of chronic pain, whereas the underlying molecular mechanisms remain elusive, which invited investigation of potential involvement of MKP1 in mPFC in mice in neuropathic pain, and its cellular and molecular mechanisms.Materials and methods: Neuropathic pain model was established in adult male Kunming mice via chronic constrictive injury (CCI) of the sciatic nerve. Paw withdrawal latency (PWL) was measured at the plantar area by radiant heat test. Stereotaxic microinjection was applied in mice as per the atlas of Mouse Brain in Stereotaxic Coordinates. mRNA levels of MKP1 in mPFC in CCI mice were assessed by RT-PCR; protein expressions of MKP1, p-p38, p-JNK and p-ERK in mPFC in CCI mice were analyzed by Western blotting, and expressions of the c-Fos in mPFC in CCI mice evaluated by immunohistochemistry. Moreover, Lenti-MKP1 particles or BCI treatment was employed to inhibit MKP1 in mPFC contralateral to the injury.Results: MKP1 was activated and persistently upregulated in mPFC neurons in CCI mice. Inhibition of MKP1 in the mPFC contralateral to the injury could reverse CCI-induced pain behavior and neuronal activity either via employment of Lenti-MKP1 particles or BCI treatment. MKP1 in the mPFC modulated neuropathic pain via dephosphorization of p38 and JNK1/2.Conclusion: The findings demonstrated that MKP1 in mPFC could play a paramount role in the modulation of neuropathic pain, which might be associated to the increased neuronal excitability in the mPFC and downregulated p-p38 and p-JNK expression.

Zi Shen Huo Luo Formula Enhances the Therapeutic Effects of Angiotensin-Converting Enzyme Inhibitors on Hypertensive Left Ventricular Hypertrophy by Interfering With Aldosterone Breakthrough and Affecting Caveolin-1/Mineralocorticoid Receptor Colocalization and Downstream Extracellular Signal-Regulated Kinase Signaling.

Left ventricular hypertrophy (LVH) is an important characteristic of hypertensive heart disease. Renin-angiotensin system (RAS) blockers have been shown to be effective drugs for the reversal of LVH. Clinical and experimental studies have shown that Zi Shen Huo Luo Formula (ZSHLF) can improve the efficacy of perindopril in the treatment of hypertensive LVH, but its mechanism is unclear. This study aimed to investigate the possible mechanism to improve the efficacy of perindopril. First, we identified 23 compounds in ZSHLF by ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis, among which ferulic acid, caffeic acid, vanillic acid, berberine, rutin, quercetin, kaempferol, stachydrine, and tiliroside have been reported to reduce blood pressure and exhibit cardioprotective effects. Second, we treated spontaneously hypertensive rats (SHRs) with perindopril and ZSHLF for 12 continuous weeks and found that chronic use of perindopril could increase the aldosterone (ALD) levels and cause aldosterone breakthrough (ABT). ZSHLF combined with perindopril reduced the ALD levels, interfered with ABT, decreased blood pressure, improved left ventricular diastolic dysfunction, and decreased the collagen volume fraction; these effects were superior to those of perindopril alone. In vitro experiments, ALD-induced cardiomyocytes (H9c2 cells) and cardiac fibroblasts were treated with ZSHLF-containing serum, which suppressed ALD-induced cardiomyocyte hypertrophy and cardiac fibroblast proliferation, increased mineralocorticoid receptor (MR) and Cav-1 colocalization and decreased phosphorylated epidermal growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase (pERK) protein expression the cells. In conclusion, ZSHLF can interfere with ABT and affect the pathological role of ALD by affecting MR and Cav-1 interactions and EGFR/ERK signaling pathway. These effects represent a possible mechanism by which ZSHLF improves the efficacy of angiotensin-converting enzyme inhibitors (ACEIs) in hypertensive LVH treatment. However, the major bioactive components or metabolites responsible for the effects and the implications of these findings in patients need further verification.

