Quercetin Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis by Inhibiting the Glucose Regulatory Protein 78 Activation.

Background: The inflammatory bowel diseases (IBD) are significantly influenced by apoptosis and endoplasmic reticulum (ER) stress. Aims: To investigate the effects of quercetin on ER stress-mediated apoptosis in a trinitrobenzene sulfonic acid (TNBS) induced experimental IBD model. Study Design: In vivo animal experimental study. Methods: To demonstrate the effect of quercetin in an experimental colitis model, Control, TNBS, and TNBS+quercetin groups were created with 24 Wistar Albino rats. Colitis was induced by intrarectal administration of 25 mg TNBS. In the TNBS+quercetin group, intragastrically 100 mg/kg quercetin was given for 7 days, immediately after colitis induction. In the TNBS-induced experimental IBD model, we evaluated the effects of quercetin on colonic epithelial cell apoptosis, oxidative stress, ER stress, the mitogen-activated protein kinase c-Jun N-terminal kinase, and the nuclear factor kappa B immunoreactivities, the levels of myeloperoxidase and tumor necrosis factor-α, the disease activity index with colonic histopathologic changes. Results: TNBS administration induced an elevated level of disease activity and oxidative stress indices, inflammation markers, and an increase in the immunoreactivities of nuclear factor kappa B and the mitogen-activated protein kinase c-Jun N-terminal kinase in the colon of the colitis group. Glucose regulatory protein 78, caspase-12 immunoreactivities, and epithelial cell apoptosis also were shown in the colon. However, quercetin improved TNBS-induced histopathological alterations, apoptosis, inflammation, oxidative stress, and ER stress. Conclusion: This study suggests that quercetin has a regulatory effect on ER stress-mediated apoptosis, and thus may be beneficial in treating IBD.

Sesamin attenuates UVA-induced keratinocyte injury via inhibiting ASK-1-JNK/p38 MAPK pathways.

BACKGROUND: Ultraviolet (UV) exposure-stimulated reactive oxygen species (ROS) formation in keratinocytes is a crucial factor in skin aging. Phytochemicals have become widely popular for protecting the skin from UV-induced cell injury. Sesamin (SSM) has been shown to play a role in extensive pharmacological activity and exhibit photoprotective effects. AIM: To assess the protective effect of SSM on UVA-irradiated keratinocytes and determine its potential antiphotoaging effect. METHODS: HaCaT keratinocytes pretreated with SSM were exposed to UVA radiation at 8 J/cm2 for 10 min. Cell viability and oxidative stress indicators were evaluated using a cell counting kit-8 and lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) assay kits. Apoptosis and intracellular ROS levels were analyzed using annexin V-fluorescein isothiocyanate/propyridine iodide and dichlorodihydrofluorescein diacetate staining, respectively. Protein levels of matrix metalloprotein-1 (MMP-1), MMP-9, Bax/Bcl-2, and mitogen-activated protein kinase (MAPK) pathway proteins, phospho-apoptosis signal-regulating kinase-1 (p-ASK-1)/ASK-1, phospho-c-Jun N-terminal protein kinase (p-JNK)/JNK, and p-p38/p38 were determined using western blotting. RESULTS: Sesamin showed no cytotoxicity until 160 µmol/L on human keratinocytes. Sesamin pretreatment (20 and 40 µM) reversed the suppressed cell viability, increased LDH release and MDA content, decreased cellular antioxidants GSH and SOD, and elevated intracellular ROS levels, which were induced by UVA irradiation. Additionally, SSM inhibited the expression of Bax, MMP-1, and MMP-9 and stimulated Bcl-2 expression. In terms of the regulatory mechanisms, we demonstrated that SSM inhibits the phosphorylation of ASK-1, JNK, and p38. CONCLUSION: The results suggest that SSM attenuates UVA-induced keratinocyte injury by inhibiting the ASK-1-JNK/p38 MAPK pathways.

The role of mitochondrial reactive oxygen species in chondrocyte mechanotransduction.

