Decreased Endoplasmic Reticulum Stress Research Articles

Protective effects of dietary protein against nitrite stress in grass carp (Ctenopharyngodon idella): A new perspective

Protein constitutes the predominant essential nutrient in fish feed. Despite extensive research efforts on the dietary protein of fish, limited attention has focused on its potential antistress properties. Therefore, this study sought to examine the impact of dietary protein on the liver structure of grass carp (Ctenopharyngodon idella) and its underlying molecular mechanism after nitrite stress, with the objective of elucidating the role of dietary protein in the stress response of fish. Specifically, various levels of dietary protein (16 %, 19 %, 22 %, 25 %, 28 % and 31 %) were investigated for their effects on liver tissue damage, oxidative stress, endoplasmic reticulum (ER) stress, autophagy and apoptosis in grass carp after exposure to a nitrite solution at a concentration of 10 mg/L for a duration of 96 h.Our study revealed that exposure to nitrite solution resulted in liver structural damage in grass carp. However, 22–25 % dietary protein levels might attenuate the impact of nitrite stress by decreasing liver contents of nitric oxide (NO), peroxynitrite anion (ONOO-), reactive oxygen species (ROS), protein carbonyl (PC), and malondialdehyde (MDA), which is reflected in serum activities of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). Histological analysis further supported the protective role of appropriate protein levels (22–28 %) in alleviating nitrite-induced liver tissue damage. Subsequent investigations demonstrated that optimal dietary protein levels decreased ER stress and autophagy in fish by modulating the expression of gene and protein involved in 78 kDa glucose regulated protein (GRP78) pathway and autophagosome formation process, respectively. Meanwhile, appropriate dietary protein levels decreased the activities of caspase-3, caspase-8 and caspase-9 by inhibiting both death receptor pathway and mitochondrial pathway associated with apoptosis. Finally, based on regression analysis of liver ONOO− content, the dietary protein requirement for grass carp (721–1381 g) under nitrite stress was determined to be 25.07 %. Overall, this study provides novel evidence that optimal protein levels can enhance the resistance of grass carp to nitrite stress and mitigate liver tissue damage by reducing oxidative stress cascade damage.

Ginsenoside Rb1 reduced ischemic stroke-induced apoptosis through endoplasmic reticulum stress-associated IRE1/TRAF2/JNK pathway.

The neuroprotective function of ginsenoside Rb1 (GRb1) in cerebral ischemia-reperfusion (I/R) was lately emphasized. However, whether GRb1 plays a regulatory role on endoplasmic reticulum (ER) stress-associated pathway in cerebral I/R damage is still unclear. The aim of this study is to explore the function of GRb1 in cerebral ischemia-induced ER stress and the underlying mechanism related to IRE1/TRAF2/JNK pathway. Longa method, cerebral infarct volume, and HE staining were used to evaluate the efficacy of GRb1 in mice with a mouse model of middle cerebral artery occlusion reperfusion (MCAO/R). We also investigated the effect and mechanism of GRb1 against ischemic stroke using in vitro oxygen-glucose deprivation reperfusion (OGD/R) model. We found that GRb1 could improve neurological scores, infarct volume, and histological injury in ischemic mice. Ischemic attack also activated neuronal apoptosis and ER stress, and this effect was attenuated by GRb1. In addition, GRb1 significantly reduced I/R-induced IRE1-TRAF2 interaction, IRE1, and JNK phosphorylation. The present study also confirmed that GRb1 significantly improved OGD/R-induced PC12 cells injury. GRb1 could decrease ER stress in OGD/R-injured PC12 cells, which was reflected by the decreased expression of GRP78 and CHOP. The ER stress inducer tunicamycin partially prevented the effects of GRb1 on cell viability, ER stress, and apoptosis after OGD/R, whereas the ER stress inhibitor 4-PBA exerted the opposite effect. Moreover, GRb1 markedly decreased IRE1-TRAF2 interaction, IRE1, and JNK phosphorylation in the presence of OGD/R insult. Furthermore, JNK inhibitor SP600125 and IRE1 inhibitor DBSA pretreatment further promoted the inhibition of GRb1 on ER stress induction and cell damage induced by OGD/R. Molecular docking further elucidated that the mechanism by which GRb1 improves cerebral ischemia maybe related to its direct binding to the kinase domain of IRE1, which in turn inhibited the phosphorylation of IRE1. Collectively, these results demonstrated that GRb1 reduced ischemic stroke-induced apoptosis through the ER stress-associated IRE1/TRAF2/JNK pathway and GRb1 has the potential as a protective drug for the treatment of cerebral ischemia.

