Expression Of Endoplasmic Reticulum Stress-related Proteins Research Articles

PDZD8 Augments Endoplasmic Reticulum-Mitochondria Contact and Regulates Ca2+ Dynamics and Cypd Expression to Induce Pancreatic β-Cell Death during Diabetes.

Diabetes mellitus (DM) is a chronic metabolic disease that poses serious threats to human physical and mental health worldwide. The PDZ domain-containing 8 (PDZD8) protein mediates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) formation in mammals. We explored the role of PDZD8 in DM and investigated its potential mechanism of action. High-fat diet (HFD)- and streptozotocin-induced mouse DM and palmitic acid (PA)-induced insulin 1 (INS-1) cell models were constructed. PDZD8 expression was detected using immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. MAM formation, interactions between voltage-dependent anion-selective channel 1 (VDAC1) and inositol 1,4,5-triphosphate receptor type 1 (IP3R1), pancreatic β-cell apoptosis and proliferation were detected using transmission electron microscopy (TEM), proximity ligation assay (PLA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, immunofluorescence staining, and Western blotting. The mitochondrial membrane potential, cell apoptosis, cytotoxicity, and subcellular Ca2+ localization in INS-1 cells were detected using a JC-1 probe, flow cytometry, and an lactate dehydrogenase kit. PDZD8 expression was up-regulated in the islets of HFD mice and PA-treated pancreatic β-cells. PDZD8 knockdown markedly shortened MAM perimeter, suppressed the expression of MAM-related proteins IP3R1, glucose-regulated protein 75 (GRP75), and VDAC1, inhibited the interaction between VDAC1 and IP3R1, alleviated mitochondrial dysfunction and ER stress, reduced the expression of ER stress-related proteins, and decreased apoptosis while increased proliferation of pancreatic β-cells. Additionally, PDZD8 knockdown alleviated Ca2+ flow into the mitochondria and decreased cyclophilin D (Cypd) expression. Cypd overexpression alleviated the promoting effect of PDZD8 knockdown on the apoptosis of β-cells. PDZD8 knockdown inhibited pancreatic β-cell death in DM by alleviated ER-mitochondria contact and the flow of Ca2+ into the mitochondria.

Endoplasmic reticulum stress is attenuated by glycolysis in lymphatic malformations.

This study aims to investigate the role of endoplasmic reticulum stress (ER stress) in human dermal lymphatic endothelial cells (HDLECs) and lymphatic malformations (LMs) and its relationship with aerobic glycolysis and inflammation. The proliferation and apoptosis of HDLECs were examined with lipopolysaccharide (LPS) treatment. ER stress-associated proteins and glycolysis-related markers were detected by western blot. Glycolysis indexes were detected by seahorse analysis and lactic acid production assay kits. Immunohistochemistry was used to reveal the ER stress state of lymphatic endothelial cells (LECs) in LMs. LPS induced ER stress in HDLECs but did not trigger detectable apoptosis. Intriguingly, LPS-treated HDLECs also showed increased glycolysis flux. Knockdown of Hexokinase 2, a key enzyme for aerobic glycolysis, significantly inhibited the ability of HDLECs to resist ER stress-induced apoptosis. Moreover, compared to normal skin, glucose-regulated protein 78 (GRP78/BIP), and phosphorylation protein kinase R-like kinase (p-PERK), two key ER stress-associated markers, were upregulated in LECs of LMs, which was correlated with the inflected state. In addition, excessively activated ER stress inhibited the progression of LMs in rat models. These data indicate that glycolysis could rescue activated ER stress in HDLECs, which is required for the accelerated development of LMs. Inflammation enhances both ER stress and glycolysis in LECs while glycolysis is required to attenuate the pro-apoptotic effect of ER stress. Endoplasmic reticulum (ER) stress is activated in lymphatic endothelial cells (LECs) of LMs, especially in inflammatory condition. The expression of ER stress-related proteins is increased in LMs and correlated with Hexokinase 2 expression. Pharmacological activation of ER stress suppresses the formation of LM lesions in the rat model. ER stress may be a promising and effective therapeutic target for the treatment of LMs.

