Thalidomide Mitigates Apoptosis via Endoplasmic Reticulum Stress in Diabetic Nephropathy

Background: Previous studies have shown that endoplasmic reticulum (ER) stress is related to the apoptosis in the development of diabetic nephropathy (DN) and thalidomide (Thd) has renal-protective effects by suppressing inflammation and proliferation of MCs in DN. However, the effect of Thd on the apoptosis of MCs in DN remains largely unclear. The present research is designed to explore the effect of Thd on apoptosis in DN and the related mechanisms. Objective: The study is designed to examine the effect and mechanism of Thd on apoptosis in type 2 diabetic mice and high glucose (HG)-induced MCs. Method: We first evaluated the ER stress markers and apoptosis-related proteins with the treatment of Thd in type 2 diabetic mice and MCs in vitro under HG conditions. MTT assay was used to assess cell viability. Additionally, we evaluated the effect of Thd treatment upon MC apoptosis through flow cytometry. Real-time polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate genes and protein expression related to ER stress and apoptosis. Results: The levels of blood urea BUN, CREA, Urine albumin, and UACR in diabetic mice were significantly reduced after 8 weeks of intervention with Thd. And also, there were upregulated glucose-regulated protein 78 (GRP78), Caspase-12, and downregulated B-cell lymphoma 2 (Bcl-2) in glomeruli of DN mice. In vitro, compared with the HG group, MC apoptosis reduced dramatically with Thd treatment along with upregulation of Bcl-2 and downregulation of Bax. At the same time, ER stress markers GRP78, C/EBP homologous protein (CHOP), and Caspase-12 were also mitigated following the Thd treatment. Conclusion: The present study indicates that Thd might reduce the ER stress in DN via downregulating of GRP78, CHOP, and Caspase12 expression, ultimately mitigating MCs apoptosis.

A study on ER stress-induced apoptosis pathway in cervical cancer HeLa cells treated with biosynthesized gold nanoparticles

Background The expression of apoptotic family of protein plays a major role in induction of programmed cell death. There are six major apoptotic proteins such as Caspase 12, Bcl 2, BAX, Cytochrome c, PARP3 and Mcl1. All these proteins have crucial role in the regulation of apoptosis through mitochondrial degradation, DNA damage, nuclear condensation and eventually cell death of the cancerous cells. It was observed that the apoptotic pathway has been initiated in the cancer cells from the expression of the apoptotic proteins. The results emphasized that the apoptotic cell death has been induced by the nanomaterials against cervical cancer HeLa cell line. Methods Initially, the nanomaterials were individually checked for potential anticancer activities through MTT assay. The cervical cancer HeLa cell line was treated with nanoparticles, nanoconjugates, nano-dox conjugate and chitosan–nano-dox conjugates. The cell lysates were processed for SDS–PAGE followed by Western blotting. The apoptotic expression has been studied for six major apoptotic proteins such as Caspase 12, Bcl 2, BAX, Cytochrome c, PARP3 and Mcl 1. Results In the present study, the biosynthesized gold nanoparticles, nanoconjugates, nano-dox conjugate, chitosan–nano-dox conjugate were treated against cervical cancer HeLa cell line. The results demonstrated anticancer effects of the nanocompounds implying nanoparticles induced apoptotic pathway in the cancer cells. Further apoptotic expression was studied for six major apoptotic proteins such as Caspase 12, Bcl 2, BAX, Cytochrome c, PARP3 and Mcl 1. The present study was focussed on anticancer efficiency of biosynthesized nanomaterials. Conclusions The in vitro anticancer study showed that the nanomaterials induced cell death over the treated cervical cancer cells. In the process of apoptotic cell death, the caspase cascade pathway was activated. The gene expression was checked in line with some of the genes involved in apoptosis, cell death. The expression was checked for Caspase 12, BAX, Bcl2, cyt c, PARP3 and Mcl 1. The expression of apoptotic proteins suggested that the cancer cell death was mediated through ER stress-induced pathway involving the major apoptotic proteins.

High doses of dexamethasone induce endoplasmic reticulum stress-mediated apoptosis by promoting calcium ion influx-dependent CHOP expression in osteoblasts

Background The long-term use of dexamethasone (Dex), a well-known immunosuppressant, leads to an imbalance in bone metabolism and rapid decline of bone mineral density due to apoptosis of osteoblasts. The molecular mechanisms by which Dex induces osteoblast apoptosis remain unclear. Materials and methods MC3T3-E1 cells were treated with 0, 10−8, 10−6, and 10−4 M Dex for 24 h. ATF6, phosphorylated PERK, PERK, phosphorylated IRE1, and IRE1 expression, cell apoptosis, and caspase-12 and caspase-3 activity were measured. CHOP expression and calcium ion influx rate were measured in cells treated with 0 and 10−4 M Dex for 24 h. The effect of 2-APB treatment was assessed in cells treated with 0 or 10−4 M Dex. Results Levels of ATF6 and phosphorylated PERK and IRE1 increased in a dose-dependent manner in MC3T3-E1 cells treated with 10−8, 10−6, and 10−4 M Dex, compared to the control group (P < 0.05). Cells treated with 10−6 and 10−4 M Dex had significantly increased apoptotic rates and caspase-12 and caspase-3 activities (P < 0.05). Cells treated with 10−4 M Dex had significantly increased CHOP levels and calcium ion influx rates (P < 0.05). Combined treatment with 10−4 M Dex and 2-APB abrogated the observed increases in cell apoptosis and caspase-12 and caspase-3 activities (P < 0.05). Conclusions High doses of Dex induce CHOP expression by promoting calcium ion influx-dependent induction of ATF6, phosphorylated PERK and phosphorylated IRE1, which induce endoplasmic reticulum stress-mediated apoptosis in osteoblasts. 2-APB protects the osteoblasts from the effects of Dex, preventing endoplasmic reticulum stress-mediated apoptosis.

