Effects of Natural Products through Inhibiting Endoplasmic Reticulum Stress on Attenuation of Idiopathic Pulmonary Fibrosis.

With ever-increasing intensive studies of idiopathic pulmonary fibrosis (IPF), significant progresses have been made. Endoplasmic reticulum stress (ERS)/unfolded protein reaction (UPR) is associated with the development and progression of IPF, and targeting ERS/UPR may be beneficial in the treatment of IPF. Natural product is a tremendous source of new drug discovery, and accumulating studies have reported that many natural products show potential therapeutic effects for IPF via modulating one or more branches of the ERS signaling pathway. Therefore, this review focuses on critical roles of ERS in IPF development, and summarizes herbal preparations and bioactive compounds which protect against IPF through regulating ERS.

Targeting Cancer Mitochondria by Inducing an Abnormal Mitochondrial Unfolded Protein Response Leads to Tumor Suppression.

The mitochondrial unfolded protein response (UPRmt) is a pivotal cellular mechanism that ensures mitochondrial homeostasis and cellular survival under stress conditions. This study investigates the role of UPRmt in modulating the response of nasopharyngeal carcinoma cells to cisplatin-induced stress. We report that the inhibition of UPRmt via AEB5F exacerbates cisplatin cytotoxicity, as evidenced by increased lactate dehydrogenase (LDH) release and apoptosis, characterized by a surge in TUNEL-positive cells. Conversely, the activation of UPRmt with oligomycin attenuates these effects, preserving cell viability and reducing apoptotic markers. Immunofluorescence assays reveal that UPRmt activation maintains mitochondrial membrane potential and ATP production in the presence of cisplatin, countering the rise in reactive oxygen species (ROS) and inhibiting caspase-9 activation. These findings suggest that UPRmt serves as a cytoprotective mechanism in cancer cells, mitigating cisplatin-induced mitochondrial dysfunction and apoptosis. The data underscore the therapeutic potential of modulating UPRmt to improve the efficacy and reduce the side effects of cisplatin chemotherapy. This study provides a foundation for future research on the exploitation of UPRmt in cancer treatment, with the aim of enhancing patient outcomes by leveraging the cellular stress response pathways.

Metformin induction of heat shock factor 1 activation and the mitochondrial unfolded protein response alleviate cardiac remodeling in spontaneously hypertensive rats.

Heart failure and cardiac remodeling are both characterized by mitochondrial dysfunction. Healthy mitochondria are required for adequate contractile activity and appropriate regulation of cell survival. In the mammalian heart, enhancement of the mitochondrial unfolded protein response (UPRmt) is cardioprotective under pressure overload conditions. We explored the UPRmt and the underlying regulatory mechanism in terms of hypertension-induced cardiac remodeling and the cardioprotective effect of metformin. Male spontaneously hypertensive rats and angiotensin II-treated neonatal rat cardiomyocytes were used to induce cardiac hypertrophy. The results showed that hypertension induced the formation of aberrant mitochondria, characterized by a reduced mtDNA/nDNA ratio and swelling, as well as lower levels of mitochondrial complexes I to V and inhibition of the expression of one protein subunit of each of complexes I to IV. Such changes eventually enlarged cardiomyocytes and increased cardiac fibrosis. Metformin treatment increased the mtDNA/nDNA ratio and regulated the UPRmt, as indicated by increased expression of activating transcription factor 5, Lon protease 1, and heat shock protein 60, and decreased expression of C/EBP homologous protein. Thus, metformin improved mitochondrial ultrastructure and function in spontaneously hypertensive rats. In vitro analyses revealed that metformin reduced the high levels of angiotensin II-induced mitochondrial reactive oxygen species in such animals and stimulated nuclear translocation of heat shock factor 1 (HSF1). Moreover, HSF1 small-interfering RNA reduced the metformin-mediated improvements in mitochondrial morphology and the UPRmt by suppressing hypertrophic signals and cardiomyocyte apoptosis. These results suggest that HSF1/UPRmt signaling contributes to the beneficial effects of metformin. Metformin-mediated targeting of mitochondrial protein homeostasis and modulation of HSF1 levels have potential therapeutic implications in terms of cardiac remodeling.

