C/EBP Homologous Protein mRNA Expression Research Articles

Palmitic acid-induced cell death: impact of endoplasmic reticulum and oxidative stress, mitigated by L-citrulline.

Palmitic acid (PA), the most abundant saturated free fatty acids, induces apoptosis in bovine mammary epithelial cells. It is suggested that oxidative stress and endoplasmic reticulum (ER) stress are key mechanisms underlying PA-induced cell death. This study aimed to investigate the interaction between ER stress and oxidative stress during PA-induced cell death in MAC-T cells. Additionally, we examined whether L-citrulline can protect against PA-induced damage of MAC-T cells. MAC-T cells were treated with 4-phenyl butyric acid (4-PBA) or N-acetyl-L-cysteine (NAC) to inhibit PA-induced ER stress and oxidative stress, respectively. MAC-T cells were pretreated with or without L-citrulline for 48 h followed by PA treatment. Cell viability was measured with MTT assays. Intracellular reactive oxygen species (ROS) levels in MAC-T cells were assessed using 5-(and-6)-chloromethyl- 2`,7`-dichlorodihydrofluorescein diacetate acetyl ester dye. Real-time qPCR was used to explore the regulation of genes associated with oxidative stress, and ER stress genes. Western blotting analysis was also carried out. 4-PBA significantly reduced PA-induced mRNA expressions of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and intracellular ROS levels. Furthermore, NAC dramatically reduced PA-induced ROS levels and the mRNA expressions of NRF2, ATF4, and CHOP. L-citrulline pretreatment effectively rescued cell viability decreased by PA. Moreover, L-citrulline pretreatment significantly downregulated the PA-induced upregulation of GRP78, ATF4, and CHOP mRNA expression, and protein expression of p-PERK and cleaved caspase-3. PA increased intracellular ROS levels and NRF2 mRNA expression, whereas L-citrulline pretreatment remarkably reduced these levels. Both ER and oxidative stresses interact during PA-induced cell death in MAC-T cells, and L-citrulline could attenuate this cell death by inhibiting ER and oxidative stresses. Therefore, L-citrulline may be a promising supplement for protecting against PA-induced cell death in bovine MECs during the lactation period of dairy cows.

Regular Kratom (Mitragyna speciosa Korth.) Use and Its Association With Endoplasmic Reticulum Stress Response

This study determined the association between expression of the endoplasmic reticulum (ER) stress sensor mRNA in the peripheral leukocytes and the patterns of kratom use and evaluated the correlations between the levels of the ER stress sensor mRNA and the severity of kratom dependence and kratom induced depressive symptoms among people who use kratom (PWUK). A total of 20 PWUK and 20 age matched non-kratom using healthy controls were recruited. Data collected from PWUK included patterns of kratom use, severity of kratom dependence, and severity of depressive symptoms during abstinence from kratom. The mRNA expression of binding immunoglobulin protein ( BiP ), X-box binding protein 1, activating transcription factor 4, and C/-EBP homologous protein ( CHOP ) (major indicators of ER stress response) were analyzed using quantitative reverse transcription polymerase chain reaction in leucocyte-derived total RNA sample of the participants. PWUK regardless of their pattern of kratom use recorded significantly higher expression of BiP mRNA compared with controls. Expression of CHOP mRNA was only significantly higher in those who first consumed kratom at the age of 18 years and above and those who have been using kratom for longer than 6 years, compared with controls. Higher expression of BiP , ATF4 , and CHOP mRNA were significantly positive correlated with greater severity of kratom dependence. Although only higher expression of BiP and CHOP mRNA were significantly positively correlated with greater severity of depressive symptoms. Regular kratom consumption may activate the ER stress pathway and there may be a link between altered ER stress response and kratom dependence and kratom induced depressive symptoms.

Mechanistic Insights into Ameliorating Effect of Geraniol on d-Galactose Induced Memory Impairment in Rats

