Stress Inhibitor 4-phenylbutyric Acid Research Articles

Epstein-Barr virus-induced gene 3 commits human mesenchymal stem cells to differentiate into chondrocytes via endoplasmic reticulum stress sensor.

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1β inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1β and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1β, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α.

Wolfram syndrome 1b mutation suppresses Mauthner-cell axon regeneration via ER stress signal pathway

Wolfram Syndrome (WS) is a fatal human inherited disease with symptoms of diabetes, vision decreasing, and neurodegeneration caused by mutations in the endoplasmic reticulum (ER)-resident protein WFS1. WFS1 has been reported to play an important role in glucose metabolism. However, the role of WFS1 in axonal regeneration in the central nervous system has so far remained elusive. Herein, we established a model of the wfs1b globally deficient zebrafish line. wfs1b deficiency severely impeded the Mauthner-cell (M-cell) axon regeneration, which was partly dependent on the ER stress response. The administration of ER stress inhibitor 4-Phenylbutyric acid (4-PBA) promoted M-cell axon regeneration in wfs1b−/− zebrafish larvae, while the ER stress activator Tunicamycin (TM) inhibited M-cell axon regeneration in wfs1b+/+ zebrafish larvae. Moreover, complementation of wfs1b at the single-cell level stimulated M-cell axon regeneration in the wfs1b−/− zebrafish larvae. Altogether, our results revealed that wfs1b promotes M-cell axon regeneration through the ER stress signal pathway and provide new evidence for a therapeutic target for WS and axon degeneration.

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.

Epimedokoreanin B inhibits the growth of lung cancer cells through endoplasmic reticulum stress-mediated paraptosis accompanied by autophagosome accumulation

Epimedokoreanin B (EKB) is a prenylated flavonoid isolated from Epimedium koreanum. In this article, we described the anti-cancerous effects of EKB and its underlying mechanism in human non-small cell lung cancer (NSCLC) A549 and NCI–H292 cells. EKB treatment inhibited cell proliferation and migration accompanied by cytoplasmic vacuolation in both cell lines. The cell death induced by EKB lacked the features of apoptosis like chromatin condensation, phosphatidyl serine exposure and caspase cleavage. The vacuoles stimulated by EKB predominantly derived from endoplasmic reticulum (ER) and mitochondria dilation, which are the characteristics of paraptosis. Down-regulation of Alix and up-regulation of ER stress-related proteins after EKB treatment further supported the occurrence of paraptosis. ER stress inhibitor 4-phenylbutyric acid (4-PBA) and protein synthesis inhibitor cycloheximide (CHX) treatment antagonized the vacuoles formation as well as cell death induced by EKB, indicating that ER stress was involved in EKB induced paraptosis. In addition, autophagosome accumulation accompanied with autophagy flux blocking was observed in EKB treated cells, this was consistent with the occurrence of ER stress. Collectively, EKB was demonstrated as a paraptosis-like cell death inducer in A549 and NCI–H292 cells. The inhibitory effect of EKB on lung cancer cell proliferation was further demonstrated in a zebrafish xenograft model. These findings raise the possibility that paraptosis inducers may be considered as alternative choices for lung cancer therapy.

The uses of transcriptomics and lipidomics indicated that direct contact with graphene oxide altered lipid homeostasis through ER stress in 3D human brain organoids

