Phosphorylation Of c-Jun N-terminal Kinase Research Articles

Anti-Melanogenesis Effect of Polysaccharide from Saussurea involucrata on Forskolin-Induced Melanogenesis in B16F10 Melanoma Cells.

As one of the prominent medicinal plants listed in the Chinese pharmacopoeia (2020), Saussurea involucrata (Kar. et Kir.) Sch.-Bip was demonstrated to possess various therapeutic effects. In our recent research, we extracted the polysaccharides from S. involucrata (SIP) at optimal conditions and conducted further structure elucidation on the main fraction as well as the confirmation of its possible anti-inflammatory activity. Hence, in this work, we assessed the in vitro antioxidant activity and anti-melanogenesis effects of the crude SIP in forskolin-induced B16F10 melanoma cells. The results show that SIP possessed strong antioxidant activity and was effective in concentration-dependently decreasing melanin formation and inhibiting tyrosinase activity in forskolin-induced B16F10 cells. Based on these results, the inhibitory mechanism of melanogenesis was investigated by measuring Tyrosinase (TYR), Tyrosinase related protein-1 (TRP-1), Tyrosinase related protein-2 (TRP-2), Microphthalmia-associated transcription factor (MITF), cAMP-response element binding protein (CREB), mitogen-activated protein kinases (MAPK) signaling protein members, and β-catenin degradation in forskolin-induced B16F10 cells. The anti-melanogenesis response of SIP might be attributed to the regulation of c-Jun N-terminal kinase (JNK) phosphorylation and β-catenin degradation pathways. These results suggest that polysaccharides from S. involucrata possess a strong anti-melanogenic effect, and thus could be used as a high-value natural material for skin whitening in cosmeceutical industries.

Protection against Lipopolysaccharide-Induced Endotoxemia by Terrein Is Mediated by Blocking Interleukin-1β and Interleukin-6 Production.

Terrein is a fungal metabolite and has been known to exert anti-melanogenesis, anti-cancer, and anti-bacterial activities. However, its role in endotoxemia has never been investigated until now. In the present study, we examined the effect of terrein on lipopolysaccharide (LPS)-induced endotoxemia in mice and characterized the potential mechanisms of action. Treatment with terrein increased the survival of mice and decreased the production of inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6) in an LPS-induced endotoxemia model. In addition, terrein suppressed the LPS-induced production of IL-1β and IL-6 in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expression of IL-1β and IL-6 was also inhibited by terrein in LPS-stimulated RAW 264.7 cells. Further study demonstrated that terrein blocked LPS-induced phosphorylation of p65 subunit of nuclear factor (NF)/κB and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) was also suppressed by terrein treatment. Collectively, these results suggest that terrein exerts a protective effect again LPS-induced endotoxemia in mice by blocking the production of inflammatory cytokines. Our results also suggest that the anti-inflammatory effect of terrein might be mediated, at least in part, by blocking the activation of NF-κB, JNK, and p38 MAPK signaling pathways.

Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells

We have investigated the effects of melatonin on major pathways related with cellular proliferation and energetic metabolism in pancreatic stellate cells. In the presence of melatonin (1 mM, 100 µM, 10 µM, or 1 µM), decreases in the phosphorylation of c-Jun N-terminal kinase and of p44/42 and an increase in the phosphorylation of p38 were observed. Cell viability dropped in the presence of melatonin. A rise in the phosphorylation of AMP-activated protein kinase was detected in the presence of 1 mM and 100 µM melatonin. Treatment with 1 mM melatonin decreased the phosphorylation of protein kinase B, whereas 100 µM and 10 µM melatonin increased its phosphorylation. An increase in the generation of mitochondrial reactive oxygen species and a decrease of mitochondrial membrane potential were noted following melatonin treatment. Basal and maximal respiration, ATP production by oxidative phosphorylation, spare capacity, and proton leak dropped in the presence of melatonin. The expression of complex I of the mitochondrial respiratory chain was augmented in the presence of melatonin. Conversely, in the presence of 1 mM melatonin, decreases in the expression of mitofusins 1 and 2 were detected. The glycolysis and the glycolytic capacity were diminished in cells treated with 1 mM or 100 µM melatonin. Increases in the expression of phosphofructokinase-1 and lactate dehydrogenase were noted in cells incubated with 100 µM, 10 µM, or 1 µM melatonin. The expression of glucose transporter 1 was increased in cells incubated with 10 µM or 1 µM melatonin. Conversely, 1 mM melatonin decreased the expression of all three proteins. Our results suggest that melatonin, at pharmacological concentrations, might modulate mitochondrial physiology and energy metabolism in addition to major pathways involved in pancreatic stellate cell proliferation.