Geraniol (GE), an important ingredient in several essential oils, displayed pleiotropic biological activities through targeting multiple signaling cascades. In the current study, we aimed to examine the protective effect of GE on d-galactose (d-gal) induced cognitive impairment and explore the underlying mechanisms. Forty male Wistar rats (8 weeks old) were randomly categorized into 4 groups; Group I (saline + vehicle [edible oil]), group II (saline + geraniol) (100 mg/kg/day orally), group III (d-galactose) (100 mg/kg/day subcutaneously injected), and group IV (d-galactose + geraniol). Behavioral impairments were evaluated. Brain levels of malondialdehyde (MDA) and reduced glutathione (GSH) as well as superoxide dismutase (SOD) and acetylcholinesterase (AchE) activities were estimated. The levels of inflammatory markers [tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and nuclear factor kappa beta (NF-kβ)], endoplasmic reticulum stress sensors [inositol requiring protein 1(IRE1) and protein kinase RNA–like endoplasmic reticulum kinase (PERK)], brain-derived neurotrophic factor (BDNF), and mitogen-activated protein kinases (MAPK) pathway were measured by ELISA. Also, hippocampal histopathological assessment and immunohistochemical analysis of glial fibrillary acidic protein (GFAP) and caspase-3 were performed. Glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) mRNA expression and protein levels were assessed. GE effectively ameliorated aging-related memory impairment through increasing GSH, BDNF, Ach levels, and SOD activity. Additionally, GE treatment caused a decrease in the levels of MDA, inflammatory mediators, and ER stress sensors as well as the AchE activity together with concomitant down-regulation of GRP78 and CHOP mRNA expression. Moreover, GE improved neuronal architecture and rat's spatial memory; this is evidenced by the shortened escape latency and increased platform crossing number. Therefore, GE offers a unique pharmacological approach for aging-associated neurodegenerative disorders.Graphical

Upregulation of Zip14 correlates with induction of endoplasmic reticulum stress (ERS) in hypertrophied hearts of Dahl saltsensitive rats

Zinc is a trace element involved in maintaining cellular structure and function. Although zinc is associated with left ventricular hypertrophy (LVH), there have been few reports on this association. This study aimed to evaluate the correlation between Zip14 and expression of endoplasmic reticulum stress (ERS) associated molecules in hypertrophied hearts of rats. Dahl salt-sensitive rats were fed a high salt diet to establish a left ventricular hypertrophy (LVH) rat model. RT-PCR was used to determine Zip14, activating transcription factor (ATF4), ATF6, x-box-binding protein 1 (xBP1), C/EBP homologous protein (CHOP), immunoglobulin-binding protein (BiP) mRNA expression. Western blotting was used to evaluate Zip14, BiP, CHOP, GAPDH expression. Zinc levels were measured by Inductively Coupled Plasma Optical Emission Spectroscopy. The results indicated that compared with the Control group, Zip14 mRNA and protein expression in LVH rat hearts were markedly increased (P < 0.01). Zinc content in rat heart tissue was significantly increased in the LVH group compared with the Control group (P < 0.05). ATF4, ATF6, xBP1 mRNA expressions were increased in LVH rat hearts compared with Control hearts (P < 0.001). Compared with the Control group, CHOP and BiP mRNA and protein expression were markedly increased in LVH rat hearts (P < 0.05, P < 0.01). Linear regression models showed that Zip14 mRNA expressions were positively correlated with zinc concentration, ATF4 and ATF6 mRNA expressions in Control hearts (P = 0.0005, P = 0.0052, P = 0.0026, respectively) and LVH rat hearts (P < 0.0001, P = 0.0119, P = 0.0033, respectively). In conclusion, upregulation of Zip14 in LVH rat hearts correlated with zinc accumulation and induction of ERS.

Upregulation of Zip14 correlates with induction of endoplasmic reticulum stress (ERS) in hypertrophied hearts of Dahl saltsensitive rats

Zinc is a trace element involved in maintaining cellular structure and function. Although zinc is associated with left ventricular hypertrophy (LVH), there have been few reports on this association. This study aimed to evaluate the correlation between Zip14 and expression of endoplasmic reticulum stress (ERS) associated molecules in hypertrophied hearts of rats. Dahl salt-sensitive rats were fed a high salt diet to establish a left ventricular hypertrophy (LVH) rat model. RT-PCR was used to determine Zip14, activating transcription factor (ATF4), ATF6, x-box-binding protein 1 (xBP1), C/EBP homologous protein (CHOP), immunoglobulin-binding protein (BiP) mRNA expression. Western blotting was used to evaluate Zip14, BiP, CHOP, GAPDH expression. Zinc levels were measured by Inductively Coupled Plasma Optical Emission Spectroscopy. The results indicated that compared with the Control group, Zip14 mRNA and protein expression in LVH rat hearts were markedly increased (P < 0.01). Zinc content in rat heart tissue was significantly increased in the LVH group compared with the Control group (P < 0.05). ATF4, ATF6, xBP1 mRNA expressions were increased in LVH rat hearts compared with Control hearts (P < 0.001). Compared with the Control group, CHOP and BiP mRNA and protein expression were markedly increased in LVH rat hearts (P < 0.05, P < 0.01). Linear regression models showed that Zip14 mRNA expressions were positively correlated with zinc concentration, ATF4 and ATF6 mRNA expressions in Control hearts (P = 0.0005, P = 0.0052, P = 0.0026, respectively) and LVH rat hearts (P < 0.0001, P = 0.0119, P = 0.0033, respectively). In conclusion, upregulation of Zip14 in LVH rat hearts correlated with zinc accumulation and induction of ERS.

