Endoplasmic Reticulum Stress Markers Research Articles

AMPK/Autophagy-Mediated Alleviation of Tendinopathy by IL-38: A Novel Strategy for the Treatment of Obesity-Related Tendinopathy

The effect of interleukin-38 (IL-38), a recently identified member of the IL-1 family with potential applications in various inflammation-related conditions, on ER stress has not been explored. Furthermore, its role in obesity-associated tendinopathy has not been investigated. In this study, human primary tenocytes were treated with palmitate (200 or 400μM) and palmitate plus IL-38 (0-50ng/mL) for 24h. Western blotting was used to assess ER stress and tendinopathogenic markers in tenocytes. Monodansylcadaverine (MDC) staining was used to evaluate autophagosomes. Apoptosis was determined by cell viability assays, caspase 3 activity assays and TUNEL assays. Cell migration was evaluated by a cell scratch assay. Small interfering (si) RNA transfection was used for target gene silencing. Treatment of tenocytes with IL-38 attenuated apoptosis, restored the balance between MMPs and TIMP-1, and alleviated ER stress under palmitate conditions. IL-38 treatment enhanced AMPK phosphorylation and promoted the expression of autophagy markers related to LC3 conversion, p62 degradation, and autophagosome formation in cultured tenocytes. The effects of IL-38 on ER stress, apoptosis, and MMP-9, MMP-13, and TIMP-1 expression in palmitate-treated tenocytes were abrogated by AMPK siRNA or 3-methyladenine (3MA). These results suggest that IL-38 alleviates ER stress through the AMPK/autophagy pathway, thereby reducing apoptosis and preventing extracellular matrix (ECM) degradation in tenocytes under hyperlipidemic conditions. This study provides a promising therapeutic avenue for treating obesity-related tendinopathy using an endogenous compound such as IL-38.

Mild endoplasmic reticulum stress alleviates FB1-triggered intestinal pyroptosis via the Sec62-PERK pathway

Fumonisin B1 (FB1), a water-soluble mycotoxin released by Fusarium moniliforme Sheld, is widely present in corn and its derivative products, and seriously endangers human life and health. Recent studies have reported that FB1 can lead to pyroptosis, however, the mechanisms by which FB1-induced pyroptosis remain indistinct. In the present study, we aim to investigate the mechanisms of pyroptosis in intestinal porcine epithelial cells (IPEC-J2) and the relationship between FB1-induced endoplasmic reticulum stress (ERS) and pyroptosis. Our experimental results showed that the pyroptosis protein indicators in IPEC-J2 were significantly increased after exposure to FB1. The ERS markers, including glucose-regulated Protein 78 (GRP78), PKR-like ER kinase protein (PERK), and preprotein translocation factor (Sec62) were also significantly increased. Using small interfering RNA silencing of PERK or Sec62, the results demonstrated that upregulation of Sec62 activates the PERK pathway, and activation of the PERK signaling pathway is upstream of FB1-induced pyroptosis. After using the ERS inhibitor 4-PBA reduced the FB1-triggered intestinal injury by the Sec62-PERK pathway. In conclusion, we found that FB1 induced pyroptosis by upregulating Sec62 to activate the PERK pathway, and mild ERS alleviates FB1-triggered damage. It all boils down to one fact, the study provides a new perspective for further, and improving the toxicological mechanism of FB1.Graphical

Endoplasmic Reticulum Stress Induced Senescence in Human Lung Fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging, however their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 years) and old (>60 years) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide or salubrinal. Afterwards, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence and ECM changes using protein analysis, proliferation assay and senescence-associated beta galactosidase (SA-β-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1) and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-β-Gal, p21, Cav-1), ER stress (PERK) and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, CXCL8 and IL-6 levels were increase in the supernatants of MG132 and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments, however salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.

