Effect Of Endoplasmic Reticulum Stress Inhibition Research Articles

Inhibition of Endoplasmic Reticulum Stress Improves Acetylcholine-Mediated Relaxation in the Aorta of Type-2 Diabetic Rats.

Endoplasmic reticulum (ER) stress contributes to insulin resistance and macro- and microvascular complications associated with diabetes. This study aimed to evaluate the effect of ER stress inhibition on endothelial function in the aorta of type-2 diabetic rats. Type-2 diabetes was developed in male Sprague–Dawley rats using a high-fat diet and low-dose streptozotocin. Rat aortic tissues were harvested to study endothelial-dependent relaxation. The mechanisms for acetylcholine-mediated relaxation were investigated using pharmacological blockers, Western blotting, oxidative stress, and inflammatory markers. Acetylcholine-mediated relaxation was diminished in the aorta of diabetic rats compared to control rats; supplementation with TUDCA improved relaxation. In the aortas of control and diabetic rats receiving TUDCA, the relaxation was mediated via eNOS/PI3K/Akt, NAD(P)H, and the KATP channel. In diabetic rats, acetylcholine-mediated relaxation involved eNOS/PI3K/Akt and NAD(P)H, but not the KATP channel. The expression of ER stress markers was upregulated in the aorta of diabetic rats and reduced with TUDCA supplementation. The expression of eNOS and Akt were lower in diabetic rats but were upregulated after supplementation with TUDCA. The levels of MDA, IL-6, and SOD activity were higher in the aorta of the diabetic rats compared to control rats. This study demonstrated that endothelial function was impaired in diabetes, however, supplementation with TUDCA improved the function via eNOS/Akt/PI3K, NAD(P)H, and the KATP channel. The improvement of endothelial function was associated with increased expressions of eNOS and Akt. Thus, ER stress plays a crucial role in the impairment of endothelial-dependent relaxation. Mitigating ER stress could be a potential strategy for improving endothelial dysfunction in type-2 diabetes.

Tendon microstructural disruption promotes tendon-derived stem cells to express chondrogenic genes by activating endoplasmic reticulum stress.

The erroneous differentiation of tendon-derived stem cells (TDSCs) into adipocytes, chondrocytes, and osteoblasts is believed to play an important role in the development of tendinopathy. However, the regulatory mechanisms of TDSC differentiation remain unclear. The aim of this study is to investigate the contribution and mechanism of the tendon microstructural disruption to the differentiation of TDSCs. Bovine Achilles tendons were sliced. The tendon slices were stretched with different tensile strains to mimic the tendon structure alteration at various scales. The TDSCs were cultured on the tendon slices. The differentiation of TDSCs and endoplasmic reticulum (ER) stress in the TDSCs were investigated with quantitative reverse transcription polymerase chain reaction, immunostaining and western blot. The effect of ER stress inhibition on chondrogenic differentiation of the TDSCs was further investigated. The structural alteration did not affect the viability of TDSCs. However, the structural alteration of tendon slices with 6.4% strain promoted TDSCs to express the chondrogenic genes. ER stress-related markers, ATF-4 and PERK, were also upregulated. With the inhibition of ER stress, the expression of ATF-4 and the chondrogenic gene SOX9 of TDSCs were inhibited. The study indicated that tendon microdamage could induce the chondrogenic differentiation of TDSCs through triggering ER stress to activate ATF-4 and SOX9 subsequently.

Inhibition of endoplasmic reticulum stress combined with activation of angiotensin-converting enzyme 2: novel approach for the prevention of endothelial dysfunction in type 1 diabetic rats.