The potential uses of graphene-based nanomaterials (NMs) in various fields lead to the concern about their neurotoxicity, considering that graphene-based NMs are capable to cross blood brain barrier (BBB) and enter central nervous system (CNS). Although previous studies reported the possibility of graphene-based NM exposure to alter lipid homeostasis in animals or cultured neurons, recent studies suggested the need to use 3D human brain organoids for mechanism-based toxicological studies as this model might better recapitulate the complex human brains. Herein, we used multi-omics techniques to investigate the mechanisms of graphene oxide (GO) on lipid homeostasis in a novel 3D brain organoid model. We found that 50 μg/mL GO induced cytotoxicity but not superoxide. RNA-sequencing data showed that 50 μg/mL GO significantly up-regulated and down-regulated 80 and 121 genes, respectively. Furthermore, we found that GO exposure altered biological molecule metabolism pathways including lipid metabolism. Consistently, lipidomics data supported dose-dependent alteration of lipid profiles by GO in 3D brain organoids. Interestingly, co-exposure to GO and endoplasmic reticulum (ER) stress inhibitor 4-phenylbutyric acid (4-PBA) decreased most of the lipid classes compared with the exposure of GO only. We further verified that exposure to GO promoted ER stress marker GRP78 proteins, which in turn activated IRE1α/XBP-1 axis, and these changes were partially or completely inhibited by 4-PBA. These results proved that direct contact with GO disrupted lipid homeostasis through the activation of ER stress. As 3D brain organoids resemble human brains, these data might be better extrapolated to humans.

PDIA2 Bridges Endoplasmic Reticulum Stress and Metabolic Reprogramming During Malignant Transformation of Chronic Colitis.

BackgroundChronic inflammation contributes to approximately 20% of cancers; the underlying mechanisms are still elusive. Here, using an animal model of colitis to colon-cancerous transformation, we demonstrated that endoplasmic reticulum (ER) stress couples with metabolic reprogramming to promote a malignant transformation of chronic inflammation.MethodsThe animal model for chronic colitis to colon-cancerous transformation was established in C57BL/6N mice by azoxymethane (AOM) and dextran sodium sulfate (DSS) treatments. The differential proteins in control and AOM/DSS-treated colon mucosa were determined using proteomic analysis; the kinetics of metabolic modifications were monitored by mitochondrial oxygen flux, extracellular acidification, and targeted metabolomics; the molecule linker between ER stress and metabolic modifications were identified by coimmunoprecipitation, KEGG pathway analysis, and the subcutaneous tumor model using gene-specific knockdown colon cancer cells. Tissue array analysis were used to evaluate the differential protein in cancer and cancer-adjacent tissues.ResultsAOM/DSS treatment induced 38 tumors in 10 mice at the 14th week with the mean tumor size 9.35 ± 3.87 mm2, which was significantly decreased to 5.85 ± 0.95 mm2 by the ER stress inhibitor 4-phenylbutyric acid (4PBA). Seven differential proteins were determined from control (1,067 ± 48) and AOM/DSS-treated mucosa (1,077 ± 59); the level of ER protein PDIA2 (protein disulfide isomerase-associated 2) was increased over 7-fold in response to AOM/DSS treatment. PDIA2 interacted with 420 proteins that were involved in 8 signaling pathways, in particular with 53 proteins in metabolic pathways. PDIA2 translocated from ER to mitochondria and interacted with the components of complexes I and II to inhibit oxophosphorylation but increase glycolysis. Knockdown PDIA2 in colon cancer cells restored the metabolic imbalance and significantly repressed tumor growth in the xenograft animal model. 4PBA therapy inhibited the AOM/DSS-mediated overexpression of PDIA2 and metabolic modifications and suppressed colon cancer growth. In clinic, PDIA2 was overexpressed in colon cancer tissues rather than cancer-adjacent tissues and was related with the late stages and lymph node metastasis of colon cancer.ConclusionsPersistent ER stress reprograms the metabolism to promote the malignant transformation of chronic colitis; PDIA2 serves as a molecule linker between ER stress and metabolic reprogramming. The inhibition of ER stress restores metabolic homeostasis and attenuates the cancerous transformation of chronic inflammation.

Crassolide Induces G2/M Cell Cycle Arrest, Apoptosis, and Autophagy in Human Lung Cancer Cells via ROS-Mediated ER Stress Pathways.