Reversibility of Endoplasmic Reticulum Stress Markers During Long-Term Glucose Starvation in Astrocytes

Previous studies have demonstrated a brain volume decrease linked to long-term starvation in patients with anorexia nervosa (AN). Food intake is critically diminished in this disorder, leading to one of the highest mortality rates within the psychiatric disease spectrum. As reported in animal models, astrocytes seem to be the most affected cell type in AN. In a recently established primary cell culture model, an elevated unfolded protein response (UPR) was observed in long-term glucose semi-starved astrocytes. A well-functioning protein machinery is essential for every cell, and prolonged UPR will lead to cell death. As a nucleic acid stress-sensing pathway with the activator located in the endoplasmic reticulum, the regulation of the cGAS-STING pathway (cyclic GMP-AMP synthase/stimulator of interferon genes) was additionally investigated in the starvation context. In the current study, a glucose semi-starvation protocol of 15 days, during which cells were supplied with 2 mM glucose in the medium, was prolonged with an additional 6-day long recovery period. Our findings showed that increased UPR mRNA expression was reversible after re-establishing the standard glucose concentration of 25 mM. Furthermore, we were able to verify the presence of cGAS and STING in astrocytes with a characteristic presence of cGAS in the astrocyte nucleus during starvation. A correlation between STING and the glial fibrillary acidic protein (GFAP) could be established, hinting at a conditional presence of STING with a specific astrocyte phenotype.Graphical

Cytotoxic sigma-2 ligands trigger cancer cell death via cholesterol-induced-ER-stress

Sigma-2-ligands (S2L) are characterized by high binding affinities to their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

Abstract PO4-18-03: Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis

Abstract Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) has been the most challenging subtype of BC, which consists of 20% of BC with an apparent correlation with poor prognosis. Despite that pyrotinib, a new HER2 inhibitor, has led to dramatic improvements in prognosis outcome, the efficacy of pyrotinib as monotherapy remain largely restricted due to its acquired resistance. Therefore, we aim at identifying a potent antitumor drug incorporated with pyrotinib for amplifying therapeutic efficacy for treating HER2-positive BC. Here, we reported a novel incorporation of pyrotinib in combination with chrysin, and explored its antitumor efficacy and the underlying mechanisms on HER2+ breast cancer. We determined that pyrotinib combined with chrysin yielded a potent synergistic effect to induce apoptosis and inhibit BT-474 and SK-BR-3 tumor cells, and suppressed in vivo tumor growth in tumor-bearing mice models. This may be mechanistically attributed to the induction of enhanced endoplasmic reticulum stress to increase the autophagy level. Furthermore, it was demonstrated that the combined treatment with pyrotinib and chrysin induced ubiquitination and G6PD degradation by regulating zinc finger and BTB/POZ domain-containing family protein 16 (ZBTB16) in tumorigenesis of BC. Besides, we identified that miR-16-5p is a potential upstream regulatory target of ZBTB16. Blocking miR-16-5p overexpression could inhibit HER2-positive tumorigenesis and significantly potentiate the efficacy of pyrotinib in combination with chrysin. Together, these findings demonstrate the utility of combined treatment with pyrotinib and chrysin as a potential option in the target treatment of HER2-positive BC through an unrecognized miR-16-5p/ZBTB16/G6PD axis. The miR-16-5p/ZBTB16/G6PD axis plays a crucial role in the pyrotinib plus chrysin-enabled anti HER2-positive BC Fig.1 a Cell viability of SK-BR-3 cells received various treatments. b Detection of cell cycle arrest of SK-BR-3 cells after various treatments. c Schematic diagram of the established protocol of the animal models. Photograph of resected tumor tissues, tumor volume (d), and tumor weight (e) of mice in different treatment groups during the whole testing period. f H&E, Ki67, and Tunel immunohistochemical staining of tumor sections of mice in various treatment groups. g Autophagy flux of SK-BR-3 cells labeled with mRFP-GFP-LC3 in the different treatment groups. h Expression level of ER stress markers in SK-BR-3 cells after various treatments via RT-qPCR and western blot. *P <0.05, compared with control group (DMSO), #P <0.05, compared with chrysin group, & P<0.05, compared with pyrotinib group. i Western blot analysis of the expression of G6PD in different treatment groups. j Fluorescence images of SK-BR-3 cells subjected to G6PD overexpression for the detection of autophagy level. k Ubibrowser database predicting the E3 ubiquitin ligases that may be involved in the regulation of G6PD ubiquitination. l Western blot analysis of G6PD protein half-life in SK-BR-3 cells with ZBTB16 silence. Cells were co-incubated with cycloheximide (CHX, 50μg/ml) for the indicated time. m Determination of the ubiquitination of G6PD in cells pretreated with 10 μM MG-132 for 3 h. Cells were transfected with ubiquitin after different treatments. The ubiquitinated G6PD was subjected to immunoprecipitation before western blot with ubiquitin antibody. n Dual-luciferase report verifying the targeting of ZBTB16 and miR-16-5p. o Transmission electron microscopy images of autophagosomes of tumors in various treatment groups (× 20000). p Immunohistochemical staining images of tumor slices for the determination of G6PD. q Schematic diagram for the underlying mechanism of pyrotinib combined chrysin against HER2-positive BC. Citation Format: Ting Luo, Xiaorong Zhong, Ping He, Dan Zheng, Yan Cheng, Kunrui Zhu. Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-03.

