Phenylbutyrate Research Articles

GRP75 Modulates Endoplasmic Reticulum-Mitochondria Coupling and Accelerates Ca2+-Dependent Endothelial Cell Apoptosis in Diabetic Retinopathy.

Endoplasmic reticulum (ER) and mitochondrial dysfunction play fundamental roles in the pathogenesis of diabetic retinopathy (DR). However, the interrelationship between the ER and mitochondria are poorly understood in DR. Here, we established high glucose (HG) or advanced glycosylation end products (AGE)-induced human retinal vascular endothelial cell (RMEC) models in vitro, as well as a streptozotocin (STZ)-induced DR rat model in vivo. Our data demonstrated that there was increased ER-mitochondria coupling in the RMECs, which was accompanied by elevated mitochondrial calcium ions (Ca2+) and mitochondrial dysfunction under HG or AGE incubation. Mechanistically, ER-mitochondria coupling was increased through activation of the IP3R1-GRP75-VDAC1 axis, which transferred Ca2+ from the ER to the mitochondria. Elevated mitochondrial Ca2+ led to an increase in mitochondrial ROS and a decline in mitochondrial membrane potential. These events resulted in the elevation of mitochondrial permeability and induced the release of cytochrome c from the mitochondria into the cytoplasm, which further activated caspase-3 and promoted apoptosis. The above phenomenon was also observed in tunicamycin (TUN, ER stress inducer)-treated cells. Meanwhile, BAPTA-AM (calcium chelator) rescued mitochondrial dysfunction and apoptosis in DR, which further confirmed of our suspicions. In addition, 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, was shown to reverse retinal dysfunction in STZ-induced DR rats in vivo. Taken together, our findings demonstrated that DR fueled the formation of ER-mitochondria coupling via the IP3R1-GRP75-VDAC1 axis and accelerated Ca2+-dependent cell apoptosis. Our results demonstrated that inhibition of ER-mitochondrial coupling, including inhibition of GRP75 or Ca2+ overload, may be a potential therapeutic target in DR.

Ascorbic acid 6-palmitate modulates microglia M1/M2 polarization in lipopolysaccharide-stimulated BV-2 cells via PERK/elF2α mediated endoplasmic reticulum stress

BackgroundNeuroinflammation-mediated microglia polarization is a major process in various central nervous system (CNS) diseases. Endoplasmic reticulum (ER) stress contributes to the inflammatory signals as well as to microglia polarization in lipopolysaccharide (LPS) induced neuroinflammation. Ascorbic acid 6-palmitate (L-AP) has been broadly used as a dietary antioxidant in foods and demonstrated a strong inhibitory effect on 5-LOX; however, the specific anti-inflammation mechanisms remain unclear. In this study, we investigated the effects and possible mechanisms of L-AP on LPS-induced neuroinflammation in BV-2 cells.MethodsImmortalized murine microglia cell line BV-2 cells were employed to assess the effect of L-AP to modulate microglia M1/M2 polarization in vivo, and the molecular mechanism was evaluated by qRT-PCR and Western blotting analysis. Molecular docking was used to predict the binding activity of L-AP with protein kinase R-like ER kinase (PERK).ResultsL-AP at 62.5 µM significantly modulated LPS-induced microglia M1/M2 polarization (increases of interleukin (IL)-10 and arginase-1 (Arg-1) transcriptions) independent of cell growth. Besides, L-AP at 62.5 µM significantly down-regulated glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding homologous protein (CHOP) mRNA levels. Similar data were shown in the tunicamycin (TM) induced ER stress cells model. Moreover, the protective effect of L-AP on TM-induced microglia M1/M2 polarization was similar to that of 4-phenyl butyric acid (4-PBA), the ER stress inhibitor. Molecular docking results indicated L-AP might directly bind with PERK, with a binding affinity of -7.7 kcal/mol. A further study unveiled that L-AP notably inhibited LPS-induced PERK/ eukaryotic initiation factor 2α (elf2α) activation.ConclusionTogether, this study revealed that L-AP possessed its effect on the reconstruction of microglia M1/M2 polarization balance in LPS-stimulated BV-2 cells via modulating PERK/elF2α mediated ER stress.

