Expression Levels Of p-ERK Research Articles

Sinularin, an Anti-Cancer Agent Causing Mitochondria-Modulated Apoptosis and Cytoskeleton Disruption in Human Hepatocellular Carcinoma.

Liver cancer remains a leading cause of death, despite advances in anti-cancer therapies. To develop novel drugs, natural products are being considered as a good source for exploration. In this study, a natural product isolated from a soft coral was applied to evaluate its anti-cancer activities in hepatocellular carcinoma SK-HEP-1 cells. Sinularin was determined to have half-maximal inhibitory concentration (IC50) values of ~10 μM after 24, 48, and 72 h. The TUNEL assay and annexin V/PI staining results showed that sinularin induced DNA fragmentation and apoptosis, respectively. An investigation at the molecular level demonstrated that the expression levels of cleaved caspases 3/9 were significantly elevated at 10 μM sinularin. Mitochondrial and intracellular reactive oxygen species (ROS) levels were significantly increased following sinularin treatment, which also affected the mitochondrial membrane potential. In addition, it significantly lowered the mitochondrial respiration parameters and extracellular acidification rates at 10 μM. Further investigation showed that sinularin significantly attenuated wound healing, cell migration, and potential colony formation at 10 μM. Fluorescence microscopic observations showed that the distribution of F-actin filaments was significantly altered at 10 μM sinularin. Supported by Western blot analyses, the expression levels of AKT, p-ERK (extracellular-signal-related kinase), vimentin and VEGF were significantly down-regulated, whereas p-p38, pJNK and E-cadherin were significantly increased. Overall, at the IC50 concentration, sinularin was able to significantly affect SK-HEP-1 cells.

Activation of the extracellular-signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) signal pathway and osteogenic factors in subchondral bone of patients with knee osteoarthritis.

BackgroundThe objectives of this study was to explore the activation of the extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathway and osteogenesis-related factors in the subchondral bone of patients with knee osteoarthritis (OA).MethodsTen patients with primary OA who underwent total knee arthroplasty in the Department of Arthritis Surgery of our hospital were enrolled, and subchondral bone tissue samples were obtained during the operation. He staining and saffron staining were used to observe the arrangement of chondrocytes in the patient tissues. The protein expression levels of JNK, p-JNK, ERK, p-ERK, Runx2 and OMD in subchondral bone were detected by Western Blot. Knee osteoarthritis mice were established. He staining was used to observe the arrangement of subchondral bone cells in the knee joint of mice. Cellular mineralized nodules were determined by alizarin red staining.ResultsFirstly, in general and staining, it was observed that the subchondral bone lesions of knee OA participants were obvious. Compared with normal knee joints, the levels of phosphorylation-c-Jun N-terminal kinase (P-JNK) and phosphorylation-extracellular-signal-regulated kinase (P-ERK) in the subchondral bone of knee arthritis participants were significantly increased (P<0.05). The level of osteomodulin (OMD) was significantly reduced (P<0.05). Secondly, compared with normal mice, the levels of JNK, P-JNK, OMD, ERK, and P-ERK in the model group were significantly different (P<0.05). At 2–8 weeks, the JNK and P-JNK levels in the mice model group increased significantly over time (P<0.05), and the OMD level decreased significantly over time (P<0.05). The levels of ERK and P-ERK fluctuated over time. Thirdly, osteoblasts were treated with different concentrations of anisomycin, and stained with alizarin red after continuous culture for 24 and 48 h, respectively. It was found that all the cells were stained with orange-red mineralized nodules. As the concentration of anisomycin was increased, the number of cell mineralization nodules was significantly larger, and the positive rate of chemical nodules increased. Different concentrations of anisomycin were given to interfere with the osteoblasts of mice. When anisomycin was administered at a dose of 25 ng, the OMD level reached the highest level. When the concentration of anisomycin was increased, the osteocalcin (OCN) level also showed an upward trend.ConclusionsThe process by which the JNK signaling pathway regulates OMD may be closely related to the pathological changes of subchondral bone in patients with knee OA, and is involved in the occurrence and development of knee arthritis.

