Expression Of ERK Research Articles

ERK1/2 gene expression and hypomethylation of Alu and LINE1 elements in patients with type 2 diabetes with and without cataract: Impact of hyperglycemia-induced oxidative stress.

This study aimed to delineate the effect of hyperglycemia on the Alu/LINE-1 hypomethylation and in ERK1/2 genes expression in type 2 diabetes with and without cataract. This study included 58 diabetic patients without cataracts, 50 diabetic patients with cataracts, and 36 healthy controls. After DNA extraction and bisulfite treatment, LINE-1 and Alu methylation levels were assessed using Real-time MSP. ERK1/2 gene expression was analyzed through real-time PCR. Total antioxidant capacity (TAC), and fasting plasma glucose (FPG) were measured using colorimetric methods. Statistical analysis was performed with SPSS23, setting the significance level at P < 0.05. The TAC levels were significantly lower for cataract and diabetic groups than controls (259.31 ± 122.99, 312.43 ± 145.46, 372.58 ± 132.95 nanomole of Trolox equivalent) with a significant correlation between FPG and TAC levels in both the cataract and diabetic groups (P < 0.05). Alu and LINE-1 sequences were found to be statistically hypomethylated in diabetic and cataract patients compared to controls. In these groups, TAC levels were directly correlated with Alu methylation (P < 0.05) but not LINE-1. ERK1/2 gene expression was significantly higher in diabetic and cataract patients, showing increases of 2.41-fold and 1.43-fold for ERK1, and 1.27-fold and 1.5 for ERK2, respectively. ERK1 expression correlated significantly with FPG levels. A reverse correlation was observed between TAC levels and ERK1/2 expression. Our findings indicate that hyperglycemia-induced oxidative stress may alter ERK1/2 gene expression patterns and induce aberrant hypomethylation in Alu and LINE-1 sequences. These aberrant changes may play a contributing role in diabetic complications such as cataracts.

Exposure to MC-LR activates the RAF/ERK signaling pathway, leading to renal inflammation and tissue damage in mice

ABSTRACT Exposure to microcysatin-LR (MC-LR) is known to result in kidney damage, however the underlying mechanisms involved in MC-LR-initiated renal injury are not known. Thus, the aim of this study was to examine the effects of exposure to MC-LR on human embryo kidney (HEK 293) cell in vitro and male C57BL/6 in vivo. In the in vitro study, HEK 293 cells were incubated with MC-LR (20 µM) for 24 hr. Treatment with MC-LR significantly increased the protein expression of RAF and ERK as well as mRNA expression levels of inflammatory cytokines TNF-α, IL-6, and IL-1β. These findings were confirmed when HEK 293 cells were co-incubated with ERK inhibitor U0126 and MC-LR demonstrating a decrease in protein expression of RAF, ERK, and mRNA levels of pro-inflammatory cytokines. Male C57BL/6 mice were intraperitoneally (ip) injected with MC-LR (20 µg/kg) daily for 21 days. Histopathological analysis demonstrated significant glomerular and tubular damage with inflammatory infiltration. The expression levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were significantly elevated following MC-LR treatment. Administration of MC-LR asignificantly enhanced the protein phosphorylation levels of RAF and ERK. Data demonstrated that exposure to MC-LR induced morphological tissue damage and renal inflammatory reactions by activating the RAF/ERK signaling pathway.

Sodium butyrate mediates the MAPK signaling pathway and apoptosis and modulates intestinal flora to alleviate glycinin-induced intestinal injury in Cyprinus carpio.

The study investigated the potential alleviating effect of sodium butyrate (SB) on intestinal injuries caused by glycinin in the diet of common carp. Fish were divided into six groups: a control group (without glycinin and SB), a Gly group (with glycinin), and four groups supplemented with different doses of SB (0.75, 1.50, 2.25, and 3.00 g/kg) based on the Gly group. All diets were isonitrogenous and isoenergetic, and the fish were fed these diets for 8 weeks. The results indicated that glycinin activated the mitogen-activated protein kinase (MAPK) signaling pathway, leading to upregulating ERK, JNK, and p38 gene expression in the intestine. However, SB2 and SB3 groups were able to inhibit this pathway. Furthermore, glycinin upregulated the expression of proapoptotic genes (Bax, Caspase-3, Caspase-8, and Caspase-9) while downregulating the antiapoptotic gene Bcl2. The SB2 and SB3 groups were found to alleviate glycinin-induced apoptosis. Additionally, dietary glycinin significantly decreased the expression of tight junction genes (ZO-1, Claudin3, Claudin7, and Occludin1) in the intestine, whereas the SB2 and SB3 groups improved intestinal barrier function. Glycinin also elevated serum levels of d-lactate, diamine oxidase, serotonin, and endothelin, resulting in intestinal damage and increased permeability. In contrast, the SB2 and SB3 groups reduced these serum levels, thereby regulating intestinal permeability. Moreover, glycinin disrupted the intestinal morphology, which was mitigated by the SB2 and SB3 groups by increasing the height and width of intestinal villi folds. Lastly, dietary glycinin altered the intestinal microecological balance by increasing Proteobacteria abundance and decreasing Clostridium and Bacteroidetes abundance. The SB2 and SB3 groups modulated the composition of dominant taxa by increasing Firmicutes and Acidobacteria abundance. Overall, SB was found to mediate the MAPK signaling pathway, apoptosis, upregulation of tight junction genes, maintenance of the intestinal physical barrier, and regulation of intestinal flora, thereby alleviating glycinin-induced intestinal damage.