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.

Senescence and Mitochondrial Dysfunction in Lung Trisomy 21 Fibroblast

Rationale: Lung associated diseases are one of the leading causes of death in Down syndrome/Trisomy21 (DS/T21). DS is often considered a progeroid syndrome, associated with increased cellular senescence and oxidative stress in dermal T21 fibroblasts. Senescence plays a crucial role in development and organogenesis. We recently demonstrated that T21 lung anomalies are initiated in utero. We aim to better understand the role of senescence in lung disease in T21. Methods: Fresh T21 and, age- and sex-matched non-T21 human fetal lungs were collected, dissociated, and human fetal lung fibroblast (HFLF) cultures were established using differential adhesion. Baseline senescence levels were determined in whole tissue and isolated fibroblasts using TAF staining for tissue only and RT-qPCR and immunofluorescence (IF) for both. β-galactosidase, CellROX/MitoSOX, and calcium release (FURA2/AM imaging) assays were performed on T21 and non-T21 fibroblasts, cultured in the absence or presence of an ER stress inhibitor (4-PBA, 2mM) or a mitochondriogenesis stimulator (PQQ, 20μM). Results: Whole tissue sections demonstrated increased TAF and γ-H2AFX staining in T21 vs non-T21. RT-qPCR demonstrated significantly increased expression of several senescence markers including CDKN1A, CDKN2A, TP53, CDKN2B and PAI1 (p<0.05; n=6) at baseline in T21 HFLF as compared to non-T21. IF for γ-H2AFX and P21 were also significantly increased in T21 cultures vs non-T21 (p<0.01, n=6). Upregulated senescence was further confirmed in T21 HFLF by increased β-galactosidase activity (6-fold vs non-T21, p<0.05, n=4) as well as increased ER and mitochondria stress markers (CellROX and mitoSOX, p<0.05, n=6) in T21 cultures. Fura2/AM assay revealed that T21 HFLF exhibit greater intracellular calcium release (p<0.01, n=3) than non-T21 HFLF at both baseline and post Acetylcholine (10uM) challenge, an indicator of increased ER stress. In addition, several mitochondria morphology genes ( FIS1, DNML1 and MFN1) increased in T21 HFLF (p<0.05, n=6). Both 4-PBA or PQQ treatments reversed senescence in T21 HFLF as shown by decreased β-galactosidase staining (p<0.01; n = 4). No differences were observed in non-T21 HFLF after treatment. However, gene expression analyses of CDKN1A, CDKN2A, TP53 and CDKN2B demonstrated attenuated expression in T21 HFLF only following PQQ treatment but not 4-PBA (p<0.05, n=4). Conclusion: In this study, we established that fibroblasts from T21 lungs present with both ER and mitochondria-associated senescence. However, only PQQ, the mitochondriogenesis stimulator, was able to completely reverse the senescence observed at baseline in T21 HFLF. Understanding this increase in senescence in the lungs of individuals with Down Syndrome may help address the respiratory issues that arise after birth. CIRM (EDUC4-12837) Postdoctoral Training Grant (RB) NIH/NHLBI R01HL141856 (DA); NIH/NHLBI Offce of The Director, National Institutes of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DA). 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.

