The unknown mechanism that controls intestinal barrier dysfunction in individuals with Crohn\'s disease (CD) plays a crucial role in the onset of intestinal inflammation. Testin, an intercellular linker protein, has the potential to protect epithelial barrier function. This study aimed to analyse the effects of Testin on CD-like colitis and explore the possible underlying mechanism. Colon samples from CD patients and trinitrobenzene-sulfonic acid (TNBS)-treated mice were collected to examine changes in Testin expression. To assess the therapeutic effects of Testin on CD-like colitis in mice, we examined the symptoms of enteritis, performed histological analysis, and evaluated intestinal barrier permeability. The ability of Testin to stabilize tight junction (TJ) proteins was investigated via immunofluorescence and western blotting. We conducted in vivo and in vitro experiments using colonic organoids and blocking techniques to explore how Testin safeguards the integrity of the intestinal barrier. Testin expression was downregulated in the colons of CD patients and TNBS-treated mice. Increasing Testin expression led to amelioration of colitis symptoms and reduced the production of inflammatory cytokines in the colons of TNBS-induced colitis model mice. Furthermore, increased Testin expression resulted in decreased depletion of TJ proteins (ZO-1 and Claudin-1) and promoted the effectiveness of the intestinal barrier in mice with TNBS-induced colon damage and in lipopolysaccharide (LPS)-stimulated colonic organoids. Elevated Testin levels inactivated the JNK/P38 signalling pathway, potentially contributing to the beneficial impact of Testin on the intestinal barrier. Testin can inhibit the loss of TJ proteins in CD mice by inactivating the JNK/P38 pathway. These findings help to clarify how Testin alleviates CD-like colitis in mice by protecting intestinal barrier function. These findings could lead to the use of a new treatment approach for CD in clinical practice.

Excessive fat deposition leads to obesity and cardiovascular diseases with abnormal metabolism. Pantothenic acid (PA) is a major B vitamin required for energy metabolism. However, the effect of PA on lipid metabolism and obesity has not been explored. We investigated the effects and molecular mechanism of PA on fat accumulation as well as the influence of adipogenic marker genes in both adult male mice and primary adipocytes. First, we demonstrated that PA attenuates weight gain in mice fed high-fat diet (HFD). Besides, PA supplementation substantially improved glucose tolerance and lipid metabolic disorder in obese mice. Furthermore, PA significantly inhibited white adipose tissue (WAT) deposition as well as fat droplets visualized by magnification in both chow and HFD group. More importantly, PA obviously suppressed the mRNA levels of CD36, IL-6, and TNF-α to alleviate inflammation and reduced the levels of PPARγ, aP2, and C/EBPα genes that are related to lipid metabolism in inguinal white adipose tissue (ing-WAT) and epididymal white adipose tissue (ei-WAT). In vitro, PA supplementation showed a lower lipid droplet aggregation as well as reduced expression levels of adipogentic genes. Finally, we identified that PA inhibits the phosphorylation levels of p38 and JNK in murine primary adipocytes. Collectively, our data demonstrated for the first time that PA attenuates lipid metabolic disorder as well as fat deposition by JNK/p38 MAPK signaling pathway.

Pathological cardiac hypertrophy (CH) is driven by maladaptive changes in myocardial cells in response to pressure overload or other stimuli. CH has been identified as a significant risk factor for the development of various cardiovascular diseases, ultimately resulting in heart failure. Melanoma differentiation-associated protein 5 (MDA5), encoded by interferon-induced with helicase C domain 1 (IFIH1), is a cytoplasmic sensor that primarily functions as a detector of double-stranded ribonucleic acid (dsRNA) viruses in innate immune responses; however, its role in CH pathogenesis remains unclear. Thus, the aim of this study was to examine the relationship between MDA5 and CH using cellular and animal models generated by stimulating neonatal rat cardiomyocytes with phenylephrine and by performing transverse aortic constriction on mice, respectively. MDA5 expression was upregulated in all models. MDA5 deficiency exacerbated myocardial pachynsis, fibrosis, and inflammation in vivo, whereas its overexpression hindered CH development in vitro. In terms of the underlying molecular mechanism, MDA5 inhibited CH development by promoting apoptosis signal-regulating kinase 1 (ASK1) phosphorylation, thereby suppressing c-Jun N-terminal kinase/p38 signaling pathway activation. Rescue experiments using an ASK1 activation inhibitor confirmed that ASK1 phosphorylation was essential for MDA5-mediated cell death. Thus, MDA5 protects against CH and is a potential therapeutic target.