Crassolide, a cembranoid diterpene extracted from the soft coral Lobophytum crissum, has been proven to possess antioxidant and immunomodulatory properties. In the present study, we assessed the anticancer effects of crassolide on human H460 non-small-cell lung cancer (NSCLC) cells. We found that crassolide exerted cytotoxic effects on H460 cancer cells in vitro, inducing G2/M phase arrest and apoptosis. In addition, in H460 cells exposed to crassolide, the expression of the autophagy-related proteins LC3-II and beclin was increased, while the expression of p62 was decreased. Moreover, inhibiting autophagy with chloroquine (CQ) suppressed the crassolide-induced G2/M arrest and apoptosis of H460 cells. Moreover, we also found that crassolide induced endoplasmic reticulum (ER) stress in lung cancer cells by increasing the expression of ER stress marker proteins and that the crassolide-induced G2/M arrest, apoptosis, and autophagy were markedly attenuated by the ER stress inhibitor 4-phenylbutyric acid (4-PBA). Furthermore, we found that crassolide promoted reactive oxygen species (ROS) production by H460 cells and that the ROS inhibitor N-acetylcysteine (NAC) decreased the crassolide-induced ER stress, G2/M arrest, apoptosis, and autophagy. In conclusion, our findings show that crassolide inhibits NSCLC cell malignant biological behaviors for the first time, suggesting that this effect may be mechanistically achieved by inducing G2/M arrest, apoptosis, and autophagy through ROS accumulation, which activates the ER stress pathway. As a result of our findings, we now have a better understanding of the molecular mechanism underlying the anticancer effect of crassolide, and we believe crassolide might be a candidate for targeted cancer therapy.

Endoplasmic reticulum stress promotes blood-testis barrier impairment in mice with busulfan-induced oligospermia through PERK-eIF2α signaling pathway

Busulfan, a chemotherapeutic agent for cancer, has detrimental effects on germ cells and fertility, yet the specific mechanisms remain largely uncertain. The blood-testis barrier (BTB) maintains a suitable microenvironment for germ cells self-renewal and spermatogenesis by blocking the interference and damage of deleterious substances. Therefore, we hypothesized that BTB abnormalities might be involved in busulfan-induced oligospermia. To verify the hypothesis, thirty male Balb/c mice were randomly administered with busulfan (at a total dose of 40 mg/kg body weight) by intraperitoneal injection for 4 weeks to establish the model of oligospermia. The results displayed that busulfan caused testicular histopathological lesions and spermatogenesis disorder. Meanwhile, busulfan disrupted BTB integrity and lessened the expressions of BTB junction proteins, including Occludin, Claudin-11 and Connexin-43. Furthermore, busulfan activated the endoplasmic reticulum (ER) stress and PERK-eIF2α signaling pathway, reflected by the increased protein expressions of GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. Finally, to evaluate whether the ER stress is involved in busulfan-induced BTB destruction, the ER stress inhibitor 4-Phenylbutyric acid (4-PBA, 1 mM) was used to intervene in busulfan-exposed TM4 cells. The results displayed that inhibition of ER stress alleviated the reduction of BTB junction protein expressions induced by busulfan in TM4 cells. These data collectively indicated that busulfan-induced BTB impairment was mediated by triggering ER stress and activation of the PERK-eIF2α signaling pathway, thereby damaging the spermatogenesis, providing a new therapeutic target for male infertility induced by busulfan.

Endoplasmic reticulum stress contributes to autophagy and apoptosis in cantharidin-induced nephrotoxicity

