Occurrence Of Endoplasmic Reticulum Stress Research Articles

Chronic Epinephrine-Induced Endoplasmic Reticulum and Oxidative Stress Impairs Pancreatic β-Cells Function and Fate.

Epinephrine influences the function of pancreatic β-cells, primarily through the α2A-adrenergic receptor (α2A-AR) on their plasma membrane. Previous studies indicate that epinephrine transiently suppresses insulin secretion, whereas prolonged exposure induces its compensatory secretion. Nonetheless, the impact of epinephrine-induced α2A-AR signaling on the survival and function of pancreatic β-cells, particularly the impact of reprogramming after their removal from sustained epinephrine stimulation, remains elusive. In the present study, we applied MIN6, a murine insulinoma cell line, with 3 days of high concentration epinephrine incubation and 2 days of standard incubation, explored cell function and activity, and analyzed relevant regulatory pathways. The results showed that chronic epinephrine incubation led to the desensitization of α2A-AR and enhanced insulin secretion. An increased number of docked insulin granules and impaired Syntaxin-2 was found after chronic epinephrine exposure. Growth curve and cell cycle analyses showed the inhibition of cell proliferation. Transcriptome analysis showed the occurrence of endoplasmic reticulum stress (ER stress) and oxidative stress, such as the presence of BiP, CHOP, IRE1, ATF4, and XBP, affecting cellular endoplasmic reticulum function and survival, along with UCP2, OPA1, PINK, and PRKN, associated with mitochondrial dysfunction. Consequently, we conclude that chronic exposure to epinephrine induces α2A-AR desensitization and leads to ER and oxidative stress, impairing protein processing and mitochondrial function, leading to modified pancreatic β-cell secretory function and cell fate.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

Marsdenia tenacissima extract induces endoplasmic reticulum stress-associated immunogenic cell death in non-small cell lung cancer cells through targeting AXL

Ethnopharmacological relevanceMarsdenia Tenacissima (Roxb.) Wight et Arn. is a traditional Chinese medicine. Its standardized extract (MTE), with the trade name Xiao-Ai-Ping injection, is widely used for cancer treatment. The pharmacological effects of MTE-inducing cancer cell death have been primarily explored. However, whether MTE triggers tumor endoplasmic reticulum stress (ERS)-associated immunogenic cell death (ICD) is unknown. Aim of the studyTo determine the potential role of endoplasmic reticulum stress in the anti-cancer effects of MTE, and uncover the possible mechanisms of endoplasmic reticulum stress-associated immunogenic cell death induced by MTE. Material and methodsThe anti-tumor effects of MTE on non-small cell lung cancer (NSCLC) were examined through CCK-8 and wound healing assay. Network pharmacology analysis and RNA-sequencing (RNA seq) were performed to confirm the biological changes of NSCLCs after MTE treatment. Western blot, qRT-PCR, reactive oxygen species (ROS) assay, and mitochondrial membrane potential (MMP) assay were used to explore the occurrence of endoplasmic reticulum stress. Immunogenic cell death-related markers were tested by ELISA and ATP release assay. Salubrinal was used to inhibit the endoplasmic reticulum stress response. SiRNA and bemcentinib (R428) were used to impede the function of AXL. AXL phosphorylation was regained by recombinant human Gas6 protein (rhGas6). The effects of MTE on endoplasmic reticulum stress and immunogenic cell death response were also proved in vivo. The AXL inhibiting compound in MTE was explored by molecular docking and confirmed by Western blot. ResultsMTE inhibited cell viability and migration of PC-9 and H1975 cells. Enrichment analysis identified that differential genes after MTE treatment were significantly enriched in endoplasmic reticulum stress-related biological processes. MTE decreased mitochondrial membrane potential (MMP) and increased ROS production. Meanwhile, endoplasmic reticulum stress-related proteins (ATF6, GRP-78, ATF4, XBP1s, and CHOP) and immunogenic cell death-related markers (ATP, HMGB1) were upregulated, and the AXL phosphorylation level was suppressed after MTE treatment. However, when salubrinal (an endoplasmic reticulum stress inhibitor) and MTE were co-treated cells, the inhibitory effects of MTE on PC-9 and H1975 cells were impaired. Importantly, inhibition of AXL expression or activity also promotes the expression of endoplasmic reticulum stress and immunogenic cell death-related markers. Mechanistically, MTE induced endoplasmic reticulum stress and immunogenic cell death by suppressing AXL activity, and these effects were attenuated when AXL activity recovered. Moreover, MTE significantly increased the expression of endoplasmic reticulum stress-related markers in LLC tumor-bearing mouse tumor tissues and plasma levels of ATP and HMGB1. Molecular docking illustrated that kaempferol has the strongest binding energy with AXL and suppresses AXL phosphorylation. ConclusionMTE induces endoplasmic reticulum stress-associated immunogenic cell death in NSCLC cells. The anti-tumor effects of MTE are dependent upon endoplasmic reticulum stress. MTE triggers endoplasmic reticulum stress-associated immunogenic cell death by inhibiting AXL activity. Kaempferol is an active component that inhibits AXL activity in MTE. The present research revealed the role of AXL in regulating endoplasmic reticulum stress and enriched the anti-tumor mechanisms of MTE. Moreover, kaempferol may be considered a novel AXL inhibitor.