The role of calcium regulation in the expression of PERK protein induced by sorafenib in hepatoma cells

AimThis study aimed to determine whether calcium signal participates in the sorafenib induced ER stress process and has an impact on the action of sorafenib for the hepatoma.MethodsLiver cancer HepG2 cell lines were maintained in DMEM with 10% fetal bovine serum and subcultured in plates or dishes at different densities for the following designed studies. HepG2 cells were basically treated with different doses of sorafenib (5–200 μM) for different time (0–48h) in the present or absent of trans‐Ned‐19 (a NAADP inhibitor, 100μM ) . The intracellular calcium signal was simultaneously recorded by confocal microscopy, and the expression level of PERK and caspase3 protein was detected by westernblot in HepG2 cells. The cell growth and apoptosis in these cells were analyzed by flow cytometry as well.ResultsConfocal microscopy showed that sorafenib (20–200 μM) induced substantial calcium release in HepG2 cells in a concentration‐dependent manner. Compared with the sorafenib intervention alone, trans‐Ned‐19 significantly attenuated sorafenib‐induced intracellular calcium elevation. Meanwhile, westernbolt assay showed that sorafenib time‐dependently but doses‐independently induced an increment in PERK expression in HepG2 cells. However, in the present of trans‐Ned‐19 was found to significantly enhance this sorafenib‐induced PERK and caspase3 levels, as well as apoptosis rate of HepG2 cells (P&lt;0.05). For further treatment with trans‐Ned‐19 for 24 h, microscopic observation showed that trans‐Ned‐19 significantly inhibited cell growth and caused abnormal cell morphology changes compared with the sorafenib alone group.ConclusionOur preliminary data showed that NAADP‐mediated intracellular calcium release was importantly involved in sorafenib‐induced perk protein expression and cell apoptosis. Blocking NAADP‐mediated calcium channels can significantly enhance the efficacy of sorafenib in the hepatoma cells, suggesting that the combination of NAADP analogues and sorafenib might be a potential strategy in the treatment of liver cancer.Support or Funding InformationSupport or Funding InformationSupported by NSFC Grants 81360077, 81860654, 30630145, 81172260 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang

Articular Cartilage Stem Cells Influence the Postoperative Repair of Hip Replacement by Regulating Endoplasmic Reticulum Stress in Chondrocytes via PERK Pathway.

ObjectiveEndoplasmic reticulum stress (ERS) is present in chondrocytes of osteoarthritis, and the intensity of ERS is related to the degree of cartilage degeneration. In vitro intervention strategies can change the status of ERS and induce the inhibition of ERS‐related pathway. Therefore, this study is designed to explore the role and molecular mechanism of cartilage stem cells (ACSCs) of ERS in chondrocytes after hip replacement.MethodsHuman cartilage cell lines C28/I2 were cultured as the control group. The ERS inducer was added into C28/I2 as ERS group. The third ERS + stem cells group was formed by adding cartilage stem cells into ERS group, and further transfection of si‐PERK was defined as si‐PERK + ERS + stem cells group. Cell cycle and apoptosis in the four groups were determined by flow cytometry. The protein expression of GRP78, PERK, ATF4, TMEM119, CDK4, Cyclin D, and BMP6 in chondrocytes in the four groups were investigated by western blot, and the distribution of PERK, TMEM119, and BMP6 in chondrocytes were observed by immunofluorescence assay. In addition, the transcriptional levels of Bcl2, Bax, and Caspase 3 were also determined by RT‐PCR.ResultsIn cell cycle assay, ERS increased the accumulation of cells in G0/G1 and G2/M, while cartilage stem cells weakened the effects. The apoptosis rates in control group, ERS, ERS + stem cells, si‐PERK + ERS + stem cells were 0%, 21.3%, 18.9%, and 15.9%, respectively, and the difference of apoptosis rate between the latter three groups and control group was statistically significant (P < 0.01). Stem cells could weaken the ERS‐induced cell apoptosis, especially reducing the number of cells in the late stage of apoptosis from 5.4% to 1.1%. The protein level of GRP78, PERK, ATF4, TMEM119, and BMP6 in the group of ERS, ERS + stem cells, and si‐PERK + ERS + stem cells were all significantly higher than those in control group, and the group of ERS + stem cells was the highest, all of the differences were significant (P < 0.01). However, the protein level of CDK4 and Cyclin D presented an absolutely opposite trend and the difference was still significant (P < 0.05). The group of si‐PERK + ERS + stem cell was lower than those in the group of ERS + stem cell but higher than those in the group of ERS (P < 0.05). The level of Caspase 3 in the latter three groups was significantly higher than those in the control group, and the group of ERS was the highest (P < 0.01). Besides, the relative level of Bcl‐2/Bax in control group was 1, but the group of ERS was about 0.5, and there was significant difference (P < 0.01). The ratio of Bcl‐2/Bax in the group of ERS + stem cells was more than 2 and significantly higher than those of other groups.ConclusionACSCs could reduce ERS‐induced chondrocyte apoptosis by PERK and Bax/Bcl‐2 signaling pathway.