Vanillin oxime inhibits lung cancer cell proliferation and activates apoptosis through JNK/ERK-CHOP pathway.

Lung cancer despite advancement in the medical field continues to be a major threat to human lives and accounts for a high proportion of fatalities caused by cancers globally. The current study investigated vanillin oxime, a derivative of vanillin, against lung cancer cells for development of treatment and explored the mechanism. Cell viability changes by vanillin oxime were measured using MTT assay. Vanillin oxime-mediated apoptosis was detected in A549 and NCI-H2170 cells at 48 h of exposure by flow cytometry. The CEBP homologous protein (CHOP) and death receptor 5 (DR5) levels were analysed by RT-PCR and protein levels by Western blotting. Vanillin oxime in concentration-dependent way suppressed A549 and NCI-H2170 cell viabilities. On exposure to 12.5 and 15 μM concentrations of vanillin oxime elevated Bax, caspase-3, and -9 levels in A549 and NCI-H2170 cells were observed. Vanillin oxime exposure suppressed levels of Bcl-2, survivin, Bcl-xL, cFLIP, and IAPs proteins in A549 and NCI-H2170 cells. It stimulated significant elevation in DR4 and DR5 levels in A549 and NCI-H2170 cells. In A549 and NCI-H2170 cells vanillin oxime exposure caused significant (p < 0.05) enhancement in CHOP and DR5 mRNA expression. Vanillin oxime exposure of A549 and NCI-H2170 cells led to significant (p < 0.05) enhancement in levels of phosphorylated extracellular-signal-regulated kinase and c-Jun N-terminal kinase. Thus, vanillin oxime inhibits pulmonary cell proliferation via induction of apoptosis through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated pathway. Therefore, vanillin oxime may be studied further to develop a treatment for lung cancer.

Rhubarb extract relieves constipation by stimulating mucus production in the colon and altering the intestinal flora

BackgroundConstipation, mainly characterized by the difficulty in defecation, is a clinical symptom caused by a variety of factors. It can be manifested as normal or slow colonic transport abnormalities, which can occur alone or concurrently with defecation disorders. As there is not uniform definition and assessment standard, no clear plan could be used for the treatment of constipation. Although rhubarb, a traditional Chinese medicine, plays a therapeutic role in diseases involving constipation symptoms, the detailed mechanism of it in treating constipation remains unclear. MethodsA model of constipation-induced by diphenoxylate was prepared. Immunofluorescent staining was used to detect the expression of mucin 2 (MUC2), calnexin and chymase in colon. Western blotting was used to detect changes of tryptase and calnexin in the colon. And real-time polymerase chain reaction (PCR) was utilized to detect the changes of immunoglobulin-binding protein (Bip), X-box binding protein 1 (Xbp1) and C/EBP homologous protein (CHOP) of colonic goblet cells in mRNA levels. ELISA and biochemical kits were utilized to detect the changes of MUC2, Trefoil factor 3 (TFF3), acetylcholine, histamine and C-C motif chemokine ligand 5 (CCL5) in the colon. And the changes of colonic mucosa and intestinal flora of constipation model mice caused by rhubarb extract (RE) were analyzed to identify the mechanism of RE on the treatment of constipation. ResultsRE promotes the secretion of colonic mucus by recruiting mast cells and enhancing the content of histamine and Ach in the mice colon. In the process, RE causes up-regulation of Bip and CHOP mRNA expression and down-regulation of Xbp1 and Xbp1s mRNA expression that induces ER stress of colonic epithelium associated with changes in the intestinal flora diversity and short-chain fatty acids content. ConclusionRE could relieve constipation by promoting the secretion of colonic mucus via mast cells activation and improving the intestinal microenvironment.

Nogo-A Is Critical for Pro-Inflammatory Gene Regulation in Myocytes and Macrophages

Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, previous studies revealed that Nogo-A was upregulated in skeletal muscles of Amyotrophic lateral sclerosis (ALS) patients. Additionally, experiments showed that endoplasmic reticulum (ER) stress marker, C/EBP homologous protein (CHOP), was upregulated in gastrocnemius muscle of a murine model of ALS. We therefore hypothesized that Nogo-A might relate to skeletal muscle diseases. According to our knocking down and overexpression results in muscle cell line (C2C12), we have found that upregulation of Nogo-A resulted in upregulation of CHOP, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while downregulation of Nogo-A led to downregulation of CHOP, IL-6 and TNF-α. Immunofluorescence results showed that Nogo-A and CHOP were expressed by myofibers as well as tissue macrophages. Since resident macrophages share similar functions as bone marrow-derived macrophages (BMDM), we therefore, isolated macrophages from bone marrow to study the role of Nogo-A in activation of these cells. Lipopolysaccharide (LPS)-stimulated BMDM in Nogo-KO mice showed low mRNA expression of CHOP, IL-6 and TNF-α compared to BMDM in wild type (WT) mice. Interestingly, Nogo knockout (KO) BMDM exhibited lower migratory activity and phagocytic ability compared with WT BMDM after LPS treatment. In addition, mice experiments data revealed that upregulation of Nogo-A in notexin- and tunicamycin-treated muscles was associated with upregulation of CHOP, IL-6 and TNF-α in WT group, while in Nogo-KO group resulted in low expression level of CHOP, IL-6 and TNF-α. Furthermore, upregulation of Nogo-A in dystrophin-deficient (mdx) murine model, myopathy and Duchenne muscle dystrophy (DMD) clinical biopsies was associated with upregulation of CHOP, IL-6 and TNF-α. To the best of our knowledge, this is the first study to demonstrate Nogo-A as a regulator of inflammation in diseased muscle and bone marrow macrophages and that deletion of Nogo-A alleviates muscle inflammation and it can be utilized as a therapeutic target for improving muscle diseases.

Carpinus turczaninowii Extract May Alleviate High Glucose-Induced Arterial Damage and Inflammation.

Hyperglycemia-induced oxidative stress triggers severe vascular damage and induces an inflammatory vascular state, and is, therefore, one of the main causes of atherosclerosis. Recently, interest in the natural compound Carpinus turczaninowii has increased because of its reported antioxidant and anti-inflammatory properties. We investigated whether a C. turczaninowii extract was capable of attenuating high glucose-induced inflammation and arterial damage using human aortic vascular smooth muscle cells (hASMCs). mRNA expression levels of proinflammatory response [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)], endoplasmic reticulum (ER) stress [CCAAT-enhancer-binding proteins (C/EBP) homologous protein (CHOP)], and adenosine monophosphate (AMP)-protein activated kinase α2 (AMPK α2)], and DNA damage [phosphorylated H2.AX (p-H2.AX)] were measured in hASMCs treated with the C. turczaninowii extracts (1 and 10 μg/mL) after being stimulated by high glucose (25 mM) or not. The C. turczaninowii extract attenuated the increased mRNA expression of IL-6, TNF-α, and CHOP in hASMCs under high glucose conditions. The expression levels of p-H2.AX and AMPK α2 induced by high glucose were also significantly decreased in response to treatment with the C. turczaninowii extract. In addition, 15 types of phenolic compounds including quercetin, myricitrin, and ellagic acid, which exhibit antioxidant and anti-inflammatory properties, were identified in the C. turczaninowii extract through ultra-performance liquid chromatography-quadrupole-time of flight (UPLC-Q-TOF) mass spectrometry. In conclusion, C. turczaninowii may alleviate high glucose-induced inflammation and arterial damage in hASMCs, and may have potential in the treatment of hyperglycemia-induced atherosclerosis.

Temporal and spatial profile of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in ischemic stroke in mice.

Although T cells play important roles in the pathophysiology of ischemic stroke, the dynamics of T cells remains unclear. In cancer, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) contribute to the maintenance of the tumor microenvironment by suppressing T cells. However, the presence of these cells has never been examined in ischemic brain. Therefore, we examined the temporal and spatial profiles of PMN-MDSCs, which are defined as the CD11b+Ly6ClowLy6G+ cells with higher expression levels of Nox2 and C/EBP Homologous Protein (CHOP) mRNA than normal neutrophil. Fluorescence-activated cell sorter (FACS) analysis showed that the count of CD11b+Ly6ClowLy6G+ cells was increased in the ischemic hemisphere and bone marrow at 72 hours, as well as in the spleen 24 hours after transient middle cerebral artery occlusion in mice. In contrast, the contralateral hemisphere, normal bone marrow, and normal spleen contained few CD11b+Ly6ClowLy6G+ cells. Real-time reverse transcription polymerase chain reaction revealed that CD11b+Ly6ClowLy6G+ cells sorted from brain and spleen 72 hours after ischemia had greater expression of Nox2 and CHOP mRNA than neutrophils in bone marrow, suggesting that these cells constitute PMN-MDSCs. Immunohistochemistry showed that CD11b+Ly6G+ cells were located in the ischemic core and border zone, indicating that PMN-MDSCs might be endemic to these regions. Although neutrophils are believed to invade infarct regions 48–72 hours after ischemia, the present study suggested that some of these cells are in fact PMN-MDSCs. Further studies on the function of PMN-MDSCs might unveil the unknown mechanisms of T cell activation and recruitment in ischemic stroke.