Andrographolide and 4-Phenylbutyric Acid Administration Increase the Expression of Antimicrobial Peptides Beta-Defensin-1 and Cathelicidin and Reduce Mortality in Murine Sepsis.

Antibiotic resistance is a global threat and requires the search for new treatment strategies. Natural antimicrobial peptides (AMPs) have pronounced antibacterial, antiviral, antifungal, and antitumor activity. AMPs' clinical use is complicated by the high synthesis costs and rapid proteolytic degradation. The search for small molecules, inducers of endogenous AMP expression, could become a new approach. Here, we investigated for the first time the effect of seven small molecules (andrographolide, levofloxacin, azithromycin, montelukast, 4-phenylbutyric acid, rosuvastatin and valsartan) on AMP (beta-defensin-1, hBD-1 and cathelicidin, LL-37) serum levels in rats. In control groups, the level of hBD-1 was 295.0 (292.9-315.4) pg/mL, and for LL-37, it was 223.8 (213.3-233.6) pg/mL. Andrographolide (ANDR) and 4-phenylbutyric acid (4-PHBA) administration significantly enhanced the level of both AMPs. The hBD-1 level was 581.5 (476.3-607.7) pg/mL for ANDR and 436.9 (399.0-531.6) pg/mL for 4-PHBA. The LL-37 level was 415.4 (376.2-453.8) pg/mL for ANDR and 398.9 (355.7-410.1) pg/mL for 4-PHBA. Moreover, we have shown that these compounds reduce mortality in a murine model of sepsis caused by a carbapenem-resistant Klebsiella aerogenes isolate. From our point of view, these small molecules are promising candidates for further study as potent AMP inducers. The data obtained allow the development of new strategies to combat antibiotic resistance and infectious diseases.

4-phenylbutyric acid-Identity crisis; can it act as a translation inhibitor?

Loss of proteostasis can occur due to mutations, the formation of aggregates, or general deficiency in the correct translation and folding of proteins. These phenomena are commonly observed in pathologies, but most significantly, loss of proteostasis characterizes aging. This loss leads to the chronic activation of stress responses and has a generally deleterious impact on the organism. While finding molecules that can alleviate these symptoms is an important step toward solutions for these conditions, some molecules might be mischaracterized on the way. 4-phenylbutyric acid (4PBA) is known for its role as a chemical chaperone that helps alleviate endoplasmic reticulum (ER) stress, yet a scan of the literature reveals that no biochemical or molecular experiments have shown any protein refolding capacity. Here, we show that 4PBA is a conserved weak inhibitor of mRNA translation, both in vitro and in cellular systems, and furthermore-it does not promote protein folding nor prevents aggregation. 4PBA possibly alleviates proteostatic or ER stress by inhibiting protein synthesis, allowing the cells to cope with misfolded proteins by reducing the protein load. Better understanding of 4PBA biochemical mechanisms will improve its usage in basic science and as a drug in different pathologies, also opening new venues for the treatment of different diseases.

Inhibition of USP1 activates ER stress through Ubi-protein aggregation to induce autophagy and apoptosis in HCC

