JNK Inhibitor Research Articles

Treatment with TNFα and lipolysis-stimulated lipoprotein receptor (LSR) antibody in the presence of HDAC inhibitors promotes apoptosis in human salivary duct adenocarcinoma

ABSTRACT Lipolysis-stimulated lipoprotein receptor (LSR), a lipid metabolism-related factor localized in tricellular tight junctions (tTJs), plays an important role in maintaining the epithelial homeostasis. LSR is highly expressed in well-differentiated cancers, and its expression decreases during malignancy. The LSR antibody inhibits cell growth and promotes apoptosis in some cancers. Histone deacetylases (HDACs) are thought to play a crucial role in carcinogenesis, and HDAC inhibitors promote differentiation and prevent cell proliferation and migration in cancers. HDAC inhibitors together with TNFα also induce apoptosis via TNFα-related apoptosis-inducing ligand (TRAIL) in some cancers. In this study, we investigated the apoptosis signaling induced by an anti-LSR antibody in human salivary duct adenocarcinoma (SDC) cell line A253, compared to TRAIL-induced apoptosis. A253 cells were treated with human recombinant TNFα with or without HDAC inhibitor trichostatin A (TSA) and quisinostat (JNJ-26481585). Treatment using TNFα with HDAC inhibitors markedly induced apoptosis in A253 cells and the anti-TNFα antibody prevented the induced apoptosis. A253 cells were treated with an antibody against the extracellular N-terminal domain of human LSR (LSR-N-ab) with or without HDAC inhibitors. Treatment with HDAC inhibitors induced LSR expression in the membranes of A253 cells. Treatment using LSR-N-ab with HDAC inhibitors markedly promoted apoptosis in A253 cells. The tricellular signaling pathway JNK inhibitor SP600125 and Hippo pathway MST1/2 inhibitor XMU-MP-1 prevented the apoptosis induced by treatment using TNFα or LSR-N-ab with HDAC inhibitors. Our findings indicated that treatment with TNFα or LSR-N-ab with HDAC inhibitors might be useful in the therapy for human SDC by enhancing apoptosis.

Human Umbilical Cord Mesenchymal Stem Cells Alleviate Diabetic Nephropathy by Inhibiting Ferroptosis via the JNK/KEAP1/NRF2 Signaling Pathway.

Aims: Diabetic nephropathy (DN) is a major cause of end-stage renal disease, with no therapeutic interventions available to control its progression. Ferroptosis, an iron-dependent regulated cell death characterized by lipid peroxidation, plays a pivotal role in the pathogenesis of DN. Human umbilical cord mesenchymal stem cells (hUCMSCs) are an effective treatment modality for DN; however, the underlying mechanism of action remains unclear. The aim of the present study was to investigate whether hUCMSCs alleviate DN via inhibiting ferroptosis and its molecular mechanisms in type 2 diabetic mice and high-glucose and palmitate-stimulated human renal tubular epithelial cell (HK-11) models. Results: Our findings revealed that hUCMSCs improved the renal structure and function and tubular injuries. HUCMSC treatment can inhibit ferroptosis by decreasing iron content, reducing reactive oxygen species, malondialdehyde and 4-hydroxynonenal generation, decreasing the expression of positive ferroptosis mediator transferrin receptor 1 and long-chain acyl-CoA synthetase 4, and enhancing the expression of negative ferroptosis mediators (i.e., ferritin heavy chain, glutathione peroxidase 4, and system Xc-cystine/glutamate reverse transporter). Mechanistically, hUCMSC treatment inhibited c-Jun N-terminal kinase (JNK) and Kelch-like ECH-associated protein 1 (KEAP1) activation while increasing the expression of nuclear factor erythroid 2-related factor 2 (NRF2). Furthermore, pretreatment of HK-11 cells with NRF2 siRNA, the JNK inhibitor SP600125, or the JNK agonist anisomycin demonstrated the regulation of the JNK/KEAP1/NRF2 signaling pathway by hUCMSCs. Innovation and Conclusion: HUCMSCs inhibit ferroptosis in DN via the JNK/KEAP1/NRF2 signaling pathway, providing a new perspective and scientific evidence for treating DN. Antioxid. Redox Signal. 00, 000-000.