Development of TRIB3-Based Therapy as a Gene-Independent Approach to Treat Retinal Degenerative Disorders.

Inherited retinal degeneration (RD) constitutes a heterogeneous group of genetic retinal degenerative disorders. The molecular mechanisms underlying RD encompass a diverse spectrum of cellular signaling, with the unfolded protein response (UPR) identified as a common signaling pathway chronically activated in degenerating retinas. TRIB3 has been recognized as a key mediator of the PERK UPR arm, influencing various metabolic pathways, such as insulin signaling, lipid metabolism, and glucose homeostasis, by acting as an AKT pseudokinase that prevents the activation of the AKT → mTOR axis. This study aimed to develop a gene-independent approach targeting the UPR TRIB3 mediator previously tested by our group using a genetic approach in mice with RD. The goal was to validate a therapeutic approach targeting TRIB3 interactomes through the pharmacological targeting of EGFR-TRIB3 and delivering cell-penetrating peptides targeting TRIB3 → AKT. The study employed rd10 and P23H RHO mice, with afatinib treatment conducted in p15 rd10 mice through daily intraperitoneal injections. P15 P23H RHO mice received intraocular injections of cell-penetrating peptides twice at a 2-week interval. Our study revealed that both strategies successfully targeted TRIB3 interactomes, leading to an improvement in scotopic A- and B-wave ERG recordings. Additionally, the afatinib-treated mice manifested enhanced photopic ERG amplitudes accompanied by a delay in photoreceptor cell loss. The treated rd10 retinas also showed increased PDE6ß and RHO staining, along with an elevation in total PDE activity in the retinas. Consequently, our study demonstrated the feasibility of a gene-independent strategy to target common signaling in degenerating retinas by employing a TRIB3-based therapeutic approach that delays retinal function and photoreceptor cell loss in two RD models.

Thapsigargin and Tunicamycin Block SARS-CoV-2 Entry into Host Cells via Differential Modulation of Unfolded Protein Response (UPR), AKT Signaling, and Apoptosis.

BACKGROUND: SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. METHODS: Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. RESULTS: TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. CONCLUSIONS: These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Polydatin and Nicotinamide Rescue the Cellular Phenotype of Mitochondrial Diseases by Mitochondrial Unfolded Protein Response (mtUPR) Activation.

Primary mitochondrial diseases result from mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) genes, encoding proteins crucial for mitochondrial structure or function. Given that few disease-specific therapies are available for mitochondrial diseases, novel treatments to reverse mitochondrial dysfunction are necessary. In this work, we explored new therapeutic options in mitochondrial diseases using fibroblasts and induced neurons derived from patients with mutations in the GFM1 gene. This gene encodes the essential mitochondrial translation elongation factor G1 involved in mitochondrial protein synthesis. Due to the severe mitochondrial defect, mutant GFM1 fibroblasts cannot survive in galactose medium, making them an ideal screening model to test the effectiveness of pharmacological compounds. We found that the combination of polydatin and nicotinamide enabled the survival of mutant GFM1 fibroblasts in stress medium. We also demonstrated that polydatin and nicotinamide upregulated the mitochondrial Unfolded Protein Response (mtUPR), especially the SIRT3 pathway. Activation of mtUPR partially restored mitochondrial protein synthesis and expression, as well as improved cellular bioenergetics. Furthermore, we confirmed the positive effect of the treatment in GFM1 mutant induced neurons obtained by direct reprogramming from patient fibroblasts. Overall, we provide compelling evidence that mtUPR activation is a promising therapeutic strategy for GFM1 mutations.

Hepatic IRE1α-XBP1 signaling promotes GDF15-mediated anorexia and body weight loss in chemotherapy.

