Alleviate Endoplasmic Reticulum Stress Research Articles

Empagliflozin suppresses hedgehog pathway, alleviates ER stress, and ameliorates hepatic fibrosis in rats

Worldwide mortality from hepatic fibrosis remains high, due to hepatocellular carcinoma and end stage liver failure. The progressive nature of hepatic fibrosis from inflammation to cicatrized tissues warrants subtle intervention with pharmacological agents that hold potential. Empagliflozin (Empa), a novel hypoglycemic drug with antioxidant and anti-inflammatory properties, has lately been proposed to have additional antifibrotic activities. In the current study, we examined the antifibrotic effect of the Empa through modulating the activity of hepatic stellate cells by hedgehog (Hh) pathway. We also assessed the markers of inflammatory response and endoplasmic reticulum (ER) stress. Male Albino rats were treated with either CCl4 (0.4 mg/kg twice/week) and/or Empa (10 mg/kg/day) for eight weeks. In this study, CCl4 rats had active Hh signaling as indicated by overexpression of Patched 1, Smoothened and Glioblastoma-2. CCl4 induced ER stress as CHOP expression was upregulated and ERAD was downregulated. CCl4-induced inflammatory response was demonstrated through increased levels of TNF-α, IL-6 and mRNA levels of IL-17 while undetectable expression of IL-10. Conversely, Empa elicited immunosuppression, suppressed the expression of Hh markers, and reversed markers of ER stress. In conclusion, Empa suppressed CCl4-induced Hh signaling and proinflammatory response, meanwhile embraced ER stress in the hepatic tissues, altogether provided hepatoprotection.

LAMC2 mitigates ER stress by enhancing ER-mitochondria interaction via binding to MYH9 and MYH10.

Highly proliferative and metastatic tumors are constantly exposed to both intrinsic and extrinsic factors that induce adaptation to stressful conditions. Chronic adaptation to endoplasmic reticulum (ER) ER stress is common to many different types of cancers, and poses a major challenge for acquired drug resistance. Here we report that LAMC2, an extracellular matrix protein upregulated in many types of cancers, is localized in the ER of lung, breast, and liver cancer cells. Under tunicamycin-induced ER stress, protein level of LAMC2 is upregulated. Transfection of cancer cells with LAMC2 resulted in the attenuation of ER stress phenotype, accompanied by elevation in mitochondrial membrane potential as well as reduction in reactive oxygen species (ROS) levels and apoptosis. In addition, LAMC2 forms protein complexes with MYH9 and MYH10 to promote mitochondrial aggregation and increased ER-mitochondria interaction at the perinuclear region. Moreover, overexpression of LAMC2 counteracts the effects of ER stress and promotes tumor growth in vivo. Taken together, our results revealed that in complex with MYH9 and MYH10, LAMC2 is essential for promoting ER-mitochondria interaction to alleviate ER stress and allow cancer cells to adapt and proliferate under stressful conditions. This study provides new insights and highlights the promising potential of LAMC2 as a therapeutic target for cancer treatment.

Inhibition of TLR4/NF-κB pathway and endoplasmic reticulum stress by overexpressed S100A4 ameliorates retinal ischemia-reperfusion injury of mice.

Retinal ischemia exists in various ischemic retinopathies including glaucoma, contributing to the death of retinal neurons. Calcium binding protein S100A4 is important in tumors, and our previous study found that S100A4 protects retinal ganglion cells (RGCs) against retinal ischemia-reperfusion (I/R) injury. This study was aimed to further discuss the neuroprotection and mechanisms of S100A4 in retinal I/R of mice. The rAAV-EF1α-s100a4-EGFP-WPRE or rAAV-EF1α-EGFP-WPRE-Pa was injected intravitreally 4 weeks before I/R. S100A4, molecules in TLR4 signaling pathway and endoplasmic reticulum (ER) stress branches, inflammatory molecules, and surviving RGCs and cholinergic amacrine (ChAT) cells were determined by quantitative PCR, western blot, or immunofluorescent staining. The apoptosis and necrosis of retinal neurons induced by I/R were inhibited by overexpressed S100A4. RGCs, ChAT cells, and the retinal function were preserved by S100A4 overexpressing 7 days after I/R. Mechanistically, the beneficial effects of S100A4 may be mediated by inhibiting the activation of TLR4 signaling pathway and alleviating ER stress, leading to the attenuation of inflammatory response of the retina after I/R. Our findings indicated that S100A4 has neuroprotective effectagainst retinal I/R injury, and promoting S100A4 expression may be an effective strategy to inhibit retinal neurons from degeneration in ischemic retinopathy.

