CHOP mRNA Research Articles

Effect of Voluntary Wheel Running on Anxiety- and Depression-Like Behaviors in Fluoride-Exposed Mice.

Fluoride, an environmental toxicant, could induce endoplasmic reticulum stress (ERS) in neuronal cells ultimately leading to apoptosis and emotional dysfunction. Meanwhile, voluntary wheel running contributes to mitigate anxiety and depression. Our investigation aimed to study the effect of voluntary wheel running on anxiety- and depression-like behaviors in fluoride-exposure mice. The results showed that exposure to 100 mg/L sodium fluoride (NaF) for 6 months can induce anxiety- and depression-like behavior in mice. Fluorosis mice subjected to voluntary wheel running have less anxiety- and depression-like behaviors. Nissl and TUNEL staining demonstrated that fluoride led to a reduced proportion of Nissl body area in the cerebral cortex and an increased apoptotic ratio of nerve cells in the cerebral cortex. In contrast, these pathologic damages were improved in voluntary wheel running mice exposed to NaF. Moreover, the expressions of mRNA in the cerebral cortex GABA, GAD65, GAD67, DR, vGLU, 5-HT1A, BDNF, NMDAR1, and Bcl2 were downregulated and the levels of c-fos, GRP78, PERK, eIF2α, CHOP, Caspase-12, and Caspase-3 mRNA were upregulated in mice exposed to fluoride. NaF treatment had increased the PERK, ATF6, IRE1, p-eIF2α, and Caspase-3 protein levels and reduced the expressions of proteins, including GAD67, VGAT, BDNF, NMDAR1, PSD95, and SYN. By contrast, fluorosis mice subjected to voluntary wheel running enhanced the expression of GAD65, GAD67, VGAT, and neuroplasticity-related proteins in mice and inhibited the PERK-CHOP pathway. It is worth noting that the correlation between the amount of exercise and the behavioral indicators as well as neurotransmitter levels was found. In conclusion, voluntary wheel running inhibits the fluoride-induced ERS and GRP78 expression through the PERK-CHOP pathway and plays an anti-apoptotic role, ultimately ameliorating emotional dysfunction in NaF-exposed mice.

Cannabinoid combination targets NOTCH1-mutated T-cell acute lymphoblastic leukemia through the integrated stress response pathway.

In T-cell acute lymphoblastic leukemia (T-ALL), more than 50% of cases display autoactivation of Notch1 signaling, leading to oncogenic transformation. We have previously identified a specific chemovar of Cannabis that induces apoptosis by preventing Notch1 maturation in leukemia cells. Here, we isolated three cannabinoids from this chemovar that synergistically mimic the effects of the whole extract. Two were previously known, cannabidiol (CBD) and cannabidivarin (CBDV), whereas the third cannabinoid, which we termed 331-18A, was identified and fully characterized in this study. We demonstrated that these cannabinoids act through cannabinoid receptor type 2 and TRPV1 to activate the integrated stress response pathway by depleting intracellular Ca2+. This is followed by increased mRNA and protein expression of ATF4, CHOP, and CHAC1, which is hindered by inhibiting the upstream initiation factor eIF2α. The increased abundance of CHAC1 prevents Notch1 maturation, thereby reducing the levels of the active Notch1 intracellular domain, and consequently decreasing cell viability and increasing apoptosis. Treatment with the three isolated molecules resulted in reduced tumor size and weight in vivo and slowed leukemia progression in mice models. Altogether, this study elucidated the mechanism of action of three distinct cannabinoids in modulating the Notch1 pathway, and constitutes an important step in the establishment of a new therapy for treating NOTCH1-mutated diseases and cancers such as T-ALL.

Protective Effects of Syringic Acid Against Oxidative Damage, Apoptosis, Autophagy, Inflammation, Testicular Histopathologic Disorders, and Impaired Sperm Quality in the Testicular Tissue of Rats Induced by Mercuric Chloride.

Mercury (Hg) is one of the most toxic heavy metals that damage testicular tissue. Mercury chloride (HgCl2) is one of the most toxic forms of mercury that can easily cross biological membranes. Syringic acid (SA) is a natural flavonoid found in many vegetables and fruits. In this study, the effects of SA against HgCl2-induced testicular damage in rats were determined by biochemical, histopathological, and spermatological analyses. For this study, a total of 35 Spraque Dawley rats were used. Rats were divided into five groups as control, HgCl2, SA 50, HgCl2 + SA 25, and HgCl2 + SA 50. HgCl2 was administered intraperitoneal (IP) at a dose of 1.23 mg/kg/bw, while SA was administered by oral gavage at doses of 25 and 50 mg/kg/bw. The rats were then sacrificed, and testicular tissues were removed. HgCl2 caused an increase in MDA level and a decrease in SOD, CAT, and GPx activity and GSH level in the testicular tissue of rats. HgCl2 is involved in the increase of eIF2-α, PERK, ATF-4, ATF-6, CHOP, NF-κB, TNF-α, IL-1β, Apaf-1, Bax, and Caspase-3 mRNA expression. HgCl2 caused a decrease in sperm motility, an increase in the rate of abnormal sperm and sperm DNA fragmentation in rats. However, SA oral administration dose-dependently inhibited endoplasmic reticulum stress, oxidative stress, inflammation, and apoptosis and preserved epididymal sperm quality and testicular histoarchitectures. In conclusion, SA had protective effects against HgCl2-induced testicular oxidative damage, inflammation, endoplasmic reticulum stress, and apoptosis.