Caki-1 Spheroids as a 3D Model for Studying Free Fatty Acid-Induced Proximal Tubule Lipotoxicity

Three-dimensional (3D) multicellular spheroid shows a biological complexity resembling native tissue in comparison to conventional monolayer cultures, making it a better model system to assess drug effcacy and cytotoxicity. The main aim of this study was to examine the usefulness of Caki-1, a human proximal tubule (PT) representative cell line, as a 3D model system for studying free fatty acid-induced PT lipotoxicity. Caki-1 spheroids were generated and maintained on ultra-low attachment plates and characterized regarding time-dependent changes in morphology and functionality. In optimal 3D culture conditions, Caki-1 cells formed well-defined large compact 3D spheroids with uniform morphology, good circularity, and increased diameter from days 2-10. There was no change when the spheroids were incubated with lipid-free bovine serum albumin (BSA, 0.5%) from day 6 to 10. Chronic exposure to palmitate (300 μM) resulted in dispersed and flattened spheroid morphology, with a larger number of dead cells in the peripheral layers and decreased spheroid core. Moreover, palmitate-treated spheroids showed a significant increase in caspase-3 activity at the external layers of the spheroids, suggesting that the detached cells underwent apoptosis. Simultaneous addition of oleate (200 μM) prevents palmitate-induced spheroid disintegration and cell death in Caki-1 3D culture. Mechanistically, palmitate triggered endoplasmic reticulum (ER) stress and autophagy dysfunction, as evidenced by significantly increased protein levels of ER stress markers AFT4 and CHOP, and autophagy substrate p62. Oleate upregulated adipose differentiation-related protein (ADRP, a lipid droplet-associated protein), and promoted lipid droplet formation in palmitate-treated Caki-1 cells. These results indicate that Caki-1 3D spheroids are a simple and reproducible in vitro system for lipotoxicity study. It can potentially be used for evaluating human renal proximal tubular toxicity and drug screening. NIH/NIGMS R16GM149499, NIH TL1DK136047. 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.

GRP78 blockade overcomes acquired resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer

AimsWhile significant upregulation of GRP78 has been documented in lung cancer patients, its association with resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) remains underexamined. Our study aimed to elucidate the functional importance of GRP78 in acquired resistance to EGFR-TKIs in non-small cell lung cancer (NSCLC) and to evaluate its potential as a therapeutic target. Main methodsImmunoblot analysis or flow cytometry was employed to assess several markers for endoplasmic reticulum (ER) stress and apoptosis. Ru(II) complex I and HA15, two known GRP78 inhibitors, were used to evaluate the functional role of GRP78. A Xenograft assay was performed to evaluate the in vivo anti-cancer effects of the GRP78 inhibitors. Key findingsWe validated a significant increase in GRP78 protein levels in HCC827-GR, H1993-GR, and H1993-ER cells. The EGFR-TKI-resistant cells overexpressing GRP78 exhibited significantly higher cell proliferation rates than did their parental counterparts. Notably, GRP78 inhibition resulted in a more profound anti-proliferative and apoptotic response via heightened ER stress and subsequent reactive oxygen species (ROS) production in EGFR-TKI-resistant cell lines compared with their parental cells. In xenograft models implanted with HCC827-GR, both Ru(II) complex I and HA15 significantly suppressed tumor growth and reduced tumor weight. Additionally, we confirmed that GRP78 plays a critical role in the proliferation of H1975, an EGFR-TKI-resistant T790M-mutant cell line, relative to other NSCLC cell lines. SignificanceOur findings strongly support targeting of GRP78 as a promising therapeutic strategy for NSCLC patients with acquired resistance to EGFR-TKIs.