Chondrocytes are mechanosensitive cells able to sense and respond to external mechanical stimuli through the process of mechanotransduction. Previous studies have demonstrated that mechanical stimulation causes mitochondrial deformation leading to mitochondrial reactive oxygen species (ROS) release in a dose-dependent manner. For this reason, we focused on elucidating the role of mitochondrial ROS as anabolic signaling molecules in chondrocyte mechanotransduction. Chondrocyte-seeded agarose gels were subjected to mechanical stimuli and the effect on matrix synthesis, ROS production, and mitogen-activated protein kinases (MAPK) signaling was evaluated. Through the use of ROS-specific staining, superoxide anion was the primary ROS released in response to mechanical stimuli. The anabolic effect of mechanical stimulation was abolished in the presence of electron transport chain inhibitors (complexes I, III, and V) and superoxide anion scavengers. Subsequent studies were centered on the involvement of MAPK pathways (ERK1/2, p38, and JNK) in the mechanotransduction cascade. While disruption of the ERK1/2 pathway had no apparent effect, the anabolic effect of mechanical stimulation was abolished in the presence of p38 and JNK pathway inhibitors. This suggest the involvement of apoptosis stimulating kinase 1 (ASK1), an upstream redox-sensitive MAP3K shared by both the JNK and p38 pathways. Future experiments will focus on the involvement of the thioredoxin-ASK1 complex which disassociates in the presence of oxidative stress, allowing ASK1 to phosphorylate several MAP2Ks. Overall, these findings indicate superoxide anion as the primary ROS released in response to mechanical stimuli and that the resulting anabolic effect on chondrogenic matrix biosynthesis arises from the ROS-dependent activation of the p38 and JNK MAPKs.

A patent review of MAPK inhibitors (2018 - present).

INTRODUCTION: The mitogen-activated protein kinase (MAPK) family consist of p38 MAP kinases, c-Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs). They are involved in a multitude of diseases, including inflammatory, autoimmune, neurodegenerative, and metabolic diseases as well as cancer. In recent years, further developments in the field of MAPK-inhibitors have been reported, including an isoform or downstream target selective inhibition of MAPKs as well as target protein degradation approaches. AREAS COVERED: This review summarizes newly patented MAPK-inhibitors that were claimed between 2018 and early 2023. Presented are the patents as well as their corresponding publications, the storyline of development, and clinical trials involving these compounds. This article elaborates a total of 27 patents, which were identified using established search engines. EXPERT OPINION: Although industrial research on MAPK-inhibitors has been ongoing for more than 20 years, novel clinical trials of MAPK-inhibitors as potential drug candidates are still being conducted in the period under review. Recently reported inhibitors show an excellent selectivity profile and are even achieving selectivity between closely related isoforms. This progression offers the possibility to eliminate unwanted side effects and may finally lead to the approval of the first MAPK-inhibitor.

Prussian blue nanozymes coated with Pluronic attenuate inflammatory osteoarthritis by blocking c-Jun N-terminal kinase phosphorylation.

Osteoarthritis (OA) is a degenerative joint disorder associated with inflammation, functional disability, and high socioeconomic costs. The development of effective therapies against inflammatory OA has been limited owing to its complex and multifactorial nature. The efficacy of Prussian blue nanozymes coated with Pluronic (PPBzymes), US Food and Drug Administration-approved components, and their mechanisms of action have been described in this study, and PPBzymes have been characterized as a new OA therapeutic. Spherical PPBzymes were developed via nucleation and stabilization of Prussian blue inside Pluronic micelles. A uniformly distributed diameter of approximately 204 nm was obtained, which was maintained after storage in an aqueous solution and biological buffer. This indicates that PPBzymes are stable and could have biomedical applications. In vitro data revealed that PPBzymes promote cartilage generation and reduce cartilage degradation. Moreover, intra-articular injections with PPBzymes into mouse joints revealed their long-term stability and effective uptake into the cartilage matrix. Furthermore, intra-articular PPBzymes injections attenuated cartilage degradation without exhibiting cytotoxicity toward the synovial membrane, lungs, and liver. Notably, based on proteome microarray data, PPBzymes specifically block the JNK phosphorylation, which modulates inflammatory OA pathogenesis. These findings indicate that PPBzymes might represent a biocompatible and effective nanotherapeutic for obstructing JNK phosphorylation.

Design and Synthesis of a Mitochondrial-Targeted JNK Inhibitor and Its Protective Effect on Parkinson's Disease Phenotypes.

C-Jun N-terminal kinase (JNK) is a key mediator involved in a variety of physiological processes. JNK activation is regulated in a complex manner by upstream kinases and phosphatases, and plays an important role in physiological processes such as the immune response and neuronal function. Therefore, JNK has become a therapeutic target for neurodegenerative diseases, ankylosing spondylitis, psoriasis, arthritis and other diseases. Inhibition of JNK activation in mitochondria holds great potential for Parkinson's disease (PD) therapy. However, no specific mitochondrial-targeted JNK inhibitor has been reported. We have developed a mitochondrial-targeted JNK inhibitor, P2, by linking a mitochondrial-specific cell-penetrating peptide to SP600125 (SP), a commercialized specific inhibitor of JNK. We found that P2 specifically inhibited mitochondrial JNK phosphorylation instead of nuclear JNK signaling. Further studies showed that P2 effectively rescued PD phenotypes both in vitro and in vivo, thus indicating that it is a potential therapeutic for PD.