Immortalization and Characterization of GFAP-expressing Glial Cells from the Adult Mouse Hypothalamus, Cortex, and Brain Stem

The generation of astrocyte cell lines from the hypothalamus is key to study glial involvement in hypothalamic physiology, including energy homeostasis. As such, we immortalized astrocytes from the hypothalamus of an adult male CD-1 mouse using SV40 T-antigen to generate the mHypoA-Ast1 cell line. A comparative approach was taken with two other murine GFAP-expressing cell lines that were also generated in this study: a mixed glial cell line from the cortex (mCortA-G1) and an oligodendrocyte cell line from the brainstem (mBstA-Olig1), as well as an established microglial cell line (IMG). mHypoA-Ast1 cells express GFAP, alongside other astrocytic markers such as Aldh1l1, Aqp4, Glt1 and S100b, and express low levels of microglial, ependymal and oligodendrocyte markers. 100 ng/mL lipopolysaccharide (LPS) elevated mRNA levels of Il6, Il1b, Tnfα and Cxcl5 in mHypoA-Ast1 cells after 4 h, while 50 μM palmitate increased Il6 and Chop mRNA, demonstrating the ability of these cells to respond to inflammatory and nutrient signals. Interestingly, co-culture of mHypoA-Ast1 cells with mHypoE-N46 hypothalamic neuronal cells prevented the palmitate-mediated increase in orexigenic neuropeptide Agrp mRNA in mHypoE-N46 cells, suggesting that this cell line can alter neuronal responses to nutrients. In conclusion, we report mHypoA-Ast1 cells representing a functional astrocyte cell line from the adult mouse brain that can be used to study the complex interactions of hypothalamic cells, as well as dysregulation that may occur in disease states, providing a key tool for neuroendocrine research.

The Involvement of the microRNAs miR-466c and miR-340 in the Palmitate-Mediated Dysregulation of Gonadotropin-Releasing Hormone Gene Expression.

Diets high in saturated fatty acids are associated with obesity and infertility. Palmitate, the most prevalent circulating saturated fatty acid, is sensed by hypothalamic neurons, contributing to homeostatic dysregulation. Notably, palmitate elevates the mRNA levels of gonadotropin-releasing hormone (Gnrh) mRNA and its activating transcription factor, GATA binding protein 4 (Gata4). GATA4 is essential for basal Gnrh expression by binding to its enhancer region, with Oct-1 (Oct1) and CEBP-β (Cebpb) playing regulatory roles. The pre- and post-transcriptional control of Gnrh by palmitate have not been investigated. Given the ability of palmitate to alter microRNAs (miRNAs), we hypothesized that palmitate-mediated dysregulation of Gnrh mRNA involves specific miRNAs. In the mHypoA-GnRH/GFP neurons, palmitate significantly downregulated six miRNAs (miR-125a, miR-181b, miR-340, miR-351, miR-466c and miR-503), and the repression was attenuated by co-treatment with 100 μM of oleate. Subsequent mimic transfections revealed that miR-466c significantly downregulates Gnrh, Gata4, and Chop mRNA and increases Per2, whereas miR-340 upregulates Gnrh, Gata4, Oct1, Cebpb, and Per2 mRNA. Our findings suggest that palmitate may indirectly regulate Gnrh at both the pre- and post-transcriptional levels by altering miR-466c and miR-340, which in turn regulate transcription factor expression levels. In summary, palmitate-mediated dysregulation of Gnrh and, consequently, reproductive function involves parallel transcriptional mechanisms.