Autophagy triggers endoplasmic reticulum stress and C/EBP homologous protein-mediated apoptosis in OGD/R-treated neurons in a caspase-12-independent manner

We reported that a high level of autophagy was initiated by oxygen-glucose deprivation (OGD) and was maintained in neurons even after oxygen-glucose deprivation followed by reoxygenation (OGD/R), accompanied by neuronal apoptosis. This study focused on autophagy-induced apoptosis and its signaling network, especially the role of endoplasmic reticulum stress (ERS). Analysis of primary cultured cortical neurons from mice showed that the autophagy-induced apoptosis depended on Caspase-8 and -9 but not Caspase-12. This finding did not mean that the endoplasmic reticulum did not participate in this process. Increases in the levels of endoplasmic reticulum (ER) biomarkers and Binding immunoglobulin protein (BiP) were induced by autophagy in OGD/R-treated neurons. In addition, as an apoptotic transcription factor induced by ER stress, C/EBP homologous protein (CHOP) expression was significantly increased in neurons after OGD/R. This result suggested that the autophagy-Bip-CHOP-caspase (8 and 9)-dependent apoptotic signaling pathway at least partly participated in autophagy-induced apoptosis in primary cortical neurons. It revealed that ER induced apoptosis in neurons suffering from OGD/R injury in an ER stress-CHOP-dependent manner rather than a caspase-12-dependent manner. However, more research on signaling or cross-linking networks and intermediate links are needed. The realization of caspase-12-independent BiP-CHOP neuronal apoptosis pathway has expanded our understanding of the neuronal apoptosis network, which may eventually provide endogenous interventional strategies for OGD/R injury after stroke.

Electroacupuncture Regulates Endoplasmic Reticulum Stress and Ameliorates Neuronal Injury in Rats with Acute Ischemic Stroke

Ischemic stroke is a common cause of morbidity, mortality, and disability worldwide. Electroacupuncture (EA) is an effective method for alleviating brain damage after ischemic stroke. However, the underlying mechanism has not been fully elucidated. This study aimed to determine whether endoplasmic reticulum stress (ERS) could contribute to the protective effects of EA in cerebral ischemia/reperfusion injury (CIRI) to provide a rationale for the widespread clinical use of EA. Rats were divided into the sham-operated (sham) group, the CIRI (model) group, and the EA group. Rats in the model group were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by 72 h of reperfusion. Rats with CIRI were treated daily with EA at GV20 and ST36 for a total of 3 days. The Longa scoring system and adhesive removal somatosensory test were applied to evaluate neurological deficits. Then, 2,3,5-triphenyltetrazolium chloride (TTC) staining was performed to measure the infarct volume. Immunofluorescence staining for NeuN and GFAP and terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling (TUNEL) staining were performed to detect apoptotic cells in brain tissue. Immunohistochemistry, quantitative real-time polymerase chain reaction (qPCR), and western blotting were used to measure the levels of ERS indicators (GRP78, CHOP/GADD153, p-eIF2α, and caspase 12). The results showed that EA significantly reduced the cerebral infarct volume, improved neurological function, and inhibited neuronal apoptosis. In the EA group compared with the model group, the mRNA expression levels of GRP78 were significantly increased, and the expression levels of proapoptotic proteins (CHOP/GADD153, p-eIF2α, and caspase 12) were significantly decreased. These results suggest that the possible mechanism by which EA protects cells against neuronal injury in CIRI may involve inhibiting endoplasmic reticulum stress.

Dahuang Danshen Decoction Inhibits Pancreatic Fibrosis by Regulating Oxidative Stress and Endoplasmic Reticulum Stress