KIAA1429 promotes tumorigenesis and gefitinib resistance in lung adenocarcinoma by activating the JNK/ MAPK pathway in an m6A-dependent manner.

Non-small cell lung cancer is the leading cause of cancer related mortality worldwide, and lung adenocarcinoma (LUAD) is one of the most common subtypes. The role of N6-methyladenosine (m6A) modification in tumorigenesis and drug resistance in LUAD remains unclear. In this study, we evaluated the effects of vir-like m6A methyltransferase-associated protein (KIAA1429) depletion on proliferation, migration, invasion, and drug resistance of LUAD cells, and identified m6A-dependent downstream genes influenced by KIAA1429. We found that KIAA1429 activated Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway as a novel signaling event, which is responsible for tumorigenesis and resistance to gefitinib in LUAD cells. KIAA1429 and MAP3K2 showed high expression in LUAD patients' tissues. Knockdown of KIAA1429 inhibited MAP3K2 expression in an m6A methylation-dependent manner, restraining the progression of LUAD cells and inhibiting growth of gefitinib-resistant HCC827 cells. KIAA1429 positively regulated MAP3K2 expression, activated JNK/ MAPK pathway, and promoted drug resistance in gefitinib-resistant HCC827 cells. We reproduced the in vitro results in nude mouse xenografted with KIAA1429 knockdown cells. Our study showed that the mechanism of m6A KIAA1429-mediated gefitinib resistance in LUAD cells occurs by activating JNK/ MAPK signaling pathway. These findings provide potential targets for molecular therapy and clinical treatment in LUAD patients with gefitinib resistance.

Effects of Holliday Junction-Recognition Protein-Mediated C-Jun N-Terminal Kinase/ Signal Transducer and Activator of Transcription 3 Signaling Pathway on Cell Proliferation, Cell Cycle and Cell Apoptosis in Bladder Urothelial Carcinoma.

The Holliday Junction-Recognition Protein (HJURP) was upregulated in several tumors, which was associated with poor outcome. This study investigated the effects of the HJURP-mediated c-Jun N-terminal kinase (JNK)/ signal transducer and activator of transcription 3 (STAT3) pathway on bladder urothelial carcinoma (BLUC). Online databases were used to analyze HJURP expression in BLUC and the correlation of HJURP to JNK1 [mitogen-activated protein kinase 8 (MAPK8)], JNK2 (MAPK9), STAT3, marker of proliferation Ki-67 (MKI67), proliferating cell nuclear antigen (PCNA), cyclin dependent kinase 2 (CDK2), CDK4 and CDK6. HJURP expression was detected in BLUC cells and human normal primary bladder epithelial cells (BdECs). BLUC cells were treated with HJURP lentivirus activation /shRNA lentivirus particles or JNK inhibitor SP600125. HJURP was upregulated in BLUC tissues and correlated with poor prognosis of patients (all P < 0.05). HJURP in tumor positively correlated with MAPK8 (R = 0.30), MAPK9 (R = 0.30), STAT3 (R = 0.15), MKI67 (R = 0.60), PCNA (R = 0.46), CDK2 (R = 0.39), CDK4 (R = 0.24) and CDK6 (R = 0.21). The JNK inhibitor SP600125 decreased p-JNK/JNK and p-STAT3/STAT3 in BLUC cells, which was reversed by HJURP overexpression (P < 0.05). The HJURP-mediated JNK/STAT3 pathway promoted BLUC cell proliferation and inhibited cell apoptosis (P < 0.05). HJURP reversed the arrested G0/G1 phase of BLUC cells by SP600125. HJURP acted as an oncogene to regulate BLUC cell proliferation, apoptosis and the cell cycle by mediating the JNK/STAT3 pathway. Therefore, HJURP targeting might be an attractive novel therapeutic target for early diagnosis and treatment in BLUC.

Macleaya cordata extract modulates inflammation via inhibition of the NF-κB and MAPK signaling pathways in porcine alveolar macrophages induced by Glaesserella parasuis.