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

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

Impact of sub-chronic polystyrene nanoplastics exposure on hematology, histology, and endoplasmic reticulum stress-related protein expression in Nile tilapia (Oreochromis niloticus)

Nanoplastics (NPs) are one of the most hazardous marine litters, having the potential to cause far-reaching impacts on the environment and humankind. The effect of NPs on fish health has been studied, but their impact on the subcellular organelles remains unexplored. The present investigation studied the possible implications of polystyrene-nanoplastics (PS-NPs) on the hematology, tissue organization, and endoplasmic reticulum (ER) stress-related proteins in Nile tilapia (Oreochromis niloticus). Fish were exposed to ∼100 nm PS-NPs at environmentally relevant (0.1 mg/L), and sublethal (1, 10 mg/L) concentrations for 14 days through water exposure. The growth performance and hematological parameters such as erythrocytes, hemoglobin, hematocrit, and leucocytes decreased, while thrombocytes increased with PS-NPs dose-dependently. The gills, liver, kidney, and heart tissues displayed increasing degrees of pathology with increased concentrations of PS-NPs. The gills showed severe epithelial hyperplasia and lamellar fusion. The liver had an abstruse cellular framework, membrane breakage, and vacuolation. While glomerular and tubular atrophy was the most prominent pathology in the kidney tissue, the heart displayed extensive myofibrillar loss and disorderly arranged cardiac cells. The ER-stress-related genes such as bip, atf6, ire1, xbp1, pkr, and apoptotic genes such as casp3a, and bax were over-expressed, while, the anti-apoptotic bcl2 was under-expressed with increasing concentrations of PS-NPs. Immunohistochemistry and blotting results of GRP78, CHOP, EIF2S, and ATF6 in gills, liver, kidney, and heart tissues affirmed the translation to ER stress proteins. The results revealed the sub-lethal adverse effects and the activation of the ER-stress pathway in fish with sub-chronic exposure to PS-NPs.

Morroniside Protects C2C12 Myoblasts from Oxidative Damage Caused by ROS-Mediated Mitochondrial Damage and Induction of Endoplasmic Reticulum Stress.

Oxidative stress contributes to the onset of chronic diseases in various organs, including muscles. Morroniside, a type of iridoid glycoside contained in Cornus officinalis, is reported to have advantages as a natural compound that prevents various diseases. However, the question of whether this phytochemical exerts any inhibitory effect against oxidative stress in muscle cells has not been well reported. Therefore, the current study aimed to evaluate whether morroniside can protect against oxidative damage induced by hydrogen peroxide (H2O2) in murine C2C12 myoblasts. Our results demonstrate that morroniside pretreatment was able to inhibit cytotoxicity while suppressing H2O2-induced DNA damage and apoptosis. Morroniside also significantly improved the antioxidant capacity in H2O2-challenged C2C12 cells by blocking the production of cellular reactive oxygen species and mitochondrial superoxide and increasing glutathione production. In addition, H2O2-induced mitochondrial damage and endoplasmic reticulum (ER) stress were effectively attenuated by morroniside pretreatment, inhibiting cytoplasmic leakage of cytochrome c and expression of ER stress-related proteins. Furthermore, morroniside neutralized H2O2-mediated calcium (Ca2+) overload in mitochondria and mitigated the expression of calpains, cytosolic Ca2+-dependent proteases. Collectively, these findings demonstrate that morroniside protected against mitochondrial impairment and Ca2+-mediated ER stress by minimizing oxidative stress, thereby inhibiting H2O2-induced cytotoxicity in C2C12 myoblasts.

Combining cisplatin with Pinellia pedatisecta Schott lipid-soluble extract induces tumor immunogenic cell death in cervical cancer