The involvement of oxidative stress mediated endoplasmic reticulum pathway in apoptosis of Golden Pompano (Trachinotus blochii) liver under PS-MPs stress

Globally, microplastics (MPs) are highly prevalent, especially in coastal areas. Unfortunately, golden pompano as a major marine fish in China is typically raised in floating marine cages near coasts, facing these MPs sources. However, toxicological studies on Golden Pompano which farm in coastal areas and face actual microplastic exposure are rare. Therefore, golden pompano were exposed to 10.0 μg/L, 100.0 μg/L, and 1000.0 μg/L polystyrene MPs (PS-MPs) for 14 days to study the potential impact of the microplastics on the Golden Pompano. Fish show slowed growth after 14 days of exposure. Histopathology shows irregular shaped nuclei and nuclear and cytoplasmic vacuolation traits in liver. Oxidative stress-related enzyme activity and gene expression data show that oxidative damage occurs in the high-concentrations (100.0 μg/L and 1000.0 μg/L) of PS-MPs exposures. Up-regulation of Grp78, Xbp-1, Eif-2α and chop gene expression indicates the occurrence of endoplasmic reticulum stress, and the western blot results also confirmed this. Severe oxidative stress also caused ERS, which ultimately increased BAX/Bcl-2 ratios and induces apoptosis. Furthermore, up-regulated anaerobic respiration, altered lipid metabolism, and immune disturbance were exhibited during PS-MPs stress. Therefore, oxidative stress appeared to be the main toxicity issue caused by MPs, while ERS-mediated apoptosis, metabolic alterations, and immune responses were induced by this stress. Notably, endoplasmic reticulum stress and apoptosis are a self-protective mechanism, which may be an intermediate link in the toxicity of microplastics. This study highlights the role of endoplasmic reticulum stress in MPs toxicology and assesses the adverse effects of microplastics on Golden Pompano.

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

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

Taurine Alleviates Cadmium-Induced Toxicity via Genetically Specific Strategies in Two Strains of Gibel Carp (Carassius gibelio)

Our previous studies in gibel carp (Carassius gibelio) have shown that cadmium (Cd) exposure elicits deleterious effects depending on the genetic background, and thus we hypothesized that mitigation via nutritional intervention may vary between strains. Therefore, two gibel carp strains (the A and F strains) were fed diets supplemented with 0% or 1% taurine for 8 weeks prior to 96 h Cd exposure, and the responses of antioxidant pathways, endoplasmic reticulum (ER) stress, autophagy, and apoptosis were investigated. The results showed that taurine supplementation had no effect on the growth performance of gibel carp. After Cd exposure, histological damage to mitochondria and ER, induction of oxidative stress and antioxidant responses, occurrence of ER stress, and apoptotic signals were observed in the livers. Upon the diet effects, taurine supplementation alleviated the ER-stress-induced autophagy and apoptosis after Cd exposure and stimulated antioxidant pathways. Regarding the difference between strains, taurine played a protective role in alleviating Cd toxicity through the antioxidant response, ER stress, and autophagy in the F strain, whereas such effects were achieved by the attenuation of apoptosis in the A strain. Taken together, our results demonstrate the potential use of taurine in the mitigation of heavy metal toxicity in aquatic organisms.