Icaritin Alleviates Glutamate-Induced Neuronal Damage by Inactivating GluN2B-Containing NMDARs Through the ERK/DAPK1 Pathway

Excitatory toxicity due to excessive glutamate release is considered the core pathophysiological mechanism of cerebral ischemia. It is primarily mediated by N-methyl-D-aspartate receptors (NMDARs) on neuronal membranes. Our previous studies have found that icaritin (ICT) exhibits neuroprotective effects against cerebral ischemia in rats, but the underlying mechanism is unclear. This study aims to investigate the protective effect of ICT on glutamate-induced neuronal injury and uncover its possible molecular mechanism. An excitatory toxicity injury model was created using rat primary cortical neurons treated with glutamate and glycine. The results showed that ICT has neuroprotective effects on glutamate-treated primary cortical neurons by increasing cell viability while reducing the rate of lactate dehydrogenase (LDH) release and reducing apoptosis. Remarkably, ICT rescued the changes in the ERK/DAPK1 signaling pathway after glutamate treatment by increasing the expression levels of p-ERK, p-DAPK1 and t-DAPK1. In addition, ICT also regulates NMDAR function during glutamate-induced injury by decreasing the expression level of the GluN2B subunit and enhancing the expression level of the GluN2A subunit. As cotreatment with the ERK-specific inhibitor U0126 and ICT abolishes the beneficial effects of ITC on the ERK/DAPK1 pathway, NMDAR subtypes and neuronal cell survival, ERK is recognized as a crucial mediator in the protective mechanism of ICT. In conclusion, our findings demonstrate that ICT has a neuroprotective effect on neuronal damage induced by glutamate, and its mechanism may be related to inactivating GluN2B-containing NMDAR through the ERK/DAPK1 pathway. This study provides a new clue for the prevention and treatment of clinical ischemic cerebrovascular diseases.

Psoralen induces liver injuries through endoplasmic reticulum stress signaling in female mice

Psoralen is the main coumarin component of Fructus psoraleae. Previously, we have found that psoralen induced hepatocytes apoptosis via PERK and ATF6 related ER stress pathways in vitro. In this study, we investigated the toxicity and ER stress induced by psoralen in female C57 mice. Mice were fed with 80 mg/kg of psoralen intra-gastrically for either 3, 7, or 21 days. Liver and kidney were weighed and their coefficients were calculated. The serum was isolated to examine the biochemical parameters including alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, alkaline phosphatase (ALP) activity, blood urea nitrogen (BUN), total bile acid (TBA), total bilirubin (TBIL), and creatinine (CRE). The transcription and expression of ER stress-related markers were determined by Wes-automated Protein Simple system, Western blot and RT-PCR. Psoralen administration for 3 days significantly increased liver coefficients but decreased kidney coefficients of mice. Histopathological examination showed minimal inflammatory cell foci and vacuolar degeneration in the liver. Besides, serum levels of ALT, TBA, BUN, and CRE were markedly altered by psoralen. Moreover, psoralen significantly increased expression and transcription levels of ER stress related markers, including Grp78, PERK, eIF2α, ATF4, IRE1α, ATF6, and XBP1. These results illustrated that psoralen induced liver injuries through ER stress signaling in female mice.

4-Phenylbutyric acid protects islet \u03b2 cell against cellular damage induced by glucocorticoids

This study, using the MIN6 cell line, examines the effect of glucocorticoids (GCs) on the expression and protein levels of endoplasmic reticulum stress (ERS) related genes. Furthermore, we evaluated the protective role of 4-phenylbutyric acid (4-PBA) on the aforesaid GCs induced changes. Pancreatic islet MIN6 cells were treated with dexamethasone (DEX) at distinct concentrations (0.1 μmol/L and 0.5 μmol/L) for different periods (1 h, 4 h, 12 h, and 24 h). The mRNA and protein levels of ERS related genes were measured using real-time qPCR (qRT-PCR) and western blotting. Similar evaluations were also carried out for the cells treated with 4-PBA combined with DEX. Upon DEX intervention which induces the unfolded protein response (UPR), the expression levels of BIP, ATF6, IRE1, and PERK increased in the MIN6 cells, both in concentration and time-dependent manner. Similarly, ERS associated gene CHOP, which is involved in the apoptotic pathway, also showed increased levels both in concentration and time-dependent manner. However, treatment with 4-PBA decreased the expression levels of ERS related proteins. Quantitative analysis found that all these results were statistically significant (P < 0.05). GCs markedly activates the ERS in the MIN6 cell line in vitro, however, this effect can be significantly alleviated upon treatment with 4-PBA.

