Glucose-regulated Protein Research Articles

Linum corymbulosum Protects Rats against CCl4-Induced Hepatic Injuries through Modulation of an Unfolded Protein Response Pathway and Pro-Inflammatory Intermediates.

Liver fibrosis is a major pathological feature of chronic liver disease and effective therapies are limited at present. The present study focuses on the hepatoprotective potential of L. corymbulosum against carbon tetrachloride (CCl4)-induced liver damage in rats. Analysis of Linum corymbulosum methanol extract (LCM) using high-performance liquid chromatography (HPLC) revealed the presence of rutin, apigenin, catechin, caffeic acid and myricetin. CCl4 administration lowered (p < 0.01) the activities of antioxidant enzymes and reduced glutathione (GSH) content as well as soluble proteins, whereas the concentration of H2O2, nitrite and thiobarbituric acid reactive substances was higher in hepatic samples. In serum, the level of hepatic markers and total bilirubin was elevated followed by CCl4 administration. The expression of glucose-regulated protein (GRP78), x-box binding protein-1 total (XBP-1 t), x-box binding protein-1 spliced (XBP-1 s), x-box binding protein-1 unspliced (XBP-1 u) and glutamate-cysteine ligase catalytic subunit (GCLC) was enhanced in CCl4-administered rats. Similarly, the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemo attractant protein-1 (MCP-1) was strongly increased with CCl4 administration to rats. Co-administration of LCM along with CCl4 to rats lowered (p < 0.05) the expression of the above genes. Histopathology of the liver showed hepatocyte injury, leukocyte infiltration and damaged central lobules in CCl4-treated rats. However, LCM administration to CCl4-intoxicated rats restored the altered parameters towards the levels of control rats. These outcomes indicate the existence of antioxidant and anti-inflammatory constituents in the methanol extract of L. corymbulosum.

Astaxanthin treatment ameliorates ER stress in polycystic ovary syndrome patients: a randomized clinical trial

Astaxanthin (ASX), as a natural carotenoid compound, exists in various types of seafood and microorganisms. It has several possible beneficial therapeutic effects for patients with polycystic ovary syndrome (PCOS). Patients with PCOS also suffer from endoplasmic reticulum (ER) stress. In the present work, it was hypothesized that ER stress could be improved by ASX in PCOS patients. Granulosa cells (GCs) were obtained from 58 PCOS patients. The patients were classified into ASX treatment (receiving 12 mg/day for 60 days) and placebo groups. The expression levels of ER stress pathway genes and proteins were explored using Western blotting and quantitative polymerase chain reaction. To assess oxidative stress markers, follicular fluid (FF) was gained from all patients. The Student’s t test was used to perform statistical analysis. After the intervention, ASX led to a considerable reduction in the expression levels of 78-kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and X-box-binding protein 1 compared to the placebo group, though the reduction in the messenger RNA (mRNA) expression level of activating transcription factor 6 was not statistically significant. However, ASX significantly increased the ATF4 expression level. GRP78 and CHOP protein levels represented a considerable decrease in the treatment group after the intervention. In addition, a statistically significant increase was found in the FF level of total antioxidant capacity in the treatment group. Based on clinical outcomes, no significant differences were found between the groups in terms of the oocyte number, fertilization rate, and fertility rate, but the ASX group had higher rates of high-quality oocytes, high-quality embryo, and oocyte maturity compared to the placebo group. Our findings demonstrated that ER stress in the GCs of PCOS patients could be modulated by ASX by changing the expression of genes and proteins included in the unfolding protein response.Trial registration This study was retrospectively registered on the Iranian Registry of Clinical Trials website (www.irct.ir; IRCT-ID: IRCT20201029049183N, 2020-11-27).

ATF6β Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6β, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6β deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6β deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6β could play a role in psychiatric conditions in the emotional centers of the brain.

Protective effect of Buddha's Temple extract against tert-butyl hydroperoxide stimulation-induced oxidative stress in DF-1 cells.