Protective effects of tauroursodeoxycholate against radiation-induced intestinal injury in a mouse model

In patients with high-level radiation exposure, gastrointestinal injury is the main cause of death. Despite the severity of damage to the gastrointestinal tract, no specific therapeutic option is available. Tauroursodeoxycholic acid (TUDCA) is a conjugated form of ursodeoxycholic acid that suppresses endoplasmic reticulum (ER) stress and regulates various cell-signaling pathways. We investigated the effect of TUDCA premedication in alleviating intestinal damage and enhancing the survival of C57BL/6 mice administered a lethal dose (15Gy) of focal abdominal irradiation. TUDCA was administered to mice 1 h before radiation exposure, and reduced apoptosis of the jejunal crypts 12 h after irradiation. At later timepoint (3.5 days), irradiated mice manifested intestinal morphological changes that were detected via histological examination. TUDCA decreased the inflammatory cytokine levels and attenuated the decrease in serum citrulline levels after radiation exposure. Although radiation induced ER stress, TUDCA pretreatment decreased ER stress in the irradiated intestinal cells. The effect of TUDCA indicates the possibility of radiation therapy for cancer in tumor cells. TUDCA did not affect cell proliferation and apoptosis in the intestinal epithelium. TUDCA decreased the invasive ability of the CT26 metastatic colon cancer cell line. Reduced invasion after TUDCA treatment was associated with decreased matrix metalloproteinase (MMP)-7 and MMP-13 expression, which play important roles in invasion and metastasis. This study shows a potential role of TUDCA in protecting against radiation-induced intestinal damage and inhibiting tumor cell migration without any radiation and radiation therapy effect.

Polygonatum sibiricum (Huang Jing) polysaccharide reduces diabetic cardiomyopathy through increasing cyclic guanosine monophosphate-protein kinase G signaling in diabetic mice.

Diabetic cardiomyopathy (DCM) is a prevalent condition among individuals with diabetes, and is associated with a high mortality rate. The anti-oxidant properties of Jing Huang or Polygonatum sibiricum polysaccharide (PSP) have been extensively used to treat diabetes-related disorders; however, its potential effectiveness against DCM remains unknown. This study aimed to investigate PSP's therapeutic effects on DCM in an experimental diabetic mouse model. To induce insulin resistance, mice were fed a high-fat diet for 3 months, followed by intraperitoneal streptozotocin injection to induce slight hyperglycemia and develop DCM. Both DCM and control mice were given PSP orally for 3 weeks. Western blotting was used to detect the protein expressions of protein kinase G, C/EBP homologous protein, glucose-regulated protein 78, phosphodiesterase type 5, protein kinase R-like endoplasmic reticulum (ER) kinase, and phospho-protein kinase R-like endoplasmic reticulum kinase in heart tissue. The results showed a reduction in bodyweight and blood glucose levels in the PSP therapy group compared with DCM group. PSP also improved cardiac function and had a negligible effect on malondialdehyde activity. Furthermore, the findings showed that PSP alleviated ER and oxidative stress observed in DCM mice hearts, leading to the inhibition of cyclic guanosine monophosphate-specific phosphodiesterase type 5 and cardiac cyclic guanosine monophosphate reactivation. Phosphodiesterase type 5 inhibition reduced high-fat diet-induced cardiac dysfunction and decreased ER stress. PSP could effectively protect diabetic myocardium by inhibiting endoplasmic reticulum stress. These findings provide crucial insights into the potential of PSP to ameliorate DCM conditions in diabetic mice by decreasing ER and oxidative stress, and enhancing cyclic guanosine monophosphate protein kinase G signaling.

