Melatonin inhibits bovine viral diarrhea virus replication by ER stress-mediated NF-κB signal pathway and autophagy in MDBK cells.

Bovine viral diarrhea-mucosal disease (BVD-MD) is a contagious disease in cattle, caused by the bovine viral diarrhea virus (BVDV). This virus continues to spread globally, exerting pressure on both public health and the economy. Despite its impact, there are currently no effective drugs for treating BVDV. This study utilized Madin-Darby bovine kidney (MDBK) cells as a model to investigate the antiviral effects of melatonin against Bovine Viral Diarrhea Virus (BVDV) and its connection with endoplasmic reticulum (ER) stress. Our results show that melatonin can suppress BVDV proliferation in MDBK cells by modulating the endoplasmic reticulum (ER) stress-mediated NF-κB pathway and autophagy. Specifically, melatonin alleviated ER stress, inhibited the activation of IκBα and p65, regulated autophagy, and reduced the expression levels of pro-inflammatory cytokines. Further, when we treated BVDV-infected cells with the ER stress inducer thapsigargin, it led to significant activation of the NF-κB pathway and autophagy. Conversely, treating the cells with the ER stress inhibitor 4-phenylbutyric acid reversed these effects. These findings suggest that melatonin exerts its antiviral effects primarily through the PERK-eIF2α-ATF4 of ER stress-mediated NF-κB pathway and autophagy. Overall, our study underscores the potential of melatonin as an effective protective and therapeutic option against BVDV, offering insights into its anti-infective mechanisms.

Inhibition of mitophagy via the EIF2S1-ATF4-PRKN pathway contributes to viral encephalitis.

INTRODUCTION: Mitophagy, a selective form of autophagy responsible for maintaining mitochondrial homeostasis, regulates the antiviral immune response and acts as viral replication platforms to facilitate infection with various viruses. However, its precise role in herpes simplex virus 1 (HSV-1) infection and herpes simplex encephalitis (HSE) remains largely unknown. OBJECTIVES: We aimed to investigate the regulation of mitophagy by HSV-1 neurotropic infection and its role in viral encephalitis, and to identify small compounds that regulate mitophagy to affect HSV-1 infection. METHODS: The antiviral effects of compounds were investigated by Western blot, RT-PCR and plaque assay. The changes of Parkin (PRKN)-mediated mitophagy and Nuclear Factor kappa B (NFKB)-mediated neuroinflammation were examined by TEM, RT-qPCR, Western blot and ELISA. The therapeutic effect of taurine or PRKN-overexpression was confirmed in the HSE mouse model by evaluating survival rate, eye damage, neurodegenerative symptoms, immunohistochemistry analysis and histopathology. RESULTS: HSV-1 infection caused the accumulation of damaged mitochondria in neuronal cells and in the brain tissue of HSE mice. Early HSV-1 infection led to mitophagy activation, followed by inhibition in the later viral infection. The HSV-1 proteins ICP34.5 or US11 deregulated the EIF2S1-ATF4 axis to suppress PRKN/Parkin mRNA expression, thereby impeding PRKN-dependent mitophagy. Consequently, inhibition of mitophagy by specific inhibitor midiv-1 promoted HSV-1 infection, whereas mitophagy activation by PRKN overexpression or agonists (CCCP and rotenone) attenuated HSV-1 infection and reduced the NF-κB-mediated neuroinflammation. Moreover, PRKN-overexpressing mice showed enhanced resistance to HSV-1 infection and ameliorated HSE pathogenesis. Furthermore, taurine, a differentially regulated gut microbial metabolite upon HSV-1 infection, acted as a mitophagy activator that transcriptionally promotes PRKN expression to stimulate mitophagy and to limit HSV-1 infection both in vitro and in vivo. CONCLUSION: These results reveal the protective function of mitophagy in HSE pathogenesis and highlight mitophagy activation as a potential antiviral therapeutic strategy for HSV-1-related diseases.

Context-dependent roles of mitochondrial LONP1 in orchestrating the balance between airway progenitor versus progeny cells.