This study aimed to determine the protective efficacy of Buddha's Temple (BT) extract against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in Gallus gallus chicken embryo fibroblast cell line (DF-1) and its effects on the cell lipid metabolism. In this experimental study, Gallus gallus DF-1 fibroblast cells were pretreated with BT 10-7 for 24 hours, followed by their six-hour exposure to t-BHP (100 μM). Water-soluble tetrazolium salt-8 (WST-8) assays were performed, and the growth curve was computed. The intracellular gene expression changes caused by BT extract were confirmed through quantitative polymerase chain reaction (qPCR). Flow cytometry, oil red O staining experiment, and thin-layer chromatography were performed for the detection of intracellular metabolic mechanism changes. The WST-8 assay results showed that the BT pretreatment of Gallus gallus DF-1 fibroblast cell increased their cell survival rate by 1.08%±0.04%, decreased the reactive oxygen species (ROS) level by 0.93%±0.12% even after exposure to oxidants, and stabilized mitochondrial activity by 1.37%±0.36%. In addition, qPCR results confirmed that the gene expression levels of tumor necrosis factor α (TNFα), TIR domain-containing adapter inducing IFN-beta (TICAM1), and glucose-regulated protein 78 (GRP78) were regulated, which contributed to cell stabilization. Thin-layer chromatography and oil red O analyses showed a clear decrease in the contents of lipid metabolites such as triacylglycerol and free fatty acids. In this study, we confirmed that the examined BT extract exerted selective protective effects on Gallus gallus DF-1 fibroblast cells against cell damage caused by t-BHP, which is a strong oxidative inducer. Furthermore, we established that this extract significantly reduced the intracellular ROS accumulation due to oxidative stress, which contributes to an increase in poultry production and higher incomes.

Immunoexpression Pattern of Autophagy Markers in Developing and Postnatal Kidneys of Dab1−/−(yotari) Mice

The purpose of this study was to compare the immunofluorescence patterns of autophagic markers: Light chain 3 beta (LC3B), Glucose regulating protein 78 (GRP78), Heat shock cognate 71 (HSC70) and Lysosomal-associated membrane protein 2A (LAMP2A) in the developing and postnatal kidneys of Dab1-/- (yotari) mice to those of wild-type samples. Embryos were obtained on gestation days 13.5 and 15.5 (E13.5 and E15.5), and adult animals were sacrificed at postnatal days 4, 11 and 14 (P4, P11, and P14). After fixation and dehydration, paraffin-embedded kidney tissues were sectioned and incubated with specific antibodies. Using an immunofluorescence microscope, sections were analyzed. For statistical analysis, a two-way ANOVA test and a Tukey's multiple comparison test were performed with a probability level of p < 0.05. A significant increase in GRP78 and LAMP2A expression was observed in the renal vesicles and convoluted tubules of yotari in embryonic stages. In postnatal kidneys, all observed proteins showed higher signal intensities in proximal and distal convoluted tubules of yotari, while a higher percentage of LC3B-positive cells was also observed in glomeruli. Our findings suggest that all of the examined autophagic markers play an important role in normal kidney development, as well as the potential importance of these proteins in renal pathology, where they primarily serve a protective function and thus may be used as diagnostic and therapeutic targets.

Expression of heat shock proteins HSPA1A, HSPA1B and TP53 in vulval lichen planus and vulval lichen sclerosus.

Heat shock proteins (HSPs) are proteins involved in protein folding and maturation. HSP expression is induced by heat shock or other stressors including cellular damage and hypoxia. The major groups, which are classified based on their molecular weight, include HSP27, HSP40, HSP60, HSP70, HSP90, and large HSP (HSP110 and glucose-regulated protein 170). The comparison of heat shock proteins and TP53 expression is yet not well studied in both vulval lichen sclerosus and lichen planus. Our aim was to assess the HSP and TP53 gene expression in women suffering from LS or LP and compare it within these groups and also healthy controls. The inclusion criteria were willingness to donate vulval biopsies, not currently or in the prior two weeks received any local nor systemic treatment for vulval disorder, age > 18 years old. The exclusion criteria were lack of consent, current vaginal infection confirmed with microbiological studies, current local or systemic treatment for vulval disease. 45 consecutive women were recruited into the study. All appropriate vulval samples were process by genetic analysis. The mean expression (± SD) of HPSA1A for controls was 5.52 ± 3.18, for LS was 7.44 ± 2.16 and for LP was 7.89 ± 2.48. The mean expression (± SD) of HPSA1B for controls was 6.54 ± 3.41, for LS was 9.94 ± 6.88 and for LP was 9.43 ± 2.31. The mean expression (± SD) of TP53 for controls was 9.11 ± 1.14, for LS was 9.94 ± 1.27 and for LP was 10.41 ± 2.00. HSPA1A expression was 3,8 higher in women with lichen sclerosus than in control group. Heat shock protein-70 is more often expressed in LS than in healthy controls. HSP-70 not only supports tumor growth and metastasis, but on the other hand mat help to develop immune-driven treatment strategies.