Mylabris, as a natural product of traditional Chinese medicine (TCM), exhibiting typical antitumor activity, and cantharidin (CTD) is the major bioactive component. However, drug-induced nephrotoxicity (DIN) extremely limited its clinical application. In this study, we proved that activation of the endoplasmic reticulum (ER) stress-dependent PERK/CHOP pathway exerts a toxic role in rats and HK-2 cells through inducing autophagy and apoptosis. Results showed that CTD could cause renal function damage, cytotoxicity, and apoptosis. The ER dilatation and autolysosomes were observed after CTD treatment. Furthermore, the distribution of LC3, ATF4, and CHOP proteins was observed in the nucleus and cytoplasm. In addition, the mRNA levels of ER stress-regulated genes (PERK, eIF2α, CHOP, and ATF4) were increased, and the expression levels of GRP78, ATF4, CHOP, LC3, Beclin-1, Atg3, Atg7, Caspase 3, and Bax/Bcl-2 proteins were increased both in vitro and in vivo. Consistently, this upregulation could be inhibited by an ER stress inhibitor 4-Phenylbutyric acid (4-PBA), indicating that ER stress is partly responsible for activation of autophagy and apoptosis in CTD-induced DIN. In conclusion, CTD could induce DIN by triggering ER stress, further activating autophagy and apoptosis both in vivo and in vitro.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

Abstract TMP116: Pharmacological Inhibition Of Endoplasmic Reticulum Stress Prevents Intracranial Aneurysm Rupture

Background: The endoplasmic reticulum (ER) is responsible for cellular protein synthesis and folding. Cellular stimuli that perturb ER homeostasis create an imbalance between the protein-folding load and capacity of ER, causing unfolded or misfolded proteins to accumulate in the ER lumen, known as ER stress. Recent studies suggested that ER stress plays significant roles in the pathogenesis of inflammatory vascular diseases. Since inflammation is emerging as a vital component of the pathophysiology of intracranial aneurysms, we hypothesized that ER stress promotes the development of aneurysm rupture by inducing sustained vascular wall inflammation. We tested this hypothesis utilizing pharmacological approaches in mice. Methods: We used 10-week-old male C57BL/6J mice and induced intracranial aneurysms by combining an elastase injection and hypertension. We tested the effects of an ER stress activator (Tunicamycin) and inhibitor (4-phenylbutyric acid, 4-PBA) on the development of aneurysmal rupture. In addition, we assessed the roles of ER stress induced via deactivation of epidermal growth factor receptor (EGFR) by Erlotinib on aneurysm rupture. Results: The pharmacological inhibition of ER stress significantly decreased the rupture rate (P<0.05, Fig. A). Meanwhile, there was a trend for the ER stress activator to increase the rupture rate (P=0.17, Fig.B). Moreover, the pharmacological inhibition of the EGFR pathway with Erlotinib significantly reduced the rupture rate (P<0.05, Fig. C). In addition, Erlotinib treatment reduced the mRNA expression of ER stress markers GRP78 and CHOP (Fig.D-E). Conclusions: The pharmacological inhibition of ER stress decreased aneurysmal rupture in a mouse model of intracranial aneurysm. In addition, inhibition of EGFR decreased aneurysm rupture through the reduction of ER stress. Our findings suggest that ER stress and its upstream EGFR pathway may serve as novel therapeutic targets for preventing aneurysm rupture.

Interactions between Endoplasmic Reticulum Stress and Autophagy: Implications for Apoptosis and Neuroplasticity-Related Proteins in Palmitic Acid-Treated Prefrontal Cells.

Lipotoxicity of palmitic acid (PA) or high-fat diets has been reported to increase endoplasmic reticulum (ER) stress and autophagy in peripheral tissue as well as apoptotic cell death. It also can lead to an AD-like pathological pattern. However, it has been unknown that PA-induced ER stress and autophagy are involved in the regulation of neuroplastic abnormalities. Here, we investigated the roles of ER stress and autophagy in apoptosis and neuroplasticity-related protein expression in PA-treated prefrontal cells. Prefrontal cells dissected from newborn Sprague-Dawley rats were treated with PA compound with ER stress inhibitor 4-phenylbutyric acid (4-PBA) and autophagy inhibitor 3-methyladenine (3-MA) or PA alone. PA promoted ER stress and autophagy and also cause apoptosis as well as a decline in the expression of neuroplasticity-related proteins. Inhibition of ER stress decreased the expressions of neuroplasticity-related proteins and reduced autophagy activation and apoptosis in PA-treated prefrontal cells. Inhibition of autophagy exacerbated apoptosis and enhanced ER stress in PA-treated prefrontal cells. The present study illustrated that both ER stress and autophagy could be involved in apoptosis and decreased neuroplasticity-related proteins, and the interaction between ER stress and autophagy may play a critical role in apoptosis in PA-treated prefrontal cells. Our results provide new insights into the molecular mechanisms in vitro of lipotoxicity in obesity-related cognitive dysfunction.