MON-201 ER Stress Activated by Androgen Enhances Apoptosis of Granulosa Cells via Induction of Death Receptor 5 in PCOS

PCOS is associated with hyperandrogenism and growth arrest of antral follicles. Excess androgens work as a stage-specific inhibitor of a follicular growth suppressing the growth of late-stage follicles. Previously, we found that endoplasmic reticulum (ER) stress is activated in granulosa cells of antral follicles in PCOS (1). This is evidenced by the activation of various unfolded protein response (UPR) genes, including a pro-apoptotic UPR transcription factor, C/EBP homologous protein (CHOP). Death receptor (DR) 5, which harbors a CHOP-binding site in its promoter region, plays a crucial role in ER stress-induced apoptosis by increasing autocrine death-ligand signal, independent of its extracellular ligand TNF-related apoptosis-inducing ligand (TRAIL) (2). Based on these observations, we hypothesized that ER stress is activated by androgen in granulosa cells of antral follicles in PCOS and activated ER stress promotes apoptosis via induction of CHOP and subsequent activation of DR5. Using RT-PCR (qPCR), we showed that testosterone induced the mRNA expression of various UPR factors including XBP1(S), HSPA5, ATF4 and CHOP along with DR5 in cultured human granulosa-lutein cells (GLCs). Treatment with an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), inhibited testosterone-induced mRNA and protein expression of DR5 and CHOP, with a concomitant reduction in apoptosis examined by qPCR, western blotting and flow cytometry respectively. Furthermore, knockdown of CHOP or DR5 by RNA interference inhibited testosterone-induced apoptosis and knockdown of CHOP reduced testosterone-induced DR5 mRNA expression. Treatment with an androgen receptor (AR) antagonist, flutamide, as well as the knockdown of AR, decreased testosterone-induced DR5 and CHOP mRNA expression and apoptosis. In addition, expression of DR5 and CHOP was upregulated in GLCs of PCOS patients and granulosa cells of antral follicles in ovarian sections obtained from both PCOS patients and dehydroepiandrosterone (DHEA)-induced PCOS mice, proven by qPCR and immunohistochemistry (IHC). Treatment of PCOS mice with TUDCA decreased apoptosis and DR5 expression in granulosa cells of antral follicles, with a concomitant reduction in CHOP expression, shown by TUNEL and IHC staining. Our findings indicate that ER stress activated by hyperandrogenism in PCOS promotes apoptosis of granulosa cells of antral follicles via induction of DR5. Reference: (1) Takahashi N, Harada M, et al. Sci Rep 2017, 7:10824 (2) Lu M et al. Science 2014;345:98-101.

Expression of LRP1 and CHOP genes associated with peripheral neuropathy in type 2 diabetes mellitus: Correlations with nerve conduction studies

AimDiabetic peripheral neuropathy (DPN) is a frequent and debilitating complication of diabetes mellitus. The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional cell surface receptor playing critical roles in lipoprotein metabolism and several cell signaling processes. C/EBP homologous protein (CHOP) is a main conduit to endoplasmic reticulum stress-induced apoptosis. We aimed to investigate LRP1 and CHOP gene expression in peripheral blood cells of type 2 diabetes mellitus (T2DM) subjects to clarify its possible relation to DPN pathogenesis. MethodThe study included 20 non-complicated T2DM subjects, 20 subjects with DPN and 20 healthy controls. Quantitative real time PCR was used to study gene expression. ResultsThere was a significant reduction in LRP1 mRNA expression and a significant increase in CHOP mRNA expression in subjects with DPN compared to non-complicated group and healthy controls. Both LRP1 and CHOP expression levels were inversely correlated, and both showed significant correlation with HbA1c, hyperlipidemia, hs-CRP, and different electrophysiological parameters. Receiver operating characteristics (ROC) analysis suggested that both LRP1 and CHOP mRNA expression and hs-CRP levels had great potential advantages to predict the progression of DPN. ConclusionLRP1 and CHOP might be involved in DPN pathogenesis and progression, thus providing opportunities for early detection and treatment.

Endoplasmic reticulum stress contributes to the pathogenesis of stress urinary incontinence in postmenopausal women.

ObjectiveTo investigate the relationship between endoplasmic reticulum stress (ERS) and the pathogenesis of stress urinary incontinence (SUI) in postmenopausal women.MethodsAnterior vaginal wall tissue was collected from postmenopausal women with SUI and control subjects. Western blotting was performed for glucose-regulated protein (GRP78), inositol-requiring enzyme 1(IRE1), protein kinase-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), C/EBP-homologous protein (CHOP), and B-cell lymphoma 2 (Bcl-2). Additionally, mRNA expression levels of PERK, activating transcription factor 4 (ATF4), and CHOP were examined by real-time polymerase chain reaction.ResultsGRP78 protein and mRNA expression levels were significantly lower in women with SUI, compared with control subjects. PERK and p-PERK expression levels were higher in women with SUI than in control subjects. However, no differences in IRE1 or ATF6 expression levels were observed in either group. Notably, higher CHOP and lower Bcl-2 protein expression levels were detected in women with SUI, compared with control subjects. Furthermore, PERK, ATF4, and CHOP mRNA expression levels were significantly higher in women with SUI than in control subjects.ConclusionsAlterations of ERS markers in SUI suggest that ERS may be involved in the development of SUI in postmenopausal women.