Glässer's disease in pigs is associated with infection by Glaesserella parasuis and is characterized by pneumonia-like symptoms, fibrinous polyserositis, polyarthritis, and meningitis. Macleaya cordata, a commonly used traditional Chinese medication, has been shown to have anti-inflammatory, antiviral, antioxidative, antimicrobial, insecticidal, and antitumor properties. However, the anti-inflammatory effects of M. cordata on G. parasuis stimulation are still poorly understood. This study explored the anti-inflammatory effects and mechanisms of M. cordata extract on G. parasuis-induced inflammatory responses, via the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, in porcine alveolar macrophages (PAMs). Porcine alveolar macrophages, when stimulated with G. parasuis, initiated transcription of interleukin (IL)-1α, IL-1ß, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α). Furthermore, p65, IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) phosphorylation were upregulated via the NF-κB and MAPK signaling pathways. However, treatment with M. cordata extract inhibited transcription of IL-1α, IL-1ß, IL-6, IL-8, and TNF-α and reduced p65, IκBα, p38, ERK, and JNK phosphorylation, by inhibiting activation of the NF-κB and MAPK signaling pathways in PAMs induced by G. parasuis. These findings reveal that M. cordata extract can reverse the inflammatory effect initiated by G. parasuis in vitro and that it possesses significant immunosuppression activity; thus, it may offer a novel strategy for controlling and treating G. parasuis infection.

La maladie de Glässer chez les porcs est associée avec une infection par Glaesserella parasuis et est caractérisée par des symptômes similaires à une pneumonie, une polysérosite fibrineuse, une polyarthrite et une méningite. Macleaya cordata, un médicament utilisé couramment en médecine traditionnelle chinoise, a été montré comme ayant des propriétés anti-inflammatoire, antivirale, anti-oxydative, antimicrobienne, insecticide et anti-tumeur. Toutefois, les effets anti-inflammatoires de M. cordata sur une stimulation par G. parasuis sont toujours peu compris. La présente étude explore les effets et mécanismes anti-inflammatoires d'un extrait de M. cordata sur les réponses inflammatoires induites par G. parasuis, via le facteur nucléaire-kappa B (NF-κB) et la voie de signalisation de la protéine kinase activée par les mitogènes (MAPK), dans les macrophages alvéolaires porcins (PAMs). Les PAMs, lorsque stimulés par G. parasuis, ont initié la transcription des interleukines (IL)-1α, IL-1ß, IL-6, IL-8, et le facteur de nécrose des tumeurs alpha (TNF-α). Également, la phosphorylation de p65, IκBα, p38, de la kinase régulée par signal extracellulaire (ERK), et de la kinase c-Jun N-terminal (JNK) était régulée à la hausse via les voies de signalisation NF-κB and MAPK. Toutefois, le traitement avec l'extrait de M. cordata a inhibé la transcription d'IL-1α, IL-1ß, IL-6, IL-8, et TNF-α et a diminué la phosphorylation de p65, IκBα, p38, ERK, et JNK, en inhibant les voies de signalisation de NF-κB et MAPK dans les PAMs induits par G. parasuis. Ces trouvailles révèlent qu'un extrait de M. cordata peut renverser l'effet inflammatoire initié par G. parasuis in vitro et qu'il possède une activité immunosuppressive significative; ainsi, ceci pourrait offrir une nouvelle stratégie pour limiter et traiter l'infection par G. parasuis.(Traduit par Docteur Serge Messier).

Cyclooxygenase-2 activates the free radical-mediated apoptosis of polymorphonuclear leukocytes in the maneb- and paraquat-intoxicated rats.

Overproduction of free radicals and inflammation could lead to maneb (MB)- and paraquat (PQ)-induced toxicity in the polymorphonuclear leukocytes (PMNs). Cyclooxygenase-2 (COX-2), an inducible COX, is imperative in the pesticides-induced pathological alterations. However, its role in MB- and PQ-induced toxicity in the PMNs is not yet clearly deciphered. The current study explored the contribution of COX-2 in MB- and PQ-induced toxicity in the PMNs and the mechanism involved therein. Combined MB and PQ augmented the production of free radicals, lipid peroxides and activity of superoxide dismutase (SOD) in the rat PMNs. While combined MB and PQ elevated the expression of COX-2 protein, activation of nuclear factor-kappa B (NF-κB) and phosphorylation of c-Jun N-terminal kinase (JNK), release of mitochondrial cytochrome c and levels of procaspase-3/9 were attenuated in the PMNs. Celecoxib (CXB), a COX-2 inhibitor, ameliorated the combined MB and PQ-induced modulations in the PMNs. MB and PQ augmented the free radical generation, COX-2 protein expression, NF-κB activation and JNK phosphorylation and reduced the cell viability of cultured rat PMNs and human leukemic HL60. MB and PQ elevated mitochondrial cytochrome c release and poly (ADP-ribose) polymerase cleavage whilst procaspase-3/9 levels were attenuated in the cultured PMNs. MB and PQ also increased the levels of phosphorylated c-jun and caspase-3 activity in the HL60 cells. CXB; SP600125, a JNK-inhibitor and pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, rescued from MB and PQ-induced changes in the PMNs and HL60 cells. However, CXB offered the maximum protection among the three. The results show that COX-2 activates apoptosis in the PMNs following MB and PQ intoxication, which could be linked to NF-κB and JNK signaling.

