Activating Transcription Factor Research Articles

Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor β (TGFβ)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFβ-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFβ-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFβ-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.

The Role of Endoplasmic Reticulum Stress in Metabolic Diseases.

The endoplasmic reticulum (ER), the center of protein folding, also controls the cell's life-and-death signaling mechanisms. ER stress caused by unfolded or misfolded proteins leads to the activation of the unfolded protein response (UPR) in the cell. The UPR utilizes three main signaling pathways to restore disrupted ER homeostasis. These signaling pathways are protein kinase R-like endoplasmic reticulum kinase, inositol-requiring enzyme 1, and activating transcription factor 6. Studies have reported that ER stress (ERS) plays a role in the pathogenesis of metabolic disorders such as diabetes, obesity, atherosclerosis, and nonalcoholic liver disease. This review will briefly discuss the ERS response in these metabolic diseases.

Effects of electroacupuncture of different intensities and durations on PERK/ATF4/CHOP signaling pathway in liver of non-alcoholic fatty liver disease rats.

To analyze the effects of electroacupuncture (EA) at "Fenglong" (ST40) and "Zusanli" (ST36) of different intensities and durations on rats with non-alcoholic fatty liver disease (NAFLD) based on the protein kinase R-like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) signaling pathway, so as to explore its mechanism underlying improvement of NAFLD. SD rats were randomly divided into normal diet group, high-fat model group, sham EA group, strong stimulation EA (SEA) group, and weak stimulation EA (WEA) group, with 15 rats in each group. Each group was further divided into 2, 3, and 4-week subgroups. NAFLD rat model was established by feeding a high-fat diet. After successful modeling, rats in the SEA and WEA groups received EA at bilateral ST40 and ST36 with dense and sparse waves (4 Hz/20 Hz) at current intensities of 4 mA (SEA group) and 2 mA (WEA group), lasting for 20 minutes, once a day, 5 days a week with 2 days of rest. The sham EA group only had the EA apparatus connected without electricity. Different duration subgroups were intervened for 2, 3, and 4 weeks. After the intervention, the contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rats were detected by an automatic biochemical analyzer；liver morphological changes were observed by Oil Red O staining；real-time fluorescence quantitative PCR and Western blot were used to detect the expression of PERK, ATF4, and CHOP mRNAs and proteins in the rat liver tissue. In the high-fat model group, there was a significant accumulation of red lipid droplets in the liver cells, which was reduced significantly in the SEA group at the 4th week. Compared with the normal diet group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and proteins in the liver tissue were elevated (P<0.01) in the high-fat model group . Compared with the high-fat model group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, CHOP mRNAs and proteins in the liver tissue were decreased (P<0.01, P<0.05) in the SEA and WEA groups. Compared with the sham EA group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs were decreased (P<0.01, P<0.05) in the SEA and WEA groups, the expression of PERK, ATF4, and CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA group at the 2nd, 3rd, and 4th week, the expression of PERK and CHOP proteins at the 2nd, 3rd, 4th week and ATF4 protein at 2nd week in the liver tissue were decreased (P<0.01, P<0.05) in the WEA group. Compared with the SEA group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and proteins in the liver tissue were elevated (P<0.05, P<0.01) in the WEA group. Compared with the 2-week time point within the groups, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and PERK proteins in the liver tissue were decreased (P<0.01, P<0.05) in the SEA and WEA groups at 3rd and 4th week, the expression of ATF4 proteins in the liver tissue was decreased (P<0.01) in the SEA group at 3rd and 4th week, and the expression of CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA group at 4th week and in the WEA group at 3rd and 4th week. Compared with the 3-week time point within the groups, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs were significantly decreased (P<0.05, P<0.01) in the SEA and WEA groups at 4th week, the expression of PERK and CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA and WEA groups at 4th week, and the expression of ATF4 protein in the liver tissue was decreased (P<0.05) in the SEA group at 4th week. EA at ST40 and ST36 can significantly improve liver function in NAFLD rats, and its mechanism of action may involve inhibiting PERK expression thereby targeting the downstream ATF4/CHOP signaling pathway to suppress endoplasmic reticulum stress, exerting a liver protective effect；the optimal effect was observed with EA intensity of 4 mA for 4 weeks.