Persistent hyperglycemia in type 1 diabetes triggers numerous signaling pathways, which may prove deleterious to the endothelium. As hyperglycemia damages the endothelial layer via multiple signaling pathways, including enhanced oxidative stress, downregulation of angiotensin-converting enzyme 2 signaling, and exacerbation of endoplasmic reticulum (ER) stress, it becomes difficult to prevent injury using monotherapy. Thus, the present study was conceived to evaluate the combined effect of ER stress inhibition along with angiotensin-converting enzyme 2 activation, two major contributors to hyperglycemia-induced endothelial dysfunction, in preventing endothelial dysfunction associated with type 1 diabetes. Streptozotocin-induced diabetic animals were treated with either diminazene aceturate (5mg·kg-1 per day, p.o.) or tauroursodeoxycholic acid, sodium salt (200mg·kg-1 per day i.p.), or both for 4 weeks. Endothelial dysfunction was evaluated using vasoreactivity assay, where acetylcholine-induced relaxation was assessed in phenylephrine pre-contracted rings. Combination therapy significantly improved vascular relaxation when compared with diabetic control as well as monotherapy. Restoration of nitrite levels along with prevention of collagen led to improved vasodilatation. Moreover, there was an overall reduction in aortic oxidative stress. We conclude that by simultaneously inhibiting ER stress and activating angiotensin-converting enzyme 2 deleterious effects of hyperglycemia on endothelium were significantly alleviated. This could serve as a novel strategy for the prevention of endothelial dysfunction.

4-phenylbutyric acid mediates therapeutic effect in systemic lupus erythematosus: Observations in an experimental murine lupus model.

Impaired function of regulatory T cells (Tregs) contributes to the pathogenesis of systemic lupus erythematosus (SLE). Our previous study demonstrated aberrant responses of T lymphocytes to endoplasmic reticulum (ER) stress in patients with SLE. The present study investigated whether ER stress inhibition by 4-phenylbutyric acid (4-PBA) ameliorated lupus manifestations in an experimental lupus model and the effect of ER stress inhibition on the frequency and function of Tregs. A murine lupus model was induced through a 4-week treatment with Resiquimod, a toll-like receptor (TLR) 7 agonist. From the 8th week, the mice were treated with 4-PBA for 4 weeks. 4-PBA significantly decreased the levels of anti-dsDNA antibodies and serum TNF-α. A significant decrease in glomerulonephritis score was also observed in the 4-PBA-treated group. ER stress inhibition decreased the activated T and B lymphocytes population of splenocytes; however, the population of Tregs was not significantly different between the vehicle and 4-PBA group. However, a markedly enhanced suppressive capacity of Treg was detected in the 4-PBA-treated group. The present results suggest that ER stress inhibition attenuated disease activity in an experimental model by improving the suppressive capacity of Tregs. Therefore, reduction of ER stress could be used as a beneficial therapeutic strategy in SLE.

Inhibition of endoplasmic reticulum stress ameliorates cardiovascular injury in a rat model of metabolic syndrome

Metabolic (Met) syndrome is characterized by hypertension, insulin resistance and dyslipidaemia with high risk of cardiovascular disease. Endoplasmic reticulum (ER) stress is a key contributor in the pathogenesis of Met syndrome. The current study investigates the effect of Tauroursodeoxycholate (TUDCA), an ER stress inhibitor, on Met syndrome-induced cardiovascular complications and the possible underlying signalling mechanisms. Met syndrome was induced in rats, which were then treated with TUDCA. Body weight, blood pressure, glucose tolerance and insulin tolerance tests were performed. ER stress, survival and oxidative stress markers were measured in heart and aorta tissue. The results showed that TUDCA improved metabolic parameters in rats with Met syndrome. Treatment mitigated the Met syndrome-induced cardiovascular complications through upregulating survival markers and downregulating ER and oxidative stress markers. These results highlight the protective effect of ER stress inhibition as a potential target in the management of cardiovascular complications associated with Met syndrome.

Endoplasmic reticulum stress inhibition blunts the development of essential hypertension in the spontaneously hypertensive rat.