BACKGROUND: Traumatic brain injury (TBI) is a significant worldwide public health concern that necessitates attention. Apoptosis signal-regulating kinase 1 (ASK1), a key player in various central nervous system (CNS) diseases, has garnered interest for its potential neuroprotective effects against ischemic stroke and epilepsy when deleted. Nonetheless, the specific impact of ASK1 on TBI and its underlying mechanisms remain elusive. Notably, mutation of ATP-binding sites, such as lysine residues, can lead to catalytic inactivation of ASK1. To address these knowledge gaps, we generated transgenic mice harboring a site-specific mutant ASK1 Map3k5-e (K716R), enabling us to assess its effects and elucidate potential underlying mechanisms following TBI.
METHODS: We employed the CRIPR/Cas9 system to generate a transgenic mouse model carrying the ASK1-K716R mutation, aming to investigate the functional implications of this specific mutant. The controlled cortical impact method was utilized to induce TBI. Expression and distribution of ASK1 were detected through Western blotting and immunofluorescence staining, respectively. The ASK1 kinase activity after TBI was detected by a specific ASK1 kinase activity kit. Cerebral microvessels were isolated by gradient centrifugation using dextran. Immunofluorescence staining was performed to evaluate blood-brain barrier (BBB) damage. BBB ultrastructure was visualized using transmission electron microscopy, while the expression levels of endothelial tight junction proteins and ASK1 signaling pathway proteins was detected by Western blotting. To investigate TBI-induced neuroinflammation, we conducted immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analyses. Additionally, immunofluorescence staining and electrophysiological compound action potentials were conducted to evaluate gray and white matter injury. Finally, sensorimotor function and cognitive function were assessed by a battery of behavioral tests.
RESULTS: The activity of ASK1-K716R was significantly decreased following TBI. Western blotting confirmed that ASK1-K716R effectively inhibited the phosphorylation of ASK1, JNKs, and p38 in response to TBI. Additionally, ASK1-K716R demonstrated a protective function in maintaining BBB integrity by suppressing ASK1/JNKs activity in endothelial cells, thereby reducing the degradation of tight junction proteins following TBI. Besides, ASK1-K716R effectively suppressed the infiltration of peripheral immune cells into the brain parenchyma, decreased the number of proinflammatory-like microglia/macrophages, increased the number of anti-inflammatory-like microglia/macrophages, and downregulated expression of several proinflammatory factors. Furthermore, ASK1-K716R attenuated white matter injury and improved the nerve conduction function of both myelinated and unmyelinated fibers after TBI. Finally, our findings demonstrated that ASK1-K716R exhibited favorable long-term functional and histological outcomes in the aftermath of TBI.
CONCLUSIONS: ASK1-K716R preserves BBB integrity by inhibiting ASK1/JNKs pathway in endothelial cells, consequently reducing the degradation of tight junction proteins. Additionally, it alleviates early neuroinflammation by inhibiting the infiltration of peripheral immune cells into the brain parenchyma and modulating the polarization of microglia/macrophages. These beneficial effects of ASK1-K716R subsequently result in a reduction in white matter injury and promote the long-term recovery of neurological function following TBI.

BACKGROUND: The C-terminal tetrapeptide Lys-Asp-Glu-Leu receptors (KDELRs) are transmembrane proteins that regulate ER stress (ERS) response, growth, differentiation, and immune responses. There is an association between KDELR2and promotion of glioblastoma tumorigenesis. The aim of the present study was to explore the functional mechanism of KDELR2 in glioma and during response to chemotherapy to temozolomide (TMZ).
METHODS: The expression of KDELR2 in glioma tissues and cells was evaluated by immunohistochemistry, western blot and RT-qPCR assay. Then role of KDELR2 was demonstrated by CCK8, colony formation, flow cytometry and Hochest 33258 assays. The expression of genes (ATF4, ATF6, PERK, eIF2-α, GRP78 and CHOP) in U373 cells was evaluated by RT-qPCR. The protein expression of genes (cleaved caspase 3, caspase 3, cleaved PARP, PARP, Bax, Bcl-2, JNK, p-JNK, p38, p-p38, ATF4, ATF6, XBP-1s, PERK, p-PERK, GRP78 and CHOP) was measured by western blot assay.
RESULTS: The expression of KDELR2 was upregulated in high-grade gliomas tissues. KDELR2 knockdown suppressed cell proliferation but increased cell apoptosis. Further, Knockdown of KDELR2 also activated the ER stress (ERS)-dependent CHOP pathway, and resulted in increased levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38. Moreover, the combination of KDELR2 knockdown and TMZ application showed a synergistic cytotoxic effect in U373 cells through the ERS-dependent CHOP and JNK/p38 pathways.
CONCLUSIONS: KDELR2 knockdown induces apoptosis and sensitizes glioma cells to TMZ, which is mediated by the ERS-dependent CHOP and JNK/p38 pathways.