Mitigation of ROS-triggered endoplasmic reticulum stress by upregulating Nrf2 retards diabetic nephropathy

Endoplasmic reticulum stress (ERS) plays a crucial role in the pathogenesis of diabetic nephropathy (DN), and it is often accompanied by an increase in reactive oxygen species (ROS) production. However, the precise relationship between NFE2-related factor-2 (Nrf2), a key regulator of ROS balance, and ERS in DN remains elusive. This study aimed to investigate the impact of Nrf2 on ERS and its therapeutic potential in DN. Herein, ERS-related changes, including increased activating transcription factor-6 (ATF6), glucose-regulated protein 78 (GRP78), and transcription factor C/EBP homologous protein (CHOP) expression, were observed in the renal tissues of streptozotocin-induced DN mice and high glucose cultured human renal proximal tubular (HK-2) cells. Nrf2 knockdown increased the sensitivity of HK-2 cells to ERS under high glucose conditions, underscoring the regulatory role of Nrf2 in ERS modulation. Notably, upregulating Nrf2 in ezetimibe-treated diabetic mice restored ERS markers and ameliorated albuminuria, glomerular hypertrophy, mesangial expansion, and tubulointerstitial fibrosis. Furthermore, the inhibition of ERS in HK-2 cells by the ROS scavenger, N-acetylcysteine, highlights the interplay between ROS and ERS. This study, for the first time, elucidates that the upregulation of Nrf2 may alleviate the negative influence of ROS-mediated ERS, presenting a promising therapeutic avenue for delaying the progression of DN. These findings suggest a potential strategy for targeting Nrf2 and ERS in developing novel therapeutic interventions for DN.

The IRE1α-XBP1 arm of the unfolded protein response is a host factor activated in SARS-CoV-2 infection

SARS-CoV-2 infection can cause severe pneumonia, wherein exacerbated inflammation plays a major role. This is reminiscent of the process commonly termed cytokine storm, a condition dependent on a disproportionated production of cytokines. This state involves the activation of the innate immune response by viral patterns and coincides with the biosynthesis of the biomass required for viral replication, which may overwhelm the capacity of the endoplasmic reticulum and drive the unfolded protein response (UPR). The UPR is a signal transduction pathway composed of three branches that is initiated by a set of sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These sensors control adaptive processes, including the transcriptional regulation of proinflammatory cytokines. Based on this background, the role of the UPR in SARS-CoV-2 replication and the ensuing inflammatory response was investigated using in vivo and in vitro models of infection. Mice and Syrian hamsters infected with SARS-CoV-2 showed a sole activation of the Ire1α-Xbp1 arm of the UPR associated with a robust production of proinflammatory cytokines. Human lung epithelial cells showed the dependence of viral replication on the expression of UPR-target proteins branching on the IRE1α-XBP1 arm and to a lower extent on the PERK route. Likewise, activation of the IRE1α-XBP1 branch by Spike (S) proteins from different variants of concern was a uniform finding. These results show that the IRE1α-XBP1 system enhances viral replication and cytokine expression and may represent a potential therapeutic target in SARS-CoV-2 severe pneumonia.

The Interplay Between Endoplasmic Reticulum Stress and Oxidative Stress in Chondrocyte Catabolism.

Oxidative stress and endoplasmic reticulum (ER) stress play pivotal roles in disrupting the homeostasis of chondrocytes by producing catalytic proteases and enhancing chondrocyte senescence, consequently contributing to the progression of osteoarthritis (OA). Despite their close interaction, the underlying molecular mechanisms remain poorly understood. Here, we show that ER stress and oxidative stress reciprocally modulate each other to promote cartilage degradation. Primary chondrocytes were obtained from the articular cartilage of 5-day-old C57BL/6J mice by excising distal femur and proximal tibia. Tunicamycin was applied to induce ER stress in primary chondrocytes. Surgical OA was induced in 12-week-old male C57BL/6J mice by destabilizing the medial meniscus (DMM). Tunicamycin-induced ER stress led to an increase in the production of reactive oxygen species (ROS) and catalytic proteases, including MMP13 and Adamts5, in primary chondrocytes, and it was primarily dependent on the NADPH oxidase (NOX) system. ER stress directly increased the expression of NOX2, NOX3, NOX4, and p22phox. Specifically, the protein kinase RNA-like ER kinase (PERK) pathway is involved in the expression of NOX4 and p22phox, the inositol-requiring enzyme 1 alpha (IRE1α) pathway in NOX2 and NOX3 expression, and the activating transcription factor 6 (ATF6) pathway influences NOX3 expression in chondrocytes. Conversely, inhibiting NOX function significantly reduced both ER stress sensor-related signaling and chondrocyte catabolism, thereby decelerating the progression of surgically induced OA in vivo. Our findings highlight the positive feedback loop between ER stress and oxidative stress in OA pathogenesis, suggesting that targeting NOX isoforms is a promising therapeutic strategy for OA.