Inhibition of extracellular signal-regulated kinase/calpain-2 pathway reduces neuroinflammation and necroptosis after cerebral ischemia-reperfusion injury in a rat model of cardiac arrest

BackgroundCerebral ischemia–reperfusion injury (CIRI) is the leading cause of poor neurological prognosis after cardiopulmonary resuscitation (CPR). We previously reported that the extracellular signal-regulated kinase (ERK) activation mediates CIRI. Here, we explored the potential ERK/calpain-2 pathway role in CIRI using a rat model of cardiac arrest (CA). MethodsAdult male Sprague-Dawley rats suffered from CA/CPR-induced CIRI, received saline, DMSO, PD98059 (ERK1/2 inhibitor, 0.3 mg/kg), or MDL28170 (calpain inhibitor, 3.0 mg/kg) after spontaneous circulation recovery. The survival rate and the neurological deficit score (NDS) were utilized to assess the brain function. Hematoxylin stain, Nissl staining, and transmission electron microscopy were used to evaluate the neuron injury. The expression levels of p-ERK, ERK, calpain-2, neuroinflammation-related markers (GFAP, Iba1, IL-1β, TNF-α), and necroptosis proteins (TNFR1, RIPK1, RIPK3, p-MLKL, and MLKL) in the brain tissues were determined by western blotting and immunohistochemistry. Fluorescent multiplex immunohistochemistry was used to analyze the p-ERK, calpain-2, and RIPK3 co-expression in neurons, and RIPK3 expression levels in microglia or astrocytes. ResultsAt 24 h after CA/CPR, the rats in the saline-treated and DMSO groups presented with injury tissue morphology, low NDS, ERK/calpain-2 pathway activation, and inflammatory cytokine and necroptosis protein over-expression in the brain tissue. After PD98059 and MDL28170 treatment, the brain function was improved, while inflammatory response and necroptosis were suppressed by ERK/calpain-2 pathway inhibition. ConclusionInflammation activation and necroptosis involved in CA/CPR-induced CIRI were regulated by the ERK/calpain-2 signaling pathway. Inhibition of that pathway can reduce neuroinflammation and necroptosis after CIRI in the CA model rats.

Deciphering the effective combinatorial components from Si-Miao-Yong-An decoction regarding the intervention on myocardial hypertrophy

Ethnopharmacological relevanceSi-Miao-Yong-An decoction (SMYAD), a classical traditional Chinese medicine (TCM) formula, has been used to treat various cardiovascular diseases in clinics. Aim of the studyThe aim of this study is to investigate the effective combinatorial components from SMYAD and its mechanism regarding the intervention on myocardial hypertrophy. Materials and methodsSMYAD constituents absorbed in rat plasma and heart were identified using UHPLC Q-Exactive-Orbitrap MS/MS. The identified constituents in SMYAD were further analyzed using ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction and molecular docking. The effective constituents were identified using isoproterenol (ISO)-induced H9c2 cardiomyocyte hypertrophy, and neochlorogenic acid (NCA), chlorogenic acid (CA), cryptochlorogenic acid (CCA), isochlorogenic acid C (ICAC), angoroside C (AGDC), isochlorogenic acid A (ICAA), sweroside (SRD), and harpagide (HPD) in SMYAD extract were quantified by HPLC for compatibility. Finally, anti-hypertrophic activities of candidate effective combinatorial components, which were prepared according to the determined molar concentration ratio of effective constituents using reference substance solution, were analyzed using immunofluorescence staining and Quantitative real-time PCR. The expression levels of PI3Kα, p-ERK, p-Akt, Akt, p-mTOR, mTOR and HIF-1α were measured using Western blot. Results32 prototypes of SMYAD were identified from plasma and heart tissue of rat. Combining with ADMET prediction, 31 dominant constituents were focused. Based on HIF-1 pathway identified in preliminary result, 17 targets were focused, which were used to dock with 31 constituents. 27 constituents were therefore hit as the potential effective constituents of SMYAD in inhibiting myocardial hypertrophy. Bioactivity evaluation showed that NCA, CA, CCA, ICAC, AGDC, ICAA, SRD, and HPD significantly inhibited the increase of H9c2 cell surface area induced by ISO. Except for ICAA and AGDC, the remaining 6 effective constituents, showing a certain inhibitory effect on ISO-induced ANP mRNA overexpression at high and low concentrations, participated in compatibility based on the molar concentration ratio determined by HPLC. Effective combinatorial components composed of the 6 effective constituents (effective combinatorial components ABC) showed significant inhibitory effect on the increase of cell surface area, and the overexpression of ANP and β-MHC mRNA in H9c2 cells induced by ISO. Moreover, effective combinatorial components ABC significantly inhibited the protein overexpressions of p-Akt, p-mTOR and HIF-1α. Based on the results, we put forward the strategy of “Focusing constituents” and “Focusing targets” for the effective constituents research of TCM formula. ConclusionEffective combinatorial components ABC composed of NCA, CA, CCA, ICAC, SRD and HPD from SMYAD inhibited ISO-induced cardiomyocyte hypertrophy and down-regulated expression of ANP and β-MHC mRNA through the inactivation of Akt/mTOR/HIF-1α pathway.

Sclareol inhibits RANKL-induced osteoclastogenesis and promotes osteoblastogenesis through promoting CCN1 expression via repressing the MAPK pathway.