Abstract 6952: The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC)

Abstract Cancer-associated cachexia (CAC), characterized by loss of skeletal muscle and involuntary body weight loss, is responsible for increased chemotherapy toxicity, reduced quality of life, and impaired survival in cancer patients. While interleukin-11 (IL11), one of the IL6 family cytokines, was originally thought to be involved in hematopoiesis, studies increasingly suggest its involvement in tumorigenesis including chemoresistance, metastasis, and survival of cancer cells. However, the precise role of IL11 in CAC has not been determined. Our lab has previously shown that IL11 treatment suppressed the myogenic differentiation of C2C12 myoblasts, suggesting that IL11 may interfere with differentiation and subsequent regeneration of muscle. In this study, we investigated the role of IL11 in cancer-induced muscle wasting using cell culture and mouse models. The mRNA expression of IL11 was highly up-regulated in C26 and Lewis lung carcinoma (LLC) cells, two well-known cachectic mouse cancer cell lines, while its expression was low in a non-cachectic MC38 mouse colon cancer cell line. Knockdown of IL11 in C26 cells using two independent lentiviral shRNAs significantly reduced the mRNA levels of the cytokines involved in CAC including GDF15, LIF, and VEGFA, while the mRNA level of IL6 was not significantly altered. Additionally, IL11 knockdown significantly reduced the mRNA and protein levels of ATF4, CHOP, and TRIB3, suggesting that IL11 may be responsible for the activation of unfolded protein response, an adaptive mechanism for growth, survival, and angiogenesis of cancer. Knockdown of IL11 in C26 and LLC cell lines also significantly reduced the volume of tumors in syngeneic mouse models, while over-expression of IL11 in MC38 cells increased tumor size. Moreover, the knockdown of IL11 in cachectic cancer cells partially prevented the loss of muscle and body weight in tumor-bearing mice, although the amelioration of muscle loss by IL11 knockdown may be associated with the decreased tumor burden. Together, these results indicate that IL11 may contribute to muscle wasting and provide novel insight into the previously unestablished role of IL11 in CAC. Citation Format: Kimberly Drinkwater, Ashlyn Meyers, Samin Kargari, Natalie Crisan, Aaron Mody, Minsub Shim. The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6952.

Stem cell factor restrains endoplasmic reticulum stress-associated apoptosis through c-Kit receptor activation of JAK2/STAT3 axis in hippocampal neuronal cells.

Alzheimer's disease (AD) is a common elderly disorder characterized by cognitive decline. Endoplasmic reticulum (ER) stress has been implicated in various neurodegenerative diseases, including AD. Stem cell factor (SCF) performs its biological functions by binding to and activating receptor tyrosine kinase c-Kit. We aimed to investigate the effects of SCF/c-Kit and JAK2/STAT3 on ER stress and apoptosis in AD. The study employed L-glutamic acid (L-Glu)-treated HT22 cells as sporadic AD cell model and APP/PS1 mice as an animal model of familiar AD. SCF, c-Kit inhibitor ISCK03 or JAK2/STAT3 inhibitor WP1066 was treated to verify the effects of SCF/c-Kit and JAK2/STAT3 on ER stress and apoptosis of L-Glu-exposed HT22 cells. Cell viability was assessed by MTT. BrdU detected cell proliferation. Flow cytometry measured cell apoptosis. The expression levels of ER stress markers GRP78, PERK, CHOP, and apoptosis protein caspase3 were determined by western blot. The effect on the mRNA of ER stress markers GRP78, PERK, CHOP and apoptotic caspase3 were quantified by RT-qPCR in primary cultured hippocampal neurons from APP/PS1 transgenic mice. Administration of SCF significantly augmented the activity and proliferation of hippocampal neuronal cells, protecting cells against L-Glu induced ER stress-associated apoptosis. Moreover, the addition of ISCK03 (c-Kit inhibitor) or WP1066 (JAK2/STAT3 inhibitor) reversed SCF effects on ER stress and apoptosis in vitro. We found that SCF inhibits L-Glu-induced ER stress-associated apoptosis via JAK2/STAT3 axis in HT22 hippocampal neuronal cells, as well as in primary hippocampal neurons from APP/PS1 mice, which provides valuable insights into the molecular mechanisms underlying the pathogenesis of AD and explores novel therapeutic targets for both sporadic and familial AD.

Antagonistic effect of selenium on programmed necrosis of testicular Leydig cells caused by cadmium through endoplasmic reticulum stress in chicken.

Cadmium (Cd) is a widely distributed environmental contaminant that is highly toxic to animals and humans. However, detailed reports on Cd-induced programmed necrosis have not been seen in chicken testicular Leydig cells. Selenium (Se) is a trace element in the human body that has cytoprotective effects in a variety of pathological damages caused by heavy metals. This study investigated the potential mechanisms of Cd-induced programmed cell necrosis and the antagonistic effect of Se on Cd toxicity. Chicken testis Leydig cells were divided into six groups, namely, control, Se (5µmol/L Na2SeO3), Cd (20µmol/L CdCl2), Se + Cd (5µmol/L Na2SeO3 and 20µmol/L CdCl2), 4-phenylbutyric acid (4-PBA) + Cd (10mmol/L 4-phenylbutyric acid and 20µmol/L CdCl2), and Necrostatin-1 (Nec-1) + Cd (60µmol/L Necrostatin-1 and 20µmol/L CdCl2). The results showed that Cd exposure decreased the activity of CAT, GSH-Px, and SOD and the concentration of GSH, and increased the concentration of MDA and the content of ROS. Relative mRNA and protein expression of GRP78, PERK, ATF6, IRE1, CHOP, and JNK increased in the Cd group, and mRNA and protein expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 significantly increased in the Cd group, while Caspase-8 mRNA and protein expression significantly decreased. The abnormal expression of endoplasmic reticulum stress-related proteins was significantly reduced by 4-PBA pretreatment; the increased expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 caused by Cd toxicity was alleviated; and the expression of caspase-8 was upregulated. Conversely, the increased mRNA and protein expression of endoplasmic reticulum stress marker genes (GRP78, ATF6, PERK, IRE1, CHOP, JNK) caused by Cd was not affected after pretreatment with Nec-1. We also found that these Cd-induced changes were significantly attenuated in the Se + Cd group. We clarified that Cd can cause programmed necrosis of chicken testicular Leydig cells through endoplasmic reticulum stress, and Se can antagonize Cd-induced programmed necrosis of chicken testicular Leydig cells.

