Expression Of Endoplasmic Reticulum Stress Proteins Research Articles

Effects of Epothilone D on Social Defeat Stress-induced Changes in Microtubule-related and Endoplasmic Reticulum Stress Protein Expression

Effects of Epothilone D on Social Defeat Stress-induced Changes in Microtubule-related and Endoplasmic Reticulum Stress Protein Expression

Effects of glial-derived neurotrophic factor on remodeling and mitochondrial function in human airway smooth muscle cells.

Airway smooth muscle (ASM) cells play important roles in airway remodeling of asthma. Our previous studies show that in vivo administration of glial-derived neurotrophic factor (GDNF) in mice induces thickening and collagen deposition in bronchial airways, whereas chelation of GDNF by GFRα1-Fc attenuates airway remodeling in the context of allergen exposure. To determine whether GDNF has direct effects on ASM, in this study, we examined GDNF in ASM cells from normal versus asthmatic humans. We found that GDNF treatment of human ASM cells had only minor effects on cell proliferation and migration, intracellular expression or extracellular deposition of collagen I (COL1), collagen III (COL3), and fibronectin. Endoplasmic reticulum (ER) stress response and mitochondrial function have been implicated in asthma. We investigated whether GDNF regulates these aspects in human ASM. We found that GDNF treatment did not affect ER stress protein expression in normal or asthmatic cells. However, GDNF treatment impaired mitochondrial morphology in ASM but without significant effects on mitochondrial respiration. Thus, it is likely that in vivo effects of GDNF on airway remodeling per se involve cell types other than those on ASM, and thus ASM may serve more as a source of GDNF rather than a target.NEW & NOTEWORTHY Our previous study suggests that glial-derived neurotrophic factor (GDNF) is involved in allergen-induced airway hyperreactivity and remodeling in vivo. Here, we show that GDNF has no direct effects in remodeling of human airway smooth muscle (ASM) but GDNF dysregulates mitochondrial morphology in human ASM in the context of asthma.

Mito-LND and (E)-Akt inhibitor-IV: novel compounds inducing endoplasmic reticulum stress and ROS accumulation against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. Although multi-kinase inhibitors can prolong the overall survival of late-stage HCC patients, the emergence of drug resistance diminishes these benefits, ultimately resulting in treatment failure. Therefore, there is an urgent need for novel and effective drugs to impede the progression of liver cancer. This study employed a concentration gradient increment method to establish acquired sorafenib or regorafenib-resistant SNU-449 cells. Cell viability was assessed using the cell counting kit-8 assay. A library of 793 bioactive small molecules related to metabolism screened compounds targeting both parental and drug-resistant cells. The screened compounds will be added to both the HCC parental cells and the drug-resistant cells, followed by a comprehensive assessment. Intracellular adenosine triphosphate (ATP) levels were quantified using kits. Flow cytometry was applied to assess cell apoptosis and reactive oxygen species (ROS). Real-time quantitative PCR studied relative gene expression, and western blot analysis assessed protein expression changes in HCC parental and drug-resistant cells. A xenograft model in vivo evaluated Mito-LND and (E)-Akt inhibitor-IV effects on liver tumors, with hematoxylin and eosin staining for tissue structure and immunohistochemistry staining for endoplasmic reticulum stress protein expression. From the compound library, we screened out two novel compounds, Mito-LND and (E)-Akt inhibitor-IV, which could potently kill both parental cells and drug-resistant cells. Mito-LND could significantly suppress proliferation and induce apoptosis in HCC parental and drug-resistant cells by upregulating glycolytic intermediates and downregulating those of the tricarboxylic acid (TCA) cycle, thereby decreasing ATP production and increasing ROS. (E)-Akt inhibitor-IV achieved comparable results by reducing glycolytic intermediates, increasing TCA cycle intermediates, and decreasing ATP synthesis and ROS levels. Both compounds trigger apoptosis in HCC cells through the interplay of the AMPK/MAPK pathway and the endoplasmic reticulum stress response. In vivo assays also showed that these two compounds could significantly inhibit the growth of HCC cells and induce endoplasmic reticulum stress. Through high throughput screening, we identified that Mito-LND and (E)-Akt inhibitor-IV are two novel compounds against both parental and drug-resistant HCC cells, which could offer new strategies for HCC patients.

Activation of NFE2L2 Alleviates Endoplasmic Reticulum Stress-Mediated Pyroptosis in Murine Hippocampus: A Bioinformatics Analysis and Experimental Validation.