Berberine Ameliorates Insulin Resistance by Inhibiting IKK/NF-κB, JNK, and IRS-1/AKT Signaling Pathway in Liver of Gestational Diabetes Mellitus Rats.

Introduction: Berberine is derived from rhizoma coptidis, a well-known Traditional Chinese herbal Medicine that has been found to be effective in the treatment of type 2 diabetes mellitus in recent years. The aim of the present study was to investigate the effects of berberine on a gestational diabetes mellitus (GDM) rat model and the related mechanisms. Methods: GDM was induced in Sprague-Dawley rats using a high-fat diet before and during pregnancy. Berberine (100 mg/kg/day) was administered from the 7th to 20th day of pregnancy. Insulin resistance (IR), glucose tolerance, and maternal, fetal, and placental weight were determined. Liver histopathological analysis, as well as analysis of C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), inhibitor kappa B kinaseß (IKKß), nuclear factor kappa-B (NF-κB), c-Jun N-terminal kinase (JNK), insulin receptor substrate-1 (IRS-1), and protein kinase B (AKT), was performed at the end of pregnancy. Results: Treatment of GDM rats with berberine markedly decreased IR, the number of dead and absorptive fetuses, maternal body weight gain, and fetal and placental weight compared with GDM without berberine. Furthermore, berberine decreased CRP and TNF-α levels, IKKß expression, NF-κB P65 nuclear translocation, and changed the phosphorylation of JNK, IRS-1, and AKT in the liver of GDM rats. Conclusions: Berberine improved IR and maternal-fetal outcomes of GDM rats, possibly through modulation of IKK/NF-κB, JNK, and IRS-1/AKT signaling pathways in the liver. Therefore, berberine may be a potential GDM treatment.

[Mechanism of paeoniflorin inhibiting apoptosis of hippocampal neurons of rats induced by lead acetate].

Objective: To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate. Methods: In September 2020, primary hippocampal neuronal cells were isolated and cultured from fetal rats, and identified using cellular immunofluorescent. MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis. MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate. According to the results, different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells, after 24 h, lead acetate was added to the cells, meanwhile, blank and model groups were set up, the content of reactive oxygen species (ROS) , superoxide dismutase (SOD) , lactate dehydrogenase (LDH) , malondialdehyde (MDA) and Caspase-3 were measured. Extracellular signal regulated kinase (ERK) , phosphorylated ERK (p-ERK) , p38 mitogen -activated protein kinases (p38MAPK) , phosphorylated p38MAPK (p-p38MAPK) , c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) protein expression in hippocampal neuronal cells were determined by Western blotting. Results: The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate, MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25 µmol/L. Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80 µmol/L. Compared with the model group, the activity of hippocampal neuronal cells was significantly increased after treating with 20, 40 or 80 µmol/L paeoniflorin (P<0.05) . Compared with the blank group, the ROS activity, LDH release level, MDA content and caspase-3 content were significantly increased (P<0.01) , and the SOD activity was significantly decreased (P< 0.01) in the hippocampal neuronal cells of the model group. Compared with the model group, the ROS activity, LDH release level, MDA content and caspase-3 content were obviously decreased (P<0.05) , SOD activity was significantly increased (P <0.01) after hippocampal neuronal cells were treated with 40 or 80 µmol/L paeoniflorin. Relative to the model group, the ratio of p-ERK/ERK were significantly up-regulated (P<0.01) , while the ratios of p-p38MAPK/p38MAPK and p-JNK/JNK were significantly down-regulated after hippocampal neuronal cells were treated with 40 or 80 µmol/L paeoniflorin (P<0.05) . Conclusion: Paeoniflorin may down-regulate the expression of p-p38MAPK and p-JNK protein, up-regulate the expression of p-ERK protein, and inhibit the apoptosis of hippocampal neurons induced by lead acetate through the MAPK signaling pathway.

Hyperoside exerts osteoprotective effect on dexamethasone-induced osteoblasts by targeting NADPH Oxidase 4 (NOX4) to inhibit the reactive oxygen species (ROS) accumulation and activate c-Jun N-terminal kinase (JNK) pathway.

