Phosphorylation Of Extracellular Regulated Protein Kinases Research Articles

An Integrated Approach Using Network Pharmacology and Experimental Validation to Reveal the Therapeutic Mechanism of Weifuchun in Treating Gastric Cancer.

Gastric cancer (GC) is a prevalent malignancy affecting the gastrointestinal tract. Weifuchun (WFC), a Chinese herbal prescription comprising red ginseng, Isodon amethystoides, and Fructus aurantii, is widely used in China for various chronic stomach disorders. However, its therapeutic role and mechanisms in treating GC remain unexplored. In a randomized, controlled, single-blind trial involving postoperative stages II and III GC patients, we compared adjuvant chemotherapy plus WFC (chemo plus WFC group) to adjuvant chemotherapy alone (chemo group) over 6 months. We assessed recurrence and metastasis rates and used systematic pharmacology to predict WFC's active components, screen target genes, and construct network interaction maps, were validated through in vitro experiments. The combined therapy significantly reduced 2-year recurrence and metastasis rates. We identified 67 active ingredients, 211 drug target proteins, 1539 disease targets, 105 shared targets, and 188 signaling pathways associated with WFC. WFC impacted cell apoptosis, proliferation, and the inflammatory response, with top tumor-related signaling pathways involving 5'-adenosine monophosphate-activated protein kinase (AMPK), mitogen-activated protein kinase, nuclear factor kappa-B (NFKB), and apoptosis. In vitro, WFC inhibited proliferation and migration while inducing apoptosis in GC cells, reduced VEGFA, TNFa, and IL6 expressions. Immunocytochemistry showed increased p-AMPK staining, and molecular analysis revealed decreased NFKB and phosphorylation of extracellular-regulated protein kinase 1/2 (ERK1/2) levels, increased p-AMPK and BAX protein levels in WFC-treated cells, effects reversed by Compound C. WFC's antitumor effects involve AMPK-dependent ERK1/2 and NFKB pathways, regulating proliferation, migration, and apoptosis in GC cells.

Effective constituents and protective effect of Mudan granules against Schwann cell injury

Ethnopharmacological relevanceDiabetic peripheral neuropathy (DPN) is the most common complication of diabetes. Mudan granules (MD) is a Chinese patent medicine for treating DPN, which is composed of nine Chinese medicinal herbs, including the radix of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. (Huangqi in Chinese), rhizome of Corydalis yanhusuo W.T. Wang (Yanhusuo), radix and rhizome of Panax notoginseng (Burk.) F. H. Chen (Sanqi), radix of Paeonia lactiflora Pall. or Paeonia veitchii Lynch (Chishao), radix and rhizome of Salvia miltiorrhiza Bge. (Danshen), rhizome of Ligusticum chuanxiong Hort. (Chuanxiong), flowers of Carthamus tinctorius L. (Honghua), lignum of Caesalpinia sappan L. (Sumu), and caulis of Spatholobus suberectus Dunn (Jixueteng). MD was reported to have a protective effect on Schwann cell (SC) that is considered as an important therapeutic target of DPN. However, the constituents of MD have not been reported, and the effective constituents and protective pathways for MD against SC injury remain unclear. Aim of the studyThis study aimed to identify the constituents in MD, and to investigate the effective constituents and protective pathways of MD against high-glucose/lipid injury in SC. Materials and methodsThe chemical constituents of MD were identified using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Protective effect and effective constituents screening were performed in an in vitro SC injury model induced by high glucose and lipid levels. The protective pathways of MD and its effective constituents were investigated by western blotting assay of related proteins. ResultsA total of 136 constituents were identified in MD. MD downregulated the phosphorylation of extracellular-regulated protein kinases 1/2 (ERK1/2) and expression of cyclooxygenase-2 (COX-2) and upregulated the expression of sirtuin 2 (SIRT2). Seven effective constituents were screened out, including three from Sanqi [20(R)-ginsenoside Rh2, 20(S)-ginsenoside Rh2, and ginsenoside Rk3], one from Huangqi (astragaloside II), one from Danshen (danshensu), and two from Chuanxiong (chlorogenic and cryptochlorogenic acid). Six of the seven compounds, excluding danshensu, inhibited the phosphorylation of ERK1/2. Both astragaloside II and chlorogenic acid upregulated the expression of SIRT2, and cryptochlorogenic acid and danshensu downregulated the expression of COX-2. ConclusionsThe constituents of MD were firstly identified, and seven effective constituents were found. MD can protect SC against high-glucose and -lipid injury by downregulating ERK1/2 phosphorylation and COX-2 expression and upregulating SIRT2 expression. Seven effective constituents regulated the expression of these proteins. This study presented an important advance toward elucidating the chemical constituents, and the effective constituents and protective pathways of MD against high-glucose/lipid injury in SC, which is very helpful for investigating the action mechanism of MD on treating DPN, and could ultimately inform the development of effective quality control procedures for MD production.