SchizandrinA induces the apoptosis and suppresses the proliferation, invasion and migration of gastric cancer cells by activating endoplasmic reticulum stress.

Apart from its basic antioxidant and anti-inflammatory properties, schizandrin A (SchA), which is isolated from Fructus schisandra, can exert anticancer effects on multiple cancer types. However, to the best of our knowledge, there has been no study identifying the impacts of SchA on gastric cancer (GC). Therefore, the aim of the present study was to identify how SchA functioned to affect the progression of GC. To investigate the role of SchA in GC development, Cell Counting Kit-8, colony formation, wound healing and Transwell assays were conducted to assess the viability, proliferation, migration and invasion of AGS cells, respectively. Then, the apoptosis rate and apoptosis- and endoplasmic reticulum (ER) stress-related protein expression levels in AGS cells exposed to SchA were detected via TUNEL assays and western blotting, respectively. Subsequently, the aforementioned functional assays were performed again in AGS cells exposed to both SchA and the ER stress inhibitor 4-phenylbutyric acid (4-PBA) for the confirmation of the effect of SchA on ER stress in GC. It was found that SchA markedly decreased the viability, proliferation, migration and invasion, while it induced the apoptosis of AGS cells. Moreover, the markers of ER stress were elevated by SchA treatment in AGS cells. Nevertheless, 4-PBA reversed the effects of SchA on the viability, proliferation, migration, invasion and apoptosis of AGS cells, accompanied by decreased expression of ER stress markers. In conclusion, the present study demonstrated that SchA induced the apoptosis and suppressed the proliferation, invasion and migration of GC cells by activating ER stress, which provides a theoretical basis for the use of SchA in the treatment of GC.

Abstract MP59: Soluble Protein Oligomers Induce Endoplasmic Reticulum Stress In Mesenteric Resistance Arteries From Male And Female Mice

Amyloid β proteins, including toxic soluble oligomers (SPOs) are not only found in the brain duringAlzheimer’s, but also in the peripheral vascular system. The precise mechanism linking increasedcirculating levels of SPOs and vascular dysfunction remains unknown. We hypothesized that SPOslead to endoplasmic reticulum (ER) stress, further release of SPOs and vascular injury. Mesentericresistance arteries (MRAs) from 14 weeks old, male and female C57BL/6 mice were used forvascular function. Agonists were acetylcholine and phenylephrine (1nM-10mM). In acuteconditions, SPOs (0.1μM) caused pathologically exacerbated endothelium-dependent vasodilationcompared to vehicle (F12 media) [Male: EC50: SPOs: -7.0 ± 0.1 (n=4), vs. Vehicle -6.6 ± 0.1 (n=7)p=0.03; Female: EC50: SPOs: -7.3 ± 0.06 (n=5) vs. Vehicle -6.7 ± 0.1 (n=6), p=0.001]. Thisphenotype was similar to the positive control tunicamycin (5mg/ml) [Male: EC50: Tunicamycin: -7.3(n=4), vs. Vehicle -6.6 (n=7) p=0.2; Female: EC50: Tunicamycin: -7.7 (n=4) vs. Vehicle -6.8 (n=5)p=0.04]. To determine whether SPO’s cause ER stress, arteries were treated with ER stressinhibitor 4-Phenylbutyric acid (2mM). The ER stress inhibitor prevented the exacerbatedvasodilation induced by SPOs showing SPOs trigger ER stress in acute conditions independent ofsex. To determine whether SPOs are a consequence of ER stress, arteries were incubated withtunicamycin in the presence of the SPO inhibitor K01-162 (10mM). Interestingly, K01-162 did notprevent the tunicamycin-induced exacerbated vasodilation in arteries from male mice. However,this response was decreased in arteries from female mice showing that inducing ER stress leadsto the release of SPOs, escalating a feed-forward mechanism of further SPO release. There wereno changes in vascular contraction with tunicamycin or SPOs irrespective of sex. ER stress wasconfirmed with anti-KDEL antibody staining, specific for ER resident chaperones Grp78/94 andvisualized with multiphoton fluorescent confocal microscopy. These results demonstrate that SPO’sexacerbate endothelium-dependent vasodilation acutely and may contribute to brain and peripheralvascular edema and loss of autoregulation observed during cardiovascular and Alzheimer’sdisease.