Wnt5a promotes Kupffer cell activation in trichloroethylene-induced immune liver injury.

Trichloroethylene (TCE) is a volatile, colorless liquid that is widely used as a chlorinated organic vehicle in industrial production and processing industries. Many workers exposed to trichloroethylene may develop trichloroethylene hypersensitivity syndrome (THS). However, the underlying mechanism of THS is still unclear, especially liver injury. The present study aimed to investigate whether Wnt5a/c-Jun N-terminal kinase (JNK) is involved in and regulates liver injury caused by TCE exposure and to provide new directions for the prevention and treatment in clinical settings of liver injury caused by TCE exposure. We used 6- to 8-week-old SPF-grade BALB/c female mice to establish a TCE sensitization model and explored the mechanism through inhibitor intervention. We found that the expression of Wnt5a/JNK was significantly elevated in the liver of TCE sensitization-positive mice. Inhibitors of Wnt Production 2 (IWP-2) are known antagonists of the Wnt pathway. TCE-sensitization mice treated with IWP-2 showed downregulated Wnt5a/JNK expression, reduced Kupffer cell activation, and decreased liver injury. At the same time, we found that phosphorylated JNK in TCE-sensitization mouse livers and extracted Kupffer cells showed a significant downward trend after inhibition of Wnt5a function. We also found that a specific JNK inhibitor, SP600125, decreased the secretion of cytokines and chemokines and decreased Kupffer cell activation. We demonstrated that Wnt5a/JNK was involved in the regulation of liver injury in TCE-sensitization mice and that it exacerbated liver injury by activating Kupffer cells and releasing chemokines. We therefore hypothesized that Kupffer cell activation was affected by JNK, which reduced chemokine and cytokine secretion and attenuated liver injury in TCE-sensitization mice.

SP600125, a selective JNK inhibitor, is a potent inhibitor of NAD(P)H: quinone oxidoreductase 1 (NQO1).

The c-Jun N-terminal kinases (JNKs) has been identified as a critical modulator in multiple cellular processes, including stress stimulus, inflammation, cell proliferation, apoptosis, etc. SP600125 is a widely used ATP-competitive reversible JNKs inhibitor. NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavoprotein mediated two or four electron-reduction of quinones. Here, we showed that SP600125 bind to the active pocket of NQO1 and inhibit NQO1 activity. SP600125 exhibits comparable inhibitory effects on NQO1-mediated quinone bioactivation, H2O2 generation, and cell death, as the specific NQO1 inhibitor dicoumarol (DIC). Importantly, the inhibitory effects of SP600125 on NQO1 are independent of JNKs inhibition. These results suggested that SP600125 is a novel NQO1 inhibitor, which provides new insights into the mechanism of action of SP600125. Furthermore, SP600125 should be used more cautiously as a JNKs inhibitor, especially when NQO1 is highly expressed. SP600125 competed with β-Lap (NQO1-bioactivated drugs) for binding to NQO1, and inhibited NQO1-dependent cell death.

Probenecid Inhibits Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase Mitogen-Activated Protein Kinase Pathways in Regulating Respiratory Syncytial Virus Response.