Osteoclasts are crucial cellular components of bone and are the cause of various bone problems like osteoporosis. Various biological activities such as anti-tumorous, anti-inflammatory, antibacterial, and immunomodulatory function are influenced by Sclareol, as a natural diterpene compound. However, studies on the effect and mechanism of Sclareol on osteoporosis are rare. In the current research, the influence of Sclareol on osteoclastogenesis and osteoblastogenesis was targeted to be discovered in ovariectomy (OVX)-induced animal models and in vitro. The expression levels of osteoclast-related genes such as c-Fos, NFATc1, and CTSK were detected by RT-qPCR and western blotting to understand the inhibition of Sclareol on the creation of osteoclast. The influence of Sclareol on osteoblastogenesis and the expression of osteoblastogenic markers were also examined. Sclareol inhibited the osteoclastogenesis caused by receptor activator of nuclear factor-κB ligand (RANKL) which promoted osteoblastogenesis through upregulating the expression of cysteine-rich protein 61 (CYR61/CCN1), which is a matricellular protein of the CCN family. The p-ERK and p-P38 protein expression levels were considerably downregulated by Sclareol. Furthermore, CCN1 overexpression partially mimicked the inhibitory effect of Sclareol, while the opposite results were obtained after CCN1 silencing. Additionally, Sclareol protected against loss of bones in an osteoporosis mouse model generated by OVX. The acquired results indicated that Sclareol represses RANKL-induced osteoclastogenesis and promotes osteoblastogenesis via promoting the expression of CCN1 by constraining the mitogen-activated protein kinase (MAPK) pathway. Our findings proposed that for the avoidance and treatment of osteoclast-linked disorders, Sclareol is a potentially effective drug. A proposed model for mediated regulation of osteoclastogenesis and osteoblastogenesis by Sclareol. The basic model of the process by which Sclareol prevents osteoclastogenesis and promotes osteoblastogenesis. Sclareol may increase the expression of CCN1 through inhibiting the MAPK pathway, thereby inhibiting osteoclast differentiation and attenuating bone resorption. Sclareol represses the expression of c-Fos, which stimulates the formation of osteoclast. In contrast, Sclareol promotes osteoblast differentiation by upregulating Runx2 expression, thereby improving the formation of bones. Consequently, Sclareol protects against loss of bones by regulating the stability of bone makeover via inhibition of bone formation and stimulation of bone resorption. Graphical Headlights 1. Sclareol represses RANKL-induced osteoclastogenesis. 2. Sclareol promotes osteoblast differentiation. 3. Sclareol inhibits the MAPK pathway through induction of CCN1. 4. Sclareol protects against bone loss by regulating the balance of bone remodeling via inhibition of bone formation and stimulation of bone resorption.

The Regulatory Effect of ERK1 Pathway in the DNA Hypomethylation of MRL/lpr Mice

Background: Defective ERK1 activity may regulate the DNA hypomethylation in T cells of lupus mice. Aims: To investigate the function of ERK1 in regulating DNA hypomethylation and explore the potential molecular mechanisms involved in lupus mice. Methods: CD4+T cells were isolated from MRL/lpr and BALB/c mice, activated in vitro in the presence or absence of 5-azacytidine (5-Aza) (a DNA methyltransferase inhibitor)/U0126 (a selective inhibitor of the ERK signaling pathway) respectively. The positive rate of CD4+T cell was measured by flow cytometry, and expression levels of CD70, ERK1, DNA transferase 1 (DNMT1) were estimated by RT-PCR and Western blot. The methylated DNA is detected using the Capture and Detection antibodies, then quantified colorimetrically. Results: The expression level of p-ERK1 in MRL/lpr mice was significantly lower than healthy control (P<0.05); Meanwhile, down regulation of DNMT1 and DNA hypomethylation were found in MRL/lpr mice T cells. In contrary, the expression levels of CD70 increased when compared with healthy control (P<0.05). The same changes were seen in healthy CD4+T cells treated with U0126. Conclusions: DNA hypomethylation regulating mechanisms in lupus T cells were associated with ERK1 signal pathway. U0126 could inhibit ERK1 signal transduction pathway in normal T cells, and induce DNA hypomethylation by regulating the expression of methylation sensitive gene CD70.