Revealing the mechanism of ethyl acetate extracts of Semen Impatientis against prostate cancer based on network pharmacology and transcriptomics

Ethnopharmacological relevanceProstate cancer (PCa) is the most common malignancy of the male genitourinary system and currently lacks effective treatment. Semen Impatientis, the dried ripe seed of Impatiens balsamina L., is described by the Chinese Pharmacopoeia as a traditional Chinese medicine (TCM) and is used in clinical practice to treat tumors, abdominal masses, etc. In our previous study, the ethyl acetate extracts of Semen Impatientis (EAESI) was demonstrated to be the most effective extract against PCa among various extracts. However, the biological effects of EAESI against PCa in vivo and the specific antitumor mechanisms involved remain unknown. Aim of the studyIn this study, we aimed to investigate the antitumor effect of EAESI on PCa in vitro and in vivo by performing network pharmacology analysis, transcriptomic analysis, and experiments to explore and verify the underlying mechanisms involved. Materials and methodsThe antitumor effect of EAESI on PCa in vitro and in vivo was investigated via CCK-8, EdU, flow cytometry, and wound healing assays and xenograft tumor models. Network pharmacology analysis and transcriptomic analysis were employed to explore the underlying mechanism of EAESI against PCa. Activating transcription factor 3 (ATF3) and androgen receptor (AR) were confirmed to be the targets of EAESI against PCa by RT‒qPCR, western blotting, and rescue assays. In addition, the interaction between ATF3 and AR was assessed by coimmunoprecipitation, immunofluorescence, and nuclear–cytoplasmic separation assays. ResultsEAESI decreased cell viability, inhibited cell proliferation and migration, and induced apoptosis in AR+ and AR− PCa cells. Moreover, EAESI suppressed the growth of xenograft tumors in vivo. Network pharmacology analysis revealed that the hub targets of EAESI against PCa included AR, AKT1, TP53, and CCND1. Transcriptomic analysis indicated that activating transcription factor 3 (ATF3) was the most likely critical target of EAESI. EAESI downregulated AR expression and decreased the transcriptional activity of AR through ATF3 in AR+ PCa cells; and EAESI promoted the expression of ATF3 and exerted its antitumor effect via ATF3 in AR+ and AR− PCa cells. ConclusionsEAESI exerts good antitumor effects on PCa both in vitro and in vivo, and ATF3 and AR are the critical targets through which EAESI exerts antitumor effects on AR+ and AR− PCa cells.

Inhibition of HDAC6 with CAY10603 alleviates acute and chronic kidney injury by suppressing the ATF6 branch of UPR

BackgroundHistone deacetylase 6 (HDAC6) inhibitor CAY10603 has been identified as a potential therapeutic agent for the treatment of diabetic kidney disease (DKD). The objective of this study was to investigate the therapeutic effects of CAY10603 in mice with acute kidney injury (AKI) and chronic kidney diseases (CKD). MethodsRenal immunohistology was performed to assess the expression levels of HDAC6 in both human and mouse kidney samples. C57BL/6J mice were intraperitoneal injected with lipopolysaccharide (LPS) to induce AKI; CD-1 mice were fed with adenine diet to induce adenine-nephropathy as CKD model. Serum creatinine, blood urea nitrogen and uric acid were measured to reflect renal function; renal histology was applied to assess kidney damage. Western blot and immunohistology were used to analyze the unfolded protein response (UPR) level. ResultsHDAC6 was significantly upregulated in renal tubular epithelial cells (RTECs) of both AKI and CKD patients as well as mice. In the murine models of AKI induced by LPS and adenine-induced nephropathy, CAY10603 exhibited notable protective effects, including improvement in biochemical indices and pathological changes. In vivo and in vitro studies revealed that CAY10603 effectively suppressed the activation of activating transcription factor 6 (ATF6) branch of UPR triggered by thapsigargin (Tg), a commonly employed endoplasmic reticulum (ER) stressor. Consistent with these findings, CAY10603 also displayed substantial inhibition of ATF6 activation in RTECs from both murine models of LPS-induced AKI and adenine-induced nephropathy. ConclusionsCollectively, these results suggest that CAY10603 holds promise as a potential therapeutic agent for both acute and chronic kidney injury.