We have previously shown that probenecid inhibits RSV, influenza virus, and SARS-CoV-2 replication in vitro in preclinical animal models and in humans. In a Phase two randomized, placebo-controlled, single-blind, dose range-finding study using probenecid to treat non-hospitalized patients with symptomatic, mild-to-moderate COVID-19, we previously showed that a 1000 mg twice daily treatment for 5 days reduced the median time to viral clearance from 11 to 7 days, and a 500 mg twice daily treatment for 5 days reduced the time to viral clearance from 11 to 9 days more than the placebo. In this study, we sought to determine the mechanism of action of the probenecid inhibition of RSV replication in human respiratory epithelial (A549) cells. We show that probenecid inhibits the RSV-induced phosphorylation of JNKs and ERKs and the downstream phosphorylation of c-jun, a component of the AP-1 transcription complex needed for virus replication. The inhibition of JNKs by probenecid reversed the repression of transcription factor HNF-4. The probenecid inhibition of JNK and ERK phosphorylation involves the MAPK pathway that precludes virus replication.

Prevention of liver metastasis via the pharmacological suppression of AMIGO2 expression in tumor cells

AMIGO2 adheres to liver endothelium and induces liver metastasis. We revealed that genetically altering AMIGO2 expression in tumor cells affects their liver metastatic potential, depending on the AMIGO2 expression level. The aim of this study was to prevent liver metastasis by pharmacologically suppressing AMIGO2 expression. For screening, we used the mouse LV12 cells because of their affinity to adhere to liver endothelium and metastasize the liver owing to elevated AMIGO2 expression. Of the 285 compounds tested, 17 reduced AMIGO2 mRNA expression. We subsequently screened for compounds that inhibited tumor cell adhesion to liver endothelium and identified five compounds that inhibit three signaling pathways (MEK, JAK, and JNK). Treatment with these compounds inhibited liver metastasis of LV12 cells. Next, we used clinically available signal inhibitors (MEK inhibitor trametinib, JAK inhibitor ruxolitinib, and JNK inhibitor SP600125), and found that ruxolitinib inhibits AMIGO2 expression more stably. Furthermore, ruxolitinib inhibited the adhesion of LV12 cells to liver endothelium and suppressed liver metastasis. Using the MKN45 gastric cancer cells, we confirmed that ruxolitinib could prevent liver metastasis of human cancer cells. These results demonstrate that pharmacological inhibition of AMIGO2 expression in tumor cells is a promising novel strategy to prevent and control liver metastasis.

AMTAC-19, a Spiro-Acridine Compound, Induces In Vitro Antitumor Effect via the ROS-ERK/JNK Signaling Pathway.

Colorectal cancer remains a significant cause of mortality worldwide. A spiro-acridine derivative, (E)-1'-((4-bromobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-19), showed significant cytotoxicity in HCT-116 colorectal carcinoma cells (half maximal inhibitory concentration, IC50 = 10.35 ± 1.66 µM) and antioxidant effects after 48 h of treatment. In this study, Molegro Virtual Docker v.6.0.1 software was used to investigate the interactions between AMTAC-19 and the Extracellular Signal-Regulated Kinase 1 (ERK1), c-Jun N-terminal Kinase 1 (JNK1), and p38 Mitogen-Activated Protein Kinase α (p38α MAPK). In vitro assays were conducted in HCT-116 cells to evaluate the effect of AMTAC-19 on the modulation of these proteins' activities using flow cytometry. Furthermore, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in the presence or absence of ERK1/2, JNK, and p38 MAPK inhibitors was used to evaluate the involvement of these enzymes in AMTAC-19 cytotoxicity. ROS production was assessed using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay at various incubation times (30 min, 1 h, 6 h, 12 h, and 24 h), and the MTT assay using N-acetyl-L-cysteine (NAC) was performed. In silico results indicated that AMTAC-19 interacts with ERK1, JNK1, and p38α MAPK. Additionally, AMTAC-19 activated ERK1/2 and JNK1 in HCT-116 cells, and its cytotoxicity was significantly reduced in the presence of ERK1/2 and JNK inhibitors. AMTAC-19 also induced a significant increase in ROS production (30 min and 1 h), while NAC pretreatment reduced its cytotoxicity. These findings support AMTAC-19's in vitro antitumor effect through ROS-dependent activation of ERK and JNK pathways.

Resveratrol and Its Derivatives Diminish Lipid Accumulation in Adipocytes In Vitro-Mechanism of Action and Structure-Activity Relationship.