Phosphoproteome Reveals Extracellular Regulated Protein Kinase Phosphorylation Mediated by Mitogen-Activated Protein Kinase Kinase-Regulating Granulosa Cell Apoptosis in Broody Geese.

Geese have strong brooding abilities, which severely affect their egg-laying performance. Phosphorylation is widely involved in regulating reproductive activities, but its role in goose brooding behavior is unclear. In this study, we investigated differences in the phosphoprotein composition of ovarian tissue between laying and brooding geese. Brooding geese exhibited ovarian and follicular atrophy, as well as significant oxidative stress and granulosa cell apoptosis. We identified 578 highly phosphorylated proteins and 281 lowly phosphorylated proteins, and a KEGG pathway analysis showed that these differentially phosphorylated proteins were mainly involved in cell apoptosis, adhesion junctions, and other signaling pathways related to goose brooding behavior. The extracellular regulated protein kinase (ERK)-B-Cell Lymphoma 2(BCL2) signaling pathway was identified as playing an important role in regulating cell apoptosis. The phosphorylation levels of ERK proteins were significantly lower in brooding geese than in laying geese, and the expression of mitogen-activated protein kinase kinase (MEK) was downregulated. Overexpression of MEK led to a significant increase in ERK phosphorylation and BCL2 transcription in H2O2-induced granulosa cells (p < 0.05), partially rescuing cell death. Conversely, granulosa cells receiving MEK siRNA exhibited the opposite trend. In conclusion, geese experience significant oxidative stress and granulosa cell apoptosis during brooding, with downregulated MEK expression, decreased phosphorylation of ERK protein, and inhibited expression of BCL2.

Experimental Study on the Inhibition of RANKL-Induced Osteoclast Differentiation In Vitro by Metformin Hydrochloride.

Objective Establishment of an in vitro osteoclast induction model under nuclear factor-κB receptor activator ligand (RANKL) induction for investigating the effect of metformin hydrochloride (Met) on osteoclast differentiation. Methods RANKL induced the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts in vitro, and Met was added at different concentrations for intervention during the induction process. After 5 d of culture and fixation, the number of osteoclasts was counted by tartrate-resistant acid phosphatase (TRAP) staining and F-actin staining, and the function of osteoclasts was examined with hydroxyapatite-coated plates. Real-time fluorescence quantitative PCR was performed to detect the expression of Cathepsin K, osteoclast associated receptor (OSCAR), and TRAP, and the effect of Met on Mitogen-activated protein kinases (MAPK) signaling pathway was detected by Western blot. Results Met significantly reduced osteoclast formation, F-actin ring formation, bone resorption, and the expression of relevant genes Cathepsin K, OSCAR, and TRAP. The Western blotting study demonstrated that Met inhibited the MAPK signaling pathway by decreasing the phosphorylation of extracellular regulated protein kinase (ERK), which plays important roles in osteoclast formation. Conclusion Metformin hydrochloride inhibited the differentiation of osteoclasts, decreased the bone resorption area, and suppressed phosphorylation of ERK in vitro.

Expression changes of c-Fos and D1R/p-ERK1/2 signal pathways in nucleus accumbens of rats after ketamine abuse

Ketamine is a commonly used dissociative anesthetic in clinical applications. However, the abuse potential has posted limits to its use and the mechanism remains to be studied. We aimed to investigate the changes of dopamine D1 receptors (D1R), phosphorylation of extracellular-regulated protein kinase 1/2 (p-ERK1/2), and c-Fos expression in the nucleus accumbens (NAc) of ketamine abuse rats. Ketamine induced severe anxiety in rats, as shown by an open field test. Nissl staining demonstrated clearly different morphologies between neurons of ketamine abuse rats and normal rats. The molecular expression changes were examined using immunohistochemistry assay and western blotting. D1R, p-ERK1/2, and c-Fos were significantly highly-expressed in NAc during ketamine exposure and were decreased by D1R antagonist SCH23390 and MAPK kinases inhibitor U0126. Taken together, the results suggest that ketamine abuse may induce the overexpression of c-Fos in NAc by up-regulating the expression of D1R and p-ERK1/2.

Protection of toll-like receptor 9 against lipopolysaccharide-induced inflammation and oxidative stress of pulmonary epithelial cells via MyD88-mediated pathways.