Comparing PI3K/Akt Inhibitors Used in Ovarian Cancer Treatment.

Epithelial ovarian carcinoma (EOC) is the most lethal gynecological malignancy. Herein, we sought to determine the efficacy of phosphoinositide 3-kinase (PI3K)/Akt inhibition using three AZD compounds in a NOD-SCID xenograft mouse model and Akt regulation in a panel of eight ovarian cancer cell lines. Elevated Akt phosphorylation on Ser473 but not on Thr308 in cancerous tissues correlated with short progression-free survival (PFS), overall survival (OS), and death. AZD8835 and AZD8186 inhibited Akt phosphorylation while AZD5363 augmented its phosphorylation on Ser473. To add, all compounds inhibited the Akt downstream effectors 4E-BP1 and p70S6 kinase. AZD8835 and AZD5363 sensitized chemoresistant ovarian cancer cells to cisplatin and paclitaxel treatment. Only AZD5363 could inhibit COL11A1 mRNA and promoter activity, which are important factors in Akt regulation and chemoresistance in ovarian cancer. By using a mouse xenograft model, AZD8835 and AZD5363, but not AZD8186, caused a significant reduction in tumor formation. AZD compounds did not change the mRNA expression of BRCA1/BRCA in ovarian cancer cells, but AZD8835 inhibited BRCA1/BRCA2 mRNA expression and p-ERK protein expression in OVCAR-8 cells with the KRAS mutation. This study highlights the importance of PI3K/Akt in ovarian tumor progression and chemoresistance and the potential application of AZD compounds, especially AZD8835 and AZD5363, as therapeutic agents for the treatment of ovarian cancer.

The Effects and Mechanisms of Dihydroartemisinin on Influenza A Virus H1N1 Induces TNF-α and IL-6 Expression in Bronchial Epithelial Cells

To investigate the effects of dihydroartemis (DHA) on influenza A virus (IAV) A/PR/8/34 (H1N1) induces the pro-inflammatory factor and protein of extracellular signal regulated kinase (ERK) signaling pathway expression in bronchial epithelial cells. The BEAS-2B cells were treated with different concentrations of DHA (i.e.,0， 12.5, 25，50 and 100 μmol/L) for 24 h and the effect of DHA on the viability of BEAS-2B cells were measure by CCK8 method. The BEAS-2B cells were absorbed with IAV for 1 h, and then were treated with different concentrations of DHA (i.e., 12.5, 25 and 50 μmol/L) for 24 h, meanwhile, the normal control group and IAV group were established. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were measured by real time quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), the expression levels of phospho-ERK (p-ERK) proteins were tested by Western blot (WB). Then, an ERK agonist (20 ng/mL) was used to treat BEAS-2B cells (the groups were divided into normal control group, DHA group, DHA+IAV group, ERK agonist group and DHA+IAV+ERK agonist group) for 24 h, and to observe the effect of DHA on inhibiting IAV induce the TNF-α, IL-6 and p-ERK expression in the BEAS-2B cells. The BEAS-2B cells viability was not significantly different from that of the normal control group after treatment with DHA (i.e., 12.5, 25, and 50 μmol/L). The expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein in IAV group were significantly up-regulated compared with that in the normal control group ( P<0.05), meanwhile, compared with the IAV group, the expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein showed dose-dependent decrease in IAV+DHA group ( P<0.05). However, ERK agonists attenuated the DHA inhibit IAV induced the proinflammatory factors TNF-α, IL-6 secretion and the p-ERK protein expression of ERK signaling pathway in BEAS-2B cells. These data suggest that DHA can inhibit IAV induces the TNF-α and IL-6 expression in BEAS-2B cells through ERK signaling pathway.

Erythropoietin Derived Peptide Improved Endoplasmic Reticulum Stress and Ischemia-Reperfusion Related Cellular and Renal Injury.