Regulatory effects of Trichinella spiralis and a serine protease inhibitor on the endoplasmic reticulum stress response of intestinal epithelial cells

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause an endoplasmic reticulum stress (ERS) response. If ERS continues or cannot be alleviated, it will cause the production of proapoptotic factors and eventually lead to apoptosis. Therefore, this study mainly explored whether Trichinella spiralis Kazal-type serine protease inhibitor (TsKaSPI) contributed to the invasion of intestinal epithelial cells during the infectious stage of T. spiralis by regulating ERS. First, in the T. spiralis infection model, H&E staining was used to analyse the damage to jejunum tissue, a TUNEL assay was used to examine cell apoptosis, and the expression of ERS-related and apoptosis-related molecules was also measured. The results showed that ERS occurred during the intestinal phase of T. spiralis infection, while remission began during the cyclic phase. Then, we selected TsKaSPI, one of the important components of T. spiralis ES antigens, for in vitro experiments. The results showed that TsKaSPI could induce apoptosis in a porcine small intestinal epithelial cell line (IPEC cells) by activating ERS and promote activation of the NF-κB signalling pathway. Inhibition experiments confirmed that the occurrence of ERS was accompanied by the activation of NF-κB, and the two processes regulated each other. Finally, we conducted in vivo experiments and administered TsKaSPI to mice. The results confirmed that TsKaSPI could activate ERS and lead to apoptosis in intestinal epithelial cells. In conclusion, T. spiralis infection and TsKaSPI can promote cell apoptosis by activating the ERS response in intestinal epithelial cells and activate the NF-κB signalling pathway to promote the occurrence and development of inflammation.

Mannan-binding lectin deficiency augments hepatic endoplasmic reticulum stress through IP3R-controlled calcium release

The aberrant release of endoplasmic reticulum (ER) calcium leads to the disruption of intracellular calcium homeostasis, which is associated with the occurrence of ER stress and closely related to the pathogenesis of liver damage. Mannan-binding lectin (MBL) is a soluble calcium-dependent protein synthesized primarily in hepatocytes and is a pattern recognition molecule in the innate immune system. MBL deficiency is highly prevalent in the population and has been reported to be associated with susceptibility to several liver diseases. We here showed that genetic MBL ablation strongly sensitized mice to ER stress-induced liver injury. Mechanistic studies established that MBL directly interacted with ER-resident chaperone immunoglobulin heavy chain binding protein (BiP), and MBL deficiency accelerated the separation of PKR-like ER kinase (PERK) from BiP during hepatic ER stress. Moreover, MBL deficiency led to enhanced activation of the PERK-C/EBP-homologous protein (CHOP) pathway and initiates an inositol 1,4,5-trisphosphate receptor (IP3R)-mediated calcium release from the ER, thereby aggravating the hepatic ER stress response. Our results demonstrate an unexpected function of MBL in ER calcium homeostasis and ER stress response, thus providing new insight into the liver injury related to ER stress in patients with MBL deficiency.

Maimendong Decoction Improves Pulmonary Function in Rats With Idiopathic Pulmonary Fibrosis by Inhibiting Endoplasmic Reticulum Stress in AECIIs.

This study was designed to investigate the mechanism by which MMDD improves lung function, and observe the effect of MMDD on endoplasmic reticulum stress(ERS) in alveolar type II epithelial cells (AECIIs) of pulmonary fibrosis rats. pulmonary fibrosis animal model was established by intratracheal injection of BLM at a dose of 6mg/kg body weight. Overall, Thirty male SPF Sprague-Dawley rats were randomly divided into control group, BLM group and BLM+MMDD group. BLM+MMDD group rats were fed 24 g/kg over three weeks for twice a day on the fourteenth day after model establishment. MMDD improves pulmonary function of fibrotic rats and reduces the occurrence of endoplasmic reticulum stress in AECIIs. MMDD could significantly improve the forced vital capacity (FVC) of bleomycin-induced pulmonary fibrosis in rats. MMDD reduced the expression of GRP78 and CHOP in AECIIs, increased the secretion of surfactant protein C (SPC) by AECIIs. Moreover, the apoptosis of the fibrosis zone in the lung tissue was remarkably mitigated by administration of MMDD. The finding of this study revealed that MMDD can improve lung function in rats with pulmonary fibrosis by reducing the occurrence of ERS and cell apoptosis of AECIIs. It may provide a new method for the treatment of pulmonary fibrosis.

