Xinyang tablet alleviated cardiac dysfunction in a cardiac pressure overload model by regulating the receptor-interacting serum/three-protein kinase 3/FUN14 domain containing 1-mediated mitochondrial unfolded protein response and mitophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Xinyang tablet (XYT) has been used for heart failure (HF) for over twenty years in clinical practice, but the underlying molecular mechanism remains poorly understood. AIMS OF THE STUDY: In the present study, we aimed to explore the protective effects of XYT in HF in vivo and in vitro. MATERIALS AND METHODS: Transverse aortic constriction was performed in vivo to establish a mouse model of cardiac pressure overload. Echocardiography, tissue staining, and real-time quantitative PCR (qPCR) were examined to evaluate the protective effects of XYT on cardiac function and structure. Adenosine 5'-triphosphate production, reactive oxygen species staining, and measurement of malondialdehyde and superoxide dismutase was used to detect mitochondrial damage. Mitochondrial ultrastructure was observed by transmission electron microscope. Immunofluorescence staining, qPCR, and Western blotting were performed to evaluate the effect of XYT on the mitochondrial unfolded protein response and mitophagy, and to identify its potential pharmacological mechanism. In vitro, HL-1 cells and neonatal mouse cardiomyocytes were stimulated with Angiotensin II to establish the cell model. Western blotting, qPCR, immunofluorescence staining, and flow cytometry were utilized to determine the effects of XYT on cardiomyocytes. HL-1 cells overexpressing receptor-interacting serum/three-protein kinase 3 (RIPK3) were generated by transfection of RIPK3-overexpressing lentiviral vectors. Cells were then co-treated with XYT to determine the molecular mechanisms. RESULTS: In the present study, XYT was found to exerta protective effect on cardiac function and structure in the pressure overload mice. And it was also found XYT reduced mitochondrial damage by enhancing mitochondrial unfolded protein response and restoring mitophagy. Further studies showed that XYT achieved its cardioprotective role through regulating the RIPK3/FUN14 domain containing 1 (FUNDC1) signaling. Moreover, the overexpression of RIPK3 successfully reversed the XYT-induced protective effects and significantly attenuated the positive effects on the mitochondrial unfolded protein response and mitophagy. CONCLUSIONS: Our findings indicated that XYT prevented pressure overload-induced HF through regulating the RIPK3/FUNDC1-mediated mitochondrial unfolded protein response and mitophagy. The information gained from this study provides a potential strategy for attenuating mitochondrial damage in the context of pressure overload-induced heart failure using XYT.

Quercetin inhibits necroptosis in cardiomyocytes after ischemia-reperfusion via DNA-PKcs-SIRT5-orchestrated mitochondrial quality control.

We investigated the mechanism by which quercetin preserves mitochondrial quality control (MQC) in cardiomyocytes subjected to ischemia-reperfusion stress. An enzyme-linked immunosorbent assay was employed in the in vivo experiments to assess myocardial injury markers, measure the transcript levels of SIRT5/DNAPK-cs/MLKL during various time intervals of ischemia-reperfusion, and observe structural changes in cardiomyocytes using transmission electron microscopy. In in vitro investigations, adenovirus transfection was employed to establish a gene-modified model of DNA-PKcs, and primary cardiomyocytes were obtained from a mouse model with modified SIRT5 gene. Reverse transcription polymerase chain reaction, laser confocal microscopy, immunofluorescence localization, JC-1 fluorescence assay, Seahorse energy analysis, and various other assays were applied to corroborate the regulatory influence of quercetin on the MQC network in cardiomyocytes after ischemia-reperfusion. In vitro experiments demonstrated that ischemia-reperfusion injury caused changes in the structure of the myocardium. It was seen that quercetin had a beneficial effect on the myocardial tissue, providing protection. As the ischemia-reperfusion process continued, the levels of DNA-PKcs/SIRT5/MLKL transcripts were also found to change. In vitro investigations revealed that quercetin mitigated cardiomyocyte injury caused by mitochondrial oxidative stress through DNA-PKcs, and regulated mitophagy and mitochondrial kinetics to sustain optimal mitochondrial energy metabolism levels. Quercetin, through SIRT5 desuccinylation, modulated the stability of DNA-PKcs, and together they regulated the "mitophagy-unfolded protein response." This preserved the integrity of mitochondrial membrane and genome, mitochondrial dynamics, and mitochondrial energy metabolism. Quercetin may operate synergistically to oversee the regulation of mitophagy and the unfolded protein response through DNA-PKcs-SIRT5 interaction.