Bacillus subtilis HW2 enhances growth performance and alleviates gut injury via attenuation of endoplasmic reticulum stress and regulation of gut microbiota in broilers under necrotic enteritis challenge

This study investigated the effects of Bacillus subtilis HW2 on the growth performance, immune response, endoplasmic reticulum (ER) stress, and intestinal health in broilers with necrotic enteritis. Three hundred 1-day-old male Cobb 500 broilers (33.88 ± 2.34 g) were randomly allocated to 5 groups including non-infected control (NC group), basal diet + necrotic enteritis challenge (NE group), basal diet + 1 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (L-Pro group), basal diet + 5 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (M-Pro group), and basal diet + 1 × 107 CFU/g B. subtilis HW2 + necrotic enteritis challenge (H-Pro group), with 6 replicates per group. All broilers except NC group were orally given with sporulated coccidian oocysts at day 14 and Clostridium perfringens from days 19 to 21. Results showed that L-Pro and M-Pro groups improved growth performance and intestinal morphology in necrotic enteritis-challenged broilers, and L-Pro, M-Pro, and H-Pro groups improved intestinal barrier function and immune response and decreased ER stress in necrotic enteritis-challenged broilers. Analysis of the gut microbiota revealed that L-Pro group increased the abundances of Alistipes, Coprobacter, Barnesiella, and Limosilactobacillus, decreased Erysipelatoclostridium abundance on day 42 in necrotic enteritis-challenged broilers. M-Pro group increased Turicibacter abundance on day 28 and the abundances of Alistipes, Barnesiella, and Limosilactobacillus on day 42 in necrotic enteritis-challenged broilers. H-Pro group decreased Romboutsia abundance on day 28 and unidentified_Clostridia abundance on day 42 in necrotic enteritis-challenged broilers. Analysis of short-chain fatty acids (SCFAs) revealed higher isobutyric acid and isovaleric acid levels in L-Pro and M-Pro groups than NE group. Correlation analysis revealed the correlations between the biochemical parameters and gut microbiota as well as SCFAs, especially Romboutsia, Barnesiella, Coprobacter, isobutyric acid, and isovaleric acid. Overall, our results indicated that B. subtilis HW2 supplementation could ameliorate necrotic enteritis infection-induced gut injury. The optimal dietary supplementation dosage of Bacillus subtilis HW2 was 5 × 106 CFU/g.

Administration of the GLP-1 receptor agonist exenatide in rats improves functional recovery after spinal cord injury by reducing endoplasmic reticulum stress

After spinal cord injury (SCI), endoplasmic reticulum (ER) stress has been reported to be an integral part of the secondary injury process that causes apoptosis of glial cells, leading to remyelination failure. This report focuses on exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist widely used to treat diabetes, as a potential agent to improve functional outcome after SCI by improving the ER stress response. Exenatide administered subcutaneously immediately after injury and 7 days later in a rat model of moderate contusive SCI revealed significant improvement in hindlimb function without any hypoglycemia. Changes in the expression of glucose regulatory protein 78 (GRP78), an endoplasmic reticulum chaperone that protects against ER stress, and C/EBP homologous transcription factor protein (CHOP), a pro-apoptotic transcription factor in the apoptosis pathway were examined as indices of ER stress. We found that administration of exenatide after SCI suppressed CHOP while increasing GRP78 in the injured spinal cord, leading to a significant decrease in tissue damage and a significant increase in oligodendrocyte progenitor cell survival. This study suggests that administration of exenatide after SCI decreases ER stress and improves functional recovery without any apparent side-effects.

Sex-Specific Effects of Buprenorphine on Endoplasmic Reticulum Stress, Abnormal Protein Accumulation, and Cell Loss After Pediatric Mild Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) in children often leads to poor developmental outcomes attributable to progressive cell loss caused by secondary injuries, including endoplasmic reticulum (ER) stress. Buprenorphine (BPN) is commonly used in children for pain management; however, the effects of BPN on ER stress in the pediatric population are still inconclusive. This study investigated the sex-specific effects of BPN on ER stress, abnormal protein accumulation, and cell loss in a mouse impact acceleration model of pediatric TBI. On post-natal day 20-21 (P20-21), male and female littermates were randomized into sham, TBI + saline and TBI + BPN groups. BPN (0.075 mg/kg) was administered to TBI + BPN mice at 30 min after injury and then every 6-12 h for 2 days. The impact of BPN was evaluated at 1, 3, and 7 days post-injury. We found that TBI induced more prominent ER stress pathway activation at 1 and 3 days post-injury in males, compared to females, whereas abnormal protein accumulation and cell loss were more severe in females at 7 days post-injury, compared with males. Although BPN partially ameliorated abnormal protein accumulation and cell loss in both males and females, BPN only decreased ER stress pathway activation in males, not in females. In conclusion, BPN exhibits sex-specific effects on ER stress, abnormal protein accumulation, and cell loss in a time-dependent manner at the acute phase after pediatric TBI, which provides the rationale to assess the potential effects of BPN on long-term outcomes after pediatric TBI in both males and females.