Dietary Betaine Attenuates High-Carbohydrate-Diet-Induced Oxidative Stress, Endoplasmic Reticulum Stress, and Apoptosis in Mandarin Fish (Siniperca chuatsi).

To investigate the impact of betaine on high-carbohydrate-diet-induced oxidative stress and endoplasmic reticulum (ER) stress, mandarin fish (Siniperca chuatsi) (23.73 ± 0.05 g) were fed with control (NC), betaine (BET), high carbohydrate (HC), and high carbohydrate + betaine (HC + BET) diets for 8 weeks. The results showed that betaine significantly promoted the growth of mandarin fish irrespective of the dietary carbohydrate levels. The HC diet induced oxidative stress, as evidenced by significantly elevated MDA levels. The HC diet significantly stimulated the mRNA levels of genes involved in ER stress (ire1, perk, atf6, xbp1, eif2α, atf4, chop), autophagy (ulk1, becn1, lc3b), and apoptosis (bax). However, betaine mitigated HC-diet-induced oxidative stress by modulating antioxidant enzymes and alleviated ER stress by regulating the mRNA of genes in the PERK-eIF2a-ATF4 pathway. Additionally, betaine significantly reduced the mRNA levels of becn1 and bax, along with the apoptosis rate, indicating a mitigating effect on autophagy and apoptosis. Overall, dietary betaine improved growth, attenuated HC-diet-induced oxidative stress and ER stress, and ultimately alleviated apoptosis in mandarin fish. These findings provide evidence for the use of betaine in aquafeeds to counter disruptive effects due to diets containing high carbohydrate levels.

Circ-Slain2 Alleviates Cartilage Degradation and Inflammation of TMJOA

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease with the cessation of matrix anabolism and aggravation of inflammation, which results in severe pain and impaired joint function. However, the mechanisms are not well understood. Circular RNAs (circRNAs) are reported to have various biological functions and participate in the development, diagnosis, prognosis, and treatment of different diseases. This study aimed to investigate the roles and mechanisms of circ-slain2 in TMJOA. We first established TMJOA mouse models and found circ-slain2 was lowly expressed in the cartilage of TMJOA through sequencing data. We observed that circ-slain2 is predominantly localized in the cytoplasm and downregulated in mouse condylar chondrocytes (mCCs) treated with tumor necrosis factor α (TNFα) and interferon γ (IFNγ). Micro–computed tomography and histological examination showed that intra-articular injection of circ-slain2 overexpressing adeno-associated virus could alleviate cartilage catabolism and synovial inflammation to relieve TMJOA in vivo. In addition, elevated circ-slain2 also showed anticatabolic and anti-inflammatory effects on IFNγ- and TNFα-stimulated mouse condylar chondrocytes (mCCs). Functional enrichment analysis indicated that protein processing in endoplasmic reticulum (ER) was associated with TMJOA, and further functional experiments confirmed that circ-slain2 could suppress ER stress in OA mCCs. RNA binding protein immunoprecipitation assay revealed an overt interaction between activating transcription factor 6 (ATF6) and circ-slain2. Inhibition of the expression of both ATF6 and circ-slain2 resulted in dilation of the ER and enhanced the expression of ER stress markers, whose ER stress level was higher than inhibition of ATF6 but lower than knockdown of circ-slain2 expression. Collectively, our research demonstrated that circ-slain2 could regulate ATF6 to relieve ER stress, reducing temporomandibular joint cartilage degradation and synovial inflammation. These findings provide prospects for developing novel osteoarthritis therapies based on circ-slain2 by focusing on reducing the inflammation of synovium and the imbalance between matrix synthesis and degradation.

CTRP4 attenuates apoptosis and epithelial-mesenchymal transition markers in podocytes through an AMPK/autophagy-dependent pathway

