Free Fatty Acids Induce Endoplasmic Reticulum Stress-Mediated Apoptosis of Macrophages in Dairy Cows with Ketosis.

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.

Objective To investigate the role of endoplasmic reticulum stress (ERS) pathway involving protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) in apoptosis in lungs of rats with bronchopulmonary dysplasis (BPD). Methods Forty-eight premature SD rats were divided into BPD group and control group according to random number table.Rats in BPD group were continually exposed to O2 with volumetric concentration factor of 850 mL/L, while rats in control group were exposed to air.Lung tissues in each group were obtained in 7, 14 and 21 days respectively.The apoptosis in lung cells was evaluated by terminal dexynucleotifyl transferase-mediated dUTP nick end labeling (TUNEL) assay.The mRNA levels of glucose regulated protein 78 (GRP78), PERK, ATF4 and CHOP were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of GRP78, phosphorylated PERK (pho-PERK), ATF4 and CHOP were detected by using Western blot. Results Compared with control group, the lung cells of the rats in BPD group developed more serious apoptosis.Furthermore, the apoptosis index (AI) in lung cells of the rats increased rapidly with the hyperoxia exposure time.This had been statistically verified by comparison with the control group at different timing(7 d: 15.50±0.58 vs 1.25±0.50, 14 d: 27.75±1.71 vs 3.25±0.96, 21 d: 50.50±3.70 vs 4.00±1.15; t=57.00, 20.58, 25.16, all P<0.01). The mRNA levels of GRP78, PERK, ATF4 and CHOP in BPD group increased significantly compared to the control group [GRP78: 7 d (33.88±3.73 )vs(11.65±1.00), 14 d (54.50±2.18)vs(12.84±1.41), 21 d (95.34±7.61)vs(12.43±0.59); PERK: 7 d (5.23±0.92)vs(1.45±0.46), 14 d (7.60±1.56)vs(2.18±0.97), 21 d (16.55±0.50)vs(2.90±1.18); ATF4: 7 d (23.04±2.45)vs(12.56±2.81), 14 d (28.66±2.66)vs(15.18±2.92), 21 d (36.63±2.99)vs(15.14±2.09); CHOP: 7 d (2.21±0.19)vs(0.81±0.02), 14 d (4.19±0.17)vs(0.90±0.08), 21 d (6.08±0.38)vs(0.88±0.10); all P<0.05]. The protein levels of GRP78, pho-PERK, ATF4 and CHOP in BPD group increased significantly as well [GRP78: 7 d (1.33±0.03 )vs(0.85±0.04), 14 d (1.31±0.02)vs(0.92±0.01), 21 d (1.82±0.28)vs(0.87±0.01); pho-PERK: 7 d (0.68±0.02)vs(0.54±0.01), 14 d (1.04±0.01)vs(0.65±0.01), 21 d (1.29±0.02)vs(0.73±0.01); ATF4: 7 d (1.26±0.01)vs(0.83±0.01), 14 d (1.39±0.02)vs(0.87±0.02), 21 d (1.67±0.02)vs(0.94±0.02); CHOP: 7 d (1.37±0.01)vs(0.47±0.06), 14 d (1.50±0.04)vs(0.74±0.05), 21 d (1.61±0.03)vs(0.55±0.02); all P<0.05]. Positive correlation was demonstrated between the expression levels of CHOP protein and AI, PERK, ATF4 in the BPD group (r=0.87, 0, 92, 0.93 respectively, all P<0.05). Conclusion PERK-ATF4-CHOP mediated ERS may participate in and contribute to the apoptosis mechanism in lungs of rats with BPD. Key words: Endoplasmic reticulum stress; Protein kinase R-like ER kinase; CCAAT/enhancer binding protein homologous protein; Bronchopulmonary dysplasis; Apoptosis