While all eukaryotic cells are dependent on mitochondria for function, in a complex tissue, which cell type and which cell behavior are more sensitive to mitochondrial deficiency remain unpredictable. Here, we show that in the mouse airway, compromising mitochondrial function by inactivating mitochondrial protease gene Lonp1 led to reduced progenitor proliferation and differentiation during development, apoptosis of terminally differentiated ciliated cells and their replacement by basal progenitors and goblet cells during homeostasis, and failed airway progenitor migration into damaged alveoli following influenza infection. ATF4 and the integrated stress response (ISR) pathway are elevated and responsible for the airway phenotypes. Such context-dependent sensitivities are predicted by the selective expression of Bok, which is required for ISR activation. Reduced LONP1 expression is found in chronic obstructive pulmonary disease (COPD) airways with squamous metaplasia. These findings illustrate a cellular energy landscape whereby compromised mitochondrial function could favor the emergence of pathological cell types.

Activation of PERK/eIF2α/ATF4/CHOP branch of endoplasmic reticulum stress response and cooperation between HIF-1α and ATF4 promotes Daprodustat-induced vascular calcification.

Introduction: Vascular calcification is accelerated in patients with chronic kidney disease (CKD) and increases the risk of cardiovascular events. CKD is frequently associated with anemia. Daprodustat (DPD) is a prolyl hydroxylase inhibitor for the treatment of CKD-associated anemia that enhances erythropoiesis through the activation of the hypoxia-inducible factor 1 (HIF-1) pathway. Studies showed that DPD promotes osteogenic differentiation of human aortic smooth muscle cells (HAoSMCs) and increases aorta calcification in mice with CKD. HIF-1 activation has been linked with endoplasmic reticulum (ER) stress; therefore, here we investigated the potential contribution of ER stress, particularly activating transcription factor 4 (ATF4), to the pro-calcification effect of DPD. Methods: Here, we used an adenine-induced CKD mouse model and HAoSMCs as an in vitro vascular calcification model to study the effect of DPD. Results: DPD treatment (15 mg/kg/day) corrects anemia but increases the expression of hypoxia (Glut1, VEGFA), ER stress (ATF4, CHOP, and GRP78), and osteo-/chondrogenic (Runx2, Sox9, BMP2, and Msx2) markers and accelerates aorta and kidney calcification in CKD mice. DPD activates the PERK/eIF2α/ATF4/CHOP pathway and promotes high phosphate-induced osteo-/chondrogenic differentiation of HAoSMCs. Inhibition of ER stress with 4-PBA or silencing of ATF4 attenuates HAoSMC calcification. DPD-induced ATF4 expression is abolished in the absence of HIF-1α; however, knockdown of ATF4 does not affect HIF-1α expression. Conclusion: We concluded that DPD induces ER stress in vitro and in vivo, in which ATF4 serves as a downstream effector of HIF-1 activation. Targeting ATF4 could be a potential therapeutic approach to attenuate the pro-calcific effect of DPD.

OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response.

Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.

FGF21 upregulation by hepatitis C virus via the eIF2α-ATF4 pathway: implications for interferon signaling suppression and TRIM31-mediated TSC degradation.

Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases and is known to induce endoplasmic reticulum (ER) stress, which alters cellular homeostasis and metabolic processes. While ER stress is implicated in HCV-related diseases, its precise role remains unclear. This study identifies fibroblast growth factor 21 (FGF21) as a key host factor significantly upregulated by HCV infection. Mechanistic analyses reveal that the activation of the FGF21 promoter by HCV is primarily mediated by the transcription factor ATF4, which is upregulated through the phosphorylation of eIF2α induced by ER stress. Additionally, CREBH activation further enhances ATF4 expression, contributing to increased FGF21 levels. TRIB3, upregulated by ATF4, acts as a negative regulator of FGF21 expression. The study also identifies FGF21-dependent upregulation of SOCS2 and TRIM31 in HCV-infected cells. SOCS2 contributes to the suppression of type 1 interferon signaling, aiding viral persistence, while TRIM31 promotes the degradation of the tumor suppressor protein TSC, activating the mTORC1 pathway and potentially promoting liver cell proliferation. These findings suggest that FGF21 upregulation in HCV-infected cells may play a role in both immune response regulation and cell proliferation, contributing to sustained viral infection and disease progression.

Nephroprotective effects of Aralia taibaiensis in a high-fat diet-streptozotocin rat model of diabetic nephropathy.