A-007 Cardiomyocyte-monocyte Cross-talk Exploits Cellular Stress Pathways Leading to Myocyte Apoptosis in Dilated Cardiomyopathy of Idiopathic Origin

Abstract Background Dilated cardiomyopathy (DCM) is the most prevalent form of cardiovascular diseases (CVDs) results in dilation and disruption of pumping mechanism of heart. Most strikingly, about 50% DCM cases do not have any identified aetiology, hence known as idiopathic DCM (IDCM). The aim of this study is to investigate major inflammatory markers and cellular stress pathways in the circulating leukocytes and cardiomyocytes to find out the cross-talk between these cell types in vitro condition by mimicking the in vivo inflammatory milieu. Methods The study is divided into two parts, first part comprising patient study and the second part is the in vitro cell culture based mechanistic study. 50 IDCM patients and 30 age and sex matched control subjects were recruited from our in-house Ramakrishna Mission Seva Pratishthan (RKMSP) hospital based on their NYHA discomfort criteria followed by intra thoracic echocardiography. Exclusively the IDCM subjects were selected for this study. Blood was collected from the patients for biochemical and inflammatory parameters and to isolate PBMCs. mRNA and protein expressions were studied by real time PCR and immunoblotting respectively. H9C2 cardiomyocyte cells alone or with LPS-activated THP-1 monocyte/macrophage line were co-cultured in presence of the major inflammatory cytokines in the same in vivo concentration as found in the IDCM patients in presence or absence of ER stress agonist or antagonists. Immunofluorescence, RT-PCR and immunoblotting were done for markers specific to myocyte stress, inflammation and apoptosis. Results Inflammatory and cardiac blood biomarkers NLR, hs-CRP, IL-6, pro-BNP and troponin-I were found to be significantly high (P < 0.05) in IDCM. Subsequently, the mRNA expressions of pro-inflammatory genes TNF-α (2.6 fold), IFN-γ (10.2 fold), IL-1β (8.5 fold) and NF-κβ (2.427 fold) were all significantly increased (P < 0.05). Looking into the cellular stress pathways, we further found upregulations of antioxidant genes SOD (2.59 fold) and catalase (2.17 fold) and ER stress markers Grp78 (2.794 fold), CHOP (3.407 fold), IRE1-α (2.104 fold) and ATF-6 (40.618 fold) mRNA expression in the PBMCs. When we stimulated the H9C2 cardiomyocytes with IL-1B and TNF-α at comparable conc. as found in the IDCM patients, the ER stress and inflammation were observed. Further, when we co-cultured these H9C2 with LPS stimulated THP-1 macrophages an exaggerated inflammation and myocyte stress was observed that led to apoptosis of the cells. Interestingly using ER stress inhibitor TUDCA, remarkably blocked the apoptosis and promotes cell survival. Conclusion Our results showed that a systemic inflammation caused by stressed PBMCs leads to cardiac deterioration and dilation in the unknown DCM cases. So far we have established that an exaggerated inflammation induce the circulating PBMCs to migrate and infiltrate the cardiac musculature where they induce oxidative stress and ER stress to the cardiomyocytes leading to apoptosis of the later. In long run these unknown forms of dilated cardiomyopathy patients suffer from a dilated heart due to a decline in the myocyte population. Our findings may warrant for a treatment strategy that alleviate the intracellular stress of the circulating leukocytes and cardiomyocytes to minimize the inflammatory changes of the heart.

Mitochondria-mediated apoptosis and endoplasmic reticulum stress are involved in the toxicity induced by copper in the porcine spleen.