Enhancing Antibody-Specific Productivity: Unraveling the Impact of XBP1s Overexpression and Glutamine Availability in SP2/0 Cells.

Augmentation of glycoprotein synthesis requirements induces endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR) and triggering unconventional XBP1 splicing. As a result, XBP1s orchestrates the expression of essential genes to reduce stress and restore homeostasis. When this mechanism fails, chronic stress may lead to apoptosis, which is thought to be associated with exceeding a threshold in XBP1s levels. Glycoprotein assembly is also affected by glutamine (Gln) availability, limiting nucleotide sugars (NS), and preventing compliance with the increased demands. In contrast, increased Gln intake synthesizes ammonia as a by-product, potentially reaching toxic levels. IgA2m(1)-producer mouse myeloma cells (SP2/0) were used as the cellular mammalian model. We explored how IgA2m(1)-specific productivity (qIgA2m(1)) is affected by (i) overexpression of human XBP1s (h-XBP1s) levels and (ii) Gln availability, evaluating the kinetic behavior in batch cultures. The study revealed a two and a five-fold increase in qIgA2m(1) when lower and higher levels of XBP1s were expressed, respectively. High h-XBP1s overexpression mitigated not only ammonia but also lactate accumulation. Moreover, XBP1s overexpressor showed resilience to hydrodynamic stress in serum-free environments. These findings suggest a potential application of h-XBP1s overexpression as a feasible and cost-effective strategy for bioprocess scalability.

Metabolic rescue of α-synuclein-induced neurodegeneration through propionate supplementation and intestine-neuron signaling in C. elegans.

Microbial metabolites that can modulate neurodegeneration are promising therapeutic targets. Here, we found that the short-chain fatty acid propionate protects against α-synuclein-induced neuronal death and locomotion defects in a Caenorhabditis elegans model of Parkinson's disease (PD) through bidirectional regulation between the intestine and neurons. Both depletion of dietary vitamin B12, which induces propionate breakdown, and propionate supplementation suppress neurodegeneration and reverse PD-associated transcriptomic aberrations. Neuronal α-synuclein aggregation induces intestinal mitochondrial unfolded protein response (mitoUPR), which leads to reduced propionate levels that trigger transcriptional reprogramming in the intestine and cause defects in energy production. Weakened intestinal metabolism exacerbates neurodegeneration through interorgan signaling. Genetically enhancing propionate production or overexpressing metabolic regulators downstream of propionate in the intestine rescues neurodegeneration, which then relieves mitoUPR. Importantly, propionate supplementation suppresses neurodegeneration without reducing α-synuclein aggregation, demonstrating metabolic rescue of neuronal proteotoxicity downstream of protein aggregates. Our study highlights the involvement of small metabolites in the gut-brain interaction in neurodegenerative diseases.

Low levels of Cd2+ combined with procymidone may cause ovarian damage in mice via unfolded protein response.

As no study about the combined effect of low levels of Cd2+ with procymidone (PCM) on organs and organisms, we investigated their actions on mouse-ovary in vivo and in vitro. Four-week mice were treated with corn oil for the control group, corn oil + 0.0045 mg/L Cd2+ (CdCl2 was dissolved in ultrapure water and freely consumed by mice) for Cd2+ group, 50 mg/kg/d PCM (suspended in corn oil and administered orally to mice) for PCM group, and 50 mg/kg/d PCM + 0.0015 (0.0045 and 0.0135) mg/L Cd2+ for L+ (M+ and H+) PCM group for 21 days. For in vitro experiment, the cultured ovaries were treated with acetone for the control group, 0.1% acetone + 8.4 µg/L Cd2+ for the Cd2+ group, 0.63 mg/L PCM (dissolved in acetone) for the PCM-group, and 0.63 mg/L PCM + 2.8 (8.4 and 25.2) µg/L Cd2+ for L+ (M+ and H+) PCM group for 7 days. Mouse body weight in each treatment group, the weight and volume of ovaries in all PCM groups were lower than the control. Both in vivo and in vitro, all-stage follicle numbers were lower in M+PCM and H+PCM groups, whereas the atretic follicles and CASPASE3/8 were higher; meanwhile, lower estradiol and progesterone and higher unfolded protein response (UPR) members in all PCM groups. L+, M+, and H+PCM groups had further ovarian damage and stronger UPR than PCM groups, as did M+PCM groups over Cd2+ groups. It is hypothesized low-level PCM and Cd2+ may mutually promote each other's triggered UPR and exacerbate ovarian damage.