Citrinin exposure disrupts organelle distribution and functions in mouse oocytes

Citrinin (CTN) is a secondary fungal metabolite produced by several species of Aspergillins and Penicillins, and it is widely found in vegetable-derived foods such as cereals and fermented rice-based food supplements. Previous studies indicated that CTN had immunotoxicity, hepatotoxicity, nephrotoxicity, and reproductive toxicity, which caused severe effects on human and animal health. However, the potential toxicity of CTN on the organelles of mouse oocytes is still unclear. In this study, we showed that the exposure to 30 μM CTN significantly reduced the developmental capacity of mouse oocytes. Our results revealed that mitochondria exhibited abnormal distribution and mitochondrial membrane potential decreased under CTN exposure. And the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, which is accompanied by the occurrence of ER stress, showing with increased GRP78 expression. We also found that similar with ER, the Golgi apparatus showed homogenous localization pattern after CTN exposure, and the vesicle transport was disturbed, showing with aberrant expression and localization of Rab11a. Moreover, our results indicated that CTN exposure increased the expression of LAMP2, indicating the induction of lysosomal damage. In summary, our study showed that CTN exposure to mouse oocytes was toxic to the distribution and functions of organelles, which further led to a decrease of oocyte quality.

Chronic Microcystin-LR Exposure Induces Abnormal Lipid Metabolism via Endoplasmic Reticulum Stress in Male Zebrafish

In order to explore effects of low levels of continuous microcystin-LR (MC-LR) (a cyanotoxin) exposure on hepatic lipid metabolism on the basis of the endoplasmic reticulum stress (ERS) pathway, we exposed adult male zebrafish to MC-LR (0, 1, 5, and 25 μg/L) for 60 days, and hepatic histopathology as well as lipid metabolic parameters were determined with mRNA levels of ERS signal molecules and downstream factors, along with genes associated with lipid metabolism in zebrafish liver. The results revealed that prolonged exposure to MC-LR remarkably altered the levels of hepatic total cholesterol and triglyceride and led to hepatic steatosis, which was also confirmed by hepatic cytoplasmic vacuolization in Hematoxylin/eosin (H&E) stain and lipid droplet accumulation in Oil Red O stain. The severity of hepatic damage and lipidation was increased in a dose-related manner. MC-LR exposure significantly upregulated transcriptional levels of ERS markers including hspa5, mapk8, and chop, indicating the occurrence of ERS in the liver of zebrafish. Concurrently, MC-LR significantly improved mRNA expression of unfolded protein response (UPR) pathway-related genes including atf6, eif2ak3, ern1, and xbp1s, suggesting that all of the three UPR branches were activated by MC-LR. MC-LR also induced significant upregulation of downstream lipid metabolism-related factors and genes including srebf1, srebf2, fatty acid synthase (fasn), acetyl-CoA carboxylase (acaca), stearoyl-CoA desaturase (scd), HMG CoA reductase (hmgcra), and HMG CoA synthase (hmgcs1), and downregulation of genes associated with lipolysis such as triglyceride hydrolase gene (atgl), hormone-sensitive enzyme gene (hsla), and carnitine palmitoyltransferase gene (cpt1aa). Our present results indicated that the cause of hepatic lipid accumulation by MC-LR was mainly by upregulating lipogenic and cholesterol genes but downregulating the expression of lipolytic genes through the induction of srebf1 and srebf2, which were involved in the activation of ERS signal pathways.

Perfluorooctanoic Acid Exposure Assessment on Common Carp Liver through Image and Ultrastructural Investigation.

Perfluorooctanoic acid (PFOA) poses particular concern as an emerging pollutant in both surface and ground waters. Fish, as a natural inhabitant of these waters and being highly representative of vertebrates, represents an ideal animal model to assess the toxic effects of PFOA. Hereby, liver microscopic texture was comparatively evaluated in individuals of common carp subchronically exposed to PFOA using grayscale differential box counting, a fractal analysis method. Furthermore, liver cytoplasmic glycogen areas and ultrastructure were also evaluated and compared to the image analysis findings. Redundancy Analysis was performed to assess, in summary, how much the variation of fractal dimension and lacunarity was explained by the concentration of PFOA in liver, the mass of liver and the number of proliferating cell nuclear antigen (PCNA)-immunoreactive nuclei. Treatment group ordination was better determined by fractal dimension than lacunarity. Interestingly, a significant complexity increase was associated with the modification of liver microscopic texture due to PFOA exposure. This complexity increase was related to “cloudy swelling”, possibly representing a primarily adaptive strategy against PFOA challenge, rather than a slight, reversible form of degeneration as traditionally proposed. The occurrence of endoplasmic reticulum stress, unfolded protein reaction and hormetic response was proposed and discussed.

