p-p38MAPK Protein Expression Research Articles

Periplaneta americana extract CII-3 triggers cell senescence through activating ROS-p38 MAPK-p53 signaling pathway in SKOV3 cells

This study aimed to investigate effect of Periplaneta americana extract CII-3 (CII-3) in senescence of SKOV3 cells. Proliferation, colony forming and cell senescence of SKOV3 cells were determined. ROS production was evaluated by flow cytometry. Transcription of telomerase (TERT), p38 MAPK and p53 gene and protein expression of p-p38 MAPK and p-p53, were identified. CII-3 at different concentrations significantly inhibited SKOV3 proliferation, and 80 μg/ml demonstrated the highest inhibitory effect. CII-3 significantly blocked cell cycle in G0/G1 phase (P<0.01) and reduced colony forming efficiency (P<0.001) of SKOV3 cells compared to those in Control group. CII-3 significantly increased SA-β-Gal positive staining SKOV3 cells (P<0.001) and reduced mitochondrial membrane potential (P<0.01) compared to those in Control group. CII-3 markedly decreased TERT gene transcription of SKOV3 cells compared to that in Control group (P<0.001). CII-3 also triggered significantly higher ROS levels in SKOV3 cells compared to that in Control group (P<0.001). CII-3 significantly increased p-p38 MAPK (P<0.001), p-p53 (P<0.001) and p21 (P<0.001) expressions of SKOV3 cells compared to those in Control group. In conclusion, CII-3 triggered cell senescence of SKOV3 cells through activating ROS-p38 MAPK-p53 signaling pathway. This study would provide a promising strategy for inhibiting cancer cell proliferation by including cell senescence.

Effect of acupoint catgut embedding on p38 MAPK pathway in the lung tissue of asthmatic rats.

To observe the effect of catgut embedding at "Feishu"(BL13), "Dingchuan" (EX-B1) and "Danzhong" (CV17) on expression of phosphorylated p38 mitogen activated protein kinase (p-p38 MAPK), interleukin-4 (IL-4), interferon-γ (IFN-γ) and changes of airway epithelial cells (AEC) in the lung tissue of bronchial asthma (BA) rats, so as to explore its mechanisms underlying improvement of BA. Forty male Wistar rats were randomly and equally divided into blank control, model, dexamethasone (DEX) and catgut embedding groups. The BA model was established by intraperitoneal injection of suspension of ovalbumin and aluminum hydroxide. Rats of the DEX group received intraperitoneal injection of DEX (1.5 mg/kg), once daily for 2 weeks, and those of the catgut embedding group received catgut embedding at BL13, EX-B1 and CV17 only one time. The rats' sneezing times per miniute in each group were recorded. H.E. staining was used to observe the histopathological changes of the lung tissue under light microscope. A transmission electron microscope (TEM) was used to observe the ultrastructural changes of AEC in the lung tissue, including the thickness of bronchial wall and bronchial smooth muscle by using an image analysis software. The protein expressions of p-p38 MAPK, IL-4 and INF-γ in the lung tissue were determined using Western blot. Morphological observation revealed that in the model group, light microscope showed deformed and swollen bronchial tube wall with increased folds and thickened bronchial smooth muscle；and TEM showed a large number of autophagy vesicles containing swollen and deformed organelles in the AEC, and apparent reduction of intracellular mitochondria, these situations were obviously milder in both DEX and catgut embedding groups. Compared with the blank control group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle in the model group were significantly increased (P<0.01), and the expressions of p-p38 MAPK and IL-4 in lung tissue were significantly increased (P<0.01), while the expression of IFN-γ was significantly decreased (P<0.01) in the model group. In comparison with the model group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle, protein expressions of p-p38 MAPK and IL-4 were significantly decreased (P<0.01), while the expression of IFN-γ was obviously increased (P<0.01) in both the DEX and catgut embedding groups. Acupoint catgut embedding can reduce the expression of IL-4 and increase the expression of IFN-γ by inhibiting p38 MAPK signal pathway of lung tissues in BA rats, which may contribute to its effect in alleviating the degree of airway epithelial cells damage.

Effects of PM2.5 exposure on clock gene BMAL1 and cell cycle in human umbilical vein endothelial cells.