Dietary conjugated linoleic acid downregulates the AlCl3-induced hyperactivation of compensatory and maladaptive signalling in the mouse brain cortex.

Oxidative stress, hyperactivation of compensatory mechanisms (unfolded protein response, UPR; nuclear factor erythroid 2-related factor 2, Nrf2) and the stimulation of maladaptive response (inflammation/apoptosis) are interconnected pathogenic processes occurring during Alzheimer's disease (AD) progression. The neuroprotective ability of dietary Conjugated linoleic acid (CLAmix) in a mouse model of AlCl3-induced AD was recently described but, the effects of AlCl3 or CLAmix intake on these pathogenic processes are still unknown. The effects of dietary AlCl3 or CLAmix - alone and in combination - were examined in the brain cortex of twenty-eight BalbC mice divided into 4 groups (n = 7 each). The neurotoxic effects of AlCl3 were investigated in animals treated for 5 weeks with 100 mg/kg/day (AL). CLAmix supplementation (600 mg/kg bw/day) for 7 weeks (CLA) was aimed at evaluating its modulatory effects on the Nrf2 pathway while its co-treatment with AlCl3 during the last 5 weeks of CLAmix intake (CLA + AL) was used to investigate its neuroprotective ability. Untreated mice were used as controls. In the CLA group, the NADPH oxidase (NOX) activation in the brain cortex was accompanied by the modulation of the Nrf2 pathway. By contrast, in the AL mice, the significant upregulation of oxidative stress markers, compensatory pathways (UPR/Nrf2), proinflammatory cytokines (IL-6, TNFα) and the proapoptotic protein Bax levels were found as compared with control. Notably, in CLA + AL mice, the marked decrease of oxidative stress, UPR/Nrf2 markers and proinflammatory cytokines levels were associated with the significant increase of the antiapoptotic protein Bcl2. The involvement of NOX in the adaptive response elicited by CLAmix along with its protective effects against the onset of several pathogenic processes triggered by AlCl3, broadens the knowledge of the mechanism underlying the pleiotropic activity of Nrf2 activators and sheds new light on their potential therapeutic use against neurodegenerative disorders.

Bioinformatics-driven discovery of silica nanoparticles induces apoptosis and renal damage via the unfolded protein response in NRK-52E cells and rat kidney.

Silica nanoparticles (SiNPs) are nanomaterials with widespread applications in drug delivery and disease diagnosis. Despite their utility, SiNPs can cause chronic kidney disease, hindering their clinical translation. The molecular mechanisms underlying SiNP-induced renal toxicity are complex and require further investigation. To address this challenge, we employed bioinformatics tools to predict the potential mechanisms underlying renal damage caused by SiNPs. We identified 1627 upregulated differentially expressed genes (DEGs) and 1334 downregulated DEGs. Functional enrichment analysis and protein-protein interaction network revealed that SiNP-induced renal damage is associated with apoptosis. Subsequently, we verified that SiNPs induced apoptosis in an in vitro model of NRK-52E cells via the unfolded protein response (UPR) in a dose-dependent manner. Furthermore, in an in vivo rat model, high-dose SiNP administration via tracheal drip caused hyalinization of the renal tubules, renal interstitial lymphocytic infiltration, and collagen fiber accumulation. Concurrently, we observed an increase in UPR-related protein levels at the onset of renal damage. Thus, our study confirmed that SiNPs induce apoptosis and renal damage through the UPR, adding to the theoretical understanding of SiNP-related kidney damage and offering a potential target for preventing and treating kidney injuries in SiNP clinical applications.