Pyroptosis has been implicated in many pathologic processes, including endoplasmic reticulum stress (ERS). However, the underlying mechanisms and molecular targets of ERS affecting pyroptosis still need further exploration. We obtained gene sets associated with ERS and pyroptosis, and the common genes were regarded as crosstalk genes linking ERS and pyroptosis. Protein-protein interaction (PPI) network was constructed, and the hub genes were obtained via Cytoscape. Moreover, to validate the efficacy of the therapeutic target, neurological tests, brain water content measurements, Nissl staining, Western blot, ELISA, TUNEL analyses, and transmission electron microscopy were performed in a mouse model. A total of 13 crosstalk genes were acquired, and enrichment analysis revealed that these genes were mainly enriched in stress-associated cellular processes and pathways, including KEAP1-NFE2L2 pathway. The hub gene, NFE2L2, was identified by Cytoscape, and tert-butylhydroquinone (tBHQ) was screened as candidate drug to activate NFE2L2. Western blot and ELISA results showed that activation of NFE2L2 could attenuate the expression of ERS and pyroptosis-related proteins by promoting nuclear translocation of Nrf2 (encoded by NFE2L2). Pathological evaluation by Nissl staining and TUNEL assay reflected a similar trend. Furthermore, activation of NFE2L2 ameliorated neurological deficits and reduced brain edema. In conclusion, our bioinformatic analysis results established the theoretical foundation of NFE2L2 as a promising therapeutic target. Moreover, in the mouse model, tBHQ pretreatment further confirmed the effectiveness of this target. We hypothesized NFE2L2 may play a key role in the progression of ERS-mediated pyroptosis. These findings may inspire new ideas to treat neurological disorders.

Impact of Aging and Disuse on Markers of Endoplasmic Reticulum Stress and the Unfolded Protein Response

Aging and disuse can impair skeletal muscle responses to stimuli and are key drivers in dysregulated proteostasis. Such dysfunction can lead to an accumulation of misfolded proteins resulting in endoplasmic reticulum stress (ERS) and the unfolded protein response (UPR). While the UPR blunts global translation and enhances the production of chaperones and foldases, little is known about ERS responses across the lifespan and in disuse contexts. Therefore, the purposes of this study were to examine if: i) the expression of ERS and UPR effector proteins differ across the lifespan in rats, and ii) age-associated patterns were recapitulated by 10 days of hindlimb casting in a separate cohort of rats. We hypothesized that aging would enhance the expression of ERS related proteins, and this expression pattern would be mimicked by 10 days of limb disuse. Male Fischer 344 rats were sacrificed at 3, 6, 12, 18, and 24 months (mo) of age, and plantaris (PLT) and soleus (SOL) muscles were collected for analysis. Six mo female Wistar rats were sacrificed after either 10 days of hindlimb casting or continued ambulation (age matched control), after which whole gastrocnemius muscles were collected for analysis. The effector eukaryotic initiation factor 2 alpha (eIF2a) was different across the lifespan (phospho/pan) in PLT and SOL muscles (p<0.001) and was upregulated in the PLT at 24 mo as compared to 3 mo (P=0.006). The downstream UPR effector C/EBP homologous protein (CHOP) was differentially expressed across the lifespan in PLT and SOL (p<0.001). CHOP was upregulated ~2.79 and 3.08-fold at 18 and 24 mo respectively in the SOL (p<0.001), and 2.62 and 2.44-fold at 18 and 24 mo respectively in the PLT. Finally, principal effector Activating Transcription Factor 6 (ATF6) was differentially expressed across the lifespan (cleaved/pan) in the PLT and SOL (P≤0.024) whereby a 1.23-fold up-regulation was evident at 18 mo in the SOL (P=0.035) and 1.34-fold at 18 mo in the PLT (P=0.026). The CHOP and ATF6 proteins were similarly dysregulated in our disuse model, whereby 1.83-fold (p<0.001) and 1.30-fold (P=0.002) greater values were observed in hind limb casted versus control rats. Effectors of the UPR pathway are upregulated in skeletal muscle across the lifespan in rats, and this effect is recapitulated with disuse. These findings warrant further research for potential therapeutics related to age- and disuse-related skeletal muscle atrophy. Funding for this study was provided by discretionary laboratory funds from MDR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Txnrd2 Attenuates Early Brain Injury by Inhibition of Oxidative Stress and Endoplasmic Reticulum Stress via Trx2/Prx3 Pathway after Intracerebral Hemorrhage in Rats