The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1) plays a role in the progression of various tumors, emerging as a potential therapeutic target. This study aimed to determine the role of USP1 as a therapeutic target in hepatocellular carcinoma (HCC). We detected USP1 expression in the tumor and adjacent tissues of patients with HCC using immunohistochemical staining. We evaluated the effect of the USP1 inhibitor ML-323 on HCC cell proliferation and cell cycle using a CCK-8 cell-counting kit and plate cloning assays, and propidium iodide, respectively. Apoptosis was detected by annexin V-FITC/Propidium Iodide (PI) staining and caspase 3 (casp3) activity. Transmission electron microscopy and LC3B immunofluorescence were used to detect autophagy. Western blotting was used to detect the accumulation of ubiquitinated proteins, the expression of endoplasmic reticulum (ER) stress-related proteins, and the AMPK-ULK1/ATG13 signaling pathway. We demonstrated that ML-323 inhibits the growth of HCC cells and induces G1 phase cell cycle arrest by regulating cyclin expression. ML-323 treatment resulted in the accumulation of ubiquitinated proteins, induced ER stress, and triggered Noxa-dependent apoptosis, which was regulated by the Activating Transcription Factor 4(ATF4). Moreover, active ER stress induces protective autophagy by increasing AMPK phosphorylation; therefore, we inhibited ER stress using 4-Phenylbutyric acid (4-PBA), which resulted in ER stress reduction, apoptosis, and autophagy in ML-323-treated HCC cells. In addition, blocking autophagy using the AMPK inhibitor compound C (CC), chloroquine (CQ), or bafilomycin A1 (BafA1) enhanced the cytotoxic effect of ML-323. Our findings revealed that targeting USP1 may be a potential strategy for the treatment of HCC.

Abstract 9482: Coronary Spasm in a Patient With Argininosuccinic Aciduria

Case presentation: A 39-year-old male patient was referred for the treatment of hypertension. The patient had been treated for argininosuccinic aciduria (ASA) since eight months of age. Therapeutic drugs including, L-arginine, sodium phenylbutyrate and antiepileptic drugs had been administered. A detailed medical history taking revealed that the patient sometimes complained of chest discomfort under psychological stress. A 12-lead electrocardiogram showed abnormal q waves in lead III and aVF. Transthoracic echocardiography showed hypokinesis of left ventricular posterior wall. Endothelial function was quantified with brachial artery flow-mediated dilatation (FMD). The patient’s FMD value was 2.4% (normal cut off value 7.0%). Coronary computed tomography angiography showed no significant coronary stenosis. Cardiac magnetic resonance revealed transmural late gadolinium enhancement in the mid-ventricular level of the left ventricular posterior wall. The patient was suspected to have prior myocardial infarction. The patient was started on amlodipine and irbesartan, however, he continued to complain of chest discomfort at rest. Cardiac catheterization was performed to make a definitive diagnosis. The acetylcholine spasm provocation test revealed diffuse left and right coronary vasospasm. Transient ST-segment elevation with chest discomfort was observed. The patient was diagnosed as having coronary spastic angina. Chest discomfort was disappeared after additional administration of isosorbide mononitrate. Discussion: ASA is a genetic disorder of the urea cycle, caused by deficiency of argininosuccinate lyase (ASL). Specific laboratory findings are hyperammonemia, argininosuccinic acidemia and low plasma arginine levels. ASL is required to utilize extracellular arginine for NOS-dependent NO synthesis in endothelial cell. Reduction of ASL enzymatic activity leads to decline in NO production even if arginine is supplemented. Efficacy of NO supplementation for treatment of long-term complications in ASA patients has been reported. The precise mechanisms of coronary spasm remain to be elucidated. Our case report might support the significance of endothelial function in the pathogenesis of coronary spasm.

Fluoride-Induced Cortical Toxicity in Rats: the Role of Excessive Endoplasmic Reticulum Stress and Its Mediated Defective Autophagy.

The cerebral cortex is closely associated with learning and memory, and fluoride is capable of inducing cortical toxicity, but its mechanism is unclear. This study aimed to investigate the role of endoplasmic reticulum stress and autophagy in fluoride-induced cortical toxicity. Rats exposed to sodium fluoride (NaF) were used as an in vivo model. The results showed that NaF exposure impaired the learning and memory capacities and increased urinary fluoride levels in rats. In addition, NaF exposure induced excessive endoplasmic reticulum stress and associated apoptosis, as evidenced by elevated IRE1α, GRP78, cleaved caspase-12, and cleaved caspase-3, as well as defective autophagy, as evidenced by increased expression of Beclin1, LC3-II, and p62 in cortical areas. Importantly, the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated endoplasmic reticulum stress as well as defective autophagy, thus confirming the critical role of endoplasmic reticulum stress and autophagy in fluoride-induced cortical toxicity. Taken together, these results suggest that excessive endoplasmic reticulum stress and its mediated defective autophagy lead to fluoride-induced cortical toxicity. This provides new insights into the mechanisms of fluoride-induced neurotoxicity and a new theoretical basis for the prevention and treatment of fluoride-induced neurotoxicity.