Hyperglycemia, characterized by high blood glucose levels resulting from pancreatic beta cell dysfunction or impaired insulin signaling, is a contributing factor in the development of diabetic nephropathy. This study aimed to investigate the effects of C1q/TNF-related protein 4 (CTRP4), known for its anti-obesity and anti-inflammatory properties in various disease models, on podocyte apoptosis and endoplasmic reticulum (ER) stress in the presence of elevated glucose levels. The expression levels of various proteins in podocytes and adipocytes were evaluated by Western blotting. Autophagosomes in podocytes were stained by MDC. Chromatin condensation in podocytes was examined by Hoechst staining. The research revealed increased expression of CTRP4 in 3T3-L1 adipocytes and CIHP-1 podocytes exposed to high glucose (HG) conditions. Treatment with CTRP4 effectively mitigated HG-induced apoptosis and ER stress and normalized epithelial-to-mesenchymal transition (EMT) markers in CIHP-1 cells. Furthermore, elevated levels of AMPK phosphorylation and autophagy were observed in CIHP-1 cells treated with CTRP4. Silencing of AMPK or the use of 3-methyl adenine (3 MA) reduced the impacts of CTRP4 on apoptosis, EMT markers and ER stress in CIHP-1 cells. In conclusion, these findings suggest that CTRP4 alleviates ER stress in podocytes under hyperglycemic conditions, leading to the suppression of apoptosis and the restoration of EMT through AMPK/autophagy-mediated signaling. These insights provide valuable information for the development of therapeutic strategies for diabetic nephropathy.

Ginsenoside F4 Alleviates Skeletal Muscle Insulin Resistance by Regulating PTP1B in Type II Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with increasing morbidity. Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of the insulin signaling cascade and has attracted intensive investigation in the T2DM study. Ginseng is widely used to treat metabolic diseases, while the effects of ginsenoside F4 (F4) on T2DM have remained unknown. Here, we identify F4 as an inhibitor of skeletal muscle insulin resistance. The results showed that F4 significantly improved the hyperglycemic state of db/db mice, alleviated dyslipidemia, and promoted skeletal muscle glucose uptake. This phenomenon was closely related to the inhibition of the PTP1B activity. On the one hand, the inhibition of PTP1B activity by F4 resulted in increased insulin receptor (INSR) and insulin receptor substrate 1 tyrosine phosphorylation and enhanced insulin sensitivity. On the other hand, F4 as a PTP1B inhibitor inhibited the inositol-requiring enzyme 1 (IRE-1)/recombinant TNF receptor associated factor 2 (TRAF2)/c-Jun N-terminal kinase signaling pathway and alleviated skeletal muscle endoplasmic reticulum (ER) stress, thereby reducing IRS-1 serine phosphorylation. Both finally activated the PI3K/AKT signaling pathway and promoted glucose transporter protein 4 translocation to the cell membrane for glucose uptake. Taken together, our experiments demonstrate that F4 activates the insulin signaling pathway by inhibiting the activity of PTP1B while inhibiting the IRE-1/TRAF2/JNK signaling pathway, enhancing insulin sensitivity, and alleviating ER stress in the skeletal muscle of db/db mice. Our results indicate that F4 can be used as a PTP1B inhibitor for the treatment of T2DM.

Ufmylation on UFBP1 alleviates non-alcoholic fatty liver disease by modulating hepatic endoplasmic reticulum stress

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease characterized by lipid accumulation and endoplasmic reticulum (ER) stress, while effective therapies targeting the specific characteristics of NAFLD are limited. Ufmylation is a newly found post-translational modification process that involves the attachment of the Ubiquitin-fold modifier 1 (UFM1) protein to its substrates via ufmylation modification system. Ufmylation regulates ER stress via modifying UFM1 binding protein 1 (UFBP1), suggesting a potential role for ufmylation in NAFLD pathogenesis. However, the precise role of ufmylation in NAFLD remains unclear. Herein, we aim to elucidate the impact of ufmylation on UFBP1 in NAFLD and explore the underlying mechanisms involved. We observed increased expression of UFM1-conjugated proteins and ufmylation modification system components in livers with steatosis derived from NAFLD patients and NAFLD models. Upregulation of ufmylation on hepatic proteins appeared to be an adaptive response to hepatic ER stress in NAFLD. In vitro, knocking down UFBP1 resulted in increased lipid accumulation and lipogenesis in hepatocytes treated with free fatty acids (FFA), which could be rescued by wild-type UFBP1 (WT UFBP1) but not by a mutant form of UFBP1 lacking the main ufmylation site lys267 (UFBP1 K267R). In vivo, ufmylation on UFBP1 ameliorated obesity, hepatic steatosis, hepatic lipogenesis, dyslipidemia, insulin resistance and liver damage in mice with NAFLD induced by a high fat diet (HFD). We also demonstrated that the downregulation of UFBP1 induced ER stress, whereas the reintroduction or overexpression of UFBP1 alleviated ER stress in a manner dependent on ufmylation in NAFLD. This mechanism could be responsible for the amelioration of aberrant hepatic lipogenesis and insulin resistance in NAFLD. Our data reveal a protective role of ufmylation on UFBP1 against NAFLD and offer a specific target for NAFLD treatment.