Thioredoxin-reductase 2 (Txnrd2) belongs to the thioredoxin-reductase family of selenoproteins and is a key antioxidant enzyme in mammalian cells to regulate redox homeostasis. Here, we reported that Txnrd2 exerted a major influence in brain damage caused by Intracerebral hemorrhage (ICH) by suppressing endoplasmic reticulum (ER) stress oxidative stress and via Trx2/Prx3 pathway. Furthermore, we demonstrated that pharmacological selenium (Se) rescued the brain damage after ICH by enhancing Txnrd2 expression. Primarily, expression and localization of Txnrd2, Trx2 and Prx3 were determined in collagenase IV-induced ICH model. Txnrd2 was then knocked down using siRNA interference in rats which were found to develop more severe encephaledema and neurological deficits. Mechanistically, we observed that loss of Txnrd2 leads to increased lipid peroxidation levels and ER stress protein expression in neurons and astrocytes. Additionally, it was revealed that Se effectively restored the expression of Txnrd2 in brain and inhibited both the activity of ER stress protein activity and the generation of reactive oxygen species (ROS) by promoting Trx2/Prx3 kilter when administrating sodium selenite in lateral ventricle. This study shed light on the effect of Txnrd2 in regulating oxidative stress and ER stress via Trx2/Prx3 pathway upon ICH and its promising potential as an ICH therapeutic target.

Xingnaojing injection alleviates cerebral ischemia/reperfusion injury through regulating endoplasmic reticulum stress in Vivo and in Vitro

BackgroundXingnaojing (XNJ) injection, an extract derived from traditional Chinese medicine, is commonly used to treat ischemic stroke (IS). Previous studies have shown that XNJ has the ability to alleviate apoptosis in cerebral ischemia-reperfusion injury. However, the potential mechanisms have not been clarified. ObjectiveTo identify the neuroprotective effect of XNJ and explore whether XNJ inhibits cell apoptosis associated with endoplasmic reticulum stress (ERS) after IS. MethodsIn this study, cultured hippocampal neurons from mouse embryos and Sprague-Dawley rats were assigned randomly to four groups: sham, model, XNJ, and edaravone. The treatment groups were administered 2 h after modelling. Neurological deficit scores and motor performance tests were performed after 24 h of modelling. Additionally, pathomorphology, cell apoptosis and calcium content were evaluated. To ascertain the expression of ERS proteins, western blotting and polymerase chain reaction were employed. ResultsThe results indicated that XNJ treatment resulted in a notable decrease in infarct volume, apoptosis and missteps compared with the model group. XNJ also exhibited improvements in neurological function, grip strength and motor time. The calcium content significantly reduced in XNJ group. The XNJ administration resulted in a reduction in the levels of proteins associated with ERS including CHOP, GRP78, Bax, caspase-12, caspase-9, and cleaved-caspase-3, but an increase of the Bcl-2/Bax ratio. Furthermore, the downregulation of mRNA expression of CHOP, GRP78, caspase-12, caspase-9, and caspase-3 was confirmed in both cultured neurons and rat model. ConclusionThese findings suggest that XNJ may alleviate apoptosis by modulating the ERS-induced apoptosis pathway, making it a potential novel therapeutic approach for ischemic stroke.

Cholesterol induction in CD8+ T cell exhaustion in colorectal cancer via the regulation of endoplasmic reticulum-mitochondria contact sites.

Hypercholesterolemia is one of the risk factors for colorectal cancer (CRC). Cholesterol can participate in the regulation of human T cell function and affect the occurrence and development of CRC. To elucidate the pathogenesis of CRC immune escape mediated by CD8+ T cell exhaustion induced by cholesterol. CRC samples (n = 217) and healthy individuals (n = 98) were recruited to analyze the relationship between peripheral blood cholesterol levels and the clinical features of CRC. An animal model of CRC with hypercholesterolemia was established. Intraperitoneal intervention with endoplasmic reticulum stress (ERS) inhibitors in hypercholesterolemic CRC mice was performed. CD69, PD1, TIM-3, and CTLA-4 on CD8+ T cells of spleens from C57BL/6J mice were detected by flow cytometry. CD8+ T cells were cocultured with MC38 cells (mouse colon cancer cell line). The proliferation, apoptosis, migration and invasive ability of MC38 cells were detected by CCK-8 assay, Annexin-V APC/7-AAD double staining, scratch assay and transwell assay, respectively. Transmission electron microscopy was used to observe the ER structure of CD8+ T cells. Western blotting was used to detect the expression of ERS and mitophagy-related proteins. Mitochondrial function and energy metabolism were measured. Immunoprecipitation was used to detect the interaction of endoplasmic reticulum-mitochondria contact site (ERMC) proteins. Immunofluorescence colocalization was used to detect the expression and intracellular localization of ERMC-related molecules. Peripheral blood cholesterol-related indices, including Tc, low density lipoproteins (LDL) and Apo(a), were all increased, and high density lipoprotein (HDL) was decreased in CRCs. The proliferation, migration and invasion abilities of MC38 cells were enhanced, and the proportion of tumor cell apoptosis was decreased in the high cholesterol group. The expression of IL-2 and TNF-α was decreased, while IFN-γ was increased in the high cholesterol group. It indicated high cholesterol could induce exhaustion of CD8+ T cells, leading to CRC immune escape. Hypercholesterolemia damaged the ER structure of CD8+ T cells and increased the expression of ER stress molecules (CHOP and GRP78), lead to CD8+ T cell exhaustion. The expression of mitophagy-related proteins (BNIP3, PINK and Parkin) in exhausted CD8+ T cells increased at high cholesterol levels, causing mitochondrial energy disturbance. High cholesterol enhanced the colocalization of Fis1/Bap31, MFN2/cox4/HSP90B1, VAPB/PTPIP51, VDAC1/IPR3/GRP75 in ERMCs, indicated that high cholesterol promoted the intermolecular interaction between ER and mitochondrial membranes in CD8+ T cells. High cholesterol regulated the ERS-ERMC-mitophagy axis to induce the exhaustion of CD8+ T cells in CRC.