Diabetic nephropathy (DN) has emerged as the foremost cause of end-stage renal disease (ESRD) globally. Endoplasmic reticulum (ER) stress plays a critical role in DN progression. Triterpenoid saponin from Aralia taibaiensis (sAT) has been reported to possess anti-diabetic and anti-oxidant effects. The aim of this study was to examine the influence of sAT on DN treatment and elucidate potential underlying mechanisms. A high-fat diet (HFD) and Streptozotocin (STZ) were employed to induce DN in male Sprague Dawley (SD) rats which were subsequently treated with varying concentrations of sAT for 8 weeks. Our findings reveal that different doses of sAT significantly mitigated hyperglycemia, reduced urinary albumin excretion, and decreased plasma creatinine and blood urea nitrogen levels in DN rats. Moreover, sAT administration improved body weight, alleviated renal fibrosis and histopathological changes in the diabetic kidneys. Notably, sAT treatment partially restored increased Bax expression and decreased Bcl-2 expression. Additionally, sAT inhibited ER stress-related proteins, including GRP78, p-PERK, ATF4 and CHOP in kidneys of DN rats. These results suggest that sAT ameliorated experimental diabetic nephropathy, at least in part, through ER stress pathway. These findings provide a scientific basis for the potential development of sAT as a therapeutic agent for DN treatment.

Overcoming Cancer Persister Cells by Stabilizing the ATF4 Promoter G-quadruplex.

Persister cells (PS) selected for anticancer therapy have been recognized as a significant contributor to the development of treatment-resistant malignancies. It is found that imposing glutamine restriction induces the generation of PS, which paradoxically bestows heightened resistance to glutamine restriction treatment by activating the integrated stress response and initiating the general control nonderepressible 2-activating transcription factor 4-alanine, serine, cysteine-preferring transporter 2 (GCN2-ATF4-ASCT2) axis. Central to this phenomenon is the stress-induced ATF4 translational reprogramming. Unfortunately, directly targeting ATF4 protein has proven to be a formidable challenge because of its flat surface. Nonetheless, a G-quadruplex structure located within the promoter region of ATF4 (ATF4-G4) is uncovered and resolved, which functions as a transcriptional regulator and can be targeted by small molecules. The investigation identifies the natural compound coptisine (COP) as a potent binder that interacts with and stabilizes ATF4-G4. For the first time, the high-resolution structure of the COP-ATF4-G4 complex is determined. The formation of this stable complex disrupts the interaction between transcription factor AP-2 alpha (TFAP2A) and ATF4-G4, resulting in a substantial reduction in intracellular ATF4 levels and the eventual death of cancer cells. These seminal findings underscore the potential of targeting the ATF4-G4 structure to yield significant therapeutic advantages within the realm of persister cancer cells induced by glutamine-restricted therapy.

ATF4-mediated different mode of interaction between autophagy and mTOR determines cell fate dependent on the level of ER stress induced by Cr(VI).

Hexavalent chromium [Cr(VI)] exists widely in occupational environments. The mechanistic target of rapamycin (mTOR) has been well-documented to regulate autophagy negatively. However, we found that low concentration of Cr(VI) (0.2 µM) elevated both mTOR and autophagy and promote cell survival. Conversely, high concentration of Cr(VI) (6 µM) caused cell death by inhibiting mTOR and subsequently inducing autophagy. Tunicamycin (Tm), as an Endoplasmic reticulum (ER) stress activator was used to induce mild ER stress at 0.1 µg/ml and it activated both autophagy and mTOR, which also caused cell migration in a similar manner to that observed with low concentration of Cr(VI). Severe ER stress caused by Tm (2 µg/ml) decreased mTOR, increased autophagy and then inhibited cell migration, which was the same as 6 µM Cr(VI) treatment, although Cr(VI) in high concentration inhibited ER stress. Activating transcription factor 4 (ATF4), a downstream target of ER stress, only increased under mild ER stress but decreased under severe ER stress and 6 µM Cr(VI) treatment. Chromatin immunoprecipitation (ChIP) experiment indicated that ATF4 could bind to the promoter of ATG4B and AKT1. To sum up, our data revealed that mild ER stress induced by low concentration of Cr(VI) could enhance transcriptional regulation of ATG4B and AKT1 by ATF4, which induced both autophagy and mTOR to promote cell viability.

Loss of Elp3 blocks intestinal tuft cell differentiation via an mTORC1-Atf4 axis.