Essential hypertension is the leading cause of premature death worldwide. However, hypertension's cause remains uncertain. endoplasmic reticulum (ER) stress has recently been associated with hypertension, but it is unclear whether ER stress causes hypertension. To clarify this question, we examined if ER stress occurs in blood vessels before the development of hypertension and if ER stress inhibition would prevent hypertension development. We used the spontaneously hypertensive rat (SHR) as a model of human essential hypertension and the Wistar-Kyoto (WKY) rat as its normotensive control. Resistance arteries collected from young rats determined that ER stress was present in SHR vessels before the onset of hypertension. To assess the effect of ER stress inhibition on hypertension development, another subset of rats were treated with 4-phenylbutyric acid (4-PBA; 1 g·kg-1·day-1) for 8 wk from 5 wk of age. Blood pressure was measured via radiotelemetry and compared with untreated SHR and WKY rats. Mesenteric resistance arteries were collected and assessed for structural and functional changes associated with hypertension. Systolic and diastolic blood pressures were significantly lower in the 4-PBA-treated SHR groups than in untreated SHRs. Additionally, 4-PBA significantly decreased the media-to-lumen ratio and ER stress marker expression, improved vasodilatory response, and reduced contractile responses in resistance arteries from SHRs. Overall, ER stress inhibition blunted the development of hypertension in the SHR. These data add evidence to the hypothesis that a component of hypertension in the SHR is caused by ER stress. NEW & NOTEWORTHY In this study, 4-phenylbutyric acid's (4-PBA's) molecular chaperone capability was used to inhibit endoplasmic reticulum (ER) stress in the small arteries of young spontaneously hypertensive rats (SHRs) and reduce their hypertension. These effects are likely mediated through 4-PBA's effects to reduce resistant artery contractility and increase nitric oxide-mediated endothelial vasodilation through a process preventing endothelial dysfunction. Overall, ER stress inhibition blunted the development of hypertension in this young SHR model. This suggests that a component of the increase in blood pressure found in SHRs is due to ER stress. However, it is important to note that inhibition of ER stress was not able to fully restore the blood pressure to normal, suggesting that a component of hypertension may not be due to ER stress. This study points to the inhibition of ER stress as an important new physiological pathway to lower blood pressure, where other known approaches may not achieve blood pressure-lowering targets.

Endoplasmic Stress Inhibition during Oocyte Maturation Improves Preimplantation Development of Cloned Pig Embryos

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Zn2+ and mPTP Mediate Endoplasmic Reticulum Stress Inhibition-Induced Cardioprotection Against Myocardial Ischemia/Reperfusion Injury

The purpose of this study was to determine whether Zn2+ is involved in endoplasmic reticulum (ER) stress inhibition-induced cardioprotection against ischemia/reperfusion (I/R) injury by modulation of the mitochondrial permeability transition pore (mPTP) opening. Isolated rat hearts were subjected to 30-min regional ischemia followed by 2h of reperfusion. Expression of glucose regulated protein 78 (GRP 78 or BIP), an ER homeostasis marker, was not increased during ischemia but was increased upon reperfusion, indicating that ER stress was initiated upon reperfusion but not during ischemia. The ER stress inhibitor tauroursodeoxycholic acid (TUDCA) given at reperfusion resulted in a significant reduction of GRP78 expression 30 and 60min after the onset of reperfusion, an effect that was reversed by the zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN). The immunofluorescence study also showed that the effect of TUDCA on GRP78 expression was reversed by TPEN. TUDCA reduced infarct size and this was reversed by the mPTP opener atractyloside, indicating that ER stress inhibition may induce cardioprotection by modulating the mPTP opening. Experiments with transmission electron microscopy and hematoxylin-eosin staining also revealed that TUDCA prevented endoplasmic reticulum and mitochondrial damages at reperfusion, which was blocked by TPEN. Exposure of cardiac H9c2 cells to H2O2 increased GRP 78 and GRP 94 expressions, suggesting that oxidative stress can induce ER stress. Cells treated with H2O2 showed a significant decrease in tetramethylrhodamine ethyl ester (TMRE) fluorescence, indicating that H2O2 triggers the mPTP opening. In contrast, TUDCA prevented the loss of TMRE fluorescence, the effect that was blocked by TPEN, indicating a role of Zn in the preventive effect of ER stress inhibition on the mPTP opening. In support, TUDCA significantly increased intracellular free zinc. These data suggest that reperfusion but not ischemia initiates ER stress and inhibition of ER stress protects the heart from reperfusion injury through prevention of the mPTP opening. Increased intracellular free Zn accounts for the cardioprotective effect of ER stress inhibition.