BackgroundPinellia pedatisecta Schott extract (PE) is extracted from Pinellia pedatisecta Schott (PPS), a traditional Chinese medicinal plant with the potential for direct anticancer effects or eliciting an anti-tumor response by activating the immune system. PurposeTo explore PE's ability and mechanism to reconstruct cisplatin's immunogenicity. MethodsCervical cancer cells were treated with cisplatin (CDDP) and/or PE. The exposure of calreticulin (CRT) on cell membrane was investigated by flow cytometry. The extracellular of ATP and HMGB1 was investigated by Western blot analysis, immunofluorescence and ELISA assay. Changes in immune profiles were using flow cytometry in vaccination and anti-tumor assays in vivo. Lastly, the mechanism of PE influenced the ROS/ERS pathway was examined by ROS assay kit, flow cytometry and Western blotting. ResultsPE treatment induced translocation of CRT from the endoplasmic reticulum to the cell membrane of tumor cells, concomitantly triggering immunogenic cell death (ICD). In terms of mechanisms, endoplasmic reticulum (ER) stress relievers could impede the ability of PE to induce immunogenicity. This indicates that PE is activated by ER stress, leading to subsequent induction of ICD. Upon analyzing RNA-seq data, it was observed that PE primarily induces programmed cell death in tumors by impeding upstream antioxidant mechanisms. Additionally, it transforms dying tumor cells into vaccines, activating a series of immune responses. ConclusionsThis study observed for the first time that PE-induced CRT exposure on the membrane of cervical cancer cells compensates for the defect of nonimmunogenic cell death inducer CDDP thereby stimulating potent ICD. This ability restores the immunogenicity of CDDP through ER stress induced by the ROS signal. ROS played a role in PE's ability to induce ICD, leading to increased expression of ER stress-related proteins, including ATF3 and IRE-1α. PE exerted anti-cancer effects by increasing the ROS levels, and ROS/ERS signaling may be a potential avenue for cervical cancer treatment. Hence, the synergistic use of PE and CDDP holds potential for enhancing immunochemotherapy in cancer treatment.

MHY446 induces apoptosis via reactive oxygen species-mediated endoplasmic reticulum stress in HCT116 human colorectal cancer cells

This study investigated the potential of a newly synthesized histone deacetylase (HDAC) inhibitor, MHY446, in inducing cell death in HCT116 colorectal cancer cells and compared its activity with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. The results showed that MHY446 increased the acetylation of histones H3 and H4 and decreased the expression and activity of HDAC proteins in HCT116 cells. Additionally, MHY446 was confirmed to bind more strongly to HDAC1 than HDAC2 and inhibit its activity. In vivo experiments using nude mice revealed that MHY446 was as effective as SAHA in inhibiting HCT116 cell-grafted tumor growth. This study also evaluated the biological effects of MHY446 on cell survival and death pathways. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) confirmed that ROS play a role in MHY446-induced cell death by reducing poly(ADP-ribose) polymerase cleavage. MHY446 also induced cell death via endoplasmic reticulum (ER) stress by increasing the expression of ER stress-related proteins. NAC treatment decreased the expression of ER stress-related proteins, indicating that ROS mediate ER stress as an upstream signaling pathway and induce cell death. While MHY446 did not exhibit superior HDAC inhibition efficacy compared to SAHA, it is anticipated to provide innovative insights into the future development of therapeutic agents for human CRC by offering novel chemical structure-activity relationship-related information.

Resistance training up-regulates Smyd1 expression and inhibits oxidative stress and endoplasmic reticulum stress in the heart of middle-aged mice

Persistent oxidative stress and endoplasmic reticulum (ER) stress are the primary mechanisms of age-related cardiovascular diseases. Although exercise training is viewed as an effective anti-aging approach, further exploration is needed to identify the mechanisms and functional targets. In this study, the impact of resistance training (RT) on the expression of Smyd1, the levels of reactive oxygen species (ROS) and the expression of ER stress-related protein in the hearts of mice of different ages were assessed. In addition, the role of Smyd1 in the aging-induced oxidative stress and ER stress were evaluated in d-galactose (D-gal)-treated H9C2 cells. We demonstrated that RT in middle age increased the expression of Smyd1 and restricted heart aging-induced ER stress. Overexpression of Smyd1 restrained oxidative stress and ER stress in D-gal-treated H9C2 cells, while the inhibition of Nrf2 and Smyd1 escalated ER stress. These findings demonstrate that Smyd1 has significant impact in regulating age-related ER stress. RT in middle age can up-regulates Smyd1 expression and inhibits oxidative stress and ER stress in the heart.