Platinum-based chemotherapy drugs can lead to the development of anorexia, a detrimental effect on the overall health of cancer patients. However, managing chemotherapy-induced anorexia and subsequent weight loss remains challenging due to limited effective therapeutic strategies. Growth differentiation factor 15 (GDF15) has recently gained significant attention in the context of chemotherapy-induced anorexia. Here, we report that hepatic GDF15 plays a crucial role in regulating body weight in response to chemo drugs cisplatin and doxorubicin. Cisplatin and doxorubicin treatments induce hepatic Gdf15 expression and elevate circulating GDF15 levels, leading to hunger suppression and subsequent weight loss. Mechanistically, selective activation by chemotherapy of hepatic IRE1α-XBP1 pathway of the unfolded protein response (UPR) upregulates Gdf15 expression. Genetic and pharmacological inactivation of IRE1α is sufficient to ameliorate chemotherapy-induced anorexia and body weight loss. These results identify hepatic IRE1α as a molecular driver of GDF15-mediated anorexia and suggest that blocking IRE1α RNase activity offers a therapeutic strategy to alleviate the adverse anorexia effects in chemotherapy.

Gelsenicine disrupted the intestinal barrier of Caenorhabditis elegans.

Gelsemium elegans Benth. (G. elegans) is a traditional medicinal herb that has anti-inflammatory, analgesic, sedative, and detumescence effects. However, it can also cause intestinal side effects such as abdominal pain and diarrhea. The toxicological mechanisms of gelsenicine are still unclear. The objective of this study was to assess enterotoxicity induced by gelsenicine in the nematodes Caenorhabditis elegans (C. elegans). The nematodes were treated with gelsenicine, and subsequently their growth, development, and locomotion behavior were evaluated. The targets of gelsenicine were predicted using PharmMapper. mRNA-seq was performed to verify the predicted targets. Intestinal permeability, ROS generation, and lipofuscin accumulation were measured. Additionally, the fluorescence intensities of GFP-labeled proteins involved in oxidative stress and unfolded protein response in endoplasmic reticulum (UPRER) were quantified. As a result, the treatment of gelsenicine resulted in the inhibition of nematode lifespan, as well as reductions in body length, width, and locomotion behavior. A total of 221 targets were predicted by PharmMapper, and 731 differentially expressed genes were screened out by mRNA-seq. GO and KEGG enrichment analysis revealed involvement in redox process and transmembrane transport. The permeability assay showed leakage of blue dye from the intestinal lumen into the body cavity. Abnormal mRNAs expression of gem-4, hmp-1, fil-2, and pho-1, which regulated intestinal development, absorption and catabolism, transmembrane transport, and apical junctions, was observed. Intestinal lipofuscin and ROS were increased, while sod-2 and isp-1 expressions were decreased. Multiple proteins in SKN-1/DAF-16 pathway were found to bind stably with gelsenicine in a predictive model. There was an up-regulation in the expression of SKN-1:GFP, while the nuclear translocation of DAF-16:GFP exhibited abnormality. The UPRER biomarker HSP-4:GFP was down-regulated. In conclusion, the treatment of gelsenicine resulted in the increase of nematode intestinal permeability. The toxicological mechanisms underlying this effect involved the disruption of intestinal barrier integrity, an imbalance between oxidative and antioxidant processes mediated by the SKN-1/DAF-16 pathway, and abnormal unfolded protein reaction.

The modulation of autophagy and unfolded protein response by ent-kaurenoid derivative CPUK02 in human colorectal cancer cells.