Abstract 14997: Zn2+ and mPTP Mediate ERS Inhibition-induced Cardioprotection Against Ischemia/Reperfusion Injury

While the role of endoplasmic reticulum stress (ERS) in myocardial ischemia/reperfusion (I/R) injury has been extensively investigated, the precise mechanism by which inhibition of ERS induces cardioprotection remains unclear. We aimed to explore the mechanism of ERS inhibition-induced cardioprotection against I/R injury, focusing on the role of Zn 2+ and the mitochondrial permeability transition pore (mPTP). Exposure of H9c2 cells to 800 μM H 2 O 2 for 20 min increased GRP78 and GRP94 expressions (296.2 ± 41.0 and 150.6 ± 13.5 %, respectively), suggesting that H 2 O 2 can induce ERS. Cells treated with H 2 O 2 showed a significant decrease (40.6 ± 7.4 %) in TMRE fluorescence compared to the normal group (92.6 ± 0.1 %), indicating that H 2 O 2 can induce the mPTP opening. In contrast, ERS inhibitor TUDCA (30 μM) prevented the loss of TMRE fluorescence (77.8 ± 6.8 %), implying that inhibition of ERS can prevent the mPTP opening. This effect of TUDCA was blocked by zinc chelator TPEN (37.7 ± 13.0 %), indicating a role of Zn 2+ in the action of TUDCA on the mPTP opening. In support, TUDCA increased intracellular free zinc, as indicated by a marked increase in Newport Green DCF fluorescence intensity. In isolated rat hearts, GRP78 expression was not increased during ischemia but was increased upon reperfusion (1.3, 1.5, 1.9, and 1.6-fold increases at 10, 30, 60, and 120 min of reperfusion). Hearts treated with TUDCA showed a significant reduction of GRP78 expression 30 and 60 min after the onset of reperfusion, an effect that was reversed by TPEN. The immunofluorescence study also showed that the effect of TUDCA on GRP78 expression was reversed by TPEN. TUDCA reduced infarct size, and this was reversed by the mPTP opener atractyloside, indicating that ERS inhibition may protect the heart by modulating the mPTP opening. Experiments with transmission electron microscopy and hematoxylin-eosin staining revealed that TUDCA prevented endoplasmic reticulum and mitochondrial damages at reperfusion, which was blocked by TPEN. In conclusion, reperfusion but not ischemia initiates ERS and inhibition of ERS protects the heart from reperfusion injury through prevention of the mPTP opening. Increased intracellular free Zn 2+ accounts for the cardioprotective effect of ERS inhibition.

Endoplasmic reticulum stress is increased after spontaneous labor in human fetal membranes and myometrium where it regulates the expression of prolabor mediators.

Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In nongestational tissues, several markers of the unfolded protein response (UPR) have been shown to regulate the inflammatory response. Thus, the aim of this study was to determine the effect of human labor on markers of ER stress in fetal membranes and myometrium. In addition, the effect of ER stress inhibition on the expression and secretion of proinflammatory and prolabor mediators was also assessed. The markers of ER stress, GRP78, IRE1, and spliced XBP1 (XBP1s), were significantly increased in fetal membranes and myometrium after term and preterm labor compared to nonlaboring samples. Given that inflammation is considered to be one of the leading causes of spontaneous preterm birth, here we used bacterial endotoxin lipopolysaccharide (LPS) as a model for infection-induced preterm birth. In term nonlabored fetal membranes and myometrium, LPS induced UPR activation as evidenced by a significant increase in the expression of GRP78, IRE1, and XBP1s in fetal membranes and myometrium. The use of the chemical chaperones 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA) alleviated ER stress induced by LPS. 4-PBA and TUDCA also ameliorated the increase in LPS-induced prolabor mediators. Our data suggest that the UPR may regulate the inflammatory responses associated with labor or infection in fetal membranes and myometrium of pregnant term and preterm women. Thus, the use of ER stress inhibitors, in particular 4-PBA or TUDCA, may be a potential therapeutic strategy for the prevention of infection-mediated spontaneous preterm birth.