Betulinic Acid Inhibits the Stemness of Gastric Cancer Cells by Regulating the GRP78-TGF-β1 Signaling Pathway and Macrophage Polarization

Cancer stemness is the process by which cancer cells acquire chemoresistance and self-renewal in the tumor microenvironment. Glucose-regulated protein 78 (GRP78) is a biomarker for gastric cancer and is involved in cancer stemness. By inducing cancer stemness in various types of cancer, the polarization of macrophages into tumor-associated macrophages (TAMs) controls tumor progression. Betulinic acid (BA) is a bioactive natural compound with anticancer properties. However, whether GRP78 regulates TAM-mediated cancer stemness in the tumor microenvironment and whether BA inhibits GRP78-mediated cancer stemness in gastric cancer remain unknown. In this study, we investigated the role of GRP78 in gastric cancer stemness in a tumor microenvironment regulated by BA. The results indicated that BA inhibited not only GRP78-mediated stemness-related protein expression and GRP78-TGF-β-mediated macrophage polarization into TAMs, but also TAM-mediated cancer stemness. Therefore, BA is a promising candidate for clinical application in combination-chemotherapy targeting cancer stemness.

Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.

Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly foldedsecretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis.

Glucose-Regulated Protein 78 Is a Potential Serum and Imaging Marker for Early Detection of Ovarian Cancer.

Understanding malignant transformation associated with ovarian cancer (OVCA) is important to establish early detection tests. This study examined whether expression of glucose-regulated protein 78 (GRP78, marker of cellular stress) increases during OVCA development, and whether GRP78 can be detected by targeted-transvaginal ultrasound (TVUS) imaging. Normal ovaries (n = 10), benign (n = 10) and malignant ovarian tumors at early (n = 8) and late stages (n = 16), hens with and without ovarian tumors at early and late stages (n = 10, each) were examined for GRP78 expression during OVCA development by immunohistochemistry, immunoblotting, gene expression and immunoassay. Feasibility of GRP78-targeted TVUS imaging in detecting early OVCA was examined. Compared with normal ovaries and benign tumors, intensity of GRP78 expression was higher (p < 0.0001) in OVCA patients. Compared with normal (9007.76 ± 816.54 pg/mL), serum GRP78 levels were significantly higher (p < 0.05) in patients with early (12,730.59 ± 817.35 pg/mL) and late-stage OVCA (13,930.12 ± 202.35) (p < 0.01). Compared with normal (222.62 ± 181.69 pg/mL), serum GRP78 levels increased (p < 0.05) in hens with early (590.19 ± 198.18 pg/mL) and late-stage OVCA (1261.38 ± 372.85) (p < 0.01). Compared with non-targeted, GRP78-targeted imaging enhanced signal intensity of TVUS (p < 0.0001). Tissue and serum levels of GRP78 increase in association with OVCA. GRP78 offers a potential serum and imaging marker for early OVCA detection.

Forsythiaside A ameliorates sepsis-induced acute kidney injury via anti-inflammation and antiapoptotic effects by regulating endoplasmic reticulum stress