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

This study was to explore whether activating transcription factor 6 (ATF6), an important sensor to endoplasmic reticulum (ER) stress, would mediate oxidized low-density lipoprotein (ox-LDL)- induced cholesterol accumulation and apoptosis in cultured macrophages and the underlying molecular mechanisms. Intracellular lipid droplets and total cholesterol levels were assayed by oil red O staining and enzymatic colorimetry, respectively. Cell viability and apoptosis were determined using MTT assay and AnnexinV-FITC apoptosis detection kit, respectively. The nuclear translocation of ATF6 in cells was detected by immunofluorescence analysis. Protein and mRNA levels were examined by Western blot analysis and real time-PCR, respectively. ATF6 siRNA was transfected to RAW264.7 cells by lipofectamin. Exposure of cells to ox-LDL induced glucose-regulated protein 78 (GRP78). C/EBP homologous protein (CHOP), a key-signaling component of ER stress-induced apoptosis, was up-regulated in ox-LDL-treated cells. ATF6, a factor that positively regulates CHOP expression, was activated by ox-LDL in a concentration- and time- dependent manner. The role of the ATF6-mediated ER stress pathway was further confirmed through the siRNA-mediated knockdown of ATF6, which attenuated ox-LDL-induced upregulation of CHOP, cholesterol accumulation and apoptosis in macrophages. In addition, the phosphorylation of double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), another factor that positively regulates CHOP expression, was induced in the presence of ox-LDL, and PERK-specific siRNA also inhibited the ox-LDL-induced upregulation of CHOP and apoptosis in RAW264.7 cells. These results demonstrate that ER stress-related proteins, particularly ATF6 and its downstream molecule CHOP, are involved in ox-LDL-induced cholesterol accumulation and apoptosis in macrophages.

Ketosis is a common metabolic disorder in high-yielding cows and is characterized by high concentrations of BHB and free fatty acids (FFA). High concentrations of FFA induce endoplasmic reticulum (ER) stress in multiple organs including mammary tissue, and result in reduced milk production and lower milk quality. In non-ruminants, loss of nuclear factor erythroid 2-related factor 1 (NFE2L1) results in ER stress. The physiological functions and molecular mechanisms controlled by NFE2L1 in bovine mammary tissue are poorly understood. Thus, the present study aimed to elucidate the role of the NFE2L1 on proteasomal homeostasis and ER stress in mammary tissue from early-lactation (DIM 6 to 14) healthy cows (CON, blood concentration of BHB <1.2 mM, n = 10) and cows with clinical ketosis (CK blood concentration of BHB >3 mM, n = 10). Compared with CON, serum concentration of glucose was lower due to CK, while serum concentrations of BHB and FFA were greater. Protein and mRNA abundance of NFE2L1 along with abundance of proteasomal subunits (PSMD1, PSMD14, PSMA1, PSMB1, and PSMB5 genes and PSMB4 and PSMB6 proteins) were lower in cows with CK, indicating that expression of NFE2L1 and proteasomal homeostasis was impaired by ketosis. In vitro, primary bovine mammary epithelial cells were exposed to various concentrations of FFA (0, 0.3, 0.6, or 1.2 mM). Compared with the 0 mM FFA, the ratio of phosphorylated (p)-protein kinase R-like ER kinase (PERK)/PERK along with the expression of inositol-requiring enzyme 1 (IRE1) α, activating transcription factor 6 (ATF6), glucose regulated protein 78 (GRP78), and C/EBP homologous protein (CHOP) was higher with 1.2 mM FFA. A similar response was observed for ER stress-associated genes (CHOP, GRP78, and XBP1) indicating that high concentrations of FFA induced ER stress. In line with in vivo results, 1.2 mM FFA downregulated the protein and mRNA abundance of NFE2L1, the abundance of PSMB6 protein, and PSM genes (PSMC1, PSMC3, and PSMD1), and increased the accumulation of ubiquitin. This suggested a marked negative effect of high FFA on NFE2L1 and proteasomal homeostasis. Silencing of NFE2L1 triggered upregulation of ER stress-associated genes as well as protein abundance of GRP78 and CHOP. Further, compared with CON-siRNA, the abundance of PSM genes was downregulated in the NFE2L1-siRNA group. In contrast, abundance of markers of ER stress and PSM genes and proteins indicated that overexpression of NFE2L1 relieved the FFA-induced ER stress and improved 26S proteasome homeostasis. Our data suggested that the mammary gland experiences ER stress during ketosis partly due to disruption of proteasomal homeostasis from the excess FFA. As such, NFE2L1 could represent a target for potential therapeutic applications in the field to alleviate the accumulation of malformed proteins that may impair the long-term lactogenic capacity of the udder.