Abstract 224: 4-phenylbutyric Protects The Heart From Heart Failure, Cardiac Rupture And Remodeling By Alleviating Cardiac Apoptosis And Fibrosis In A Mouse Model Of Myocardial Infaction

Evidences showed that endoplasmic reticulum (ER) stress has involved in cardiac hypertrophy and inhibition of ER stress exerts an effective cardioprotective effect. But little is known about the effect of ER stress inhibition on myocardial infarction (MI). The aim of this study is to investigate whether direct inhibition of ER stress by 4-phenylbutyric acid (4-PBA), an ER stress chemical chaperone, could attenuate MI induced by left coronary artery (LCA) ligation in male C57BL/6 mice. Either NaCl or 4-PBA (20 mg/kg/d) alone was intra-peritoneal injected to the mice immediately after MI for 4 weeks. Heart failure, cardiac rupture and remodeling occurred after MI. There was 52.5% vs 24% death rate in control group vs 4-PBA group during the early phase after MI (2-7 days). The survivals underwent cardiac remodeling with impaired systolic and diastolic functions at the end. The ventricular aneurysm and fibrosis were much smaller in 4-PBA group and the heart functions were improved. Western blot showed that the ER stress markers, Bax, Caspase 3, TGFβ1 and Smad 2/3 in the heart tissues of the control group were significantly increased after MI, in which Bax and Caspase 3 were increased markedly in the early phase of MI, while TGFβ1 and Smad 2/3 were increased obviously in the late phase. 4-PBA decreased those protein levels in the early and late phases of MI respectively. These findings indicate that inhibition of ER stress using 4-PBA could be an effective therapeutic agent protects the heart from post-infarcted heart failure, cardiac rupture and remodeling.

Abstract 518: Inhibition of Endoplasmic Reticulum Stress Reduces Aneurysm Induction in Different Aneuryms Models

Abdominal aorta aneurysm (AAA) is a major cause of mortality in men. The role of endoplasmic reticulum (ER) stress in the pathogenesis of AAA is unknown. Human AAA specimens from diabetic patients were found to have increased expression of ER stress markers. The inhibition of ER stress signaling with (taurine-conjugated ursodeoxycholic acid, Tudca) administration reduces the development of aneurysm in Ang-II infused and calcium chloride (CaCl2)-induced mouse models. Type 2 diabetic (db-/db-) and control (db-/db+) mice were infused with angiotensin II (Ang-II, 1000ng/kg/min) and treated with or without the ER stress inhibitor Tudca for 4 weeks. Mice infused with Ang-II displayed an increase in blood pressure, plasma cholesterol and CRP levels, which were reduced after ER stress inhibition. Human AAA and Ang-II-induced AAA are characterized by an increase in lumen, cross sectional area and were associated with increased macrophage infiltration, expression of the ER stress marker CHOP and elastic fiber disruption, and these indicators of aneurysm disease were reduced by Tudca treatment. To determine the effect of ER stress inhibition on aneurysm formation in a model that did not exhibit blood pressure and metabolic changes, we exposed the adventitial surface of carotid arteries of db-/db- and db-/db+ mice to CaCl2 and then treated the mice with or without Tudca. Our results indicate that in the CaCl2 induced carotid artery aneurysm model that treatment with Tudca significantly reduced carotid artery aneurysm formation. These results provide evidence in support of a role for that ER stress activation in the induction of aneurysm in type 2 diabetes.