Ischemia-reperfusion (IR) injury often affects transplant and native kidneys alike. IR injury is one of the main causes of acute kidney injury (AKI) and further associated with the progression of chronic kidney disease. Our previous study revealed the renoprotection of erythropoietin derived cyclic helix-B surface peptide (CHBP) against IR injury. However, the precise role and underlying mechanism of endoplasmic reticulum stress (ERS) in the injury and the renoprotection induced by IR or CHBP, respectively, have not been fully defined. This study using mouse kidney epithelial cells (TCMK-1) revealed that the level of CHOP (a key marker of ERS), PERK, and JNK (regulators of CHOP) was gradually increased by the prolonged time of hydrogen peroxide (H2O2) stimulation. In addition, CHOP mRNA and protein were significantly reduced by small interfering RNA (siRNA) target CHOP, as were apoptotic and inflammatory mediator caspase-3 and HMGB-1, and early apoptosis. Furthermore, CHOP mRNA was correlated positively with PERK protein, active caspase-3, HMGB-1 and apoptosis, but negatively with cell viability in vitro, while CHOP protein was also correlated positively with the level of tubulointerstitial damage and active caspase-3 protein in vivo. Finally, CHBP improved the viability of TCMK-1 cells subjected to H2O2 stimulation time-dependently, with reduced level of CHOP mRNA. CHBP also inhibited the increase of CHOP protein, not only in TCMK-1 cells, but also in the IR injury kidneys at 2 weeks. Moreover, CHBP reduced the expression of PERK mRNA and protein, JNK and HMGB-1 protein, as well as early and later apoptosis. In addition, raised CHOP at 12 h post IR injury might be an early time window for intervention. In conclusion, the differential role of ERS and CHBP in IR-related injury was proved in mouse TCMK-1 cells and kidneys, in which the mechanistic signaling pathway was associated with CHOP/PERK/JNK, HMGB-1/caspase-3, and apoptosis. CHOP might be a potential biomarker and CHBP might be therapeutic drug for IR-induced AKI.

MicroRNA-126 protects against vascular injury by promoting homing and maintaining stemness of late outgrowth endothelial progenitor cells

BackgroundEndothelial progenitor cells (EPCs) contribute to reendothelialization and neovascularization and protect against vascular injury and ischemia of various organs. We have previously shown downregulation of microRNA (miR)-126 in EPCs from diabetic patients, which contributes to dysfunction of EPCs including impaired migratory ability. The aims of the present study were to examine (1) in vitro the effects of miR-126 on the homing and stemness of late outgrowth EPCs (LOCs), along with relevant signaling pathways, and (2) in vivo the effects of modulating LOCs by manipulating miR-126 expression on LOC homing and reendothelialization of injured arteries in GK rats (a non-obese diabetes model).MethodsRat bone marrow-derived LOCs were transfected with miR-126 inhibitor or lentiviral vectors expressing miR-126. LOC migration was determined by transwell migration assay. CXCR4 expression was measured by real-time PCR, Western blotting, and confocal microscopy while related signaling pathway proteins were measured by Western Blotting. Stemness gene expression, and gene and protein expression and promoter activity of KLF-8 were also measured. LOCs transfected with lenti-miR-126 or miR-126 inhibitor were injected into GK rats with carotid artery injury, and then vascular reendothelialization and the extent of intimal hyperplasia were examined.ResultsLenti-miR-126 increased while miR-126 inhibitor decreased LOC migration and CXCR4 expression on LOCs. miR-126 positively regulated p-ERK, VEGF, p-Akt, and eNOS protein expression, and inhibitors of these proteins blocked miR-126-induced CXCR4 expression and also reduced LOC migration. Overexpression of miR-126 promoted while inhibition of miR-126 suppressed stemness gene expression in LOCs. miR-126 also inhibited gene and protein expression and promoter activity of KLF-8 while shRNA-mediated knockdown of KLF-8 increased stemness gene expression. Upregulation of stemness gene expression by miR-126 overexpression was completely abrogated by co-transfection of lenti-KLF-8 and lenti-miR-126 into LOCs. In GK rats, transplantation of LOCs overexpressing miR-126 enhanced LOC homing and reendothelialization and decreased intimal hyperplasia of injured arteries.ConclusionOur results indicate that miR-126 protects against vascular injury by promoting CXCR4 expression and LOC homing via ERK/VEGF and Akt/eNOS signaling pathways and maintaining stemness via targeting KLF-8.

Alzheimer's drug Memantine inhibits metastasis and p-Erk protein expression on 4T1 breast cancer cells.