Knockdown of Tripartite motif-containing 22 (TRIM22)relieved the apoptosis of lens epithelial cells by suppressing the expression of TNF receptor-associated factor 6 (TRAF6)

ABSTRACT Cataract is a disease that causes severe visual impairment in patients. Recent studies have found that lens epithelial cell apoptosis caused by oxidative damage is the critical cause of cataract. Moreover, TRIM22 could alleviate the ubiquitination of TRAF6. The expression of TRAF6 could activate the p38/MAPK pathway and aggravate the oxidative stress induced damage of lens epithelial cells. However, whether the TRIM22 could alleviate the oxidative stress induced damage of lens epithelial cells by regulating the expression of TRAF6 and p38/MAPK pathway is unclear. In this study, we stimulated the lens epithelial cells with the H2O2 and established the TRIM22 knockdown cells. Next, proliferation of these cells was determined by CCK-8 and EdU assays. Apoptosis of these cells was detected with the TUNEL assays. Levels of ROS was explored with the DCFH-DA staining. Finally, the expression levels of TRAF6, p-p38 and p-ERK were determined with the western blotting. According to the results, we found that knockdown of TRIM22 suppressed the proliferation and relieved the H2O2 induced DNA double-strand break and apoptosis of these cells. Inhibition of TRIM22 inhibited the production of ROS in these cells. Moreover, restriction of TRIM22 induced the decreased levels of TRAF6, p-p38 and p-ERK in lens epithelial cells. We concluded that inhibition of TRIM22 relieved the apoptosis of lens epithelial cells by suppressing the expression of TRAF6, p-p38 and p-ERK.

The protective effect of flesinoxan on myelin damage caused by maladaptation to stress in mice

Our previous study demonstrated that the maintenance of hippocampal myelination contributes to the development of stress adaptation. Further, we showed that 5-HT1A receptor in the hippocampus may be involved in the protection of major myelin proteins loss induced by unadaptable excessive stress. However, the mechanism by which activation of 5-HT1A receptor promote myelination remains unknown. In the present study, we investigated the protective effects of 5-HT1A receptor agonist flesinoxan on unadaptable stress-induced myelin damage and its molecular mechanisms in mice. Western blot analysis revealed that administration of flesinoxan significantly increased the expression level of BDNF, p-ERK, p-CREB, and Olig2 in the hippocampus of stress-maladaptive mice. Furthermore, in the immunohistochemical analysis, a significant increase in the number of Olig2+/BrdU+ positive cells were observed in the dentate gyrus of the hippocampus of flesinoxan-treated, but not vehicle-treated, stress-maladaptive mice. The present findings indicate that 5-HT1A receptor-mediated enhancement of oligogenesis via activation of ERK/CREB/BDNF signaling pathways may be involved in the protection of myelin loss induced by unadaptable excessive stress.

Enhanced cellular osteogenic differentiation on CoFe2O4/P(VDF-TrFE) nanocomposite coatings under static magnetic field

Cellular responses can be regulated and manipulated through combining stimuli-responsive biomaterial with external stimulus. In this present, the magneto-responsive CoFe2O4/P(VDF-TrFE) nanocomposite coatings were designed to understand cell behaviors of preosteoblasts, as well as get insight into the underlying mechanism of osteogenic differentiation under static magnetic field (SMF). CoFe2O4/P(VDF-TrFE) nanocomposite coatings with differential magnetic property (low, medium and high magnetization) were prepared by incorporation of different mass fraction of CoFe2O4 nanoparticles (6%, 13 %, 20 %) into P(VDF-TrFE) matrix. Cell experiments indicated that all nanocomposite coatings with the assistance of SMF could promote the cell attachment, proliferation and osteogenic differentiation of MC3T3-E1 cells. Among different nanocomposite coatings, low magnetization coating (6%) showed a higher ALP activity and gene expression of Runx2, Col-I, OCN. Molecular biology assays demonstrated that the combination of nanocomposite coatings and SMF could significantly up-regulate the expression level of α2β1 integrin and p-ERK. Whereas, the addition of inhibitor U0126 down-regulated sharply the expression level of p-ERK, which indicated that cellular osteogenic differentiation of MC3T3-E1 cells was governed through α2β1 integrin-mediated MEK/ERK signaling pathways during CoFe2O4/P(VDF-TrFE) nanocomposite coatings were combined with SMF. This work provided a promising strategy to enhance cellular osteogenic differentiation through a remote-control manner, which exhibited great potential in the application of bone tissue repair and regeneration.

Soluble CD40 ligand inhibits the growth of non-Hodgkin's lymphoma cells through the JNK signaling pathway.

The incidence of non-Hodgkin's lymphoma (NHL) has been increasing annually and has become a serious threat to human health. However, the pathogenesis of NHL remains unclear. The present study aimed to investigate the effect of soluble CD40 ligand (sCD40L) on NHL cells and its underlying mechanism. Cell Counting kit-8 assay and flow cytometry apoptosis experiments were conducted to investigate the effects of sCD40L on cell proliferation and apoptosis. Western blotting was performed to detect the protein expression levels of BAX, Bcl-2, ERK, p-ERK, JNK, p-JNK, p38, p-p38 and c-JUN. The results of the present study demonstrated that exogenous sCD40L significantly inhibited the proliferation and promoted the apoptosis of Raji and CA46 cells. Additionally, exogenous sCD40L promoted the apoptosis of lymphoma cells by activating the JNK signaling pathway.