Tooth Loss Induces Memory Impairment and Glial Activation in Young Wild-Type Mice.

Tooth loss is closely associated with Alzheimer's disease (AD). Previously, we reported that tooth loss induced memory impairment in amyloid precursor protein knock-in mice by decreasing neuronal activity and synaptic protein levels and increasing glial activation, neuroinflammation, and pyramidal neuronal cell loss without altering amyloid-β levels in the hippocampus. However, the effects of tooth loss in young wild-type mice have not been explored yet. We investigated the effects of tooth loss on memory impairment, neuronal activity, synaptic protein levels, glial activation, and pyramidal neuronal cell loss in young wild-type mice. Two-month-old wild-type mice were randomly divided into control and tooth loss groups. In the tooth loss group, maxillary molar teeth on both sides were extracted, whereas no teeth were extracted in the control group. Two months after tooth extraction, we performed a novel object recognition test to evaluate memory function. Glial activation, neuronal activity, synaptic protein levels, and the number of pyramidal neurons were evaluated using immunofluorescence staining, immunohistochemistry, and western blotting. The tooth loss group exhibited memory impairment and decreased neuronal activity and the levels of synaptic proteins in both the hippocampus and cortex. Moreover, tooth loss increased the activation of phosphorylated c-Jun N-terminal kinase (JNK), heat shock protein 90 (HSP90), and glial activation and reduced the number of pyramidal neurons in the hippocampus. Tooth loss in the young wild-type mice will attenuate neuronal activity, decrease synaptic protein levels, and induce pyramidal neuronal loss, and eventually lead to memory impairment.

Effects of diacylglycerol O-acyltransferase 1 (DGAT1) on endoplasmic reticulum stress and inflammatory responses in adipose tissue of ketotic dairy cows.

Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of β-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 μM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 μM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

Neochamaejasmine A Promotes Apoptosis and Cell Cycle Arrest in B16F10 Melanoma Cells via JNK and p38 MAPK Signaling Pathway.

The incidence of melanoma has been increasing over the last 30 years. The most common treatments, such as surgery, chemotherapy, and radiotherapy, frequently cause serious damage to the body. It is therefore critical to develop a new therapeutic strategy for the treatment of melanoma. This research aims to evaluate the anti-tumor effect of Neochamaejasmine A (NCA) on B16F10 melanoma cells and the underlying molecular mechanisms. The CCK-8 kit was utilized to assay the influence of NCA on the vitality of B16F10 cells. Modifications in B16F10 cells morphology were observed using a phase-contrast microscope. Apoptosis of B16F10 melanoma cells was assessed by Hoechst 33258, Annexin V and propidium iodide staining. Cell cycle was detected using a commercial kit by flow cytometry. The mRNA and protein expression levels associated with apoptosis and cell cycle arrest were detected by RT-PCR and Western blot. The expression level of pathway proteins was assessed using Western blot. It was found that the proliferation of B16F10 cells was inhibited by NCA in concentration- and time-dependent manners. NCA promoted apoptosis by halting the cell cycle at the G2/M phase. After treatment with NCA, cell apoptosis was confirmed by Hoechst 33258 staining. NCA triggered the cell cycle to seize at the G2/M stage by downregulating cyclin B1 and cyclin-dependent kinase 2 (CDC2) expression. Moreover, the mRNA and protein expression of cleaved caspase- 9 and Bcl-2-associated X-protein (Bax) were increased, whereas there was a decline in the expression of B-cell lymphoma 2 (Bcl-2). The p-p38/p38 and phosphorylated c-Jun N-terminal kinase (p-JNK/JNK) ratio were also elevated by NCA. The apoptosis and G2/M cell cycle arrest were inhibited in cells co-treated with the p38 inhibitor SB203580 and JNK inhibitor SP600125. The expression of apoptosis-related proteins Bax was decreased, and Bcl-2 was increased. The findings of this study showed that NCA could induce apoptosis and cell cycle arrest in B16F10 melanoma cells by activating JNK and p38 MAPK signaling pathway.