Protective effect of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P<0.05 or P<0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P<0.05 or P<0.01), while Bcl-2 was significantly up-regulated(P<0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P<0.01), whereas Bcl-2 was significantly up-regulated(P<0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

Soluble Protein Oligomers induce Endoplasmic Reticulum Stress in Acute Conditions in Mesenteric Resistance Arteries from Male and Female Mice

Background While amyloid β proteins, including toxic soluble oligomers, are a well established pathogenic mechanism of alzheimer's disease, SPOs are also found in the peripheral vascular system where their accumulation is considered to be a fundamental mechanism that underlies vascular dysfunction and cell death. However, the precise mechanism linking increased circulating levels of SPOs and vascular dysfunction remains unknown. We therefore hypothesized that SPOs are recognized by the vasculature leading to endoplasmic reticulum (ER) stress which further induces the release of SPOs and vascular injury. Methods & results Mesenteric resistance arteries (MRAs) from male and female wild type C57BL/6 mice (14-20 weeks old) were isolated to measure vascular function via a wire myograph. Concentration response curves to acetylcholine (10-9-10-5mol/L) and phenylephrine (10-9-10-5 mol/L) were performed. We observed that in acute conditions, and in arteries from both male and female mice, incubation with SPOs (0.1mM) caused exacerbated endothelium-dependent vasodilation compared to vehicle (F12 media, 5mL) [Male: EC50: Vehicle -6.6 ± 0.1 (n=7) vs. SPOs: -7.0 ± 0.1 (n=4), p=0.03; Female: EC50: Vehicle -6.7 ± 0.1 (n=6) vs. SPOs: -7.3 ± 0.06 (n=5), p=0.001]. A similar phenotype was observed in arteries treated with ER stress inducer tunicamycin (5mg/ml, positive control). This suggests that SPOs and ER stress exacerbate endothelium-dependent vasodilation, independent of sex. To determine whether SPO's are a cause of ER stress, arteries were treated with SPOs or vehicle for 15 min prior to incubation with an ER stress inhibitor 4-Phenylbutyric acid (4-PBA) (2mM) for 30 min. We observed that incubation with 4-PBA prevented the exacerbated vasodilation induced by SPOs in both male and female arteries, indicating that SPOs trigger ER stress in acute conditions and this is also independent of sex (Fig 1). On the other hand, to determine whether SPOs are a consequence of ER stress, arteries were incubated with ER stress inducer tunicamycin (5mg/ml) or vehicle for 15 min prior to incubation with the oligomer inhibitor K01-162 (10mM) for 30 min. Interestingly, the SPO inhibitor K01-162 did not prevent the exacerbated vasodilation induced by tunicamycin in arteries from male mice. However, this response was restored in arteries from female mice suggesting that ER stress induces the release of SPOs in arteries from female but not from male mice in acute conditions. Phenylephrine-induced vascular contraction did not change in the presence of tunicamycin or SPOs irrespective of sex. Conclusion We concluded that SPOs induce ER stress and subsequently exacerbate endothelium-dependent vasodilation in acute conditions irrespective of sex. However, ER stress induction in arteries from females also leads to the release of SPOs, escalating a feed-forward mechanism of further SPO release. Collectively, these results demonstrate that SPO's exacerbate endothelium-dependent vasodilation acutely and may contribute to vascular damage.