CT-Scan-Assessed Body Composition and Its Association with Tumor Protein Expression in Endometrial Cancer: The Role of Muscle and Adiposity Quantities.

Background: Endometrial cancer is strongly associated with obesity, and tumors often harbor mutations in major cancer signaling pathways. To inform the integration of body composition into targeted therapy paradigms, this hypothesis-generating study explores the association between muscle mass, body fat, and tumor proteomics. Methods: We analyzed data from 113 patients in The Cancer Genome Atlas (TCGA) and Cancer Proteomic Tumor Analysis Consortium (CPTAC) cohorts and their corresponding abdominal CT scans. Among these patients, tumor proteomics data were available for 45 patients, and 133 proteins were analyzed. Adiposity and muscle components were assessed at the L3 vertebral level on the CT scans. Patients were stratified into tertiles of muscle and fat mass and categorized into three groups: high muscle/low adiposity, high muscle/high adiposity, and low muscle/all adiposities. Linear and Cox regression models were adjusted for study cohort, stage, histology type, age, race, and ethnicity. Results: Compared with the high-muscle/low-adiposity group, both the high-muscle/high-adiposity (HR = 4.3, 95% CI = 1.0-29.0) and low-muscle (HR = 4.4, 95% CI = 1.3-14.9) groups experienced higher mortality. Low muscle was associated with higher expression of phospho-4EBP1(T37 and S65), phospho-GYS(S641) and phospho-MAPK(T202/Y204) but lower expression of ARID1A, CHK2, SYK, LCK, EEF2, CYCLIN B1, and FOXO3A. High muscle/high adiposity was associated with higher expression of phospho-4EBP1 (T37), phospho-GYS (S641), CHK1, PEA15, SMAD3, BAX, DJ1, GYS, PKM2, COMPLEX II Subunit 30, and phospho-P70S6K (T389) but with lower expression of CHK2, CRAF, MSH6, TUBERIN, PR, ERK2, beta-CATENIN, AKT, and S6. Conclusions: These findings demonstrate an association between body composition and proteins involved in key cancer signaling pathways, notably the PI3K/AKT/MTOR, MAPK/ERK, cell cycle regulation, DNA damage response, and mismatch repair pathways. These findings warrant further validation and assessment in relation to prognosis and outcomes in these patients.

Evaluating the effect of acute myeloblastic leukemia-derived exosomes on the human bone marrow mesenchymal stromal cell proliferation.

The progression of leukemia is substantially associated with the interactions of leukemic cells with surrounding cells within the bone marrow microenvironment (BMM), and these interactions were facilitated using exosomes as vital mediators. The current study aimed to examine the proliferative effects of exosomes derived from the HL-60 cell line, a representative of acute myeloblastic leukemia (AML), on the cell cycle progression of human bone marrow mesenchymal stromal cells (hBM-MSCs), a key element of the BMM. hBM-MSCs were treated with different concentrations of AML-derived exosomes from the HL-60 cell line. The results were obtained from MTT, cell proliferation, cell cycle, and RT-qPCR evaluations. In the current study, we found that the proliferation effects of AML-derived exosomes relied on the dose and the time, and the optimal effects of exosomes were seen in 50μg/ml, 48h treatment. Flow cytometry analysis revealed a significant increase in the G1 phase, showing a 1.6-fold change compared to the control group (p value < 0.0001). RT-qPCR results demonstrated a significant upregulation of CCND1 (3.3-fold, p value < 0.0001), CDK4 (3.7-fold, p value < 0.0001), CDK6 (3.3-fold, p value < 0.0001), RAS (3.2-fold, p value < 0.0001), and Erk (3.4-fold, p value < 0.0001) expression levels, along with increased Ki-67 (2.6-fold, p value < 0.0001) levels. Moreover, treatment with 50μg/ml, 48h of AML-derived exosomes resulted in a notable reduction in BM-MSC apoptosis both in early (p value < 0.0001) and late (p value < 0.0001) apoptosis rate compared to control group. The findings will be of interest to AML-derived exosomes, which were able to potentiate the activation of the signaling pathways involved in the survival and proliferation of hBM-MSCs. Our findings suggest their specific targeting as a potential therapeutic strategy against cancer progression and metastasis.

Suppression of KLF5 targets RREB1 to restrain the proliferation of ovarian cancer cells through ERK/MAPK signaling pathway.