Fine particulate matter (PM2.5) exposure has been closely associated with cardiovascular diseases, which are relevant to cell cycle arrest. Brain and muscle aryl-hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) not only participates in regulating the circadian clock but also plays a role in modulating cell cycle. However, the precise contribution of the circadian clock gene BMAL1 to PM2.5-induced cell cycle change remains unclear. This study aims to explore the impact of PM2.5 exposure on BMAL1 expression and the cell cycle in human umbilical vein endothelial cells (HUVECs). HUVECs was exposed to PM2.5 for 24 hours at different concentrations ((0, 12.5, 25, 75 and 100 μg.mL-1) to elucidate the potential toxic mechanism. Following exposure to PM2.5, cell viability, ROS, cell cycle, and the expression of key genes and proteins were detected. A remarkable decrease in cell viability is observed in the PM2.5-exposed HUVECs, as well as a significant increase in ROS production. In addition, PM2.5-exposed HUVECs have cycle arrest in G0/G1 phase, and the gene expression of p27 is also markedly increased. The protein expression of BMAL1 and the gene expression of BMAL1 are increased significantly. Moreover, the protein expressions of p-p38 MAPK and p-ERK1/2 exhibit a marked increase in the PM2.5-exposed HUVECs. Furthermore, following the transfection of HUVECs with siBMAL1 to suppress BMAL1 expression, we observed a reduction in both the protein and gene expression of the MAPK/ERK pathway in HUVECs exposed to PM2.5. Overall, our results indicate that PM2.5 exposure significantly upregulates the circadian clock gene expression of BMAL1 and regulates G0/G1 cell cycle arrest in HUVECs through the MAPK/ERK pathway, which may provide new insights into the potential molecular mechanism regarding BMAL1 on PM2.5-induced cardiovascular diseases.

Riboflavin deficiency reduces bone mineral density in rats by compromising osteoblast function

Insufficient riboflavin intake has been associated with poor bone health. This study aimed to investigate the effect of riboflavin deficiency on bone health in vivo and in vitro. Riboflavin deficiency was successfully developed in rats and osteoblasts. The results indicated that bone mineral density, serum bone alkaline phosphatase, bone phosphorus, and bone calcium were significantly decreased while serum ionized calcium and osteocalcin were significantly increased in the riboflavin-deficient rats. Riboflavin deficiency also induced the reduction of Runx2, Osterix, and BMP-2/Smad1/5/9 cascade in the femur. These results were further verified in cellular experiments. Our findings demonstrated that alkaline phosphatase activities and calcified nodules were significantly decreased while intracellular osteocalcin and pro-collagen I c-terminal propeptide were significantly increased in the riboflavin-deficient osteoblasts. Additionally, the protein expression of Osterix, Runx2, and BMP-2/Smad1/5/9 cascade were significantly decreased while the protein expression of p-p38 MAPK were significantly increased in the riboflavin-deficient cells compared to the control cells. Blockage of p38 MAPK signaling pathway with SB203580 reversed these effects in riboflavin-deficient osteoblastic cells. Our data suggest that riboflavin deficiency causes osteoblast malfunction and retards bone matrix mineralization via p38 MAPK/BMP-2/Smad1/5/9 signaling pathway.

Effects of muscle derived-IL-6 on insulin resistance of skeletal muscle cells and its mechanisms

To study the effects of exogenous calcium-load on promoting muscle-derived IL-6 secretion, and regulating AMPK and p38MAPK signal pathway to improve insulin resistance. C2C12 cell lines and palmitic acid-induced insulin resistance C2C12 cell lines were selected as the experimental objects. Preliminary experiment was aimed to determinate the glucose concentrations of culture solutions and observe contraction status of cells under microscope following different calcium concentrations culture 24 h. In the first official experiment, cells were divided into four groups: control group (A group, normal culture solution), IR group(B group, 0.6 mmol/L palmitic acid culture cells 24 h), 1 000 ng/ml IL-6 culture IR B group cells 48 h(IL-6+IR group) and IL-6 shRNA culture A group cells (IL-6shRNA group). In the second official experiment, cells were divided into three groups: IR group(A group), 100 μmol/L CaCl2 culture IR group cells 48 h(CaCl2+IR group) and 100 μmol/L CaCl2 and IL-6shRNA co- culture IR group cells 48 h(CaCl2+IL-6shRNA+IR group). The expression levels of GLUT4 mRNA and IL-6 mRNA were measured by real-time PCR, the protein expression levels of p-AMPK, p-p38MAPK, p-IRS-1 and p-PI-3K were measured by Western blot. Preliminary experiment results showed that compared with 0 μmol/L CaCl2 group, the glucose concentrations were decreased significantly after cells treated with CaCl2, at different concentrations. The cell contractions were observed under microscope and the cell contraction was most obvious treated with 100 μmol/L CaCl2. The first official experiment results showed that compared with IR group, the contents of p-AMP-activated protein kinase(p-AMPK), p-insulin receptor substrate 1(p-IRS-1), p-phosphoinositide-3 kinase(p-PI-3K), the expression level of glucose transporter 4(GLUT4) mRNA and the glucose uptake of IL-6+IR group were increased significantly(P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was decreased significantly (P＜0.01) ; Compared with control group, the expression levels of p-AMPK, P-IRS-1, p-PI-3K, the expression level of GLUT4 mRNA and the glucose uptake of IL-6shRNA group were decreased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was increased significantly (P＜0.01). The second official experiment results showed that compared with IR group, the expression levels of p-AMPK, P-IRS-1, p-PI-3K, the level of GLUT4 mRNA of CaCl2+IR group were increased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was decreased significantly (P＜0.01); Compared with CaCl2+IR group, the contents of p-AMPK, P-IRS-1, p-PI-3K, the expression level of GLUT4 mRNA and the glucose uptake of CaCl2+IL-6 shRNA+IR group were decreased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was increased significantly (P＜0.01). Exogenous Ca-load can stimulate muscle cells contraction, and exercise-induced IL-6 improves insulin resistance by activating AMPK, PI-3Kand inhibiting p38MAPK signal pathway.