"Adjusting internal organs and dredging channel" electroacupuncture treatment prevents the development of diabetic peripheral neuropathy by downregulating glucose-related protein 78 (GRP78) and caspase-12 in streptozotocin-diabetic rats.

The clinical efficacy of electroacupuncture in treating diabetic peripheral neuropathy (DPN) is significant, but the underlying mechanism of action is not clear. Considering that glucose-regulated protein 78 (GRP78) and caspase-12 are major proteins participating in cell apoptosis, we investigated the effects of "adjusting internal organs and dredging channel" electroacupuncture therapy on GRP78 and caspase-12 levels in streptozotocin (STZ)-diabetic rats to elucidate the mechanism of action. Rats were first divided into two groups: one group was rendered diabetic with a single injection of 50 mg/kg STZ, whereas the other normal control group was injected with an equivalent volume of citrate buffer. The STZ-diabetic rats were randomly divided into three groups: model control and electroacupuncture- and mecobalamin-treated groups. After 12 weeks treatment, the therapeutic efficacy of electroacupuncture was assessed using sciatic nerves isolated from rats. In the electroacupuncture group, rats were treated by electroacupuncture for 20 minutes once daily for 6 days each week, with 1 day off, for 12 consecutive weeks. The selected acupressure points include bilateral acupressure points of BL13 (Fehu), BL20 (Pishu), BL23 (Shenshu), LI4 (Hegu), LR3 (faichong), ST36 (Zusanli), and SP6 (Sanyiniiao). Acupressure points were stimulated using a HuaTuo SDZ-V Electric Acupuncture Therapy Apparatus. The acupressure points of BL13 and BL23, as well as SP6 and LR3, were connected on the same side with a dilatational wave of 3 Hz (frequency ratio of 1 : 5) to stimulate the parts of the body to the extent that could be tolerated by the rat. As for the mecobalamin-treated groups, mecobalamin was administrated to rats intragastrically at a dose of 20 mg/kg once daily for 12 consecutive weeks. Immunofluorescence and western blot analysis were used to determine GRP78 and caspase-12 levels in sciatic nerves. In addition, cell apoptosis in sciatic nerves was determined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. Electroacupuncture markedly reduced the pathological injury to sciatic nerves in STZ-diabetic rats. Moreover, electroacupuncture significantly downregulated GRP78 and caspase-12 and reduced cell apoptosis of sciatic nerves in DPN rats. Electroacupuncture improved DPN by downregulating GRP78 and caspase-12 and reducing cell apoptosis of sciatic nerves in STZ-diabetic rats, and further inhibited the occurrence of endoplasmic reticulum stress, thus preventing sciatic nerve injuries.

The effect of endoplasmic reticulum stress on detrusor muscle apoptosis in diabetic cystopathy

Objective To observe the effect of Endoplasmic Reticulum Stress (ERS) on detrusor muscle apoptosis in Streptozotocin (STZ)-induced diabetic rats. Methods At 4, 8, 12, and 16 weeks after induction of diabetic rat models, the sections of detrusor muscle tissue were made and were stained with Van Gieson solution, the collagen fibers of each group were observed and their proportions were calculated; the sub-cellular ultrastructure was observed by Transmission Electron Microscopy (TEM). Proliferating Cell Nuclear Antigen (PCNA) assay kit and Strept Avidin-Biotin Complex (SABC) staining were used for the evaluation of Detrusor Smooth Muscle (DSM) cell proliferation. Moreover, the expression of three hallmarks of ERS-associated apoptosis, including Glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and cysteinyl aspartate-specific protease (Caspase)-12, was detected by Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Results The proportion of collagen fibers showed a significant climbing trend [(13.38±2.65)%, (18.14±1.93)%, (19.71±3.05)% vs. (7.86±0.72)%, P<0.05] since DM8w group, except DM4w group [(8.02±0.43)%]. With the time prolonged, the swelled and fused cisternaes started to show; the mild particle fusion and degranulation also could be seen in the zone of ER; the distorted or pyknotic nucleus contained clusters of unidentified dark-stained granules that could be observed. Compared with the control group, the increased PCNA-positive percentages [(20.34±4.55)%, (46.88±7.12)%, (39.78±1.93)%, (22.15±5.64)% vs. (16.26±2.43)%, P<0.05] and apoptosis indexes [(17.12±2.78)%, (28.12±4.09)%, (28.12±4.09)%, (43.39±6.82)% vs. (6.32±1.23)%, P<0.05] can be seen in model groups. Elevated expression of GRP78, CHOP, and Caspase-12 at both protein and mRNA levels in a time-dependent fashion were detected (P<0.05). Conclusion Morphological and ultrastructural damages to the DSM as well as the cell proliferation and apoptosis indicate the time-dependent alterations. The occurrence of ERS may be involved in the development of diabetic cystopathy (DCP) and may contribute to the diabetic detrusor muscle apoptosis. Key words: Endoplasmic reticulum stress; Diabetic cystopathy; Detrusor; Apoptosis