Baicalein stimulates fibroblast growth factor 21 expression by up-regulating retinoic acid receptor-related orphan receptor α in C2C12 myotubes

Retinoic acid receptor-related orphan receptor (ROR) α has been implicated in various physiological functions, including the immune system, inflammation, and circadian rhythms. In the present study, the synthetic RORα/γ agonist SR1078 stimulated the production and gene expression of fibroblast growth factor 21 (FGF21) in C2C12 myotubes. FGF21, a member of the FGF family, plays important roles in the regulation of peripheral glucose tolerance and lipid metabolism and improves metabolic health. The mRNA expression and secretion of FGF21 was significantly weaker in Rora-silenced cells than in cells transfected with non-targeting control siRNA. SR1078 significantly up-regulated C/EBP homologous protein (CHOP), an established marker of ER stress, in a dose-dependent manner in C2C12 myotubes, while CHOP expression was decreased in Rora-silenced C2C12 cells, suggesting that RORα is involved in the regulation of FGF21 expression and stimulates ER stress in C2C12 myotubes. The naturally occurring compound baicalein up-regulated FGF21 expression and secretion in C2C12 myotubes. Additionally, the up-regulation of CHOP mRNA and protein expression was observed in C2C12 myotubes after the baicalein treatment. Furthermore, the knockdown of RORα prevented the augmentation of FGF21 and up-regulation of CHOP in response to baicalein in C2C12 cells. Collectively, these results suggest that baicalein stimulates the ER stress response and FGF21 expression through an RORα-dependent mechanism in C2C12 myotubes, and indicate the potential of baicalein as an effective anti-obesity therapy via its ability to enhance FGF21 production.

Astragaloside attenuates the progression of prostate cancer cells through endoplasmic reticulum stress pathways.

Astragaloside (As) has been demonstrated extensively to serve roles in a variety of tumor types, including glioma, lung cancer, colorectal cancer, breast cancer and cervical cancer, and has therefore been widely used in Traditional Chinese Medicine. To the best of our knowledge, the present study was the first to investigate the efficacy of the Traditional Chinese Medicine astragaloside on tumor growth and the apoptosis of prostate cancer cells. In addition, further investigation into the underlying molecular mechanisms via the endoplasmic reticulum (ER) stress pathway was also performed. In the present study, the human prostate cancer DU-145 cell line was employed as an experimental model in vitro and cells were divided into five treatment groups: Dimethyl sulfoxide (DMSO) group (control), low-dose astragaloside group (L-As; 20 nmol/l), moderate-dose astragaloside group (M-As; 50 nmol/l), high-dose astragaloside group (H-As, 100 nmol/l) and ER stress suppressor group (tauroursodeoxycholic acid; TUDCA). The proliferative ability and apoptosis rate of the DU-145 cells were detected via Cell Counting kit-8 methods and flow cytometry, respectively. Furthermore, the ER stress factors [binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP) and caspase-12] were assessed through reverse transcription polymerase chain reaction. Additionally, the protein expression levels of inositol-requiring enzyme 1 (IRE1), phosphorylated protein kinase R-like ER kinase (p-PERK), iron-regulated transcriptional activator Aft (AFT)4 and AFT6 were measured detected by western blot analysis. Administration of As significantly reduced the cell viability and promoted apoptosis (P<0.05) in a dose-dependent manner. Expression of ER-stressed genes BiP, CHOP and caspase-12 mRNA was increased by As administration, while TUDCA treatment led to a lower mRNA expression of these genes, compared with the control group. Results of western blot analysis indicated that the protein expression of IRE1, AFT4 and AFT6 was upregulated in the H-As group, and that the ratio of p-PERK/PERK was also higher than in the other groups. The administration of As demonstrated significant therapeutic effects on the proliferation of prostate cancer cells, as well as the expression of related proteins and genes. The results of the present study suggested future clinical potential of As for the treatment of prostate cancer.