Effect of beraprost sodium on renal function and p38MAPK signaling pathway in rats with diabetic nephropathy

Purpose: To investigate the effect of beraprost sodium (BPS) on renal function and P38MAPK pathway in diabetic nephropathy (DN) rats.Methods: Sprague Dawley (SD) rats (n = 30) were randomly divided into three groups, viz, normal control (NC), diabetic nephropathy (DN) and beraprost sodium (BPS). Creatinine (Cr), blood urea nitrogen (BUN) and fasting blood glucose (FBG), were determined by Hitachi 7020 automatic biochemical analyzer, while low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG) and total cholesterol (TC) were measured by Olympus 400 automatic biochemical analyzer. Western blot analysis was performed to examine protein expression. Interleukin-6 (IL-6), hs-CRP, and TNF-α levels were evaluated using enzyme linked immunosorbent assay (ELISA).Results: After 8 weeks of treatment, renal function indices (urine output, KW/BW, UAlb/24 h, Cr and BUN), blood lipid indices (FBG, LDL-C, TG and TC) and inflammatory factors levels (IL-6, hs-CRP and TNF-α) in DN group were higher than NC group (p &lt; 0.05). In BPS group, renal function and blood lipid indices and inflammatory factor levels decreased when compared to DN group (p &lt; 0.05). Furthermore, BPS inhibited the protein expression of p-P38MAPK, TGF-β1 and COX-2.Conclusion: Beraprost sodium improves renal function in DN rats by inhibiting P38MAPK signalingpathway.

Resveratrol Treats UVB-Induced Photoaging by Anti-MMP Expression, through Anti-Inflammatory, Antioxidant, and Antiapoptotic Properties, and Treats Photoaging by Upregulating VEGF-B Expression.

UVB exposure is one of the primary factors responsible for the development of photoaging, and the aim of this study was to investigate the mechanism involved in the photoprotective properties of resveratrol (RES) in UVB-induced photoaging. Photoaging models of Hacat cells and ICR mice were established by UVB irradiation. The effect of RES on cell viability was then assessed using the MTT assay. The effect of RES on reactive oxygen species (ROS) production was detected through a fluorescent probe assay. The effect of RES on oxidized glutathione (GSSH) content, and superoxide dismutase (SOD) activity in photoaging Hacat cells, were measured separately, using kits. An enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of RES on IL-6 secretion. The effect of VEGF-B on RES photoprotection was examined through the RT-qPCR method, after silencing VEGF-B through siRNA transfection. For animal experiments, the relative water content of the skin of ICR mice was determined using the Corneometer CM825 skin moisture tester. Starting from the third week of the study, the back skin of photoaging ICR mice was photographed weekly using the TIVI700 camera, and the depth of skin wrinkles in photoaging ICR mice was also analyzed. The thickness of the epidermis in photoaging ICR mice was assessed by the hematoxylin-eosin (HE) staining method. The content of collagen fibers in the skin dermis of photoaging ICR mice was measured by the Masson trichrome staining method. The content of collagen III in the dermis of the skin in photoaging ICR mice was measured through immunohistochemistry (IHC) techniques. The effect of RES on the mRNA expression levels of MMP-1, MMP-9, HO-1, GPX-4, IL-6, TNF-α, VEGF-B, caspase9, and caspase3 in photoaging Hacat cells, and that of MMP-3, Nrf2, HO-1, NQO1, SOD1, GPX-4, caspase9, caspase3, and IL-6 in the skin of photoaging ICR mice, was measured by RT-qPCR. The effects of RES on caspase3, Nrf2 (intranuclear), COX-2, P-ERK1/2, ERK1/2, P-P38MAPK, and P38MAPK in photoaging Hacat cells, and on MMP-9, caspase3, COX-2, P-JNK, P-ERK1/2, and P-P38MAPK protein expression in the skin of photoaging ICR mice, were assayed by the WB method. The results of this study, therefore, show that RES has a protective effect against UVB-induced photoaging in both Hacat cells and ICR mice. Its mechanism of action may include reducing the expression of MMPs and the secretion of collagen and inflammatory factors by inhibiting the ROS-mediated MAPK and COX-2 signaling pathways, balancing oxidative stress in the skin of Hacat cells and ICR mice by promoting the Nrf2 signaling pathway, inducing antiapoptotic effects by inhibiting caspase activation, and exerting antioxidant and antiapoptotic effects by targeting the VEGF-B, demonstrating its photoprotective effects against UVB irradiation-induced photoaging.