Compound Qiying Granules alleviates diabetic peripheral neuropathy by inhibiting endoplasmic reticulum stress and apoptosis

BackgroundDiabetic peripheral neuropathy (DPN) is a major complication of diabetes. This study aimed to investigate the therapeutic effects and molecular mechanisms of Compound Qiying Granules (CQYG) for DPN.MethodsRats and RSC96 cells of DPN models were established to evaluate the therapeutic effects of CQYG. Then the morphology and apoptotic changes of sciatic nerves were detected. Further, tandem mass tag based quantitative proteomics technology was used to identify differentially expressed proteins (DEPs) and the underlying molecular mechanisms. Protein expression of key signaling pathways was also detected.ResultsCQYG treatment significantly improved blood glucose and oxidative stress levels, and further reduced nerve fiber myelination lesions, denervation, and apoptosis in DPN rats. Further, 2176 DEPs were found in CQYG treated DPN rats. Enrichment analysis showed that protein processing in the endoplasmic reticulum (ER), and apoptosis were all inhibited after CQYG treatment. Next, CQYG treatment reduced inflammatory factor expression, mitochondrial damage, and apoptosis in RSC96 cells which induced by high glucose. Transmission electron microscopy results found that CQYG treatment improved the morphology of nerve myelin, mitochondria, and ER. CQYG treatment decreased ER stress and apoptosis pathway proteins that were highly expressed in DPN models. In addition, we also predicted the potential targets of CQYG in DEPs.ConclusionsCQYG exerts neuroprotective effects in experimental diabetic neuropathy through anti-ER stress and anti-apoptosis.

Silibinin protects GLUTag cells from PA-induced injury via suppressing endoplasmic reticulum stress

ABSTRACT Silibinin is a natural flavonoid with anti-diabetic activity. Glucagon-like peptide−1 (GLP−1), an intestinal hormone mainly secreted from L cells, which regulates insulin production and sensitivity, appears to be a potential therapeutic strategy for T2DM. The current study aims to determine the protective effect of silibinin against palmitic acid (PA)-induced damage in GLUTag cells. The results revealed that PA triggered endoplasmic reticulum (ER) stress and apoptosis in GLUTag cells, while silibinin attenuated PA-induced lipotoxicity. Based on the estrogen-like effects of silibinin and the role of estrogen receptors in regulating glycolipid metabolism, the involvement of estrogen receptors in the protective effects of silibinin in GLUTag cells was further investigated. The results showed that estrogen receptor α- and β-specific inhibitors reversed the inhibitory impact of silibinin on ER stress. Our study demonstrated that silibinin protects GLUTag cells from PA-induced injury by decreasing ER stress under the regulation of estrogen receptors α and β.

ADP-ribose transferase PARP16 mediated-unfolded protein response contributes to neuronal cell damage in cerebral ischemia/reperfusion.

Ischemic stroke is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis, leading to impairment of endoplasmic reticulum (ER) function and activating the unfolded protein response (UPR). PARP16 is an active (ADP-ribosyl)transferase known tail-anchored ER transmembrane protein with a cytosolic catalytic domain. Here, we find PARP16 is highly expressed in ischemic cerebral hemisphere and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated immortalized hippocampal neuronal cell HT22. Using an adeno-associated virus-mediated PARP16 knockdown approach in mice, we find PARP16 knockdown decreases infarct demarcations and has a better neurological outcome after ischemic stroke. Our data indicate PARP16 knockdown decreases ER stress and neuronal death caused by OGD/R, whereas PARP16 overexpression promotes ER stress-mediated cell damage in primary cortical neurons. Furthermore, PARP16 functions mechanistically as ADP-ribosyltransferase to modulate the level of ADP-ribosylation of the corresponding PERK and IRE1α arm of the UPR, and such modifications mediate activation of PERK and IRE1α. Indeed, pharmacological stimulation of the UPR using Brefeldin A partly counteracts PARP16 knockdown-mediated neuronal protection upon OGD/R treatment. In conclusion, PARP16 plays a crucial role in post-ischemic UPR and PARP16 knockdown alleviates brain injury after ischemic stroke. This study demonstrates the potential of the PARP16-PERK/IRE1α axis as a target for neuronal survival in ischemic stroke.

Endoplasmic Reticulum Stress and Cancer: Could Unfolded Protein Response Be a Druggable Target for Cancer Therapy?