Pristimerin synergistically sensitizes conditionally reprogrammed patient derived-primary hepatocellular carcinoma cells to sorafenib through endoplasmic reticulum stress and ROS generation by modulating Akt/FoxO1/p27kip1 signaling pathway

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. Sorafenib (SORA), as a first-line therapeutic drug, has been used to treat HCC, but resistance poses a major limitation on the efficacy of SORA chemotherapy. Pristimerin (PRIS), a natural bioactive component isolated from various plant species in the Celastraceae and Hippocrateaceae families, has been reported to exhibit outstanding antitumor effects in several types of cells in vitro. PurposeThe aim of this study was to investigate whether PRIS can exert synergistic anti-tumor effects with the combination of SORA, and if so, through what mechanism. MethodsConditionally reprogrammed patient derived-primary hepatocellular carcinoma cells (CRHCs) were isolated from human liver cancer tissues and treated with SORA and PRIS. Cell proliferation, apoptosis, migration and tube formation ability were detected by DNA content quantification, flow cytometry, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively. ResultsInitially, we observed that the combination of the two drugs had a much stronger inhibitory effect on CRHCs growth than either drug alone. Moreover, the combination of 2 µM SORA and 1 µM PRIS exhibited a significant anti‑migrative and anti-invaded effect on CRHCs, and remarkably inhibited capillary structure formation of Human Umbilical Vein Endothelial Cells (HUVECs). Furthermore, the combined treatment with SORA and PRIS synergistically induced intrinsic apoptosis in CRHCs, involving a caspase-4‐dependent mechanism paralleled by an increased Bax/Bcl‐xL ratio. These activities were mediated through ROS generation and the induction of endoplasmic reticulum (ER) stress and mitochondrial dysfunction. GRP78 silencing or ER stress inhibitor 4-phenylbutyric acid administration was revealed to abolish the anticancer effects of PRIS, indicating the critical role of GRP78 in mediating the bioactivity of PRIS. The present study also provides mechanistic evidence that PRIS modulated the Akt/FoxO1/p27kip1 signaling pathway, which is required for mitochondrial-mediated intrinsic apoptosis, activation of ER stress, and stimulation of caspase-4 induced by PRIS, and, consequently resulting in suppressed cell viability, migration and angiogenesis co-treated with SORA in CRHCs. ConclusionOur results suggest the use of PRIS as sensitizers of chemotherapy paving the way for innovative and promising targeted chemotherapy-based therapeutic strategies in human HCC.

Endothelial Nox4 dysfunction aggravates atherosclerosis by inducing endoplasmic reticulum stress and soluble epoxide hydrolase

Background and aimsOur previous findings have demonstrated the protective effect of endothelial Nox4-based NADPH oxidase on atherosclerosis. One of the possible mechanisms is the inhibition of soluble epoxide hydrolase (sEH), a proinflammatory and atherogenic factor. Our goal was to investigate whether in vivo inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) alleviates endothelial Nox4 dysfunction caused atherosclerosis and the regulatory mechanism of endothelial Nox4 on sEH. Methods& results: We used endothelial human Nox4 dominant-negative (EDN) transgenic mice in ApoE deficient background to mimic the dysfunction of endothelial Nox4 in atherosclerosis-prone conditions. In EDN aortic endothelium, sEH and the inflammatory marker vascular cell adhesion molecule 1 (VCAM1) were upregulated. TPPU reduced atherosclerotic lesions in EDN mice. In EDN endothelial cells (ECs), the endoplasmic reticulum (ER) stress markers (BIP, IRE1α, phosphorylation of PERK, ATF6) were upregulated, and they can be suppressed by ER stress inhibitor 4-phenyl butyric acid (4-PBA). In EDN ECs, 4-PBA downregulated the expression of sEH and VCAM1, suppressed inflammation, and its application in vivo reduced atherosclerotic lesions of EDN mice. ConclusionsEndothelial Nox4 dysfunction upregulated sEH to enhance inflammation, probably by its induction of ER stress. Inhibition of ER stress or sEH is beneficial to alleviate atherosclerosis caused by endothelial Nox4 dysfunction.