Effect of Jinzhen granule on two coronaviruses: The novel SARS-CoV-2 and the HCoV-229E and the evidences for their mechanisms of action

BackgroundAlthough severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) pose a huge threat to human public health, no specific treatment is available. Jinzhen granule (JZ) is a traditional eight ingredients-Chinese medicine with prominent efficacy for treating viral-induced diseases. However, little is known about the antiviral effect and mechanism of JZ against SARS-CoV-2 and HCoV-229E. PurposeThis study aimed to reveal the antiviral effects of JZ against SARS-CoV-2 and HCoV-229E, and to further explore the underlying mechanisms regulating the host immune response. MethodsThe chromatographic separation of JZ was performed using a Shimadzu analytical high-performance liquid chromatograph with UV detection and Alltech ELSD 2000ES. We conducted cytopathic effect (CPE) and plaque reduction assays to evaluate the antiviral effect of JZ. A lethal human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 was established to determine the protective effect of JZ on mortality and lung virus titers. Real-time quantitative PCR assays were used to analyze the expression of proinflammatory cytokines in vitro and in vivo. Western blotting was further performed to determine the activities on regulating the nuclear factor kappa B (NF-κB)/MAPK pathway. Finally, mitochondrial membrane potential assays, flow cytometry analysis and western blotting were used to assess the anti-apoptotic potency toward HCoV-229E infection. ResultsThe results showed that 13 chemical components were identified and five peaks were determined and quantitated (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g). We found that JZ exerted inhibitory potency against SARS-CoV-2 and HCoV-229E in vitro by using CPE and plaque reduction assays, and it was further found that JZ protected mice infected by SARS-CoV-2 from death and inhibited lung virus titers. JZ also significantly decreased the induction of inflammatory cytokines (IL-1α, IL-6, CCL-5 and MIP-1β), similar to the observed in vitro effect. Moreover, JZ suppressed the release of inflammatory cytokines in vitro and it decreased the protein expression of p-p38 MAPK, p-JNK, p-NF-κB p65 and p-IκBα induced by HCoV-229E and increased the expression of IκBα. Notably, JZ significantly protected HCoV-229E-infected Huh-7 cells from mitochondrial damage and decreased apoptotic cells. The activation of the mitochondria-mediated apoptotic pathway was inhibited by JZ, as shown by the reduced expression of cleaved caspase-9, caspase-3 and p-PARP. ConclusionsIn conclusion, JZ (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g) exhibited antiviral activities against SARS-CoV-2 and HCoV-229E by regulating the NF-κB/MAPK pathway and the mitochondria-mediated apoptotic pathway. These findings demonstrated the efficacy of JZ against CoVs and suggested JZ treatment as a novel clinical therapeutic strategy for COVID-19.

Effect of Regorafenib on P2X7 Receptor Expression and Different Oncogenic Signaling Pathways in a Human Breast Cancer Cell Line: A Potential of New Insight of the Antitumor Effects of Regorafenib.

Background: Breast cancer is the most common malignancy in women worldwide. P2X7 is a transmembrane receptor expressed in breast cancer and activated by the ATP tumor microenvironment, driving cell proliferation, angiogenesis, and metastasis via different signaling pathways. The role of the P2X7 receptor, hypoxia, and autophagy in regulating tumor progression is controversial. The multikinase inhibitor regorafenib prevents the activation of numerous kinases involved in angiogenesis, proliferation, and metastasis. The present study aimed to evaluate the modulatory effect of regorafenib on the hypoxia/angiogenesis/P2X7R/autophagy axis on the MCF7 breast cancer cell line and its impact on different signaling pathways involved in breast cancer pathogenesis. Methods: The levels of VEGF, VEGFR, PI3K, NF-κB, HIF-1α, and LC3-II were analyzed using ELISA, and caspase-3 activity was also assessed colorimetrically. Phosphorylated (p)-p38 MAPK and purinergic ligand-gated ion channel 7 (P2X7) receptor protein expression levels were analyzed via Western blotting. Reverse transcription-quantitative PCR was used to determine the mRNA expression levels of Beclin 1 (BECN1), LC3-II, and sequestosome 1 (p62). Results: Regorafenib reduced MCF7 cell viability in a dose-dependent manner. Furthermore, regorafenib significantly reduced levels of PI3K, NF-κB, VEGF, VEGFR, P2X7 receptor, and p-p38 MAPK protein expression, and markedly reduced p62 mRNA expression levels. However, regorafenib significantly increased caspase-3 activity, as well as BECN1 and LC3-II mRNA expression levels. Conclusions: Regorafenib was demonstrated to possibly exhibit antitumor activity on the breast cancer cell line via modulation of the P2X7/HIF-1α/VEGF, P2X7/P38, P2X7/ERK/NF-κB, and P2X7/beclin 1 pathways.