Hyperoside (Hyp) is a flavonoid active compound deriving from Chinese herbal medicines. Increasing studies have implicated that Hyp may serve as a predominant promoting factor in osteoblast differentiation. This paper investigates whether Hyp could relieve glucocorticoid-induced osteonecrosis of the femoral head (GONFH) via promoting osteoblast survival and differentiation as well as to uncover its potential mechanism. GONFH cell model was induced by treating MC3T3-E1 cells with dexamethasone (DEX). The viability, apoptosis, and osteogenic differentiation of DEX-induced cells with the presence or absence of Hyp were assessed by CCK-8, Tunel, ALP assay, and ARS staining, respectively. The NADPH Oxidase 4 (NOX4) overexpression was performed by transfection with overexpression vector. Besides, western blot was used to determine the levels of apoptosis-, osteogenic differentiation-, and c-Jun N-terminal kinase (JNK) signaling-related proteins. It was noticed that Hyp caused no significant effects on the viability of MC3T3-E1 cells without any treatment but significantly enhanced the viability of DEX-induced cells. Besides, Hyp inhibited the apoptosis in DEX-induced cells but enhanced ALP activity and calcium nodule formation. Additionally, Hyp declined NOX4 expression in DEX-induced cells. However, NOX4 overexpression partially reversed the impacts of Hyp on DEX-exposed MC3T3-E1 cells. Finally, Hyp suppressed the activation of ROS/JNK pathway in DEX-induced cells, which was then counteracted by NOX4 overexpression. In conclusion, Hyp could promote the survival and differentiation of DEX-induced osteoblasts by targeting NOX4 to inhibit the ROS/JNK pathway. These results provide evidence for the application of Hyp in treating GONFH.

Pollen typhae total flavone suppresses A7r5 cell proliferation promoted by insulin involving the MEK1/2-ERK1/2 cascades.

Pollen typhae, a traditional medicine in China, performs an anti-diabetic function and has anti-atherosclerosis effects involving suppression of vascular smooth muscle cell proliferation. However, the potential mechanisms keep to be revealed. The present study intended to investigate the influences of Pollen typhae extract named Pollen typhae total flavone (PTF) on A7r5 cell proliferation promoted by insulin and to uncover the underlying mechanisms. Proliferation and viability were evaluated by CCK-8 method. Western blotting was adopted to analyze the protein expression. Insulin promoted A7r5 cell proliferation, while PTF suppressed insulin-promoted proliferation in a concentration-dependent fashion. Although PTF did not change c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38MAPK) or MAPK kinase 1/2 (MEK1/2) protein expression and failed to affect the phosphorylation of JNK and p38MAPK, PTF remarkably inhibited extracellular signal-regulated kinase 1 and 2 (ERK1/2) protein expression and reduced ERK1/2 and MEK1/2 phosphorylation in A7r5 cells stimulated by insulin. Insulin-induced proliferation of A7r5 cells was abolished by inhibiting ERK1/2, which was in line with PTF. These findings indicate that PTF suppresses insulin-promoted proliferation of A7r5 cells involving the MEK1/2-ERK1/2 cascades, providing new insight into the potential uses of PTF for treatment of diabetic atherosclerosis.

Mechanism of paeoniflorin inhibiting apoptosis of hippocampal neurons of rats induced by lead acetate / 中华劳动卫生职业病杂志

Objective: To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate. Methods: In September 2020, primary hippocampal neuronal cells were isolated and cultured from fetal rats, and identified using cellular immunofluorescent. MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis. MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate. According to the results, different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells, after 24 h, lead acetate was added to the cells, meanwhile, blank and model groups were set up, the content of reactive oxygen species (ROS) , superoxide dismutase (SOD) , lactate dehydrogenase (LDH) , malondialdehyde (MDA) and Caspase-3 were measured. Extracellular signal regulated kinase (ERK) , phosphorylated ERK (p-ERK) , p38 mitogen -activated protein kinases (p38MAPK) , phosphorylated p38MAPK (p-p38MAPK) , c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) protein expression in hippocampal neuronal cells were determined by Western blotting. Results: The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate, MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25 μmol/L. Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80 μmol/L. Compared with the model group, the activity of hippocampal neuronal cells was significantly increased after treating with 20, 40 or 80 μmol/L paeoniflorin (P<0.05) . Compared with the blank group, the ROS activity, LDH release level, MDA content and caspase-3 content were significantly increased (P<0.01) , and the SOD activity was significantly decreased (P< 0.01) in the hippocampal neuronal cells of the model group. Compared with the model group, the ROS activity, LDH release level, MDA content and caspase-3 content were obviously decreased (P<0.05) , SOD activity was significantly increased (P <0.01) after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin. Relative to the model group, the ratio of p-ERK/ERK were significantly up-regulated (P<0.01) , while the ratios of p-p38MAPK/p38MAPK and p-JNK/JNK were significantly down-regulated after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin (P<0.05) . Conclusion: Paeoniflorin may down-regulate the expression of p-p38MAPK and p-JNK protein, up-regulate the expression of p-ERK protein, and inhibit the apoptosis of hippocampal neurons induced by lead acetate through the MAPK signaling pathway.