Expansion of white adipose tissue causes systemic inflammation and increased risk of metabolic diseases due to its endocrine function. Resveratrol was suggested to be able to prevent obesity-related disorders by mimicking caloric restriction; however, its structure-activity relationships and molecular targets are still unknown. We aimed to compare the effects of resveratrol and its analogues on adipocyte metabolism and lipid accumulation in vitro. Mouse embryonic fibroblasts were differentiated to adipocytes in the absence or presence of resveratrol or its derivatives (oxyresveratrol, monomethylated resveratrol, or trimethylated resveratrol). Intracellular lipid content was assessed by Oil Red O staining. Glucose uptake and its response to insulin were estimated by 2-NBDG, and mitochondrial activity was assayed via resazurin reduction. Involvement of potential molecular pathways was investigated by concurrent treatment with their inhibitors. Although lipid accumulation was significantly reduced by all analogues without altering protein content, oxyresveratrol was the most potent (IC50 = 4.2 μM), while the lowest potency was observed with trimethylated resveratrol (IC50 = 27.4 μM). Increased insulin-stimulated glucose uptake was restored by each analogue with comparable efficiency. The enhanced mitochondrial activity was normalized by resveratrol and its methylated derivatives, while oxyresveratrol had a minor impact on it. Among the examined pathways, inhibition of SIRT1, PGC-1α, and JNK diminished the lipid-reducing effect of the compounds. Autophagy appeared to play a key role in the effect of all compounds but oxyresveratrol. Resveratrol and its analogues can mimic caloric restriction with complex mechanisms, including activation of SIRT1, PGC-1α, and JNK, making them possible drug candidates to treat obesity-related diseases.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases

Abstractc‐Jun N‐terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen‐activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3‐selective covalent inhibitor JC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine in the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160‐fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long‐lasting inhibition against cellular JNK3. In addition, its alkyne‐containing probe JC‐P1 could label JNK3 in SH‐SY5Y cell lysate and living cells, with good proteome‐wide selectivity. JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform‐selective and cell‐active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3‐targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases.

c-Jun N-terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen-activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3-selective covalent inhibitor JC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine in the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160-fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long-lasting inhibition against cellular JNK3. In addition, its alkyne-containing probe JC-P1 could label JNK3 in SH-SY5Y cell lysate and living cells, with good proteome-wide selectivity. JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform-selective and cell-active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3-targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Myocardial ischemia-reperfusion injury upregulates nucleostemin expression via HIF-1α and c-Jun pathways and alleviates apoptosis by promoting autophagy

Myocardial ischemia-reperfusion (I/R) injury, often arising from interventional therapy for acute myocardial infarction, leads to irreversible myocardial cell death. While previous studies indicate that nucleostemin (NS) is induced by myocardial I/R injury and mitigates myocardial cell apoptosis, the underlying mechanisms are poorly understood. Here, our study reveals that NS upregulation is critical for preventing cardiomyocyte death following myocardial I/R injury. Elevated NS protein levels were observed in myocardial I/R injury mouse and rat models, as well as Hypoxia/reoxygenation (H/R) cardiac cell lines (H9C2 cells). We identified binding sites for c-Jun and HIF-1α in the NS promoter region. Inhibition of JNK and HIF-1α led to a significant decrease in NS transcription and protein expression. Furthermore, inhibition of autophagy and NS expression promoted myocardial cell apoptosis in H/R. Notably, the cell model showed reduced LC3I transformation to LC3II, downregulated Beclin1, upregulated p62, and altered expression of autophagy-related proteins upon NS interference in H/R cells. These findings suggest that NS expression, driven by c-Jun and HIF-1α pathways, facilitates autophagy, providing protection against both myocardial I/R injury and H/R-induced cardiomyocyte apoptosis.