The overexpression of Kruppel-like factor 5 (KLF5) appears in several types of cancer. KLF5 may be an effective therapeutic target for treating OC, but its function in ovarian cancer (OC) remains unknown. The KLF5 mRNA expression levels in several OC cell lines were analyzed using RT-qPCR. Then, NC-siRNA or KLF5-siRNA was transfected into SK-OV-3 and OVCAR-3 cells. RT-qPCR and WB were used to detect the efficiency of KLF5 silence, CCK-8, colony formation assay, IHC staining, flow cytometry, and WB were performed to investigate the KLF5 function on OC cell proliferation and the activation of the extracellular signal-regulated Kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway. Next, a dual-luciferase and IF assay were used to determine the relationship between KLF5 and the Ras response element-binding protein (RREB1). SK-OV-3 and OVCAR-3 cells were treated with KLF5-siRNA and C16-PAF + EGF (MAPK agonist), separately or in combination. Proteins including KLF5, RREB1, p-p38, p-ERK1/2, ERK5, p-ERK5, CyclinD1, CDK4, and CDK6 were quantified by WB. Finally, CCK-8, colony formation assay, and flow cytometry were employed again. KLF5 is highly expressed in OC cells compared with normal cells. When KLF5 knockdowns in SK-OV-3 and OVCAR-3 cells, the cell proliferation restrains, andthe G1 phase prolongs. In addition, KLF5 silence caused a decrease of CyclinD1, CDK4, CDK6, p-p38, p-ERK1/2, and p-ERK5/ERK5 expression levels. However, these statuses could be revised by C16-PAF + EGF. Results also found that when the ERK/MAPK signaling is activating, RREB1 is expressed low. The KLF5 silence could up-regulate the RREB1 expression. The KLF5 silence could restrain the OC cell proliferation and cell cycle. KLF5-siRNA may target upregulating RREB1 expression, thereby inhibiting the activation of the ERK/MAPK signaling pathway in OC cells.

Isoginkgetin Inhibits RANKL-induced Osteoclastogenesis and Alleviates Bone Loss

Osteoporosis is characterized by excessive osteoclast activity leading to bone loss, decreased bone mineral density, and increased susceptibility to fractures. Through in vivo/vitro experiments, along with network pharmacology analysis, we aimed to explore the underlying mechanisms of Isoginkgetin (IGG) in inhibiting osteoclastogenesis, providing valuable insights for further research in the future. Firstly, we ascertained the safe concentration of IGG stimulation on BMMs, followed by a systematic exploration of the concentration gradient at which IGG inhibited osteoclastogenesis using TRAP analysis. An osteoporosis model was established to further validate the in vitro experimental findings by combining Micro-CT and immunohistochemical analysis. The results show that IGG did not exhibit cytotoxicity or proliferative effects on BMMs at concentrations equal to or less than 10 μM. Additionally, IGG inhibited the activity of osteoclastogenesis and bone resorption function at lower concentrations. RT-PCR and Western Blot results demonstrated that IGG could downregulate genes and proteins associated with osteoclastogenesis. The Western Blot results also showed that IGG inhibited the phosphorylation expression of P38, ERK, and P65 in the MAPK and NF-κB pathways. At the same time, it rescued the degradation of IκB-α at 15 and 60 min. IGG can also impact the relative expression levels of oxidative proteins such as SOD-1, HO-1, and catalase, thereby influencing cellular equilibrium and stress levels, ultimately inhibiting the formation of mature OC. In vivo experiments demonstrated that IGG alleviated bone loss caused by osteoclasts and improved relevant parameters of trabecular bone. So, IGG effectively attenuated osteoclastogenesis, and improved bone density, thereby portraying its role in osteoporosis management.

Gandou Bushen Decoction improves spermatogenesis and promotes spermatogenic cell proliferation in Wilson disease TX mice by activating testicular ERK signaling pathway

To investigate the therapeutic mechanism of Gandou Bushen Decoction (GDBSD) for improving reproductive disorders in male mouse models of Wilson disease (WD). Sixty male homozygous TX mice were randomized equally into 4 groups and treated with daily gavage of saline (WD model group), penicillamine (0.09 g/kg), or GDBSD (0.2 mL/10 g), or with intraperitoneal injection of U0126 (20 mg/kg) in addition to GDBSD gavage, with 15 male DL mice as control. After 4 weeks of treatment, copper content in testicular tissue of the mice was detected, and histopathology of the testes and epididymis was examined using HE staining and electron microscopy. TUNEL staining was used to identify apoptotic cells in the testes. The protein expressions of Bcl-2, Cytc, caspase-3, ERK, and p-ERK in the testicular tissue were evaluated with Western blotting, and BrdU-positive cells were detected with immunohistochemical labeling. Sperm density, viability, malformation rate and fertility levels of male mice were studied. Treatment with penicillamine and GDBSD obviously improved pathological changes of the testis, increased sperm density and motility, lowered sperm abnormality rate, fertility levels and increased testicular JOHNSEN score of TK mice, but the therapeutic effect of GDBSD was blocked by U0126. GDBSD treatment significantly lowered Cytc and caspase-3 expressions and increased Bcl-2 expression in the testicular tissue of TX mice (P < 0.05), while U0126 treatment significantly lowered testicular Bcl-2 expression level. No significant differences were found in total protein expression levels of ERK1/2 among the 5 groups, but p-ERK protein expression was significantly reduced in WD and U0126 groups and increased in penicillamine and GDBSD groups. GDBSD can improve spermatogenesis and enhance fertility of male TX mice with WD possibly by activating the ERK signaling pathway to enhance proliferation and reduce apoptosis of the spermatogenic cells.