Reduction of excessive unfolded protein response by 4-phenylbutyric acid may mitigate procymidone-induced testicular damage in mice by changing the levels of circRNA Scar and circZc3h4.

Procymidone (PCM) exposure below the no-observed-effect level triggers changes in circRNA Scar and circZc3h4 and overactivation of the unfolded protein response (UPR) in mice, culminating in testicular injury. The 4-phenyl butyric acid (4-PBA) is known to stabilize proteins and reduce the UPR. This study employed an in vitro system in which mouse testes were cultured with 1 × 10-5 M PCM and varying concentrations (0, 20, 40, and 80 mM) of 4-PBA; 4-week-old male mice were subsequently treated with 100 mg/kg/d PCM (suspended in corn oil) and/or 100 mg/kg/d 4-PBA for 21 d, consecutively. The treatments were as follows: the negative control (NC) group was orally administered corn oil; the positive control (PC) group was orally administered PCM; the 4-PBA group was intraperitoneally injected with 4-PBA; the 4-PBA-I group was orally administered PCM and 4-PBA simultaneously; the 4-PBA-II group received daily administration of 4-PBA 24 h prior to PCM; and the 4-PBA-III group was intraperitoneally injected with 4-PBA for 7 d after 21 d of PCM administration. However, the 4-PBA intervention groups showed no considerable changes in the overall or testicular appearance of mice. In vitro, 4-PBA inhibited the PCM-induced testicular injury, with the most significant effect observed at 80 mM. In vivo, the 4-PBA-III group exhibited the best in vivo effects. Our findings indicate that 4-PBA conferred testicular protection by decreasing PCM-induced circRNA Scar, elevating circZc3h4, and suppressing UPR both in vitro and in vivo. It has been hypothesized that 4-PBA mitigates testicular damage by reducing excessive UPR levels.

L-Proline Prevents Endoplasmic Reticulum Stress in Microglial Cells Exposed to L-azetidine-2-carboxylic Acid.

L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that shares structural similarities with its proteogenic L-proline amino acid counterpart. For this reason, AZE can be misincorporated in place of L-proline, contributing to AZE toxicity. In previous work, we have shown that AZE induces both polarization and apoptosis in BV2 microglial cells. However, it is still unknown if these detrimental effects involve endoplasmic reticulum (ER) stress and whether L-proline co-administration prevents AZE-induced damage to microglia. Here, we investigated the gene expression of ER stress markers in BV2 microglial cells treated with AZE alone (1000 µM), or co-treated with L-proline (50 µM), for 6 or 24 h. AZE reduced cell viability, nitric oxide (NO) secretion and caused a robust activation of the unfolded protein response (UPR) genes (ATF4, ATF6, ERN1, PERK, XBP1, DDIT3, GADD34). These results were confirmed by immunofluorescence in BV2 and primary microglial cultures. AZE also altered the expression of microglial M1 phenotypic markers (increased IL-6, decreased CD206 and TREM2 expression). These effects were almost completely prevented upon L-proline co-administration. Finally, triple/quadrupole mass spectrometry demonstrated a robust increase in AZE-bound proteins after AZE treatment, which was reduced by 84% upon L-proline co-supplementation. This study identified ER stress as a pathogenic mechanism for AZE-induced microglial activation and death, which is reversed by co-administration of L-proline.

Potent carotenoid astaxanthin expands the anti-cancer activity of cisplatin in human prostate cancer cells.

Prostate cancer (PCa) is the second most common type of cancer and the sixth cause of death in men worldwide. Radiotherapy and immunotherapy are commonly used in treating PCa, but understanding the crosstalk mechanisms of carcinogenesis and new therapeutic approaches is essential for supporting poor diagnosis and existing therapies. Astaxanthin (ASX) is a member of the xanthophyll family that is an oxygenated derivative of carotenoids whose synthesis is in plant extracts from lycopene. ASX has protective effects on various diseases, such as Parkinson's disease and cancer by showing potent antioxidant and anti-inflammatory properties. However, there is an ongoing need for a detailed investigation of the molecular mechanism of action to expand its therapeutic use. In the present study, we showed the new regulatory role of ASX in PCa cells by affecting the unfolded protein response (UPR) signaling, autophagic activity, epithelial-mesenchymal transition (EMT) and regulating the expression level of angiogenesis-related protein vascular endothelial growth factor A (VEGF-A), proto-oncogene c-Myc and prostate-specific antigen (PSA). Additionally, we determined that it exhibited synergistic action with cisplatin and significantly enhanced apoptotic cell death in PCa cells. Present findings suggest that ASX may be a potent adjuvant therapeutic option in PCa treatment when used alone or combined with chemotherapeutics. Schematic illustration of the biochemical activity of astaxanthin and its combination with cisplatin.