Unfolded protein response (UPR) is an adaptive response which is used for re-establishing protein homeostasis, and it is triggered by endoplasmic reticulum (ER) stress. Specific ER proteins mediate UPR activation, after dissociation from chaperone Glucose-Regulated Protein 78 (GRP78). UPR can decrease ER stress, producing an ER adaptive response, block UPR if ER homeostasis is restored, or regulate apoptosis. Some tumour types are linked to ER protein folding machinery disturbance, highlighting how UPR plays a pivotal role in cancer cells to keep malignancy and drug resistance. In this review, we focus on some molecules that have been revealed to target ER stress demonstrating as UPR could be a new target in cancer treatment.

Pan-Src kinase inhibitor treatment attenuates diabetic kidney injury via inhibition of Fyn kinase-mediated endoplasmic reticulum stress

Src family kinases (SFKs) have been implicated in the pathogenesis of kidney fibrosis. However, the specific mechanism by which SFKs contribute to the progression of diabetic kidney disease (DKD) remains unclear. Our preliminary transcriptome analysis suggested that SFK expression was increased in diabetic kidneys and that the expression of Fyn (a member of the SFKs), along with genes related to unfolded protein responses from the endoplasmic reticulum (ER) stress signaling pathway, was upregulated in the tubules of human diabetic kidneys. Thus, we examined whether SFK-induced ER stress is associated with DKD progression. Mouse proximal tubular (mProx24) cells were transfected with Fyn or Lyn siRNA and exposed to high glucose and palmitate (HG-Pal). Streptozotocin-induced diabetic rats were treated with KF-1607, a novel pan-Src kinase inhibitor (SKI) with low toxicity. The effect of KF-1607 was compared to that of losartan, a standard treatment for patients with DKD. Among the SFK family members, the Fyn and Lyn kinases were upregulated under diabetic stress. HG-Pal induced p70S6 kinase and JNK/CHOP signaling and promoted tubular injury. Fyn knockdown but not Lyn knockdown inhibited this detrimental signaling pathway. In addition, diabetic rats treated with KF-1607 showed improved kidney function and decreased ER stress, inflammation, and fibrosis compared with those treated with losartan. Collectively, these findings indicate that Fyn kinase is a specific member of the SFKs implicated in ER stress activation leading to proximal tubular injury in the diabetic milieu and that pan-SKI treatment attenuates kidney injury in diabetic rats. These data highlight Fyn kinase as a viable target for the development of therapeutic agents for DKD.

Endoplasmic-reticulum-stress-induced lipotoxicity in human kidney epithelial cells.

Accumulation of lipids and their intermediary metabolites under endoplasmic reticulum (ER) stress instigates metabolic failure, described as lipotoxicity, in the kidney. This study aimed to determine ER-stress-related sphingolipid and polyunsaturated fatty acid (PUFA) changes in human kidney cells. Tunicamycin (TM) was employed to induce ER stress and an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), was given to minimize cytotoxicity. Cell viability was determined by MTT assay. Sphingomyelin (SM), ceramide (CER), and PUFA levels were measured by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78), cleaved caspase-3 and cyclooxygenase-1 (COX-1) levels were assessed by immunofluorescence. Cytosolic phospholipase A2 (cPLA2), total COX, and prostaglandin E2 (PGE2) were measured to evaluate changes in enzyme activity. Decreased cell viability was observed in TM treated cells. Administration of TUDCA following TM treatment significantly increased cell viability compared to TM treatment alone. Tunicamycin-induced ER stress was confirmed by significantly increased protein levels of GRP78. A significant increase was observed in C18-C24 CERs and caspase-3 activity, while a significant decrease occurred in sphingosine-1-phosphate (S1P) and cPLA2 activity in cells treated with TM versus controls. The decrease in cPLA2 activity was accompanied by significantly increased PUFA levels in TM treated cells. TUDCA treatment in conjunction with TM significantly decreased ER stress, C18-C24 CERs, caspase 3 activity, and increased S1P levels. Results show the buildup of long chain CERs and PUFAs in kidney cells undergoing ER stress alongside increased apoptotic activity. TUDCA administration, along with TM treatment alleviated the buildup of CERs and TM-induced apoptotic activity in kidney epithelial cells.

Inhibition of TRPA1 Ameliorates Periodontitis by Reducing Periodontal Ligament Cell Oxidative Stress and Apoptosis via PERK/eIF2α/ATF-4/CHOP Signal Pathway.