Methyl Paraben Affects Porcine Oocyte Maturation Through Mitochondrial Dysfunction.

Parabens are widely used in various industries, which are including chemical, pharmaceutical, food, cosmetic, and plastic processing industries. Among these, methyl paraben (MP) serves as an antimicrobial preservative in processed foods, pharmaceuticals, and cosmetics, and it is particularly detected in baby care products. Studies indicate that MP functions as an endocrine-disrupting compound with estrogenic properties, negatively affecting mitochondrial bioenergetics and antioxidant activity in testicular germ cells. However, limited information exists regarding studies on the effects of MP in oocytes. The aim of this study was to investigate the specific mechanism and the toxic effects of MP during oocyte maturation cultured in vitro using a porcine oocyte model. The results indicated that MP (50 μM) inhibited oocyte expansion, significantly reducing the expression of expansion-related genes MAPK1 and ERK1, and decreased the first polar body extrusion significantly as well. ATP levels decreased, reactive oxygen species (ROS) levels remained unchanged, and glutathione (GSH) levels decreased significantly, resulting in an elevated ROS/GSH ratio. The expression of antioxidant genes SOD1 and GPX was significantly decreased. Additionally, a significant decrease in levels of mitochondrial production and biosynthesis protein PGC1α+β, whereas levels of antioxidant-related protein Nrf2 and related gene expression were significantly increased. Autophagy protein LC3B and gene expression significantly decreased, and apoptosis assay indicated a significant increase in levels of caspase3 protein and apoptosis-related genes. These results demonstrated the negative effect of MP on oocyte maturation. In conclusion, our findings indicate that MP disrupts redox balance and induces mitochondrial dysfunction during meiosis in porcine oocytes, resulting in the inhibition of meiotic progression. The present study reveals the mechanism underlying the effects of methyl para-hydroxybenzoate on oocyte maturation.

DNAR-05. VALIDATING THE ERK5 PROTAC OS11 AS A POTENTIAL NOVEL THERAPEUTIC INEX VIVO GLIOMA STEM CELL MODELS OF INTRATUMOURAL HETEROGENEITY AND RESIDUAL DISEASE

Abstract Glioblastoma is a grade IV brain tumour with poor survival rates of 15 months post-diagnosis, and 5-year survival rates of 10%. Limited progress over the past 40 years to improve the current treatment regimen of surgery and chemo-/radio-therapy are mainly due to tumour heterogeneity that drive resistance mechanisms. The persistence of glioma stem-like cells (GSCs) sub-populations, particularly within post-surgical residual tumour tissue. ERK5 is an emerging oncology drug target that we previously highlighted as a potential target for combined therapy with temozolomide (TMZ) in glioblastoma due to heightened ERK5 expression, which is associated with poor survival. This current project aims to expand on our previous findings by establishing whether targeting ERK5 can sensitise glioblastoma cells to TMZ using primary derived ex vivo GSC models of intratumor heterogeneity and residual disease. Primary GSC models were derived from patients at the Royal Hallamshire Hospital. The PROTAC OS11 (CRUK &amp; University of Manchester) was used to degrade ERK5. DNA damage and cell survival were evaluated following treatment of TMZ alone and in combination with OS11. Consistent with our findings in established glioma cell lines, OS11 was able to robustly degrade ERK5 in a 3D GSC model for at least 72hrs. Combining OS11 and TMZ in GSCs led to a significant increase in DNA damage, with MGMT negative cells exhibiting a greater increase in DNA damage compared to MGMT positive cells. However, unlike our previous findings in established cell lines, this did not lead to reduced cell survival in combination with TMZ. These findings further support the preclinical use of ex vivo models that better represent intratumoural heterogeneity and post-surgical GCS niches. Overall, these data highlight the value of preclinical validation in more clinically relevant models to identify the best possible novel therapeutic approaches to pursue further towards clinical delivery.

Lancao decoction in the treatment of alzheimer's disease via activating PI3K/AKT signaling to promote ERK involving in enhancing neuronal activities in the hippocampus