Selenite as a dual apoptotic and ferroptotic agent synergizes with EGFR and KRAS inhibitors with epigenetic interference.

BACKGROUND: Selenium, an essential trace element, has previously been investigated as a pro-apoptotic and DNA demethylation agent. It sensitizes the response to chemotherapy in patients who were refractory to cytotoxic agents. Meanwhile, ferroptosis is a novel approach to cancer treatment by triggering cell death and reversing drug resistance. The role of selenium in treating cancer cells harboring druggable oncogenic alterations and its underlying mechanism are largely unknown. RESULTS: We treated lung adenocarcinoma cell lines-EGFR-mutant H1975 (H1975 EGFR p.L858R and p.T790M) and KRAS-mutant H358 (H358 KRAS p.G12C), with sodium selenite to examine its effect on cell apoptosis, ferroptosis, and DNA methylation, as well as its interaction with existing targeted therapy, osimertinib, and adagrasib. We observed selenite to be a dual apoptotic and ferroptotic agent on lung cancer cells, associated with the activation of p38-ATF4-DDIT3 axis in the unfolded protein response. Ferroptosis induction was more remarkable in H1975 than H358. Selenite also altered cellular DNA methylation machinery through downregulating DNMT1 and upregulating TET1, though not as a major mechanism of its activity. Low-dose selenite synergized with osimertinib in EGFR-mutant H1975, and with adagrasib in KRAS-mutant H358, with stronger synergism observed in H1975. CONCLUSION: These results suggest that selenite is a potential apoptotic and ferroptotic drug candidate for the treatment of especially EGFR- and potentially KRAS-mutant lung cancer.

Endoplasmic Reticulum Stress in Elderly Patients with COVID-19: Potential of Melatonin Treatment.

Aging processes, including immunosenescence, inflammation, inflammasome formation, genomic instability, telomeric attrition, and altered autophagy, are involved in viral infections and they may contribute to increased pathophysiological responses to the SARS-CoV-2 infection in the elderly; this poses additional risks of accelerated aging, which could be found even after recovery. Aging is associated with oxidative damage. Moreover, SARS-CoV-2 infections may increase the production of reactive oxygen species and such infections will disturb the Ca++ balance via an endoplasmic reticulum (ER) stress-mediated unfolded protein response. Although vaccine development and anti-inflammation therapy lower the severity of COVID-19, the prevalence and mortality rates are still alarming in some countries worldwide. In this review, we describe the involvement of viral proteins in activating ER stress transducers and their downstream signals and in inducing inflammation and inflammasome formation. Furthermore, we propose the potential of melatonin as an ER stress modulator, owing to its antioxidant, anti-inflammatory, and immunoregulatory effects in viral infections. Considering its strong safety profile, we suggest that additive melatonin supplementation in the elderly could be beneficial in treating COVID-19.

Alisma Shugan Decoction attenuates hepatic fibrosis and endoplasmic reticulum stress in mice with carbon tetrachloride-induced fibrosis.