Kennedy disease, a degenerative disorder caused by an expanded glutamine tract, is mediated by misfolding of the mutant androgen receptor (AR) protein, a process that may disrupt proteasome function. We hypothesized that this might lead to endoplasmic reticulum (ER) stress and induction of the unfolded protein response (UPR), a complex physiologic pathway that regulates cell survival. To test this hypothesis, we used aminoterminal fragments of wild type (AR16Q) or mutant (AR112Q) AR that triggered glutamine length-dependent cell death and activated an ER stress-inducible promoter. To evaluate the role of the UPR, we examined the contributions of three proximal sensors of ER stress: activating transcription factor 6 (ATF6), inositol requiring 1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK). AR112Q toxicity was significantly increased by a dominant negative ATF6 mutant and significantly decreased by a constitutively active ATF6 mutant, indicating that ATF6 promoted cell survival. In contrast, co-transfection with three separate IRE1alpha dominant negative mutants failed to alter glutamine length-dependent toxicity, suggesting that this arm of the UPR did not significantly affect AR112Q induced cell death. Activation of PERK, an ER transmembrane protein that functions as the third proximal UPR sensor, promoted glutamine length-dependent toxicity. Although nuclear localization sequence- and nuclear export sequence-targeted proteins both activated the UPR, this pathway more potently influenced toxicity when proteins were targeted to the cytoplasm. Taken together, our data demonstrate that the UPR is activated in cells expressing long glutamine tracts and that this pathway modulates polyglutamine toxicity.

BackgroundEndoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration.MethodsPrimary cultured cardiomyocytes from neonatal rats were exposed to TG (1 µmol/L) treatment for 24 h, following PQS pre-treatment (160 µg/mL) for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase (Si-PERK) for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 (GRP78), calreticulin, PERK, eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were assayed by western blotting.ResultsBoth PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect.ConclusionsOur data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.

Endoplasmic reticulum (ER) dysfunction caused by excessive ER stress is a crucial mechanism underlying seizures-induced neuronal injury. Studies have shown that mitochondrial reactive oxygen species (ROS) are closely related to ER stress, and our previous study showed that activating transcription factor 5 (ATF5)-regulated mitochondrial unfolded protein response (mtUPR) modulated mitochondrial ROS generation in a hippocampal neuronal culture model of seizures. However, the effects of ATF5-regulated mtUPR on ER stress and the underlying mechanisms remain uncertain in epilepsy. In this study, ATF5 upregulation by lentivirus infection attenuated seizures-induced neuronal damage and apoptosis in a rat model of pilocarpine-induced epilepsy, whereas ATF5 downregulation by lentivirus infection had the opposite effects. ATF5 upregulation potentiated mtUPR by increasing the expression of mitochondrial chaperone heat shock protein 60 (HSP60) and caseinolytic protease proteolytic subunit (ClpP) and reducing mitochondrial ROS generation in pilocarpine-induced seizures in rats. Additionally, upregulation of ATF5 reduced the expression of glucose-regulated protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), suggesting suppression of ER stress; Moreover, ATF5 upregulation attenuated apoptosis-related proteins such as B-cell lymphoma-2 (BCL2) downregulation, BCL2-associated X (BAX) and cleaved-caspase-3 upregulation. However, ATF5 downregulation exerted the opposite effects. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO attenuated the harmful effects of ATF5 downregulation on ER stress and neuronal apoptosis by reducing mitochondrial ROS generation. Overall, our study suggested that ATF5-regulated mtUPR exerted neuroprotective effects against pilocarpine-induced seizures in rats and the underlying mechanisms might involve mitochondrial ROS-mediated ER stress.