Endoplasmic reticulum stress and alterations of peroxiredoxins in aged hearts

Aging-related cardiovascular disease is influenced by multiple factors, with oxidative stress being a key contributor. Aging-induced endoplasmic reticulum (ER) stress exacerbates oxidative stress by impairing mitochondrial function. Furthermore, a decline in antioxidants, including peroxiredoxins (PRDXs), augments the oxidative stress during aging. To explore if ER stress leads to PRDX degradation during aging, young adult (3 mo.) and aged (24 mo.) male mice were studied. Treatment with 4-phenylbutyrate (4-PBA) was used to alleviate ER stress in young adult and aged mice. Aged hearts showed elevated oxidative stress levels compared to young hearts. However, treatment with 4-PBA to attenuate ER stress reduced oxidative stress in aged hearts, indicating that ER stress contributes to increased oxidative stress in aging. Moreover, aging resulted in reduced levels of peroxiredoxin 3 (PRDX3) in mitochondria and peroxiredoxin 4 (PRDX4) in myocardium. While 4-PBA treatment improved PRDX3 content in aged hearts, it did not restore PRDX4 content in aged mice. These findings suggest that ER stress not only leads to mitochondrial dysfunction and increased oxidant stress but also impairs a vital antioxidant defense through decreased PRDX3 content. Additionally, the results suggest that PRDX4 may contribute an upstream role in inducing ER stress during aging.

Uranium affects nitrogen metabolism and endoplasmic reticulum protein homeostasis in plants

The toxicity of uranium (U) to plants makes the phytoremediation effect of U contaminated soil unsatisfactory, and the understanding of the mechanism of U toxicity to plants is limited. In the present study, the effects of U on nitrogen (N) metabolism and endoplasmic reticulum (ER) protein homeostasis in radish (Raphanus sativus) and broad bean (Vicia faba) were analyzed. The results showed that the content of amine N was significantly increased in R. sativus root after U treatment, but significantly decreased in both root and leaf of V. faba, indicating that U had an adverse effect on the N nutrition in V. faba. After U treatment, the nitrate reductase (NR) activity in R. sativus roots and leaves was increased to 2.9 and 4.8 times of that in control group, but decreased by 26.9% and 74.3% in V. faba, respectively. The transcriptome sequencing results showed that 3 NR genes were up-regulated in R. sativus root, while 1 was down-regulated in V. faba after U treatment. In addition, U interfered with ER protein homeostasis and induced ER stress. The bZIP17/28 and bZIP60-mediated unfolded protein response (UPR) pathways were activated in R. sativus root. This promoted downstream ER associated degradation (ERAD) pathway, facilitating the degradation of unfolded (or misfolded) proteins, thereby alleviating ER stress. While in V. faba, U triggered severe programmed cell death through the induction of ER stress, which was significantly alleviated by exogenous addition of the chemical chaperone 4-phenylbutyric acid. Overall, more efficient N assimilation activity and ER protein homeostasis maintenance mechanism (especially ERAD) may be one of the key mechanisms for R. sativus tolerance to U stress. This study for the first time revealed the toxicological mechanism of U in plants in terms of N metabolism and ER protein homeostasis.

Hypothermic oxygenated perfusion attenuates DCD liver ischemia–reperfusion injury by activating the JAK2/STAT3/HAX1 pathway to regulate endoplasmic reticulum stress