Ethnopharmacological relevanceSepsis is a systemic inflammatory response syndrome caused by an infection in the body, and accompanying acute kidney injury (AKI) is a common complication of sepsis. It is associated with increased mortality and morbidity. Forsythia Fructus, the dried fruit of Forsythia suspensa (Thunb.) Vahl, is a commonly used traditional Chinese medicine.Aims of the studyThis study aimed to elucidate the protective effect of Forsythiaside A (FTA) on sepsis-induced AKI by downregulating inflammatory and apoptotic responses, and exploring its underlying mechanism.MethodsSeptic AKI was induced through intraperitoneal injection of LPS (10 mg/kg) using male C57BL/6 mice and pretreated with FTA or control saline. First, we assessed the degree of renal injury by creatinine, blood urea nitrogen measurement, and HE staining of renal tissue; secondly, the inflammation and apoptosis were measured byELISA, qPCR, and TUNEL immunofluorescence; finally, the mechanism was explored by computer molecular docking and Western blot.ResultsOur data showed that FTA markedly attenuated pathological kidney injuries, alleviated the elevation of serum BUN and Creatinine, suggesting the renal protective effect of FTA. Notably, FTA significantly inhibited the renal expression of proinflammatory cytokine IL-1β, IL-6, and TNF-α both at protein and mRNA levels and attenuated cell apoptosis in the kidney, as measured by caspase-3 immunoblot and TUNEL assay, indicating its anti-Inflammation and antiapoptotic properties. Mechanistically, administration of LPS resulted in robust endoplasmic reticulum (ER) stress responses in the kidney, evidenced by glucose-regulated protein 78(GRP78) upregulation, protein kinase RNA–like endoplasmic reticulum kinase (PERK) activation, eukaryotic initiation factor 2 alpha (elF2α) phosphorylation and C/EBP homologous protein (CHOP) overexpression, which could be significantly blocked by FTA pretreatment. Dynamic simulation and molecular docking were performed to provide further insight.ConclusionsCollectively, our data suggest that FTA ameliorates sepsis-induced acute kidney injury via its anti-inflammation and antiapoptotic properties by regulating PERK signaling dependent ER stress responses.

The clinical significance of circulating glucose-regulated protein 78, Caspase-3, and C/EBP homologous protein levels in patients with heart failure

Background and aimsThe destruction of endoplasmic reticulum (ER) homeostasis leads to heart failure (HF), which further aggravates ER stress. Limited data are available on the levels of ER stress markers in HF patients in clinical practice. This study aimed to determine the clinical significance of the ER stress markers, glucose-regulated protein 78 (GRP78), Caspase-3, and C/EBP homologous protein (CHOP), in predicting HF and its severity. Materials and methodsA total of 62 patients with HF and 44 healthy controls were enrolled in the study, and all participants were followed-up for 2 years. ResultsSerum GRP78, Caspase-3, and CHOP levels were significantly higher in patients with HF than those in healthy controls. The level of GRP78 increased with the severity of HF. GRP78 levels were negatively correlated with left ventricular ejection fraction, and positively correlated with N-terminal B-type natriuretic peptide, D-dimer, and lactic acid. Serum GRP78 and Caspase-3 levels showed moderate predictive values for HF patients. ConclusionER stress markers, GRP78 and Caspase-3, had a certain predictive value in HF and can be used as serum biomarkers for the diagnosis of HF. Additionally, GRP78 showed a certain predictive value in HF severity.

Is GRP78 (Glucose-regulated protein 78) a prognostic biomarker in differents types of cancer? A systematic review and meta-analysis.

GRP78 is a chaperone with anti-apoptotic function associated with aggressive tumors. This systematic review aimed to evaluate GRP78 expression in cancer and its relation to prognosis outcomes. This review was conducted in different databases searching for human cancer studies assessing GRP78 immunohistochemical levels on tissue samples. A total of 98 manuscripts were included. In 62% of the studies, GRP78 was associated with a worse prognosis. A meta-analysis included 29 studies that detected a significantly higher expression of GRP78 in cancer tissues (RR= 2.35, 95% CI 1.75-3.15) compared to control. A meta-analysis of 3 and 5-years Overall Survival revealed an increased risk of death for tumors with high expression of GRP78 (RR=1.36, 95%CI 1.16-1,59, I2 = 57%) and (RR=1.65, 95%CI 1.22-2.21, I2 =64%), respectively. GRP78 is an important prognostic biomarker for different types of cancer and a promising therapeutic target.