Magnetic-Field-Assisted Cellular Osteogenic Differentiation on Magnetic Zinc Ferrite Coatings via MEK/ERK Signaling Pathways

Combining an external stimulus and stimuli-responsive biomaterials can regulate cellular behaviors. In this paper, a magneto-responsive zinc ferrite (ZnFe2O4) coating was designed to gain insight into the preosteoblasts behaviors and osteogenic differentiation mechanism under a static magnetic field (SMF). ZnFe2O4 coatings with distinct magnetization (low, medium, and high magnetizations) were prepared by being annealed at different temperatures. Cellular biology experiments indicated that all ZnFe2O4 coatings with the assistance of SMF could promote the early proliferation (3 days) and osteogenic differentiation of MC3T3-E1 cells. Among different ZnFe2O4 samples, low and medium magnetization of ZnFe2O4 showed a higher osteogenesis-related gene expression (Runx2, Col-I, OCN) than that of high magnetization ZnFe2O4 under SMF, while cellular adhesion and proliferation cultured on different ZnFe2O4 samples presented insignificant differences. Molecular biology tests showed that the combination of ferromagnetic ZnFe2O4 and SMF could significantly improve the expression level of α2β1 integrin and p-ERK. However, the addition of the inhibitor U0126 sharply reduced the expression level of p-ERK, which indicated that α2β1 integrin-mediated MEK/ERK signaling pathways play a key role in SMF-assisted cellular osteogenic differentiation over ZnFe2O4 coatings. This work provides an attractive strategy to enhance cellular osteogenic differentiation in a remote-control way, which exhibited enormous potential in the field of bone tissue repair and regeneration.

Sarpogrelate and rosuvastatin synergistically ameliorate aortic damage induced by hyperlipidemia in apolipoprotein E-deficient mice.

The current study aimed to investigate whether sarpogrelate and rosuvastatin possess anti-arterial injury, and attempted to elucidate the mechanism of action underlying this activity. Sarpogrelate, a 5-hydroxytryptamine type 2A antagonist, is extensively used to prevent arterial thrombosis; however, its effects on atherosclerosis remain unknown. In the present study, sarpogrelate combined with rosuvastatin or rosuvastatin alone were administered to male ApoE-/- mice fed a high-fat diet (HFD) for 8 weeks. Metabolic parameters in the blood samples were analyzed using an automatic analyzer. Aortic tissues were stained with hematoxylin and eosin for morphological analysis. The expression levels of oxidized-low density lipoprotein (LDL) specific scavenging receptors, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and cluster of differentiation 68 were detected via immunostaining. mRNA expression levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α were determined via reverse transcription-quantitative PCR analysis, while protein expression levels of LOX-1 and phosphor(p)-ERK were determined via western blot analysis. The results demonstrated that sarpogrelate combined with rosuvastatin treatment significantly decreased total cholesterol and LDL cholesterol levels in the serum, and alleviated intimal hyperplasia and lipid deposition, accompanied by decreased inflammatory cell infiltration and lower expression levels of inflammatory cytokines, compared with rosuvastatin monotherapy or HFD treatment. Furthermore, sarpogrelate combined with rosuvastatin treatment significantly decreased the expression levels of LOX-1 and p-ERK. Taken together, these results suggest that the positive effects of sarpogrelate combined with rosuvastatin treatment on aortic injury may be associated with the regulation of the LOX-1/p-ERK signaling pathway. Sarpogrelate and rosuvastatin synergistically decreased aortic damage in ApoE-/- HFD mice, and thus provide a basis for the treatment of aortic injury caused by hyperlipidemia with sarpogrelate.

Zeaxanthin Induces Apoptosis via ROS-Regulated MAPK and AKT Signaling Pathway in Human Gastric Cancer Cells.