Microcystin-leucine arginine exposure contributes to apoptosis and follicular atresia in mice ovaries by endoplasmic reticulum stress-upregulated Ddit3

Microcystin-leucine arginine (MC-LR), an intracellular toxin to cause reproduction toxicity, is produced by blooming cyanobacteria and widely distributed in eutrophic waters. It is revealed that MC-LR-induced female reproductive toxicity is more severe than male reproductive toxicity. Previous studies mainly focused on male reproductive toxicity, and the molecular mechanisms of MC-LR-induced apoptosis, follicular atresia and infertility in female remain largely unclear. Here, it was found that MC-LR treatment could induce apoptosis, inflammation, follicular atresia, and decrease of gonadal index in mice ovaries. RNA-Seq data showed that the up-regulation of DNA-damage inducible transcript 3 (Ddit3) under endoplasmic reticulum (ER) stress had predominantly regulatory role in MC-LR-induced apoptotic pathway. Furthermore, MC-LR exposure promoted cleavage of activating transcription factor 6 (ATF6, 50kd), inositol-requiring enzyme 1 (Ire1) expression, phosphorylation of IRE1, mitogen-activated protein kinase 5 (Map3k5) and Ddit3 expression, which was accompanied by the upregulation of death receptor 5 (Dr5) and active-caspase-3, and a decrease in Bcl-2 expression. ER stress inhibitor 4-Phenyl butyric acid (4-PBA) ameliorated these MC-LR-induced changes in protein or mRNA level. More importantly, knockdown of Ddit3 suppressed MC-LR-induced cell apoptosis and follicular atresia by directly regulating Dr5 and Bcl-2. Additionally, it was also found that MC-LR increased Map3k5 phosphorylation by inhibiting protein phosphatase 2A (PP2A) activity, and then promoted Ddit3 expression. In short, our data suggests that Ddit3 promotes MC-LR-induced mice ovarian cells apoptosis and follicular atresia via ER stress activation, which provides a new insight into the relation between infertility in females and the emerging water pollutant MC-LR.

Social defeat stress induces myocardial injury by modulating inflammatory factors.

ObjectivesWe investigated the endoplasmic reticulum (ER) stress markers C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP) 78, as well as the inflammatory factors nuclear factor (NF)-κB and IκBα, to assess how social defeat stress induces myocardial injury. Furthermore, we evaluated the protective effects of the ER stress inhibitor 4-phenylbutyric acid (PBA) on myocardial injury in mice.MethodsAdult mice were divided into control, control + PBA, social defeat, and social defeat + PBA groups. The social defeat and social defeat + PBA groups were exposed to social defeat stress for 10 days. Cardiac tissues from all groups were analyzed after social defeat stress. H9C2 cells were used to detect the role of the ER stress agonist thapsigargin on expression of ER stress and inflammatory markers.ResultsSocial defeat stress promoted apoptosis of cardiomyocytes, increased CHOP, NF-κB and, phospho-NF-κB protein expression, and decreased GRP78 and IκBα protein expression. Moreover, PBA significantly reversed these changes and attenuated thapsigargin-induced increased expression of CHOP and phospho-NF-κB, and decreased IκBα expression in H9C2 cells.ConclusionsSocial defeat stress initiates ER stress, promotes expression of inflammatory factors, and induces myocardial injury. Inhibiting ER stress could protect the myocardium from social defeat stress-induced myocardial injury.