Genipin Attenuates Sepsis-Induced Splenocyte Apoptosis via the Inhibition of Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) dysfunction is characterized by ER stress, which can be triggered by sepsis. Recent studies have reported that lessening ER stress is a promising therapeutic approach to improving the outcome of sepsis. Genipin is derived from gardenia fruit, which is a traditional Chinese medicinal herb for anti-inflammation. Here, mice were treated with genipin (2.5 mg/kg) intravenously to assess its biological effects and underlying mechanism against polymicrobial sepsis. Furthermore, the present study focused on detecting the levels of ER stress-related proteins, including protein kinase R-like ER kinase (PERK), glucose-regulated protein of 78 kDa (GRP78), phosphorylated-eukaryotic initiation factor 2α (p-eIF2α), and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP). The results demonstrated that genipin significantly decreased the serum concentrations of tumor necrosis factor-α and interleukin-6, alleviated histopathological damage to the lungs, livers and spleens, and even improved the survival rates of septic mice. Moreover, sepsis significantly upregulated the protein expression levels of splenic GRP78, PERK, p-eIF2α and CHOP, but their levels were significantly suppressed by genipin. Furthermore, genipin also significantly downregulated cleaved caspase-3 expression levels and reduced sepsis-induced splenocyte apoptosis. In conclusion, genipin potentially improved the survival rate of sepsis and attenuated sepsis-induced organ injury and an excessive inflammatory response in mice. The effects of genipin against sepsis were potentially associated with decreased splenocyte apoptosis via the attenuation of sepsis-induced ER stress to further inhibit ER stress-induced apoptosis.

Endoplasmic Reticulum Stress-Activated Neuronal and Microglial Autophagy Contributes to Postoperative Cognitive Dysfunction in Neonatal rats.

Surgery and anesthesia in neonates may lead to cognitive impairment or abnormal behaviors. It has been shown that autophagy plays a critical role in neuropsychiatric disorders, while the role of autophagy in postoperative cognitive impairment in neonates is not known. Here, we determined this role and the involvement of endoplasmic reticulum (ER) stress in regulating brain cell autophagy after surgery. Seven-day old neonatal rats (P7) had right common carotid artery exposure under anesthesia with 3% sevoflurane for 2h. Learning and memory were tested using Barnes maze (BM) and fear conditioning (FC) on P31-42 and P42-44, respectively. In another experiment, rat brains were harvested for biochemical studies. The ratio of microtubule-associated protein 1 light chain 3 (LC3) BII/I was increased and sequestosome 1 (P62/SQSTM1) levels were decreased in the brain 24h after surgery and anesthesia in neonatal rats. Immunofluorescent staining of LC3B was co-localized with a neuronal or a microglial marker but was not co-localized with a marker for astrocytes in rats with surgery. These rats had a poorer performance in the BM and FC tests than control rats when they were adolescent. Pretreatment with an autophagy inhibitor, 3-methyladenine, attenuated the poor performance. Surgery and anesthesia increased the expression of 78kDa glucose-regulated protein (BIP/GRP78), an indicator of ER stress, 6h after surgery and anesthesia. The ER stress activator tunicamycin and inhibitor tauroursodeoxycholic acid increased the markers for autophagy in control rats and decreased the autophagy markers in rats with surgery, respectively. Our results suggest that surgery in neonatal rats induces ER stress that then activates neuronal and microglial autophagy, which contributes to learning and memory impairment later in life.

Effects of light on growth, feeding rate, digestion, and antioxidation in juvenile razor clams Sinonovacula constricta

In the present study, we explored the effects of seven light treatments (white, violet, blue, cyan, green, yellow, and red) on juvenile Sinonovacula constricta (length, 693.79 ± 82.91 μm), employing dark treatment as a control. A rearing experiment was conducted for one week in which the feeding rate was examined daily, e.g., one, two and three h after feeding. At the conclusion of the experiment, overall growth was calculated, and enzymatic activity related to digestion, superoxide dismutase, catalase, and glutathione peroxidase pertaining to antioxidant biomarkers, as well as expression levels of genes related to antioxidation and stress response, were examined. The results showed that the highest growth rates were observed in the yellow light group, while the lowest growth rates were found in the violet and blue light groups; on the other hand, no significant differences were found among the other groups. Meanwhile, when exposed to white, yellow, or green light, juveniles exhibited high feeding rates, while those in the violet and blue light groups showed low feeding rates. Regarding enzymatic activity related to digestion, those of α-amylase, lipase, pepsin, and trypsin were relatively higher in the yellow light group than in the other groups. Regarding oxidative stress, the yellow light group showed higher superoxide dismutase, catalase, and glutathione peroxidase activities than the other groups. In contrast, the lowest malonaldehyde level was found in the white light group, followed by yellow light, while there were no significant differences among the other groups. In addition, the expression levels of genes related to antioxidation and stress response (heat shock protein 70 and glucose-regulated protein 78) in juveniles generally exhibited higher levels in the red light group, whereas they showed relatively lower levels in the yellow light group. Collectively, these results suggest that the application of yellow light may facilitate the growth of juvenile S. constricta, while red and violet light have opposite effects.