Octanoic acid-rich enteral nutrition prevented lipopolysaccharide-induced acute liver injury through c-Jun N-terminal kinase-dependent autophagy.

BACKGROUND: Acute liver injury (ALI) is an essential component of sepsis associated with poor outcomes. Octanoic acid (OA), a medium-chain fatty acid, has a protective effect on sepsis-induced organ damage, and autophagy is an adaptive response to sepsis. However, the underlying mechanism by which OA prevents ALI remains unknown. Therefore, we investigated whether OA-rich enteral nutrition (EN) prevented lipopolysaccharide (LPS)-induced ALI through the c-Jun N-terminal kinase (JNK)-dependent autophagy. METHODS: Firstly, Sprague Dawley rats were randomly divided into four groups (sham, LPS, LPS + EN, and LPS + EN + OA) to detect the effect of OA-rich EN on LPS-induced ALI. Then, rats were randomly divided into five groups (sham, LPS, LPS + EN + OA, LPS + EN + OA + anisomycin (AN), and LPS + SP600125) to explore the mechanism by which OA-rich EN prevented ALI. EN and OA-rich EN were conducted through gastric tubes for 3 days. The liver protective effects were measured by liver histopathological changes, enzymes, inflammatory cytokines of serum and liver, the levels of autophagy, and JNK activity. RESULTS: OA-rich EN inhibited JNK activity, up-regulated autophagy and prevented LPS-induced ALI. Inhibition of JNK activity conferred by SP promoted autophagy and prevented LPS-induced ALI. Moreover, the protective effect of autophagy and inhibition of JNK activity conferred by OA-rich EN on ALI was counteracted by AN. CONCLUSION: OA-rich EN prevented LPS-induced ALI through JNK-dependent autophagy. This result suggested that OA-rich EN may be a therapeutic potential for ALI in patients with sepsis.

Trehalose and N-Acetyl Cysteine Alleviate Inflammatory Cytokine Production and Oxidative Stress in LPS-Stimulated Human Peripheral Blood Mononuclear Cells.

BACKGROUND: Evidence has shown that inflammation and oxidative stress are implicated in the development of a great number of human diseases. Trehalose possesses various biological effects including antioxidant and anti-inflammatory activities. However, there is little data on the effects of trehalose on human cells including peripheral blood mononuclear cells (PBMCs). Here, we aimed to investigate whether trehalose could attenuate oxidative stress and inflammation induced by lipopolysaccharides (LPS) in PBMCs. METHODS: The enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to assess the levels of inflammatory cytokines. To investigate the phosphorylation of c-Jun N-terminal kinase (JNK) and NF-κB, western blot analysis was utilized. Oxidant-antioxidant markers were assessed using ELISA and colorimetric procedures. RESULTS: The results revealed that trehalose significantly mitigated the effect of LPS on the phosphorylation of JNK and NF-κB-P65 (p < .00). This mitigation was associated with significantly reduced levels of inflammatory cytokines IL-6, TNF-α, and IL-1ß and increased levels of anti-inflammatory cytokine IL-10 (P < .05). The antioxidant N-acetyl cysteine (NAC) also showed similar effects on JNK and NF-κB-P65 phosphorylation and inflammatory cytokines (p < .00). Furthermore, trehalose alleviated oxidative stress in LPS-stimulated PBMCs as it reversed the altered levels of malondialdehyde and total thiols (p ≤ .05) and restored the activity of antioxidant enzymes glutathione peroxidase and manganese superoxide dismutase (p < .001). CONCLUSION: The results of this study indicated that trehalose prevented inflammation and oxidative stress in the LPS-stimulated PBMCs, providing evidence for the benefits of trehalose as a potential therapeutic agent in inflammatory conditions. ABBREVIATIONS: LPS: Lipopolysaccharide; NAC: N-Acetyl cysteine; ROS: Reactive oxygen species; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; MDA: Malondialdehyde; MAPK: Mitogen-activated protein kinases; JNK: c-Jun N-terminal kinase; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells.

Irisin Recovers Osteoarthritic Chondrocytes In Vitro.