Intestinal tuft cells are critical for anti-helminth parasite immunity because they produce IL-25, which triggers IL-13 secretion by activated group 2 innate lymphoid cells (ILC2s) to expand both goblet and tuft cells. We show that epithelial Elp3, a tRNA-modifying enzyme, promotes tuft cell differentiation and is consequently critical for IL-25 production, ILC2 activation, goblet cell expansion and control of Nippostrongylus brasiliensis helminth infection in mice. Elp3 is essential for the generation of intestinal immature tuft cells and for the IL-13-dependent induction of glycolytic enzymes such as Hexokinase 1 and Aldolase A. Importantly, loss of epithelial Elp3 in the intestine blocks the codon-dependent translation of the Gator1 subunit Nprl2, an mTORC1 inhibitor, which consequently enhances mTORC1 activation and stabilizes Atf4 in progenitor cells. Likewise, Atf4 overexpression in mouse intestinal epithelium blocks tuft cell differentiation in response to intestinal helminth infection. Collectively, our data define Atf4 as a negative regulator of tuft cells and provide insights into promotion of intestinal type 2 immune response to parasites through tRNA modifications.

Nitidine chloride inhibits mTORC1 signaling through ATF4-mediated Sestrin2 induction and targets IGF2R for lysosomal degradation.

AIMS: Nitidine chloride (NC), a natural phytochemical alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits multiple bioactivities, including antitumor, anti-inflammatory, and other therapeutic effects. However, the primary targets of NC and the mechanism of action (MOA) have not been explicitly defined. METHODS: We explored the effects of NC on mTORC1 signaling by immunoblotting and fluorescence microscopy in wild-type and gene knockout cell lines generated by the CRISPR/Cas9 gene editing technique. We identified IGF2R as a direct target of NC via the drug affinity-responsive target stability (DARTS) method. We investigated the antitumor effects of NC using a mouse melanoma B16 tumor xenograft model. KEY FINDINGS: NC inhibits mTORC1 activity by targeting amino acid-sensing signaling through activating transcription factor 4 (ATF4)-mediated Sestrin2 induction. NC directly binds to IGF2R and promotes its lysosomal degradation. Moreover, NC displayed potent cytotoxicity against various cancer cells and inhibited B16 tumor xenografts. SIGNIFICANCE: NC inhibits mTORC1 signaling through nutrient sensing and directly targets IGF2R for lysosomal degradation, providing mechanistic insights into the MOA of NC.

Polystyrene microplastics trigger testosterone decline via GPX1.

As an emerging environmental endocrine disruptor, polystyrene microplastics (PS-MPs) are considered to have the anti-androgenic feature and impair male reproductive function. To explore the adverse effects of PS-MPs on testosterone synthesis and male reproduction and further elucidate underlying mechanisms, BALB/c mice and Leydig cells were employed in the present work. The results indicated that 50 µm PS-MPs accumulated in mouse testes and were internalized into the cytoplasm. This not only damaged the testicular histomorphology and ultrastructure, but also reduced the viability of Leydig cells and the serum level of GnRH, FSH, LH, and testosterone. After PS-MPs exposure, the ubiquitination degradation and miR-425-3p-targeted modulation synergistically contributed to the suppression of GPX1, which induced oxidative stress and subsequently activated the PERK-EIF2α-ATF4-CHOP pathway of endoplasmic reticulum (ER) stress. The transcription factor CHOP positively regulated the expression of SRD5A2 by directly binding to its promoter region, thereby accelerating testosterone metabolism and ultimately lowing testosterone levels. Besides, PS-MPs compromised testosterone homeostasis via interfering with the hypothalamic-pituitary-testis (HPT) axis. Taken together, PS-MPs possess an anti-androgenic characteristic and exert male reproductive damage effects. The antioxidant enzyme GPX1 plays a crucial role in the PS-MPs-mediated testosterone decline.

Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer.

Metabolic reprogramming is a hallmark of human cancer, and cancer-specific metabolism provides opportunities for cancer diagnosis, prognosis, and treatment. However, the underlying mechanisms by which metabolic pathways affect the initiation and progression of colorectal cancer (CRC) remain largely unknown. Here, we demonstrate that cysteine is highly enriched in colorectal tumors compared to adjacent non-tumor tissues, thereby promoting tumorigenesis of CRC. Synchronously importing both cysteine and cystine in colorectal cancer cells is necessary to maintain intracellular cysteine levels. Hypoxia-induced reactive oxygen species (ROS) and ER stress regulate the co-upregulation of genes encoding cystine transporters (SLC7A11, SLC3A2) and genes encoding cysteine transporters (SLC1A4, SLC1A5) through the transcription factor ATF4. Furthermore, the metabolic flux from cysteine to reduced glutathione (GSH), which is critical to support CRC growth, is increased due to overexpression of glutathione synthetase GSS in CRC. Depletion of cystine/cysteine by recombinant cyst(e)inase effectively inhibits the growth of colorectal tumors by inducing autophagy in colorectal cancer cells through mTOR-ULK signaling axis. This study demonstrates the underlying mechanisms of cysteine metabolism in tumorigenesis of CRC, and evaluates the potential of cysteine metabolism as a biomarker or a therapeutic target for CRC.