Pharmacological inhibition of lysosomal two-pore channel 2 (TPC2) confers neuroprotection in stroke via autophagy regulation

Lysosomal function and organellar Ca2+ homeostasis become dysfunctional in Stroke causing disturbances in autophagy, the major process for the degradation of abnormal protein aggregates and dysfunctional organelles. However, the role of autophagy in Stroke is controversial since excessive or prolonged autophagy activation exacerbates ischemic brain injury. Of note, glutamate evokes NAADP-dependent Ca2+ release via lysosomal TPC2 channels thus controlling basal autophagy. Considering the massive release of excitotoxins in Stroke, autophagic flux becomes uncontrolled with abnormal formation of autophagosomes causing, in turn, disruption of excitotoxins clearance and neurodegeneration. Here, a fine regulation of autophagy via a proper pharmacological modulation of lysosomal TPC2 channel has been tested in preclinical Stroke models. Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia was induced in rats by transient middle cerebral artery occlusion (tMCAO). Under these conditions, TPC2 protein expression as well as autophagy and endoplasmic reticulum (ER) stress markers were studied by Western blotting, while TPC2 localization and activity were measured by immunocytochemistry and single-cell video-imaging, respectively. TPC2 protein expression and immunosignal were highly modulated in primary cortical neurons exposed to extreme hypoxic conditions causing dysfunction in organellar Ca2+ homeostasis, ER stress and autophagy-induced cell death. TPC2 knocking down and pharmacological inhibition by Ned-19 during hypoxia induced neuroprotection. The effect of Ned-19 was reversed by the permeable form of TPC2 endogenous agonist, NAADP-AM. Of note, Ned-19 prevented ER stress, as measured by GRP78 (78 kDa glucose-regulated protein) protein reduction and caspase 9 downregulation. In this way Ned-19 restored organellar Ca2+ level. Interestingly, Ned-19 reduced the infarct volume and neurological deficits in rats subjected to tMCAO and prevented hypoxia-induced cell death by blocking autophagic flux. Collectively, the pharmacological inhibition of TPC2 lysosomal channel by Ned-19 protects from focal ischemia by hampering a hyperfunctional autophagy.

Secreted glucose regulated protein78 ameliorates DSS-induced mouse colitis.

The secreted form of 78-kDa glucose-regulated protein (sGRP78) has been widely reported for its property in aiding resolution of inflammatory. However, little is known on its potential in the treatment of colitis. To investigate the expression pattern and functional outcome of GRP78 in ulcerative colitis, its expression was measured in human and murine colitis samples. It was found that GRP78 was spontaneously secreted to a high level in gut, which is a physiological site of immune tolerance. During the active phase of DSS-induced colitis, the sGRP78 level was significantly reduced but rebounded quickly during resolving phase, making it a potential candidate for the treatment of colitis. In the following experiments, the administration of sGRP78 was proved to decrease susceptibility to experimental colitis, as indicated by an overall improvement of intestinal symptoms, restoration of TJ integrity, decreased infiltration of immune cells and impaired production of inflammatory cytokines. And specific cleavage of endogenous sGRP78 could aggravate DSS colitis. Adoptive transfer of sGRP78-conditioned BMDMs reduced inflammation in the gut. We linked sGRP78 treatment with altered macrophage biology and skewed macrophage polarization by inhibiting the TLR4-dependent MAP-kinases and NF-κB pathways. Based on these studies, as a naturally occurring immunomodulatory molecule, sGRP78 might be an attractive novel therapeutic agent for acute intestinal inflammation.

Induction of autophagy improves skin and hair conditions in dogs with underlying diseases.