Background: Over the years, Alisma Shugan Decoction (ASD), because of its potent anti-inflammation activity, has been used in traditional Chinese medicine (TCM) for treatment of many inflammation-associated disorders including those of the heart, blood vessel and brain. Methods: Herein, we examined the probable therapeutic effect of ASD in carbon tetrachloride (CCl4)-induced liver injury and fibrosis mice models. Results: Our results demonstrate that ASD dose-dependently reduced the fibrosis-related increased collagen deposition secondary to liver tissue exposure to CCl4. Data from our biochemical analyses showed significantly less liver damage biomarkers including ALT, AST and hydroxyproline in the ASD-treated samples, suggesting hepato-protective effect of ASD. Furthermore, we demonstrated that treatment with ASD significantly reversed CCl4-induced elevation of TNF-α, IL-6, IL-1ß and MP-1. Interestingly, NF-κB signalling, a principal regulator of inflammation was markedly suppressed by ASD treatment. In addition, treatment with ASD deregulated stress signalling pathways by suppressing the expression of markers of unfolded protein response, such as ATF6, IRE and GRP78. Conclusion: In conclusion, the present study provides preclinical evidence for the use of ASD as an efficacious therapeutic option in cases of chemical-induced liver damage and/or fibrosis. Further large-cohort validation of these findings is warranted.

Comprehensive Transcriptome Analysis Reveals Genome-Wide Changes Associated with Endoplasmic Reticulum (ER) Stress in Potato (Solanum tuberosum L.).

We treated potato (Solanum tuberosum L.) plantlets with TM and performed gene expression studies to identify genome-wide changes associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). An extensive network of responses was identified, including chromatin remodeling, transcriptional reprogramming, as well as changes in the structural components of the endomembrane network system. Limited genome-wide changes in alternative RNA splicing patterns of protein-coding transcripts were also discovered. Significant changes in RNA metabolism, components of the translation machinery, as well as factors involved in protein folding and maturation occurred, which included a broader set of genes than expected based on Arabidopsis research. Antioxidant defenses and oxygen metabolic enzymes are differentially regulated, which is expected of cells that may be experiencing oxidative stress or adapting to protect proteins from oxidation. Surges in protein kinase expression indicated early signal transduction events. This study shows early genomic responses including an array of differentially expressed genes that have not been reported in Arabidopsis. These data describe novel ER stress responses in a solanaceous host.

Lactiplantibacillus plantarum 299v supplementation modulates ß-cell ER stress and antioxidative defense pathways and prevents type 1 diabetes in gluten-free BioBreeding rats.

The increasing incidence of Type 1 diabetes has coincided with the emergence of the low-fiber, high-gluten Western diet and other environmental factors linked to dysbiosis. Since Lactiplantibacillus plantarum 299 v (Lp299v) supplementation improves gut barrier function and reduces systemic inflammation, we studied its effects in spontaneously diabetic DRlyp/lyp rats provided a normal cereal diet (ND) or a gluten-free hydrolyzed casein diet (HCD). All rats provided ND developed diabetes (62.5±7.7 days); combining ND with Lp299v did not improve survival. Diabetes was delayed by HCD (72.2±9.4 days, p = .01) and further delayed by HCD+Lp299v (84.9±14.3 days, p < .001). HCD+Lp299v pups exhibited increased plasma propionate and butyrate levels, which correlated with enriched fecal Bifidobacteriaceae and Clostridiales taxa. Islet transcriptomic and histologic analyses at 40-days of age revealed that rats fed HCD expressed an autophagy profile, while those provided HCD+Lp299v expressed ER-associated protein degradation (ERAD) and antioxidative defense pathways, including Nrf2. Exposing insulinoma cells to propionate and butyrate promoted the antioxidative defense response but did not recapitulate the HCD+Lp299v islet ERAD transcriptomic profile. Here, both diet and microbiota influenced diabetes susceptibility. Moreover, Lp299v supplement modulated antioxidative defense and ER stress responses in ß-cells, potentially offering a new therapeutic direction to thwart diabetes progression and preserve insulin secretion.

Reduction in Insulin Mediated ERK Phosphorylation by Palmitate in Liver Cells Is Independent of Fatty Acid Induced ER Stress.

