LncRNA-UC.25+ ShRNA Alleviates P2Y14 Receptor Mediated Diabetic Neuropathic Pain Via STAT1

Diabetic neuropathic pain (DNP) is a common complication of diabetes, and its complicated pathogenesis as well as clinical manifestations has brought great troubles to clinical treatment. The spinal cord is an important part of regulating the occurrence and development of DNP. Spinal microglia can regulate the activity of spinal cord neurons and have a regulatory effect on chronic pain. P2Y12 receptor is involved in DNP. P2Y14 and P2Y12 receptor belong to the Gi subtype of P2Y receptors, but there is no report that P2Y14 receptor is involved in DNP. Closely related to many human diseases, the dysregulation of lncRNA has the effect of promoting or inhibiting the occurrence and development of diseases. The aim of this research is to investigate the function of spinal cord P2Y14 receptor in type 2 DNP and to understand the function as well as the possible mechanism of lncRNA-UC.25+ (UC.25+) in rat spinal cord P2Y14 receptor-mediated DNP. Our results showed that P2Y14 shRNA can reduce the expression of P2Y14 in DNP rats, thereby restraining the activation of microglia, decreasing the expression of inflammatory factors and the level of p38 MAPK phosphorylation. At the same time, UC.25+ shRNA can down-regulate the expression of P2Y14 receptor, reduce the release of inflammatory factors, and diminish the p38 MAPK phosphorylation, indicating that UC.25+ can alleviate spinal cord P2Y14 receptor-mediated DNP. The RNA immunoprecipitation result showed that UC.25+ enriched STAT1 and positively regulated its expression. The chromatin immunoprecipitation result indicated that STAT1 combined to the promoter region of P2Y14 receptor and positively regulated the expression of P2Y14 receptor. Therefore, we infer that UC.25+ may alleviate DNP in rats by regulating the expression of P2Y14 receptor in spinal microglia via STAT1.

A Combination Extract of Gardeniae Fructus and Perillae Folium Exerts Anti-Inflammatory Effects on LPS-Activated RAW 264.7 Mouse Macrophages via an ER Stress-Induced CHOP Pathway

The aim of this study is to investigate the effects of a combination extract of Gardeniae Fructus and Perillae Folium (GP) on inflammatory reactions in lipopolysaccharide (LPS)-activated mouse macrophages RAW 264.7 cells. Multiplex cytokine assay, Fluo-4 calcium assay, Flow cytometry assay for phospho-P38 MAPK, and quantitative PCR were carried out. GP significantly reduced LPS-induced productions of macrophage inflammatory protein (MIP)-1α and monokine induced by gamma interferon (MIG) and release of intracellular calcium in LPS-activated RAW 264.7 cells. GP also significantly inhibited P38 MAPK phosphorylation and mRNA levels of Chop, Camk2a, Stat1, Stat3, Jak2, Fas, Nos2, and Ptgs2 in LPS-activated RAW 264.7 cells. Taken together, this study represents that GP exerts anti-inflammatory effects on LPS-activated RAW 264.7 cells via ER stress-induced CHOP pathway.

The antioxidant N-acetylcysteine promotes immune response and inhibits epithelial-mesenchymal transition to alleviate pulmonary fibrosis in chronic obstructive pulmonary disease by suppressing the VWF/p38 MAPK axis

Background/aim N-Acetylcysteine (NAC) demonstrates applications in the prevention of exacerbation of chronic obstructive pulmonary disease (COPD). COPD is often characterized by fibrosis of the small airways. This study aims at investigating the physiological mechanisms by which NAC might mediate the pulmonary fibrosis in COPD. Methods A total of 10 non-smokers without COPD and 10 smokers with COPD were recruited in this study, and COPD rat models were established. Cigarette smoke extract (CSE) cell models were constructed. The gain- or loss-of-function experiments were adopted to determine the expression of VWF and the extent of p38 MAPK phosphorylation, levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and immunoglobulins (IgG, IgM and IgA) in the serum of COPD rats and supernatant of alveolar epithelial cells and to detect cell invasion and migration and the ratio of CD3+, CD4+, CD8+ and CD4+/CD8+T lymphocytes. Results Expression of VWF and the extent of p38 MAPK phosphorylation were increased in COPD. NAC inhibited p38 MAPK phosphorylation by reducing the VWF expression. NAC could inhibit cell migration and invasion, elevate E-cadherin expression, the ratio of CD3+, CD4+, CD8+ and CD4+/CD8+T lymphocytes, and levels of IgG, IgA, and IgM, and reduce N-cadherin expression and levels of IL-6 and TNF-α in CSE cells and serum of COPD rats. NAC promoted immune response and suppressed epithelial-mesenchymal transformation (EMT) to relieve COPD-induced pulmonary fibrosis in vitro and in vivo by inhibiting the VWF/p38 MAPK axis. Conclusions Collectively, NAC could ameliorate COPD-induced pulmonary fibrosis by promoting immune response and inhibiting EMT process via the VWF/p38 MAPK axis, therefore providing us with a potential therapeutic target for treating COPD.