Orientin inhibits the progression of fibroblast-like synovial cells in rheumatoid arthritis by regulating MAPK-signaling pathway.

Natural compounds are found to play an essential role in diverse inflammatory diseases, including rheumatoid arthritis (RA). Orientin, a flavonoid compound, is closely related to diverse pathological processes. Nevertheless, the role of orientin in RA is still unknown. The cell viability was tested through cell counting kit 8 (CCK-8) assay, and the number of cell colonies was calculated via colony formation assay. In addition, flow cytometry assay was employed to detect apoptosis rate in human RA fibroblast-like synoviocytes (RA-FLS). Besides, Transwell assay was introduced to determine cell migratory and invasive abilities. Moreover, the level of cytokines (IL-8, IL-1β, and IL-6) was estimated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent serologic assay. Furthermore, western blotting analysis was used to test the protein levels of cleaved-caspase-3, Bax, BCL-2, matrix metalloproteinase (MMP)-2, MMP-9, phosphorylated c-Jun N-terminal kinase, p-P38, and phospho-extracellular signal-related kinase. Orientin inhibited cell viability, migration as well as invasion in a concentration- dependent manner in human RA-FLS. Additionally, treatment of orientin facilitated apoptosis and decreased the secretion of cytokines induced by tumor necrosis factor alpha (TNF-α) in human RA-FLS. Moreover, orientin inactivated mitogen-activated protein kinase (MAPK)-related signaling pathway, notably in human RA-FLS. These findings confirmed that orientin inhibited human RA-FLS development and decreased TNF-α-induced inflammatory factors, at least partly, by modulating MAPK-signaling pathway, which implied that orientin might be an effective agent for treating RA.

WITHDRAWN:Prussian blue nanozymes coated with pluronic attenuate inflammatory osteoarthritis by blocking c-Jun N-terminal kinase phosphorylation

This article has been withdrawn: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/policies/article-withdrawal). This article has been withdrawn at the request of the editor and publisher. The publisher regrets that an error occurred which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error.

Bullatine A exerts anti-inflammatory effects by inhibiting the ROS/JNK/NF-κB pathway and attenuating systemic inflammatory responses in mice

Context Aconiti brachypodi Radix (Xue-shang-yi-zhi-hao) is a traditional Chinese herbal medicine that is capable of anti-analgesic and anti-inflammatory effects. Bullatine A (BA) is one of the major active ingredients of this plant, and most of the previous studies reported that it has anti-analgesic effects. However, the mechanism of BA anti-inflammatory remains unclear. Objective This study investigates the anti-inflammatory activities of BA, both in vitro and in vivo, and elucidates its mechanism. Materials and methods In vitro, BA (10, 20, 40 and 80 μM) was added to 1 µg/mL of lipopolysaccharide (LPS)-activated microglia BV2 cells and immortalized murine bone marrow-derived macrophages, respectively. After 6 h, the mRNA and protein levels of inflammatory factors were determined by real-time quantitative PCR and western blotting. In vivo, C57BL/6 mice were randomly divided into control, model (5 mg/kg dose of LPS) and treated groups (LPS with 5, 10 or 20 mg/kg dose of BA) to evaluate the anti-inflammatory efficacy of BA. Results BA significantly inhibited LPS-induced expression of inflammatory factors, such as IL-1β, IL-6, TNF-α, inducible nitric oxide synthase (iNOS) and COX-2. Further investigations showed that BA reduced the translocation of NF-κB p65 (38.5%, p < 0.01). BA also reduced the phosphorylation of c-Jun N-terminal kinase (JNK) (11.2%, p < 0.05) and reactive oxygen species (ROS) generation (24.2%, p < 0.01). Furthermore, BA treatment attenuated the LPS-primed inflammatory response and liver and lung damage in vivo. Conclusions BA can inhibit the inflammatory response in part through the ROS/JNK/NF-κB signalling pathway, providing a theoretical basis for the clinical application of BA in the treatment of periphery inflammatory diseases.