Rare protective variants and glaucoma-relevant cell stressors modulate Angiopoietin-like 7 expression.

Rare missense and nonsense variants in the Angiopoietin-like 7 (ANGPTL7) gene confer protection from primary open-angle glaucoma (POAG), though the functional mechanism remains uncharacterized. Interestingly, a larger variant effect size strongly correlates with in silico predictions of increased protein instability (r = -0.98), suggesting that protective variants lower ANGPTL7 protein levels. Here, we show that missense and nonsense variants cause aggregation of mutant ANGPTL7 protein in the endoplasmic reticulum (ER) and decreased levels of secreted protein in human trabecular meshwork (TM) cells; a lower secreted:intracellular protein ratio strongly correlates with variant effects on intraocular pressure (r = 0.81). Importantly, accumulation of mutant protein in the ER does not increase expression of ER stress proteins in TM cells (P > 0.05 for all variants tested). Cyclic mechanical stress, a glaucoma-relevant physiologic stressor, also significantly lowers ANGPTL7 expression in primary cultures of human Schlemm's canal (SC) cells (-2.4-fold-change, P = 0.01). Collectively, these data suggest that the protective effects of ANGPTL7 variants in POAG stem from lower levels of secreted protein, which may modulate responses to physiologic and pathologic ocular cell stressors. Downregulation of ANGPTL7 expression may therefore serve as a viable preventative and therapeutic strategy for this common, blinding disease.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

Cyanidin-3-O-glucoside plays a protective role against renal ischemia/ reperfusion injury via the JAK/STAT pathway.

To investigate the role of cyanidin-3-O-glucoside (C3G) in renal ischemia/reperfusion (I/R) injury and the potential mechanisms. Mouse models were established by clamping the left renal vessels, and in vitro cellular models were established by hypoxic reoxygenation. Renal dysfunction and tissue structural damage were significantly higher in the I/R group. After treatment with different concentrations of C3G, the levels of renal dysfunction and tissue structural damage decreased at different levels. And its protective effect was most pronounced at 200 mg/kg. The use of C3G reduced apoptosis as well as the expression of endoplasmic reticulum stress (ERS)-related proteins. Hypoxia/reoxygenation (H/R)-induced apoptosis and ERS are dependent on oxidative stress in vitro. In addition, both AG490 and C3G inhibited the activation of JAK/STAT pathway and attenuated oxidative stress, ischemia-induced apoptosis and ERS. The results demonstrated that C3G blocked renal apoptosis and ERS protein expression by preventing reactive oxygen species (ROS) production after I/R via the JAK/STAT pathway, suggesting that C3G may be a potential therapeutic agent for renal I/R injury.

The neuroprotective effect of Dl-3-n-butylphthalide in epileptic rats via inhibiting endoplasmic reticulum stress.