PTX promotes breast cancer migration and invasion by recruiting ATF4 to upregulate FGF19.

BACKGROUND: Widely-spread among women, breast cancer is a malignancy with fatalities, and chemotherapy is a vital treatment option for it. Recent studies have underscored the potential of chemotherapeutic agents such as paclitaxel, adriamycin, cyclophosphamide, and gemcitabine, among others, in facilitating tumor metastasis, with paclitaxel being extensively researched in this context. The molecular mechanism of these genes and their potential relevance to breast cancer is noteworthy. METHOD: Clinical tissue specimens were used to analyze the expression and clinical significance of FGF19 or P-FGFR4 in patients with breast cancer before and after chemotherapy. qRT-PCR, ELISA, immunofluorescence and Western blotting were used to detect the expression level of FGF19 in breast cancer cells. The biological impacts of paclitaxel, FGF19, and ATF4 on breast cancer cells were assessed through CCK8, Transwell, and Western blot assays. The expression of ATF4 in breast cancer cells was determined through database analysis, Western blot analysis, qRT-PCR, and immunofluorescence. The direct interaction between FGF19 and ATF4 was confirmed by a luciferase assay, and Western blotting was used to assess the levels of key proteins in the stress response pathway. To confirm the effects of PTX and FGF19 in vivo, we established a lung metastasis model in nude mice. RESULTS: FGF19 expression was increased in breast cancer patients after chemotherapy. Paclitaxel can boost the migration and invasion of breast cancer cells, accompanied by an increase in FGF19 expression. ATF4 might be involved in facilitating the enhancing effect of FGF19 on breast cancer cell migration. Finally, stimulation during paclitaxel treatment could trigger a stress response, influencing the expression of FGF19 and the migration of breast cancer cells. CONCLUSION: These data suggest that paclitaxel regulates FGF19 expression through ATF4 and thus promotes breast cancer cell migration and invasion.

Tetrahydrocurcumin Attenuates Polymyxin B Sulfate-Induced HK-2 Cells Apoptosis by Inhibiting Endoplasmic Reticulum Stress-Mediated PERK/eIF2α/ATF4/CHOP Signaling Pathway Axis.

The clinical application of polymyxin B (PMB) is limited by its nephrotoxic effects, making the reduction of PMB-induced nephrotoxicity has become a pressing concern for clinicians. Tetrahydrocurcumin (THC), known for its beneficial characteristics in biological functions, presents an attractive option for intervention therapy to mitigate PMB-induced nephrotoxicity. However, the underlying mechanism of how THC mitigates PMB-induced nephrotoxicity is still poorly understood. Here, we first evaluated the potential of THC intervention therapy to mitigate PMB-induced nephrotoxicity in an in vitro model of PMB-induced cell injury. Moreover, we demonstrated that THC effectively protected HK-2 cells from PMB-induced apoptosis by using cell counting kit-8 and flow cytometry assay. THC could also suppress PMB-induced endoplasmic reticulum (ER) stress via PERK/eIF2α/ATF4/CHOP pathway. In addition, using PERK inhibitor GSK2606414 to inhibit ER stress also alleviated PMB-induced apoptosis. Taken together, these findings provide novel insights that THC possesses the ability to alleviate PMB-induced nephrotoxicity by inhibiting the ER stress-mediated PERK/eIF2α/ATF4/CHOP axis, which sheds light on the benefits of THC as an intervention strategy to reduce PMB-induced nephrotoxicity, thus providing a potential avenue for improved clinical outcomes in patients receiving PMB treatment.

Induction of the PERK-eIF2α-ATF4 Pathway in M1 Macrophages under Endoplasmic Reticulum Stress.

Translation inhibition can activate two cell death pathways. The first pathway is activated by translational aberrations, the second by endoplasmic reticulum (ER) stress. In this work, the effect of ribosome-inactivating protein type II (RIP-II) viscumin on M1 macrophages derived from the THP-1 cell line was investigated. The number of modified ribosomes was evaluated by real-time PCR. Transcriptome analysis revealed that viscumin induces the ER stress activated by the PERK sensor.