Transgenic Rice Seed Extracts Exert Immunomodulatory Effects by Modulating Immune-Related Biomarkers in RAW264.7 Macrophage Cells.

Protopanaxadiol (PPD), a native active triterpenoid present in Panax ginseng, has been reported to exert immune-related effects. We previously created PPD-producing transgenic rice by introducing the P. ginseng protopanaxadiol synthase and dammarenediol-II synthase genes into Dongjin rice. In the present study, the seeds of the T4 generation of this transgenic rice were tested for their immunomodulatory effects in RAW264.7 macrophage cells. Treatment with transgenic rice seed extract in RAW264.7 cells (i) significantly enhanced nitric oxide (NO) production in a dose-dependent manner without any cytotoxicity (up to 100 µg/mL), (ii) upregulated the expression of immune-related genes and increased production of the inflammation mediator prostaglandin E2 (PGE2), and (iii) activated nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) by promoting the phosphorylation of NF-κB p65, p38 MAPK, and c-Jun N-terminal protein kinase (JNK). In lipopolysaccharide (LPS)-treated RAW264.7 cells used to mimic the inflammation condition, treatment with transgenic rice seed extract significantly reduced NO production, proinflammatory cytokine expression, and PGE2 production, all of which are LPS-induced inflammation biomarkers, by inhibiting the phosphorylation of NF-κB p65, p38 MAPK, and JNK. Collectively, these results indicate that PPD-producing transgenic rice has immunomodulatory effects.

Huangqi decoction attenuates renal interstitial fibrosis transforming growth factor-β1/mitogen-activated protein kinase signaling pathways in 5/6 nephrectomy mice.

To investigate the effect of Huangqi decoction on renal interstitial fibrosis and its association with the transforming growth factor-β1 (TGF-β1) / mitogen-activated protein kinase (MAPK) signaling pathway. 120 C57/BL mice were randomly divided into six groups: sham group, Enalapril (20 mg/kg) group, 5/6 nephrectomy model group, and 5/6 nephrectomy model plus Huangqicoction (0.12, 0.36 and 1.08 g/kg respectively) groups. Detecting 24hours urinary protein, blood pressure, serum creatinine, urea nitrogen content changes. Periodic Acid-Schiff stain (PAS) and Masson's trichrome staining was used to observe the renal tissue pathological changes. Protein expression of TGF-β1, Phosphorylated P38 mitogen activated protein kinases (P-P38), Phosphorylated c-jun N-terminal kinase (P-JNK), Phosphorylated extracellular regulated proteinhnase (P-ERK), Fibroblast-specific protein-1 (FSP-1), Alpha smooth muscle actin (α-SMA), Type III collagen (Collagen III), Connective tissue growth factor (CTGF), Bcl-2 Assaciated X protein (Bax) and B cell lymphoma 2 (Bcl-2) were measured with western blot and immunohistochemical. Both Huangqi decoction and Enalapril improved the kidney function, 24 h urinary protein and the fibrosis in 5/6 nephrectomy mice, Huangqi decoction downregulated the expressions of TGF-β1, FSP-1, α-SMA, Collagen III and CTGF in a dose-dependent manner, and it has a significant difference ( 0.01) compared with model group.Huangqi decoction downregulated the expressions of P-P38, P-JNK, P-ERK and Bcl-2 in a dose-dependent manner, while upregulated the expression of Bax. The protective effect of Huangqi decoction for renal interstitial fibrosis in 5/6 nep-hrectomized mice the inhibition of Epithelial-Mesenchymal Transitions and downregulating the TGF-β1/ MAPK signaling pathway.

Reactive oxygen species/c-Jun N-terminal kinase/nuclear factor kappa-B signaling molecules are involved in pe-riodontitis-induced liver injury by regulating apoptosis.