Copper (Cu) is one of the common heavy metal pollutants in the environment, and its toxic mechanisms have been extensively studied. However, the immunotoxicity induced by Cu remains rarely reported, and the effects of Cu on endoplasmic reticulum stress and mitochondria-mediated apoptosis have been little studied in the spleen. In this study, pigs were fed with different contents of Cu (10, 125, and 250mg/kg Cu) for 80days to establish a toxicity model. The results showed the Cu exposure triggered endoplasmic reticulum stress in the spleen, as evidenced by increased mRNA and protein levels of GRP94, GRP78, CHOP, XBP1, ATF6, and JNK; the positive rate of GRP78 increased by immunofluorescence analysis. Additionally, mitochondrial fission and fusion homeostasis were disrupted, the expression levels of mitochondrial dynamics-related genes Mfn1, Mfn2, and OPA1 decreased, DRP1 increased, and the positive rate of Mfn1 decreased by immunofluorescence analysis. Furthermore, Cu exposure could induce apoptosis, as demonstrated by the increased expression level of related proteins and genes Bak, Bax, Caspase-3, P53, and Cytc. In conclusion, these results suggest chronic Cu exposure can lead to endoplasmic reticulum stress and imbalance in mitochondrial dynamics and induced apoptosis of pig spleen, and these results provided new insights into the underlying mechanism of Cu exposure caused splenic toxicity, which has public health implications where humans and animals are exposed to copper contamination.

Evaluation of linagliptin and insulin combined therapy on unfolded protein response in type 1 diabetic mouse heart.

The aim of this study is to reveal the effects of the use of linagliptin, a DPP-4 inhibitor due to its beneficial cardiovascular effects, on endoplasmic reticulum stress (ERS) signaling, which is involved in the pathogenesis of cardiovascular complications related to type 1 diabetes. BALB/c female mice (n = 72) were divided into six groups: control, diabetes+insulin, diabetes+linagliptin, diabetes+linagliptin+insulin, diabetes+TUDCA, and diabetes+TUDCA+insulin. Immunohistochemistry and western blot method, qRT-PCR, ELISA method, and malondialdehyde (MDA) measurements were performed. Linagliptin administered to the type 1 diabetic mouse heart significantly reduced the expression levels of the total and cleaved forms of ATF6, ATF4, and p-JNK, caspase 3. Immunohistochemical and western blot analyses revealed that cleaved caspase 3 protein expression was significantly increased in the diabetes+insulin group compared to the other groups. According to ELISA findings, TUDCA was more effective in reducing NOX 1 and MDA levels than linagliptin. While linagliptin decreased the Chop mRNA level, no change was observed in the Grp78 mRNA level. Our findings showed that there was not much difference between the administration of linagliptin alone or in combination with insulin. Our study reveals that linagliptin is an effective therapeutic agent on ERS and apoptotic UPR in type 1 diabetic hearts.

Endoplasmic reticulum stress inhibition ameliorated WFS1 expression alterations and reduced pancreatic islets’ insulin secretion induced by high-fat diet in rats

Endoplasmic reticulum (ER) stress is involved in the development of glucose homeostasis impairment. When ER stress occurs, the unfolded protein response (UPR) is activated to cope with it. One of the UPR components is WFS1 (Wolfram syndrome 1), which plays important roles in ER homeostasis and pancreatic islets glucose-stimulated insulin secretion (GSIS). Accordingly and considering that feeding high-fat food has a major contribution in metabolic disorders, this study aimed to investigate the possible involvement of pancreatic ER stress in glucose metabolism impairment induced by feeding high-fat diet (HFD) in male rats. After weaning, the rats were divided into six groups, and fed on normal diet and HFD for 20 weeks, then 4-phenyl butyric acid (4-PBA, an ER stress inhibitor) was administered. Subsequently, in all groups, after performing glucose tolerance test, the animals were dissected and their pancreases were removed to extract ER, islets isolation and assessment of GSIS. Moreover, the pancreatic ER stress [binding of immunoglobulin protein (BIP) and enhancer-binding protein homologous protein (CHOP)] and oxidative stress [malondialdehyde (MDA), glutathione (GSH) and catalase] biomarkers as well as WFS1 expression level were evaluated. HFD decreased pancreatic WFS1 protein and GSH levels, and enhanced pancreatic catalase activity, MDA content, BIP and CHOP protein and mRNA levels as well as Wfs1 mRNA amount. Accordingly, it increased BIP, CHOP and WFS1 protein levels in the extracted ER of pancreas. In addition, the HFD caused glucose intolerance, and decreased the islets’ GSIS and insulin content. However, 4-PBA administration restored the alterations. It seems that, HFD consumption through inducing pancreatic ER stress, altered WFS1 expression levels, reduced the islets’ GSIS and insulin content and finally impaired glucose homeostasis.