BackgroundZeaxanthin, a carotenoid commonly found in plants, has a variety of biological functions including anti-cancer activity.PurposeThis study aimed to investigate the potential mechanisms of zeaxanthin in human gastric cancer cells.MethodsCCK-8 assay was used to examine the cytotoxic effect of zeaxanthin on human gastric cancer cells. Flow cytometry was used to analyse AGS cell cycle distribution and apoptosis status. Western blot analysis was used to detect the expression levels of cycle-related proteins (Cyclin A, Cyclin B1, CDK1/2, p21, and p27), apoptosis-related proteins (Bcl-2, Bad, caspase-3, PARP), MAPK, AKT, STAT3, and NF-κB.ResultsCCK-8 assay showed that zeaxanthin has obvious cytotoxic effects on 12 types of human gastric cancer cells, but no obvious toxic effect on normal cells. In addition, flow cytometry and Western blotting results showed that zeaxanthin induces apoptosis by reducing mitochondrial membrane potential; increasing Cytochrome C, Bax, cleaved-caspase-3 (cle-cas-3), and cleaved-PARP (cle-PARP) expression levels; and decreasing Bcl-2, pro-caspase-3 (pro-cas-3), and pro-PARP expression levels. Additionally, zeaxanthin caused cell cycle arrest at the G2/M phase by increasing the levels of p21 and p27 and reduced the levels of AKT, Cyclin A, Cyclin B1, and Cyclin-dependent kinase 1/2 (CDK1/2). Furthermore, after zeaxanthin treatment, the expression levels of reactive oxygen species (ROS), p-JNK, p-p38, and I-κB increased, and the expression levels of p-ERK, p-AKT, STAT3, and NF-κB decreased. However, the ROS scavenger N-acetylcysteine (NAC) and MAPK inhibitors inhibited zeaxanthin-induced apoptosis, and under the action of zeaxanthin, MAPK regulated NF-κB and STAT3, and reduced their protein expression levels.ConclusionZeaxanthin has a potential effect against gastric cancer cells through the ROS-mediated MAPK, AKT, NF-κB, and STAT3 signaling pathways, and it is expected to become a new drug for the treatment of human gastric cancer.

Combination of Tanshinone IIA and Cisplatin Inhibits Esophageal Cancer by Downregulating NF-κB/COX-2/VEGF Pathway.

Cisplatin (DDP) represents one of the common drugs used for esophageal squamous cell carcinoma (ESCC), but side effects associated with DDP and drug resistance lead to the failure of treatment. This study aimed to understand whether tanshinone IIA (tan IIA) and DDP could generate a synergistic antitumor effect on ESCC cells. Tan IIA and DDP are demonstrated to restrain ESCC cell proliferation in a time- and dose-dependent mode. Tan IIA and DDP at a ratio of 2:1 present a synergistic effect on ESCC cells. The combination suppresses cell migration and invasion abilities, arrests the cell cycle, and causes apoptosis in HK and K180 cells. Molecular docking indicates that tan IIA and DDP could be docked into active sites with the tested proteins. In all treated groups, the expression levels of E-cadherin, β-catenin, Bax, cleaved caspase-9, P21, P27, and c-Fos were upregulated, and the expression levels of fibronectin, vimentin, Bcl-2, cyclin D1, p-Akt, p-ERK, p-JNK, P38, COX-2, VEGF, IL-6, NF-κB, and c-Jun proteins were downregulated. Among these, the combination induced the most significant difference. Our results suggest that tan IIA could be a novel treatment for combination therapy for ESCC.

THE PROTECTIVE EFFECT AND MECHANISM OF MESENCHYMAL STEM CELLS ON DIABETIC PULMONARY FIBROSIS IN RATS

Background and Objective: Diabetes affects a variety of organs such as the kidneys, eyes, and liver, and there is increasing evidence that the lung is also one of the target organs of diabetes and imbalance of Sirt3-mediated stress responses such as inflammation, oxidative stress, apoptosis, autophagy, and ER stress may contribute to diabetic pulmonary fibrosis. Although previous studies have reported that mesenchymal stem cells (MSCs) have beneficial effects on various diabetic complications, the effect and mechanisms of MSCs on diabetes-induced lung injury is not clear. Therefore, the present study aims to investigate the protective effect and possible mechanisms of MSCs against diabetic pulmonary fibrosis in rats. Methods and Results: In this study, the STZ-induced diabetes model was constructed in rats, and the effect and potential mechanisms of bone marrow MSCs on diabetic pulmonary fibrosis were investigated. The results revealed that fibrotic changes in the lung were successfully induced in the diabetic rats, while MSCs significantly inhibited or even reversed the changes. Specifically, MSCs up-regulated the expression levels of Sirt3 and SOD2 and then activated the Nrf2/ARE signaling pathway, thereby controlling MDA, GSH content and iNOS, NADPH oxidase subunit p22phox expression levels in the lung tissue. Meanwhile, high levels of Sirt3 and SOD2 induced by MSCs reduced the expression levels of IL-1β, TNF-α, ICAM-1 and MMP9 by suppressing the NF-κB/HMGB1/NLRP3/Caspase1 signaling pathway, as well as regulated the expression levels of cleaved caspasese3, Bax and Bcl2 by up-regulating the expression level of P-Akt, thereby inhibiting the apoptosis of the lung tissue. In addition, MSCs also regulated the expression levels of LC3, P62, BiP, Chop, and PERK, thereby enhancing autophagy and attenuating endoplasmic reticulum stress. Conclusions: In conclusion, our study demonstrates that MSCs effectively ameliorate diabetic pulmonary fibrosis via regulating Sirt3-mediated stress responses, including enhancement of autophagy and inhibition of oxidative stress, inflammation, apoptosis, and endoplasmic reticulum stress. These results not only shed new light on the pathogenesis of diabetic lung injury but also provide a theoretical foundation for further exploration of diabetic pulmonary fibrosis therapeutics. Keywords: Mesenchymal stem cells, diabetic pulmonary fibrosis, Sirt3, stress response This work was supported by the National Natural Science Foundation of China (grant numbers 81870609, 31571474, 81802210).