The occurrence and development of periodontitis and nonalcoholic fatty liver disease (NAFLD) are closely related to the accumulation of reactive oxygen species (ROS). ROS are involved in regulating the activation of c-Jun N-terminal kinase (JNK)/nuclear factor kappa-B (NF-κB) signaling molecules. When the signaling molecules are overactivated by ROS, the internal environment of the body can be disturbed. Therefore, this study aimed to explore the mechanism by which ROS/JNK/NF-κB signaling molecules are involved in periodontitis-induced liver injury. Twelve SPF male Wistar rats were randomly divided into control and periodontitis groups. The perio-dontitis model of rats was established by wire ligation in the neck of bilateral maxillary first molars. After 8 weeks, the periodontal clinical indexes of the rats were examined, and the rats were sacrificed. Micro-CT reconstruction of a three-dimensional alveolar bone structure and analysis of alveolar bone absorption were conducted. Pathological changes in the periodontal and liver tissues were analyzed by histopathology. MitoSOX red reagent was used to detect the ROS content in liver tissue. Biochemical kits were used to detect liver function and oxidative stress biomarkers. The mRNA expression levels ofinterleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), NF-κB, BCL2-associated X (Bax), and B-cell lymphoma-2 (Bcl-2) in liver tissue were detected through quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of phosphorylated c-Jun N-terminal kinase (P-JNK), JNK, NF-κB, Caspase-3, Bax, and Bcl-2 in liver tissue were detected by Western blot. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Micro-CT results showed that the mice in the periodontitis group had obvious alveolar bone resorption and significantly greater distance from the cemento-enamel junction to the alveolar bone crest than those in the control group. Histopathological results showed that a large number of inflammatory cells were infiltrated in the periodontal tissue of the periodontitis group. In addition, the resorption of alveolar ridge bone was obvious and liver tissue structure was destroyed, with balloon-like changes and red lipid droplets. MitoSOX red staining results showed that the ROS level was significantly higher in the liver tissue of the periodontitis group than in that of the control group. Biochemical test results showed that the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the serum of the periodontitis group were higher than those in the serum of the control group. The levels of superoxide dismutase (SOD) and glutathione (GSH) in liver tissue decreased, whereas the that of malondialdehyde (MDA) increased. Western blot and qRT-PCR results revealed that the mRNA levels of IL-6, TNF-α, Bax, and NF-κB and the protein levels of P-JNK/JNK, NF-κB, Caspase-3, and Bax were significantly higher in the liver tissue of the perio-dontitis group than in that of the control group. Meanwhile, the mRNA and protein levels of Bcl-2 were lower in the periodontitis group than in the control group. TUNEL staining showed that the number of apoptotic cells was significantly higher in the periodontitis group than in the control group. ROS/JNK/NF-κB signaling molecules are involved in periodontitis-induced liver injury by regulating apoptosis.

IL-17A Promotes Epithelial ADAM9 Expression in Cigarette Smoke-Related COPD.

BackgroundIt has been reported that a disintegrin and metalloproteinase 9 (ADAM9) is involved in the pathogenesis of cigarette smoke (CS)-associated chronic obstructive pulmonary disease (COPD). But how CS exposure leads to upregulation of ADAM9 remains unknown.MethodsPatients who underwent lobectomy for a solitary pulmonary nodule were enrolled and divided into three groups: non-smokers with normal lung function, smokers without COPD and smoker patients with COPD. Immunoreactivity of interleukin (IL)-17A and ADAM9 in small airways and alveolar walls was measured by immunohistochemistry. Wild-type and Il17a−/− C57BL/6 mice were exposed to CS for six months, and ADAM9 expression in the airway epithelia was measured by immunoreactivity. In addition, the protein and mRNA expression levels of IL-17A and ADAM9 were assessed in CS extract (CSE) and/or IL-17A-treated human bronchial epithelial (HBE) cells.ResultsThe immunoreactivity of ADAM9 was increased in the airway epithelia and alveolar walls of patients with COPD compared to that of the controls. The expression of IL-17A was also upregulated in airway epithelial cells of patients with COPD and correlated positively with the level of ADAM9. The results from the animal model showed that Il17a−/− mice were protected from emphysema induced by CS exposure, together with a reduced level of ADAM9 expression in the airway epithelia, suggesting a possible link between ADAM9 and IL-17A. Consistently, our in vitro cell model showed that CSE stimulated the expression of ADAM9 and IL-17A in HBE cells in a dose- and time-dependent manner. Recombinant IL-17A induced ADAM9 upregulation in HBE cells and had a synergistic effect with CSE, whereas blocking IL-17A inhibited CSE-induced ADAM9 expression. Further analysis revealed that IL-17A induced c-Jun N-terminal kinase (JNK) phosphorylation, thereby increasing ADAM9 expression.ConclusionOur results revealed a novel role of IL-17A in CS-related COPD, where IL-17A contributes to ADAM9 expression by activating JNK signaling.