An Integration of Network Pharmacology and Experimental Verification to Investigate the Mechanism of Guizhi to Treat Nephrotic Syndrome.

Background: Guizhi has the pharmacological activity of anti-inflammatory. However, the effect mechanism of Guizhi against nephrotic syndrome (NS) remains unclear. A network pharmacological approach with experimental verification in vitro and in vivo was performed to investigate the potential mechanisms of Guizhi to treat NS. Methods: Active compounds and potential targets of Guizhi, as well as the related targets of NS were obtained from the public databases. The intersecting targets of Guizhi and NS were obtained through Venny 2.1.0. The key targets and signaling pathways were determined by protein-protein interaction (PPI), genes ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis. And the overall network was constructed with Cytoscape. Molecular docking verification was carried out by AutoDock Vina. Finally, in vitro and in vivo experiments were performed to verify the mechanism of Guizhi to treat NS. Results: 63 intersecting targets were obtained, and the top five key targets mainly involed in NF- Kappa B and MAPK signaling pathway. In the overall network, cinnamaldehyde (CA) was the top one active compound with the highest degree value. The molecular docking showed that the top five key targets were of good binding activity with the active components of Guizhi. To in vitro experiment, CA, the main active component of Guizhi, inhibited the secretion of IL-1β, IL-6, TNF-α in LPS challenged RAW264.7 cells, and down regulated the protein expression of p-NF-κB p65 and p-p38 MAPK in LPS challenged RAW264.7 cells. In vitro experiment showed that, 24 urinary protein and renal function were increased in ADR group. To western blot, CA down regulated the protein expression of p-p38 MAPK in rats of adriamycin-induced nephropathy. Conclusion: CA might be the main active component of Guizhi to treat NS, and the underlying mechanism might mainly be achieved by inhibiting MAPK signaling pathway.

Nur77 Increases Glucose Transport in Mouse Skeletal Muscle Cells by Activating p38MAPK Under Lipotoxicity

Diabetes Mellitus (DM) is a common disease in clinic and belongs to a metabolic disease. Related studies have shown that Nur77 regulates sugar metabolism in a variety of tissue cells. Nur77 (orphan nuclear receptor) is a transcription factor and involves in glucose metabolism are different in skeletal muscle cells via p38MAPK signaling pathway. In the study, an IR model was to explore the mechanism of skeletal muscle IR. L6 myocytes were cultured and control, Nur77 knockout and Nur77 overexpression group was set followed by analysis of L6 muscle cell morphology, activity by MTT assay, glucose concentration by glucose oxidase-peroxidase (GOD-POD) p-p38MAPK and skeletal muscle glucose transporter-4 (GLUT4) level by western blot. After 8 to 12 hours of culture, the boundaries between cells were unknown with irregular order and no muscle-shaped structure. The cell morphology was mainly spindle-shaped and triangular. After 2 to 3 days, cell arrangement was regular and morphology was mainly spindle-shaped. The cell activity and glucose concentration in control group at different time points showed no differences which were significantly different in knockout and overexpression group with significantly higher cell activity and glucose concentration for overexpression group than knockout and control group (P &lt; 0.05). In addition, overexpression group also showed significantly increased expression of p-p38MAPK4 and GLUT4. Nur77 can increase glucose transport under lipotoxic state by activating p38MAPK signaling pathway and increasing the protein expression of p-p38MAPK and GLUT4 in muscle cells.

Protective Effect of Isoflurane on Myocardial Ischemia-Reperfusion Injury in Rats Through P38MAPK Signaling Pathway

Objective: Abnormal p38 MAPK activation involves in ischemia-reperfusion (IR) injury. Isoflurane (ISO) is a clinically used inhaled anesthetic and protects myocardial I-R injury. Our study assessed whether ISO exerts a protective role in myocardial I-R injury. Methods: Rat myocardial I-R injury model was set followed by analysis of p-p38 MAPK expression in myocardial tissue by western blot, caspase-3 activity, as well as MDA and SOD content. Rats were assigned into Sham group, IR group, I-R + ISO group, followed by measuring p-p38 MAPK expression, caspase-3 activity, MDA and SOD, cell apoptosis and ROS content. Results: Compared with Sham group, MDA content, caspase-3 activity and p-p38 MAPK protein expression as well as ROS content and apoptosis rate in I-R model rats were significantly increased and SOD activity was significantly decreased. ISO pretreatment significantly reduced MDA content, caspase-3 activity, ROS content and apoptosis rate in I-R model rats, increased SOD activity and reduced p-p38 MAPK expression. Conclusion: Activation of p38MAPK signaling plays a role in mediating myocardial IR injury and cardiomyocyte apoptosis. ISO pretreatment inhibits oxidative stress and cardiomyocyte apoptosis and protects myocardial IR injury via inhibiting p38MAPK signaling.