Hyperglycemia promotes myocardial dysfunction via the ERS-MAPK10 signaling pathway in db/db mice

AbstractRecent studies have demonstrated that hyperglycemia is a major risk factor for the development and exacerbation of cardiovascular disease (CVD). However, the molecular mechanisms involved in diabetic cardiomyopathy (DCM) have not been fully elucidated. In this study, we focused on the underlying mechanism of DCM. Leptin receptor-deficient db/db mice were used to model a type 2 diabetes mellitus (T2DM) model in our study. WT mice and db/db mice received 4-phenylbutyric acid (4-PBA) (25 mg/kg/day) and saline by intraperitoneal injection every other day for 4 weeks. WT and db/db mice were given tail vein injections of 100 μL of rAAV9-Sh-MAPK10 and rAAV9-Sh-GFP at the age of 6–8 weeks. Echocardiography was performed to measure cardiac function, histological examinations were used to evaluate ventricular hypertrophy and fibrosis. Quantitative RT–qPCR was used to assess the mRNA expression of Jun N-terminal kinase 3 (JNK3, MAPK10), atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and collagen I and III. Immunoblotting was performed to measure the levels of cardiac hypertrophy-related proteins, fibrosis-related proteins, endoplasmic reticulum stress (ERS)-related proteins and apoptosis-related proteins. TUNEL staining was performed to examine cardiomyocyte apoptosis. In contrast to 12-week-old db/db mice, 16-week-old db/db mice showed the most severe myocardial dysfunction. The DCM induced by hyperglycemia was largely alleviated by 4-PBA (25 mg/kg/day, intraperitoneal injection). Similarly, tail vein injection of rAAV9-Sh-MAPK10 reversed the phenotype of the heart in db/db mice including cardiac hypertrophy and apoptosis in db/db mice. The mechanistic findings suggested that hyperglycemia initiated the ERS response through the negative regulation of sirtuin 1 (SIRT1), leading to the occurrence of myocardial dysfunction, and specific knockdown of MAPK10 in the heart directly reversed myocardial dysfunction induced by hyperglycemia. We demonstrated that hyperglycemia promotes DCM in db/db mice through the ERS-MAPK10 signaling pathway in diabetic mice.

Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer’s disease mouse model: a role for ER-mitochondria interaction

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer’s disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

Depletion and Reversal of Hepatocellular Carcinoma Inducing CTL through ER Stress-Dependent PERK-CHOP Signaling Pathway.

Aims In this report, it was investigated that hepatoma cells can cause downregulation of cytotoxic T lymphocyte (CTL) function and tea polyphenols (TPs) can reverse downregulation of CTL function. Methods The expression of GRP78, PD-1, and TIM-3 was detected by western blotting in CTLL-2 cocultured with Hepa1-6 cells. Moreover, perforin (PRF1) and granzyme B (GzmB) protein levels and ER morphology were examined by ELISA and TEM, respectively. After 4-phenylbutyric acid (4-PBA) or tunicamycin (TM) treatment, programmed cell death protein 1 (PD-1), and mucin domain 3 (TIM-3), PRF1, and GzmB were measured by western blotting and ELISA. After sh-CHOP or GSK2656157 (PERK inhibitor) stimulation, the activation of the PERK-CHOP pathway was detected in CTLL-2 cells. Finally, changes in PD-1, TIM-3, PRF1, and GzmB levels were detected to verify the reversal of CTL depletion by TP. Results The expression of GRP78, PD-1, and TIM-3 clearly increased, and swelling was observed for the endoplasmic reticulum (ER) in CTLL-2 cells cocultured with hepatoma cells. Concurrently, the levels of PRF1 and GzmB decreased. CTLL-2 depletion was induced after stimulation with TM and differed from 4-PBA stimulation. Treatment with sh-CHOP or GSK2656157 caused a decrease in PD-1 and TIM-3 expression, whereas the expression of PRF1 and GzmB clearly increased. After adding TP, the function of CTLs increased markedly. Conclusion Hepatoma cells induced the depletion of CTLs through the ER stress PERK-CHOP pathway, and TP reversed this depletion by downregulating ER stress.