The purpose of this study is to investigate whether Dl-3-n-Butylphthalide (NBP) has a neuroprotective effect on pilocarpine-induced epileptic (EP) rats through endoplasmic reticulum stress (ERS)-mediated apoptosis. The Sprague-Dawley rats were divided into four groups: control (CON), EP, EP + NBP 60 (NBP 60 mg/kg) and EP + NBP 120 (NBP 120 mg/kg) groups. After the successful establishment of the temporal lobe EP model using the lithium-pilocarpine, the rats were given NBP for 28 consecutive days in EP + NBP 60 and EP + NBP 120 groups. Then, the spontaneous recurrent seizure (SRS) latency, SRS frequency and seizure duration were observed in each group. In order to observe the abnormal discharge of rats, the intracranial electrodes were implanted to monitor the electroencephalogram. Nissl staining was used to observe the damage to the hippocampal CA1 neurons, TUNEL staining was employed to observe hippocampal neuronal apoptosis. Western blot was used to detect the expression of ERS and ERS-mediated apoptotic proteins. NBP 60 and NBP 120 decreased SRS frequency (all p < 0.05), shortened seizure duration (all p < 0.05), and reduced the abnormal discharge of the brain. Nissl staining and TUNEL staining results show that NBP protected the hippocampal neurons from damage (all p < 0.05) and inhibited hippocampal neuronal apoptosis in EP rats (all p < 0.05). NBP 60 and NBP 120 could reduce ERS and ERS-mediated apoptotic protein expression in EP rats (all p < 0.05). In addition, the therapeutic effect of NBP on epilepsy in rats is dose-dependent. The SRS frequency of the EP + NBP 120 group was lower, and the seizure duration was shorter than in the EP + NBP 60 group (all p < 0.05), and there were more neurons in the EP + NBP 120 group than in the EP + NBP 60 group ( p < 0.05). NBP had a significant neuroprotective effect in EP rats. Large doses of NBP are more effective than low doses. The mechanism may be associated with the inhibition of ERS and ERS-mediated apoptosis.

Purpurogallin improves neurological functions of cerebral ischemia and reperfusion mice by inhibiting endoplasmic reticulum stress and neuroinflammation

BackgroundPurpurogallin (PPG) has been testified to have neuroprotective effects. This study intends to probe the neuroprotection of PPG on cerebral ischemia/reperfusion (I/R) injury and its potential mechanism. MethodsC57/B6 mice, BV2 microglia and HT22 hippocampal neurons were used for in-vivo and in-vitro experiments. I/R injury models were constructed using middle cerebral artery occlusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R), respectively. The expression of apoptosis and inflammatory proteins, and endoplasmic reticulum (ER) stress proteins were gauged by Western blotting (WB). The contents of inflammatory cytokines in OGD/R-induced BV2 microglia were testified by enzyme-linked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8), TUNEL assay and flow cytometry (FCM) were utilized to examine the viability and apoptosis of cells. The neurological, learning and memory functions were evaluated by the modified neurological severity score (mNSS) and water maze experiment. 2, 3, 5-triphenyltetrazole chloride (TTC) staining was utilized to calculate the volume of cerebral infarction and cerebral edema in the peri-infarct area. Apoptosis-related proteins, inflammation-related proteins and ER stress proteins were gauged by WB. ELISA was conducted to verify inflammatory cytokines. ResultsPPG treatment notably abated the expression of ER stress proteins and inflammatory factors in OGD/R-induced BV2 microglia and boosted HT22 neuron's viability and eased their apoptosis in comparison to the control group. In vivo, PPG treatment signally lessened cerebral infarct area, cerebral edema, and neurological deficit scores in MCAO/R mice. Additionally, PPG caused a dramatic decline in neuronal apoptosis and levels of ER stress proteins and inflammatory factors in the brain’s peri-infarct region of MCAO/R mice. Mechanically, PPG blocked the TLR4/NF-κB pathway in OGD/R-induced BV2, HT22 neurons, and the MCAO/R mice. ConclusionPPG attenuates brain I/R damage probably by suppressing ER stress and neuroinflammation via inactivation of the TLR4/NF-κB pathway, suggesting that PPG may be a candidate drug for treating cerebral I/R injury.

MTOR Modulates the Endoplasmic Reticulum Stress-Induced CD4+ T Cell Apoptosis Mediated by ROS in Septic Immunosuppression.

Introduction When sepsis attacks the body, the excessive reactive oxygen species (ROS) production can result to endoplasmic reticulum stress (ERS) and eventually cause lymphocyte apoptosis. The mammalian target of rapamycin (mTOR) is essential for regulating lymphocyte apoptosis; we hypothesized that it mediates CD4+ T cell apoptosis during ROS-related ERS. Method We, respectively, used ROS and ERS blockers to intervene septic mice and then detected ERS protein expression levels to verify the relationship between them. Additionally, we constructed T cell-specific mTOR and TSC1 gene knockout mice to determine the role of mTOR in ROS-mediated, ERS-induced CD4+ T cell apoptosis. Results Blocking ROS significantly suppressed the CD4+ T cell apoptosis associated with the reduction in ERS, as revealed by lower levels of GRP78 and CHOP. ERS rapidly induced mTOR activation, leading to the induction of CD4+ T cell apoptosis. However, mTOR knockout mice displayed reduced expression of apoptotic proteins and less ER vesiculation and expansion than what was observed in the wild-type sepsis controls. Conclusion By working to alleviate ROS-mediated, ERS-induced CD4+ T cell apoptosis, the mTOR pathway is vital for CD4+ T cell survival in sepsis mouse model.