Feasibility exploration of GSH in the treatment of acute hepatic encephalopathy from the aspects of pharmacokinetics, pharmacodynamics, and mechanism.

Our previous study highlighted the therapeutic potential of glutathione (GSH), an intracellular thiol tripeptide ubiquitous in mammalian tissues, in mitigating hepatic and cerebral damage. Building on this premise, we posited the hypothesis that GSH could be a promising candidate for treating acute hepatic encephalopathy (AHE). To verify this conjecture, we systematically investigated the feasibility of GSH as a therapeutic agent for AHE through comprehensive pharmacokinetic, pharmacodynamic, and mechanistic studies using a thioacetamide-induced AHE rat model. Our pharmacodynamic data demonstrated that oral GSH could significantly improve behavioral scores and reduce hepatic damage of AHE rats by regulating intrahepatic ALT, AST, inflammatory factors, and homeostasis of amino acids. Additionally, oral GSH demonstrated neuroprotective effects by alleviating the accumulation of intracerebral glutamine, down-regulating glutamine synthetase, and reducing taurine exposure. Pharmacokinetic studies suggested that AHE modeling led to significant decrease in hepatic and cerebral exposure of GSH and cysteine. However, oral GSH greatly enhanced the intrahepatic and intracortical GSH and CYS in AHE rats. Given the pivotal roles of CYS and GSH in maintaining redox homeostasis, we investigated the interplay between oxidative stress and pathogenesis/treatment of AHE. Our data revealed that GSH administration significantly relieved oxidative stress levels caused by AHE modeling via down-regulating the expression of NADPH oxidase 4 (NOX4) and NF-κB P65. Importantly, our findings further suggested that GSH administration significantly regulated the excessive endoplasmic reticulum (ER) stress caused by AHE modeling through the iNOS/ATF4/Ddit3 pathway. In summary, our study uncovered that exogenous GSH could stabilize intracerebral GSH and CYS levels to act on brain oxidative and ER stress, which have great significance for revealing the therapeutic effect of GSH on AHE and promoting its further development and clinical application.

Crosstalk between ferroptosis and necroptosis in cerebral ischemia/reperfusion injury and Naotaifang formula exerts neuroprotective effect via HSP90-GCN2-ATF4 pathway.

BACKGROUND: Cerebral ischemia/reperfusion injury (CIRI) is a sequence of pathophysiological processes after blood recanalization in the patients with ischemic stroke, and has become the hinder for the rehabilitation. Naotaifang formula (NTF) has exhibited the clinical effectiveness for this disease. However, its action effects and molecular mechanisms against CIRI are not fully elucidated. PURPOSE: The research was to clarify the crosstalk between ferroptosis and necroptosis in CIRI, and uncover the mechanism underlying the neuroprotection of NTF. METHODS: This study established MCAO/R rat models with various reperfusion times. Western blot, transmission electron microscope, laser speckle imaging, immunofluorescence, immunohistochemistry and pathological staining were conducted to detect and analyze the obtained results. Subsequently, various NTF doses were used to intervene in MCAO/R rats, and biology experiments, such as western blot, Evans blue, immunofluorescence and immunohistochemistry, were used to analyze the efficacy of NTF doses. The effect of NTF was further clarified through in vitro experiments. Eventually, HT22 cells that suffered OGD/R were subjected to pre-treatment with plasmids overexpressing HSP90, MLKL, and GPX4 to indicate the interaction among ferroptosis and necroptosis. RESULTS: There was a gradual increase in the Zea Longa score and cerebral infarction volume following CIRI with prolonged reperfusion. Furthermore, the expression of factors associated with pro-ferroptosis and pro-necroptosis was upregulated in the cortex and hippocampus. NTF alleviated ferroptosis and necroptosis in a dose-dependent manner, downregulated HSP90 levels, reduced blood-brain barrier permeability, and thus protected nerve cells from CIRI. The results in vitro research aligned with those of the in vivo research. HSP90 and MLKL overexpression promoted necroptosis and ferroptosis while activating the GCN2-ATF4 pathway. GPX4 overexpression had no effect on necroptosis or the associated signaling pathway. The administration of NTF alone, as well as its combination with the overexpression of HSP90, MLKL, or GPX4 plasmids, decreased the expression levels of factors associated with pro-ferroptosis and pro-necroptosis and reduced the protein levels of the HSP90-GCN2-ATF4 pathway. Moreover, the regulatory effects of the NTF alone group on GSH, ferrous iron, and GCN2 were more significant compared with those of the HSP90 overexpression combination group. CONCLUSION: Ferroptosis and necroptosis were gradually aggravated following CIRI with prolonged reperfusion. MLKL overexpression may promote ferroptosis and necroptosis, while GPX4 overexpression may have little effect on necroptosis. HSP90 overexpression accelerated both forms of cell death via the HSP90-GCN2-ATF4 pathway. NTF alleviated ferroptosis and necroptosis to attenuate CIRI by regulating the HSP90-GCN2-ATF4 pathway. Our research provided evidence for the potential of drug development by targeting HSP90, MLKL, and GPX4 to protect against ischemic stroke.