Disodium Fumarate Alleviates Endoplasmic Reticulum Stress, Mitochondrial Damage, and Oxidative Stress Induced by the High-Concentrate Diet in the Mammary Gland Tissue of Hu Sheep

The long-term feeding of the high-concentrate diet (HC) reduced rumen pH and induced subacute rumen acidosis (SARA), leading to mammary gland tissue damage among ruminants. Disodium fumarate enhanced rumen bufferation and alleviated a decrease in rumen pH induced by the HC diet. Therefore, the purpose of this study was to investigate whether disodium fumarate could alleviate endoplasmic reticulum (ER) stress, mitochondrial damage, and oxidative stress induced by the high-concentrate diet in the mammary gland tissue of Hu sheep. In this study, 18 Hu sheep in mid-lactation were randomly divided into three groups: one fed with a low-concentrate diet (LC) diet, one fed with a HC diet, and one fed with a HC diet with disodium fumarate (AHC). Each sheep was given an additional 10 g of disodium fumarate/day. The experiment lasted for eight weeks. After the experiment, rumen fluid, blood, and mammary gland tissue were collected. The results show that, compared with the LC diet, the HC diet could reduce rumen pH, and the pH below 5.6 was more than 3 h, and the LPS content of blood and rumen fluid in HC the diet was significantly higher than in the LC diet. This indicates that the HC diet induced SARA in Hu sheep. However, the supplementation of disodium fumarate in the HC diet increased the rumen pH and decreased the content of LPS in blood and rumen fluid. Compared with the LC diet, the HC diet increased Ca2+ content in mammary gland tissue. However, the AHC diet decreased Ca2+ content. The HC diet induced ER stress in mammary gland tissue by increasing the mRNA and protein expressions of GRP78, CHOP, PERK, ATF6, and IRE1α. The HC diet also activated the IP3R-VDAC1-MCU channel and lead to mitochondrial damage by inhibiting mitochondrial fusion and promoting mitochondrial division, while disodium fumarate could alleviate these changes. In addition, disodium fumarate alleviated oxidative stress induced by the HC diet by activating Nrf2 signaling and reducing ROS production in mammary gland tissue. In conclusion, the supplementation of disodium fumarate at a daily dose of 10 g/sheep enhanced rumen bufferation by maintaining the ruminal pH above 6 and reduced LPS concentration in ruminal fluid and blood. This reaction avoided the negative effect observed by non-supplemented sheep that were fed with a high-concentrate diet involving endoplasmic reticulum stress, oxidative stress, and mitochondrial damage in the mammary gland tissue of Hu sheep.

Tunicamycin induces depression-like behaviors in male rats, accompanied by initiated chaperon-mediated autophagy and decreased synaptic protein expression in the hippocampus

Background and aimEndoplasmic reticulum (ER) stress participates in the occurrence and development of depression, but the underlying mechanism is not fully understood. This study aimed to investigate the behavioral performance and intracerebral molecular changes in an ER stress model of male rats. MethodsIntrahippocampal injection of tunicamycin (TM) was performed on male rats as a model of ER stress. The body weight was determined, and behavioral tests, including sucrose preference test (SPT), open field test (OFT), and forced swimming test (FST), were performed to evaluate depressive and anxiety-like phenotypes within 8 days after injection. The levels of chaperone-mediated autophagy (CMA), synaptic proteins, and neuroinflammation related factors in this model were measured via real-time quantitative PCR and Western blot analysis. ResultsIntrahippocampal injection of TM (2 or 1 μg) induced depression-like behaviors in rats, as indicated by the reduced body weight, sucrose preference in SPT, central time in OFT, and increased immobility time in FST. The mRNA and protein levels of GRP78, ATF4, CHOP, LAMP2A, IL-1β, IL-6, and TNF-α were significantly increased, while the expressions of MEF2D, PSD95, SYN, p-CREB (Ser133), and BDNF were significantly decreased in the hippocampus in the model group compared with the sham group. ConclusionsThese results confirmed that intrahippocampal injection of TM was a valid method to induce an ER stress rat model with depression-like behaviors accompanied by decreased synaptic protein expression and neuroinflammation. The alteration in CMA-related proteins in this ER stress depression model indicated the involvement of CMA in the development of depression.

Allosteric Inhibition of c-Abl to Induce Unfolded Protein Response and Cell Death in Multiple Myeloma.

Endoplasmic reticulum stress activates inositol-requiring enzyme 1α (IRE1α) and protein kinase, R-like endoplasmic reticulum kinase (PERK), the two principal regulators of the unfolded protein response (UPR). In multiple myeloma, adaptive IRE1α signaling is predominantly activated and regulates cell fate along with PERK. Recently, we demonstrated that GNF-2, an allosteric c-Abl inhibitor, rheostatically enhanced IRE1α activity and induced apoptosis through c-Abl conformational changes in pancreatic β cells. Herein, we analyzed whether the pharmacological modulation of c-Abl conformation resulted in anti-myeloma effects. First, we investigated the effects of GNF-2 on IRE1α activity and cell fate, followed by an investigation of the anti-myeloma effects of asciminib, a new allosteric c-Abl inhibitor. Finally, we performed RNA sequencing to characterize the signaling profiles of asciminib. We observed that both GNF-2 and asciminib decreased cell viability and induced XBP1 mRNA splicing in primary human myeloma cells and myeloma cell lines. RNA sequencing identified the induction of UPR- and apoptosis-related genes by asciminib. Asciminib re-localized c-Abl to the endoplasmic reticulum, and its combination with a specific IRE1α inhibitor, KIRA8, enhanced cell death with the reciprocal induction of CHOP mRNA expression. Together, the allosteric inhibition of c-Abl-activated UPR with anti-myeloma effects; this could be a novel therapeutic target for multiple myeloma.