Celecoxib and sulindac sulfide elicit anticancer effects on PIK3CA-mutated head and neck cancer cells through endoplasmic reticulum stress, reactive oxygen species, and mitochondrial dysfunction

Gain-of-function mutation in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) is a significant factor in head and neck cancer (HNC). Patients with HNC harboring PIK3CA mutations receive therapeutic benefits from the use of non-steroidal anti-inflammatory drugs (NSAIDs). However, the molecular mechanisms underlying these effects remain unknown. Here, we examined the Detroit562 and FaDu cell lines as HNC models with and without a hyperactive PIK3CA mutation (H1047R), respectively, regarding their possible distinct responses to the NSAIDs celecoxib and sulindac sulfide (SUS). Detroit562 cells exhibited relatively high PI3K/Akt pathway-dependent cyclooxygenase-2 (COX-2) expression, associated with cell proliferation. Celecoxib treatment restricted cell proliferation and upregulated endoplasmic reticulum (ER) stress-related markers, including GRP78, C/EBP-homologous protein, activating transcription factor 4, death receptor 5, and reactive oxygen species (ROS). These effects were much stronger in Detroit562 cells than in FaDu cells and were largely COX-2-independent. SUS treatment yielded similar results. Salubrinal (an ER stress inhibitor) and N-acetyl-L-cysteine (a ROS scavenger) prevented NSAID-induced ROS generation and ER stress, respectively, indicating crosstalk between ER and oxidative stress. In addition, celecoxib and/or SUS elevated cleaved caspase-3 levels, Bcl-2-associated X protein/Bcl-2-interacting mediator of cell death expression, and mitochondrial damage, which was more pronounced in Detroit562 than in FaDu cells. Salubrinal and N-acetyl-L-cysteine attenuated celecoxib-induced mitochondrial dysfunction. Collectively, our results suggest that celecoxib and SUS efficiently suppress activating PIK3CA mutation-harboring HNC progression by inducing ER and oxidative stress and mitochondrial dysfunction, leading to apoptotic cell death, further supporting NSAID treatment as a useful strategy for oncogenic PIK3CA-mutated HNC therapy.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

NCI 159456 PERK Inhibitor as a Targeted Therapy for Lung Cancer: An In Vitro Study.

Non-small cell lung cancer (NSCLC) represents the most common histological type of lung cancer, characterized by a five-year survival rate of 15% and poor prognosis. Accumulating evidence indicates a prominent role of endoplasmic reticulum (ER) stress and the protein kinase RNA-like ER kinase (PERK)-dependent pathway of the unfolded protein response (UPR) in the pathogenesis of NSCLC. Increased expression of downstream targets of PERK was observed in various subtypes of NSCLC, and it was associated with a more aggressive phenotype, high risk of recurrence, and poor prognosis. Therefore, the present study aimed to investigate the biological effect of the selective PERK inhibitor NCI 159456 on A549 NSCLC cells and Human Pulmonary Fibroblasts (HPF) in vitro. Treatment of both normal and ER-stressed A549 cells with NCI 159456 resulted in a significant increase in the mRNA expression level of pro-apoptotic genes like activating transcription factor 4 (ATF4), DNA damage inducible transcript 3 (DDIT3), and BCL2 Associated X, Apoptosis Regulator (BAX) as well as a decreased level of the anti-apoptotic gene B-cell lymphoma 2 (Bcl-2). Cytotoxicity and genotoxicity analyses revealed that NCI 159456 significantly decreased viability and increased DNA damage in A549 cells under normal and ER stress conditions. Caspase-3 and reactive oxygen species (ROS) detection assays demonstrated that NCI 159456 significantly induced apoptosis and increased the ROS level in normal and ER-stressed A549 cells. Importantly, treatment with the inhibitor did not affect substantially normal HPF cells at any used concentration. The results indicate that PERK inhibitors could potentially be applied as a targeted therapy for NSCLC.