BackgroundHepatic ischemia–reperfusion injury (IRI) in donation after cardiac death (DCD) donors is a major determinant of transplantation success. Endoplasmic reticulum (ER) stress plays a key role in hepatic IRI, with potential involvement of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway and the antiapoptotic protein hematopoietic-lineage substrate-1-associated protein X-1 (HAX1). In this study, we aimed to investigate the effects of hypothermic oxygenated perfusion (HOPE), an organ preservation modality, on ER stress and apoptosis during hepatic IRI in a DCD rat model.MethodsTo investigate whether HOPE could improve IRI in DCD livers, levels of different related proteins were examined by western blotting and quantitative real-time polymerase chain reaction. Further expression analyses, immunohistochemical analyses, immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and transmission electron microscopy were conducted to analyze the effects of HOPE on ER stress and apoptosis. To clarify the role of the JAK2/STAT3 pathway and HAX1 in this process, AG490 inhibitor, JAX1 plasmid transfection, co-immunoprecipitation (CO-IP), and flow cytometry analyses were conducted.ResultsHOPE reduced liver injury and inflammation while alleviating ER stress and apoptosis in the DCD rat model. Mechanistically, HOPE inhibited unfolded protein responses by activating the JAK2/STAT3 pathway, thus reducing ER stress and apoptosis. Moreover, the activated JAK2/STAT3 pathway upregulated HAX1, promoting the interaction between HAX1 and SERCA2b to maintain ER calcium homeostasis. Upregulated HAX1 also modulated ER stress and apoptosis by inhibiting the inositol-requiring enzyme 1 (IRE1) pathway.ConclusionsJAK2/STAT3-mediated upregulation of HAX1 during HOPE alleviates hepatic ER stress and apoptosis, indicating the JAK2/STAT3/HAX1 pathway as a potential target for IRI management during DCD liver transplantation.Graphical

Protective Effect of Water-Soluble Acacetin Prodrug on APAP-Induced Acute Liver Injury Is Associated with Upregulation of PPARγ and Alleviation of ER Stress.

A water-soluble acacetin prodrug has been synthesized and reported by our group previously. Acetaminophen (APAP) overdose is a leading cause of acute liver injury. We found that subcutaneous injection of acacetin prodrug (5, 10, 20 mg/kg) decreased serum ALT, AST, and ALP, corrected the abnormal MDA and GSH in liver, and improved intrahepatic hemorrhage and destruction of liver structures in APAP (300 mg/kg)-treated mice. Molecular mechanism analysis revealed that the expressions of endoplasmic reticulum (ER) stress markers ATF6, CHOP, and p-PERK, apoptosis-related protein BAX, and cleaved caspase 3 were decreased by acacetin in a dose-dependent manner in vivo and in vitro. Moreover, via the acacetin-upregulated peroxisome-proliferator-activated receptor gamma (PPARγ) of HepG2 cells and liver, the suppressive effect of acacetin on ER stress and apoptosis was abolished by PPARγ inhibitor (GW9662) or PPARγ-siRNA. Molecular docking revealed that acacetin can bind to three active pockets of PPARγ, mainly by hydrogen bond. Our results provide novel evidence that acacetin prodrug exhibits significant protective effect against APAP-induced liver injury by targeting PPARγ, thereby suppressing ER stress and hepatocyte apoptosis. Acacetin prodrug is likely a promising new drug candidate for treating patients with acute liver injury induced by APAP.

HMMR alleviates endoplasmic reticulum stress by promoting autophagolysosomal activity during endoplasmic reticulum stress-driven hepatocellular carcinoma progression.

The mechanism of hepatitis B virus (HBV)-induced carcinogenesis remains an area of interest. The accumulation of hepatitis B surface antigen in the endoplasmic reticulum (ER) of hepatocytes stimulates persistent ER stress. Activity of the unfolded protein response (UPR) pathway of ER stress may play an important role in inflammatory cancer transformation. How the protective UPR pathway is hijacked by cells as a tool for malignant transformation in HBV-related hepatocellular carcinoma (HCC) is still unclear. Here, we aimed to define the key molecule hyaluronan-mediated motility receptor (HMMR) in this process and explore its role under ER stress in HCC development. An HBV-transgenic mouse model was used to characterize the pathological changes during the tumor progression. Proteomics and transcriptomics analyses were performed to identify the potential key molecule, screen the E3 ligase, and define the activation pathway. Quantitative real-time PCR and Western blotting were conducted to detect the expression of genes in tissues and cell lines. Luciferase reporter assay, chromatin immunoprecipitation, coimmunoprecipitation, immunoprecipitation, and immunofluorescence were employed to investigate the molecular mechanisms of HMMR under ER stress. Immunohistochemistry was used to clarify the expression patterns of HMMR and related molecules in human tissues. We found sustained activation of ER stress in the HBV-transgenic mouse model of hepatitis-fibrosis-HCC. HMMR was transcribed by c/EBP homologous protein (CHOP) and degraded by tripartite motif containing 29 (TRIM29) after ubiquitination under ER stress, which caused the inconsistent expression of mRNA and protein. Dynamic expression of TRIM29 in the HCC progression regulated the dynamic expression of HMMR. HMMR could alleviate ER stress by increasing autophagic lysosome activity. The negative correlation between HMMR and ER stress, positive correlation between HMMR and autophagy, and negative correlation between ER stress and autophagy were verified in human tissues. This study identified the complicated role of HMMR in autophagy and ER stress, that HMMR controls the intensity of ER stress by regulating autophagy in HCC progression, which could be a novel explanation for HBV-related carcinogenesis.