Dexmedetomidine Reduce Multiple Organ Injury Induced by Lung Ischemia/ Reperfusion of Mice Through Inhibiting Endoplasmic Reticulum Stress Response

To investigate the effect of Dexmedetomidine reduce brain, myocardium and kidney injury induced by lung ischemia/reperfusion of mice through inhibiting endoplasmic reticulum stress response. SPF C57BL/6J male mice were randomly divided into 5 groups (n = 10): the sham operation group (group sham), the ischemia reperfusion Group (Group I/R), the Atipamezole Group (Group Atip), dexmedetomidine Group (Group-DEX) and dexmedetomidine+AtipamezoleGroup (Group DA). Lung ischemia reperfusion models were established with the method of 30min in-vivo left hilus occlusion followed by 180min reperfusion. In addition to the steps same as Group I/R, the mice in Groups Atip, Dex and DA were given Atip(250μg/kg), Dex(20μg/kg), and Dex+ Atip(20μg/kg+250μg/kg) respectively by intraperitoneal injection 30min before hilar occlusion. After reperfusion, the blood was taken from the eye-orbit to determine the activity of serum CK-MB and LDH and the levels of serum creatinine and urea nitrogen. The brain tissue was taken to determine the brain water content. The tissue of myocardium, brain and kidney was taken to observe the cellular morphology change under the light microscope. The expression of Caspase3 activity in heart, brain and kidney was determined with Thermos Scientific Microplate Reader. The TUNEL method was used to detect the apoptotic index of heart, brain and kidney. The Western blot and RT-PCR were used to determine the expression of proteins and mRNA of JNK, Caspase12, CHOP and GRP78. Compared with Group Sham, obvious injury in the tissue of myocardium, brain and kidney was observed in the other four groups under the light microscope. The activity of CKMB and LDH, and the levels of serum creatinine, urea nitrogen and brain water content were significantly increased as well. The apoptotic index, Caspase3 activity, and the expression of the proteins and mRNA of JNK, Caspase12, CHOP and GRP78 were also significantly elevated. Compared with Group I/R, Atip and DA, the mitigated injury in the myocardium, brain and kidney was observed in Group DEX under the light microscope. The activity of CK-MB and LDH, and the levels of serum creatinine, urea nitrogen and brain water content were decreased and the apoptotic index, Caspase3 activity, and the expression of the proteins and mRNAs of JNK, Caspase12 and CHOP were also significantly decreased in Group DEX, but the proteins and mRNA expression of GRP78 were significantly elevated. For the comparison among theGroup I/R, Atip and DA, there is no significant difference was found.(grp78).The pre-administration of DEX can mitigate the heart, brain and kidney injury induced by lung ischemia reperfusion. The mechanism may be related to the activation of α2-adrenergic receptor and inhibition of endoplasmic reticulum over-response.

비만유도 흰쥐에서 반복적 절식과 운동부하가 골격근내 세포사멸 및 당수송체에 미치는 영향

The purpose of this study is to rely on an extremely limited diet weight loss is a heavy negative effect on intracellular lipid metabolism and reduced insulin secretion induced by inhibition of intracellular homeostasis was thought to be a factor. For therefore investigate the physiological effects of exercise was conducted. Twenty-two Sparague Dawley rats were divided into 4 groups as follows: high fat diet group(HF, n=8), high fat diet with repeated fasting and refeeding group (HFFR, n=8), repeated fasting and refeeding and exercise after high fat diet group(HFFR＋EX, n=8) and pair feding to HFFR group (HFFR＋PF, n=8). Diet-induced obesity was fed a high-fat for 8 weeks, and obesity intervention was drived by treadmill running exercise program or food intake restriction. As a result, through repeated food restriction were decreased body weight, body fat reduction and improvement blood lipid profiles. And when combined exercise training was confirmed this trend more pronounced(p.05). Furthermore, the expression of GLUT4 expression in skeletal muscle decreases due to restricted movement, but it was confirmed to be increased through exercise training. Expression of apoptosis-related CHOP mRNA were combined exercise and dietary restriction significantly decreased. In conclusion, the results of this study, including obesity, diabetes and other metabolic syndrome disorders in a course of treatment being carried out mainly in the dietary restriction and exercise programs to the specific direction is expected to be present.