Saturated free fatty acids (FFAs) such as palmitate in the circulation are known to cause endoplasmic reticulum (ER) stress and insulin resistance in peripheral tissues. In addition to protein kinase B (AKT) signaling, extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance. However, there are conflicting data regarding role of ERK signaling in ER stress-induced insulin resistance. In this study, we investigated the effects of ER stress on insulin resistance and ERK phosphorylation in Huh-7 cells and evaluated how oleate prevents palmitate-mediated ER stress. Treatment with insulin resulted in an increase of 38-45% in the uptake of glucose in control cells compared to non-insulin-treated control cells, along with an increase in the phosphorylation of AKT and ERK. We found that treatment with palmitate increased the expression of ER stress genes, including the splicing of X box binding protein 1 (XBP1) mRNA. At the same time, we observed a decrease in insulin-mediated uptake of glucose and ERK phosphorylation in Huh-7 cells, without any change in AKT phosphorylation. Supplementation of oleate along with palmitate mitigated the palmitate-induced ER stress but did not affect insulin-mediated glucose uptake or ERK phosphorylation. The findings of this study suggest that palmitate reduces insulin-mediated ERK phosphorylation in liver cells and this effect is independent of fatty-acid-induced ER stress.

Exposure to automobile exhaust-derived PM2.5 induces spermatogenesis dysfunction by damaging UPRmt of prepubertal rats.

Automobile exhaust-derived particulate matter 2.5 (PM2.5) can cause spermatogenic cell damage, potentially resulting in male infertility. This study uses male prepubertal Sprague Dawley (SD) rats to explore the molecular mechanisms by which automobile exhaust-derived PM2.5 causes spermatogenic cell damage and induces spermatogenesis dysfunction during sexual maturity by disrupting the mitochondrial unfolded protein response (UPRmt) in spermatogenic cells. Male prepubertal SD rats were randomly divided into four groups: control (intratracheal instillation of normal saline), low-dose PM2.5 (5 mg/kg), high-dose PM2.5 (10 mg/kg), and PM2.5 10 mg/kg +Vit (100 mg/kg of vitamin C and 50 mg/kg of vitamin E). The rats were treated for four weeks, with five consecutive treatment days and two non-treatment days, followed by cohabitation. Testicular and epididymal tissues were harvested for analysis. The mitochondria in spermatogenic cells were observed under an electron microscope. UPRmt-, oxidative stress-, and apoptosis-related markers in spermatogenic cells were examined. Spermatogenic cell numbers and conception rate declined significantly with increasing PM2.5 dose, with their mitochondria becoming vacuolated, swollen, and degenerated to varying degrees. The apoptosis of spermatogenic cells was abnormally enhanced in PM2.5 exposed groups compared to the control group. Spermatogenic cell numbers of conception rate gradually recovered, mitochondrial damage in spermatogenic cells was alleviated, and spermatogenic cell apoptosis was significantly reduced after vitamin intervention. In addition, protein levels of superoxide dismutase 1 (Sod1), nuclear factor erythroid 2-related factor 2 (Nrf2), and B-cell lymphoma 2 (Bcl-2) were significantly lower, while those of Bcl2-associated X apoptosis regulator (Bax), cleaved caspase 3 (Casp3), and cytochrome c (Cyt-c) and malondialdehyde (MDA) levels were significantly higher in the high-dose PM2.5 group than in the control group. The levels of UPRmt-related proteins C/EBP homologous protein (Chop), heat shock protein 60 (Hsp60), and activating transcription factors 4 (Atf4) and 5 (Atf5) were higher in the low-dose PM2.5 group, lower in the high-dose PM2.5 group, and gradually recovered in PM2.5 10 mg/kg +Vit group. Our results show that exposure to automobile exhaust-derived PM2.5 induces oxidative stress responses, leads to post-sexual maturation UPRmt dysfunction and mitochondrial impairment, and abnormally enhances spermatogenic cell apoptosis in prepubertal rats, resulting in male infertility.

The joint action of unfolded protein response, circZc3h4, and circRNA Scar in procymidone-induced testicular injury in adolescent mice.