Irisin attenuates ventilator-induced diaphragmatic dysfunction by inhibiting endoplasmic reticulum stress through activation of AMPK.

Mechanical ventilation (MV) is an essential life-saving technique, but prolonged MV can cause significant diaphragmatic dysfunction due to atrophy and decreased contractility of the diaphragm fibres, called ventilator-induced diaphragmatic dysfunction (VIDD). It is not clear about the mechanism of occurrence and prevention measures of VIDD. Irisin is a newly discovered muscle factor that regulates energy metabolism. Studies have shown that irisin can exhibit protective effects by downregulating endoplasmic reticulum (ER) stress in a variety of diseases; whether irisin plays a protective role in VIDD has not been reported. Sprague-Dawley rats were mechanically ventilated to construct a VIDD model, and intervention was performed by intravenous administration of irisin. Diaphragm contractility, degree of atrophy, cross-sectional areas (CSAs), ER stress markers, AMPK protein expression, oxidative stress indicators and apoptotic cell levels were measured at the end of the experiment.Our findings showed that as the duration of ventilation increased, the more severe the VIDD was, the degree of ER stress increased, and the expression of irisin decreased.ER stress may be one of the causes of VIDD. Intervention with irisin ameliorated VIDD by reducing the degree of ER stress, attenuating oxidative stress, and decreasing the apoptotic index. MV decreases the expression of phosphorylated AMPK in the diaphragm, whereas the use of irisin increases the expression of phosphorylated AMPK. Irisin may exert its protective effect by activating the phosphorylated AMPK pathway.

Dysregulation of miR-146a is associated with exacerbated inflammation, oxidative and endoplasmic reticulum stress in the progression of diabetic foot ulcer.

Recent evidence has implicated the role of microRNA-146a (miR-146a) in regulating inflammatory responses. In the present study, we investigated the role of miRNA-146a in the progression of diabetic foot ulcer (DFU) in type 2 diabetes mellitus patients (T2DM) and studied its correlation with stress mediators such as Endoplasmic Reticulum (ER) and oxidative stress. Ninety subjects were enrolled and evenly distributed among three groups: Controls (n = 30), T2DM without complications (n = 30) and T2DM with foot ulcers (n = 30). Subsequently, each group was further subdivided based on the University of Texas classification. Peripheral blood was collected from all the study subjects, while tissue biopsies were taken only from DFU patients. Total RNA from both PBMCs and wound tissues were isolated using miRNA isolation kit and qPCR was performed to check the expression of miR-146a, ER stress and oxidative stress markers. Our findings revealed a significant decrease in miR-146a expression among T2DM patients with Grade 2 and Grade 3 DFUs compared with those with Grade 0 and Grade 1 DFUs. Notably, inflammatory genes regulated by miR-146a, including TRAF6, IRAK-1 and ADAM, were all upregulated in T2DM patients with Grade 2 and Grade 3 DFUs. Moreover, reduced miR-146a levels were correlated with increased markers of ER stress and oxidative stress in Grade 2 and Grade 3 DFU patients. Furthermore, our in vitro experiment using mouse 3T3 fibroblasts demonstrated a downregulation of miR-146a following induction of hyperglycaemia, ER stress and oxidative stress in these cells. These findings suggest a potential link between diminished miR-146a expression and heightened oxidative and ER stress in T2DM patients with more severe grades of DFUs. Our results imply that targeting miR-146a may hold therapeutic promise for managing disease progression in DFU patients, as it could help alleviate oxidative and ER stress associated with diabetic complications.