Anticancer Activity of Cannabidiol (CBD) in Human Colorectal Cancer Cells: A Mechanistic Study

ObjectivesCannabidiol (CBD) is a major non-psychoactive bioactive component of the phytocannabinoids abundant in cannabis. Its potential use for treatment of colorectal cancer has been proposed by several studies. This study investigated the anti-cancer activity of CBD and the mechanism of that activity in human colorectal cancer cells. MethodsHuman colorectal cancer cell lines (SW620, SW480, HCT116, and Caco-2) and a human normal colon cell line (CCD18CO) were maintained in DMEM supplemented with 10% FBS and antibiotics. Cell viability was measured by MTT assay. Cell cycle phase was measured by flow cytometry. Apoptosis was analyzed by measuring caspase 3/7 enzyme activity. Western blotting was performed to compare expression of proteins regulating cell cycle arrest, apoptosis, and endoplasmic reticulum (ER) stress. Selective inhibitors for cannabinoid receptors were used to test the relevance of each receptor for CBD-mediated cell viability repression and apoptosis induction. Different types of CBD derivatives were tested for effects on cell viability. Data were analyzed using one-way analysis of variance (ANOVA), or two-way ANOVA followed by Bonferroni post-hoc tests (Dunnett T3 tests for unequal variance) for intergroup comparisons. ResultsCBD repressed viability of SW620, SW480, HCT116, and Caco-2 human colorectal cancer cells, with respective IC50 values of 5.4, 10.4, 10.8, and 20 μM for 24-hour treatment and 4.7, 5.8, 5.7, and 13.8 μM for 48-hour treatment. Moreover, CBD treatment led to G1-phase cell cycle arrest and an increased sub-G1 population with downregulation of cyclin D1, cyclin D3, CDK2, CDK4, and CDK6. It also increased activity of caspase 3/7, production of cleaved PARP, and expression of ER stress proteins (BiP, IRE1α, p-eIF2, ATF3 and ATF4). Repression of cell viability and induction of apoptotic cell death occurred through a mechanism dependent on cannabinoid receptor type 2 (CB2). Suppression of cell viability was also observed in cells treated with other of non-psychoactive cannabinoid derivatives (cannabidivarin, cannabigerol, cannabicyclol, cannabigerovarin). ConclusionsOur data indicate that CBD and its derivatives could be promising agents for the prevention of human colorectal cancer. Funding SourcesNo funding source.

Ginsenoside-Mc1 reduces ischemia/reperfusion-induced cardiac arrhythmias through activating JAK2/STAT3 pathway and attenuating oxidative/endoplasmic reticulum stress in hyperlipidemic rats

Abstract Objectives Patients suffering from myocardial ischemia-reperfusion (IR) injuries usually have varying degrees of negatively-affecting comorbidities like hyperlipidemia. We evaluated the preconditioning effect of ginsenoside-Mc1 on reperfusion injury-induced myocardial arrhythmias, along with cardiac oxidative stress, endoplasmic reticulum stress protein expression, and histological damage in hyperlipidemic rats, and further, explore the role of JAK2/STAT3 activity. Methods Thirty-five Sprague–Dawley rats fed a high-fat diet for eight weeks. Ginsenoside-Mc1 (10 mg/dL, IP) was administered to hyperlipidemic rats daily for one month before IR injury. IR injury was induced by 35 min LAD coronary artery ligation and subsequent 60-min reperfusion. A selective JAK2 inhibitor (AG490) was injected before IR injury. Electrocardiography was recorded and myocardial arrhythmias (ventricular premature complexes, tachycardia, and fibrillation) were evaluated in the reperfusion phase according to Lambeth convention. Hematoxylin-Eosin staining, spectrophotometry, and Western blotting techniques were employed to measure the endpoints. Results IR injury in hyperlipidemic rats significantly increased the reperfusion-induced myocardial arrhythmias counts, timing, incidence, and severity. The expression of proteins of endoplasmic reticulum stress (p-PERK, p-eIF2α, CHOP), and oxidative stress marker malondialdehyde were significantly upregulated following IR induction, whereas antioxidant superoxide-dismutase and JAK2/STAT3 proteins expression significantly reduced, as compared with untreated-hyperlipidemic rats. Administration of ginsenoside-Mc1 to hyperlipidemic rats significantly corrected the arrhythmogenic feature of IR injury, reduced phosphorylation and expression of PERK, eIF2α, CHOP, and improved oxidative stress and histological changes. Interestingly, inhibition of the JAK2/STAT3 pathway via AG490 significantly abolished ginsenoside-Mc1-induced cardioprotection. Conclusions Taken together, ginsenoside-Mc1 exerts substantial anti-arrhythmogenic influences against myocardial IR injury in hyperlipidemic rats through activation of JAK2/STAT3 pathway and subsequent reduction of oxidative/endoplasmic reticulum stress.