Effect of dexmedetomidine on apoptosis and CHOP in hypoxia/reoxygenation injury A549 cell

To investigate the effects of dexmedetomidine (Dex) on injury of A549 cells induced by hypoxia/reoxygenation(H/R)and the influence of C/EBP homologous protein (CHOP) expression. Logarithmic growth phase A549 cells(it originated from alveolar type Ⅱ epithelial cell line) were randomly divided into 4 groups (n=10):normoxic control group (N), Dex group (D), hypoxia/reoxygenation group (H), hypoxia/reoxygenation + Dex group(HD). At the beginning of modeling, 1 nmol/L Dex was puted into D and HD groups. N and D groups were cultured in the normoxic incubator for 30 h. H and HD group were incubated in the anoxic cultivation for 6 h, fo llowed by normoxic culture for 24 h. Then A549 cells were observed under the inverted microscope to observe the morphological changes. Cell activity was detected by cell counting Kit-8(CCK-8) and the apoptosis index(AI) was detected by in situ end labeling (TUNEL) method. The expression of CHOP、glucose-regulated protein of molecular weight 78 kDa (Grp78)、cysteinyl aspirate-specificprotease-3 (caspase-3) protein and CHOP、Grp78 mRNA were detected by Western blot and RT-PCR. Compared with N group, the number of adherent cells in H group decreased significantly, and cell morphology changed. The absorbance value in H group decreased obviously (P<0. 01). The AI value and expression of CHOP, Grp78, caspase-3 proteins and CHOP, Grp78 mRNA were significantly increased (P<0.01). Compared with H group, the cell damage in HD group was decreased, the absorbance value increased (P<0.01), the number of apoptosis cells decreased relatively (P<0.01), the expression of CHOP, caspase-3 protein and CHOP mRNA decreased (P<0. 01). Dex has notable effects against H/R injury, which may be related to effective inhibition of apoptosis mediated by the CHOP's signal path.

Therapeutic efficacy and mechanism of action of ginsenoside Rg1 in treating acute hepatic failure in mice

Objective: To examine the regulatory effect of ginsenoside Rg1 (G-Rg1) on endoplasmic reticulum stress and its effect on hepatocellular apoptosis in carbon tetrachloride (CCl(4))-induced acute liver failure (ALF). Methods: Forty healthy, adult male C57/BL mice were randomly divided into normal saline control (NS) group, G-Rg1 blank control (G-Rg1) group, CCl(4) model (CCl(4)) group, and G-Rg1 preventive treatment (CCl(4)+G-Rg1) group, and an ALF mouse model was established by CCl(4) induction. Blood and liver specimens were collected from all mice upon sacrifice at 12 hours post-intraperitoneal injection. Serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST) and total bilirubin (TBil) levels were determined using commercial test kits. The mRNA expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was measured using real-time PCR. The protein expression of GRP78, CHOP, caspase12, and caspase3 were measured by Western blot. Histological changes in the liver were assessed by hematoxylin-eosin staining, and the expression of GRP78 and caspase3 was detected by immunohistochemistry. Hepatocyte apoptosis was determined using terminal transferase dUTP nick end labeling. Quantitative data were analyzed using one-way ANOVA, and subsequent pairwise comparisons were performed using the LSD-t method. Results: Serum ALT, AST, and TBil levels in the CCl(4)+G-Rg1 group were significantly reduced compared with those in the CCl(4) group (ALT: 691.30 ± 108.06 U/L vs 980.66 ± 110.29 U/L, F = 365.07, P < 0.05; AST: 195.40 ± 15.41 U/L vs 319.44 ± 89.32 U/L, F = 115.64, P < 0.05; TBil: 1.09 ± 0.11 mg/dl vs 1.56 ± 0.12 mg/dl, F = 211.29, P < 0.05). The relative mRNA expression of GRP78 and CHOP was significantly lower in the CCl(4) + G-Rg1 group than in the CCl(4) group (P < 0.05). The relative protein expression of caspase3, GRP78, caspase12, and CHOP was significantly reduced to different extents in the CCl(4)+G-Rg1 group compared with those in the CCl4 group (P < 0.05). The CCl(4) + G-Rg1 group showed reduced liver tissue degeneration and necrosis compared with the CCl(4) group. Furthermore, the CCl(4)+G-Rg1 group showed significantly fewer brown granules in the liver than the CCl4 group (P < 0.05), indicating that G-Rg1 preventive treatment reduced CCl(4)-induced hepatocyte apoptosis. Conclusion: G-Rg1 prophylaxis can inhibit inflammation and reduce hepatocyte necrosis and apoptosis during CCl(4)-induced ALF. Its mechanism may involve the suppression of endoplasmic reticulum stress-related signaling molecules to alleviate hepatocyte endoplasmic reticulum stress and apoptosis. The results of this study suggest that G-Rg1 may inhibit liver inflammation and hepatocyte apoptosis through multiple targets to protect liver function.

Preventive effects of total saponins of Panax japonicus on fatty liver fibrosis in mice.