Acute lung injury (ALI) caused by lipopolysaccharide (LPS) is a common, severe clinical syndrome. Injury caused by inflammation and oxidative stress in vascular endothelial and alveolar epithelial cells is a vital process in the pathogenesis of ALI. Toll-like receptor 9 (TLR9) is highly expressed in LPS-induced ALI rats. In this study, Beas-2B human pulmonary epithelial cells and A549 alveolar epithelial cells were stimulated by LPS, resulting in the upregulation of TLR9 in a concentrationdependent manner. Furthermore, TLR9 overexpression and interference vectors were transfected before LPS administration to explore the role of TLR9 in LPS-induced ALI in vitro. The findings revealed that inhibition of TLR9 reduced inflammation and oxidative stress while suppressing apoptosis of LPS-induced Beas-2B and A549 cells, whereas TLR9 overexpression aggravated these conditions. Moreover, TLR9 inhibition resulted in downregulated protein expression of myeloid differentiation protein 88 (MyD88) and activator activator protein 1 (AP-1), as well as phosphorylation of nuclear factor-?B (NF-kappaB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The phosphorylation of extracellular-regulated protein kinases 1/2 was upregulated compared to that of cells subjected to only LPS administration, and this was reversed by TLR9 overexpression. These results indicate that inhibition of TLR9 plays a protective role against LPS-induced inflammation and oxidative stress in Beas-2B and A549 cells, possibly via the MyD88/NF-kappaB and MyD88/MAPKs/AP-1 pathways.

RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells.

BackgroundThe v-raf-leukemia viral oncogene 1 (RAF1) plays an essential physiological role in reproduction and development through the mediation of steroid hormone synthesis. Follicle-stimulating hormone (FSH) signaling pathway was not involved in the majority of RAF1 studies, whether RAF1 takes part in the signaling events of gonadotropic hormones such as FSH in ovarian tissue is unknown.MethodsThe process is blocked by treating granulosa cells (GCs) with the RAF1 inhibitor, RAF709. Inhibition of RAF1 activity by RAF709 decreased extracellular regulated protein kinases (ERK) phosphorylation and suppressed the expression of the cytochrome P450 subfamily 19 member 1 (CYP19A1), which is a major rate-limiting enzyme that participates in the last step of E2 biosynthesis.ResultsWe found that RAF1, acting as a downstream molecule, mediates FSH signalling to stimulate estradiol (E2) synthesis and secretion in mouse ovarian GCs. Gene expression of RAF1 was induced by FSH and the secretion of E2 increased into the bloodstream of mice and into the supernatant of primary GCs. Our in vitro and in vivo studies clearly illustrate RAF1 plays an important medium adjusting role in the FSH signaling pathway, and RAF1 acting as a downstream molecule to trigger ERK phosphorylation to stimulate GC E2 synthesis and secretion.ConclusionsRAF1 plays a pivotal mediating role in the FSH signaling pathway by inducing the phosphorylation of ERK and promoting E2 synthesis.

Propofol via Antioxidant Property Attenuated Hypoxia-Mediated Mitochondrial Dynamic Imbalance and Malfunction in Primary Rat Hippocampal Neurons.

Background Hypoxia may induce mitochondrial abnormality, which is associated with a variety of clinical phenotypes in the central nervous system. Propofol is an anesthetic agent with neuroprotective property. We examined whether and how propofol protected hypoxia-induced mitochondrial abnormality in neurons. Methods Primary rat hippocampal neurons were exposed to propofol followed by hypoxia treatment. Neuron viability, mitochondrial morphology, mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) production were measured. Mechanisms including reactive oxygen species (ROS), extracellular regulated protein kinase (ERK), protein kinase A (PKA), HIF-1α, Drp1, Fis1, Mfn1, Mfn2, and Opa1 were investigated. Results Hypoxia increased intracellular ROS production and induced mPTP opening, while reducing ATP production, MMP values, and neuron viability. Hypoxia impaired mitochondrial dynamic balance by increasing mitochondrial fragmentation. Further, hypoxia induced the translocation of HIF-1α and increased the expression of Drp1, while having no effect on Fis1 expression. In addition, hypoxia induced the phosphorylation of ERK and Drp1ser616, while reducing the phosphorylation of PKA and Drp1ser637. Importantly, we demonstrated all these effects were attenuated by pretreatment of neurons with 50 μM propofol, antioxidant α-tocopherol, and ROS scavenger ebselen. Besides, hypoxia, propofol, α-tocopherol, or ebselen had no effect on the expression of Mfn1, Mfn2, and Opa1. Conclusions In rat hippocampal neurons, hypoxia induced oxidative stress, caused mitochondrial dynamic imbalance and malfunction, and reduced neuron viability. Propofol protected mitochondrial abnormality and neuron viability via antioxidant property, and the molecular mechanisms involved HIF-1α-mediated Drp1 expression and ERK/PKA-mediated Drp1 phosphorylation.

GluN2A/ERK/CREB Signaling Pathway Involved in Electroacupuncture Regulating Hypothalamic-Pituitary-Adrenal Axis Hyperactivity.

The hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis caused by stress will inevitably disrupt the homeostasis of the neuroendocrine system and damage physiological functions. It has been demonstrated that electroacupuncture (EA) can modulate HPA axis hyperactivity during the perioperative period. As the initiating factor of the HPA axis, hypothalamic corticotrophin-releasing hormone (CRH) is the critical molecule affected by EA. However, the mechanism by which EA reduces CRH synthesis and secretion remains unclear. Activated N-methyl-D-aspartate receptor (NMDAR) has been linked to over-secretion of hypothalamic CRH induced by stress. To determine whether NMDAR is involved in EA regulating the over-expression of CRH, a surgical model of partial hepatectomy (HT) was established in our experiment. The effect of EA on hypothalamic NMDAR expression in HT mice was examined. Then, we investigated whether the extracellular regulated protein kinases (ERK)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway mediated by NMDAR was involved in EA regulating HPA axis hyperactivity. It was found that surgery enhanced the expression of hypothalamic CRH and caused HPA axis hyperactivity. Intriguingly, EA effectively suppressed the expression of CRH and decreased the activation of GluN2A (NMDAR subunit), ERK, and CREB in HT mice. GluN2A, ERK, and CREB antagonists had similar effects on normalizing the expression of CRH and HPA axis function compared with EA. Our findings suggested that surgery enhanced the activation of the hypothalamic GluN2A/ERK/CREB signaling pathway, thus promoting the synthesis and secretion of CRH. EA suppressed the phosphorylation of GluN2A, ERK, and CREB in mice that had undergone surgery, indicating that the GluN2A/ERK/CREB signaling pathway was involved in EA alleviating HPA axis hyperactivity.

A dual-targeted molecular therapy of PP242 and cetuximab plays an anti-tumor effect through EGFR downstream signaling pathways in colorectal cancer.

Epidermal growth factor receptor (EGFR) and its downstream Ras-mitogen-activated protein kinase kinase (MAPKK, MEK)-extracellular regulated protein kinase (ERK) signaling pathway and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway play important roles in the pathogenesis of colorectal cancer (CRC). The combination therapy of anti-EGFR and anti-mTOR needs to be explored. Here we combined the anti-EGFR monoclonal antibody cetuximab (CTX) with the mTOR inhibitor PP242 in CRC cell lines and mouse xenograft models and discussed the changes of EGFR downstream signaling pathways of CRC cell lines. In HT-29 cells and Caco-2 cells, combined application of CTX and PP242 significantly inhibited the proliferation of CRC cells in vivo and in vitro. In BRAF wild-type Caco-2 cells, combined application of CTX and PP242 inhibited the activation of the EGFR and its downstream signaling pathways. Our research further demonstrates the effectiveness of the combined application of CTX and PP242 in inhibiting CRC cell lines from the perspective of cell proliferation, cell cycle, apoptosis, and mouse xenografts. We revealed that the combined application of CTX and PP242 can inhibit tumor growth and proliferation by inhibiting the phosphorylation of key molecules in EGFR downstream MEK-ERK and MEK 4/7 (MKK)-c-Jun N-terminal kinase (JNK) signaling pathways in BRAF wild-type CRC cells. In addition, we found that in BRAF mutant CRC cells, the monotherapy of PP242 resulted in negative feedback increased EGFR phosphorylation rates, accompanied by significant up-regulation of downstream MEK and ERK phosphorylation.

Proline-Serine-Threonine Phosphatase-Interacting Protein 2 Alleviates Diabetes Mellitus-Osteoarthritis in Rats through Attenuating Synovial Inflammation and Cartilage Injury.