The plasma membrane-associated transcription factor NAC091 regulates unfolded protein response in Arabidopsis thaliana

Adverse environmental stresses may cause the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), and the unfolded protein response (UPR) pathway is initiated to mitigate the ER stress. Previous studies demonstrate that NAC062, a plasma membrane-associated transcription factor, plays important roles in promoting cell survival under ER stress conditions in Arabidopsis thaliana. In this study, we identified another plasma membrane-associated transcription factor, NAC091 (also known as ANAC091/TIP), as an important UPR mediator. ER stress induces the expression of NAC091, which is mainly dependent on the ER stress regulators bZIP60 and bZIP28. In addition, NAC091 has transcriptional activation activity, and the truncated form of NAC091 devoid of the C-terminal transmembrane domain (TMD) forms a homodimer in the nucleus. Under ER stress conditions, NAC091 relocates from the plasma membrane to the nucleus and regulates the expression of canonical UPR genes involved in cell survival. Further, the loss-of-function mutant of NAC091 confers impaired ER stress tolerance. Together, these results reveal the important role of NAC091 in ER stress response in Arabidopsis, and demonstrate that NAC091 relays the ER stress signal from the plasma membrane to the nucleus to alleviate ER stress and promote cell survival in plants.

Shenlian extract protected ox-LDL-loaded macrophages against ER stress by promoting LAL-LXRα mediated cholesterol flux

Ethnopharmacological relevanceShenlian (SL) extract is consisted of extracts from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm.f.) Nees, two herbs commonly used in Chinese clinical formula to treat atherosclerosis by removing blood stasis and clearing away heat. Pharmacologically, the anti-atherosclerotic effects of these two herbs are related to unresolved inflammation and the macrophage anergy or apoptosis in lesions led by the lipid flux blockage and ER stress. However, the deeper understanding of SL extract in protecting macrophage in plaques remains unknown. Aim of the studyThis study aimed to investigate the underlying mechanism of SL extract in protecting ER-stressed macrophages from apoptosis in atherosclerosis. MethodsThe ApoE−/− atherosclerotic mice model and ox-LDL loaded macrophages model were established to assess the effect of SL extract on ER stress in vivo and in vitro. Key markers related to ER stress in plaque were determined by immunohistochemical staining. Proteins involved in apoptosis and ER stress in macrophages loaded by ox-LDL were assessed by Western blot. ER morphology was observed by electron microscope. Lipid flux was temporally and quantitatively depicted by Oil red staining. The LAL and LXRα were blocked by lalistat and Gsk 2033 respectively to investigate whether SL extract protected the function of macrophages by the activation of LAL-LXRα axis. ResultsOur study reported that, in ApoE−/− atherosclerotic mice, SL extract effectively relieved ER stress of carotid artery plaque. In lipid-overloaded macrophage models, SL extract significantly alleviated ER stress by promoting cholesterol degradation and efflux, which finally prevented apoptosis of foam cells induced by ox-LDL. Blockage of ER stress by 4-Phenylbutyric acid (4-PBA), an inhibitor of Endoplasmic Reticulum (ER) stress, largely attenuated the protective effects of SL extract on macrophage. By utilizing the selective antagonists against both LAL and LXRα, this study further revealed that the beneficial effects of SL extract in macrophages was dependent on the proper functionalization of LAL-LXRα axis. ConclusionsBy highlighting the therapeutic significance of macrophage protection in resolving atherosclerosis inflammation, our study pharmacologically provided convincing mechanistic evidence of SL extract in the activation LAL-LXRα axis and revealed its promising potential in the promotion of cholesterol turnover and prevention of ER stress induced apoptosis in lipid-loaded macrophages.