Objective In periodontitis, excessive oxidative stress combined with subsequent apoptosis and cell death further exacerbated periodontium destruction. TRPA1, an important transient receptor potential (TRP) cation channel, may participate in the process. This study is aimed at exploring the role and the novel therapeutic function of TRPA1 in periodontitis. Methods Periodontal ligament cells or tissues derived from healthy and periodontitis (PDLCs/Ts and P-PDLCs/Ts) were used to analyze the oxidative and apoptotic levels and TRPA1 expression. TRPA1 inhibitor (HC030031) was administrated in inflammation induced by P. gingivalis lipopolysaccharide (P.g.LPS) to investigate the oxidative and apoptotic levels of PDLCs. The morphology of the endoplasmic reticulum (ER) and mitochondria was identified by transmission electron microscope, and the PERK/eIF2α/ATF-4/CHOP signal pathways were detected. Finally, HC030031 was administered to periodontitis mice to evaluate its effect on apoptotic and oxidative levels in the periodontium and the relieving of periodontitis. Results The oxidative, apoptotic levels and TRPA1 expression were higher in P-PDLC/Ts from periodontitis patients and in P.g.LPS-induced inflammatory PDLCs. TRPA1 inhibitor significantly decreased the intracellular calcium, oxidative stress, and apoptosis of inflammatory PDLCs and decreased ER stress by downregulating PERK/eIF2α/ATF-4/CHOP pathways. Meanwhile, the overall calcium ion decrease induced by EGTA also exerted similar antiapoptosis and antioxidative stress functions. In vivo, HC030031 significantly reduced oxidative stress and apoptosis in the gingiva and periodontal ligament, and less periodontium destruction was observed. Conclusion TRPA1 was highly related to periodontitis, and TRPA1 inhibitor significantly reduced oxidative and apoptotic levels in inflammatory PDLCs via inhibiting ER stress by downregulating PERK/eIF2α/ATF-4/CHOP pathways. It also reduced the oxidative stress and apoptosis in periodontitis mice thus ameliorating the development of periodontitis.

Silymarin diminishes oleic acid-induced lipid accumulation in HepG2 cells by modulating the expression of endoplasmic reticulum stress markers

Background and objectivesNon-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder which presents with the accumulation of triglycerides in hepatocytes due to imbalances in the uptake, synthesis, export, and oxidation of fatty acids. Silymarin is a flavonolignan derived from milk thistle, a herbal medicine that has been used for centuries to treat liver disease. In this study, the effect of silymarin on genes involved in lipogenesis and endoplasmic reticulum (ER) stress was investigated in the cellular NAFLD model. Materials and methodsHepG2 cells were treated with 0.5 mM oleic acid (OA) in the presence or absence of silymarin for 24 h, and subsequently, lipid accumulation was assessed by staining the cells with Oil red O as well as the measurement of intracellular triglyceride (TG) content with an enzymatic method. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to measure the expression of sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), Sirtuin 1 (SIRT1), TRB3, and ATF4. ResultsLipid accumulation was increased in OA-treated hepatocytes, an effect that was attenuated by silymarin. The expression of FAS, SREBP-1, TRB3, and ATF4 was significantly increased in OA-treated HepG2 cells, while the expression of SIRT1 was decreased. Treatment with silymarin could successfully diminish SREBP-1, FAS, TRB3, and ATF4 gene expression while augmenting the expression of SIRT1. ConclusionsSilymarin not only efficiently reduces lipogenesis but also decreases ER stress, which is a critical parameter in lipogenesis. Therefore, silymarin might be considered an effective supplementary option in the treatment of liver steatosis.

Peptidylarginine deiminase 2 promotes T helper 17-like T cell activation and activated T cell-autonomous death (ACAD) through an endoplasmic reticulum stress and autophagy coupling mechanism