Plantamajoside modulates the proliferation, stemness, and apoptosis of lung carcinoma via restraining p38MAPK and AKT phosphorylation.

BackgroundPlantamajoside (PMS), an active anti-inflammatory component and antioxidant derived from Herba Plantaginis, has been reported to exert a suppressive effect in liver cancer in vivo. In this study, we tested the effects of PMS on the metastatic 95D cell line.Methods95D cells were characterized as most sensitive to PMS across several lung cancer cell lines. Cell viability within 24 h was tested with CCK-8. Different concentrations of PMS (0, 50, 100, and 200 µg/mL) and 5 µg/mL of cisplatin were established for later 24 h treatment. Relative mRNA and protein expression were assessed with PCR and Western blotting. Cell proliferation and stemness were indicated with colony and sphere formation. Cell metastasis was evaluated with wound healing and Transwell. Apoptotic cells and mitochondrial membrane potential were investigated with flow cytometry.ResultsCCK-8 assay showed PMS to inhibit the viability of 95D cells in a dose-dependent manner. PMS decreased colony formation and inhibited stemness in 95D cells. Invasion and migration were also inhibited. Moreover, PMS induced cell apoptosis, and decreased mitochondrial membrane potential. All of these effects were dose dependent. Interestingly, PMS treatment reduced the protein expression of p-p38 MAPK and p-AKT but not that of p38 MAPK and AKT.ConclusionsPMS inhibited proliferation, stemness, and migration, and initiated apoptosis in 95D cells, possibly through p38 MAPK and AKT dephosphorylation and mitochondria dysfunction. These findings support the promise of PMS as a prodrug in lung cancer treatment.

Effect of electroacupuncture on the morphological changes of the spinal dorsal horn and the expression of p38 mitogen-activated protein kinase in the injured spinal cord of rats with neuropathic pain

To investigate the effect of electroacupuncture stimulation at "Zusanli"(ST36) and "Kunlun"(BL60) on the morphological changes of the spinal dorsal horn and the expression of p38 mitogen-activated protein kinase (p38MAPK) in the injured spinal cord of rats with neuropathic pain. Male Sprague-Dawley rats were randomly divided into sham model group, model group, electroacupuncture group, and medication group, with 10 rats in each group. Spinal nerve ligation of the L5 lumbar vertebra was performed to establish a rat model of neuropathic pain. The rats in the electroacupuncture group were given electroacupuncture at ST36 and BL60 of the operation side with dilatational wave at a frequency of 2 Hz/100 Hz and an intensity of 1.5 mA once a day, 30 minutes each time, and those in the medication group were given intraperitoneal injection of 100 mg/mL Gabapentin solution (100 mg/kg) once a day; the one-week intervention was started at one week after surgery. Mechanical withdraw threshold (MWT) and thermal withdrawal latency (TWL) were observed and recorded before modeling and on days 1,3,5,7,10,12 and 14 after modeling, and the motor function of the affected hindlimb was scored. Methenamine silver stain was used to observe the morphological changes of the spinal dorsal horn, and Western blot was used to measure the relative protein expression of p38MAPK and phospho-p38MAPK(p-p38MAPK) in L4-L6 spinal segments. Compared with the sham model group, the model group had significant reductions in MWT and TWL at each time point (P<0.001) and a significant increase in motor function score (P<0.001); compared with the model group, the electroacupuncture group and the medication group had significant increases in MWT and TWL and a significant reduction in motor function score after treatment (P<0.05). The model group had obvious neuronal fibrillary tangles, particle vacuolar degeneration, and vacuoles containing argyrophilic grains in the cytoplasm of neural cells under a light microscope, while there were fewer neuronal fibrillary tangles in the electroacupuncture group and reduced vacuolar degeneration in the medication group. Compared with the sham model group, the model group had significant increases in the protein expression of p-p38MAPK in the spinal dorsal horn (P<0.001), and compared with the model group, the electroacupuncture group and the medication group had significant reductions in the protein expression of p-p38MAPK in the spinal dorsal horn(P<0.05). Electroacupuncture stimulation at ST36 and BL60 can increase pain threshold, improve the motor function of the affected hindlimb, and improve the necrosis of neurofibrils in the spinal dorsal horn in rats with neuropathic pain, possibly by regulating the expression of p-p38MAPK in the spinal dorsal horn.