Icariin Attenuation of Diabetic Kidney Disease Through Inhibition of Endoplasmic Reticulum Stress via G Protein-Coupled Estrogen Receptors.

Diabetic kidney disease (DKD) is the most common complication of diabetes mellitus and has become the primary cause of End-Stage Renal Disease (ESRD) globally. Icariin (ICA), an effective component extracted from Epimedium, has antiosteoporosis effect, antitumor effects, anti-ischemia effects, and other effects. In this study, a mouse DKD model was established, and Icariin solid nanoliposomes were administered to determine whether ICA had a protective effect on the renal function of DKD mice by regulating estrogen level and endoplasmic reticulum (ER) stress pathway. The results showed that the microalbumin/creatinine in urine, serum urea nitrogen, and CHOL in ICA cultured DKD mice significantly decreased, and mice nephropathy improved significantly. rat renal tubule epithelial cells were further tested, and the rat renal tubule epithelial cells were modeled by cultured cells with high glucose. The results showed that high glucose could promote the proliferation of renal tubular epithelial cells. Simultaneously, ICA can inhibit the proliferation of renal tubular epithelial cells and induce cell apoptosis. Furthermore, the expression of ER stress-related proteins IRE1 and XBP-1S was further detected. Additionally, to ICA intervention, a GPER antagonist (G-15) was added for intervention, the inhibitory effects of IRE1 and XBP-1S were reversed, and the ER stress pathway was activated. Cell experiments showed that ICA could promote GPER expression, while inhibiting GPER expression promoted the activation of ER stress pathway, and GPER expression was negatively correlated with ER stress protein expression. Therefore, the experiment proved that in DKD tissues, a high concentration of ICA can inhibit the ER stress response by promoting the expression of GPER, reducing the proliferation of diabetic nephropathy, and increasing the rate of tissue apoptosis.

Ginsenoside-Mc1 reduces cerebral ischemia-reperfusion injury in hyperlipidemia through mitochondrial improvement and attenuation of oxidative/endoplasmic reticulum stress

The neuroprotective effect of ginsenoside-Mc1 (GMc1) in hyperlipidemic rats in the setting of cerebral ischemiareperfusion injury (I/RI), as well as the role of mitochondrial ATP-sensitive potassium (mitoKATP) channels and oxidative/ endoplasmic reticulum (ER) stress, was investigated. Hyperlipidemia (8 weeks) was induced by a high-fat diet in Sprague Dawley rats. GMc1 (10 mg/kg, i.p.) was given to hyperlipidemic rats daily for one month before I/RI. Rat brains were subjected to 2 h of local ischemia followed by 24 h reperfusion. The cerebral infarcted injury was measured by triphenyltetrazolium chloride staining and the levels of oxidative stress indicators were detected by ELISA and spectrophotometry. A fluorometric technique was employed to evaluate mitochondrial function. Western blotting was used to detect changes in the expression of ER stress proteins. GMc1 reduced cerebral infarct volume in hyperlipidemic rats in comparison to untreated ones (P&lt;0.01). GMc1 reduced cerebral infarct volume in hyperlipidemic rats as compared to untreated rats (P&lt;0.01). GMc1 significantly decreased mitochondrial membrane depolarization, mitochondrial reactive oxygen species (mitoROS) and malondialdehyde levels (P&lt;0.01), while increasing the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx) (P&lt;0.001). GMc1 administration reduced the expression of ER stress markers, including phosphorylated (p)-endoplasmic reticulum kinase (PERK), p-eukaryotic translation initiation factor 2 subunit 1 (elF2?), and C/EBP homologous protein (CHOP). Inhibition of mitoKATP channels with hydroxydecanoate significantly eliminated the protective impacts of GMc1 in hyperlipidemic rats subjected to cerebral I/RI. The neuroprotective effect of GMc1 preconditioning was remarkably improved by increasing mitoKATP channel activity and decreasing oxidative and ER stress levels in hyperlipidemic rats, implying that this compound could be an appropriate candidate for reducing cerebral I/RI in comorbidities.