BACKGROUND: CPUK02 (15-Oxosteviol benzyl ester) is a semi-synthetic derivative of stevioside known for its anticancer effects. It has been reported that the natural compound of stevioside and its associated derivatives enhances the sensitivity of cancer cells to conventional anti-cancer agents by inducing endoplasmic reticulum (ER) stress. In response to ER stress, autophagy and unfolded protein responses (UPR) are activated to restore cellular homeostasis. Consequently, the primary aim of this study is to investigate the impact of CPUK02 treatment on UPR and autophagy markers in two colorectal cancer cell lines. METHODS: HCT116 and SW480 cell lines were treated with various concentrations of CPUK02 for 72 h. The expression levels of several proteins and enzymes were evaluated to investigate the influence of CPUK02 on autophagy and UPR pathways. These include glucose-regulated protein 78 (GRP78), Inositol-requiring enzyme 1-α (IRE1-α), spliced X-box binding protein 1 (XBP-1 s), protein kinase R-like ER kinase (PERK), C/EBP homologous protein (CHOP), Beclin-1, P62 and Microtubule-associated protein 1 light chain 3 alpha (LC3ßII). The evaluation was conducted using western blotting and quantitative real-time PCR techniques. RESULTS: The results obtained indicate that the treatment with CPUK02 reduced the expression of UPR markers, including GRP78 and IRE1-α at protein levels and XBP-1 s, PERK, and CHOP at mRNA levels in both HCT116 and SW480 cell lines. Furthermore, CPUK02 also influenced autophagy by decreasing Beclin-1 and increasing P62 and LC3ßII at mRNA levels in both HCT116 and SW480 treated cells. CONCLUSIONS: The study findings suggest CPUK02 may exert its cytotoxic effects by inhibiting UPR and autophagy flux in colorectal cancer cells.

Endoplasmic reticulum stress produced by Thapsigargin affects the occurrence of spike-wave discharge by modulating unfolded protein response pathways and activating immune responses in a dose-dependent manner.

The Endoplasmic Reticulum (ER) is associated with many cellular functions, from post-transcriptional modifications to the proper folding of proteins, and disruption of these functions causes ER stress. Although the relationship between epileptic seizures and ER stress has been reported, the contribution of ER stress pathways to epileptogenesis is still unclear. This study aimed to investigate the possible effects of ER stress-related molecular pathways modulated by mild- and high-dose Thapsigargin (Tg) on absence epileptic activity, CACNA1H and immune responses in WAG/Rij rats. For this purpose, rats were divided into four groups; mild-dose (20 ng) Tg, high-dose (200 ng) Tg, saline, and DMSO and drugs administered intracerebroventriculary. EEG activity was recorded for 1 h and 24 h after drug administration following the baseline recording. In cortex and thalamus tissues, GRP78, ERp57, GAD153 protein changes (Western Blot), Eif2ak3, XBP-1, ATF6, CACNA1H mRNA expressions (RT-PCR), NF-κB and TNF-α levels (ELISA) were measured. Mild-dose-Tg administration resulted in increased spike-wave discharge (SWD) activity at the 24th hour compared to administration of saline, and high-dose-Tg and it also significantly increased the amount of GRP78 protein, the expression of Eif2ak3, XBP-1, and CACNA1H mRNA in the thalamus tissue. In contrast, high-dose-Tg administration suppressed SWD activity and significantly increased XBP-1 and ATF6 mRNA expression in the thalamus, and increased NF-κB and TNF-α levels. In conclusion, our findings indicate that Tg affects SWD occurrence by modulating the unfolded protein response pathway and activating inflammatory processes in a dose-dependent manner.

Novel Spirocyclic Dimer, SpiD3, Targets Chronic Lymphocytic Leukemia Survival Pathways with Potent Preclinical Effects.

Chronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NFκB signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacologic agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib) have enhanced patient survival. However, resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations, and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective, imposing the need for novel therapeutics. We evaluated SpiD3, a novel spirocyclic dimer, in CLL cell lines, patient-derived CLL samples, ibrutinib-resistant CLL cells, and in the Eµ-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling, NFκB signaling, and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells, SpiD3 retained its antileukemic effects, mirrored in reduced activation of key proliferative pathways (e.g., PRAS, ERK, MYC). Translationally, we observed reduced tumor burden in SpiD3-treated Eµ-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NFκB signaling and the UPR, culminating in profound antitumor properties independent of TME stimuli. SIGNIFICANCE: SpiD3 demonstrates cytotoxicity in CLL partially through inhibition of NFκB signaling independent of tumor-supportive stimuli. By inducing the accumulation of unfolded proteins, SpiD3 activates the UPR and hinders protein synthesis in CLL cells. Overall, SpiD3 exploits critical CLL vulnerabilities (i.e., the NFκB pathway and UPR) highlighting its use in drug-resistant CLL.

Engineered Exosomes with ATF5-Modified mRNA Loaded in Injectable Thermogels Alleviate Osteoarthritis by Targeting the Mitochondrial Unfolded Protein Response.

