HDAC4 represses ER stress induced chondrocyte apoptosis by inhibiting ATF4 and attenuates cartilage degeneration in an osteoarthritis rat model.

BACKGROUND: The present study evaluated whether the lack of histone deacetylase 4 (HDAC4) increases endoplasmic reticulum stress-induced chondrocyte apoptosis by releasing activating transcription factor 4 (ATF4) in human osteoarthritis (OA) cartilage degeneration. METHODS: Articular cartilage from the tibial plateau was obtained from patients with OA during total knee replacement. Cartilage extracted from severely damaged regions was classified as degraded cartilage, and cartilage extracted from a relatively smooth region was classified as preserved cartilage. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to detect chondrocyte apoptosis. HDAC4, ATF4, and C/EBP homologous protein (CHOP) expression levels were measured using immunohistochemistry staining and real-time quantitative PCR. Chondrocytes were transfected with HDAC4 or HDAC4 siRNA for 24 h and stimulated with 300 µM H2O2 for 12 h. The chondrocyte apoptosis was measured using flow cytometry. ATF4, CHOP, and caspase 12 expression levels were measured using real-time quantitative PCR and western blotting. Male Sprague-Dawley rats (n = 15) were randomly divided into three groups and transduced with different vectors: ACLT + Ad-GFP, ACLT + Ad-HDAC4-GFP, and sham + Ad-GFP. All rats received intra-articular injections 48 h after the operation and every three weeks thereafter. Cartilage damage was assessed using Safranin O staining and quantified using the Osteoarthritis Research Society International score. ATF4, CHOP, and collagen II expression were detected using immunohistochemistry, and chondrocyte apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. RESULTS: The chondrocyte apoptosis was higher in degraded cartilage than in preserved cartilage. HDAC4 expression was lower in degraded cartilage than in preserved cartilage. ATF4 and CHOP expression was increased in degraded cartilage. Upregulation of HDAC4 in chondrocytes decreased the expression of ATF4, while the expression of ATF4 was increased after downregulation of HDAC4. Upregulation of HDAC4 decreased the chondrocyte apoptosis under endoplasmic reticulum stress, and chondrocyte apoptosis was increased after downregulation of HDAC4. In a rat anterior cruciate ligament transection OA model, adenovirus-mediated transduction of HDAC4 was administered by intra-articular injection. We detected a stronger Safranin O staining with lower Osteoarthritis Research Society International scores, lower ATF4 and CHOP production, stronger collagen II expression, and lower chondrocyte apoptosis in rats treated with Ad-HDAC4. CONCLUSION: The lack of HDAC4 expression partially contributes to increased ATF4, CHOP, and endoplasmic reticulum stress-induced chondrocyte apoptosis in OA pathogenesis. HDAC4 attenuates cartilage damage by repressing ATF4-CHOP signaling-induced chondrocyte apoptosis in a rat model of OA.

Differential effects of montelukast and zafirlukast on MDA­MB­231 triple­negative breast cancer cells: Cell cycle regulation, apoptosis, autophagy, DNA damage and endoplasmic reticulum stress.

Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple­negative breast cancer MDA­MB­231 cells. By contrast, only zafirlukast induces G0/G1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription­quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki­67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti­apoptotic protein Bcl­2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3­II and DNA damage markers, including cleaved PARP­1, phosphorylated (p)­ATM and p­histone H2AX. The number of caspase 3/7­positive cells was greater in montelukast­treated cells compared with zafirlukast­treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol­requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast­induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.

Resveratrol alleviates endoplasmic reticulum stress-induced cell death and improves functional prognosis after traumatic brain injury in mice.

Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory activity of RSV in traumatic brain injury (TBI) in mice associated with endoplasmic reticulum stress (ERS). After establishing three experimental groups (sham, TBI, and TBI+RSV), we explored the results of RSV after TBI on ERS and caspase-12 apoptotic pathways. The expression levels of C/EBP homologous protein (CHOP), glucose regulated protein 78kD (GRP78), caspase-3, and caspase-12 in cortical brain tissues were assessed by western blotting. The qPCR analysis was also performed on mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in cortical brain tissue. In addition, the expression of GRP78 in microglia (ionized calcium binding adaptor molecule 1; Iba-1) and neurons (neuronal nuclei; NeuN) was identified by immunofluorescence staining. The neurological function of mice was assessed by modified neurological severity scores (mNSS). After drug treatment, the expression of CHOP, GRP78, caspase-3 and caspase-12 decreased, and qPCR results showed that TNF-α and IL-1ß were down-regulated. Immunofluorescence staining showed down-regulation of Iba-1+/GRP78+ and NeuN+/GRP78+ cells after RSV treatment. The mNSS analysis confirmed improvement after RSV treatment. RSV improved apoptosis by downregulating the ERS signaling pathway and improved neurological prognosis in mice with TBI.

Licochalcone D exhibits cytotoxicity in breast cancer cells and enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through upregulation of death receptor 5.

Anticancer strategies using natural products or derivatives are promising alternatives for cancer treatment. Here, we showed that licochalcone D (LCD), a natural flavonoid extracted from Glycyrrhiza uralensis Fisch, suppressed the growth of breast cancer cells, and was less toxic to MCF-10A normal breast cells. LCD-induced DNA damage, cell cycle arrest, and apoptosis in breast cancer cells. Furthermore, LCD potentiated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. Mechanistically, LCD was revealed to reduce survival protein expression and to upregulate death receptor 5 (DR5) expressions. Silencing DR5 blocked the ability of LCD to sensitize cells to TRAIL-mediated apoptosis. LCD increased CCAAT/enhancer-binding protein homologous protein (CHOP) expression in breast cancer cells. Knockdown of CHOP attenuated DR5 upregulation and apoptosis triggered by cotreatment with LCD and TRAIL. Furthermore, LCD suppressed the phosphorylation of extracellular signal-regulated kinase and promoted the phosphorylation of c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Pretreatment with JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580 abolished the upregulation of DR5 and CHOP, and also attenuated LCD plus TRAIL-induced cleavage of poly(ADP-ribose) polymerase. Overall, our results show that LCD exerts cytotoxic effects on breast cancer cells and arguments TRAIL-mediated apoptosis by inhibiting survival protein expression and upregulating DR5 in a JNK/p38 MAPK-CHOP-dependent manner.

Silybin prevented avermectin-induced cardiotoxicity in carp by modulating oxidative stress, inflammation, endoplasmic reticulum stress, mitochondrial apoptosis, and autophagy.

Avermectin is one of the widely used anthelmintics in aquaculture and exhibits substantial toxicity to aquatic organisms. Silybin is extensively used for its anti-inflammatory, antioxidant and anti-apoptotic biological properties. Heart is essential for the survival of fish and plays a vital role in pumping blood oxygen and nutrients. Residual avermectin in water poses harm to carp. However, there is still insufficient research on whether silybin can mitigate the toxicity of avermectin to carp heart tissues. In this research, we established a model involving carp subjected to acute avermectin exposure and administered diets containing silybin to explore the potential protective effects of silybin against avermectin-induced cardiotoxicity. The results revealed that avermectin induced oxidative stress, inflammation, endoplasmic reticulum (ER) stress, mitochondrial pathway apoptosis and autophagy in the cardiac tissues of carp. Compared with the avermectin group, silybin significantly reduced ROS accumulation in cardiac tissues, restored antioxidant enzyme activity, inhibited mRNA transcript levels of pro-inflammatory-related factors, and attenuated ER stress, mitochondrial pathway apoptosis and autophagy. Protein-protein interaction (PPI) analysis demonstrated that silybin mitigated avermectin-induced cardiac oxidative stress, inflammation, ER stress, mitochondrial pathway apoptosis and autophagy. Silybin exerted anti-inflammatory effects through the Nuclear Factor kappa B (NF-κB) pathway, antioxidant effects through the Nuclear factor erythroid 2-related factor 2 (Nrf2) - Kelch-like ECH-associated protein 1 (Keap1) pathway, alleviated cardiac ER stress through the Glucose-regulated protein 78 (GRP78)/Activating Transcription Factor 6 (ATF6)/C/EBP homologous protein (CHOP) axis, suppressed apoptosis through the mitochondrial pathway, and inhibited excessive autophagy initiation through the PTEN-induced putative kinase 1 (PINK1)/Parkin RBR E3 ubiquitin protein ligase (PARKIN) signaling pathway. This study provided evidence supporting the protective effect of silybin against avermectin-induced cardiotoxicity in carp, highlighting its potential as a dietary additive to protect fish from adverse effects caused by avermectin exposure.