ObjectiveTo explore the possible way of proline‐serine–threonine phosphatase‐interacting protein 2 (PSTPIP2) influencing diabetes mellitus‐osteoarthritis (DM‐OA) progression.Methods In vivo, eight‐week‐old male Sprague Dawley rats were induced with DM‐OA by intraperitoneal injection of streptozotocin with high‐fat diet feeding and intra‐articular injection of monoiodoacetate. PSTPIP2 overexpression was achieved by intra‐articular injection of lentivirus vectors. PSTPIP2 expression was verified by real‐time polymerase chain reaction and Western blotting. Histological changes were examined by hematoxylin/eosin and safranin‐O/fast‐green staining. In vitro, rat synovial fibroblasts were induced DM‐OA by stimulation of high glucose (HG) and interleukin (IL)‐1β. PSTPIP2 overexpression was achieved by lentivirus infection. U0126 was added as an ERK inhibitor. Levels of tumor necrosis factor (TNF)‐α, IL‐6, and IL‐1β were detected using enzyme‐linked immunosorbent assay. Expression of matrix metalloproteinase (MMP)‐3, MMP‐13, aggrecanase‐2 (ADAMTS‐5), intercellular cell adhesion molecule (ICAM)‐1, extracellular regulated protein kinase (ERK) and phospho‐ERK (p‐ERK) was detected by Western blotting.ResultsIn DM‐OA rats, PSTPIP2 relative messenger RNA (mRNA) level was significantly decreased compared to control rats. The protein expression was also decreased obviously. Inflammation score in synovium was dramatically increased, accompanying with increased TNF‐α, IL‐6, and IL‐1β levels. Osteoarthritis research society international (OARSI) score in cartilage was markedly increased, along with increased MMP‐3, MMP‐13, ADAMTS‐5, ICAM‐1, ERK and p‐ERK expression. In PSTPIP2‐overexpressed DM‐OA rats, PSTPIP2 mRNA level and protein expression was increased compared to DM‐OA rats received negative‐control lentivirus vectors. The inflammation score, as well as TNF‐α, IL‐6, and IL‐1β levels were dramatically decreased. Also, the OARSI score and protein expression of MMP‐3, MMP‐13, ADAMTS‐5, ICAM‐1, ERK and p‐ERK were decreased. In HG+IL‐1β‐treated rat synovial fibroblasts, PSTPIP2 protein expression was decreased compared to normal glucose (NG)‐treated cells. Levels of TNF‐α, IL‐6, and IL‐1β, as well as expression of MMP‐3, MMP‐13, ADAMTS‐5, ICAM‐1, ERK and p‐ERK were increased. After cells were infected with PSTPIP2‐overexpressed lentivirus, levels of TNF‐α, IL‐6, and IL‐1β, and expression of MMP‐3, MMP‐13, ADAMTS‐5, ICAM‐1, ERK and p‐ERK were obviously decreased compared to cells infected with NC lentivirus. In addition, ERK inhibitor U0126 treatment also decreased the TNF‐α, IL‐6, and IL‐1βlevels and MMP‐3, MMP‐13, ADAMTS‐5, ICAM‐1, ERK and p‐ERK expression in HG + IL‐1β treated rat synovial fibroblasts.ConclusionOverexpression of PSTPIP2 alleviates synovial inflammation and cartilage injury during DM‐OA progression via inhibiting ERK phosphorylation.

The synergistic effects of opioid and neuropeptide B/W in rat acute inflammatory and neuropathic pain models

The use of morphine is controversial due to the incidence of rewarding behavior, respiratory depression, and tolerance, leading to increased drug dose requirements, advancing to morphine addiction. To overcome these barriers, strategies have been taken to combine morphine with other analgesics. Neuropeptide B23 and neuropeptide W23 (NPB23 and NPW23) are commonly used to relieve inflammatory pain and neuropathic pain. As NPB23 and NPW23 system shares similar anatomical basis with opioid system at least in the spinal cord we hypothesized that NPB23 or NPW23 and morphine may synergistically relieve inflammatory pain and neuropathic pain. To test this hypothesis, we demonstrated that μ opioid receptor and NPBW1 receptor (receptor of NPB23 and NPW23) are colocalized in the superficial dorsal horn of the spinal cord. Secondly, co-administration of morphine witheitherNPB23 or NPW23 synergistically attenuated inflammatory and neuropathic pain. Furthermore, either NPB23 or NPW23 significantly reduced morphine-induced conditioned place preference (CPP) and constipation. We also found that phosphorylation of extracellular-regulated protein kinase (ERK1/2) following morphine was profoundly potentiated by the application of NPB23 or NPW23. Hence, combination of morphine with either NPB23 or NPW23 reduced dose of morphine required for pain relief in inflammatory and neuropathic pain, while effectively prevented some side-effects of morphine.

PELP1 promotes the expression of RUNX2 via the ERK pathway during the osteogenic differentiation of human periodontal ligament stem cells

ObjectiveThe aim of this study was to determine the physiological function and mechanism of proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) at the molecular level in vitro. DesignDuring the osteogenic differentiation of hPDLSCs, the change of PELP1 and the osteogenic commitment markers runt-related transcription factor 2(RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) were monitored by quantitative real-time PCR (qRT-PCR) and western blots. To elucidate how PELP1 regulates RUNX2, the expression of RUNX2, the phosphorylation of extracellular regulated protein kinases (ERK) and subcellular location of PELP1 were detected under conditions that PELP1 was either knockdown by specific siRNA or overexpressed. A pharmacological inhibitor of ERK, U0126 was used while PELP1 was overexpressed, and the expression of RUNX2 was monitored by qRT-PCR. ResultsPELP1 was upregulated during the osteogenic differentiation of hPDLSCs. Knockdown of PELP1 suppressed the expression of RUNX2, whereas overexpression of PELP1 increased RUNX2 expression. Moreover, PELP1 knockdown resulted in reduced ERK phosphorylation and RUNX2 expression, and PELP1 overexpression induced RUNX2 expression was inhibited by U0126 in the hPDLSCs. ConclusionsPELP1 regulates the expression of RUNX2 during the osteogenic differentiation of hPDLSCs and that the ERK pathway is involved in this process.