β-carotene targets IP3R/GRP75/VDAC1-MCU axis to renovate LPS-induced mitochondrial oxidative damage by regulating STIM1

Endoplasmic reticulum (ER) and mitochondria are the main sites for the storage and regulation of Ca2+ homeostasis. An imbalance of Ca2+ homeostasis can cause ER stress and mitochondrial dysfunction, thereby inducing apoptosis. The store-operated calcium entry (SOCE) is the main channel for extracellular calcium influx. Mitochondria-associated endoplasmic reticulum (MAM) is an important agent for Ca2+ transfer from the ER to the mitochondria. Therefore, regulation of SOCE and MAMs has potential therapeutic value for disease prevention and treatment. In this study, bovine mammary epithelial cells (BMECs) and mice were used as models to explore the mechanisms of β-carotene to relieve ER stress and mitochondrial dysfunction. BAPTA-AM, EGTA (Ca2+ inhibitor), and BTP2 (SOCE channel inhibitor) alleviated ER stress and mitochondrial oxidative damage induced by increased intracellular Ca2+ levels after lipopolysaccharide (LPS) stimulation. Furthermore, inhibition of ER stress by 4-PBA (ER stress inhibitor), 2-APB (IP3R inhibitor), and ruthenium red (mitochondrial calcium uniporter (MCU) inhibitor) restored mitochondrial function by reducing mitochondrial ROS. Our data also confirm that β-carotene targeted STIM1 and IP3R channels to repair LPS-induced ER stress and mitochondrial disorders. Consistent with the in vitro study, in vito experiments in mice further showed that β-carotene attenuated LPS-induced ER stress and mitochondrial oxidative damage by inhibiting the expression of STIM1 and ORAI1, and reducing the level of Ca2+ in mouse mammary glands. Therefore, ER stress-mitochondrial oxidative damage mediated by the STIM1-ER-IP3R/GRP75/VDAC1-MCU axis plays an vital role in the development of mastitis. Our results provided novel ideas and therapeutic targets for the prevention and treatment of mastitis.

Ursodeoxycholic Acid Binds PERK and Ameliorates Neurite Atrophy in a Cellular Model of GM2 Gangliosidosis.

The Unfolded protein response (UPR), triggered by stress in the endoplasmic reticulum (ER), is a key driver of neurodegenerative diseases. GM2 gangliosidosis, which includes Tay-Sachs and Sandhoff disease, is caused by an accumulation of GM2, mainly in the brain, that leads to progressive neurodegeneration. Previously, we demonstrated in a cellular model of GM2 gangliosidosis that PERK, a UPR sensor, contributes to neuronal death. There is currently no approved treatment for these disorders. Chemical chaperones, such as ursodeoxycholic acid (UDCA), have been found to alleviate ER stress in cell and animal models. UDCA's ability to move across the blood-brain barrier makes it interesting as a therapeutic tool. Here, we found that UDCA significantly diminished the neurite atrophy induced by GM2 accumulation in primary neuron cultures. It also decreased the up-regulation of pro-apoptotic CHOP, a downstream PERK-signaling component. To explore its potential mechanisms of action, in vitro kinase assays and crosslinking experiments were performed with different variants of recombinant protein PERK, either in solution or in reconstituted liposomes. The results suggest a direct interaction between UDCA and the cytosolic domain of PERK, which promotes kinase phosphorylation and dimerization.

Abstract 1330: Golgi disorganization and ER stress: the mechanism underlying alcohol-mediated prostate cancer progression

Abstract The link between prostate cancer (PCa) risk and alcohol consumption has long been debated. In our recent analysis of the epidemiologic evidence for this link, we found that, since the onset of the PSA testing era, a preponderance of studies indicates ethanol (EtOH) consumption is strongly associated with PCa risk. Despite epidemiologic support for this relationship, little is known about the underlying mechanisms. Our group introduced the concept of “onco-Golgi,” where the Golgi becomes fragmented alongside activating transcription factor 6 (ATF6)-mediated Endoplasmic Reticulum (ER) stress. This results in increased plasma membrane (PM) expression of abnormally glycosylated αv Integrins by Golgi glycosyltransferase, N-acetylglucosaminyltransferase-V (MGAT5). Importantly, these MGAT5-modified integrins bind to pentameric Galectin-3, resulting in clustering and increased retention on the PM. This, in turn, modulates tumor cell behavior, including adhesion to extracellular matrix and migration, leading to acceleration of prostate tumor dissemination to lymph nodes and distant organs, including bones. We have also found the EtOH treatment causes further Golgi disorganization. PCa tissues from heavy alcoholic patients demonstrate higher MGAT5 expression and elevated Integrin αv levels on the PM. We propose that EtOH promotes PCa lethality by increasing Integrin αv-mediated PCa progression. Altered glycosylation of Integrin αv in the onco-Golgi is expected to be exacerbated by alcohol’s disorganizing effect on Golgi. We have found a positive correlation between the number of Golgi fragments and the intensity of Integrin αv on the PM in both docetaxel-resistant PC-3 and DU145 cells after EtOH treatment. Importantly, we revealed that Golgi disorganization in advanced PCa cells is an autophagy-driven process. We found that autophagy inhibitor, Hydroxychloroquine (HCQ), restores compact Golgi in both PC-3 and DU145 cells, as well as in mice orthotopic PC-3-derived tumors. The effect of HCQ was strengthened by depletion of ATF6, suggesting the synergetic effect of autophagy inhibition and alleviation of ER stress. Significantly, HCQ treatment attenuates the EtOH-induced Golgi fragmentation and restores the level of PM Integrin αv to that of control cells. We expect that our established protocol to restore Golgi morphology and inhibit ER stress by HCQ treatment and ATF6 depletion, respectively, will attenuate the impact of EtOH on PCa cells. Overall, these data shed light on alcohol-promoting effects on prostate tumor growth and metastasis and provide a potentially effective therapeutic strategy. Citation Format: Amanda Macke, Artem Pachikov, Taylor Divita, Armen Petrosyan. Golgi disorganization and ER stress: the mechanism underlying alcohol-mediated prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1330.