Peptididylarginine deiminase type 2 (PADI2) catalyzes the conversion of arginine residues to citrulline residues on proteins. We demonstrate that PADI2 induces T cell activation and investigate how PADI2 promotes activated T cell autonomous death (ACAD). In activated Jurkat T cells, overexpression of PADI2 significantly increases citrullinated proteins and induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling, ultimately resulting in the expression of autophagy-related proteins and autophagy. PADI2 promoted autophagy and resulted in the early degradation of p62 and the light chain 3B (LC3B)-II accumulation. In Jurkat T cells, silencing the autophagy-related gene (Atg) 12 protein inhibits PADI2-mediated autophagy and promotes ER stress and apoptosis, whereas overexpression of Atg12 decreased ER stress and prolonged autophagy to promote cell survival. Additionally, PADI2 regulates T cell activation and the production of Th17 cytokines in Jurkat T cells (interleukins 6, IL-17A, IL-17F, IL-21, and IL-22). In Jurkat T cells, silencing IL-6 promotes autophagy mediated by PADI2 and inhibits PADI2-induced apoptosis, whereas silencing Beclin-1 increases the activation and survival of Th17-like T cells while decreasing autophagy and apoptosis. PADI2 silencing alleviates ER stress caused by PADI2 and decreases cytokine expression associated with Th17-like T cell activation and ACAD. We propose that PADI2 was involved in Th17 lymphocyte ACAD via a mechanism involving ER stress and autophagy that was tightly regulated by PADI2-mediated citrullination. These findings suggest that inhibiting Th17 T cell activation and the development of severe autoimmune diseases may be possible through the use of novel antagonists that specifically target PADI2.

The interaction of SIRT4 and Calreticulin during ER stress in glia cells.

Endoplasmic Reticulum (ER) stress is the response that occurs after the dysfunction of ER and its structure. Activated UPR triggers a stress response using ER membrane proteins such as PERK, IRE-1, GRP78, ATF5 ve ATF6. Sirtuins are enzymes that carry out post-translational modifications such as deacetylation and ADP-ribosylation. In our previous study, we identified Calreticulin as a SIRT4-interacting protein via mass spectrometry. Calreticulin binds to misfolded proteins, prevents them from leaving ER, which results in the reduction of ER stress. In this study, we aimed to investigate the interaction between SIRT4 and Calreticulin during ER stress in glia cells (IHA-immortalized human astrocytes). To trigger ER stress in glia cells, we first optimized the dose and the duration of the Tunicamycin which is 2.5μg/ml concentration for 16h. SIRT4 gene was silenced with lentiviral particles using 4 MOI (Multiplicity of Infection). In SIRT4-silenced cells, when treated with 2.5μg/ml Tunicamycin for 16h, the increase in the expressions of ATF6, GRP78 and the ratio of spliced/unspliced XBP1 mRNA were reduced. This shows that silencing SIRT4 may decrease ER stress. SIRT4-Calreticulin interaction was shown both in control and ER-stress induced glia cells. Additionally, this interaction did not change with the ER stress. SIRT4 only ADP-ribosylates Calreticulin during ER stress. Normally, SIRT4 ADP-ribosylates and deactivates Calreticulin during ER stress condition. When SIRT4 is silenced, the ADP-ribosylation level of Calreticulin decreases resulting in the activation of Calreticulin and the reduction of ER stress. In summary, SIRT4 inhibitors may be investigated as protective agents or drug candidates in neurodegenerative diseases where ER stress mostly underlies as one of the molecular mechanisms.

Fucoidan alleviates the mitochondria and endoplasmic reticulum stresses in ischemic rat livers

BackgroundHepatic ischemia reperfusion (I/R) injury remains a major problem for liver surgery leading to graft dysfunction. The use of compounds of natural origin as fucoidan, a sulfated polysaccharide from brown seaweed, could have a potential clinical benefit in the treatment of ischemic diseases. Nevertheless, the accurate mechanisms of action of fucoidan on endoplasmic reticulum (ER) stress response and mitochondria after warm hepatic ischemia have not been yet investigated. Thus, the present study focused on the modulation of fucoidan effects on the signaling pathways involving the mitochondria and the ER in ischemic rat liver. MethodsMale Wistar rats were subjected to either sham operation or one hour of partial warm ischemia (70%) followed by two hours of reperfusion. Before ischemia, rats were treated with 0.9% NaCl (I/R group), fucoidan (100 mg/kg body weight) orally for 7 consecutive days (I/R-Fuc group), TUDCA (tauroursodeoxycholic acid, inhibitor of ER stress) (100 mg / kg body weight, i.v.) 10 min before ischemia (I/R-TUDCA group) or fucoidan with TUDCA (I/R-Fuc-TUDCA group). ResultsOur results showed that fucoidan and TUDCA reduced cytolysis and induced a significant improvement in antioxidant status, as compared to I/R. Interestingly, preconditioning with fucoidan resulted in significant decreased ER stress, as reflected by GRP78, p-PERK, ATF-6, XBP-1 and TRAF2. Furthermore, the phosphorylation of MAPKs (ERK, JUNK and P38) significantly diminished after fucoidan treatment. Rats undergoing fucoidan treatment protected liver against mitochondrial stress, which was correlated with low induction of apoptosis (caspase-3). Inhibition of ER stress by TUDCA administration boosted all the protective effects of fucoidan. ConclusionIn conclusion, fucoidan treatment may represent an effective strategy in reducing liver I/R injury through mitochondrial stress attenuation and ER inhibition.