Borax affects cellular viability by inducing ER stress in hepatocellular carcinoma cells by targeting SLC12A5.

Hepatocellular carcinoma (HCC) presents a persistent challenge to conventional therapeutic approaches. SLC12A5 is implicated in an oncogenic capacity and facilitates the progression of cancer. The objective of this investigation is to scrutinize the inhibitory effects of borax on endoplasmic reticulum (ER)-stress and apoptosis mediated by SLC12A5 in HepG2 cells. Initially, we evaluated the cytotoxic impact of borax on both HL-7702 and HepG2 cell lines. Subsequently, the effects of borax on cellular morphology and the cell cycle of these lines were examined. Following this, we explored the impact of borax treatment on the mRNA and protein expression levels of SLC12A5, C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78), activating transcription factor-6 (ATF6), caspase-3 (CASP3), and cytochrome c (CYC) in these cellular populations. The determined IC50 value of borax for HL-7702 cells was 40.8 mM, whereas for HepG2 cells, this value was 22.6 mM. The concentrations of IC50 (22.6 mM) and IC75 (45.7 mM) of borax in HepG2 cells did not manifest morphological aberrations in HL-7702 cells. Conversely, these concentrations in HepG2 cells induced observable morphological and nuclear abnormalities, resulting in cell cycle arrest in the G1/G0 phase. Additionally, the levels of SLC12A5, ATF6, CHOP, GRP78, CASP3, and CYC were elevated in HepG2 cells in comparison to HL-7702 cells. Moreover, SLC12A5 levels decreased following borax treatment in HepG2 cells, whereas ATF6, CHOP, GRP78, CASP3, and CYC levels exhibited a significant increase. In conclusion, our data highlight the potential therapeutic effects of borax through the regulation of ER stress in HCC by targeting SLC12A5.

CHOP-mediated IL-23 overexpression does not drive colitis in experimental spondyloarthritis.

HLA-B27 is a major risk factor for spondyloarthritis (SpA), yet the underlying mechanisms remain unclear. HLA-B27 misfolding-induced IL-23, which is mediated by endoplasmic reticulum (ER) stress has been hypothesized to drive SpA pathogenesis. Expression of HLA-B27 and human ß2m (hß2m) in rats (HLA-B27-Tg) recapitulates key SpA features including gut inflammation. Here we determined whether deleting the transcription factor CHOP (Ddit3-/-), which mediates ER-stress induced IL-23, affects gut inflammation in HLA-B27-Tg animals. ER stress-mediated Il23a overexpression was abolished in CHOP-deficient macrophages. Although CHOP-deficiency also reduced Il23a expression in immune cells isolated from the colon of B27+ rats, Il17a levels were not affected, and gut inflammation was not reduced. Rather, transcriptome analysis revealed increased expression of pro-inflammatory genes, including Il1a, Ifng and Tnf in HLA-B27-Tg colon tissue in the absence of CHOP, which was accompanied by higher histological Z-scores. RNAScope localized Il17a mRNA to the lamina propria of the HLA-B27-Tg rats and revealed similar co-localization with Cd3e (CD3) in the presence and absence of CHOP. This demonstrates that CHOP-deficiency does not improve, but rather exacerbates gut inflammation in HLA-B27-Tg rats, indicating that HLA-B27 is not promoting gut disease through ER stress-induced IL-23. Hence, CHOP may protect rats from more severe HLA-B27-induced gut inflammation.