MiR-30a-5p and miR-153-3p regulate LPS-induced neuroinflammatory response and neuronal apoptosis by targeting NeuroD1

Neurogenic differentiation 1 (NeuroD1) is an essential transcription factor for neuronal differentiation, maturation, and survival, and is associated with inflammation in lipopolysaccharide (LPS)-induced glial cells; however, the concrete mechanisms are still ambiguous. Therefore, we investigated whether NeuroD1-targeting miRNAs affect inflammation and neuronal apoptosis, as well as the underlying mechanism. First, we confirmed that miR-30a-5p and miR-153-3p, which target NeuroD1, reduced NeuroD1 expression in microglia and astrocytes. In LPS-induced microglia, miR-30a-5p and miR-153-3p suppressed pro-inflammatory cytokines, reactive oxygen species, the phosphorylation of c-Jun N-terminal kinase, extracellular-signal-regulated kinase (ERK), and p38, and the expression of cyclooxygenase and inducible nitric oxide synthase (iNOS) via the NF-κB pathway. Moreover, miR-30a-5p and miR-153-3p inhibited the expression of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasomes, NLRP3, cleaved caspase-1, and IL-1β, which are involved in the innate immune response. In LPS-induced astrocytes, miR-30a-5p and miR-153-3p reduced ERK phosphorylation and iNOS expression via the STAT-3 pathway. Notably, miR-30a-5p exerted greater anti-inflammatory effects than miR-153-3p. Together, these results indicate that miR-30a-5p and miR-153-3p inhibit MAPK/NF-κB pathway in microglia as well as ERK/STAT-3 pathway in astrocytes to reduce LPS-induced neuronal apoptosis. This study highlights the importance of NeuroD1 in microglia and astrocytes neuroinflammation and suggests that it can be regulated by miR-30a-5p and miR-153-3p.

Hcmv-miR-UL148D regulates the staurosporine-induced apoptosis by targeting the Endoplasmic Reticulum to Nucleus signaling 1(ERN1).

The propensity of viruses to co-opt host cellular machinery by reprogramming the host’s RNA-interference machinery has been a major focus of research, however, regulation of host defense mechanisms by virus-encoded miRNA, is an additional regulatory realm gaining momentum in the arena of host-viral interactions. The Human Cytomegalovirus (HCMV) miRNAs, regulate many cellular pathways alone or in concordance with HCMV proteins, thereby paving a conducive environment for successful infection in the human host. We show that HCMV miRNA, hcmv-miR-UL148D inhibits staurosporine-induced apoptosis in HEK293T cells. We establish that ERN1 mRNA is a bonafide target of hcmv-miR-UL148D and its encoded protein IRE1α is translationally repressed by the overexpression of hcmv-miR-UL148D resulting in the attenuation of apoptosis. Unlike the host microRNA seed sequence (6–8 nucleotides), hcmv-miR-UL148D has long complementarity to 3’ UTR of ERN1 mRNA resulting in mRNA degradation. The repression of IRE1α by the hcmv-miR-UL148D further downregulates Xbp1 splicing and c-Jun N-terminal kinase phosphorylation thus regulating ER-stress and ER-stress induced apoptotic pathways. Strikingly, depletion of ERN1 attenuates staurosporine-induced apoptosis which further suggests that hcmv-miR-UL148D functions through regulation of its target ERN1. These results uncover a role for hcmv-miR-UL148D and its target ERN1 in regulating ER stress-induced apoptosis.

Quercus acuta Thunb. Suppresses LPS-Induced Neuroinflammation in BV2 Microglial Cells via Regulating MAPK/NF-κB and Nrf2/HO-1 Pathway.