Angelica sinensis polysaccharides mitigate cadmium-induced apoptosis in layer chicken chondrocytes by inhibiting the JNK signaling pathway

Cadmium (Cd), a toxic heavy metal pollutant, inflicts widespread damage on various organs and tissues, including cartilage, where it induces chondrocyte apoptosis. Angelica sinensis polysaccharides (ASP), a key active component of the traditional Chinese medicine Angelica sinensis, have been shown to possess anti-apoptotic effects on chondrocytes. This study investigates the in vitro effects of ASP on alleviating Cd-induced apoptosis in layer chicken chondrocytes, focusing on the mitochondrial apoptosis pathway mediated by the c-Jun N-terminal kinase (JNK) signaling pathway. Chondrocytes were isolated from layer chicken embryos and confirmed to express collagen type II alpha 1 (Col2a1). We found that Cd triggered apoptosis in the chondrocytes; however, the use of the JNK inhibitor SP 600125 mitigated mitochondrial structural damage casused by Cd, indicating the involvement of JNK signaling in this process. Furthermore, ASP effectively alleviated Cd-induced apoptosis in layer chicken chondrocytes by inhibiting JNK signaling in vitro. Our findings provide a theoretical foundation for the clinical application of ASP in preventing Cd-induced cartilage diseases in poultry.

Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer.

All patients with metastatic breast cancer (MBC) expressing estrogen receptor-α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of patients, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Accumulating clinical evidence indicate adverse outcomes for these patients, beyond that expected by resistance to endocrine therapy. Here we aimed to study the role of ESR1 mutations in conferring chemoresistance in BC cells. MCF-7 cells harboring Y537S and D538G ESR1 mutations (mut-ER) were employed to study the response to chemotherapy drugs, paclitaxel and doxorubicin, using viability and apoptotic assay in vitro, and tumor growth in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter and ChIP assays. MDR1 expression was analyzed in clinical samples using IHC. Cell harboring ESR1 mutations displayed relative chemoresistance compared to WT-ER, evidenced by higher viability and reduced apoptosis as well as resistance to paclitaxel in vivo. To elucidate the underlying mechanism, MDR1 expression was examined and elevated levels were observed in mut-ER cells, and in clinical BC samples. MDR1 is regulated by the c-Jun pathway, and we showed high correlation between these two genes in BC using TCGA databases. Accordingly, we detected higher JNK/c-Jun expression and activity in ESR1-mutated cells, as well as increased occupancy of c-Jun in MDR1 promoter. Importantly, JNK inhibition decreased MDR1 expression and restored sensitivity to chemotherapy. Taken together, these data indicate that ESR1 mutations confer chemoresistance through activation of the JNK/MDR1 axis. These finding suggest a novel treatment option for BC tumors expressing ESR1 mutations.

The novel role of complement 3 in Wnt5a-mediated vascular smooth muscle cell calcification