Engineered Exosomes with ATF5-Modified mRNA Loaded in Injectable Thermogels Alleviate Osteoarthritis by Targeting the Mitochondrial Unfolded Protein Response.

Osteoarthritis (OA) progression is highly associated with chondrocyte mitochondrial dysfunction and disorders of catabolism and anabolism of the extracellular matrix (ECM) in the articular cartilage. The mitochondrial unfolded protein response (UPRmt), which is an integral component of the mitochondrial quality control (MQC) system, is essential for maintaining chondrocyte homeostasis. We successfully validated the pivotal role of activating transcription factor 5 (ATF5) in upregulating the UPRmt, mitigating IL-1β-induced inflammation and mitochondrial dysfunction, and promoting balanced metabolism in articular cartilage ECM, proving its potential as a promising therapeutic target for OA. Modified mRNAs (modRNAs) have emerged as novel and efficient gene delivery vectors for nucleic acid therapeutic approaches. In this study, we combined Atf5-modRNA (modAtf5) with engineered exosomes derived from bone mesenchymal stem cells (ExmodAtf5) to exert cytoprotective effects on chondrocytes in articular cartilage via Atf5. However, the rapid localized metabolization of ExmodAtf5 limits its application. PLGA-PEG-PLGA (Gel), an injectable thermosensitive hydrogel, was used as a carrier of ExmodAtf5 (Gel@ExmodAtf5) to achieve a sustained release of ExmodAtf5. In vitro and in vivo, the use of Gel@ExmodAtf5 was shown to be a highly effective strategy for OA treatment. The in vivo therapeutic effect of Gel@ExmodAtf5 was evidenced by the preservation of the intact cartilage surface, low OARSI scores, fewer osteophytes, and mild subchondral bone sclerosis and cystic degeneration. Consequently, the combination of ExmodAtf5 and PLGA-PEG-PLGA could significantly enhance the therapeutic efficacy and prolong the exosome release. In addition, the mitochondrial protease ClpP enhanced chondrocyte autophagy by modulating the mTOR/Ulk1 pathway. As a result of our research, Gel@ExmodAtf5 can be considered to be effective at alleviating the progression of OA.

MiR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4.

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and β-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in β- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in β- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

Deoxynivalenol and T-2 toxin cause liver damage and egg quality degradation through endoplasmic reticulum stress in summer laying hens.

The present study aimed to find whether low doses of mixed mycotoxins would affect egg quality in laying hens, and to explore the oxidative stress induced liver damage through endoplasmic reticulum during summer stress. A total of 96 Jinghong laying hens, 36 wks of age, were divided into four treatments, with eight repetitions per treatment and three hens per repetition. All the hens were raised in summer (average temperature: 31.3 ± 0.5℃; average humidity: 85.5 ± 0.2%) for 28d. One treatment was fed a basal diet as control (CON), and the other three treatments were fed the same diets containing 3.0mg/kg deoxynivalenol (DON), 0.5mg/kg T-2 toxin (T-2), and 1.5mg/kg DON + 0.25mg/kg T-2 toxin (Mix). Albumen height and Haugh unit were decreased (P < 0.05) in the Mix group on day 14 and 28. The activity of total antioxidant capacity, glutathione peroxidase, catalase, and superoxide dismutase were decreased (P < 0.05) in the DON, T-2, and Mix groups. The alkaline phosphatase level in DON, T-2, and Mix groups was significantly increased (P < 0.05). The level of interleukin-1β, interferon-γ, and tumor necrosis factor-α in the Mix group were higher (P < 0.05) than CON, DON, and T-2 groups. Mix group upregulated the mRNA expressions of protein kinase RNA-like ER kinase, activating transcription factor4, IL-1β, nuclear factor-κ-gene binding, and nuclear respiratory factor 2 in the liver (P < 0.05). The results showed that low doses of DON and T-2 toxin could cause oxidative stress in the liver, but DON and T-2 toxin have a cumulative effect on virulence, which can reduce egg quality and cause endoplasmic reticulum stress in the liver.