Procymidone (PCM) is a low toxicity fungicide, and an endocrine-disrupting chemical (EDC) that particularly damages the reproductive system of male vertebrates. In present study, adolescent mice in control, low-, medium-, and high-dose groups were orally administered 0 (equal volume of soybean oil), 50, 100, and 200 mg/kg/day PCM, respectively, for 21 days. Additionally, a three-dimensional culture of mouse testes was performed in vitro, and the control, low dose (0.33 × 10-5  M), medium dose (1 × 10-5  M), and high dose (3 × 10-5  M) PCM groups were established. We have found that, under both in vivo and in vitro conditions, all doses of PCM caused damage to mouse testes. Moreover, the levels of circZc3h4 RNA and Zc3h4 decreased while miR-212 increased in all treatment groups, with a corresponding rise in circRNA Scar and fall in Atp5b, compared to those in the control group, and all the changes showed a dose-response relationship. Besides, we have identified that low doses of PCM could activate the Ire1-Xbp1 pathway, whereas the medium and high doses activated the Perk-Elf2α-Atf4, Ire1-Xbp1, and Atf6 pathways. And it is, therefore, speculated that the unfolded protein response (UPR), circZc3h4 and circRNA Scar may have taken joint action in testicular injury in adolescent mice induced by PCM at the no observed adverse effect level (NOAEL, 100 mg/kg/day) and below NOAEL doses.

Combination of Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos improves cardiac function of diabetic cardiomyopathy mice by regulating the unfolded protein response signaling pathway.

Diabetic cardiomyopathy (DCM) is a unique clinical entity elicited by diabetes independent of other cardiovascular risk factors, of which the pathological mechanisms and treatment strategies remain largely undefined. This study aimed to clarify the role of unfolded protein response (UPR) signaling pathway in the pathogenesis of DCM, and to explore the effect of aqueous extract of Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos (DH) on DCM mice. Cardiac function of DCM mice was evaluated by echocardiography, and lipid profile of left ventricular was analyzed by untargeted lipidomics. The results showed that DH significantly improved the diabetic symptoms, cardiac dyslipidemia, and systolic dysfunction of DCM mice. UPR signaling pathway was significantly down-regulated in the left ventricular of DCM mice. DH significantly up-regulated the transcriptions of key transducers in UPR signaling pathway. Conditional knockout of Xbp1 in cardiomyocyte (a key regulator in UPR signaling pathway) eliminated the protective effect of DH on cardiac systolic function of DCM mice, which suggested that UPR signaling pathway, especially the Xbp1, was required for DH protection against DCM. In conclusion, DH improved cardiac function of DCM mice, and this effect was dependent on its regulation of UPR signaling pathway.

Evidence From Human Placenta, Endoplasmic Reticulum-Stressed Trophoblasts, and Transgenic Mice Links Transthyretin Proteinopathy to Preeclampsia.

BACKGROUND: We have demonstrated that protein aggregation plays a pivotal role in the pathophysiology of preeclampsia and identified several aggregated proteins in the circulation of preeclampsia patients, the most prominent of which is the serum protein TTR (transthyretin). However, the mechanisms that underlie protein aggregation remain poorly addressed. METHODS: We examined TTR aggregates in hypoxia/reoxygenation-exposed primary human trophoblasts (PHTs) and the preeclampsia placenta using complementary approaches, including a novel protein aggregate detection assay. Mechanistic analysis was performed in hypoxia/reoxygenation-exposed PHTs and Ttr transgenic mice overexpressing transgene-encoded wild-type human TTR or Ttr-/- mice. High-resolution ultrasound analysis was used to measure placental blood flow in pregnant mice. RESULTS: TTR aggregation was inducible in PHTs and the TCL-1 trophoblast cell line by endoplasmic reticulum stress inducers or autophagy-lysosomal disruptors. PHTs exposed to hypoxia/reoxygenation showed increased intracellular BiP (binding immunoglobulin protein), phosphorylated IRE1α (inositol-requiring enzyme-1α), PDI (protein disulfide isomerase), and Ero-1, all markers of the unfolded protein response, and the apoptosis mediator caspase-3. Blockade of IRE1α inhibited hypoxia/reoxygenation-induced upregulation of Ero-1 in PHTs. Excessive unfolded protein response activation was observed in the early-onset preeclampsia placenta. Importantly, pregnant human TTR mice displayed aggregated TTR in the junctional zone of the placenta and severe preeclampsia-like features. High-resolution ultrasound analysis revealed low blood flow in uterine and umbilical arteries in human TTR mice compared with control mice. However, Ttr-/- mice did not show any pregnancy-associated abnormalities. CONCLUSIONS: These observations in the preeclampsia placenta, cultured trophoblasts, and Ttr transgenic mice indicate that TTR aggregation is an important causal contributor to preeclampsia pathophysiology.