APOL1 risk variants cause podocytes injury through enhancing endoplasmic reticulum stress.

Two coding sequence variants (G1 and G2) of Apolipoprotein L1 (APOL1) gene have been implicated as a higher risk factor for chronic kidney diseases (CKD) in African Americans when compared with European Americans. Previous studies have suggested that the APOL1 G1 and G2 variant proteins are more toxic to kidney cells than the wild-type APOL1 G0, but the underlying mechanisms are poorly understood. To determine whether endoplasmic reticulum (ER) stress contributes to podocyte toxicity, we generated human podocytes (HPs) that stably overexpressed APOL1 G0, G1, or G2 (Vec/HPs, G0/HPs, G1/HPs, and G2/HPs). Propidium iodide staining showed that HP overexpressing the APOL1 G1 or G2 variant exhibited a higher rate of necrosis when compared with those overexpressing the wild-type G0 counterpart. Consistently, the expression levels of nephrin and podocin proteins were significantly decreased in the G1- or G2-overexpressing cells despite the maintenance of their mRNA expressions levels. In contrast, the expression of the 78-kDa glucose-regulated protein ((GRP78), also known as the binding Ig protein, BiP) and the phosphorylation of the eukaryotic translation initiation factor 1 (eIF1) were significantly elevated in the G1/HPs and G2/HPs, suggesting a possible occurrence of ER stress in these cells. Furthermore, ER stress inhibitors not only restored nephrin protein expression, but also provided protection against necrosis in G1/HPs and G2/HPs, suggesting that APOL1 risk variants cause podocyte injury partly through enhancing ER stress.

Geniposide Increases Unfolded Protein Response-Mediating HRD1 Expression to Accelerate APP Degradation in Primary Cortical Neurons.

Altered proteostasis induced by amyloid peptide aggregation and hyperphosphorylation of tau protein, is a prominent feature of Alzheimer's disease, which highlights the occurrence of endoplasmic reticulum stress and triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis. In this study, we investigated the role of geniposide in the activation of UPR induced by high glucose in primary cortical neurons. We found that high glucose induced a significant activation of UPR, and geniposide enhanced the effect of high glucose on the phosphorylation of IRE1α, the most conserved UPR signaling branch. We observed that geniposide induced the expression of HRD1, an ubiquitin-ligase E3 in a time dependent manner, and amplified the expression of HRD1 induced by high glucose in primary cortical neurons. Suppression of IRE1α activity with STF-083010, an inhibitor of IRE1 phosphorylation, prevented the roles of geniposide on the expression of HRD1 and APP degradation in high glucose-treated cortical neurons. In addition, the results from RNA interfere on HRD1 revealed that HRD1 was involved in geniposide regulating APP degradation in cortical neurons. These data suggest that geniposide might be benefit to re-establish proteostasis by enhancing the UPR to decrease the load of APP in neurons challenged by high glucose.