Suppression of PERK/eIF2α/CHOP pathway enhances oridonin-induced apoptosis by inhibiting autophagy in Small-Cell lung cancer cells.

Chinese herbs have been used to treat small-cell lung cancer (SCLC) due to their low toxicity and significant efficacy. This study focused on oridonin, a natural compound extracted from Rabdosia rubescens, and aimed to investigate its potential antitumor activity on SCLC and to evaluate the synergistic effect of combining oridonin with other small molecules. In this study, oridonin exhibited a dual effect. At lower concentrations, it suppressed the cell viability of SCLC cells (H1688 and H446). At high concentrations, oridonin induced SCLC cell apoptosis, damaged HBE cells in vitro and compromised the function of the liver and heart in vivo. The lower concentration of oridonin induced autophagy by enhancing the expression of p62 and the LC3B-II/LC3B-I ratio. This phenomenon might be associated with the activation of the protein kinase RNA-like ER kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF2α)/growth arrest and DNA damage-inducible gene 153 (CHOP/GAD153) pathway. Therefore, the combined effect of oridonin with GSK2606414 or 3- methyladenine increased apoptosis in SCLC cells and reduced tumor growth. A similar phenomenon was observed after oridonin was combined with p62 or CHOP RNA interference treatment. Simultaneously, the combination of oridonin and GSK2606414 exhibited therapeutic efficacy without manifesting adverse effects. Our findings suggest that oridonin at lower concentrations can induce autophagy by activating the PERK/eIF2α/CHOP signaling pathway. The inhibition of the PERK/eIF2α/CHOP pathway could enhance oridonin therapeutic responses by triggering apoptosis. The novel therapeutic approach of combining oridonin with a PERK inhibitor is promising as a strategy for the treatment of SCLC.

Increased ER Stress and Unfolded Protein Response Activation in Epithelial and Inflammatory Cells in Hypersensitivity Pneumonitis.

Several types of cytotoxic insults disrupt endoplasmic reticulum (ER) homeostasis, cause ER stress, and activate the unfolded protein response (UPR). The role of ER stress and UPR activation in hypersensitivity pneumonitis (HP) has not been described. HP is an immune-mediated interstitial lung disease that develops following repeated inhalation of various antigens in susceptible and sensitized individuals. The aim of this study was to investigate the lung expression and localization of the key effectors of the UPR, BiP/GRP78, CHOP, and sXBP1 in HP patients compared with control subjects. Furthermore, we developed a mouse model of HP to determine whether ER stress and UPR pathway are induced during this pathogenesis. In human control lungs, we observed weak positive staining for BiP in some epithelial cells and macrophages, while sXBP1 and CHOP were negative. Conversely, strong BiP, sXBP1- and CHOP-positive alveolar and bronchial epithelial, and inflammatory cells were identified in HP lungs. We also found apoptosis and autophagy markers colocalization with UPR proteins in HP lungs. Similar results were obtained in lungs from an HP mouse model. Our findings suggest that the UPR pathway is associated with the pathogenesis of HP.

[Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway].

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 µmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 µmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

Mechanism of apoptosis induced by the combined action of acrylamide and Elaidic acid through endoplasmic reticulum stress injury.

Thermal processing of food is likely to form acrylamide (AA) and elaidic acid (EA), which are both mainly metabolized by the liver. The two substances are associated with the pathogenesis of liver disease. In the current study, we investigated the toxic effects of the combined action of AA and EA on HSC-T6 cells, and the mechanism of apoptosis exacerbated by the co-exposure. The results showed a synergistic effect of AA and EA, which exacerbated the damage and oxidative stress (OS) in HSC-T6. Meanwhile, the expression of endoplasmic reticulum stress (ERS) proteins, such as GRP78 and CHOP, was increased, the ERS pathway was activated, and Ca2+ in cells was increased, which exacerbated mitochondrial damage, and opened IP3R-Grp75-VDAC1 channel. Both ERS and mitochondrial damage caused the process of cell apoptosis. Inhibition of ERS by 4-phenylbutyric acid (4-PBA) significantly reversed the synergistic effects on mitochondrial damage via ERS, suggesting that AA and EA exacerbated mitochondrial damage through ERS-mediated Ca2+ overload. AA and EA synergistically damaged the function of mitochondria through exacerbating ERS and led to cell apoptosis.