Naringenin Alleviates Renal Ischemia Reperfusion Injury by Suppressing ER Stress-Induced Pyroptosis and Apoptosis through Activating Nrf2/HO-1 Signaling Pathway.

Endoplasmic reticulum (ER) stress, pyroptosis, and apoptosis are critical molecular events in the occurrence and progress of renal ischemia reperfusion (I/R) injury. Naringenin (4′,5,7-trihydroxyflavanone) is one of the most widely consumed flavonoids with powerful antioxidant and anti-inflammatory activities. However, whether naringenin is able to relieve renal I/R injury and corresponding mechanisms have not been fully clarified. This study was aimed at exploring its role and relevant mechanisms in renal I/R injury. The C57Bl/6 mice were randomly assigned to receive administration with naringenin (50 mg/kg/d) or sterile saline (1.0 mL/d) for 3 d by gavage and suffered from renal I/R surgery. One specific ER stress inhibitor, 4-phenylbutyric acid (4-PBA, 100 mg/kg/d), was intraperitoneally administered to validate the regulation of ER stress on pyroptosis and apoptosis. Cultured HK-2 cells went through the process of hypoxia/reoxygenation (H/R) to perform cellular experiments with the incubation of naringenin (200 μM), 4-PBA (5 mM), or brusatol (400 nM). The animal results verified that naringenin obviously relieved renal I/R injury, while it refined renal function and attenuated tissue structural damage. Furthermore, naringenin treatment inhibited I/R-induced ER stress as well as pyroptosis and apoptosis as indicated by decreased levels of specific biomarkers such as GRP78, CHOP, caspase-12, NLRP3, ASC, caspase-11, caspase-4, caspase-1, IL-1β, GSDMD-N, BAX, and cleaved caspase-3 in animals and HK-2 cells. Besides, the upregulated expression of Nrf2 and HO-1 proteins after naringenin treatment suggested that naringenin activated the Nrf2/HO-1 signaling pathway, which was again authenticated by the usage of brusatol (Bru), one unique inhibitor of the Nrf2 pathway. Importantly, the application of 4-PBA showed that renal I/R-generated pyroptosis and apoptosis were able to be regulated by ER stress in vivo and in vitro. In conclusion, naringenin suppressed ER stress by activating Nrf2/HO-1 signaling pathway and further alleviated pyroptosis and apoptosis to protect renal against I/R injury.

HMGA2 mediates Cr (VI)-induced metabolic reprogramming through binding to mitochondrial D-Loop region.

Hexavalent chromium [Cr (VI)] exists environmentally and occupationally. It has been shown to pose a carcinogenic hazard in certain occupations. This study was to investigate the role of high mobility group A2 (HMGA2) in Cr (VI)-induced metabolism reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis in A549 and HELF cells. First, knockdown of HMGA2 by siHMGA2 significantly attenuated Cr (VI)-reduced expression of OXPHOS-related proteins (COX IV and ND1) and mitochondrial mass, indicating that HMGA2 was involved in Cr (VI)-reduced OXPHOS. Overexpression of HMGA2 by transfection of HMGA2-DNA plasmids reduced the expression of COX IV, ND1 and mitochondrial mass, suggesting the negative role of HMGA2 in OXPHOS. Secondly, both CCCP, the inhibitor of mitochondrial function, and the ER stress inhibitor, 4-phenylbutyric acid (4-PBA), decreased the level of HMGA2, indicating that the interaction of mitochondrial dysfunction and ER stress resulted in Cr (VI)-induced HMGA2 expression. Further study demonstrated that ER stress/HMGA2 axis mediated the metabolism rewiring from OXPHOS to aerobic glycolysis. Notably, Cr (VI) induced the accumulation of HMGA2 proteins in mitochondria and ChIP assay demonstrated that HMGA2 proteins could bind to D-loop region of mitochondrial DNA (mtDNA), which provided the proof for HMGA2-modulating OXPHOS. Taken together, our results suggested that the interaction of mitochondria and ER stress-enhanced HMGA2 played an important role in Cr (VI)-induced metabolic reprogramming from OXPHOS to glycolysis by binding directly to D-loop region of mtDNA. This work informs on the potential mode of action for Cr (VI)-induced tumors and builds on growing evidence regarding the contribution of cellular metabolic disruption contributing to carcinogenicity.