ATF3 characterizes aggressive drug-tolerant persister cells in HGSOC

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.

Ablation of IFNγ in myeloid cells suppresses liver inflammation and fibrogenesis in mice with hepatic small heterodimer partner (SHP) deletion

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is a common complication of obesity and, in severe cases, progresses to metabolic dysfunction-associated steatohepatitis (MASH). Small heterodimer partner (SHP) is an orphan member of the nuclear receptor superfamily and regulates metabolism and inflammation in the liver via a variety of pathways. In this study, we investigate the molecular foundation of MASH progression in mice with hepatic SHP deletion and explore possible therapeutic means to reduce MASH. MethodsHepatic SHP knockout mice (SHPΔhep) and their wild-type littermates (SHPfl/fl) of both sexes were fed a fructose diet for 14 weeks and subjected to an oral glucose tolerance test. Then, plasma lipids were determined, and liver lipid metabolism and inflammation pathways were analyzed with immunoblotting, RNAseq, and qPCR assays. To explore possible therapeutic intersections of SHP and inflammatory pathways, SHPΔhep mice were reconstituted with bone marrow lacking interferon γ (IFNγ−/−) to suppress inflammation. ResultsHepatic deletion of SHP in mice fed a fructose diet decreased liver fat and increased proteins for fatty acid oxidation and liver lipid uptake, including UCP1, CPT1α, ACDAM, and SRBI. Despite lower liver fat, hepatic SHP deletion increased liver inflammatory F4/80+ cells and mRNA levels of inflammatory cytokines (IL-12, IL-6, Ccl2, and IFNγ) in both sexes and elevated endoplasmic reticulum stress markers of Cox2 and CHOP in female mice. Liver bulk RNAseq data showed upregulation of genes whose protein products regulate lipid transport, fatty acid oxidation, and inflammation in SHPΔhep mice. The increased inflammation and fibrosis in SHPΔhep mice were corrected with bone marrow-derived IFNγ−/− myeloid cell transplantation. ConclusionHepatic deletion of SHP improves fatty liver but worsens hepatic inflammation possibly by driving excess fatty acid oxidation, which is corrected by deletion of IFNγ specifically in myeloid cells. This suggests that hepatic SHP limits fatty acid oxidation during fructose diet feeding but, in doing so, prevents pro-MASH pathways. The IFNγ-mediated inflammation in myeloid cells appears to be a potential therapeutic target to suppress MASH.

Mitochondria-associated endoplasmic reticulum membranes involve in oxidative stress-induced intestinal barrier injury and mitochondrial dysfunction under diquat exposing.

Many factors induced by environmental toxicants have made oxidative stress a risk factor for the intestinal barrier injury and growth restriction, which is serious health threat for human and livestock and induces significant economic loss. It is well-known that diquat-induced oxidative stress is implicated in the intestinal barrier injury. Although some studies have shown that mitochondria are the primary target organelle of diquat, the underlying mechanism remains incompletely understood. Recently, mitochondria-associated endoplasmic reticulum membranes (MAMs) have aroused increasing concerns among scholars, which participate in mitochondrial dynamics and signal transduction. In this study, we investigated whether MAMs involved in intestinal barrier injury and mitochondrial dysfunction induced by diquat-induced oxidative stress in piglets and porcine intestinal epithelial cells (IPEC-J2 cells). The results showed that diquat induced growth restriction and impaired intestinal barrier. The mitochondrial reactive oxygen species (ROS) was increased and mitochondrial membrane potential was decreased following diquat exposure. The ultrastructure of mitochondria and MAMs was also disturbed. Meanwhile, diquat upregulated endoplasmic reticulum stress marker protein and activated PERK pathway. Furthermore, loosening MAMs alleviated intestinal barrier injury, decrease of antioxidant enzyme activity and mitochondrial dysfunction induced by diquat in IPEC-J2 cells, while tightening MAMs exacerbated diquat-induced mitochondrial dysfunction. These results suggested that MAMs may be associated with the intestinal barrier injury and mitochondrial dysfunction induced by diquat in the jejunum of piglets.