Ethnopharmacological relevancePrevious study has demonstrated lancao decoction (LC), a traditional Chinese medicine (TCM) fomula and recorded in “Huangdineijing”, has a therapeutic effect on cognitive impairment (early clinical manifestations of alzheimer's disease (AD), which suggests that LC may have potential therapeutic advantages for AD. Whether LC has the therapeutic effect on AD and its potential mechanisms were still further indicated. Aim of the studyIn this study, we aimed to uncover the potential advantage and neuronal mechanisms of LC in the treatment of AD in APP/PS1 mice in the hippocampus. Methods and materialsWe chose APP/PS1 mice to combing with behavioral tests including morris water maze (MWM) or y-maze to determine the role of LC in the therapeutic actions of AD. Network pharmacology was used to screen potential targets and pathways involving in LC's treatments of AD. Western blot was used to detect the phosphorylated expressions of proteins in hippocampus in APP/PS1 mice in the hippocampus. Pharmacological interventions were used to elucidate the relationship between the role of LC in the treatment of AD and the pathway, as well as the upstream and downstream interactions with neuronal activities. ResultsAccording to our previous LC effective dose (2.5 g/kg), the dose was also able to significantly reduce the latency to the platform, and significantly increase the number of crossing times and time spend in the target quadrant in APP/PS1 mice in MWM, which was consistent with donepezil (DON) after 14 days chronic treatments. Network pharmacology showed that PI3K/AKT and MAPK pathways were closely associated with LC's treatments of AD, and protein autophosphorylation played a role in this process. The phosphorylated expressions of PI3K and AKT were obviously reduced in APP/PS1 mice in the hippocampus, which were both reversed by LC or DON. The phosphorylated expressions of MAPK including P38, JNK and ERK were also significantly reduced in APP/PS1 mice hippocampus, but only the phosphorylated expression of ERK was reversed by LC or DON. Inhibiting the activities of PI3K/AKT pathway by LY294002 blocked LC's improvement of behavioral deficits in APP/PS1 mice, including reducing latency to platform and increasing the number of crossings time in MWM in APP/PS1 mice, which also blunted LC's up-regulated phosphorylated expressions of PI3K, AKT and ERK in the hippocampus. Moreover, suppressing the activities of ERK by PD98059 also blocked LC's improvement of AD-related behavioral deficits including decreasing latency to new arm and increasing time in new arm in y-maze test, which also inhibited LC's enhancement of synaptic proteins (PSD95 and synapsin1) in the hippocampus and the number of EGR1-positive cells in the hippocampal dentate gyrus (DG). ConclusionsTake together, our study revealed that LC had the therapeutic effects on AD by activating the PI3K/AKT pathway to enhance ERK activity and further strengthened neuronal activities in the hippocampus.

Hepatitis B Virus-Induced Resistance to Sorafenib and Lenvatinib in Hepatocellular Carcinoma Cells: Implications for Cell Viability and Signaling Pathways.

Background/Objectives: Sorafenib and lenvatinib are tyrosine kinase inhibitors used in hepatocellular carcinoma (HCC) treatment. This study investigates how hepatitis B virus (HBV) infection affects their efficacy in HepG2 hepatoma cells. Methods: HepG2 and HBV-infected HepG2/2215 cells were treated with varying concentrations of both drugs. The cell viability, cell cycle gene expression, cycle progression, and phosphorylation levels of ERK and AKT were analyzed. Results: The HBV-infected cells showed significant alterations in their cell cycle gene expressions, with an 80-fold increase in CCND2 expression and a higher proportion of cells in the G2/M phase, indicating enhanced proliferation. While both drugs decreased HepG2 cell viability in a concentration-dependent manner, HBV infection conferred resistance, as evidenced by the increased viable cells in the HepG2/2215 cultures. Sorafenib and lenvatinib decreased key cyclin and cyclin-dependent kinase expressions in uninfected cells, with less effect on the HBV-infected cells. Both drugs lowered the pERK and pAKT levels in the HepG2 cells. In the HBV-infected cells, sorafenib reduced the pERK and pAKT levels to a lesser extent. However, treatment with lenvatinib elevated the levels of pERK and pAKT. Conclusions: In conclusion, HBV infection increases resistance to both sorafenib and lenvatinib in hepatoma cells by influencing the cell cycle regulatory genes and critical signaling pathways. However, the resistance mechanisms likely differ between the two medications.

Combined Targeting of SYK and BCL-2 in Acute Myeloid Leukaemia and Mechanism Study