P80 Natural Essence Exerts Efficient Anti-HIV-1- as Well as Adjuvant Effects in DCs

Dendritic cells (DCs), as well as complement, play a major role during human immunodeficiency virus 1 (HIV-1) entry and infection at mucosal sites. Together, DCs and complement are key points for understanding host defence against HIV-1 infection and for studying the impact of new drugs on the regulation of innate host-pathogen interactions and adaptive immunity. For this, we evaluated the antiviral effect of the P80 natural essence (Longan extract) on interactions of non- and complement-opsonized HIV-1 with DCs. In viability assays, we first illustrated the effects of P80 natural essence on DC function. We found that P80 concentrations above 1.5% caused increased cell death, while at concentrations between 0.5% and 1% the compound exerted efficient antiviral effects in DCs and illustrated an adjuvant effect regarding DC activation. DC maturation, as well as co-stimulatory capacity, were significantly improved by P80 natural essence via p38 MAPK phosphorylation in presence of the viral challenge independent of the opsonization pattern. These findings might be exploited for future therapeutic options to target DC subsets directly at mucosal sites by P80 natural essence and to block entry of both, non- and complement-opsonized HIV-1.

Noble 3,4-Seco-triterpenoid Glycosides from the Fruits of Acanthopanax sessiliflorus and Their Anti-Neuroinflammatory Effects

Acanthopanax sessiliflorus (Araliaceae) have been reported to exhibit many pharmacological activities. Our preliminary study suggested that A. sessiliflorus fruits include many bioactive 3,4-seco-triterpenoids. A. sessiliflorus fruits were extracted in aqueous EtOH and fractionated into EtOAc, n-BuOH, and H2O fractions. Repeated column chromatographies for the organic fractions led to the isolation of 3,4-seco-triterpenoid glycosides, including new compounds. Ultra-high-performance liquid chromatography (UPLC) mass spectrometry (MS) systems were used for quantitation and quantification. BV2 and RAW264.7 cells were induced by LPS, and the levels of pro-inflammatory cytokines and mediators and their underlying mechanisms were measured by ELISA and Western blotting. NMR, IR, and HR-MS analyses revealed the chemical structures of the nine noble 3,4-seco-triterpenoid glycosides, acanthosessilioside G–O, and two known ones. The amounts of the compounds were 0.01–2.806 mg/g, respectively. Acanthosessilioside K, L, and M were the most effective in inhibiting NO, PGE2, TNF-α, IL-1β, and IL-6 production and reducing iNOS and COX-2 expression. In addition, it had inhibitory effects on the LPS-induced p38 and ERK MAPK phosphorylation in both BV2 and RAW264.7 cells. Nine noble 3,4-seco-triterpenoid glycosides were isolated from A. sessiliflorus fruits, and acanthosessilioside K, L, and M showed high anti-inflammatory and anti-neuroinflammatory effects.

Sargassum Horneri (Turner) C. Agardh Regulates Neuroinflammation via Inhibition of LPS-stimulated Microglia Activation in Vitro and in Vivo

Previously we reported that Sargassum horneri (Turner) C. Agardh (S. horneri) is a brown algae species that exerts anti-inflammatory activity toward murine macrophages. However, the anti-neuroinflammatory effects and the mechanism of S. horneri on microglia cells are still unknown. We investigated the anti-neuroinflammatory effects of S. horneri extract on microglia in vitro and in vivo. In present study, we found that S. horneri was not cytotoxic to BV-2 microglia cells, and it significantly decreased lipopolysaccharide (LPS)-induced NO production. Moreover, S. horneri also diminished the protein expression of iNOS, COX-2, and cytokines production including IL-1b, TNF-a, and IL-6 on LPS-stimulated microglia activation. S. horneri elicited anti-neuroinflammatory effects by inhibiting phosphorylation of p38 MAPK and NF-kB. In addition, S. horneri inhibited astrocytes and microglia activation in LPS-challenged mice brain. Therefore, these results suggested that S. horneri exerted anti-neuroinflammatory effects on LPS-stimulated microglia cells activation by inhibiting MAPK phosphorylation and NF-kB signaling.