Zinc Accumulation Aggravates Cerebral Ischemia/Reperfusion Injury Through Inducing Endoplasmic Reticulum Stress.

Zinc is highly enriched in the central nervous system. Numerous evidences suggest that high concentration of zinc acts as a critical mediator of neuronal death in the ischemic brain, however, the possible mechanisms of neurotoxicity of zinc during cerebral ischemia/reperfusion (I/R) remain elusive. Endoplasmic reticulum (ER) is a storage location of intracellular zinc. ER stress related genes were up-regulated during zinc-induced neuronal death in vascular-type senile dementia. In the present study, we investigated whether intracellular accumulated zinc aggravates I/R injury through ER stress and ER stress-associated apoptosis. Male Sprague-Dawley rats were subjected to 90min middle cerebral artery occlusion (MCAO) and received either vehicle or zinc chelator TPEN 15mg/kg. The expression of ER stress related factors glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α), ER stress related apoptotic proteins CCAAT-enhancer-binding protein homologous protein (CHOP) and caspase-12, as well as anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) were assessed 24h after reperfusion. Our results showed that the levels of GRP78 and p-eIF2α, as well as CHOP and caspase-12, were increased in ischemic brain, indicating that cerebral I/R triggers ER stress. Furthermore, GRP78, CHOP and caspase-12 were all colocalized with the zinc-specific dyes NG, suggesting that there is certain relationship between cytosolic labile zinc and ER stress following cerebral ischemia. Chelating zinc with TPEN reversed the expression of GRP78, p-eIF2α in ischemic rats. Moreover, CHOP and NeuN double staining positive cells, as well as caspase-12 and TUNEL double staining positive cells were also decreased after TPEN treatment, indicating that chelating zinc might inhibit ER stress and decreased ER stress associated neuronal apoptosis. In addition, TPEN treatment reversed the downregulated level of Bcl-2, which localized in the ER membrane and involved in the dysfunction of ER, confirming that the anti-apoptosis effects of chelating zinc following I/R are exerted via inhibition of the ER stress. Taken together, this study demonstrated that excessive zinc activates ER stress and zinc induced neuronal cell death is at least partially due to ER stress specific neuronal apoptosis in ischemic penumbra, which may provide an important mechanism of cerebral I/R injury.

NRF2-mediated SIRT3 induction protects hepatocytes from ER stress-induced liver injury.

Chronic endoplasmic reticulum (ER) stress in hepatocytes plays a role in the pathogenesis of nonalcoholic fatty liver disease. Therefore, given the association between oxidative stress, mitochondrial dysfunction, and ER stress, our study investigated the role of NRF2-mediated SIRT3 activation in ER stress. SIRT3, a sirtuin, was predicted as the target of NRF2 based on bioinformatic analyses and animal experiments. Nrf2 abrogation diminished mitochondrial DNA content in hepatocytes with Ppargc1α and Cpt1a inhibition, whereas its overexpression enhanced oxygen consumption. Further, chromatin immunoprecipitation and luciferase reporter assays indicated that NRF2 induced SIRT3 through the antioxidant responsive element (ARE) sites comprising the -641 to -631bp and -419 to -409bp regions. In tunicamycin-induced ER stress conditions and liver injury animal models following ER stress, NRF2levels were highly correlated with SIRT3. Nrf2 deficiency enhanced the tunicamycin-mediated induction of CHOP, which was attenuated by Sirt3 overexpression. Further, Sirt3 delivery to hepatocytes in Nrf2knockout mice prevented tunicamycin from increasing mortality by decreasing ER stress. SIRT3 was upregulated in livers of patients with nonalcoholic liver diseases, whereas lower SIRT3 expression coincided with more severe disease conditions. Taken together, our findings indicated that NRF2-mediated SIRT3 induction protects hepatocytes from ER stress-induced injury, which may contribute to the inhibition of liver disease progression.