Mechanism of Isoquercitrin Inducing Apoptosis of HepG2 Cells Through PERK-eIF2a-ATF4 Signaling Pathway

Purpose: To observe the intervention effect of isoquercitrin on related factors in PERK-eIF2a-ATF4 signaling pathway after it acts on HepG2 cells. Materials and Method: HepG2 cells were tested with different concentrations of isoquercitrin. MTT assay was employed to detect the cell proliferation. Expressions of GRP78, PERK, elF2a, ATF4 and CHOP mRNA and protein were measured by qRT-PCR and Western blot. Results: Cell experiments showed that isoquercitrin inhibited the proliferation of HepG2 cells in a dose-and time-dependent manner. Compared with the control group, the expressions of GRP78, PERK, elF2a, ATF4, CHOP mRNA and the expressions of GRP78, p-PERK, elF2a, ATF4, CHOP protein were significantly increased after isoquercitrin treatment, and the expressions of PERK protein decreased significantly. Conclusion: The present study verified that isoquercitrin induce the apoptosis of HepG2 cells, and its mechanism may be related to the intervention of related factors in PERK-eIF2a-ATF4 signaling pathway.

RETRACTED: Protective effect of Huangpu Tongqiao capsule against Alzheimer's disease through inhibiting the apoptosis pathway mediated by endoplasmic reticulum stress in vitro and in vivo

ObjectivesHuangpu Tongqiao Capsule (HPTQC) is a traditional Chinese medicine (TCM) that has been used to treat Alzheimer's disease (AD). This study was to explore the pharmacological action and molecular mechanism of HPTQC in the treatment of AD. MethodsThe possible targets of HTPQC were predicted by the molecular docking technique. Intraperitoneal injection of D-galactose and bilateral injection of Aβ25-35 in hippocampus induced AD rat model. Morris water maze was used to observe learning and memory function. The primary hippocampal neurons were induced by Aβ25-35. Moreover, the apoptosis rate of hippocampal nerve cells was detected through AnnexinV/PI double standard staining. The mRNA and protein levels of GRP78, CHOP, Caspase 12, Caspase 9, and Caspase 3 were detected by PCR and western blot. ResultsThe prediction results suggest that HPTQC may act on GRP78. HPTQC significantly improved the learning and memory function, and decreased neuronal apoptosis in vivo and in vitro. In addition, HPTQC could decrease the mRNA and protein expression levels of GRP78, CHOP, Caspase12, Caspase9, and Caspase3, and the effect trend was consistent with the specific inhibitor of GRP78. ConclusionsHPTQC has a neuroprotective effect against AD by inhibiting the apoptosis pathway mediated by endoplasmic reticulum stress.

Dietary aflatoxin B1 caused the growth inhibition, and activated oxidative stress and endoplasmic reticulum stress pathway, inducing apoptosis and inflammation in the liver of northern snakehead (Channa argus)

The purpose of this study was to investigate the effects of dietary aflatoxin B1 (AFB1) on growth performance and AFB1 biotransformation, and hepatic oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and inflammation in northern snakehead (Channa argus). A total of 600 northern snakeheads (7.52 ± 0.02 g) were divided into five groups (three replicates/group) and fed the diets with AFB1 at concentrations of 0, 50, 100, 200, and 400 ppb for 8 weeks. The results demonstrated that dietary AFB1 (≥ 200 ppb) reduced FBW, WG, and SGR. 100, 200, and 400 ppb AFB1 treatment groups significantly decreased the PER, CRP, C3, C4, IgM, and LYS levels in northern snakehead, while FCR was significant increased. Moreover, dietary AFB1 (100, 200, and 400 ppb) increased cyp1a, cyp1b (except 400 ppb), and cyp3a mRNA expression levels, while reducing the GST enzymatic activity and mRNA expression levels in northern snakehead. Furthermore, AFB1 (≥ 100 ppb) increased ROS, MDA, and 8-OHdG levels, and grp78, ire1, perk, jnk, chop, and traf2 mRNA expression levels, and decreased SOD, CAT, GSH-Px, and GSH (except 100 ppb) levels and the gene expression levels of cat, gsh-px (except 100 ppb), and Cu/Zn sod. In addition, AFB1 (100, 200, and 400 ppb) up-regulated the cyt-c, bax, cas-3, and cas-9 mRNA levels in the liver, while down-regulating the bcl-2 expression levels. Meanwhile, the expression levels of nf-κb, tnf-α (except 100 ppb), il-1β, and il-8 in the liver were up-regulated in AFB1 treatment groups (≥ 100 ppb), while the iκbα mRNA levels were down-regulated. In summary, dietary AFB1 reduced growth performance and humoral immunity in northern snakehead. Meanwhile, the cyclic occurrence of oxidative stress and ER stress, and induced apoptosis and inflammation, is one of the main reasons for AFB1-induced liver injury in the northern snakehead, which will provide valuable information and a fresh perspective for further research into AFB1-induced liver injury in fish.