Biochanin A Alleviates Cerebral Ischemia/Reperfusion Injury by Suppressing Endoplasmic Reticulum Stress-Induced Apoptosis and p38MAPK Signaling Pathway In Vivo and In Vitro

We have previously shown that biochanin A exhibits neuroprotective properties in the context of cerebral ischemia/reperfusion (I/R) injury. The mechanistic basis for such properties, however, remains poorly understood. This study was therefore designed to explore the manner whereby biochanin A controls endoplasmic reticulum (ER) stress, apoptosis, and inflammation within fetal rat primary cortical neurons in response to oxygen-glucose deprivation/reoxygenation (OGD/R) injury, and in a rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) injury. For the OGD/R in vitro model system, cells were evaluated after a 2 h OGD following a 24 h reoxygenation period, whereas in vivo neurological deficits were evaluated following 2 h of ischemia and 24 h of reperfusion. The expression of proteins associated with apoptosis, ER stress (ERS), and p38 MAPK phosphorylation was evaluated in these samples. Rats treated with biochanin A exhibited reduced neurological deficits relative to control rats following MCAO/R injury. Additionally, GRP78 and CHOP levels rose following I/R modeling both in vitro and in vivo, whereas biochanin A treatment was associated with reductions in CHOP levels but further increases in GRP78 levels. In addition, OGD/R or MCAO/R were associated with markedly enhanced p38 MAPK phosphorylation that was alleviated by biochanin A treatment. Similarly, OGD/R or MCAO/R injury resulted in increases in caspase-3, caspase-12, and Bax levels as well as decreases in Bcl-2 levels, whereas biochanin A treatment was sufficient to reverse these phenotypes. Together, these findings thus demonstrate that biochanin A can alleviate cerebral I/R-induced damage at least in part via suppressing apoptosis, ER stress, and p38 MAPK signaling, thereby serving as a potent neuroprotective agent.

Peptide Based Inhibitors of Protein Binding to the Mitogen-Activated Protein Kinase Docking Groove

Mitogen-activated protein kinases (MAPK) are important regulatory units in cells and they take part in the regulation of many cellular functions such as cell division, differentiation or apoptosis. All MAPKs have a shallow docking groove that interacts with linear binding motifs of their substrate proteins and their regulatory proteins such as kinases, phosphatases, scaffolds. Inhibition of these protein–protein interactions may reduce or abolish the activity of the targeted kinase. Based on the wide range of their biological activity, this kind of inhibition can be useful in the treatment of many disorders like tumors, inflammation or undesired cell apoptosis. In this study a linear binding motif from the RHDF1 protein—a 15 amino acids long peptide—was selected for optimization to increase its cellular uptake but retaining its low micromolar binding affinity. First, we synthesized an octaarginine conjugate that showed efficient cellular uptake. Next, we set out to reduce the size of this construct. We were able to decrease the length of the original peptide, and to increase its cellular uptake with specific chemical modifications. These new constructs bound better to ERK2 and p38 kinases than the original peptide and they showed markedly increased cellular uptake. The new octaarginine conjugate and one of the minimized bicyclic derivatives could inhibit the phosphorylation of intracellular ERK or p38. However, the modulation of MAPK phosphorylation levels by these cell-penetrating peptides were complex, despite that in biochemical assays they all inhibited MAPK-substrate binding as well as phosphorylation. The optimized peptides depending on the applied concentration caused an expected decrease, but also some unexpected increase in MAPK phosphorylation patterns in the cell. This possibly reflects the complexity of MAPK docking groove mediated protein–protein interactions including bone fide MAPK clients such activator kinases, deactivating phosphatases or regulatory scaffolds. Thus, our findings with optimized cell-penetrating “inhibitory” peptides highlight the opportunities but also the pitfalls of docking peptide based MAPK activity regulation and call for a better quantitative understanding of MAPK mediated protein–protein interactions in cells.

Sprouty4 negatively regulates ERK/MAPK signaling and the transition from in situ to invasive breast ductal carcinoma

Breast ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal carcinoma (IDC). It is still unclear which DCIS will become invasive and which will remain indolent. Patients often receive surgery and radiotherapy, but this early intervention has not produced substantial decreases in late-stage disease. Sprouty proteins are important regulators of ERK/MAPK signaling and have been studied in various cancers. We hypothesized that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling and that its loss/reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry, we found reduced Sprouty4 expression in IDC patient samples compared to DCIS, and that ERK/MAPK phosphorylation had an inverse relationship to Sprouty4 expression. These observations were reproduced using a 3D culture model of disease progression. Knockdown of Sprouty4 in MCF10.DCIS cells increased ERK/MAPK phosphorylation as well as their invasive capability, while overexpression of Sprouty4 in MCF10.CA1d IDC cells reduced ERK/MAPK phosphorylation, invasion, and the aggressive phenotype exhibited by these cells. Immunofluorescence experiments revealed reorganization of the actin cytoskeleton and relocation of E-cadherin back to the cell surface, consistent with the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, MEK1/2 was pharmacologically inhibited in IDC cells. Nanomolar concentrations of MEK162/binimetinib restored an epithelial-like phenotype and reduced pericellular proteolysis, similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 acts to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions.