PBA Treatment Research Articles

4-phenylbutyric acid improves sepsis-induced cardiac dysfunction by modulating amino acid metabolism and lipid metabolism via Comt/Ptgs2/Ppara

IntroductionCardiac dysfunction after sepsis the most common and severe sepsis-related organ failure. The severity of cardiac damage in sepsis patients was positively associated to mortality. It is important to look for drugs targeting sepsis-induced cardiac damage. Our previous studies found that 4-phenylbutyric acid (PBA) was beneficial to septic shock by improving cardiovascular function and survival, while the specific mechanism is unclear.ObjectivesWe aimed to explore the specific mechanism and PBA for protecting cardiac function in sepsis.MethodsThe cecal ligation and puncture-induced septic shock models were used to observe the therapeutic effects of PBA on myocardial contractility and the serum levels of cardiac troponin-T. The mechanisms of PBA against sepsis were explored by metabolomics and network pharmacology.ResultsThe results showed that PBA alleviated the sepsis-induced cardiac damage. The metabolomics results showed that there were 28 metabolites involving in the therapeutic effects of PBA against sepsis. According to network pharmacology, 11 hub genes were found that were involved in lipid metabolism and amino acid transport following PBA treatment. The further integrated analysis focused on 7 key targets, including Comt, Slc6a4, Maoa, Ppara, Pparg, Ptgs2 and Trpv1, as well as their core metabolites and pathways. In an in vitro assay, PBA effectively inhibited sepsis-induced reductions in Comt, Ptgs2 and Ppara after sepsis.ConclusionsPBA protects sepsis-induced cardiac injury by targeting Comt/Ptgs2/Ppara, which regulates amino acid metabolism and lipid metabolism. The study reveals the complicated mechanisms of PBA against sepsis.

Chaperone interventional therapy restores proteostasis, improves cognition and increases ADAM10 in young and aged APP knockin mice

AbstractBackgroundTwo distinct phenotypes of Alzheimer’s disease are deficits in cognition and proteostasis, including chronic activation of the unfolded protein response (UPR) and aberrant Aβ production. The UPR is a signal transduction pathway activated in the endoplasmic reticulum (ER) at the onset of proteostatic stress. With age the UPR is less efficient and the expression of the key regulator, the chaperone BiP / Hspa5, also declines. It is unknown if restoring proteostasis by reducing chronic and aberrant UPR activation in AD can improve pathology and cognition. Here, we present data using an APP knock‐in mouse model of AD and several small molecule chaperone supplementation paradigms, including an early and late‐stage intervention.MethodTransgenic APP knock‐in mice were used as a model for AD. Two groups of mice and their wildtype littermates, (treatment starting early at 1‐2 mos old and late at 12 mos old) were administered the chemical chaperone 4‐phenyl butyrate (PBA) by weekly I.P. for 10 weeks. Controls were given I.P. injections of sterile saline. After 10 weeks of PBA or saline treatment, mice were subjected to the Spatial Object Recognition (SOR) cognitive tasks. A third group of APP knock‐in mice and wildtype littermates were given stereotaxic local hippocampal AAV‐BiP overexpression or AAV‐mCherry control injections. Following recovery and viral expression, mice were subjected to the SOR test. All mice were perfused, and tissue was collected for both immunohistochemistry and biochemical analyses.ResultPBA treatment improved performance in the SOR test both with early and late‐stage PBA interventions. AAV‐BiP overexpression recapitulated those results. Histological data indicates that chaperone treatment, both with PBA injections and AAV‐BiP overexpression, reduced hippocampal ER stress and was correlated with increased pCREB. Chronic PERK activation which leads to inhibition of protein translation was reduced, while XBP1s levels were increased with chaperone treatment. This was coupled with increased ADAM10, which is associated with non‐amyloidogenic cleavage of APPConclusionReducing proteostatic stress even at a late age improves cognition in a mouse model of Alzheimer’s disease. The implications of these results could have an impact on the development of therapies to inform the development of potential treatments for Alzheimer’s disease.

Physiological 4-phenylbutyrate promotes mitochondrial biogenesis and metabolism in C2C12 myotubes

Type 2 diabetes is characterized by elevated circulating blood metabolites such as glucose, insulin, and branched chain amino acids (BCAA), which often coincide with reduced mitochondrial function. 4-Phenylbutyrate (PBA), an ammonia scavenger, has been shown to activate BCAA metabolism, resolve endoplasmic reticulum (ER) stress, and rescue BCAA-mediated insulin resistance. To determine the effect of PBA on the altered metabolic phenotype featured in type 2 diabetes, the present study investigated the effect of PBA on various metabolic parameters including mitochondrial metabolism and mitochondrial biogenesis. C2C12 myotubes were treated with PBA at 0.5 mM (representing physiologically attainable blood concentrations) or 10 mM (representing physiologically unattainable/proof-of-concept levels) for up to 24 h. Mitochondrial and glycolytic metabolism were assessed via oxygen consumption and extracellular acidification rate, respectively. Mitochondrial content, lipid content, and ER stress were measured by fluorescent staining. Metabolic gene expression was measured by qRT-PCR. Both doses of PBA increased expression of indicators of mitochondrial biogenesis, though only PBA at 0.5 mM increased mitochondrial function and content while 10 mM PBA reduced mitochondrial function and content. PBA at 0.5 mM also rescued reduced mitochondrial function during insulin resistance, though PBA also caused a reduced insulin stimulated pAkt expression during insulin resistance. PBA treatment also increased extracellular BCAA accumulation during insulin resistance despite unchanged pBCKDH expression. Taken together, PBA may increase mitochondrial biogenesis, content, and function in a dose-dependent fashion which may have implications for prevention or treatment of metabolic disease such as insulin resistance.

Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas

BackgroundTargeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density.ResultsDecitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients.ConclusionThe combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.

Effects of Epigenetic Modification of PGC-1α by a Chemical Chaperon on Mitochondria Biogenesis and Visual Function in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by gradual photoreceptor death, which lacks a definitive treatment. Here, we demonstrated the effect of 4-phenylbutyric acid (PBA), a chemical chaperon that can suppress endoplasmic reticulum (ER) stress, in P23H mutant rhodopsin knock-in RP models. In the RP models, constant PBA treatment led to the retention of a greater number of photoreceptors, preserving the inner segment (IS), a mitochondrial- and ER-rich part of the photoreceptors. Electroretinography showed that PBA treatment preserved photoreceptor function. At the early point, ER-associated degradation markers, xbp1s, vcp, and derl1, mitochondrial kinetic-related markers, fis1, lc3, and mfn1 and mfn2, as well as key mitochondrial regulators, pgc-1α and tfam, were upregulated in the retina of the models treated with PBA. In vitro analyses showed that PBA upregulated pgc-1α and tfam transcription, leading to an increase in the mitochondrial membrane potential, cytochrome c oxidase activity, and ATP levels. Histone acetylation of the PGC-1α promoter was increased by PBA, indicating that PBA affected the mitochondrial condition through epigenetic changes. Our findings constituted proof of concept for the treatment of ER stress-related RP using PBA and revealed PBA’s neuroprotective effects, paving the way for its future clinical application.

Hyperglycemia-triggered ATF6-CHOP pathway aggravates acute inflammatory liver injury by \u03b2-catenin signaling

Although hyperglycemia has been documented as an unfavorable element that can further induce liver ischemia–reperfusion injury (IRI), the related molecular mechanisms remain to be clearly elaborated. This study investigated the effective manner of endoplasmic reticulum (ER) stress signaling in hyperglycemia-exacerbated liver IRI. Here we demonstrated that in the liver tissues and Kupffer cells (KCs) of DM patients and STZ-induced hyperglycemic mice, the ER stress-ATF6-CHOP signaling pathway is activated. TLR4-mediated pro-inflammatory activation was greatly attenuated by the addition of 4-phenylbutyrate (PBA), one common ER stress inhibitor. The liver IRI in hyperglycemic mice was also significantly reduced after PBA treatment. In addition, deficiency of CHOP (CHOP−/−) obviously alleviates the hepatic IRI, and pro-inflammatory effects deteriorated by hyperglycemia. In hyperglycemic mice, β-catenin expression was suppressed while the ATF6-CHOP signal was activated. In the liver tissues of PBA-treated or CHOP−/− hyperglycemic mice, the expression of β-catenin was restored. Furthermore, CHOP deficiency can induce protection against hyperglycemia-related liver IRI, which was disrupted by the knockdown of β-catenin will cause this protection to disappear. High glucose (HG) treatment stimulated ATF6-CHOP signaling, reduced cellular β-catenin accumulation, and promoted the TLR4-related inflammation of BMDMs. But the above effects were partially rescued in BMDMs with CHOP deficiency or by PBA treatment. In BMDMs cultured in HG conditions, the anti-inflammatory functions of CHOP−/− were destroyed by the knockdown of β-catenin. Finally, chimeric mice carrying WT or CHOP−/− BMDMs by bone marrow transplantation were adopted to verify the above conclusion. The current study suggested that hyperglycemia could trigger ER stress-ATF6-CHOP axis, inhibit β-catenin activation, accelerate inflammation, and deteriorate liver IRI, thus providing the treatment potential for management of sterile liver inflammation in DM patients.

4-Phenylbutyrate (PBA) treatment reduces hyperglycemia and islet amyloid in a mouse model of type 2 diabetes and obesity

Amyloid deposits in pancreatic islets, mainly formed by human islet amyloid polypeptide (hIAPP) aggregation, have been associated with loss of β-cell mass and function, and are a pathological hallmark of type 2 diabetes (T2D). Treatment with chaperones has been associated with a decrease in endoplasmic reticulum stress leading to improved glucose metabolism. The aim of this work was to investigate whether the chemical chaperone 4-phenylbutyrate (PBA) prevents glucose metabolism abnormalities and amyloid deposition in obese agouti viable yellow (Avy) mice that overexpress hIAPP in β cells (Avy hIAPP mice), which exhibit overt diabetes. Oral PBA treatment started at 8 weeks of age, when Avy hIAPP mice already presented fasting hyperglycemia, glucose intolerance, and impaired insulin secretion. PBA treatment strongly reduced the severe hyperglycemia observed in obese Avy hIAPP mice in fasting and fed conditions throughout the study. This effect was paralleled by a decrease in hyperinsulinemia. Importantly, PBA treatment reduced the prevalence and the severity of islet amyloid deposition in Avy hIAPP mice. Collectively, these results show that PBA treatment elicits a marked reduction of hyperglycemia and reduces amyloid deposits in obese and diabetic mice, highlighting the potential of chaperones for T2D treatment.

Effect of dietary supplementation with a blend of protected aromatic compounds, including benzoic acid, on growth performance and faecal microbial profile of weaned piglets as an alternative to Zinc Oxide

Organic acids (OA), especially benzoic acid, have long been used for their wide antimicrobial activity and have shown to promote intestinal health and increase average daily gain (ADG), feed intake (FI) and feed conversion ratio (FCR) of pigs. On the other side, the European Commission decided to ban the use of pharmacological levels of zinc oxide (ZnO) in diets for this animal species from 2022. The aim of this study was to investigate the effect of a blend of protected aromatic compounds including benzoic acid (PBA) as a substitute of supra-nutritional levels of ZnO in piglet diets on growth performance and on gut microbiota profile. The trial was conducted in 4 farms. For each farm, piglets were allocated to one of the two experimental groups: i) piglets receiving a diet with 2.5 g/kg of ZnO; ii) piglets receiving the same diet but with 2.5 g/kg of PBA. Piglets were blocked to each diet based on body weight and sex. 1720 piglets were allocated to the ZnO treatment and 1728 to the PBA. The animals received the treatment for a total of 6 weeks (4 weeks during suckling and 2 weeks post-weaning). Cumulative performance from weaning to 6 weeks post-weaning were calculated. Faecal pool samples were collected from 8 box replicates per group from each farm to characterize the microbial profile. Piglets receiving PBA showed higher ADG (P <0.01) and lower FCR (P <0.05) in all the farms compared to ZnO, no differences in feed intake (FI)were seen. A reduction of mortality and diarrhoea incidence was observed in pigs from the PBA treatment compared to those in the ZnO (P <0.01). The faecal microbiota was characterized by a higher alpha diversity in the PBA treatment (Shannon: 4.52 vs 4.1, P <0.01; invSimpson: 36.1 vs 24.5 P <0.01). The microbial composition was significantly influenced by treatment (P <0.01, R2= 0.03). Animals receiving PBA were characterized by a higher abundance of Ruminococcus (FDR <0.01), Prevotellaceae (FDR <0.01) and Fibrobacteraceae (FDR <0.05), known for their fiber fermenting abilities. In conclusion, the supplementation with PBA positively influenced the microbial profile of weaned piglets by increasing the alpha diversity, and by favouring the proliferation of known beneficial bacteria, related to the metabolism of polysaccharides and production of SCFA. These findings on the faecal microbial profile can contribute to explain the higher ADG and lower FCR observed in piglets supplemented with PBA.

Hepatocyte Endoplasmic Reticulum Stress Inhibits Hepatitis B Virus Secretion and Delays Intracellular Hepatitis B Virus Clearance After Entecavir Treatment.

Background: The aim of this study was to explore the effects of endoplasmic reticulum (ER) stress on hepatitis B virus (HBV) replication and the antiviral effect of entecavir (ETV).Methods: Thapsigargin (TG) and stearic acid (SA) were used to induce ER stress in HepG2.2.15 cells and HepAD38 cells that contained an integrated HBV genome, while ETV was used to inhibit HBV replication. The expression levels of glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α) were measured by western blotting. Intracellular HBV DNA was determined by qPCR; HBsAg by western blotting; HBV RNA by real-time RT-qPCR; HBsAg and HBeAg in supernatants by enzyme-linked immunosorbent assay (ELISA); and HBV DNA in supernatants by qPCR.Results: TG and SA induced ER stress in HepG2.2.15 cells and HepAD38 cells from 12 to 48 h post treatment. However, 4-phenylbutyric acid (PBA) partly alleviated the TG-induced ER stress. Moreover, TG inhibited HBsAg, HBeAg, and HBV DNA secretion from 12 to 48 h, while different concentrations of SA inhibited HBsAg and HBV DNA secretion at 48 h. TG promoted intracellular HBV DNA and HBsAg accumulation and the transcription of the HBV 3.5-kb mRNA and S mRNA. PBA treatment restored the secretion of HBsAg and HBV DNA. Finally, ER stress accelerated extracellular HBV DNA clearance but delayed intracellular HBV DNA clearance after ETV treatment.Conclusions: Hepatocyte ER stress promoted intracellular HBV DNA and HBsAg accumulation by inhibiting their secretion. Our study also suggested that hepatocyte ER stress delayed intracellular HBV DNA clearance after ETV treatment.

Chronic treatment of 4-phenylbutyric acid ameliorates cognitive impairment after focal cerebral ischemia/ reperfusion injury in rats

Objectives: Stroke, apart from causing physical disabilities, it also often leads to cognitive impairment in patients. At present, there is no effective drug available for the treatment of post-stroke cognitive impairment. The present study was undertaken to evaluate the ameliorative effect of 4-Phenylbutyric acid (PBA) against cognitive and memory deficits due to focal cerebral ischemia/reperfusion (I/R) in rats. Materials and Methods: Focal cerebral I/R injury was achieved by the middle cerebral artery occlusion (MCAO) method. PBA (100 and 300 mg/kg, i.p.) was administered once daily for 2 weeks. The neurological score was counted to evaluate the severity of neurological motor deficits. The cognitive functions, including learning and memory, were assessed using various paradigms such as Y-maze, passive avoidance task and Morris water maze. Results: The chronic treatment of PBA (100 and 300 mg/kg, i.p.) dose-dependently improved the neurological motor deficits as shown by significant decrease in neurological score in MCAO-treated rats. Besides, PBA (100 and 300 mg/kg, i.p.) treatment markedly improved working memory as shown by significant increase in the relative percentage alternations compared to untreated control MCAO rats in Y-maze. PBA also significantly decreased the transfer latency in the acquisition trial and increased in probe trial in passive avoidance task suggesting an improvement in learning and memory in MCAO rats. There was also a significant improvement in spatial learning and memory, as evidenced by the reduced escape latency in acquisition trial and the increased number of entries into the platform zone, time spent in the platform quadrant and track plot in probe trial PBA-treated MCAO rats during Morris water maze task. Conclusion: This study, thus, demonstrates the potential of PBA in ameliorating cognitive dysfunctions in focal cerebral ischemia. Since PBA is already available for the treatment of urea cycle disorders, it may also be investigated for repurposing its use in the treatment of post-stroke cognitive impairment.

4-PHENYLBUTYRATE: MOLECULAR MECHANISMS AND AGING INTERVENTION POTENTIAL

4-Phenylbutyrate (PBA) is a FDA approved drug for treating patients with urea cycle disorders. Additionally, PBA acts upon several pathways thought of as important modifiers of aging including: histone deacetylation, proteostasis as a chemical chaperone, and stress resistance by regulating expression of oxidative stress response proteins. PBA has also been shown to extend lifespan and improve markers of age-related health in Drosophila. Due to its wide range of effects PBA has been investigated for use in numerous age-related disorders including neurodegenerative and cardiovascular diseases. To better understand the effects of PBA on the molecular level, we used both in cellulo and in vivo studies. Treatment of primary mouse fibroblasts, C2C12 mouse muscle cells, and NCTC 1469 mouse liver cells with PBA demonstrated differential responses among cell lines to upregulation of oxidative stress response and histone acetylation. Specifically, upregulation of the oxidative stress response protein DJ-1 by PBA was found to have a corresponding dose response curve to histone H3 acetylation in primary fibroblasts. To study effects of PBA in vivo, four cohorts of HET3 mice were treated with PBA at different doses in drinking water for 4 weeks. PBA was well tolerated and led to different effects on body composition dependent on the sex of mice. We are currently investigating the molecular effects of PBA treatment in multiple tissues samples from these mice. The potential of PBA to alter many fundamental pathways, and specifically those related to stress responses, make it an attractive prospect for treatment of many age-related disorders.

4-PBA prevents diabetic muscle atrophy in rats by modulating ER stress response and ubiquitin-proteasome system

PurposeSkeletal muscle is severely affected in diabetes leading to muscle atrophy. Previously we reported the role of ER stress in muscle atrophy due to hyperglycemia. Hence, in the present study, we investigated the effect of a classical ER stress inhibitor, 4-phenylbutyric acid (PBA), on muscle atrophy in diabetic rats. MethodsDiabetes was induced in male rats by streptozotocin, and PBA was administered (40 mg/kg/day; intraperitoneal) after two months of diabetes for two more months. Gastrocnemius muscle is collected after four months of experimental period. The cross-sectional area of myocytes was measured on Hematoxylin and Eosin stained muscle sections. Protein levels of ER stress markers, ubiquitin-proteasome system (UPS) components, and apoptosis were analysed by immunoblot. Proteasomal activity and apoptotic cells were measured. ResultsER stress markers (GRP78, ATF6, ATF4 and CHOP) that are elevated in diabetes are decreased with PBA treatment. PBA also averted diabetes-induced alterations in UPS (higher levels of E1, atrogin-1, UCHL1 and UCHL5, accumulation of ubiquitinated proteins and increased proteasomal activity). Apoptosis mediators-p53, BAX, and cleaved caspase-3 protein levels, and TUNEL positive cells were decreased in PBA treated diabetic rats. PBA notably improved the muscle-cross sectional area. ConclusionsResults highlighted the therapeutic potential of PBA in diabetes muscle wastage.

The Chemical Chaperone 4-Phenylbutyric Acid Prevents Alcohol-Induced Liver Injury in Obese KK-Ay Mice.

Co-occurrence of metabolic syndrome and chronic alcohol consumption is increasing worldwide. The present study investigated the effect of the chemical chaperone 4-phenylbutyric acid (PBA)-which has been shown to alleviate dietary steatohepatitis caused by endoplasmic reticulum (ER) stress-on chronic-plus-binge ethanol (EtOH)-induced liver injury in a mouse model of obesity. Male KK-Ay mice (8weeks old) were fed a Lieber-DeCarli diet (5% EtOH) for 10days. Some mice were given PBA intraperitoneally (120mg/kg body weight, daily) during the experimental period. On day 11, mice were gavaged with a single dose of EtOH (4g/kg body weight). Control mice were given a dextrin gavage after being pair-fed a control diet. All mice were then serially euthanized before or at 9hours after gavage. Chronic-plus-binge EtOH intake induced massive hepatic steatosis along with hepatocyte apoptosis and inflammation, which was reversed by PBA treatment. Administration of PBA also suppressed chronic-plus-binge EtOH-induced up-regulation of ER stress-related genes including binding immunoglobulin protein (Bip), unspliced and spliced forms of X-box-binding protein-1 (uXBP1 and sXBP1, respectively), inositol trisphosphate receptor (IP3R), and C/EBP homologous protein (CHOP). Further, it blocked chronic-plus-binge EtOH-induced expression of the oxidative stress marker heme oxygenase-1 (HO-1) and 4-hydroxynonenal. Chronic EtOH alone (without binge) increased Bip and uXBP1, but it did not affect those of sXBP1, IP3R, CHOP, or HO-1. PBA reversed the prebinge expression of these genes to control levels, but it did not affect chronic EtOH-induced hepatic activity of cytochrome P450 2E1. Binge EtOH intake after chronic consumption induces massive ER stress-related oxidative stress and liver injury in a mouse model of obesity through dysregulation of the unfolded protein response. PBA ameliorated chronic-plus-binge EtOH-induced liver injury by reducing ER and oxidative stress after an EtOH binge.

Sodium 4-phenylbutyrate ameliorates particles-induced inflammatory osteolysis by promoting macrophage apoptosis and inhibiting inflammatory and osteoclastogenic reactions

Macrophages represent both the ‘initiators’ and ‘amplifiers’ of the pathogenetic cascade in inflammatory osteolysis. In this study, we investigated the therapeutic potential of ER-stress protector (PBA) on the inflammatory, osteoclastogenic and apoptotic reactions in cultured macrophages and osteolytic periosteum in particles-induced osteolysis (PIO) animal models. The results demonstrated that apoptosis-related markers, inflammation, and osteoclastogenesis were significantly up-regulated in particles-treated macrophages and osteolytic periosteum tissues. ER-stress protector down-regulated ER chaperone (IRE, Bip, CHOP), and ameliorated particles-induced osteolysis by promoting macrophage apoptosis and inhibiting the inflammatory reactions and osteoclastogenesis. During the apoptotic process, apoptotic regulators, JNK, the mitochondrial and death receptor pathways were all involved in PBA–induced macrophages apoptosis, while the ER stress pathway was not. More importantly, PBA treatment alone was of relative low toxicity and experimental tested safeness in PIO mice. Since the ER chaperone BiP migrates out of the ER into the nucleus (intracellular protein) and to the cell surface (extracellular protein). Thus, BiP conforms to the definition of a true moonlighting protein with two quite distinct roles. Besides the function that focused on preserving or restoring ER function, the BiP has also been implicated in exerting functions that are of immunologic importance, and the discovery of cell surface BiP makes it an innovative concept in biological therapy for inflammatory diseases. Therefore, future studies will need to address the many open questions about the physiological significance of the ER and Bip to improve the overall understanding of how these diseases develop and indicate how they might be treated with pharmacological interventions that modulate ER or Bip on inflammatory diseases.

Orally administered sodium 4-phenylbutyrate suppresses the development of dextran sulfate sodium-induced colitis in mice.

Sodium 4-phenylbutyrate (PBA) exerts therapeutic effects in a wide range of pathologies. A previous study by the present authors revealed that intraperitoneal administration of PBA suppresses the onset of dextran sulfate sodium (DSS)-induced colitis in mice. In the present study, the effects of orally administered PBA are investigated, as this route of administration is more clinically relevant. The therapeutic efficacy of PBA (10 mg/12 h) in mice with experimental colitis was assessed based on the disease activity index, production of inflammatory cytokines, colon length and histopathological investigations. The results of the present study demonstrated a significantly higher survival rate in the PBA-treated group compared with the PBA-untreated (DSS control) group (P=0.0156). PBA treatment improved pathological indices of experimental colitis (P<0.05). Furthermore, the oral administration of PBA significantly inhibited the DSS-induced shortening of the colon (P<0.05) and overproduction of interleukin (IL)-1β and IL-6 (both P<0.05) as measured in colonic lavage fluids. A marked attenuation of the DSS-induced overproduction of tumor necrosis factor was also observed. For histopathological analysis, a marked decrease in mature goblet cells and increase in enlarged nuclei of the absorptive cells was observed in colon lesions of DSS control mice as compared with normal untreated mice. However, in the PBA-treated mice, no such lesions were observed and the mucosa resembled that of DSS-untreated mice. The results of the present study, combined with those results of a previous study, suggest that oral and intraperitoneal administration of PBA have similar preventative effects on DSS-induced colitis, achieved by suppressing its pathogenesis.

Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.

Human islet amyloid polypeptide (hIAPP) aggregation is associated with β-cell dysfunction and death in type 2 diabetes (T2D). we aimed to determine whether in vivo treatment with chemical chaperone 4-phenylbutyrate (PBA) ameliorates hIAPP-induced β-cell dysfunction and islet amyloid formation. Oral administration of PBA in hIAPP transgenic (hIAPP Tg) mice expressing hIAPP in pancreatic β cells counteracted impaired glucose homeostasis and restored glucose-stimulated insulin secretion. Moreover, PBA treatment almost completely prevented the transcriptomic alterations observed in hIAPP Tg islets, including the induction of genes related to inflammation. PBA also increased β-cell viability and improved insulin secretion in hIAPP Tg islets cultured under glucolipotoxic conditions. Strikingly, PBA not only prevented but even reversed islet amyloid deposition, pointing to a direct effect of PBA on hIAPP. This was supported by in silico calculations uncovering potential binding sites of PBA to monomeric, dimeric, and pentameric fibrillar structures, and by in vitro assays showing inhibition of hIAPP fibril formation by PBA. Collectively, these results uncover a novel beneficial effect of PBA on glucose homeostasis by restoring β-cell function and preventing amyloid formation in mice expressing hIAPP in β cells, highlighting the therapeutic potential of PBA for the treatment of T2D.-Montane, J., de Pablo, S., Castaño, C., Rodríguez-Comas, J., Cadavez, L., Obach, M., Visa, M., Alcarraz-Vizán, G., Sanchez-Martinez, M., Nonell-Canals, A., Parrizas, M., Servitja, J.-M., Novials, A. Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.

4PBA strongly attenuates endoplasmic reticulum stress, fibrosis, and mitochondrial apoptosis markers in cyclosporine treated human gingival fibroblasts

Cyclosporine induces overgrowth of human gingiva. Previously we have shown (i) cyclosporine-inducing ER stress in human gingival fibroblasts (HGF), (ii) increased matrix protein expression, and (iii) interference with mitochondrial pro- and anti-apoptotic factors. This study was undertaken to assess the effects of melatonin (an antioxidant), 4PBA (an ER stress inhibitor), and simvastatin on the expression of ER Stress markers as well as on matrix and mitochondrial markers. HGF incubated with cyclosporine, or without melatonin/4PBA/statin. After 24 hr of incubation, mRNA expression of ER stress markers (GRP78, CHOP, XBP1, and XBPs) and matrix protein markers (like α-SMA, VEGF, TGF-β, CTGF), and mitochondrial apoptosis markers estimated and compared with housekeeping gene GAPDH. Compared to the control cyclosporine significantly augmented ER Stress and matrix proteins, which decreased significantly with the use of melatonin, 4PBA, and simvastatin. The mitochondrial proapoptotic molecule cyclophilin D, as well as Bcl2 expression also decreased after PBA treatment, paralleling an increase in cytochrome c expression. The effect of 4PBA was much more pronounced than the influence of other two. In conclusion, 4PBA could be a viable therapeutic option for drug-induced gingival overgrowth.