Osteoarthritis (OA) progression is highly associated with chondrocyte mitochondrial dysfunction and disorders of catabolism and anabolism of the extracellular matrix (ECM) in the articular cartilage. The mitochondrial unfolded protein response (UPRmt), which is an integral component of the mitochondrial quality control (MQC) system, is essential for maintaining chondrocyte homeostasis. We successfully validated the pivotal role of activating transcription factor 5 (ATF5) in upregulating the UPRmt, mitigating IL-1ß-induced inflammation and mitochondrial dysfunction, and promoting balanced metabolism in articular cartilage ECM, proving its potential as a promising therapeutic target for OA. Modified mRNAs (modRNAs) have emerged as novel and efficient gene delivery vectors for nucleic acid therapeutic approaches. In this study, we combined Atf5-modRNA (modAtf5) with engineered exosomes derived from bone mesenchymal stem cells (ExmodAtf5) to exert cytoprotective effects on chondrocytes in articular cartilage via Atf5. However, the rapid localized metabolization of ExmodAtf5 limits its application. PLGA-PEG-PLGA (Gel), an injectable thermosensitive hydrogel, was used as a carrier of ExmodAtf5 (Gel@ExmodAtf5) to achieve a sustained release of ExmodAtf5. In vitro and in vivo, the use of Gel@ExmodAtf5 was shown to be a highly effective strategy for OA treatment. The in vivo therapeutic effect of Gel@ExmodAtf5 was evidenced by the preservation of the intact cartilage surface, low OARSI scores, fewer osteophytes, and mild subchondral bone sclerosis and cystic degeneration. Consequently, the combination of ExmodAtf5 and PLGA-PEG-PLGA could significantly enhance the therapeutic efficacy and prolong the exosome release. In addition, the mitochondrial protease ClpP enhanced chondrocyte autophagy by modulating the mTOR/Ulk1 pathway. As a result of our research, Gel@ExmodAtf5 can be considered to be effective at alleviating the progression of OA.

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.

Protein quality control systems in the endoplasmic reticulum and the cytosol coordinately prevent alachlor-induced proteotoxic stress in Saccharomyces cerevisiae.

Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in the eukaryotic model organism Saccharomyces cerevisiae. Therefore, this study aimed to uncover cellular mechanisms involved in preventing alachlor-induced proteotoxicity. The findings reveal that the ubiquitin-proteasome system (UPS) plays a crucial role in eliminating alachlor-denatured proteins by tagging them with polyubiquitin for subsequent proteasomal degradation. Exposure to alachlor rapidly induced an inhibition of proteasome activity by 90 % within 30 min. The molecular docking analysis suggests that this inhibition likely results from the binding of alachlor to ß subunits within the catalytic core of the proteasome. Notably, our data suggest that nascent proteins in the endoplasmic reticulum (ER) are the primary targets of alachlor. Consequently, the unfolded protein response (UPR), responsible for coping with aberrant proteins in the ER, becomes activated within 1 h of alachlor treatment, leading to the splicing of HAC1 mRNA into the active transcription activator Hac1p and the upregulation of UPR gene expression. These findings underscore the critical roles of the protein quality control systems UPS and UPR in mitigating alachlor-induced proteotoxicity by degrading alachlor-denatured proteins and enhancing the protein folding capacity of the ER.

Boosting wound healing in diabetic rats: The role of nicotinamide riboside and resveratrol in UPR modulation and pyroptosis inhibition.