Role of Neurocellular Endoplasmic Reticulum Stress Response in Alzheimer's Disease and Related Dementias Risk.

Currently, more than 55 million people around the world suffer from dementia, and Alzheimer's Disease and Related Dementias (ADRD) accounts for nearly 60-70% of all those cases. The spread of Alzheimer's Disease (AD) pathology and progressive neurodegeneration in the hippocampus and cerebral cortex is strongly correlated with cognitive decline in AD patients; however, the molecular underpinning of ADRD's causality is still unclear. Studies of postmortem AD brains and animal models of AD suggest that elevated endoplasmic reticulum (ER) stress may have a role in ADRD pathology through altered neurocellular homeostasis in brain regions associated with learning and memory. To study the ER stress-associated neurocellular response and its effects on neurocellular homeostasis and neurogenesis, we modeled an ER stress challenge using thapsigargin (TG), a specific inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), in the induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) of two individuals from our Mexican American Family Study (MAFS). High-content screening and transcriptomic analysis of the control and ER stress-challenged NSCs showed that the NSCs' ER stress response resulted in a significant decline in NSC self-renewal and an increase in apoptosis and cellular oxidative stress. A total of 2300 genes were significantly (moderated t statistics FDR-corrected p-value ≤ 0.05 and fold change absolute ≥ 2.0) differentially expressed (DE). The pathway enrichment and gene network analysis of DE genes suggests that all three unfolded protein response (UPR) pathways, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF-6), and inositol-requiring enzyme-1 (IRE1), were significantly activated and cooperatively regulated the NSCs' transcriptional response to ER stress. Our results show that IRE1/X-box binding protein 1 (XBP1) mediated transcriptional regulation of the E2F transcription factor 1 (E2F1) gene, and its downstream targets have a dominant role in inducing G1/S-phase cell cycle arrest in ER stress-challenged NSCs. The ER stress-challenged NSCs also showed the activation of C/EBP homologous protein (CHOP)-mediated apoptosis and the dysregulation of synaptic plasticity and neurotransmitter homeostasis-associated genes. Overall, our results suggest that the ER stress-associated attenuation of NSC self-renewal, increased apoptosis, and dysregulated synaptic plasticity and neurotransmitter homeostasis plausibly play a role in the causation of ADRD.

hUCMSC-derived exosomes protect against GVHD-induced endoplasmic reticulum stress in CD4+ T cells by targeting the miR-16-5p/ATF6/CHOP axis.

Exosomes generated from mesenchymal stem cells (MSCs) are thought to be a unique therapeutic strategy for several autoimmune deficiency illnesses. The purpose of this study was to elucidate the protective effects of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exo) on CD4+ T cells dysfunction during graft-versus-host disease (GVHD) and to identify the underlying processes involved. Here, we showed that hUCMSC-Exo treatment can effectively attenuate GVHD injury by alleviating redox metabolism disorders and inflammatory cytokine bursts in CD4+ T cells. Furthermore, hUCMSC-Exo ameliorate ER stress and ATF6/CHOP signaling-mediated apoptosis in CD4+ T cells and promote the development of CD4+IL-10+ T cells during GVHD. Moreover, downregulating miR-16-5p in hUCMSC-Exo impaired their ability to prevent CD4+ T cells apoptosis and weakened their ability to promote the differentiation of CD4+IL-10+ T cells. Collectively, the obtained data suggested that hUCMSC-Exo suppress ATF6/CHOP signaling-mediated ER stress and apoptosis in CD4+ T cells, enhance the differentiation of CD4+IL-10+ T cells, and reverse the imbalance of immune homeostasis in the GVHD process by transferring miR-16-5p. Our study provided further evidence that GVHD patients can benefit from hUCMSC-Exo-mediated therapy.