Suppression of endoplasmic reticulum stress by 4-PBA enhanced atherosclerotic plaque stability via up-regulating CLOCK expression

Endoplasmic reticulum (ER) stress refers to a condition where the normal functioning of the ER is disrupted due to a variety of cellular stress factors. As a result, there is an accumulation of unfolded and misfolded proteins within the ER. Numerous studies have shown that ER stress can exacerbate inflammatory reactions and contribute to the development of various inflammatory diseases. However, the role of ER stress in the stability of atherosclerotic plaques remains poorly understood. In this study, we aimed to explore the potential impact of a specific ER stress inhibitor known as 4-phenyl butyric acid (4-PBA) on atherosclerosis in mice. The mice were fed a high-fat diet, and treatment with 4-PBA significantly improved the stability of the atherosclerotic plaques. This was evidenced by a reduction in oxidative stress and an increase in circadian locomotor output cycles kaput (CLOCK) protein and mRNA expression within the plaques. Additionally, 4-PBA reduced the expression of ER stress-related proteins and decreased apoptosis in the atherosclerotic plaques. In vitro investigation, we observed the effect of 4-PBA on vascular smooth muscle cells (VSMCs) that were exposed to oxidized low-density lipoprotein (ox-LDL), a significant contributor to the development of atherosclerosis. 4-PBA reduced reactive oxygen species (ROS) production and attenuated apoptosis, GRP78 and CHOP protein expression in ox-LDL-Induced VSMCs via up-regulating CLOCK expression. However, when the short hairpin RNA against CLOCK (sh-CLOCK) was introduced to the VSMCs, the protective effect of 4-PBA was abolished. This suggests that the up-regulation of CLOCK expression is crucial for the beneficial effects of 4-PBA on atherosclerotic plaque stability. This finding suggests that targeting ER stress and modulating CLOCK protein levels might be a promising way to enhance the stability of atherosclerotic plaques.

Caspase-3/GSDME dependent pyroptosis contributes to offspring lung injury induced by gestational PFOS exposure via PERK/ATF4 signaling.

Perfluorooctane sulfonate (PFOS) is widely used in industry and consumer products. Previous studies have showed that PFOS gestational exposure is associated with offspring lung damage in rat. However, the underlying mechanisms remain poorly understood. In this study, we investigated the role of gasdermin E (GSDME) in lung injury of offspring and its underlying mechanisms using in vivo and in vitro approaches. Pregnant SD rats were exposed to PFOS (1mg/kg BW/d) between gestational day 12-18, and the lung tissue of the offspring was evaluated on postnatal day 7. PFOS treated animals exhibited alveolar septal thickening and inflammation-related damages, with an increased expression of GSDME in alveolar type II epithelial cells (AECII). Furthermore, in vitro experiments demonstrated that PFOS exposure (with 225μM and up) upregulated the caspase-3/GSDME signaling pathway in AECII. Also, ultrastructure analysis revealed significant changes in the endoplasmic reticulum (ER) structure in PFOS-induced pyroptotic cells, which is consistent with the ER stress detected in these cells. Additionally, PFOS exposure led to increased expression of ER stress-related proteins, including p-PERK, p-eIF2α, ATF4, and CHOP. Subsequently, using specific inhibitors, we found that the PERK/ATF4 pathway acted as an upstream signal regulating GSDME-dependent pyroptosis. Overall, our findings show that GSDME-dependent pyroptosis plays a crucial role in the lung injury induced by gestational PFOS exposure, and the PERK/ATF4 pathway may function as a possible mediator of this process.

Xenon postconditioning attenuates neuronal injury after spinal cord ischemia/reperfusion injury by targeting endoplasmic reticulum stress-associated apoptosis.