Activation of p38MAPK in spinal microglia contributes to autologous nucleus pulposus-induced mechanical hyperalgesia in a modified rat model of lumbar disk herniation

Recent studies have implicated the activation of p38 mitogen-activated protein kinase (MAPK) and glial cells contribute to hyperalgesia following nerve injury or nerve compression. In our work, we investigated the underlying mechanisms of autologous nucleus pulposus (NP)-induced mechanical hyperalgesia in a modified rat model of lumbar disk herniation (LDH). Firstly, our results showed that 50% mechanical withdrawal threshold (50% MWT) decreased on postoperative day (POD) 1 and significantly minimally reduced on POD 7 and lasted for day 28 after surgery (P < 0.05). Secondly, phosphorylation of p38MAPK (p-p38MAPK) and glial cells were monitored on POD 1, 3, 7, 14 and 28 using immunofluorescence staining. P38MAPK activation, observed in the spinal cord, began to increase on POD 1, peaked on POD 3, and significantly decreased on POD 14 and POD 28 (P < 0.05). Microglia activation was initiated at day 1, maximal at day 3, and maintained until day 14 after surgery (P < 0.05). Astrocytic activation was found in 7 to 14 days after modelling (P < 0.05). Then, double immunostaining method was applied to observe the co-expression of p-p38MAPK and glial cells, and it showed that p-p38MAPK was mainly expressed in activated microglia, rarely in neurons, and none in astrocytes. Lastly, we discovered that both SB203580 (50ug, p38MAPK inhibitor) and minocycline (0.5 mg, microglial inhibitor) would inhibit the p-p38MAPK protein expression tested by western blot analysis and reduce mechanical hyperalgesia. In conclusion, current study suggest that activation or phosphorylation of p38MAPK in spinal microglia contributes to autologous NP-induced mechanical hyperalgesia in our animal model.

Effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury in newborn rats

To explore the effects of p38 MAPK signaling pathway on cognitive function and recovery of neuronal function after hypoxic-ischemic brain injury (HIBI) in newborn rats. Seventy-two healthy SPF grade SD newborn rats were randomly and equally divided into Normal group (healthy rats) and Sham group (rats underwent sham operation), Model group (HIBI model rats), p38 MAPK Inhibitor group (HIBI model rats treated with p38 MAPK inhibitor) and p38 MAPK Activator group (HIBI model rats treated with p38 MAPK activator). On postnatal day 28, Morris water maze, tail suspension test and inclined plane test were conducted on rats in each group. Twenty-four hours after modeling, the expression of p-p38 MAPK protein and apoptosis related genes in rat hippocampal tissues was detected by TUNEL staining, qRT-PCR and Western blot. Compared with Normal group, escape latency and inclined plane test time were prolonged, the number of passing through the platform and tail suspension time were reduced (all P < 0.05); Bax and Caspase-3 mRNA and protein expression levels and p-p38 MAPK protein level were increased, Bcl-2 mRNA level was decreased, and neuronal apoptosis proportion was increased in Model group (all P < 0.05). Compared with Model group, the above indicators showed reversed and enhanced trends in p38 MAPK Inhibitor and p38 MAPK Activator groups, respectively (all P < 0.05). Inhibition of p38 MAPK signaling pathway can effectively improve the learning and memory ability and motor function of newborn rats with HIBI, and reduce neuronal apoptosis in the hippocampal tissues, thereby promoting neuronal recovery.

Heat Shock Protein 70 Protects the Heart from Ischemia/Reperfusion Injury through Inhibition of p38 MAPK Signaling.

Background Heat shock protein 70 (Hsp70) has been shown to exert cardioprotection. Intracellular calcium ([Ca2+]i) overload induced by p38 mitogen-activated protein kinase (p38 MAPK) activation contributes to cardiac ischemia/reperfusion (I/R) injury. However, whether Hsp70 interacts with p38 MAPK signaling is unclear. Therefore, this study investigated the regulation of p38 MAPK by Hsp70 in I/R-induced cardiac injury. Methods Neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation for 6 h followed by 2 h reoxygenation (OGD/R), and rats underwent left anterior artery ligation for 30 min followed by 30 min of reperfusion. The p38 MAPK inhibitor (SB203580), Hsp70 inhibitor (Quercetin), and Hsp70 short hairpin RNA (shRNA) were used prior to OGD/R or I/R. Cell viability, lactate dehydrogenase (LDH) release, serum cardiac troponin I (cTnI), [Ca2+]i levels, cell apoptosis, myocardial infarct size, mRNA level of IL-1β and IL-6, and protein expression of Hsp70, phosphorylated p38 MAPK (p-p38 MAPK), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), phosphorylated signal transducer and activator of transcription3 (p-STAT3), and cleaved caspase3 were assessed. Results Pretreatment with a p38 MAPK inhibitor, SB203580, significantly attenuated OGD/R-induced cell injury or I/R-induced myocardial injury, as evidenced by improved cell viability and lower LDH release, resulted in lower serum cTnI and myocardial infarct size, alleviation of [Ca2+]i overload and cell apoptosis, inhibition of IL-1β and IL-6, and modulation of protein expressions of p-p38 MAPK, SERCA2, p-STAT3, and cleaved-caspase3. Knockdown of Hsp70 by shRNA exacerbated OGD/R-induced cell injury, which was effectively abolished by SB203580. Moreover, inhibition of Hsp70 by quercetin enhanced I/R-induced myocardial injury, while SB203580 pretreatment reversed the harmful effects caused by quercetin. Conclusions Inhibition of Hsp70 aggravates [Ca2+]i overload, inflammation, and apoptosis through regulating p38 MAPK signaling during cardiac I/R injury, which may help provide novel insight into cardioprotective strategies.