Acute myeloid leukemia (AML) is a highly heterogeneous haematological malignancy and is the most common acute leukaemia in adults, with a median age of onset of 68 years and a 5-year survival rate of only 30%. Only 60-80% of adult AML patients can achieve complete remission with the first induction chemotherapy, and 50-80% of AML patients ultimately relapse with the “3+7” first-line treatment regimen based on anthracycline and cytarabine (Ara-C), so there is an urgent need to find safer and more effective targeted therapies. There is an urgent need to find safer and more effective targeted therapies. Venetoclax (ABT-199) is an FDA-approved oral selective Bcl-2 inhibitor with limited anti-leukaemia activity as a single agent, and mechanisms of resistance include up-regulation of anti-apoptotic proteins, compensation of multiple metabolic pathways such as OXPHOS glycolysis, and mutations in BCL-2 and BAX.In 2018, the FDA approved the use of Venetoclax in combination with demethylating drugs (azacitidine ( AZA) or low-dose azacitidine AraC (LDAC)) in combination for newly diagnosed AML in older adults aged 75 years and older, and while this combination greatly improves remission rates, most patients relapse within 18 months. It has been suggested that direct or indirect inhibition of oxidative phosphorylation (OXPHOS) may be able to circumvent adaptive resistance of AML to Venetoclax+ AraC combination therapy. Therefore, an ideal candidate therapy in combination with Venetoclax should be able to antagonise its resistance mechanisms from metabolic pathways and down-regulation of MCL-1. Purpose To investigate the efficacy and related mechanisms of co-targeting SYK with BCL-2 for the treatment of acute myeloid leukaemia (AML). Experimental Design AML cell lines and primary patient samples were collected for cell proliferation assays. Firstly, cell activity was detected by MTS colorimetric assay, by Compusyn calculation software, the joint index CI value was calculated, apoptosis was detected by flow cytometry, mitochondrial membrane potential was altered, gene expression was detected by transcriptome sequencing, and the signalling pathways were analysed, followed by apoptosis-related proteins, BCL-2-related proteins by Western Blot, and glucose was detected by metabolism kit. sugar consumption,ATP and lactate content. The in vivo efficacy of the combination of Venetoclax and Entospletinib was assessed using an MV4-11-derived xenograft mouse model. Results SYK is highly expressed in AML and is associated with poor prognosis. ...The combination of Venetoclax and Entospletinib in AML cell lines and AML primary patient cells had significant synergistic growth inhibitory and apoptosis-inducing effects...Venetoclax in combination with Entospletinib down-regulated the expression of anti-apoptotic proteins, MCL-1, and BCL-XL, and down-regulated phosphorylated ERK expression, thereby affecting MCL1 post-translational protein stability rather than transcriptional levels.... Venetoclax in combination with Entospletinib downregulated the expression of anti-apoptotic proteins MCL-1, BCL-XL and phosphorylated ERK, thereby affecting the post-translational protein stability of MCL1, but not the transcriptional level. Conclusions The combination of Venetoclax and Entospletinib had significant synergistic killing effects in both AML cell lines and AML primary patient cells.Venetoclax in combination with Entospletinib exerts synergistic therapeutic effects on AML through down-regulation of ERK phosphorylation affecting the expression of anti-apoptotic proteins MCL-1, BCL-XL and reprogramming of glucose metabolism.

CSWT improves ventricular function by promoting angiogenesis through Rho/ROCK/ERK1/2 signal pathway in infarcted myocardium in rats

Abstract Objective This study aimed to investigate the effects of extracorporeal cardiac shock wave therapy (CSWT) on myocardial ischemia and cardiac function in rats with myocardial infarction, and to explore its impact on the expression of myocardial vascular endothelial growth factor and the extent of myocardial fibrosis, thereby elucidating the mechanism of angiogenesis. Methods A rat model of myocardial infarction was induced by ligating the left anterior descending branch of the coronary artery, confirmed by echocardiography. Animals were randomly assigned to five groups: sham operation group, myocardial infarction group, myocardial infarction + CSWT group, myocardial infarction + Y-27632 group, and myocardial infarction + CSWT + Y-27632 group. Cardiac function was evaluated by echocardiography on the 16th and 72nd day post-surgery. Myocardial morphology was assessed by HE staining, and the extent of myocardial fibrosis was determined by Masson's trichrome staining. Immunohistochemical staining for factor VIII was conducted to observe angiogenesis in each infarcted area. Western blotting was employed to detect the protein expression of extracellular regulated kinase (ERK1/2) and pro-angiogenic cytokines in the infarct border zone. Results CSWT treatment significantly reduced myocardial fibrosis and improved cardiac function compared to the myocardial infarction group. Additionally, CSWT upregulated ERK expression and angiogenesis-related factors, promoting angiogenesis. The beneficial effects of CSWT were abolished by inhibiting the Rho/ROCK/ERK1/2 pathway. Furthermore, the area of myocardial fibrosis was significantly reduced in the MI+Y-27632 group compared to the myocardial infarction group (p &amp;lt; 0.01), and left ventricular systolic function index was significantly higher in the myocardial infarction group. Conclusions CSWT may promote angiogenesis post-myocardial infarction by activating the Rho/ROCK/ERK1/2 pathway. Additionally, Y-27632 may improve cardiac function in rats with myocardial infarction, potentially through mitigating myocardial fibrosis.echo images of ratsprotein expression by WB

The potential role of Circular RNA New biomarkers for the prognosis of hepatocellular carcinoma