MiR-200a and miR-200b restrain inflammation by targeting ORMDL3 to regulate the ERK/MMP-9 pathway in asthma

Background Asthma is one of the most frequent and serious diseases worldwide. Inflammation has been reported to correlate with airway remodeling, which is critical for the progression of asthma. Better understanding of novel molecules modulating asthma and the underlying mechanism will benefit explorations of new treatments. Method: To explore the role of miR-200a and miR-200b in asthma, miR-200a mimics/inhibitor and miR-200b mimics/inhibitor were employed in A549 cells, respectively. Expression levels of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1β, were measured by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). A dual luciferase reporter assay was performed to identify whether miR-200a/200b directly bound to Orosomucoid 1-like 3 (ORMDL3). ERK, p-ERK and MMP-9, involved in downstream pathways of ORMDL3, were detected using qRT-PCR and western blotting. Results: MiR-200a/200b silencing significantly increased the expression of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1β, in A549 cells. ORMDL3 was the target gene of miR-200a/200b, with high expression levels in miR-200a inhibitor and miR-200b inhibitor groups. MiR-200a and miR-200b played synergistic roles in the regulation of the inflammatory effect in A549 cells. Expression levels of p-ERK and MMP-9 were significantly increased in miR-200a inhibitor and miR-200b inhibitor groups and were rescued by ERK inhibitor and MMP-9 inhibitor, respectively. Conclusion: These findings suggest that miR-200a and miR-200b are required to regulate asthma inflammation. Reduction in miR-200a/200b promotes the development of asthma inflammation by targeting ORMDL3 to activate the ERK/MMP-9 pathway. Therefore, elevating miR-200a and miR-200b and decreasing ORMDL3 might be potential strategies for inhibition of the asthma process.

Non-ionizing radiofrequency field induces unfolded protein response (UPR) in endoplasmic reticulum of mouse neuronal cells

ObjectiveTo examine whether exposure of mouse neuronal cells to radiofrequency fields used in mobile communication devices can induce stress in endoplasmic reticulum (ER) and activate unfolded protein response (UPR). MethodsHT22 mouse hippocampus neuronal cells were exposed to continuous wave 900 ​MHz radiofrequency fields (RF) at 120 ​μW/cm2 power intensity for 4 ​h/d for 5 consecutive days. The positive control cells were irradiated with 4 ​Gy of 60Co γ-rays at a dose rate of 0.5 ​Gy/min (GR). Twenty-four hours after the last exposure, cells were collected, and the expressions of sensor transmembrane proteins were detected using Western blot analysis. ResultsThe expression levels of Ire1, PERK, p-IRE1 and p-PERK, GRP78 and CHOP proteins were detected. There were no statistically significant differences in the expression levels of IRE1 and PERK proteins in control (CT), sham(SH)-, RF- and GR-exposed cells (P ​< ​0.05). The phosphorylated protein levels of p-IRE1 and p-PERK were significantly increased in cells exposed to RF and GR (P ​< ​0.05). The expression levels of GRP78 and CHOP were significantly increased in RF- and GR-exposed cells compared to CT and SH-exposed cells (P ​< ​0.05). Cells treated with 1 ​μg/ml TM for 24 ​h showed significantly increased expression levels of GRP78 and CHOP proteins compared to controls (P ​< ​0.05). In the presence of 2 ​mmol/L PBA, TM-induced increased levels of GRP78 and CHOP proteins were reduced (P ​< ​0.05). ConclusionsThe exposure of non-ionizing 900 ​MHz RF was able to cause stress in HT22 mouse neuronal cells and activated UPR in ER. Since UPR plays an important role in both cell survival (when UPR is mitigated) and apoptosis/death (under unresolvable stress conditions), further studies are required to determine the fate of the cells exposed to RF.