This study investigated the effects of total flavonoids of Dracocephalum moldavica(TFDM) on apoptosis in rat H9c2 cells induced by endoplasmic reticulum stress(ERS) established by oxygen-glucose deprivation and reoxygenation(OGD/R) injury and tunicamycin(TM), and explored the potential mechanisms. After successful modeling, the following groups were set in this experiment: control group, model(OGD/R or TM) group, and TFDM low-, medium-, and high-dose groups(12.5, 25, and 50 μg·mL~(-1)). The OGD/R injury model was constructed in vitro. Cell proliferation was assessed using the cell counting kit-8(CCK-8) method. The levels of lactate dehydrogenase(LDH) and creatine kinase MB isoenzyme(CKMB) in the cell supernatant were detected. Western blot was used to assess the expression of ERS-related proteins, including glucose regulatory protein 78(GRP78), C/EBP homologous protein(CHOP), activating transcription factor 6(ATF6), and apoptotic proteins B-cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax). Apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) method. In the TM-induced ERS model, Western blot was used to measure the expression of ERS pathway-related proteins GRP78, CHOP, inositol-requiring enzyme 1(IRE1), X-box binding protein 1(XBP1), protein kinase RNA-like endoplasmic reticulum kinase(PERK), eukaryotic initiation factor 2α(eIF2α), ATF6, p-ATF6, and apoptotic proteins Bcl-2, Bax, cysteinyl aspartate specific proteinase-12(caspase-12), and cleaved caspase-12. Gene expression of GRP78, CHOP, PERK, and ATF6 was detected by real-time fluorescence quantitative PCR(RT-qPCR). Apoptosis was again detected using the TUNEL method. The results showed that in the OGD/R model, compared with the control group, the levels of LDH and CKMB in the cell supernatant were significantly increased in the OGD/R group. Compared with the OGD/R group, the levels of LDH and CKMB in the TFDM group were significantly reduced. Western blot results revealed that compared with the control group, the expression of ERS-related proteins and Bax in the OGD/R group was significantly increased, while the expression of Bcl-2 was significantly decreased. Compared with the OGD/R group, the expression of ERS-related proteins and Bax in the TFDM groups was significantly reduced, and the expression of Bcl-2 was significantly increased. TUNEL assay showed that apoptosis was significantly decreased after TFDM treatment. In the TM-induced ERS experiment, compared with the control group, the expression of ERS-related genes, ERS-related proteins, and apoptotic proteins in the TM group was significantly increased, while the expression of Bcl-2 was significantly decreased. Compared with the TM group, the expression of ERS-related genes, ERS-related proteins, and apoptotic proteins in the TFDM group was significantly reduced, and the expression of Bcl-2 was significantly increased. These results suggest that ERS exists in the OGD/R-injured H9c2 cell model, and TFDM can effectively inhibit ERS-induced apoptosis. The mechanism may be related to the downregulation of ERS pathway-related proteins and apoptotic proteins.

Fibroblast growth factor 21 (FGF21), as an endocrine factor, is secreted into circulation by injured cardiomyocytes. Endoplasmic reticulum (ER) stress-induced apoptosis has been proposed as an important pathophysiological mechanism for cardiomyocyte injury. However, whether the enhanced expression of FGF21 in cardiomyocytes is linked to ER stress, and the effect and underlying mechanism of FGF21 on ER stress-induced cardiomyocyte apoptosis remain unclear. In the present study, it was demonstrated that mild ER stress resulted in upregulated expression levels of FGF21 and its main receptors, as a response to cell compensation, at the induction of ≤5 µM tunicamycin (TM). However, excessive ER stress (TM ≥10 µM) activated the ER stress-mediated apoptosis signaling pathways, including PKR-like ER kinase (PERK)-eukaryotic translational initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT/-enhancer-binding protein homologous protein (CHOP) and inositol-requiring kinase 1α (IRE1α)-c-Jun N-terminal kinases (JNK), as well as inhibited the expression of FGF21 and its primary receptors. In addition, FGF21 overexpression provided protection against ER stress-induced cardiomyocyte injury, as evidenced by increased cell viability and reduced apoptosis. These changes were associated with the inhibition of ER stress-mediated apoptosis signaling pathways, as well as increased phosphorylation of FGFR1 and ERK1/2. However, the protective effects of overexpressed FGF21 were abolished following treatment with FGFR1 and ERK1/2 inhibitors. Thus, mild ER stress may induce the expression of FGF21 and its primary receptors in cardiomyocytes. FGF21 inhibits ER stress-induced cardiomyocyte injury as least in part via the FGFR1-ERK1/2 signaling pathway.

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Endoplasmic reticulum (ER) stress-associated apoptosis plays a role in organ remodeling after insult. The effect of ER stress on renal tubular damage and fibrosis remains controversial. This study aims to investigate whether ER stress is involved in tubular destruction and interstitial fibrosis in vivo. Renal cell apoptosis was proven by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) stain and poly-ADP ribose polymerase expression in the unilateral ureteral obstruction (UUO) kidney. ER stress was evoked and confirmed by the upregulation of glucose-regulated protein 78 (GRP78) and the common Lys-Asp-Glu-Leu (KDEL) motif of ER retention proteins after UUO. ER stress-associated proapoptotic signals, including B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2-associated × protein (BAX) expression, caspase-12 and c-Jun N-terminal kinase (JNK) phosphorylation, were activated in the UUO kidney. Prolonged ER stress attenuated both unsplicing and splicing X-box binding protein 1 (XBP-1) protein expression, but continued to activate inositol-requiring 1α (IRE1α)-JNK phosphorylation, protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α subunit (eIF2α), activating transcription factor (ATF)-4, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleavage activating transcription factor 6 (cATF6)-CHOP signals, which induce ER stress-related apoptosis but attenuate adaptive unfolded protein responses in UUO kidneys. However, renal apoptosis and fibrosis were attenuated in candesartan-treated UUO kidney. Candesartan was associated with maintenance of XBP-1 expression and attenuated ATF4, cATF6 and CHOP protein expression. Taken together, results show that overwhelming ER stress leads to renal cell apoptosis and subsequent fibrosis; and candesartan, at least in part, restores renal integrity by blocking ER stress-related apoptosis. Reducing ER stress may present a way to attenuate renal fibrosis.