Exercise training mitigates ER stress and UCP2 deficiency-associated coronary vascular dysfunction in atherosclerosis

Endoplasmic reticulum (ER) stress and uncoupling protein-2 (UCP2) activation are opposing modulators of endothelial dysfunction in atherosclerosis. Exercise reduces atherosclerosis plaques and enhances endothelial function. Our aim was to understand how exercise affects ER stress and UCP2 activation, and how that relates to endothelial dysfunction in an atherosclerotic murine model. Wild type (C57BL/6, WT) and apolipoprotein-E-knockout (ApoEtm1Unc, ApoE KO) mice underwent treadmill exercise training (EX) or remained sedentary for 12 weeks. Acetylcholine (ACh)-induced endothelium-dependent vasodilation was determined in the presence of an eNOS inhibitor (L-NAME), UCP2 inhibitor (genipin), and ER stress inducer (tunicamycin). UCP2, ER stress markers and NLRP3 inflammasome signaling were quantified by western blotting. p67phox and superoxide were visualized using immunofluorescence and DHE staining. Nitric oxide (NO) was measured by nitrate/nitrite assay. ACh-induced vasodilation was attenuated in coronary arterioles of ApoE KO mice but improved in ApoE KO-EX mice. Treatment of coronary arterioles with L-NAME, tunicamycin, and genipin significantly attenuated ACh-induced vasodilation in all mice except for ApoE KO mice. Exercise reduced expression of ER stress proteins, TXNIP/NLRP3 inflammasome signaling cascades, and Bax expression in the heart of ApoE KO-EX mice. Further, exercise diminished superoxide production and NADPH oxidase p67phox expression in coronary arterioles while simultaneously increasing UCP2 expression and nitric oxide (NO) production in the heart of ApoE KO-EX mice. Routine exercise alleviates endothelial dysfunction in atherosclerotic coronary arterioles in an eNOS, UCP2, and ER stress signaling specific manner, and resulting in reduced TXNIP/NLRP3 inflammasome activity and oxidative stress.

Angiopoietin-like proteins 8 knockout reduces intermittent hypoxia-induced vascular remodeling in a murine model of obstructive sleep apnea

ObjectiveObstructive sleep apnea (OSA) is a major risk factor for cardiovascular mortality. Apnea-induced chronic intermittent hypoxia (CIH) is a primary pathophysiological manifestation of OSA that promotes various cardiovascular alterations, such as aortic vascular remodeling. In this study, we investigated the association between angiopoietin-like proteins 8 (ANGPTL8) and CIH-induced aortic vascular remodeling in mice. MethodsC57BL/6J male mice were divided into four groups: Normoxia group, ANGPTL8−/− group, CIH group, CIH + ANGPTL8−/− group. Mice in the normoxia group and ANGPTL8−/− group received no treatment, while mice in the CIH and CIH + ANGPTL8−/− group were subjected to CIH (21%–5% O2, 180 s/cycle, 10 h/day) for 6 weeks. At the end of the experiments, intima-media thickness (IMT), elastin disorganization, and aortic wall collagen abundance were assessed in vivo. Immunohistochemistry and Western-blot were used to detect endoplasmic reticulum stress (ERS) and aortic vascular smooth muscle cell proliferation. ANGPTL8 shRNA and ANGPL8 overexpression were used in aortic vascular smooth muscle cells to investigate the mechanism of ANGPTL8 in CIH. ResultsCompared to the control group, CIH exposure significantly increased intima-media thickness (IMT), elastic fibers disorganization, and aortic wall collagen abundance. CIH also significantly increased blood pressure, induced hyperlipidemia, as well as the expression of ERS protein activating transcription factor-6 (ATF6) and aortic vascular smooth muscle cell proliferation. Contrary, ANGPTL8−/− significantly mitigated the CIH-induced vascular remodeling; ANGPTL8−/− decreased CIH-induced hypertension and hyperlipidemia, inhibited the protein expression of ATF6, and aortic vascular smooth muscle cell proliferation. Moreover, our in vitro study suggested that CIH could induce ANGPTL8 expression via hypoxia-inducible factor (HIF-1α); ANGPTL8 induced proliferation of aortic vascular smooth muscle cells via the ERS pathway. ConclusionANGPTL8−/− can prevent CIH-induced aortic vascular remodeling, probably through the inhibition of the ERS pathway. Therefore, ANGPTL8 might be a potential target in CIH-induced aortic vascular remodeling.