Cannabinoid Receptor-2 Activation in Keratinocytes Contributes to Elevated Peripheral β-Endorphin Levels in Patients With Obstructive Jaundice.

Cholestatic diseases are often accompanied by elevated plasma levels of endogenous opioid peptides, but it is still unclear whether central or peripheral mechanisms are involved in this process, and little is known about the change of pain threshold in these patients. The purpose of this study was to determine the preoperative pain threshold, postoperative morphine consumption, and central and peripheral β-endorphin levels in patients with obstructive jaundice. This study also tests the hypothesis that activation of the cannabinoid receptor-2 (CB2R) in skin keratinocytes by endocannabinoids is the mechanism underlying circulating β-endorphin elevation in patients with obstructive jaundice. The electrical pain thresholds, 48-hour postoperative morphine consumption, concentrations of β-endorphin in plasma and cerebrospinal fluid, skin and liver β-endorphin expression, and plasma levels of endocannabinoids were measured in jaundiced (n = 32) and control (n = 32) patients. Male Sprague-Dawley rats and human keratinocytes (human immortalized keratinocyte cell line [HaCaT]) were used for the in vivo and in vitro experiments, respectively. Mechanical and thermal withdrawal latency, plasma level, and skin expression of β-endorphin were measured in CB2R-antagonist-treated and control bile duct-ligated (BDL) rats. In cultured keratinocytes, the effect of CB2R agonist AM1241-induced β-endorphin expression was observed and the phosphorylation of extracellular-regulated protein kinases 1/2, p38, and signal transducer and activator of transcription (STAT) pathways were investigated. This study found (1) the plasma level of β-endorphin (mean ± standard error of the mean [SEM]) was 193.9 ± 9.6 pg/mL in control patients, while it was significantly increased in jaundiced patients (286.6 ± 14.5 pg/mL); (2) the electrical pain perception threshold and the electrical pain tolerance threshold were higher in patients with obstructive jaundice compared with controls, while the 48-hour postoperative morphine consumption was lower in the jaundiced patients; (3) there was no correlation between plasma β-endorphin levels, electrical pain thresholds, and 48-hour postoperative morphine consumption in patients with obstructive jaundice; (4) the plasma level of the endogenous cannabinoid anandamide was increased in the jaundiced patients; (5) CB2R antagonist treatment of the BDL rats reduced β-endorphin levels in plasma and skin keratinocytes, while it did not alter the nociceptive thresholds in BDL and control rats; (6) the endocannabinoid anandamide-induced β-endorphin synthesis and release via CB2R in cultured keratinocytes; and (7) phosphorylation of extracellular-regulated protein kinases 1/2 is involved in the CB2R-agonist-induced β-endorphin expression in keratinocytes. CB2R activation in keratinocytes by the endocannabinoid anandamide may play an important role in the peripheral elevation of β-endorphin during obstructive jaundice.

Antitumor activity and mechanism of resistance of the novel HDAC and PI3K dual inhibitor CUDC-907 in pancreatic cancer.

Pancreatic cancer is a highly malignant disease with an extremely poor prognosis. The benefit of chemotherapy treatment for pancreatic cancer is very limited. Therefore, new therapeutic targets and approaches are urgently needed for this deadly disease. Multi-target therapy is a potential and feasible treatment strategy. Given the important roles that histone deacetylases (HDACs) and phosphoinositide-3-kinase (PI3K) play in pancreatic cancer, we investigated the antitumor activity and mechanism of novel HDAC and PI3K dual inhibitor CUDC-907 in pancreatic cancer. MTT assay and flow cytometric analysis were used to examine the in vitro antitumor activity of CUDC-907. A BxPC-3-derived xenograft mouse model was used to determine CUDC-907 in vivo efficacy. The TUNEL assay as used to determine apoptosis in tumors in vivo post CUDC-907 treatment. Western blots were used to determine the effect of CUDC-907 on protein levels. Our results show that CUDC-907 decreased viable cells and induced cell death in a concentration-dependent manner. Furthermore, CUDC-907 showed promising in vivo antitumor activity in the BxPC-3-derived xenograft mouse model while exhibiting tolerable toxicity. Furthermore, long-term treatment with CUDC-907 induced phosphorylation of AKT, S6 (ribosomal protein S6), and ERK (extracellular regulated protein kinase), and inhibition of PI3K (phosphatidylinositol 3-kinase), mTOR (mammalian target of rapamycin), or ERK significantly enhanced CUDC-907-induced cell deathin pancreatic cell lines. Taken together, these findings support the clinical development of CUDC-907 for the treatment of pancreatic cancer and identify compensatory activation of mTOR and MEK/ERK as a possible mechanism of resistance to CUDC-907.