Chemical Chaperone of Endoplasmic Reticulum Stress Inhibits Epithelial-Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium via the c-Src Pathway.

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF-β1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF-β1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-β1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α-SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF-β1 on migration of A549 cells and suppressed TGF-β1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-β1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF-β1 in upper airway chronic inflammatory disease such as CRS.

Sodium 4-phenylbutyric acid prevents murine acetaminophen hepatotoxicity by minimizing endoplasmic reticulum stress.

Acetaminophen (APAP) overdose induces severe oxidative stress followed by hepatocyte apoptosis/necrosis. Previous studies have indicated that endoplasmic reticulum (ER) stress is involved in the cell death process. Therefore, we investigated the effect of the chemical chaperone 4-phenyl butyric acid (PBA) on APAP-induced liver injury in mice. Eight-week-old male C57Bl6/J mice were given a single intraperitoneal (i.p.) injection of APAP (450mg/kg body weight), following which some were repeatedly injected with PBA (120mg/kg body weight, i.p.) every 3h starting at 0.5h after the APAP challenge. All mice were then serially euthanized up to 12h later. PBA treatment dramatically ameliorated the massive hepatocyte apoptosis/necrosis that was observed 6h after APAP administration. PBA also significantly prevented the APAP-induced increases in cleaved activating transcription factor 6 and phosphorylation of c-Jun N-terminal protein kinase and significantly blunted the increases in mRNA levels for binding immunoglobulin protein, spliced X-box binding protein-1, and C/EBP homologous protein. Moreover, PBA significantly prevented APAP-induced Bax translocation to the mitochondria, and the expression of heme oxygenase-1 mRNA and 4-hydroxynonenal. By contrast, PBA did not affect hepatic glutathione depletion following APAP administration, reflecting APAP metabolism. PBA prevents APAP-induced liver injury even when an APAP challenge precedes its administration. The underlying mechanism of action most likely involves the prevention of ER stress-induced apoptosis/necrosis in the hepatocytes during APAP intoxication.

Effects of 4-phenylbutyric acid on chronic cerebral hypoperfusion-induced cognitive dysfunction

Objective To explore the effects of 4-phenylbutyric acid(PBA) on cognitive dysfunction after chronic cerebral hypoperfusion and underlying mechanisms. Methods 62 male Sprague Dawley rats in clean degree were divided into sham group(n=14), chronic cerebral ischemia group(bilateral carotid arteries occlusion, 2VO group, n=24), and chronic cerebral ischemia with PBA treatment(2VO+ PBA group, n=24). The chronic cerebral ischemia models were produced by the occlusion of bilateral common caroid artery for 1 month. During the hypoperfusion, the rats were injected intraperitoneally with 11.25 mg·ml-1 PBA(90 mg·kg-1·d-1) or equal volume of saline once a day for 3 days followed by every other day for 27 days. Learning and memory abilities were tested by Morris water maze and novel object recognition test. The expression and distribution of NR2A in hippocampus were examined by Western blot and immunohistochemistry. Results Morris water maze test showed that the 2VO group had significantly longer latent time than sham group in searching platform (P 0.05). Immunohistochemistry data was consistent with the data of Western blot for NR2A level and distribution. The ratio of p-CREB/total CREB in 2VO group(0.62±0.04) was remarkably lower than that in sham group(1.00±0.07), but the ratio of p-CREB/total CREB in 2VO+ PBA group(0.97±0.07) was remarkably higher than that in 2VO group(P<0.01). Conclusion NR2A reduction is associated with chronic cerebral hypoperfusion-induced cognitive impairment, which is rescued by the PBA treatment. It suggests that PBA may have a therapeutic effect on preventing chronic cerebral hypoperfusion-induced cognitive impairment. Key words: Chronic cerebral hypoperfusion; 4-phenylbutyric acid; Cognitive dysfunction