A study on expression of GRP78 and CHOP in neutrophil endoplasmic reticulum and their relationship with neutrophil apoptosis in the development of sepsis.

We investigated the relationship between neutrophil apoptosis and endoplasmic reticulum stress (ERS) in sepsis and its mechanism. A prospective cohort study was conducted by recruiting a total of 58 patients with sepsis. Peripheral blood samples were collected on 1, 3, 5 and 7 days after admission to the ICU. The expressions of endoplasmic reticulum specific glucose regulatory protein 78 (GRP78), C/EBP homologous protein (CHOP), apoptosis signal-regulating kinase 1 (ASK1), Bcl-2-like 11 (BIM), death receptor 5 (DR5), c-Jun N-terminal kinases (JNK) and p38 were detected by Western blot and PCR. The subcellular location of CHOP and GRP78 was observed by immunofluorescence analysis. Spearman correlation was used to analyze the correlation between the expression of chop protein and the apoptosis rate of peripheral blood neutrophils. Healthy volunteers in the same period were selected as the healthy control group. The expression of GRP78 protein was significantly elevated on the first day of ICU admission and showed a decreasing trend on the third, fifth and seventh day, but was significantly higher than the corresponding healthy control group. The expression of CHOP protein reached the highest level on the third day. The expression of chop protein in each group was significantly higher than that in the corresponding healthy control group. Immunofluorescence staining clearly showed that the CHOP protein accumulated in the nucleus, with an elevation in the intensity of GRP78. The neutrophil apoptosis rate of sepsis patients on the 1st, 3rd, 5th and 7th day of ICU stay was significantly higher than that of the healthy control group, with the highest apoptosis rate on the 3rd day, and then decreased gradually. CHOP protein expression level was significantly positively correlated with neutrophil apoptosis rate in sepsis patients. Endoplasmic reticulum stress occurs in neutrophils during the development of sepsis. GRP78 protein and CHOP protein may be involved in the pathological process of neutrophil apoptosis in sepsis.

Deciphering the role of FUS::DDIT3 expression and tumor microenvironment in myxoid liposarcoma development.

BACKGROUND: Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. METHODS: To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. RESULTS: Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. CONCLUSIONS: Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

Novel insights into the protective effects of leonurine against acute kidney injury: Inhibition of ER stress-associated ferroptosis via regulating ATF4/CHOP/ACSL4 pathway.

Acute kidney injury (AKI) is a common and serious global health problem with high risks of mortality and the development of chronic kidney diseases. Leonurine is a unique bioactive component from Leonurus japonicus Houtt. and exerts antioxidant, antiapoptotic or anti-inflammatory properties. This study aimed to explore the benefits of leonurine on AKI and the possible mechanisms involved, with a particular foc on the regulation of ferroptosis and endoplasmic reticulum (ER) stress. Our results showed that leonurine exhibited prominent protective effects against AKI, as evidenced by the amelioration of histopathological alterations and reduction of renal dysfunction. In addition, leonurine significantly suppressed ferroptosis in AKI both in vivo and in vitro by effectively restoring ultrastructural abnormalities in mitochondria, decreasing ASCL4 and 4-HNE levels, scavenging reactive oxygen species (ROS), as well as increasing GPX4 and GSH levels. In parallel, leonurine also markedly mitigated ER stress via down-regulating PERK, eIF-2α, ATF4, CHOP and CHAC1. Further studies suggested that ER stress was closely involved in erastin-induced ferroptosis, and leonurine protected tubular epithelial cells in vitro by inhibiting ER stress-associated ferroptosis via regulating ATF4/CHOP/ASCL4 signalling pathway. Mechanistically, ATF4 silencing in vitro regulated CHOP and ACSL4 expressions, ultimately weakening both ER stress and ferroptosis. Notably, analyses of single-cell RNA sequencing data revealed that ATF4, CHOP and ASCL4 in renal tubular cells were all abnormally upregulated in patients with AKI compared to healthy controls, suggesting their contributions to the pathogenesis of AKI. Altogether, these findings suggest that leonurine alleviates AKI by inhibiting ER stress-associated ferroptosis via regulating ATF4/CHOP/ASCL4 signalling pathway, thus providing novel mechanisms for AKI treatment.