Beta-cypermethrin (β-CYP), a widely-used pyrethroid pesticide, is considered to have anti-androgenic effects and could impair male reproduction. To ascertain whether MAPK pathways, DNA methyltransferases (DNMTs), and miRNAs played pleiotropic roles in β-CYP-mediated testicular dysfunction, Sprague-Dawley rats and Leydig cells were employed in this study. Results showed that plasma testosterone levels were declined, testicular histomorphology and ultrastructures were abnormally altered, and Leydig cell functions were damaged after β-CYP exposure. JNK and p38/MAPK pathways were inactivated, accompanied by the decrease in c-Jun and Sp1 expressions. Specific activators/inhibitors of MAPK pathways and Co-IP demonstrated that DNMT3α was synergistically regulated by JNK/p38 pathways. The activity, mRNA and protein expressions of DNMT3α were all reduced by β-CYP. β-CYP induced expressions of intronic miR-140-5p and its host gene Wwp2, and then overexpressed miR-140-5p suppressed steroidogenic StAR, P450scc, and 3β-HSD by directly targeting SF-1. SF-1 silencing/overexpression, ChIP, and qPCR indicated that SF-1 modulated positively StAR, P450scc, and 3β-HSD expressions by directly binding to their promoter regions. Intriguingly, 5α-reductase expressions were downregulated after β-CYP exposure. Collectively, β-CYP has the anti-androgenic feature and the DNMT3α/miR-140-5p/SF-1 cascade co-regulated by JNK/p38 functions critically in β-CYP-caused testosterone declines. The downregulation of 5α-reductases may be a potential compensatory mechanism of the organism.

Spinal cord injury (SCI) is a complex pathological change that includes primary SCI and gradually evolves into secondary SCI. Accumulating evidence demonstrates that circular RNAs (circRNAs) are involved in the pathology of a variety of neurological diseases and injuries. However, the characteristics and function of circRNAs in SCI have yet to be elucidated. Although previous research demonstrated that circPrkcsh induces astrocytes to produce inflammatory factors and chemokines, the precise function and mechanism of circPrkcsh in microglia after SCI remains unknown. In this study, we constructed a mouse model of SCI by applying a SCI impactor. Quantitative Real-time PCR and Fluorescence in situ hybridization analysis revealed that circPrkcsh was upregulated in the microglia of SCI mice when compared to sham-operated mice. Gain- or loss-of-function experiments and in vivo assays further indicated that circPrkcsh promotes microglia M1 polarization both in vivo and in vitro. Furthermore, bioinformatics analysis, dual-luciferase assays, and RNA immunoprecipitation assays, confirmed that circPrkcsh serves as a competing endogenous RNA (ceRNA) to promote the expression of MEKK1 mRNA by sponging miR-488. Double knockout rescue experiments further showed that circPrkcsh regulates the MEKK1/JNK/p38 MAPK pathway via miR-488. Our research provides a better understanding of the mechanism of circPrkcsh in SCI and demonstrates that the circPrkcsh/miR-488/Mekk1 axis is a promising regulatory method for the treatment of SCI.