S-Nitroso-L-Cysteine Ameliorated Pulmonary Hypertension in the MCT-Induced Rats through Anti-ROS and Anti-Inflammatory Pathways.

Pulmonary hypertension (PH) is a progressive and life-threatening chronic disease in which increased pulmonary artery pressure (PAP) and pulmonary vasculature remodeling are prevalent. Inhaled nitric oxide (NO) has been used in newborns to decrease PAP in the clinic; however, the effects of NO endogenous derivatives, S-nitrosothiols (SNO), on PH are still unknown. We have reported that S-nitroso-L-cysteine (CSNO), one of the endogenous derivatives of NO, inhibited RhoA activity through oxidative nitrosation of its C16/20 residues, which may be beneficial for both vasodilation and remodeling. In this study, we presented data to show that inhaled CSNO attenuated PAP in the monocrotaline- (MCT-) induced PH rats and, moreover, improved right ventricular (RV) hypertrophy and fibrosis induced by RV overloaded pressure. In addition, aerosolized CSNO significantly inhibited the hyperactivation of signal transducers and activators of transduction 3 (STAT3) and extracellular regulated protein kinases (ERK) pathways in the lung of MCT-induced rats. CSNO also regulated the expression of smooth muscle contractile protein and improved aberrant endoplasmic reticulum (ER) stress and mitophagy in lung tissues following MCT induction. On the other hand, CSNO inhibited reactive oxygen species (ROS) production in vitro, which is induced by angiotensin II (AngII) as well as interleukin 6 (IL-6). In addition, CSNO inhibited excessive ER stress and mitophagy induced by AngII and IL-6 in vitro; finally, STAT3 and ERK phosphorylation was inhibited by CSNO in a concentration-dependent manner. Taken together, CSNO led to pulmonary artery relaxation and regulated pulmonary circulation remodeling through anti-ROS and anti-inflammatory pathways and may be used as a therapeutic option for PH treatment.

Norcantharidin induces G2/M arrest and apoptosis via activation of ERK and JNK, but not p38 signaling in human renal cell carcinoma ACHN cells.

Renal cell carcinoma (RCC) is generally acknowledged as the most resistant primary malignancy unresponsive to conventional radiotherapy and chemotherapy treatments. Norcantharidin (NCTD), a therapeutic compound derived from medicinal plants, has been shown to trigger apoptosis, as well as antimetastatic and antioxidant activities in several tumor cells. However, NCTD's mechanism of antitumor activity in the RCC cell line remains unclear. In this study, we report that NCTD led to a time- and dose-dependent inhibition of cell proliferation. It had also markedly induced apoptosis and G2/M phase cell cycle arrest in a dose-dependent manner by decreasing the expressions of pro-caspase-3, pro-caspase-9, cyclin B1, and pCDC25C while increasing active caspase-3, cleaved-PARP, P21, and pCDC2 levels. Interestingly, NCTD treatment provoked the phosphorylation of extracellular-regulated protein kinase (ERK) and c-Jun-N-terminal kinase (JNK), but not of p38 MAPK. Moreover, SCH772984 and SP600125, ERK and JNK inhibitors, respectively, could partially abolish NCTD-induced apoptosis and G2/M phase cell cycle arrest. Collectively, these findings suggest that NCTD might activate JNK and ERK signaling pathways, consequently inducing apoptosis and G2/M arrest through the modulation of related proteins. This study provided evidence that NCTD is a promising therapeutic drug for the treatment of RCC.

Beneficial Effects of Crocin against Depression via Pituitary Adenylate Cyclase-Activating Polypeptide.

Depression is one of the foremost psychological illness, and the exact mechanism is unclear. Recent studies have reported that the pituitary adenylate cyclase-activating polypeptide (PACAP) signaling pathway is involved in the progression of depression. In the present study, we extracted crocin from the traditional Chinese medicine (TCM), Gardenia jasminoides Ellis, to evaluate its antidepressant effect and clarify the underlying mechanism. Here, we established a chronic unpredictable mild stress (CUMS) mouse model to assess whether crocin can improve depression-like behavior in an open field test (OFT), tail suspension test (TST), forced swimming test (FST), and sucrose preference test (SPT). A corticosterone (CORT) model of PC12 was set up to explore the antidepressant mechanism of crocin. We pretreated PC12 cells with crocin for 1 hour and then stimulated the cells with CORT for 24 hours. Cell survival was detected by Hoechst staining and MTT assay. The expression of PACAP, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), and extracellular regulated protein kinases (ERK) were analyzed by western blotting. PACAP RNAi was used to interfere with PC12 cells to downregulate the content of PACAP. The results showed that crocin (30 mg/kg) significantly reversed the decrease of body weight and elevation of serum CORT, mitigated CUMS induced depression-like behaviors of mice, and crocin (12.5 μmol/L) protected PC12 cells against CORT (200 μmol/L)-induced injury. Furthermore, crocin greatly increased the protein expression of PACAP and phosphorylation of ERK and CREB in the CORT model. PACAP RNAi cancelled the neuroprotective effect of crocin. In conclusion, these results indicated that crocin exerted an antidepressant effect via upregulating PACAP and its downstream ERK and CREB signaling pathways.