Esculin suppresses the PERK-eIF2α-CHOP pathway by enhancing SIRT1 expression in oxidative stress-induced rat chondrocytes, mitigating osteoarthritis progression in a rat model.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease characterized by the gradual degeneration of chondrocytes, involving endoplasmic reticulum (ER) stress. Esculin is a natural compound with antioxidant, anti-inflammatory and anti-tumor properties. However, its impact on ER stress in OA therapy has not been thoroughly investigated. We aim to determine the efficiency of Esculin in OA treatment and its underlying mechanism. METHODS: We utilized the tert-butyl hydroperoxide (TBHP) to establish OA model in chondrocytes. The expression of SIRT1, PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins were detected by Western blot and Real-time PCR. The apoptosis was evaluated by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. X-ray imaging, Hematoxylin & Eosin staining, Safranin O staining and immunohistochemistry were used to assess the pharmacological effects of Esculin in the anterior cruciate ligament transection (ACLT) rat OA model. RESULTS: Esculin downregulated the expression of PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins, while upregulated the expression of SIRT1 and Bcl2 in the TBHP-induced OA model in vitro. It was coincident with the results of TUNEL staining and flow cytometry. We further confirmed the protective effect of Esculin in the rat ACLT-related model. CONCLUSION: Our results suggest the potential therapeutic value of Esculin on osteoarthritis. It probably inhibits the PERK-eIF2α-ATF4-CHOP pathway by upregulating SIRT1, thereby mitigating endoplasmic reticulum stress and protecting chondrocytes from apoptosis.

Deoxynivalenol leads to endoplasmic reticulum stress-mediated apoptosis via the IRE1/JNK/CHOP pathways in porcine embryos.

The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 µM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling.

Phenylbutyric acid inhibits hypoxia-induced trophoblast apoptosis and autophagy in preeclampsia via the PERK/ATF-4/CHOP pathway.

Preeclampsia (PE) is a common pregnancy complication with a high mortality rate. Abnormally activated endoplasmic reticulum stress (ERS) is believed to be responsible for the destruction of key placental cells-trophoblasts. Phenylbutyric acid (4-PBA), an ERS inhibitor, is involved in regulating the development of ERS-related diseases. At present, how 4-PBA affects trophoblasts and its mechanisms is still unclear. In this study, PE cell models were established by stimulating HTR-8/SVneo cells with hypoxia. To verify the underlying mechanisms of 4-PBA on PE, CCT020312, an activator of PERK, was also used. The results showed that 4-PBA restored hypoxia-induced trophoblast viability, inhibited HIF-1α protein expression, inflammation, and PERK/ATF-4/CHOP pathway. Hoechst 33342 staining and flow cytometry results confirmed that 4-PBA decreased hypoxia-induced apoptosis in trophoblasts. The results of the JC-1 analysis and apoptosis initiation enzyme activity assay also demonstrated that 4-PBA inhibited apoptosis related to the mitochondrial pathway. Furthermore, by detecting autophagy in trophoblasts, an increased number of autophagic vesicles, damaged mitochondria, enhanced dansylcadaverine fluorescence, enhanced levels of autophagy proteins Beclin-1, LC3II, and decreased p62 were seen in hypoxia-stimulated cells. These changes were reversed by 4-PBA. Furthermore, it was observed that CCT020312 reversed the effects of 4-PBA on the viability, apoptosis, and autophagosome number of hypoxia-induced trophoblasts. In summary, 4-PBA reduces autophagy and apoptosis via the PERK/ATF-4/CHOP pathway and mitochondrial pathway, thereby restoring the viability of hypoxic trophoblasts. These findings provide a solid evidence base for the use of 4-PBA in PE treatment and guide a new direction for improving the outcomes of patients with PE.