Rationale: Glioblastoma multiforme (GBM) almost invariably gain invasive phenotype with limited therapeutic strategy and ill-defined mechanism. By studying the aberrant expression landscape of gliomas, we find significant up-regulation of p-MAPK level in GBM and a potent independent prognostic marker for overall survival. DHHC family was generally expressed in glioma and closely related to the activation of MAPK signaling pathway, but its role and clinical significance in GBM development and malignant progression are yet to be determined. Method: Bioinformatics analysis, western blotting and immunohistochemistry (IHC) were performed to detect the expression of ZDHHC17 in GBM. The biological function of ZDHHC17 was demonstrated by a series of in vitro and in vivo experiments. Pharmacological treatment, flow cytometry, Transwell migration assay, Co- Immunoprecipitation and GST pulldown were carried out to demonstrate the potential mechanisms of ZDHHC17. Results: ZDHHC17 is up-regulated and coordinated with MAPK activation in GBM. Mechanistically, ZDHHC17 interacts with MAP2K4 and p38/JNK to build a signaling module for MAPK activation and malignant progression. Notably, the ZDHHC17-MAP2K4-JNK/p38 signaling module contributes to GBM development and malignant progression by promoting GBM cell tumorigenicity and glioma stem cell (GSC) self-renewal. Moreover, we identify a small molecule, genistein, as a specific inhibitor to disrupt ZDHHC17-MAP2K4 complex formation for GBM cell proliferation and GSC self-renewal. Moreover, genistein, identified herein as a lead candidate for ZDHHC17-MAP2K4 inhibition, demonstrated potential therapeutic effect in patients with ZDHHC17-expressing GBM. Conclusions: Our study identified disruption of a previously unrecognized signaling module as a target strategy for GBM treatment, and provided direct evidence of the efficacy of its inhibition in glioma using a specific inhibitor.

In this study, we investigated whether Quercetin has anti-cancer effects on A375SM and A375P human melanoma cells. Cell viability was assessed using an MTT assay. The proliferation of melanoma cells was measured by a wound-healing assay. Quercetin significantly decreased viability and proliferation of A375SM cells in a concentration-dependent manner. However, quercetin had no effect on A375P cells. DAPI staining showed increased chromatin condensation in a concentration-dependent manner, indicating apoptosis. Flow cytometric analysis indicated that quercetin suppressed the viability of A375SM cells by inducing apoptosis. Expression of quercetin-induced apoptosis proteins was investigated by Western blot analysis. Quercetin increased the expression of Bax, phospho-JNK, phospho-p38 and phospho-ERK1/2, cleaved poly-ADP ribose polymerase and decreased Bcl-2 in a concentration-dependent manner. We also investigated the in vivo tumor-growth inhibitory effect of quercetin. Quercetin (at 50 and 100 mg/kg) significantly decreased the A375SM tumor volume compared to the control group and increased apoptosis as assessed by the TUNEL assay. Immunohistochemistry staining revealed that the level of phosphor-JNK and phosphor-p38 increased in the quercetin-treated mice. These results indicate that quercetin inhibited the growth of A375SM melanoma cells through apoptosis and thus can be regarded as a new and effective chemo-preventive or therapeutic agent.

Numerous studies have reported that inflammation is involved in the pathophysiology of depression. Pioglitazone, a PPAR-γ agonist, has potential anti-inflammatory and antidepressive effects. However, the underlying molecular mechanisms of the antidepressant-like effect of pioglitazone on an inflammation-related mouse model of depression remain to be fully elucidated. Herein, we aimed to explore the effects of pioglitazone on depressive-like behaviours of mice exposed to lipopolysaccharides (LPS), and elucidate the underlying mechanisms. We assessed behaviour changes of mice pretreated with pioglitazone exposed to LPS. Additionally, neural apoptosis, and the expression of apoptosis-related (cleaved caspase-3, Bax, Bcl-2, cyt c) and signalling proteins (AKT, JNK, p38) were assessed in the prefrontal cortex (PFC) of these mice. Furthermore, we assessed the influence of anisomycin, a JNK/p38 agonist, and LY294002, a PI3K/AKT inhibitor, on the antidepressant-like effect of pioglitazone in mice. We show that pioglitazone pretreatment (20 mg/kg, intragastrically) attenuated LPS-induced (10 ng/μL per site) depressive-like behaviours. GW9662, a PPAR-γ antagonist, significantly blocked the antidepressant-like effect of pioglitazone. Furthermore, at the molecular level, pioglitazone significantly reversed, via PPAR-γ-dependent increase in neural apoptosis in the PFC of mice, accompanied by upregulation of the PI3K/AKT pathway and down-regulation of the JNK/p38 pathway. Moreover, both anisomycin and LY294002 abrogated the antidepressant-like effect of pioglitazone.; In conclusion, our results showed that PI3K/AKT/JNK/p38 signalling pathway-mediated neural apoptosis in the PFC of mice may be involved in the antidepressant-like effect of pioglitazone. This provides novel insights into and therapeutic targets for inflammation-related depression.