Protective effect of nano-selenium on mercury-induced prehierarchical follicular atresia in laying hens

This study investigated the effect of nano-selenium (nano-Se) in protecting laying hens from mercury (Hg)-induced prehierarchical follicular atresia. Furthermore, the endoplasmic reticulum stress (ERS) was explored to reveal the molecular mechanism. In vivo, 720 Hyline-Brown laying hens were treated with Hg and nano-Se alone or in combination. In vitro, the prehierarchical follicles were treated with Hg, nano-Se and 4-phenyl butyric acid (4-PBA) alone or in combination (Control, 25 μM Hg group, 10 μM nano-Se group, 20 μM nano-Se group, 25 μM Hg + 10 μM nano-Se group, 25 μM Hg + 20 μM nano-Se group, 25 μM Hg + 4-PBA group, and 25 μM Hg + 20 μM nano-Se + 4-PBA group). The GCs were treated with Hg and nano-Se alone or in combination (Control, 15 μM Hg group, 6 μM nano-Se group, 12 μM nano-Se group, 15 μM Hg + 6 μM nano-Se group, 15 μM Hg + 12 μM nano-Se group). The results revealed that dietary Hg significantly reduced laying performance (P < 0.05) and egg quality (P < 0.05), whereas nano-Se addition prevented these reductions (P < 0.05). Hg exposure significantly induced the accumulation of Hg in PHFs (P < 0.05), prehierarchical follicular atresia (P < 0.05) and apoptosis in PHFs, whereas nano-Se addition significantly prevented these effects (P < 0.05). The levels of sex hormones (P < 0.05) were significantly decreased after Hg exposure in vivo and in vitro, while nano-Se addition prevented the reductions. Furthermore, the RNA-Seq results showed that the key factors of the ERS presented differential expression, including C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) in GCs. Hg exposure significantly increased the key gene expression of endoplasmic reticulum stress in GCs, whereas nano-Se addition prevented the induction of expression of these genes. In addition, the protein levels of PERK, inositol requiring protein 1α (IRE1α) and ATF6 were significantly increased, whereas nano-Se addition prevented the enhancements of protein expression in GCs. In conclusion, this study shows that Hg exposure can reduce induce prehierarchical follicular atresia, whereas nano-Se can prevent these effects. Our results also elucidate a key role of ERS in these protective effects of nano-Se in laying hens.

Atractylenolide III Attenuates Apoptosis in H9c2 Cells by Inhibiting Endoplasmic Reticulum Stress through the GRP78/PERK/CHOP Signaling Pathway.

The objective of this study was to determine the effect of atractylenolide III (ATL-III) on endoplasmic reticulum stress (ERS) injury, H9c2 cardiomyocyte apoptosis induced by tunicamycin (TM), and the GRP78/PERK/CHOP signaling pathway. Molecular docking was applied to predict the binding affinity of ATL-III to the key proteins GRP78, PERK, IREα, and ATF6 in ERS. Then, in vitro experiments were used to verify the molecular docking results. ERS injury model of H9c2 cells was established by TM. Cell viability was detected by MTT assay, and apoptosis was detected by Hoechst/PI double staining and flow cytometry. Protein expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, and Caspase-3 were detected by Western blot. And mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4 were detected by RT-qPCR. Moreover, the mechanism was further studied by using GRP78 inhibitor (4-phenylbutyric acid, 4-PBA), and PERK inhibitor (GSK2656157). The results showed that ATL-III had a good binding affinity with GRP78, and the best binding affinity was with PERK. ATL-III increased the viability of H9c2 cells, decreased the apoptosis rate, downregulated Bax and Caspase-3, and increased Bcl-2 compared with the model group. Moreover, ATL-III downregulated the protein and mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4, consistent with the inhibition of 4-PBA. ATL-III also decreased the expression levels of PERK, eIF2α, ATF4, CHOP, Bax, and Caspase-3, while increasing the expression of Bcl-2, which is consistent with GSK2656157. Taken together, ATL-III could inhibit TM-induced ERS injury and H9c2 cardiomyocyte apoptosis by regulating the GRP78/PERK/CHOP signaling pathway and has myocardial protection.