Fli-1 Activation through Targeted Promoter Activity Regulation Using a Novel 3', 5'-diprenylated Chalcone Inhibits Growth and Metastasis of Prostate Cancer Cells.

The friend leukemia integration 1 (Fli-1) gene is involved in the expression control of key genes in multiple pathogenic/physiological processes, including cell growth, differentiation, and apoptosis; this implies that Fli-1 is a strong candidate for drug development. In our previous study, a 3′,5′-diprenylated chalcone, (E)-1-(2-hydroxy-4-methoxy-3,5-diprenyl) phenyl-3-(3-pyridinyl)-propene-1-one (C10), was identified as a novel anti-prostate cancer (PCa) agent. Here, we investigated the molecular mechanisms underlying the anti-cancer effects of C10 on the growth, metastasis, and invasion of PC3 cells in vitro. Our results show that C10 exhibited a strong inhibitory effect on proliferation and metastasis of PC3 cells via several cellular and flow cytometric analyses. Further mechanism studies revealed that C10 likely serves as an Fli-1 agonist for regulating the expression of Fli-1 target genes including phosphatidylinositol 3-kinase (P110), murine double minute2 (MDM2), B-cell lymphoma-2 (Bcl-2), Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP-1), and globin transcription factor-1 (Gata-1) as well as the phosphorylation of extracellular-regulated protein kinases 1 (ERK1). Further, we confirmed that C10 can regulate the expressions of vascular endothelial growth factor 1 (VEGF-1), transforming growth factor-β2 (TGF-β2), intercellular cell adhesion molecule-1 (ICAM-1), p53, and matrix metalloproteinase 1 (MMP-1) genes associated with tumor apoptosis, migration, and invasion. Thus, C10 exhibits stronger anticancer activity with novel molecular targets and regulatory molecular mechanisms, indicating its great potency for development as a novel targeted anticancer drug.

N-methyl-d-aspartate receptor subunit 2B on keratinocyte mediates peripheral and central sensitization in chronic post-ischemic pain in male rats

The spinal N-methyl-d-aspartate (NMDA) receptor, and particularly its NR2B subunit, plays a pivotal role in neuropathic pain. However, the role of peripheral NMDA receptor in neuropathic pain is less well understood. We first treated cultured human keratinocytes, HaCaT cells with NMDA or NR2B-specific antagonist, ifenprodil and evaluated the level of total and phosphorylated NR2B at 24 h using Western blot. Next, using the chronic post-ischemia pain (CPIP) model, we administered NMDA or ifenprodil subcutaneously into the hind paws of male rats. Nociceptive behaviors were assessed by measuring mechanical and thermal withdrawal thresholds. Expression and phosphorylation of NR2B on keratinocyte were analyzed at 6, 12, 18, and 24 h on day 1 (initiation of pain) as well as day 2, 6, 10 and 14 (development and maintenance of pain) after the ischemia. The level of peripheral sensitization-related proteins (nuclear factor-κB (NF-κB), extracellular regulated protein kinases (ERK), and interleukin-1β (IL-1β)) in epidermis and dorsal root ganglion (DRG) were evaluated by immunofluorescence and western blot. Central sensitization-related C-fos induction, as well as astrocytes and microglia activation in the spinal cord dorsal horn (SDH) were studied using immunofluorescence. Administration of NMDA upregulated NR2B phosphorylation on HaCaT cells. CPIP-induced mechanical allodynia and thermal hyperalgesia were intensified by NMDA and alleviated by ifenprodil. CPIP resulted in an early upregulation of NR2B (peaked at 24 h) and late phosphorylation of NR2B (peaked at 14d) in hindpaw keratinocytes. CPIP led to an upregulation and phosphorylation of NF-κB and ERK, as well as an increased IL-1β production in the ipsilateral skin and DRG. CPIP-associated c-fos induction in SDH persisted from acute to chronic stages after ischemia, while microglia and astrocyte activation were only observed in chronic phase. These CPIP-induced changes were also suppressed by ifenprodil administered subcutaneously in the hind paw. Our findings reveal a previously unrecognized role of keratinocyte NMDA receptor subunit 2B in peripheral and central nociceptive sensitization induced by CPIP.