Physical exercise favors weight loss and ameliorates articular pain and function in patients suffering from osteoarthritis. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types. This study aimed to investigate the effect of irisin on human osteoarthritic chondrocytes (hOAC) in vitro. Our hypothesis was that irisin would improve hOAC metabolism and proliferation. Cells were cultured in growing media and then exposed to either phosphate-buffered saline (control group) or human recombinant irisin (experimental group). Cell proliferation, glycosaminoglycan content, type II/X collagen gene expression and protein quantification as well as p38/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK), protein kinase B (Akt), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) involvement were evaluated. Furthermore, gene expression of interleukin (IL)-1 and -6, matrix metalloproteinase (MMP)-1 and -13, inducible nitric oxide synthase (iNOS), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -3 were investigated following irisin exposure. Irisin increased hOAC cell content and both type II collagen gene expression and protein levels, while decreased type X collagen gene expression and protein levels. Moreover, irisin decreased IL-1, IL-6, MMP-1, MMP-13 and iNOS gene expression, while increased TIMP-1 and TIMP-3 levels. These effects seemed to be mediated by inhibition of p38, Akt, JNK and NFκB signaling pathways. The present study suggested that irisin may stimulate hOAC proliferation and anabolism inhibiting catabolism through p38, Akt, JNK, and NFκB inactivation in vitro, demonstrating the existence of a cross-talk between muscle and cartilage.

RhoA-JNK Regulates the E-Cadherin Junctions of Human Gingival Epithelial Cells.

The junctional epithelium (JE) is unique with regard to its wide intercellular spaces and sparsely developed intercellular junctions. Thus, knowledge of the molecular mechanisms that regulate the formation of the intercellular junctions of the junctional epithelium may be essential to understand the pathophysiology of the JE. HOK-16B cells, a normal human gingival epithelial cell line, were used to identify the molecules involved in the regulation of the formation of intercellular E-cadherin junctions between human gingival epithelial cells. Activation of c-Jun N-terminal kinase (JNK) disrupted the intercellular junctions through the dissociation of E-cadherin. The role of JNK in the formation of these E-cadherin junctions was further confirmed by demonstrating that JNK inhibition induced the formation of intercellular E-cadherin junctions. The upstream signaling of JNK was also examined. Activation of the small GTPase RhoA disrupted the formation of E-cadherin junctions between HOK-16B cells, which was accompanied by JNK activation. Disruption of these intercellular junctions upon RhoA activation was prevented when JNK activity was inhibited. In contrast, RhoA inactivation led to HOK-16B cell aggregation and the formation of intercellular junctions, even under conditions in which the cellular junctions were naturally disrupted by growth on a strongly adhesive surface. Furthermore, the JE of mouse molars had high JNK activity associated with low E-cadherin expression, which was reversed in the other gingival epithelia, including the sulcular epithelium. Interestingly, JNK activity was increased in cells grown on a solid surface, where cells showed higher RhoA activity than those grown on soft surfaces. Together, these results indicate that the decreased formation of intercellular E-cadherin junctions within the JE may be coupled to high JNK activity, which is activated by the upregulation of RhoA on solid tooth surfaces.

Dehydroepiandrosterone prevents linoleic acid-induced endothelial cell senescence by increasing autophagy.

BACKGROUND: Autophagy has emerged as a potentially important factor in the pathogenesis of atherosclerosis. Dehydroepiandrosterone (DHEA) is an adrenal steroid of great recent interest due to its anti-aging and anti-atherogenic effects; however, little is known about its role in autophagy and endothelial senescence. OBJECTIVE: The aim of this study was to investigate whether DHEA prevents linoleic acid (LA)-induced endothelial senescence by enhancing autophagy. MATERIALS/METHODS: After pre-treatement with or without DHEA prior to LA treatment in human aortic endothelial cells (HAECs), the level of senescence was compared by senescence-associated acidic ß-galactosidase (SA-ß-Gal) staining and hyperphosphorylated pRB (ppRB) protein level. Autophagy was detected by LC3 conversion and measuring the level of p62/SQSTM1 (sequestosome 1), a protein degraded by autophagy. The fusion of autophagosome and lysosome was confirmed by fluorescence microscopy. RESULTS: Pre-treatment with DHEA inhibited LA-induced endothelial senescence. DHEA increased the conversion of LC3-I to LC3-II and decreased the level of p62 in a time- and dose-dependent manner. Although both DHEA and LA treatment increased the conversion of LC3-I to LC3-II, treatment of LA increased p62 and decreased fusion of autophagosome and lysosome, which reflected decreased autophagic flux. However, pre-treatment with DHEA restored autophagic flux inhibited by LA. When we evaluated signaling pathways, we found that JNK activation involved in LC3 conversion induced by DHEA. CONCLUSION: DHEA prevents LA-induced endothelial senescence by restoring autophagy and autophagic flux through JNK activation.