Sclerostin aggravates insulin signaling in skeletal muscle and hepatic steatosis via upregulation of ER stress by mTOR-mediated inhibition of autophagy under hyperlipidemic conditions.

Recently, sclerostin (SCL), a circulating glycoprotein, was proposed to be a novel myokine involved in developing metabolic disorders. The association between SCL levels and insulin resistance in skeletal muscle, liver, and adipose tissue was studied in individuals with aggravated glucose tolerance. Thus, we hypothesized that elevated circulating SCL might affect skeletal muscle insulin signaling and hepatic lipid metabolism, and aimed to investigate the effects of SCL on skeletal muscle insulin resistance and hepatic steatosis in obesity using in vitro and in vivo experimental models under hyperlipidemic conditions. In the current study, we found elevated SCL messenger RNA expression levels in myocytes in obese patients. In addition to a higher blood level, SCL was expressed at an elevated level in the skeletal muscle of mice fed a high-fat diet (HFD). Higher SCL release levels and expression were also noticed in palmitate-treated C2C12 myocytes. SCL suppression by in vivo transfection improves skeletal muscle insulin resistance and hepatic steatosis in HFD-fed mice. The treatment of C2C12 myocytes with recombinant SCL aggravated insulin signaling. Furthermore, treatment with SCL augmented lipogenic lipid deposition in human primary hepatocytes. Treatment with SCL upregulated mammalian target of rapamycin (mTOR) phosphorylation and suppressed autophagy markers, thereby causing endoplasmic reticulum (ER) stress. 4-Phenylbutyric acid, a pharmacological ER stress inhibitor, abolished the effects of SCL on insulin signaling in C2C12 myocytes and lipid accumulation in primary hepatocytes. In conclusion, SCL promotes skeletal muscle insulin resistance and hepatic steatosis by upregulating ER stress via the mTOR/autophagy-mediated pathway. The present study suggests that antagonizing SCL might be a novel therapeutic strategy for simultaneously managing insulin resistance and hepatic steatosis in obesity.

Effects of PB-TURSO on the transcriptional and metabolic landscape of sporadic ALS fibroblasts.

ObjectiveALS is a rapidly progressive, fatal disorder caused by motor neuron degeneration, for which there is a great unmet therapeutic need. AMX0035, a combination of sodium phenylbutyrate (PB) and taurursodiol (TUDCA, TURSO), has shown promising results in early ALS clinical trials, but its mechanisms of action remain to be elucidated. Therefore, our goal was to obtain an unbiased landscape of the molecular effects of AMX0035 in ALS patient‐derived cells.MethodsWe investigated the transcriptomic and metabolomic profiles of primary skin fibroblasts from sporadic ALS patients and healthy controls (n = 12/group) treated with PB, TUDCA, or PB–TUDCA combination (Combo). Data were evaluated with multiple approaches including differential gene expression and metabolite abundance, Gene Ontology and metabolic pathway analysis, weighted gene co‐expression correlation analysis (WGCNA), and combined multiomics integrated analysis.ResultsCombo changed many more genes and metabolites than either PB or TUDCA individually. Most changes were unique to Combo and affected the expression of genes involved in nucleocytoplasmic transport, unfolded protein response, mitochondrial function, RNA metabolism, and innate immunity. WGCNA showed significant correlations between ALS gene expression modules and clinical parameters that were abolished by Combo treatment.InterpretationThis study is the first to explore the molecular effects of Combo in ALS patient‐derived cells. It shows that Combo has a greater and distinct impact compared with the individual compounds and provides clues to drug targets and mechanisms of action, which may underlie the benefits of this investigational drug combination.