Abstract Background Although vascular calcification (VC) is a risk factor for cardiovascular and all-cause mortality, a therapy is not yet available. VC involves the transdifferentiation of vascular smooth muscle cells (VSMC) towards an osteochondrogenic lineage that results in calcium phosphate salts deposition in the arterial wall. We have previously shown an association between plasma WNT5A levels and new onset and progression of coronary artery calcification. Purpose We aimed to study the poorly understood role of Wnt5a in VSMC calcification. Methods Study 1 included 18 patients with coronary artery disease (CAD) undergoing cardiac surgery; VSMC were isolated from their saphenous veins to perform in vitro assays and microarrays. Study 2 included 11 patients with CAD; IMA were treated ex vivo with Wnt5a recombinant protein (rWnt5a) to perform western blot (WB) analyses. Study 3 included 647 patients with CAD; RNA sequencing was performed on samples from their internal mammary arteries (IMA), IMA perivascular adipose (PVAT), and thoracic adipose tissue (ThAT). VSMC and human aortic smooth muscle cells (HASMC) from healthy donors were grown in osteogenic medium in the presence or absence of rWnt5a with or without specific protein inhibitors. Calcium deposition and expression of VC markers (i.e. RUNX2, BMP2, and MSX2) were assessed with a colorimetric assay and by RT-qPCR, respectively. Phosphorylation/activation of non-canonical Wnt pathway effectors was evaluated by WB analyses. Results rWnt5a treatment significantly increased calcium deposition in both VSMC and HASMC at a concentration that activates both JNK and CaMKII, that are effectors of the non-canonical planar cell polarity pathway and the calcium-dependent pathway, respectively. Inhibition of either JNK or CaMKII could not counteract rWnt5a-mediated increase in VSMC calcium deposition; whereas, inhibition of both non-canonical pathway effectors (JNK-i and CaMKII-i) prevented rWnt5a-mediated increase in calcium deposition by VSMC (A) and HASMC. Inhibition of JNK and CaMKII also prevented rWnt5a-induced gene expression of VC markers, such as RUNX2 (B). rWnt5a treatment in IMA increased the activation of JNK and CaMKII. Patients with higher WNT5A levels in IMA had significantly higher RUNX2 levels. By intersecting RNA sequencing and microarray data complement 3 (C3) resulted as the most upregulated gene in rWnt5a-treated VSMC among those genes whose levels positively correlated in IMA with both WNT5A and RUNX2 expression (C). Patients with higher WNT5A levels in IMA, their PVAT, and ThAT had significantly higher C3 levels (D), and patients with higher levels of C3AR1, that is the gene coding for the C3a receptor, had higher VC markers expression. C3 inhibition (C3-i) with a C3AR1 antagonist counteracted Wnt5a-induced VSMC calcium deposition (E). Conclusions Non-canonical Wnt signalling promotes VSMC calcification through C3. Wnt5a may be a therapeutic target in VC.Abstract Figure

High Levels of Uric Acid Upregulate Endothelin Receptors: The Role of MAPK Pathways in an in vitro Study

IntroductionUric acid (UA) is the end product of purine compounds' metabolism. There is overwhelming evidence linking hyperuricemia (high levels of UA) and cerebrovascular diseases, but the effect of high levels of UA on cerebral vessels is not fully understood. The aim of this research is to clarify how UA affect the endothelin (ET) receptor in rat cerebral arteries and the related mechanism.Material and methodsIn an in vitro setting, segments of rat cerebral arteries (n=12) were exposed to high levels of UA, either alone or in conjunction with MAPK pathway inhibitors. ET agonists were used to induce contractions that were then measured with a myograph. ET receptor expression was measured using RT-PCR (n=6), Western Blot (n=3), or immunohistochemistry (n=3) to quantify mRNA and protein levels.ResultsThe study revealed that high levels of UA notably increase ETA and ETB receptor-induced contractions and boost the expression of ET receptors in cerebral arteries when compared to that are fresh or cultured alone, suggesting that UA enhances ETA and ETB receptors. Additionally, the up-regulation of ETB receptors induced by UA was inhibited by the p38 inhibitor SB203580, the JNK inhibitor SP600125, and the ERK1/2 inhibitor U0126. SB203580 significantly blocked the increase in ETA receptor-mediated contractions induced by UA and the upregulation of ETA receptor. Neither SP600125 nor U0126 had such effect.ConclusionsHigh levels of UA stimulate the up-regulation of ET receptors in rat cerebral arteries in vitro through MAPK pathways. This study may offer novel perspectives on hyperuricemia-associated cerebrovascular diseases.

Dectin-1 participates in the immune-inflammatory response to mouse Aspergillus fumigatus keratitis by modulating macrophage polarization.