Using an ER-specific optogenetic mechanostimulator to understand the mechanosensitivity of the endoplasmic reticulum

The ability of cells to perceive and respond to mechanical cues is essential for numerous biological activities. Emerging evidence indicates important contributions of organelles to cellular mechanosensitivity and mechanotransduction. However, whether and how the endoplasmic reticulum (ER) senses and reacts to mechanical forces remains elusive. To fill the knowledge gap, after developing a light-inducible ER-specific mechanostimulator (LIMER), we identify that mechanostimulation of ER elicits a transient, rapid efflux of Ca2+ from ER in monkey kidney COS-7 cells, which is dependent on the cation channels transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and polycystin-2 (PKD2) in an additive manner. This ER Ca2+ release can be repeatedly stimulated and tuned by varying the intensity and duration of force application. Moreover, ER-specific mechanostimulation inhibits ER-to-Golgi trafficking. Sustained mechanostimuli increase the levels of binding-immunoglobulin protein (BiP) expression and phosphorylated eIF2α, two markers for ER stress. Our results provide direct evidence for ER mechanosensitivity and tight mechanoregulation of ER functions, placing ER as an important player on the intricate map of cellular mechanotransduction.

Photodynamic Therapy of Head and Neck Cancers with Ruthenium-based Photosensitizers

SignificanceThe management of patients with head and neck cancers relies mainly on platinum-based chemotherapies. However, severe side effects and tumor resistance impair treatment efficacy, notably due to frequent mutations of the TP53 tumor suppressor. Here we propose a ruthenium-based photodynamic therapy as a p53-independent therapeutic strategy, and show that its phototoxicity relies on the induction of endoplasmic reticulum stress and immunogenic cell death. ApproachSQ20B cells were treated with the Ru I ruthenium-based photosensitizer prior to illumination. Ru I cytotoxicity and mode of action were studied using molecular and cellular read-outs. ResultsRu I showed an interesting phototoxicity and triggered the expression of key markers of endoplasmic reticulum stress and early stages of immunogenic cell death. ConclusionsRu I-PDT appears to be efficient in vitro on head and neck cancer models. Its phototoxicity seems to be associated to two p53-independent pathways, whose functional links remain to be clarified.

The proteasome inhibitor carfilzomib exerts anti-inflammatory and antithrombotic effects on the endothelium

BackgroundCarfilzomib (CFZ) is a second-generation proteasome inhibitor used to treat multiple myeloma. Potent inhibition of the proteasome results in chronic proteotoxic endoplasmic reticulum (ER) stress, leading to apoptosis. While CFZ has improved survival rates in multiple myeloma, it is associated with an increased risk of cardiovascular adverse effects. While this has been putatively linked to cardiotoxicity, CFZ could potentially also exhibit adverse effects on the endothelium. ObjectivesTo investigate the effects of CFZ on the endothelium. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with CFZ, and expression of relevant markers of ER stress, inflammation, and thrombosis was measured and functionally assessed. ResultsCFZ failed to induce ER stress in HUVECs but induced the expression of Kruppel-like factor 4, endothelial nitric oxide synthase, tissue plasminogen activator, and thrombomodulin and reduced tumor necrosis factor alpha (TNFα)-mediated intercellular adhesion molecule 1 and tissue factor expression, suggesting a potential protective effect on the endothelium. Consistent with these observations, CFZ reduced leukocyte adhesion under shear stress and reduced factor Xa generation and fibrin clot formation on the endothelium following TNFα treatment and inhibited von Willebrand factor (VWF) and angiopoietin-2 exocytosis from Weibel–Palade bodies. Subsequently, CFZ inhibited the formation of VWF-platelet strings, and moreover, media derived from myeloma cell lines induced VWF release, a process also inhibited by CFZ. ConclusionThese data demonstrate that CFZ is unable to induce ER stress in confluent resting endothelial cells and can conversely attenuate the prothrombotic effects of TNFα on the endothelium. This study suggests that CFZ does not negatively alter HUVECs, and proteasome inhibition of the endothelium may offer a potential way to prevent thrombosis.