Rosmarinic acid alleviates ultraviolet-mediated skin aging via attenuation of mitochondrial and ER stress responses.

Chronic exposure to Ultraviolet B radiation (UV-B) evokes a myriad of toxic signalling events in the irradiated skin. One of such response is ER stress, which is known to exacerbate photodamage responses. Also, recent literature has highlighted the adverse impact of environmental toxicants on mitochondrial dynamics and mitophagy. Impaired mitochondrial dynamics escalates oxidative damage and causes apoptosis. There have been evidences that support crosstalk between ER stress and mitochondrial dysfunction. However, mechanistic clarification is still needed to verify the interactions between UPR responses and mitochondrial dynamics impairment in UV-B-induced photodamage models. Lastly, plant-based natural agents have garnered attention as therapeutic agents against skin photodamage. Thus, gaining mechanistic insights of plant-based natural agents is required for their application and feasibility in clinical settings. With this aim in view, this study was performed in primary human dermal fibroblasts (HDFs) and Balb/C mice. Different parameters regarding mitochondrial dynamics, ER stress, intracellular damage and histological damage were analyzed using western blot, rt-PCR and microscopy. We demonstrated that UV-B exposure leads to induction of UPR responses, upregulation of Drp-1 and inhibition of mitophagy. Further, 4-PBA treatment leads to reversal of these noxious stimuli in irradiated HDF cells, thereby, indicating an upstream role of UPR induction in mitophagy inhibition. Also, we explored the therapeutic effect of Rosmarinic acid (RA) against ER stress and impaired mitophagy in photodamage models. RA prevents intracellular damage via alleviation of ER stress and mitophagic responses in HDFs and irradiated Balb/C mice skin. The current study summarizes the mechanistic insights into UVB-mediated intracellular damage and role of natural plant-based agent (RA) in ameliorating these toxic responses.

Hydroxysafflor Yellow A Exerts Neuroprotective Effect by Reducing Aβ Toxicity Through Inhibiting Endoplasmic Reticulum Stress in Oxygen-Glucose Deprivation/Reperfusion Cell Model.

Ischemia stroke is thought to be one of the vascular risks associated with neurodegenerative diseases, such as Alzheimer's disease (AD). Hydroxysafflor yellow A (HSYA) has been reported to protect against stroke and AD, while the underlying mechanism remains unclear. In this study, SH-SY5Y cell model treated with oxygen-glucose deprivation/reperfusion (OGD/R) was used to explore the potential mechanism of HSYA. Results from cell counting kit-8 (CCK-8) showed that 10 μM HSYA restored the cell viability after OGD 2 hours/R 24 hours. HSYA reduced the levels of malondialdehyde and reactive oxygen species, while improved the levels of superoxide dismutase and glutathione peroxidase. Furthermore, apoptosis was inhibited, and the expression of brain-derived neurotrophic factor was improved after HSYA treatment. In addition, the expression levels of amyloid-β peptides (Aβ) and BACE1 were decreased by HSYA, as well as the expression levels of binding immunoglobulin heavy chain protein, PKR-like endoplasmic reticulum (ER) kinase pathway, and activating transcription factor 6 pathway, whereas the expression level of protein disulfide isomerase was increased. Based on these results, HSYA might reduce Aβ toxicity after OGD/R by interfering with apoptosis, oxidation, and neurotrophic factors, as well as relieving ER stress.