Background. In Traditional Chinese Medicine (TCM), Dahuang Danshen decoction (DD) is used to treat pancreatic fibrosis. Pancreatic fibrosis is a typical manifestation of chronic pancreatitis (CP), which affects the digestive system. The therapeutic mechanisms of DD in pancreatic fibrosis are unclear. Aim. This study aimed to investigate the regulatory mechanisms of DD on oxidative stress and endoplasmic reticulum stress in CP. Materials and Methods. Experimental rats were intraperitoneally injected with 500 mg/kg BW of diethyldithiocarbamate (DDC) twice a week for six weeks to induce CP. At the same time, DD was administered orally at daily doses of 1.37 g/kg BW, 2.74 g/kg BW, and 5.48 g/kg BW to evaluate its treatment effects on CP. After all treatments, pancreatic tissues were harvested and subjected to H&E staining. Transmission electron microscopy (TEM) was also performed to show the endoplasmic reticulum structure in the pancreatic tissues. Immunohistochemistry was used to detect the α-SMA expression level in the pancreatic tissues. Metabolomics analysis of the serum and proteomics analysis of the pancreatic tissues were performed to reveal the changes of endogenous metabolites and proteins, respectively. Concentrations of GSH, MDA, SOD, ROS, col-1, and col-3 were determined using corresponding kits. The western blotting method was used to determine the protein levels of Keap-1, HO-1, NQO1, Nrf2, GRP, JNK, and caspase 12. The pancreatic mRNA levels of NQO1, GPX1, HO-1, GST-π, GRP, JNK, and caspase 12 were also determined by quantitative PCR. The interactions between TCM components and Keap-1 were investigated by molecular docking modeling. Results. The pathohistological results demonstrated that DD could ameliorate DDC-induced CP in vivo, indicated by reduction of α-SMA, col-1, col-3, TNF-α, and IL-6. DD increased serum levels of GSH and SOD but reduced pancreatic ROS. DD decreased cytoplasmic Keap-1 and increased Nrf2 nuclear localization. Correspondingly, DD increased the expression levels of Nrf2 downstream antioxidant genes NQO1, GPX1, HO-1, and GST-π. DD also decreased ERS hallmarks caspase 12 cleavage and GRP expression. Eventually, DD inhibited PSC activation by reducing JNK phosphorylation and MMK-3/p38 expression. Molecular docking analysis showed that salvianolic acid B and emodin had a good binding affinity toward Keap-1. Conclusions. These results demonstrated that DD could ameliorate the oxidative and endoplasmic reticulum stress through releasing Nrf2 from Keap-1 binding and inducing the downstream antioxidant enzymes. As a result, DD could thwart pancreatic fibrosis by inhibiting PSCs activation, which was induced by OS and ERS through JNK and MMK3/p38 pathways.

High Doses of Dexamethasone Induce Endoplasmic Reticulum Stress-Mediated Apoptosis by Promoting Calcium Ion Influx-Dependent CHOP Expression in Osteoblasts

Background: The molecular mechanisms by which dexamethasone (Dex) induces apoptosis in osteoblasts remain unclear.Materials and Methods: MC3T3-E1 cells were treated with 0, 10-8, 10-6, and 10-4 M Dex for 24 h. The expression of ATF6, and phosphorylated PERK and IRE1, cell apoptosis, and the activity of caspase-12 and caspase-3 were measured. The expression of CHOP and the rate of influx of calcium ions were also measured in cells treated with 0 and 10-4 M Dex for 24 h. The effect of 2-APB treatment was assessed in cells treated with 0 or 10-4 M Dex.Results: The levels of ATF6 and phosphorylated PERK and IRE1 increased in a dose-dependent manner in MC3T3-E1 cells treated with 10-8, 10-6, and 10-4 M Dex, compared to in cells treated with 0 M Dex (P <0.05). Cells treated with 10-6 and 10-4 M Dex had significantly increased cell apoptosis rates and caspase-12 and caspase-3 activity compared to the control (P <0.05). Cells treated with 10-4 M Dex had significantly increased levels of CHOP and calcium ion influx rates compared to in the control (P <0.05). Combined treatment with 10-4 M Dex and 2-APB abrogated the observed increases in cell apoptosis and the activity of caspase-12 and caspase-3 (P>0.05). Conclusion: High doses of Dex induce endoplasmic reticulum stress-mediated apoptosis by promoting calcium ion influx-dependent expression of CHOP, and the activation of caspase-12 and caspase-3 in osteoblasts. Combined treatment with 2-APB protects the cells from the effects of Dex, preventing endoplasmic reticulum stress-mediated apoptosis.

The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12−/− (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, β and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Unfolded protein response triggers differential apoptotic mechanisms in ovaries and early embryos exposed to maternal type 1 diabetes

Diabetes mellitus (DM) has profound effects on the female mammalian reproductive system, and early embryonic development, reducing female reproductive outcomes and inducing developmental programming in utero. However, the underlying cellular and molecular mechanisms remain poorly defined. Accumulating evidence implicates endoplasmic reticulum (ER)-stress with maternal DM associated pathophysiology. Yet the direct pathologies and causal events leading to ovarian dysfunction and altered early embryonic development have not been determined. Here, using an in vivo mouse model of Type 1 DM and in vitro hyperglycaemia-exposure, we demonstrate the activation of ER-stress within adult ovarian tissue and pre-implantation embryos. In diabetic ovaries, we show that the unfolded protein response (UPR) triggers an apoptotic cascade by the co-activation of Caspase 12 and Cleaved Caspase 3 transducers. Whereas DM-exposed early embryos display differential ER-associated responses; by activating Chop in within embryonic precursors and Caspase 12 within placental precursors. Our results offer new insights for understanding the pathological effects of DM on mammalian ovarian function and early embryo development, providing new evidence of its mechanistic link with ER-stress in mice.