Neuroprotective Effects of Salidroside Against Beta-Amyloid-Induced Cognitive Impairment in Alzheimer’s Disease Mice Through the PKC/p38MAPK Pathway

Alzheimer’s disease (AD) is a common neurodegenerative disease as well as the main cause of dementia. A progressive cognitive decline with age is considered as the major manifestation of AD. Amyloid beta-peptide (Aβ) is one of the primary causes leading to cognitive dysfunction in AD. Recent studies have suggested that the activation of PKC/p38MAPK pathway is related to the neurotoxicity induced by β-amyloid. Salidroside is the major active component of Rhodiola crenu-lata, has been reported with widely neuroprotective effects. The protective effects of salidroside against β-amyloid induced neural apoptosis via the MAPKs pathway has been confirmed in the vitro study. The present study aimed to investigate the neuroprotective effects of salidroside through the PKC/p38MAPK pathway in β-amyloid induced AD mice. The results by Y maze showed that salidroside improved Aβ-induced cognitive impairment. Nissl staining results showed that salidroside affected neuronal damage in hippocampus and cerebral cortex of AD mice. Western blot results revealed that salidroside enhanced protein expression of p-PKC, whereas it suppressed protein expression of p-p38MAPK, Bax and cleaved caspase-3. Thus, the present results demonstrated that salidroside ameliorated cognitive dysfunction in Aβ25–35 induced AD mice. And the effects on protein expression of p-PKC and p-p38MAPK contributed to the neuroprotective effects of salidroside against neural apoptosis in AD mice.

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.

Effect of unfractionated heparin on high mobility group box 1-mediated expression of vascular endothelial cadherin

To observe the damage of high mobility group box 1 (HMGB1) on human umbilical vein endothelial cell (HUVEC) barrier permeability and the protective effect of unfractionated heparin (UFH), and to explore the down-regulated protection effect mechanism of UFH on HMGB1-mediated vascular endothelial cadherin (VE-cadherin) expression. The trypsin-digested HUVEC were subcultured in culture flasks. When the cells were grown to 80%, they were randomly divided into four groups: phosphate buffer (PBS) control group (200 μL PBS), recombinant human high mobility group box 1 (rhHMGB1) treatment group (100 μg/L rhHMGB1), UFH control group (10 kU/L UFH), and UFH pretreatment group (10 kU/L UFH+100 μg/L rhHMGB1). The cells in each group were challenged with different reagent for 24 hours, and the activity of endothelial cells was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The permeability of endothelial cells was measured by Transwell method, and the expression and distribution of VE-cadherin was observed by immunofluorescence. The protein expressions of VE-cadherin and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) were determined by Western Blot. After treatment with 100 μg/L rhHMGB1 for 24 hours, the activity of endothelial cells was not significantly different from that of the PBS control group (A value: 0.230±0.004 vs. 0.255±0.006, P > 0.05), but the permeability was significantly increased (glucan FD40 fluorescence intensity: 11.05±0.12 vs. 6.34±0.39, P < 0.05). Compared with PBS control group, the fluorescence microscopy showed that the VE-cadherin membrane localization was reduced, the distribution was loose, and there were obvious fissures between cells in rhHMGB1 treatment group, and quantitative analysis showed the protein expression of VE-cadherin was decreased significantly (VE-cadherin/β-actin: 0.16±0.04 vs. 0.31±0.03, P < 0.05), and the expression of p-p38MAPK protein was significantly increased (p-p38MAPK/β-actin: 0.79±0.03 vs. 0.26±0.05, P < 0.05). UFH pretreatment could protect HMGB1-mediated endothelial cell injury, cell permeability was significantly reduced (glucan FD40 fluorescence intensity: 9.11±0.23 vs. 11.05±0.12), fluorescence expression of VE-cadherin was enhanced, membrane localization was significantly increased, quantitative analysis showed that VE-cadherin protein expression was significantly up-regulated (VE-cadherin/β-actin: 0.24±0.02 vs. 0.16±0.04), and p38MAPK phosphorylation level was significantly decreased (p-p38MAPK/β-actin: 0.54±0.05 vs. 0.79±0.03), the difference was statistically significant as compared with rhHMGB1 treatment group (all P < 0.05). There was no significant difference in all parameters between PBS control group and UFH control group. UFH can protect the endothelial cell barrier from the HMGB1 by regulating the expression and distribution of VE-cadherin. The mechanism may be related to the inhibition of p38MAPK phosphorylation by UFH.