The aim of this study was to investigate whether Dectin-1 influences the immune-inflammatory response in A. fumigatus keratitis by modulating macrophage polarization. 1. The models of 1-day, 3-day, and 5-day of fungal keratitis were established in SPF C57BL/6 mice after stimulation by A. fumigatus. Dectin-1 agonist (curdlan) and antagonist (laminaran) were injected separately in the mouse subconjunctivae for 1 day in the established mouse model of A. fumigatus keratitis; PBS was used as the control. Inflammation of the mouse cornea was observed under a slit lamp to obtain a clinical score. 2. The expression of M1 (TNF-α, INOS, IL-6, IL-12) and M2 (Arg-1, IL-10, Fizz-1, Ym-1) cytokine-encoding mRNAs was quantified by RT-PCR. 3. Changes in the number of macrophages and expression of M1 and M2 macrophages in mouse corneas detected by immunofluorescence and flow cytometry. 4. Pre-treatment of RAW264.7 cells with MAPK cell signaling pathway inhibitors SB203580 (p38 inhibitor, 10µM), U0126 (ERK inhibitor, 20µM), SP600125 (JNK inhibitor, 10µM) and DMSO separately for 2h, and stimulated by A. fumigatus for 12h. Changes in the mRNA expression of M1 and M2 cytokines in the macrophages were quantified by RT-PCR. 1. With curdlan pre-treatment, mouse corneal inflammation worsened, and the clinical score increased after infection. In contrast, in the laminaran pre-treated group, corneal inflammation was alleviated and the clinical score decreased significantly compared to the PBS group after infection. 2. Compared with the control group, the expression levels of macrophage phenotype-related M1 and M2 cytokine mRNAs increased significantly 1, 3, and 5 days after A. fumigatus infected the corneas of mice. 3. With curdlan pre-treatment, the expression of mRNAs encoding M1 cytokines increased, while those encoding M2 cytokines decreased in the cornea compared to the PBS group. In contrast, after infection, mRNA levels for M1 cytokines decreased significantly and those for M2 cytokines increased in the cornea of the laminaran pre-treated group compared to the PBS group. 4. The number of macrophages in the corneal stroma of mice in the curdlan pretreatment group increased significantly compared with the PBS group, while in the laminaran pretreatment group this number decreased significantly. 5. The results of flow cytometry showed that after 3 days of mouse corneal A. fumigatus infection, the number of macrophages in the mouse A. fumigatus model in the curdlan pretreatment group was increased (10.4%) and the number of macrophages in the mouse A. fumigatus model in the laminaran pretreatment group (6.31%), when compared with the AF+FBS group (7.91%). The proportion of M1-type macrophages was increased in the curdlan pretreated group (55.6%) compared to the AF+FBS group (51.2%), the proportion of laminaran pretreatment group had a decreased proportion of M1-type macrophages (46.8%); while M2-type macrophages were the opposite of M1-type: the proportion of M2-type macrophages was 49.2% in the AF+FBS group, the proportion of M2-type macrophages was decreased in the curdlan pretreatment group (44.0%), and the proportion of M2-type macrophages was increased in the laminaran pretreatment group (53.5%). 6. Expression of M1 and M2 cytokine-encoding mRNAs decreased and increased, respectively, after infection, in the RAW264.7 cells pre-treated with MAPK pathway inhibitors, compared to the control. In a mouse model of A. fumigatus keratitis, Dectin-1 can affect macrophage recruitment and polarization, may regulate macrophage phenotype-associated factor changes through the MAPK signaling pathway.

Map3k1 suppresses terminal differentiation of migratory eye progenitors in planarian regeneration.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.

JNK inhibitor and ferroptosis modulator as possible therapeutic modalities in Alzheimer disease (AD)