Drug repurposing studies enable shorter routes to the clinic by skipping the steps like in vitro in vivo screening, chemical optimization and toxicological studies. In our study, we investigated the potent anti-cancer effect of Alzheimer's drug Memantine on 4T1 breast cancer cells. Memantine's effect on proliferation of 4T1 cells was evaluated by using the MTT assay. Memantine inhibited 4T1 cell proliferation in a concentration- dependent manner at 24 and 48 hours. We investigated the drug's effect on the protein expressions of Bax, Bcl-2, Casp-3, Casp-9, E-Cad, Vimentin, B-Cat, GSK3B, p-ERK, ERK, p-GS, GS that are involved in apoptosis, metastasis and cell survival. Memantine altered the Bcl-2, Bax, Casp3, Casp-9 apoptotic protein expression levels. We found that memantine inhibited p-Erk expression and that result suggested a plausible mechanism of action for memantine's antineoplastic effect. Memantine also inhibited wound closure at 24 h, significantly (p = 0.0055). Memantine inhibited 4T1 breast cancer cell proliferation at significantly lower doses than mostly studied re-purposed drug Metformin. Therefore, we believe that memantine might hold a great promise as a new repositioned drug in cancer treatment and it is our further interest to investigate its effects in vivo (Fig. 3, Ref. 22).

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.

Role of miR-106-mediated mitogen-activated protein kinase signaling pathway in oxidative stress injury and inflammatory infiltration in the liver of the mouse with gestational hypertension.

We aim to investigate the role of miR-106-mediated mitogen-activated protein kinase (MAPK) signaling pathway in oxidative stress (OS) injury and inflammatory infiltration in the liver of the mouse with gestational hypertension (GH). Ninety specific pathogen-free mice (Kunming species) during middle to late gestation were selected for the study. Fifteen mice were used as control, while the rest were used for establishing the GH model. The mice were assigned to six groups: normal group (normal gestation), model group (GH model), negative control group (GH model, intravenously injected with negative control vector), miR-106a-mimic group (GH model, intravenously injected with vector overexpressing miR-106a, which mimics the overexpression of endogenous mature miR-106a), SB203580 group (GH model, intravenously injected with MAPK pathway inhibitor SB203580), and miR-106a-mimic+SB203580 group (GH model, intravenously injected with SB203580 and vector overexpressing miR-106a). Fourteen days after electrical stimulation, all the groups except for the normal group had elevated blood pressure vs those on day 0 and 7. Compared with the normal group, the other groups had lower levels of miR-106a expression, nitric oxide, nitric oxide synthase,catalase, superoxide dismutase, S cell ratio, and interleukin-4 (IL-4) and IL-10 in the serum and liver as opposed to increased levels of blood pressure, p38MAPK mRNA expression, p-p38MAPK positive expression rate, protein expressions of p-p38MAPK, p-ERK, and p-JNK, H2 O2 and malondialdehyde in liver, G0/G1 cell ratio, apoptosis rate, and IL-6, interferon-γ (IFN-γ), and IFN-α in the serum and liver (all P < .05). The miR-106 overexpression or inhibiting MAPK signaling pathway can attenuate OS injury and inflammatory response in the liver of the mouse with GH, and the effect can be even better if both miR-106a overexpression and inhibiting MAPK pathway are applied. In conclusion, miR-106a overexpression can inhibit OS injury and inflammatory infiltration in the liver of the mouse with GH by mediating MAPK signaling pathway.

MicroRNA-1298-5p inhibits cell proliferation and the invasiveness of bladder cancer cells via down-regulation of connexin 43.

MicroRNA (miR)-1298 is widely down-regulated in a variety of malignant tumors, which facilitates cell proliferation, invasiveness, and migration. However, the specific biological function of miR-1298 in bladder cancer (BC) is still unknown. Connexin 43 (Cx43) is often up-regulated in tumors. Identifying miRNAs that target Cx43 in the setting of BC will help to develop Cx43-based therapies for BC. In this study, the results demonstrated that the expression levels of miR-1298 and Cx43 were significantly down-regulated and up-regulated, respectively, in BC tissues. Overexpression of miR-1298 inhibited cell proliferation, migration, and invasiveness in two BC cell lines as determined using MTT assays, cell cycle assays, colony formation assays, Transwell assays, gelatin zymography, and Western blot. In addition, we found that miR-1298 decreased Cx43 expression by directly targeting the 3'-UTR. Further, we observed that the promotion of BC cell proliferation, migration, and invasiveness from Cx43 on could be partially attenuated by overexpressing miR-1298. Moreover, the protein expression of p-ERK was ameliorated after transfection with overexpressed-miR-1298. Knockdown of Cx43 reversed the promotion of cell migration and invasiveness due to decreased expression of miR-1298. All of the data from our study indicate that miR-1298 could be a diagnostic marker of BC and a potential therapeutic agent via inhibiting Cx43.