ATF4 inhibits tumor development and mediates p-GCN2/ASNS upregulation in colon cancer.

Colon cancer (CC) is a highly malignant tumor with a high incidence and poor prognosis. This study aimed to explore the function and molecular mechanisms of activating transcription factor 4 (ATF4) in CC. The expression levels of ATF4, GCN2, and ASNS in CC tissues were measured using immunohistochemistry (IHC) and reverse transcription quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), clone formation, transwell, and flow cytometry assays were conducted to assess cell viability, clonogenicity, migration, invasion, cell cycle, and apoptosis, respectively, in the ATF4 knockdown and overexpression SW480 cell lines. The effect of ATF4 on the expression of GCN2 and ASNS was detected using RT-qPCR, Chip-qPCR, and western blotting. ATF4, GCN2, and ASNS were expressed at low levels in CC tissues, and all had a significant negative correlation with tumor diameter. ATF4 knockdown promoted cell proliferation, invasion, and S-phase cell cycle and inhibited apoptosis in SW480 cells. In contrast, ATF4 overexpression had the opposite effect. Furthermore, ATF4 overexpression enhanced ATF4 binding to the ASNS promoter region. ATF4 knockdown significantly inhibited the expression of p-GCN2 and ASNS, whereas ATF4 overexpression significantly upregulated their expression. ATF4 inhibited CC cell viability, clone formation ability, migration, and invasion and promoted apoptosis, possibly by regulating the expression of p-GCN2 and ASNS. Our study provides a novel potential therapeutic target for the treatment of CC.

Niacin, an innovative protein kinase-C-dependent endoplasmic reticulum stress reticence in murine Parkinson's disease.

AIMS: Niacin (NIA) supplementation showed effectiveness against Parkinson's disease (PD) in clinical trials. The depletion of NAD and endoplasmic reticulum stress response (ERSR) are implicated in the pathogenesis of PD, but the potential role for NAD precursors on ERSR is not yet established. This study was undertaken to decipher NIA molecular mechanisms against PD-accompanied ERSR, especially in relation to PKC. METHODS: Alternate-day-low-dose-21 day-subcutaneous exposure to rotenone (ROT) in rats induced PD. Following the 5th ROT injection, rats received daily doses of either NIA alone or preceded by the PKC inhibitor tamoxifen (TAM). Extent of disease progression was assessed by behavioral, striatal biochemical and striatal/nigral histopathological/immunohistochemical analysis. KEY FINDINGS: Via activating PKC/LKB1/AMPK stream, NIA post-treatment attenuated the ERSR reflected by the decline in ATF4, ATF6 and XBP1s to downregulate the apoptotic markers, CHOP/GADD153, p-JNK and active caspase-3. Such amendments congregated in motor activity/coordination improvements in open field and rotarod tasks, enhanced grid test latency and reduced overall PD scores, while boosting nigral/striatal tyrosine hydroxylase immunoreactivity and increasing intact neurons (Nissl stain) in both SNpc and striatum that showed less neurodegeneration (H&E stain). To different extents, TAM reverted all the NIA-related actions to prove PKC as a fulcrum in conveying the drug neurotherapeutic potential. SIGNIFICANCE: PKC activation is a pioneer mechanism in the drug ERSR inhibitory anti-apoptotic modality to clarify NIA promising clinical and potent preclinical anti-PD efficacy. This kinase can be tagged as a druggable target for future add-on treatments that can assist dopaminergic neuronal aptitude against this devastating neurodegenerative disease.