MicroRNA-384-mediated Herpud1 upregulation promotes angiotensin II-induced endothelial cell apoptosis

BackgroundAngiotensin II (Ang II)-induced damage to endothelial cells (ECs) plays a crucial role in the pathogenesis of atherosclerosis. This study aimed to investigate the role of microRNA-384 (miR-384) in endothelial cell apoptosis. MethodsThe expression of five various miRNAs in Ang II-treated human umbilical vein endothelial cells (HUVECs) were detected by qPCR. The Ang II-induced apoptosis of HUVECs was determined by flow cytometry, TUNEL staining and western blot. Endoplasmic reticulum (ER) stress markers were detected by western blot analysis. The target gene of miR-384 was determined by bioinformatics analyses. qPCR, western blotting and immunofluorescence were performed to determine the expression level of homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1). ResultsmiR-384 expression level was significantly decreased in Ang II-treated HUVECs. Ang II-induced HUVEC apoptosis was accompanied by the occurrence of ER stress. A decreased rate of HUVEC apoptosis and a decreased rate of ER stress were observed following restoration of miR-384 expression. Herpud1 expression level was increased in HUVECs treated with Ang II, and miR-384 mimics effectively inhibited Herpud1 expression. Mechanistically, miR-384 directly targets the 3′-untranslated region of Herpud1. Furthermore, effects of miR-384 on HUVECs apoptosis and ER stress were at least partly reversed by knockdown of Herpud1 expression. ConclusionThe results of the present study collectively indicated that miR-384 expression level was downregulated in Ang II-treated HUVECs and miR-384 overexpression protected HUVECs against Ang II-induced apoptosis by negatively regulating Herpud1. These findings point towards new strategies by which apoptosis of ECs can be suppressed.

Potential Role of Endoplasmic Reticulum Stress in Pathogenesis of Diabetic Retinopathy.

Diabetes mellitus is a metabolic disease that leads to several complications which include retinopathy. Multiple biochemical abnormalities have been proposed to explain the development of retinopathy, including oxidative stress. Although the existence of oxidative stress has been established in the retina from long standing diabetic animals, pathogenesis and progression of retinopathy remain unclear. In order to gain insight into the pathogenesis of diabetic retinopathy, we analyzed the levels of different oxidative stress biomarkers in the retina at early stages during the progress of streptozotocin-induced diabetes. No significant changes in glutathione content, expression of NADPH-oxidase, levels of lipid peroxidation, nor production of free radicals were observed in the retina up to 45days of diabetes induction. Likewise, a transient decrease in aconitase activity, parallel to an increase in the superoxide dismutase activity was observed at 20days of hyperglycemia, suggesting a high capacity of retina to maintain its redox homeostasis, at least at early stages of diabetes. Nonetheless, we found an early and time-dependent increase in the levels of oxidized proteins, which was not affected by the administration of the antioxidant quercetin. Also, positive immunoreactivity to the reticulum stress protein CHOP was found in glial Müller cells of diabetic rat retinas. These findings suggest the occurrence of endoplasmic reticulum stress as a primary event in retina pathogenesis in diabetes.

Effects of a plant product consisting of green tea and curcuma extract on milk production and the expression of hepatic genes involved in endoplasmic stress response and inflammation in dairy cows

During the periparturient phase, cows are typically in an inflammation-like condition, and it has been proposed that inflammation associated with the induction of stress of the endoplasmic reticulum (ER) in the liver contributes to the development of fatty liver syndrome and ketosis. In the present study, the hypothesis that supplementation of dairy cows with a plant product consisting of green tea (95%) and curcuma extract (5%) rich in polyphenols attenuates inflammation and ER stress in the liver during early lactation was investigated. Twenty-seven cows were assigned to two groups, either a control group (n = 14) or a treatment group (n = 13). Both groups of cows received a total mixed ration, and the ration of the treatment group was supplemented with 0.175 g of the plant product per kg dry matter from week 3 prepartum to week 9 postpartum. Dry matter intake and energy balance during week 2 to week 9 postpartum were not different between the two groups. However, cows supplemented with the plant product had a greater amount of energy-corrected milk during week 2 to week 9 postpartum and lower concentrations of triacylglycerols and cholesterol in the liver in week 1 and week 3 postpartum than cows of the control group (p < 0.05). Cows supplemented with the plant product showed a trend towards a reduced mRNA concentration of haptoglobin (p < 0.10), while relative mRNA concentrations of eight genes of the unfolded protein response considered in the liver were not different between the two groups of cows. Relative hepatic mRNA concentration of fibroblast growth factor, a stress hormone induced by various stress conditions, was reduced at week 1 and week 3 postpartum in cows supplemented with the plant product (p < 0.05). Overall, the data of this study suggest that – although there were only minor effects on the occurrence of ER stress and inflammation – a supplementation of polyphenols might be useful to improve milk yield and prevent fatty liver syndrome in dairy cows.