背景与目的肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducting ligand, TRAIL）可诱导肿瘤细胞发生凋亡，然而相当数量的肿瘤细胞可耐受TRAIL诱导的凋亡而得以存活。本实验观察硫利达嗪（thioridazine, THZ）通过诱导内质网应激（endoplasmic reticulum stress, ER stress）介导的死亡受体5（death receptor 5, DR5）表达上调，继而增敏TRAIL对肺腺癌细胞PC9的生长抑制及凋亡诱导效应，探讨其机制。方法不同浓度硫利达嗪及TRAIL单独或联合处理PC9细胞，MTT法检测细胞活性变化，流式细胞术检测细胞表面DR5表达及细胞凋亡率，Western blotting检测内质网应激相关蛋白GRP78（glucose regulated protein 78）、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（C/EBP homologous protein, CHOP）、p-PERK（PKR-like ER kinase）、p-eIF2α（eukaryotic initiation factor-2α, eIF2α）、ATF4（activating transcription factor 4, ATF4）及凋亡相关蛋白Caspase-3、Caspase-9、Caspase-8、PARP、DR5表达变化。结果硫利达嗪对PC9细胞的增殖抑制效应呈浓度依赖性（P < 0.05），硫利达嗪可增加TRAIL对PC9细胞的抑制作用及凋亡诱导作用且可上调PC9细胞表面DR5表达水平，流式细胞术结果显示：TRAIL联合硫利达嗪组细胞凋亡率较单独TRAIL组显著增加（P < 0.05），Western blotting结果显示：TRAIL联合硫利达嗪组细胞Cleaved-caspase-8、Cleaved-PARP、DR5表达水平较单独TRAIL组明显上调。DR5表达上调及促凋亡效应是通过诱导内质网应激发生，并伴随着GRP78及CHOP表达上调发生的，且该效应可被4-苯基丁酸（4-phenylbutyric acid, 4-PBA）可抑制（P < 0.05）。结论硫利达嗪增敏TRAIL对PC9细胞的增殖抑制效应显著，其机制可能与硫利达嗪内质网应激介导的DR5上调有关。

Endoplasmic reticulum (ER) stress is a causative factor of inflammatory bowel diseases. ER stress mediators, including CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP), are elevated in intestinal epithelia from patients with inflammatory bowel diseases. The present study arose from the question of how chemical ER stress and CHOP protein were associated with nuclear factor-κB (NF-κB)-mediated epithelial inflammatory response. In a human intestinal epithelial cell culture model, chemical ER stresses induced proinflammatory cytokine interleukin-8 (IL-8) expression and the nuclear translocation of CHOP protein. CHOP was positively involved in ER-activated IL-8 production and was negatively associated with expression of peroxisome proliferator-activated receptor γ (PPARγ). ER stress-induced IL-8 production was enhanced by NF-κB activation that was negatively regulated by PPARγ. Mechanistically, ER stress-induced CHOP suppressed PPARγ transcription by sequestering C/EBPβ and limiting availability of C/EBPβ binding to the PPARγ promoter. Due to the CHOP-mediated regulation of PPARγ action, ER stress can enhance proinflammatory NF-κB activation and maintain an increased level of IL-8 production in human intestinal epithelial cells. In contrast, PPARγ was a counteracting regulator of gut inflammatory response through attenuation of NF-κB activation. The collective results support the view that balances between CHOP and PPARγ are crucial for epithelial homeostasis, and disruption of these balances in mucosal ER stress can etiologically affect the progress of human inflammatory bowel diseases.

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P&lt;0.05 or P&lt;0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P&lt;0.05 or P&lt;0.01), while Bcl-2 was significantly up-regulated(P&lt;0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P&lt;0.01), whereas Bcl-2 was significantly up-regulated(P&lt;0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.