Microglial activation-mediated neuroinflammation is associated with the pathogenesis of neurodegenerative disorders. Therefore, the management of microglial cell activation and their inflammatory response is an important therapeutic approach for preventing neurodegenerative diseases. Quercus acuta Thunb. (QA) (Fagaceae) is a tree found in Korea, China, and Japan. The current study investigated the anti-neuroinflammatory effects of QA and its mechanism of action in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Pretreatment with a methanol extract of dried QA stems (QAE) inhibited the production of nitric oxide and proinflammatory cytokines and decreased the expression of inducible nitric oxide synthase, cyclooxygenase-2 in LPS-stimulated BV2 microglial cells. Furthermore, it inhibited the phosphorylation and degradation of inhibitory κBα and decreased the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB). Moreover, QAE inhibited the phosphorylation of extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase, which is known as mitogen-activated protein kinase (MAPK). Additionally, QAE treatment increased heme oxygenase-1 (HO-1) expression by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, thereby ameliorating LPS-induced intracellular hydrogen peroxide production. Finally, it was found that catechin and taxifolin, two phytochemicals of QAE, also reduced the expression of inflammatory mediators. These findings suggest that QA is beneficial for preventing microglia-mediated neuroinflammatory response through the inhibition of NF-κB, MAPK and the activation of Nrf2/HO-1 signaling pathways.

HM-Chromanone, a Major Homoisoflavonoid in Portulaca oleracea L., Improves Palmitate-Induced Insulin Resistance by Regulating Phosphorylation of IRS-1 Residues in L6 Skeletal Muscle Cells.

This study investigated the effect of (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone) on palmitate-induced insulin resistance and elucidated the underlying mechanism in L6 skeletal muscle cells. Glucose uptake was markedly decreased due to palmitate-induced insulin resistance in these cells; however, 10, 25, and 50 µM HM-chromanone remarkably improved glucose uptake in a concentration-dependent manner. HM-chromanone treatment downregulated protein tyrosine phosphatase 1B (PTP1B) and phosphorylation of c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), which increased because of palmitate mediating the insulin-resistance status in cells. HM-chromanone promoted insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and suppressed palmitate-induced phosphorylation of IRS-1 serine. This activated phosphoinositide 3-kinase (PI3K) and stimulated protein kinase B (AKT) phosphorylation. Phosphorylated AKT promoted the translocation of Glucose transporter type 4 to the plasma membrane and significantly enhanced glucose uptake into muscle cells. Additionally, HM-chromanone increased glycogen synthesis through phosphorylating glycogen synthase kinase 3 alpha/beta (GSK3 α/β) via AKT. Consequently, HM-chromanone may improve insulin resistance by downregulating the phosphorylation of IRS-1 serine through inhibition of negative regulators of insulin signaling and inflammation-activated protein kinases in L6 skeletal muscle cells.

Apremilast ameliorates acute respiratory distress syndrome by inhibiting neutrophil-induced oxidative stress

BackgroundThe pathogenesis of acute respiratory distress syndrome (ARDS) is attributed to the dysregulation of oxidative stress and neutrophil recruitment. We aimed to investigate the anti-inflammatory effects of apremilast on human neutrophils and assess its efficacy for treating ARDS. MethodsWe analysed superoxide anion generation, integrin expression, and adhesion in activated human neutrophils using spectrophotometry, flow cytometry, and immunofluorescence microscopy. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was determined using immunoblotting. A murine lipopolysaccharide (LPS)-induced ARDS model was used to evaluate the therapeutic effects of apremilast. ResultsApremilast significantly decreased superoxide anion production, reactive oxygen species (ROS) generation, cluster of differentiation (CD)11 b expression, and neutrophil adhesion in formyl-l-methionyl-l-leucyl-l-phenylalanine activated human neutrophils. Apremilast elevated cyclic 3′,5′-adenosine monophosphate (cAMP) and protein kinase A (PKA) activity in activated neutrophils. It reduced cellular cAMP-specific phosphodiesterase (PDE) activity and selectively inhibited enzymatic PDE4 activity. The activated cAMP/PKA pathway suppressed the phosphorylation of ERK and JNK as well as Ca2+ mobilization in activated neutrophils. All inhibitory effects of apremilast on activated neutrophils were reversed by a PKA inhibitor. In vivo examinations indicated that apremilast alleviated lung neutrophil infiltration, myeloperoxidase (MPO) activity, pulmonary oedema, and alveolar damage in LPS-induced ARDS. ConclusionApremilast inhibits inflammatory responses after neutrophil activation via cAMP/PKA-dependent inhibition of ERK and JNK activation. Our study revealed apremilast suppresses oxidative stress and chemotaxis by selectively inhibiting PDE4 in neutrophils and thus protects against endotoxin-induced ARDS in mice. Apremilast can be used as an alternative off-label drug in treating acute lung damage.