IntroductionNonalcoholic fatty liver disease (NAFLD) is a condition in which excess fat accumulates in the liver of a patient without a history of alcohol abuse. Fatty liver fibrosis, a severe form of NAFLD, is a key step which can be reversed by effective medical intervention. This paper aims to describe the protective role and mechanisms of action of total saponins of Panax japonicus (SPJ) against fatty liver fibrosis in mice. In this study, fatty liver fibrosis was induced by a high-fat (HF) diet combined with intraperitoneal injection of porcine serum.Material and methodsThe fatty liver fibrosis model was induced by HF diet combined with intraperitoneal injection of porcine serum. The endoplasmic reticulum stress (ERS) response and C/EBP homologous protein (CHOP) and p-Jun N-terminal kinase (JNK)-mediated apoptosis and inflammation were assessed by serum biochemistry, hematoxylin-eosin (H + E), Masson and electronic microscopy staining, Hyp content detection, Western blotting and real time polymerase chain reaction (RT-PCR).ResultsSaponins of Panax japonicus could significantly improve liver function and decrease the lipid level in the serum. The liver steatosis, collagen fibers and inflammatory cell infiltration were significantly improved in the SPJ group according to microscope observation. The RT-PCR analysis revealed that the collagen I (Coll), α smooth muscle actin (α-SMA), tissue inhibitors of MMPs (TIMP), CHOP and GRP78 mRNA expression levels were distinctly weakened by SPJ treatment; and western blotting analysis indicated that the phosphorylated JNK (p-JNK), Coll and 78 kD glucose-regulated protein (GRP78) protein expression levels were significantly alleviated, which might be associated with the inhibition of the ERS response and the CHOP and JNK-mediated apoptosis and inflammation pathway.ConclusionsBased on this research, SPJ as a preventive medicine has great potential in prevention of liver fibrosis.

Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure

Acrylamide (AA) is a well-known neurotoxic compound in humans and experimental animals. However, intracellular stress signaling pathways responsible for the neurotoxicity of AA are still not clear. In this study, we explored the involvement of the endoplasmic reticulum (ER) stress response in AA-induced neuronal damage in vitro and in vivo. Exposure of SH-SY5Y human neuroblastoma cells to AA increased the levels of phosphorylated form of eukaryotic translation initiation factor 2α (eIF2α) and its downstream effector, activating transcription factor 4 (ATF4), indicating the induction of the unfolded protein response (UPR) by AA exposure. Furthermore, AA exposure increased the mRNA level of c/EBP homologous protein (CHOP), the ER stress-dependent apoptotic factor, and caused the accumulation of reactive oxygen species (ROS) in SH-SY5Y cells. Treatments of SH-SY5Y cells with the chemical chaperone, 4-phenylbutyric acid and the ROS scavenger, N-acetyl-cysteine reduced the AA-induced expression of ATF4 protein and CHOP mRNA, and resulted in the suppression of apoptosis. In addition, AA-induced eIF2α phosphorylation was also suppressed by NAC treatment. In consistent with in vitro study, exposure of zebrafish larvae at 6-day post fertilization to AA induced the expression of chop mRNA and apoptotic cell death in the brain, and also caused the disruption of brain structure. These findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress and subsequent eIF2α–ATF4–CHOP signaling cascade. The accumulation of ROS by AA exposure appears to be responsible for this ER stress-mediated apoptotic pathway.

Lipopolysaccharide Activates the Unfolded Protein Response in Human Periodontal Ligament Fibroblasts.

The activation of the unfolded protein response (UPR) has been demonstrated in periodontal diseases. However, the cellular and molecular mechanisms by which the UPR is induced in periodontitis remain unclear. In this study, the effects of lipopolysaccharide (LPS) on the induction of the UPR in human periodontal ligament fibroblasts (HPDLFs) in vitro are investigated. HPDLFs isolated from human PDLs were stimulated with various concentrations of Escherichia coli LPS (0.1, 1, and 10 μg/mL) for the indicated time points (0, 3, 6, 9, 12, and 18 hours). The messenger RNA (mRNA) and protein levels of molecular markers associated with UPR activation, such as glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP), were measured at different time points of LPS treatment. Apoptosis of HPDLFs was assessed by Annexin V-FITC and propidium iodide staining, followed by flow cytometry. LPS treatment of HPDLFs increased GRP78 mRNA and protein levels in a concentration-dependent manner. Additionally, LPS also induced the expression, splicing, and activation of XBP1 mRNA. Moreover, LPS-induced CHOP expression was concentration dependent: a lower concentration of LPS (0.1 μg/mL) had no effect on CHOP mRNA levels, but higher concentrations of LPS (1 and 10 μg/mL) markedly increased CHOP mRNA and protein expression without inducing apoptosis. The findings demonstrate that activating the Toll-like receptor-4 signaling pathway in HPDLFs using LPS triggers the UPR in vitro, warranting additional investigation into the precise mechanisms by which pathways promote this response under inflammatory conditions.