The purpose of the study is to explore the underlying mechanisms of xenon (Xe) which protects against spinal cord ischemia/reperfusion injury (SCIRI). A SCIRI rat model was induced by abdominal artery occlusion for 85 min and reperfusion. Xe postconditioning (50% Xe) was administered 1 h after 1 h of reperfusion. At reperfusion time points (2, 4, 6, and 24 h), rats were treated with spinal cord scans by MRI to assess the time of peak spinal cord injury after SCIRI. Subsequently, endoplasmic reticulum (ER) stress inhibitor sodium 4-phenylbutyrate (4-PBA) was administered by daily intraperitoneal injection (50 mg/kg) for 5 days before SCIRI. At 4 h after reperfusion, motor function, immunofluorescence staining, hematoxylin and eosin (HE) staining, Nissl staining, TUNEL staining, real-time reverse transcription polymerase chain (RT-PCR) reaction, and western blot analyses were performed to investigate the protective effects of Xe against SCIRI. In the rat I/R model, spinal cord edema peaked at reperfusion 4 h. SCIRI activated ER stress, which was located in neurons. Xe postconditioning remarkably alleviated hind limb motor function, reduced neuronal apoptosis rate, increased the number of normal neurons, and inhibited the expression of ER stress-related protein in spinal cord. Furthermore, the administration of the ER stress inhibitor 4-PBA strongly decreased ER stress-induced apoptosis following SCIRI. Xe postconditioning inhibits ER stress activation, which contributes to alleviate SCIRI by suppressing neuronal apoptosis.

A Native Drug-Free Macromolecular Therapeutic to Trigger Mutual Reinforcing of Endoplasmic Reticulum Stress and Mitochondrial Dysfunction for Cancer Treatment.

Drug-free macromolecular therapeutics are promising alternatives to traditional drugs. Nanomedicines with multiple organelles targeting can potentially increase the efficacy. Herein, a drug-free macromolecular therapeutic was designed to formulate endoplasmic reticulum (ER) and mitochondria dual-targeting nanoparticles (EMT-NPs), which can synergistically elicit ER stress and mitochondrial dysfunction. In vitro experiments indicated that EMT-NPs could effectively enter ER and mitochondria at an approximate ratio of 2 to 3. Subsequently, EMT-NPs could upregulate ER stress-related protein expression (IRE1α, CHOP), boosting calcium ion (Ca2+) efflux and activating the caspase-12 signaling cascade in cancer cells. In addition, EMT-NPs induced direct oxidative stress in mitochondria; some mitochondrial-related apoptotic events such as decreased mitochondrial membrane potential (MMP), upregulation of Bax, cytochrome c release, and caspase-3 activation were also observed for tumor cells upon incubation with EMT-NPs. Furthermore, the leaked Ca2+ from ER could induce mitochondrial Ca2+ overloading to further augment cancer cell apoptosis. In brief, mitochondrial and ER signaling networks collaborated well to promote cancer cell death. Extended photoacoustic and fluorescence imaging served well for the treatment of in vivo patient-derived xenografts cancer model. This drug-free macromolecular strategy with multiple subcellular targeting provides a potential paradigm for cancer theranostics in precision nanomedicine.

Pre-ischaemic Treatment with Enriched Environment Alleviates Acute Neuronal Injury by Inhibiting Endoplasmic Reticulum Stress-dependent Autophagy and Apoptosis

Enriched environment (EE) is effective in preventing cerebral ischemia–reperfusion (I/R) injury. However, little is known about the mechanism underlying the neuroprotection of EE preprocessing. Endoplasmic reticulum (ER) stress has been demonstrated to be extensively involved in I/R injury. We aimed to investigate the potential regulatory mechanism of ER stress in the neuroprotection of pre-ischemic EE. Rats were subjected to middle cerebral artery occlusion (MCAO) or sham surgery after 4 weeks of exposure in standard or enriched environments. We found that EE pretreatment alleviates acute neuronal injury after MCAO, as shown by reduced infarct volume and neurological deficit score. The expression of ER stress-related proteins, markers of autophagy, and apoptosis were detected to investigate the underlying mechanism. Our results showed that pre-ischemic EE inhibited the ER stress, as evidenced by the inactivation of activating transcription factor 6 (ATF6), protein kinase RNA (PKR)-like ER kinase (PERK), and inositol-requiring enzyme 1 (IRE1) pathways. Moreover, the rats reared in EE were detected with lower autophagic activity and apoptosis levels. The decrease in activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and phospho-c-Jun N-terminal kinases (p-JNK) expression suggested EE pretreatment might inhibit autophagy and apoptosis via modulating ER stress-mediated PERK-ATF4-CHOP and IRE1-JNK signal pathways, which provides a new idea for the prevention of the deleterious cerebral and functional consequences of ischemic stroke.

Melatonin regulates proliferation, apoptosis and invasion of trophoblasts in preeclampsia by inhibiting endoplasmic reticulum stress.