Activating transcription factor 6 alleviates secondary brain injury by increasing cystathionine γ-lyase expression in a rat model of intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) comprises primary and secondary injuries, the latter of which induces increased inflammation and apoptosis and is more severe. Activating transcription factor 6 (ATF6) is a type-II transmembrane protein in the endoplasmic reticulum (ER). ATF6 target genes could improve ER homeostasis, which contributes to cryoprotection. Hence, we predict that ATF6 will have a protective effect on brain tissue after ICH. The ICH rat model was generated through autologous blood injection into the right basal ganglia, the expression of ATF6 after ICH was determined by WB and IF. The expression of ATF6 was effectively controlled by means of intervention, and a series of measures was used to detect cell death, neuroinflammation, brain edema, blood-brain barrier and other indicators after ICH. Finally, the effects on long-term neural function of rats were measured by behavioral means. ATF6 was significantly increased in the ICH-induced brain tissues. Further, ATF6 was found to modulate the expression of cystathionine γ-lyase (CTH) after ICH. Upregulation of ATF6 attenuated neuronal apoptosis and inflammation in ICH rats, along with mitigation of ICH-induced brain edema, blood-brain barrier deterioration, and cognitive behavior defects. Conversely, ATF6 genetic knockdown induced effects counter to those aforementioned. This study thereby emphasizes the crucial role of ATF6 in secondary brain injury in response to ICH, indicating that ATF6 upregulation may potentially ameliorate ICH-induced secondary brain injury. Consequently, ATF6 could serve as a promising therapeutic target to alleviate clinical ICH-induced secondary brain injuries.

Endoplasmic Reticulum Stress and Unfolded Protein Response in Vernal Keratoconjunctivitis.

Vernal keratoconjunctivitis (VKC) is an ocular allergic disease characterized by a type 2 inflammation, tissue remodeling, and low quality of life for the affected patients. We investigated the involvement of endoplasmic reticulum (ER) stress and unfolded protein response in VKC. Conjunctival imprints from VKC patients and normal subjects (CTs) were collected, and RNA was isolated, reverse transcribed, and analyzed with the Affymetrix microarray. Differentially expressed genes between VKC patients and CTs were evaluated. Genes related to ER stress, apoptosis, and autophagy were further considered. VKC and CT conjunctival biopsies were analyzed by immunohistochemistry (IHC) with specific antibodies against unfolded protein response (UPR), apoptosis, and inflammation. Conjunctival fibroblast and epithelial cell cultures were exposed to the conditioned medium of activated U937 monocytes and analyzed by quantitative PCR for the expression of UPR, apoptosis, autophagy, and inflammatory markers. ER chaperones HSPA5 (GRP78/BiP) and HYOU1 (GRP170) were upregulated in VKC patients compared to CTs. Genes encoding for ER transmembrane proteins, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), ER-associated degradation (ERAD), and autophagy were upregulated, but not those related to apoptosis. Increased positive reactivity of BiP and ATF6 and unchanged expression of apoptosis markers were confirmed by IHC. Cell cultures in stress conditions showed an overexpression of UPR, proinflammatory, apoptosis, and autophagy markers. A significant overexpression of genes encoding for ER stress, UPR, and pro-inflammatory pathway components was reported for VKC. Even though these pathways may lead to ER homeostasis, apoptosis, or inflammation, ER stress in VKC may predominantly contribute to promote inflammation.

Spinal bestrophin-1 and anoctamin-1 channels have a pronociceptive role in the tactile allodynia induced by REM sleep deprivation in rats