A molecular phenotypic screen reveals that lobetyolin alleviates cardiac dysfunction in 5/6 nephrectomized mice by inhibiting osteopontin

BackgroundCardiovascular diseases are the major cause of mortality in patients with advanced chronic kidney diseases. The predominant abnormality observed among this population is cardiac dysfunction secondary to myocardial remodelings, such as hypertrophy and fibrosis, emphasizing the need to develop potent therapies that maintain cardiac function in patients with end-stage renal disease. AimsTo identify potential compounds and their targets as treatments for cardiorenal syndrome type 4 (CRS) using molecular phenotyping and in vivo/in vitro experiments. MethodsGene expression was assessed using bioinformatics and verified in animal experiments using 5/6 nephrectomized mice (NPM). Based on this information, a molecular phenotyping strategy was pursued to screen potential compounds. Picrosirius red staining, wheat germ agglutinin staining, Echocardiography, immunofluorescence staining, and real-time quantitative PCR (qPCR) were utilized to evaluate the effects of compounds on CRS in vivo. Furthermore, qPCR, immunofluorescence staining and flow cytometry were applied to assess the effects of these compounds on macrophages/cardiac fibroblasts/cardiomyocytes. RNA-Seq analysis was performed to locate the targets of the selected compounds. Western blotting was performed to validate the targets and mechanisms. The reversibility of these effects was tested by overexpressing Osteopontin (OPN). ResultsOPN expression increased more remarkably in individuals with uremia-induced cardiac dysfunction than in other cardiomyopathies. Lobetyolin (LBT) was identified in the compound screen, and it improved cardiac dysfunction and suppressed remodeling in NPM mice. Additionally, OPN modulated the effect of LBT on cardiac dysfunction in vivo and in vitro. Further experiments revealed that LBT suppressed OPN expression via the phosphorylation of c-Jun N-terminal protein kinase (JNK) signaling pathway. ConclusionsLBT improved CRS by inhibiting OPN expression through the JNK pathway. This study is the first to describe a cardioprotective effect of LBT and provides new insights into CRS drug discovery.

Quinolinic Acid Induces Alterations in Neuronal Subcellular Compartments, Blocks Autophagy Flux and Activates Necroptosis and Apoptosis in Rat Striatum.

Quinolinic acid (QUIN) is an agonist of N-methyl-D-aspartate receptor (NMDAr) used to study the underlying mechanism of excitotoxicity in animal models. There is evidence indicating that impairment in autophagy at early times contributes to cellular damage in excitotoxicity; however, the status of autophagy in QUIN model on day 7 remains unexplored. In this study, the ultrastructural analysis of subcellular compartments and the status of autophagy, necroptosis, and apoptosis in the striatum of rats administered with QUIN (120nmol and 240nmol) was performed on day 7. QUIN induced circling behavior, neurodegeneration, and cellular damage; also, it promoted swollen mitochondrial crests, spherical-like morphology, and mitochondrial fragmentation; decreased ribosomal density in the rough endoplasmic reticulum; and altered the continuity of myelin sheaths in axons with separation of the compact lamellae. Furthermore, QUIN induced an increase and a decrease in ULK1 and p-70-S6K phosphorylation, respectively, suggesting autophagy activation; however, the increased microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and sequestosome-1/p62 (SQSTM1/p62), the coexistence of p62 and LC3 in the same structures, and the decrease in Beclin 1 and mature cathepsin D also indicates a blockage in autophagy flux. Additionally, QUIN administration increased tumor necrosis factor alpha (TNFα) and receptor-interacting protein kinase 3 (RIPK3) levels and its phosphorylation (p-RIPK3), as well as decreased B-cell lymphoma 2 (Bcl-2) and increased Bcl-2-associated X protein (Bax) levels and c-Jun N-terminal kinase (JNK) phosphorylation, suggesting an activation of necroptosis and apoptosis, respectively. These results suggest that QUIN activates the autophagy, but on day 7, it is blocked and organelle and cellular damage, neurodegeneration, and behavior alterations could be caused by necroptosis and apoptosis activation.