Abstract Introduction/Objective Hepatocellular carcinoma (HCC) is the most commonly diagnosed cancer with related deaths worldwide. The non-coding RNAs as the functions of circRNAs in tumor progression have attracted great attention to the clinicopathological characteristics of HCC. Methods/Case Report The present study was conducted to investigate and evaluate the expression level of hsa_circ_2124, and hsa_circ_4018 downstream target gene pathways related to clinico-pathological parameters of HCC. Cell proliferation was examined by West-1 assay and protein expression levels by using western blotting analysis. Results (if a Case Study enter NA) The hsa_circ_2124 expression level was up-regulated in HepG2 and Hep3B cells, respectively compared to normal liver cells. Furthermore, hsa_circ_4018 was down-regulated in two HCC cell lines compared to the normal cell line (LO2 cells), demonstrating the heterogeneity characteristics of the circRNA expressions in different HCC cell lines. Our data showed a significant inhibition of hsa_circ_4018 in HCC cell lines which in turn inhibited cell proliferation and migration. Besides, there is an inhibition in cell proliferation after hsa_circ_2124 knockdown in the tested cell lines. In addition, our data reported that the Knockdown of hsa_circ_2124 reduced the expression of ERK, c-Myc, and β-catenin expression in Hep-G2 cells. Furthermore, hsa_circ_4018 showed significant inhibition in β-catenin and c-Myc expression levels in Hep3B cells compared to the control. The knockdown of hsa_circ_2124 induced G1-phase arrest in HepG2 cells compared to control, suggesting the role of hsa_circ_2124 in the inhibition of HCC progression. Conclusion All in all, our data revealed that hsa_circ_2124 or hsa_circ_4018 plays a role in the regulation of MAPK and Wnt/β-catenin signaling pathways, which might be potential therapeutic biomarkers for HCC.

MAPK pathway and NIS in B-CPAP human papillary thyroid carcinoma cells treated with resveratrol

BackgroundResveratrol, a herbal phytoalexin, is known to have anti-tumor effects in several tumors including thyroid cancer cells. AimThe aim of this study was to determine the effects of resveratrol on the expression of BRAF, ERK and NIS mRNA levels and protein expression in B-CPAP human thyroid papillary cancer cell line. MethodsB-CPAP cells were treated with resveratrol at concentrations of 10–100 μM for 24–48–72 h. Cell viability was assessed by XTT Cell Proliferation Assay. BRAF, ERK and NIS mRNA levels were evaluated by rt-PCR method. Protein expressions were evaluated by Western Blot method. ResultsResveratrol was found to inhibit cell proliferation in a time and dose dependent manner. The IC50 values of resveratrol were 18.7 μM and 56.8 μM after 48 h and 72 h respectively. Resveratrol treatment of B-CPAP cells resulted in up to 1.5-fold reduction in BRAF mRNA and up to 5.5 fold reduction in ERK mRNA levels. NIS mRNA levels showed up to 3-fold increase. Western Blot studies confirmed the rt- PCR results with a decrease in BRAF and ERK, and increase in NIS protein expressions. ConclusionThis study demonstrated that resveratrol inhibits thyroid papillary carcinoma cell proliferation and reduces poor prognostic BRAF and ERK mRNA and protein expressions, while increasing NIS mRNA and protein expression suggesting a redifferentiating effect. More studies are needed to evaluate resveratrol as a novel therapeutic agent in the treatment of papillary thyroid cancer.

N6‑methyladenosine methyltransferase METTL14 is associated with macrophage polarization in rheumatoid arthritis.

Rheumatoid arthritis (RA) is largely caused by the inflammatory response triggered by macrophage polarization. Through epigenetic reprogramming, the inflammatory state of macrophages can be modified. Macrophage polarization is associated with the RNA epigenetic alteration N6-methyladenosine (m6A) RNA methylation. However, the specific function and underlying mechanisms of m6A methylation in the role of macrophage polarization in RA remain to be elucidated. The mRNA expression levels of m6A methylase genes and signaling pathway components associated with RA macrophages were determined in the present study using reverse-transcription quantitative PCR. Methyltransferase 14 (METTL14) protein expression levels were determined using western blot analysis, and the levels of specific cellular secretion factors were determined using ELISA and flow cytometry. The results of the present study demonstrated that elevated METTL14 expression was associated with joint tenderness, and METTL14 expression was positively correlated with both C-reactive protein and rheumatoid factor expression levels. Moreover, METTL14 exhibited potential in the prediction of visual analogue scale. Pro-inflammatory cytokines (TNF-α) and M1 macrophage markers (CD68+CD86+) were also positively associated with METTL14 expression. The results of the Kyoto Encyclopedia of Genes and Genomes analysis revealed that METTL14 was strongly associated with the MAPK signaling pathway. Notably, JNK and ERK2 exhibited a positive correlation with the M1 macrophage marker, CD68+CD86+, which was positively associated with the pro-inflammatory factor, TNF-α. JNK and ERK2 expression levels were markedly increased in the METTL14 high-expression group, compared with in the low-expression group; however, p38 and ERK1 expression levels were not significantly different between these groups. Collectively, the results of the present study demonstrated that METTL14 expression was significantly increased in the peripheral blood and synovial tissue of patients with RA, highlighting the potential association with both immunoinflammatory markers and clinical symptoms. In addition, it was suggested that METTL14 may exacerbate the downstream inflammatory response, through mediating macrophage polarization via the MAPK pathway.

Systemic corticosterone enhances fear memory extinction in rats: Involvement of the infralimbic medial prefrontal cortex GABAA and GABAB receptors.

The infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) regulates the extinction of conditioned fear memory. Glucocorticoid and gamma-aminobutyric acid (GABA) receptors are expressed in the mPFC and are also critical in fear extinction. This study investigated the possible interactive effects of the glucocorticoids and GABAergic system in the IL on the regulation of fear extinction. The rats were trained using an auditory fear conditioning task during which they received three conditioned stimuli (tones, 30s, 4kHz, 80dB), co-terminated with the three unconditioned stimuli (footshock, 0.8mA, 1s). Extinction testing was conducted over 3 days (Ext 1-3). Thirty minutes before the first extinction trial (Ext 1), the rats received bicuculline (BIC, 1mg/kg/2mL, intraperitoneal [i.p.]) as a GABAA receptor antagonist or CGP55845 (CGP, 0.1mg/kg/2ML, i.p.) as a GABAB receptor antagonist followed by systemic injection of corticosterone (CORT, 3mg/kg/2ML, i.p.). Furthermore, separate groups of rats received a bilateral intra-IL injection of BIC (100ng/0.3µL/side) or CGP (10ng/0.3µL/side) followed by a systemic injection of CORT (3mg/kg/2ML, i.p.) before the first extinction trial (Ext 1). The extracellular signal-regulated kinase (ERK1) and cAMP response element-binding (CREB) activity in the IL was examined by Western blot analysis after Ext 1. The results indicated that systemic CORT injection facilitated fear extinction and increased the expression of ERK1 but not CREB in the IL. Both systemic and intra-IL co-injection of BIC or CGP blocked the effects of CORT on fear extinction and ERK1 expression. These findings suggest that glucocorticoids and the GABAergic system may modulate fear extinction through the ERK pathway in the IL.

Sea Buckthorn Oil Promotes the PI3K-Akt-ERK Signaling Pathway and Macrophage M2 Polarization to Reduce Radiation-induced Skin Injury.

In this work, we explored the role and mechanism of sea buckthorn oil in reducing radiation-induced skin damage. The radiation-induced rat skin injury model was established using strontium-90. Rats were treated with sea buckthorn oil twice a day postirradiation, and skin damage was observed at different times and evaluated using an injury score. Skin pathological changes were observed using hematoxylin and eosin (H&E) staining. Western blotting and immunohistochemistry were used to detect the expression of vascular growth and pathway proteins. ELISA was used to detect the secretion level of inflammatory factors. Immunohistochemistry was used to detect macrophage polarization marker proteins. We found that sea buckthorn oil can alleviate radiation-induced skin damage, accelerate skin vascular regeneration, and promote the up-regulation of vascular endothelial growth factor (VEGF) and its receptor (VEGFR). These results demonstrate the beneficial effects of sea buckthorn oil on radiation-induced skin damage. Furthermore, the levels of IL-1β and TNF-α in the sea buckthorn oil treatment group were significantly lower than those in the control group, while the levels of IL-4 and IL10 were significantly higher (P < 0.05). CD206 expression also increased in the sea buckthorn oil treatment group, while CD16 expression decreased compared to the control group (P < 0.05). Western blotting showed that PI3K, Akt and ERK expression increased in the sea buckthorn oil treatment group (P < 0.05). The beneficial effect of sea buckthorn oil in reducing the inflammatory response in irradiated rats was diminished when they were treated with PI3K inhibitor. We conclude that sea buckthorn oil may regulate macrophage M2 polarization by increasing the PI3K-Akt-ERK signaling pathway, thereby inhibiting the inflammatory response and promoting skin vascular regeneration to prevent and treat radiation-induced skin damage.

Metabolomics and 16S rDNA sequencing of intestinal flora reveal the regulation of Sparassis latifolia polysaccharides on splenic immune function in lead-exposed mice

Sparassis latifolia polysaccharides (SLPs) have immunomodulatory activity and lead excretion ability, but its regulatory mechanism through the gut microbiota-spleen axis has not been elucidated. In this study, spleen metabolomics and intestinal flora sequencing were combined to explore the regulatory mechanism of SLPs on spleen immune function in lead-exposed mice. The results showed that SLPs effectively reduced spleen lead content, alleviated spleen enlargement and oxidative stress. SLPs changed glycerophospholipid metabolism, increased lysophosphatidylcholine content and inhibited the expression of G2A, ERK2 and NF-kB genes and the phosphorylation of ERK2 and NF-kB in lead-exposed mice. Furthermore, SLPs inhibited potential intestinal pathogens such as Clostridium_sensu_stricto_1, Lachnospiraceae, Oscillospiraceae and Alistipes_indistinctus, which were positively correlated with phosphatidylethanolamine metabolites. In addition, SLPs reduced the spleen tissue damage of lead-exposed mice by co-housing, and reduced the relative abundance of Clostridium_sensu_stricto_1, Prevotellaceae, and RF39, which were positively correlated with spleen enlargement, and inhibited the expression of ERK2/NF-κB signaling pathway-related genes such as G2A, ERK2 and Fas. In summary, SLPs can reduce the relative abundance of pathogenic microorganisms by regulating the structure of intestinal flora, regulate the glycerophospholipid metabolism of spleen in lead-exposed mice, alleviate oxidative damage and inflammatory response, and restore spleen immune function.