Gambogenic acid induces apoptosis and autophagy through ROS-mediated endoplasmic reticulum stress via JNK pathway in prostate cancer cells.

Prostate cancer (PCa) is the most common malignant tumor in males, which frequently develops into castration-resistant prostate cancer (CRPC) with limited therapies. Gambogenic acid (GNA), a flavonoids compound isolated from Gamboge, exhibits anti-tumor capacity in various cancers. Our results showed that GNA revealed not only antiproliferative and pro-apoptotic activities but also the induction of autophagy in PCa cells. In addition, autophagy inhibitor chloroquine enhanced the pro-apoptosis effect of GNA. Moreover, the activation of JNK pathway and the induction of apoptosis and autophagy triggered by GNA were attenuated by JNK inhibitor SP600125. We also found that GNA significantly promoted reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress. Meanwhile, suppressing ER stress with 4-phenylbutyric acid (4-PBA) markedly blocked the activation of JNK pathway induced by GNA. Further research indicated that ROS scavenger N-acetyl-L-cysteine (NAC) effectively abrogated ER stress and JNK pathway activation induced by GNA. Furthermore, NAC and 4-PBA significantly reversed GNA-triggered apoptosis and autophagy. Finally, GNA remarkably suppressed prostate tumor growth with low toxicity in vivo. In conclusion, the present study revealed that GNA induced apoptosis and autophagy through ROS-mediated ER stress via JNK signaling pathway in PCa cells. Thus, GNA might be a promising therapeutic drug against PCa.

Determination of immobilized lipase stability depends on the substrate and activity determination condition: Stress inactivations and optimal temperature as biocatalysts stability indicators

Lipases A and B from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) and Candida rugosa have been immobilized on octyl, octyl-vinyl sulfone (blocked with ethylendiamine) and amino-glutaraldehyde. The biocatalysts exhibited different specificity versus triacetin and p-nitro phenyl butyrate. Optimal activities were determined using triacetin for all biocatalysts, and this ranged from 40 °C for CALA and TLL, to 60 °C for an amino-glutaraldehyde-CRL. The biocatalysts were inactivated at 70 and 75 °C, determining their residual activities at 25 °C or 55 °C. The inactivation courses were very different depending on the substrate; in most cases the biocatalysts maintained more activity during the thermal inactivation using triacetin (except using TLL). When determining the residual activities at 55 °C, the values increased in most cases, reaching high hyperactivation values using CALA (even 23 folds). That way, the “stability” of the different preparations was strongly influenced by the substrate and residual activity determination conditions, and did not agree in most cases with the optimal temperatures.

Amoebicidal activity of cationic carbosilane dendrons derived with 4-phenylbutyric acid against Acanthamoeba griffini and Acanthamoeba polyphaga trophozoites and cysts

Amoebae from the genus Acanthamoeba are important pathogens responsible for severe illnesses in humans such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. In the last few decades, AK diagnoses have steadily increased. Most patients suffering from AK were contact lens users and the infection was related to poor hygiene. However, therapy is not yet well established, and treatments may last for several months due to resistance. Moreover, these treatments have been described to generate cytotoxicity. Therefore, there is an urgent need to develop new therapeutic strategies against AK. In this study, the amoebicidal activity of different generation cationic carbosilane dendrons derived with 4-phenylbutyric acid was demonstrated against Acanthamoeba polyphaga and Acanthamoeba griffini trophozoites and cysts. In addition, the combination of chlorhexidine digluconate and the most effective dendron (ArCO2G2(SNMe3I)4) showed an in vitro effect against Acanthamoeba trophozoites and cysts, reducing the minimal trophozoite amoebicidal concentration as well as concentrations with cysticidal activity.