Potential Hepatoprotective Effects of Irbesartan, an Accessible Angiotensin II Receptor Blocker, Against Cisplatin-Induced Liver Injury in a Rat Model.

Drug-induced liver injury is a common adverse reaction that frequently occurs with chemotherapeutic agents, such as cisplatin (CIS). This study seeks to enhance our understanding of drug actions and their associated adverse effects by examining the toxicity of CIS on rat liver tissue. We aimed to investigate the potential hepatoprotective effects of irbesartan (IRB), an easily accessible angiotensin II receptor blocker, in mitigating CIS-induced hepatotoxicity. Wistar albino rats were divided into four groups. These groups included a control group [saline, per oral (p.o.)] for seven days, and 1 mL saline intraperitoneal [(i.p.) on the fourth day]; a CIS group (1 mL saline for seven days and 7.5 mg/kg CIS i.p. on the fourth day); a CIS + IRB group (IRB: 50 mg/kg p.o. for seven days and 7.5 mg/kg CIS i.p. on the fourth day), and an IRB group (50 mg/kg IRB p.o. for seven days). The effect of IRB on interleukin-1 beta (IL-1β) and caspase 3 levels was evaluated by immunohistochemical analysis, and its effects on mRNA expression levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and immunoglobulin-heavy-chain-binding protein (BiP) were tested by quantitative real-time polymerase chain reaction. IRB administration mitigated CIS-induced liver toxicity by inhibiting endoplasmic reticulum (ER) stress. Specifically, this drug reduced the mRNA expression of ER stress markers, including CHOP and BiP. In addition, IRB treatment decreased oxidative stress, inflammatory responses, and apoptotic markers. These findings suggest that IRB is a promising therapeutic option for preventing CIS-induced liver injury, potentially by modulating ER stress-related pathways.

Possible role of endoplasmic reticulum stress in the pathogenesis of chronic adenoiditis and adenoid hypertrophy: A prospective, parallel-group study.

Adenoid tissue is a first-line host defense secondary lymphoid organ, especially in childhood. The endoplasmic reticulum (ER) is required to maintain balanced cellular activity. With impaired ER functions, protein accumulation occurs, resulting in ER stress, which plays a role in the etiopathogenesis of many diseases. We aimed to investigate the relationship between ER stress and adenoid tissue disorders, thereby elucidating the mechanisms of immunity-related diseases. Fifty-four pediatric patients (>3 years old) who underwent adenoidectomy for chronic adenoiditis (CA) or adenoid hypertrophy (AH) were enrolled in this prospective, parallel-group clinical study. Adenoids were divided into two groups (CA or AH) based on their size and evaluated for ER stress pathway and apoptosis pathway markers by Real-time PCR and Western blot analysis. ER stress pathway markers significantly differed between the CA and AH groups. Children with CA had higher ER stress marker levels than the AH group (p < .001 for ATF-4, ATF-6, and GRP78, and p < .05 for EDEM1, CHOP, EIF2AK3, ERNI, and GRP94). Apoptosis pathway marker levels (BAX and BCL-2) were not different between groups. ER stress contributes to the etiopathogenesis of adenoid tissue diseases and the pathogenesis of adenoid tissue disorders, which are part of the immune response. These results may guide the development of new and alternative treatments for immune system disorders.