Bestrophin-1 and anoctamin-1 are members of the calcium-activated chloride channels (CaCCs) family and are involved in inflammatory and neuropathic pain. However, their role in pain hypersensitivity induced by REM sleep deprivation (REMSD) has not been studied. This study aimed to determine if anoctamin-1 and bestrophin-1 are involved in the pain hypersensitivity induced by REMSD. We used the multiple-platform method to induce REMSD. REM sleep deprivation for 48 h induced tactile allodynia and a transient increase in corticosterone concentration at the beginning of the protocol (12 h) in female and male rats. REMSD enhanced c-Fos and α2δ-1 protein expression but did not change activating transcription factor 3 (ATF3) and KCC2 expression in dorsal root ganglia and dorsal spinal cord. Intrathecal injection of CaCCinh-A01, a non-selective bestrophin-1 blocker, and T16Ainh-A01, a specific anoctamin-1 blocker, reverted REMSD-induced tactile allodynia. However, T16Ainh-A01 had a higher antiallodynic effect in male than female rats. In addition, REMSD increased bestrophin-1 protein expression in DRG but not in DSC in male and female rats. In marked contrast, REMSD decreased anoctamin-1 protein expression in DSC but not in DRG, only in female rats. Bestrophin-1 and anoctamin-1 promote pain and maintain tactile allodynia induced by REM sleep deprivation in both male and female rats, but their expression patterns differ between the sexes.

Babaodan overcomes cisplatin resistance in cholangiocarcinoma via inhibiting YAP1

Context Cholangiocarcinoma with highly heterogeneous, aggressive, and multidrug resistance has a poor prognosis. Although babaodan (BBD) combined with cisplatin improved non-small cell lung cancer efficacy, its impact on overcoming resistance in cholangiocarcinoma remains unexplored. Objective This study explored the role and mechanism of BBD on cisplatin resistance in cholangiocarcinoma cells (CCAs). Materials and methods Cisplatin-resistant CCAs were exposed to varying concentrations of cisplatin (25–400 μg/mL) or BBD (0.25–1.00 mg/mL) for 48 h. IC50 values, inhibition ratios, apoptosis levels, DNA damage, glutathione (GSH) levels, oxidized forms of GSH, total GSH content, and glutaminase relative activity were evaluated using the cell counting kit 8, flow cytometry, comet assay, and relevant assay kits. Results BBD-reduced the cisplatin IC50 in CCAs from 118.8 to 61.83 μg/mL, leading to increased inhibition rate, apoptosis, and DNA damage, and decreased expression of B-cell lymphoma-2, p-Yes-associated protein 1/Yes-associated protein 1, solute carrier family 1 member 5, activating transcription factor 4, and ERCC excision repair 1 in a dose-dependent manner with maximum reductions of 78.97%, 51.98%, 54.03%, 56.59%, and 63.22%, respectively; bcl2-associated X and gamma histone levels were increased by 0.43–115.77% and 22.15–53.39%. The impact of YAP1 knockdown on cisplatin-resistant CCAs resembled BBD. GSH, oxidized GSH species, total GSH content, and glutaminase activity in cisplatin-resistant CCAs with BBD treatment also decreased, while YAP1 overexpression countered BBD’s effects. Discussion and conclusion This study provides a scientific basis for BBD clinical application and provides a new direction for BBD biological mechanism research.

Identification of activating transcription factor 6 (ATF6) as a novel prognostic biomarker and potential target in oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is originating from oral mucosal epithelial cells. Autophagy plays a crucial role in cancer treatment by promoting cellular self-degradation and eliminating damaged components, thereby enhancing therapeutic efficacy. In this study, we aim to identify a novel autophagy-related biomarker to improve OSCC therapy. MethodsWe firstly utilized Cox and Lasso analyses to identify that ATF6 is associated with OSCC prognosis, and validated the results by Kaplan-Meier survival analysis. We further identified the downstream pathways and related genes by enrichment analysis and WGCNA analysis. Subsequently, we used short interfering RNA to investigate the effects of ATF6 knockdown on proliferation, migration, apoptosis, and autophagy in SCC-9 and SCC-15 cells through cell viability assay, transwell assay, EdU incorporation assay, flow cytometry analysis, western blot analysis and immunofluorescence analysis, etc. ResultsBioinformatics analyses showed that ATF6 overexpression was associated with prognosis and detrimental to survival. In vitro studies verified that ATF6 knockdown reduced OSCC cell proliferation and migration. Mechanistically, ATF6 knockdown could promote cellular autophagy and apoptosis. ConclusionWe propose that ATF6 holds potential as a prognostic biomarker linked to autophagy in OSCC. This study provides valuable clues for further exploration of targeted therapy against OSCC.

ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.