BACKGROUND: Diabetes-related skin ulcers provide a substantial therapeutic issue, sometimes leading to amputation, needing immediate practical treatments for efficient wound care. While the exact mechanisms are unknown, pyroptosis and deregulation of the unfolded protein response (UPR) are known to exacerbate inflammation. Nicotinamide Riboside (NR) and Resveratrol (RV), which are known for their Nicotinamide adenine dinucleotide (NAD+) boosting and anti-inflammatory properties, are being studied as potential treatments. The purpose of this study was to shed light on the underlying molecular mechanisms and explore the medical application of NR and RV in diabetic wound healing. METHODS: 54 male Sprague-Dawley rats divided into control, diabetic (DM), Gel Base, DM-NR, DM-RV, and DM-NR + RV. Rats were orally administered 50 mg/kg/day of RV and 300 mg/kg/day of NR for 5 weeks. Following diabetes induction, their wounds were topically treated with 5 % NR and RV gel for 15 days. The wound closure rate, body weight, and serum lipid profiles were examined. Gene expression study evaluated UPR and pyroptosis-related genes (BIP, PERK, ATF6, IRE1α, sXBP1, CHOP, NLRP3, caspase-1, NFκB, and IL1-ß) in wound tissues, alongside histological assessment of cellular changes. RESULTS: NR and RV treatments greatly enhanced wound healing. Molecular investigation demonstrated UPR and pyroptosis marker modifications, suggesting UPR balance and anti-inflammatory effects. Histological investigation demonstrated decreased inflammation and increased re-epithelialization. The combination of NR and RV therapy had better results than either treatment alone. CONCLUSION: This study shows that NR and RV have therapeutic promise in treating diabetic wounds by addressing UPR dysregulation, and pyroptosis. The combination therapy is a viable strategy to improving the healing process, providing a multimodal intervention for diabetic skin ulcers. These findings pave the way for additional investigation and possible therapeutic applications, giving hope for better outcomes in diabetic wound care.

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.

Inhibition of HDAC6 with CAY10603 alleviates acute and chronic kidney injury by suppressing the ATF6 branch of UPR.

BACKGROUND: Histone deacetylase 6 (HDAC6) inhibitor CAY10603 has been identified as a potential therapeutic agent for the treatment of diabetic kidney disease (DKD). The objective of this study was to investigate the therapeutic effects of CAY10603 in mice with acute kidney injury (AKI) and chronic kidney diseases (CKD). METHODS: Renal immunohistology was performed to assess the expression levels of HDAC6 in both human and mouse kidney samples. C57BL/6J mice were intraperitoneal injected with lipopolysaccharide (LPS) to induce AKI; CD-1 mice were fed with adenine diet to induce adenine-nephropathy as CKD model. Serum creatinine, blood urea nitrogen and uric acid were measured to reflect renal function; renal histology was applied to assess kidney damage. Western blot and immunohistology were used to analyze the unfolded protein response (UPR) level. RESULTS: HDAC6 was significantly upregulated in renal tubular epithelial cells (RTECs) of both AKI and CKD patients as well as mice. In the murine models of AKI induced by LPS and adenine-induced nephropathy, CAY10603 exhibited notable protective effects, including improvement in biochemical indices and pathological changes. In vivo and in vitro studies revealed that CAY10603 effectively suppressed the activation of activating transcription factor 6 (ATF6) branch of UPR triggered by thapsigargin (Tg), a commonly employed endoplasmic reticulum (ER) stressor. Consistent with these findings, CAY10603 also displayed substantial inhibition of ATF6 activation in RTECs from both murine models of LPS-induced AKI and adenine-induced nephropathy. CONCLUSIONS: Collectively, these results suggest that CAY10603 holds promise as a potential therapeutic agent for both acute and chronic kidney injury.

Stachydrine hydrochloride protects the ischemic heart by ameliorating endoplasmic reticulum stress through a SERCA2a dependent way and maintaining intracellular Ca2+ homeostasis.