Alzheimer disease (AD) is among the most prevalent neurodegenerative diseases globally, marked by cognitive and behavioral disruptions. Ferroptosis is a form of controlled cell death characterized by intracellular iron accumulation associated with lipid peroxide formation, which subsequently promotes AD initiation and progression. We hypothesized that targeting the ferroptosis pathway may help in AD management. Therefore, our study aimed to evaluate the potential neuroprotective effect of the antifungal Ciclopirox olamine (CPX-O) that acts through iron chelation. We employed CPX-O separately or in combination with the JNK inhibitor (SP600125) in a mice model of AlCl3-induced AD. Animals underwent examination for behavioral, biochemical, histological, and immunohistochemical findings. Our results revealed that AlCl3 was associated with disruptions in learning and memory parameters, neuronal degeneration in the hippocampus, increased immunoreactivity of amyloid-β and tau proteins, a significant rise in iron, nitric oxide (NO), malondialdehyde (MDA), JNK, and P53 levels, along with the significant decrease in glutathione peroxidase activity. Interestingly, the administration of CPX-O alone or in combination with SP600125 in the AlCl3-induced AD model caused an improvement in the previously described examination findings. Therefore, CPX-O may be a promising candidate for AD treatment, and future clinical trials will be required to confirm these preclinical findings.

Trehalose Attenuates In Vitro Neurotoxicity of 6-Hydroxydopamine by Reducing Oxidative Stress and Activation of MAPK/AMPK Signaling Pathways.

The effects of trehalose, an autophagy-inducing disaccharide with neuroprotective properties, on the neurotoxicity of parkinsonian mimetics 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpiridinium (MPP+) are poorly understood. In our study, trehalose suppressed 6-OHDA-induced caspase-3/PARP1 cleavage (detected by immunoblotting), apoptotic DNA fragmentation/phosphatidylserine externalization, oxidative stress, mitochondrial depolarization (flow cytometry), and mitochondrial damage (electron microscopy) in SH-SY5Y neuroblastoma cells. The protection was not mediated by autophagy, autophagic receptor p62, or antioxidant enzymes superoxide dismutase and catalase. Trehalose suppressed 6-OHDA-induced activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and AMP-activated protein kinase (AMPK), as revealed by immunoblotting. Pharmacological/genetic inhibition of JNK, p38 MAPK, or AMPK mimicked the trehalose-mediated cytoprotection. Trehalose did not affect the extracellular signal-regulated kinase (ERK) and mechanistic target of rapamycin complex 1 (mTORC1)/4EBP1 pathways, while it reduced the prosurvival mTORC2/AKT signaling. Finally, trehalose enhanced oxidative stress, mitochondrial damage, and apoptosis without decreasing JNK, p38 MAPK, AMPK, or AKT activation in SH-SY5Y cells exposed to MPP+. In conclusion, trehalose protects SH-SY5Y cells from 6-OHDA-induced oxidative stress, mitochondrial damage, and apoptosis through autophagy/p62-independent inhibition of JNK, p38 MAPK, and AMPK. The opposite effects of trehalose on the neurotoxicity of 6-OHDA and MPP+ suggest caution in its potential development as a neuroprotective agent.

Cardioprotective Action of the JNK Inhibitor Tryptanthrin Oxime in the Late Period after Myocardial Infarction.

In experiments on Wistar rats, the effect of a new selective JNK inhibitor tryptanthrin oxime (TR-Ox) on parameters of systemic hemodynamics, cardiohemodynamics, and post-infarction fibrosis was studied 4 months after acute myocardial ischemia (1 h) followed by reperfusion. TR-Ox was administered intraperitoneally at a dose of 12 mg/kg 20 min before reperfusion, and then once a day for the next 4 days. Administration of TR-Ox to animals in the acute phase of myocardial infarction contributed to more complete preservation of myocardial viability in the delayed period: a relative increase of muscle elements proportion in the scar, a decrease in the formation of connective tissue areas with complete and >50% replacement of the myocardium, and deceleration of fibrotic scarring in myocardium areas distant from the focus of injury, resulting in improved systolic and diastolic myocardial function. Four months after myocardial infarction, significant improvement in systemic hemodynamics and cardiohemodynamics parameters was observed in the group treated with TR-Ox: stroke volume, cardiac output, left ventricular systolic pressure, maximum rates of left ventricle pressure rise and fall significantly increased and the left ventricle end-diastolic pressure decreased in comparison with the corresponding parameters in the control group.