Clinical evidence indicated the activation of endoplasmic reticulum stress (ERS) in pregnant women with preeclampsia (PE), and the regulatory role of melatonin (MT) in ERS. This study aims to explore the possible effect and mechanism of MT on ERS and on the infiltration of trophoblasts in PE. The serum expression levels of MT and GRP78 in pregnant women with PE were measured. The cell proliferation, invasion, migration and apoptosis of trophoblasts were also determined. The trophoblast cell infiltration in placenta tissues was detected in EVOS image system. The expressions of ERS related proteins were measured by RT-qPCR and western blot. The PE-serum treatment on HTR-8/SVneo cells led to activated ERS and suppressed cell biological functions. PE mouse models after MT treatment or 4-PBA treatment had reduced blood pressure, proteinuria, apoptosis and increased foetus and placenta weight, in addition to enhanced cell infiltration. In vivo and in vitro evidence demonstrated MT can simultaneously suppress ERS and ASK1/JNK signal pathway in PE to promote the infiltration of trophoblasts.

Salidroside attenuates sepsis-associated acute lung injury through PPP1R15A mediated endoplasmic reticulum stress inhibition

BackgroundThis study explored the role and mechanism of salidroside in sepsis-associated acute lung injury (ALI). MethodSepsis-associated ALI in rats was induced by cecal ligation and perforation method, while lipopolysaccharide (LPS) was used to stimulate cell model. After the treatment of salidroside and dexamethasone (DEX), hematoxylin and eosin staining was applied to evaluate the rat lung injury. Next, the number of total cells and neutrophils in rat bronchoalveolar lavage fluid (BALF) was counted, and the lung wet/dry (W/D) ratio and water content were measured. The neutrophil elastase activity and inflammatory factor levels in BALF were detected by Elastase Assay kit and ELISA. The expressions of PPP1R15A and endoplasmic reticulum (ER) stress-related genes were detected by quantitative reverse transcription polymerase chain reaction and western blot. And the effects of silenced PPP1R15A and tauroursodeoxycholic acid (TUDCA) on cell viability, apoptosis and endoplasmic reticulum (ER) stress were determined by cell counting kit-8 assay, flow cytometry and western blot. ResultThe lung injury, inflammation and edema in sepsis model rats were alleviated by salidroside and DEX, meanwhile salidroside increased the viability and inhibited apoptosis in LPS-treated cells. The expression of PPP1R15A was decreased in sepsis models and increased by salidroside, and salidroside down-regulated the ER stress-related protein expressions in vitro and in vivo. Silenced PPP1R15A reversed the effect of salidroside on cell viability, apoptosis and ER stress, whereas TUDCA could counteract the above effect of silenced PPP1R15A. ConclusionSalidroside targeted PPP1R15A to ameliorate lung injury in sepsis through inhibiting ER stress.

Dendrobine inhibits dopaminergic neuron apoptosis via MANF-mediated ER stress suppression in MPTP/MPP+-induced Parkinson's disease models

BackgroundParkinson's disease (PD) is an age-related neurodegenerative disorder without effective treatments. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been suggested to be capable of protecting against PD by inhibiting endoplasmic reticulum (ER) stress-mediated neuronal apoptosis. PurposeThis study was aimed to evaluate the antiparkinsonian effect of dendrobine and reveal its underlying mechanisms from the perspective of MANF-mediated ER stress suppression. MethodsBehavioral assessments of PD mice as well as LDH/CCK-8 assay in SH-SY5Y cells and primary midbrain neurons were carried out to detect the antiparkinsonian effect of dendrobine. Immunofluorescence, western blot, flow cytometry and shRNA-mediated MANF knockdown were used to determine the apoptosis of dopaminergic neurons and the expressions of ER stress-related proteins for investigating the underlying mechanism of dendrobine. ResultsDendrobine significantly ameliorated the motor performance of PD mice and attenuated the injuries of dopaminergic neurons. Dendrobine could also relieve neuronal apoptosis, up-regulate MANF expression and inhibit ER stress, which were largely abolished by shRNA-mediated MANF knockdown in PD model. ConclusionDendrobine might protect against PD by inhibiting dopaminergic neuron apoptosis, which was achieved by facilitating MANF-mediated ER stress suppression. Our study suggested that dendrobine could act as a MANF up-regulator to protect against PD, and provided a potential candidate for exploring etiological agents of PD.

Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca2+-Regulated ERK1/2-Activating Pathway.

Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01–0.5 mM) exhibited greater cytotoxicity than ketamine (0.1–3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca2+]i). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca2+ chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca2+]i in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca2+]i-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca2+]i/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model.

Background Activating transcription factor 6 (ATF6) and receptor-interacting protein 3 (RIP3) are important signaling proteins in endoplasmic reticulum (ER) stress and necroptosis, respectively. However, their regulatory relationship and clinical significance are unknown. We investigate the impact of ATF6 on RIP3 expression, and its role in hepatocyte necroptosis in an acute liver injury model. Methods In vivo and in vitro experiments were carried out. LO2 cells were treated with thapsigargin (TG). In vivo, male BALB/c mice were treated with carbon tetrachloride (CCl4, 1 mL/kg) or tunicamycin (TM, 2 mg/kg). Then, the impact of ATF6 or RIP3 silencing on liver injury, hepatocyte necroptosis, and ER stress-related protein expression was examined. Results TG induced ER stress and necroptosis and ATF6 and RIP3 expression in LO2 cells. The knockdown of ATF6 significantly decreased RIP3 expression (p < 0.05) and increased ER stress and necroptosis. The downregulation of RIP3 significantly reduced necroptosis and ER stress (p < 0.05). Similar results were observed in CCl4 or the TM-induced mouse model. The knockdown of ATF6 significantly decreased CCl4-induced RIP3 expression and increased liver injury, necroptosis, and ER stress in mice livers (p < 0.05). In contrast, the downregulation of RIP3 significantly reduced liver injury, hepatocyte necroptosis, and ER stress. Conclusions Hepatocyte ATF6 has multiple roles in acute liver injury. It reduces hepatocyte necroptosis via negative feedback regulation of ER stress. In addition, ATF6 can upregulate the expression of RIP3, which is not helpful to the recovery process. However, downregulating RIP3 reduces hepatocyte necroptosis by promoting the alleviation of ER stress. The findings suggest that RIP3 could be a plausible target for the treatment of liver injury.

Honokiol inhibits endoplasmic reticulum stress-associated lipopolysaccharide-induced inflammation and apoptosis in bovine endometrial epithelial cells.

Honokiol (HKL) has been previously reported to exert anti-inflammatory effects in numerous diseases. However, the role of HKL in endometritis remains unclear. The present study aimed to explore and elucidate the role of HKL in a lipopolysaccharide (LPS)-induced in vitro model of endometritis. Bovine endometrial epithelial cells (bEECs) were pre-treated with HKL at doses of 1, 10 and 20 µM, followed by 1 µg/ml LPS. MTT assay was then used to detect cell viability. ELISA was utilized to measure the levels of the proinflammatory cytokines TNF-α, IL-1β and IL-6 in bEECs culture supernatants. Reverse transcription-quantitative PCR was further performed to examine the mRNA expression levels of these cytokines. Cell apoptosis was observed by TUNEL staining and the levels of Bcl-2, Bax, cleaved caspase 3 and cleaved caspase 9 were assayed by western blotting. Western blotting was also performed to detect the expression levels of endoplasmic reticulum (ER) stress-related proteins activating transcription factor 6, CCAAT-enhancer-binding protein homologous protein, inositol-requiring enzyme 1 and cleaved caspase 12 in bEECs. LPS treatment reduced cell viability and HKL treatment improved the viability of bEECs after LPS treatment. The LPS-induced inflammatory response and apoptosis in bEECs were also inhibited by HKL treatment. Additionally, the increased expression of ER stress-related proteins induced by LPS was reversed by HKL treatment. Following stimulation with the ER stress inducer tunicamycin, it was revealed that HKL attenuated ER stress and inhibited LPS-induced inflammatory response and apoptosis in bEECs. In summary, HKL inhibited ER stress associated with LPS-induced inflammation and apoptosis in bEECs, providing evidence that HKL can serve to be a novel agent for the treatment of endometritis.