This study aimed to explore the effects and mechanism of action of stachydrine hydrochloride (Sta) against myocardial infarction (MI) through sarcoplasmic/endoplasmic reticulum stress-related injury. The targets of Sta against MI were screened using network pharmacology. C57BL/6 J mice after MI were treated with saline, Sta (6 or 12 mg kg-1) for 2 weeks, and adult mouse and neonatal rat cardiomyocytes (AMCMs and NRCMs) were incubated with Sta (10-4-10-6 M) under normoxia or hypoxia for 2 or 12 h, respectively. Echocardiography, Evans blue, and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used for morphological and functional analyses. Endoplasmic reticulum stress (ERS), unfolded protein reaction (UPR), apoptosis signals, cardiomyocyte contraction, and Ca2+ flux were detected using transmission electron microscopy (TEM), western blotting, immunofluorescence, and sarcomere and Fluo-4 tracing. The ingredient-disease-pathway-target network revealed targets of Sta against MI were related to apoptosis, Ca2+ homeostasis and ERS. Both dosages of Sta improved heart function, decreased infarction size, and potentially increased the survival rate. Sta directly alleviated ERS and UPR and elicited less apoptosis in the border myocardium and hypoxic NRCMs. Furthermore, Sta upregulated sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) in both ischaemic hearts and hypoxic NRCMs, accompanied by restored sarcomere shortening, resting intracellular Ca2+, and Ca2+ reuptake time constants (Tau) in Sta-treated hypoxic ARCMs. However, 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) (25 µM), a specific SERCA inhibitor, totally abolished the beneficial effect of Sta in hypoxic cardiomyocytes. Sta protects the heart from MI by upregulating SERCA2a to maintain intracellular Ca2+ homeostasis, thus alleviating ERS-induced apoptosis.

Salubrinal promotes phospho-eIF2α-dependent activation of UPR leading to autophagy-mediated attenuation of iron-induced insulin resistance.

Identification of new mechanisms mediating insulin sensitivity is important to allow validation of corresponding therapeutic targets. In this study, we first used a cellular model of skeletal muscle cell iron overload and found that endoplasmic reticulum (ER) stress and insulin resistance occurred after iron treatment. Insulin sensitivity was assessed using cells engineered to express an Akt biosensor, based on nuclear FoxO localization, as well as western blotting for insulin signaling proteins. Use of salubrinal to elevate eIF2α phosphorylation and promote the unfolded protein response (UPR) attenuated iron-induced insulin resistance. Salubrinal induced autophagy flux and its beneficial effects on insulin sensitivity were not observed in autophagy-deficient cells generated by overexpressing a dominant-negative ATG5 mutant or via knockout of ATG7. This indicated the beneficial effect of salubrinal-induced UPR activation was autophagy-dependent. We translated these observations to an animal model of systemic iron overload-induced skeletal muscle insulin resistance where administration of salubrinal as pretreatment promoted eIF2α phosphorylation, enhanced autophagic flux in skeletal muscle and improved insulin responsiveness. Together, our results show that salubrinal elicited an eIF2α-autophagy axis leading to improved skeletal muscle insulin sensitivity both in vitro and in mice.

KTN1 mediated unfolded protein response protects keratinocytes from ionizing radiation-induced DNA damage.

BACKGROUND: The unfolded protein response (UPR) is one of the cytoprotective mechanisms against various stresses and essential for the normal function of skin. Skin injury caused by ionizing radiation (IR) is a common side effect of radiotherapy and it is unclear how UPR affects IR-induced skin injury. OBJECTIVES: To verify the effect of UPR on IR-induced DNA damage in keratinocytes and the relation between an endoplasmic reticulum (ER) protein KTN1 and UPR. METHODS: All experiments were performed on keratinocytes models: HaCaT and HEK-A. ER lumen and the expression levels of KTN1 and UPR pathway proteins (PERK, IRE1α and ATF6) were examined by transmission electron microscopy and immunoblotting, respectively. 4-PBA, an UPR inhibitor, was used to detected its effects on DNA damage and cell proliferation. Subsequently, the effects of KTN1 deletion on UPR, DNA damage and cell proliferation after IR were detected. Tunicamycin was used to reactivate UPR and then we examined its effects on DNA damage. RESULTS: UPR was activated by IR in keratinocytes. Inhibition of UPR aggravated DNA damage and suppressed cell proliferation after IR. KTN1 expression was upregulated by IR and KTN1 depletion reduced ER expansion and the expression of UPR-related proteins. Moreover, KTN1 depletion aggravated DNA damage and suppressed cell proliferation after IR could reversed by reactivation of UPR. CONCLUSION: KTN1 deletion aggravates IR-induced keratinocyte DNA damage via inhibiting UPR. Our findings provide new insights into the mechanisms of keratinocytes in response to IR-induced damage.