Autophagy was reported to play a crucial role in maintaining general and skin health. The study used a synthesized autophagy inducer (AI) (Aquatide™ cospharm Inc.; Daejeon, Korea), for evaluating the effects of autophagy on skin and hair in dogs. Twenty-two dogs with poor skin and hair which were diagnosed with canine atopic dermatitis (CAD) or pituitary-dependent hyperadrenocorticism (PDH) were included. Clinical scores using Canine Atopic Dermatitis Extent and Severity Index-04 (CADESI-04), Pruritus Visual Analog Scale (PVAS) and skin barrier function using measurement of transepidermal water loss (TEWL) were evaluated and canine keratinocytes were also used in vitro investigation of pro-inflammatory cytokines after AI treatment. In the AI group, clinical scores and skin barrier function were improved at week 8 significantly compared to in the other groups. In particular, the AI significantly improved the hair surface damage at 8 weeks compared to the baseline. In vitro, the AI reduced pro-inflammatory cytokines by activating the 78-kDa glucose-regulated protein (GRP78). AI improve skin barrier function and hair damage and reduce pro-inflammatory cytokines by inhibiting reactive oxygen species (ROS) production in dogs.

Syringic acid ameliorates ischemia/reperfusion-induced testicular injury in rats via suppressing of HMGB1/NF-κB axis and endoplasmic reticulum stress.

To investigate the possible protective role of syringic acid on torsion/detorsion-induced testicular injury using biochemical and histopathological approaches for the first time. A total of 24 rats were divided into 4 groups: sham control, torsion/detorsion, torsion/detorsion + syringic acid (50mg/kg and 100mg/kg). Tissue malondialdehyde, total oxidant status and total antioxidant status levels were determined using colorimetric methods. Tissue 8-hydroxy-2'-deoxyguanosine, superoxide dismutase, catalase, high mobility group box 1, nuclear factor kappa B protein 65, tumor necrosis factor-alpha, interleukin-6, myeloperoxidase, 78-kDa glucose-regulated protein, activating transcription factor-6, C/EBP homologous protein and caspase-3 levels were determined using commercial enzyme-linked immunosorbent assay kits. Johnsen's testicle scoring system was used for histological evaluation. Compared with the control group, the levels of oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis were significantly increased in the torsion/detorsion group (p < 0.05). Syringic acid administrations statistically significantly restored these damage in a dose dependent manner (p < 0.05). Moreover, it was found that the results of histological examinations supported the biochemical results to a statistically significant extent. The overall results suggest that syringic acid emerges as a potential compound for the treatment of testicular torsion and may be subject to clinical trials.

GRP78 promotes the osteogenic and angiogenic response in periodontal ligament stem cells.

Periodontitis is a progressive disease that ultimately leads to bone and tooth loss. A major consequence of periodontal disease is the inability to regain lost bone in the periodontium. The importance was demonstrated of glucose-regulated protein-78 (GRP78) in the osteogenic differentiation of periodontal ligament stem cells and their potential use for regeneration of the periodontium. Previous studies have shown the relationship between GRP78 and dentine matrix protein-1 (DMP1). The importance of this receptor-ligand complex in supporting the process of osteogenesis and angiogenesis was confirmed in this study. To show the function of GRP78 in mineralised tissues, transgenic periodontal ligament stem cells (PDLSCs) were generated in which GRP78 was either overexpressed or silenced. Gene expression analysis of the cells cultured under osteogenic conditions showed an increase in key osteogenic genes with the overexpression of GRP78. RNA-Seq analysis was also performed to understand the transcriptome profile associated with genotype changes. Using the database for annotation, visualisation, and integration discovery (DAVID) for the functional enrichment analysis of differentially expressed genes, the upregulation of genes promoting osteogenesis and angiogenesis with GRP78 overexpression was demonstrated. Alizarin red staining and scanning electron microscopy analysis revealed matrix mineralisation with increased calcium deposition in GRP78 overexpressing cells. The in vivo osteogenic and angiogenic function of GRP78 was shown using a subcutaneous implantation rodent model. The results suggested that GRP78 in PDLSCs can regulate the expression of both osteogenesis and angiogenesis. Therefore, GRP78 could be considered as a therapeutic target for repair of diseased periodontium.