Effects of electroacupuncture of different intensities and durations on PERK/ATF4/CHOP signaling pathway in liver of non-alcoholic fatty liver disease rats. / ä¸åŒå¼ºåº¦åŠæ—¶ç¨‹ç”µé’ˆå¹²é¢„å¯¹éžé ’ç²¾æ€§è„‚è‚ªè‚ç— å¤§é¼ è‚è„PERK/ATF4/CHOP通路的影响.

OBJECTIVES: To analyze the effects of electroacupuncture (EA) at "Fenglong" (ST40) and "Zusanli" (ST36) of different intensities and durations on rats with non-alcoholic fatty liver disease (NAFLD) based on the protein kinase R-like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) signaling pathway, so as to explore its mechanism underlying improvement of NAFLD. METHODS: SD rats were randomly divided into normal diet group, high-fat model group, sham EA group, strong stimulation EA (SEA) group, and weak stimulation EA (WEA) group, with 15 rats in each group. Each group was further divided into 2, 3, and 4-week subgroups. NAFLD rat model was established by feeding a high-fat diet. After successful modeling, rats in the SEA and WEA groups received EA at bilateral ST40 and ST36 with dense and sparse waves (4 Hz/20 Hz) at current intensities of 4 mA (SEA group) and 2 mA (WEA group), lasting for 20 minutes, once a day, 5 days a week with 2 days of rest. The sham EA group only had the EA apparatus connected without electricity. Different duration subgroups were intervened for 2, 3, and 4 weeks. After the intervention, the contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in rats were detected by an automatic biochemical analyzer；liver morphological changes were observed by Oil Red O staining；real-time fluorescence quantitative PCR and Western blot were used to detect the expression of PERK, ATF4, and CHOP mRNAs and proteins in the rat liver tissue. RESULTS: In the high-fat model group, there was a significant accumulation of red lipid droplets in the liver cells, which was reduced significantly in the SEA group at the 4th week. Compared with the normal diet group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and proteins in the liver tissue were elevated (P<0.01) in the high-fat model group . Compared with the high-fat model group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, CHOP mRNAs and proteins in the liver tissue were decreased (P<0.01, P<0.05) in the SEA and WEA groups. Compared with the sham EA group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs were decreased (P<0.01, P<0.05) in the SEA and WEA groups, the expression of PERK, ATF4, and CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA group at the 2nd, 3rd, and 4th week, the expression of PERK and CHOP proteins at the 2nd, 3rd, 4th week and ATF4 protein at 2nd week in the liver tissue were decreased (P<0.01, P<0.05) in the WEA group. Compared with the SEA group with the same treatment duration, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and proteins in the liver tissue were elevated (P<0.05, P<0.01) in the WEA group. Compared with the 2-week time point within the groups, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs and PERK proteins in the liver tissue were decreased (P<0.01, P<0.05) in the SEA and WEA groups at 3rd and 4th week, the expression of ATF4 proteins in the liver tissue was decreased (P<0.01) in the SEA group at 3rd and 4th week, and the expression of CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA group at 4th week and in the WEA group at 3rd and 4th week. Compared with the 3-week time point within the groups, the contents of serum ALT, AST, and the expression of PERK, ATF4, and CHOP mRNAs were significantly decreased (P<0.05, P<0.01) in the SEA and WEA groups at 4th week, the expression of PERK and CHOP proteins in the liver tissue was decreased (P<0.01) in the SEA and WEA groups at 4th week, and the expression of ATF4 protein in the liver tissue was decreased (P<0.05) in the SEA group at 4th week. CONCLUSIONS: EA at ST40 and ST36 can significantly improve liver function in NAFLD rats, and its mechanism of action may involve inhibiting PERK expression thereby targeting the downstream ATF4/CHOP signaling pathway to suppress endoplasmic reticulum stress, exerting a liver protective effect；the optimal effect was observed with EA intensity of 4 mA for 4 weeks.