内质网应激偶联炎性反应研究进展

内质网是真核细胞蛋白合成和折叠的主要细胞器,当内质网内蛋白合成或折叠负担增加,引起未折叠或错误折叠蛋白增多时,可激活内质网几条特定信号通路,启动未折叠蛋白反应,这对维持细胞稳态有重要意义。越来越多研究表明,多种炎性反应疾病与内质网应激有密切联系。一方面,内质网应激引起的未折叠蛋白反应可以诱发或者抑制炎症,另一方面,炎性反应也能影响蛋白折叠,从而促进或缓解内质网应激。2型糖尿病、肿瘤和动脉粥样硬化等多种重大疾病的病理机制都涉及内质网应激与炎性反应的相互作用,对该问题的深入研究不仅能加深人们对这些疾病发病机制的理解,也有助于相关药物的研发。     

Effects of naringenin on PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways in rats with myocardial ischemia/reperfusion injury

AIM To observe the effect of naringenin on cardiac injury in ischemia/reperfusion （I/R） rats, and to explore whether the role of naringenin is involved in PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways. METHODS SD rats （n=48） were randomly divided into sham operation （sham） group, model （I/R） group, naringenin treatment （NAR） group and naringenin+LY294002 （NL） group. Myocardial I/R injury model was prepared by ligation of left anterior descending coronary artery of rats for 30 min followed by reperfusion for 120 min. After reperfusion, the serum levels of cardiac troponin I （cTnI） was measured by ELISA. HE staining, TTC staining and TUNEL staining were used to detect the myocardial histopathological changes, myocardial infarction area and myocardial cell apoptotic rate. The mRNA levels of endoplasmic reticulum stress-related indicators glucose-regulated protein 78 （GRP78）, C/EBP homologous protein （CHOP） and caspase-12 were detected by RT-qPCR. The protein levels of cleaved caspase-3, GRP78, CHOP, caspase-12, p-PI3K and p-AKT were determined by Western blot. RESULTS Compared with I/R group, the serum content of cTnI, myocardial pathological damage, myocardial infarction area and myocardial cell apoptotic rate were significantly reduced （P<0.05）, the protein levels of cleaved caspase-3, GRP78, CHOP and caspase-12 were decreased （P<0.05）, and the protein levels of p-PI3K and p-AKT were increased in NAR group （P<0.05）. LY294002 attenuated the protective effect of naringenin to some extent. CONCLUSION Naringenin reduces myocardial I/R injury in rats possibly by activating PI3K/AKT signaling pathway and subsequently regulating endoplasmic reticulum stress and its related apoptotic pathways.     

Suberoylanilide hydroxamic acid induces apoptosis of HepG2 cells by endoplasmic reticulum stress apoptotic pathway

AIM: To investigate the effect of suberoylanilide hydroxamic acid (SAHA) on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells and to explore its possible mechanism. METHODS: HepG2 cells were treated with SAHA at different concentrations for 48 h. The proliferation of HepG2 cells was detected by real-time cellular analysis. The protein levels of acetylated histones H3K9 and H3K27, glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK) and p-PERK were determined by Western blot. The cell apoptosis was analyzed by flow cytometry. RESULTS: Compared with control group, treatment with SAHA at 0.1 μmol/L and 1 μmol/L for 48 h showed no significant inhibitory effect on the proliferation of HepG2 cells, while SAHA at 6 μmol/L and 12 μmol/L significantly inhibited the proliferation of HepG2 cells (P<0.05). The results of Western blot showed that the protein levels of acH3K9, acH3K27, GRP78 and p-PERK increased significantly after treated with SAHA at diffe-rent concentrations for 48 h, while the protein level of PERK was decreased significantly (P<0.05). The results of flow cytometry analysis showed that the apoptotic rates of the HepG2 cells increased with the increase in SAHA concentration. CONCLUSION: SAHA up-regulates the acetylation of H3K9 and H3K27 in the HepG2 cells and induces apoptosis of HepG2 cells by activating the endoplasmic reticulum stress-related apoptosis pathway.     

Model construction of rat coronary artery smooth muscle cell endoplasmic reticulum stress induced by thapsigargin

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress (ERS) model in CASMCs of SD rats. METHODS: CASMCs were cultured by tissue explant method. The morphological characteristics were observed under optical microscope. The marker proteins of CASMCs, including α-SMA and SM-MHC, were identified by immunofluorescence technique. The protein expression levels of BiP and CHOP, the marker molecules of ERS, were determined by Western blot. RESULTS: The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical "hill and valley" growth pattern of CASMCs at 9~10 d. The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed. The results of Western blot showed that the protein expression of BiP and CHOP in TG (1 and 2 μmol/L) treatment groups was increased compared with control group. Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION: CASMCs can be successfully cultured by tissue explant method. ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.     

Protective effect of Huangqi injection combined with puerarin injection on myocardium of type 2 diabetes mice

AIM:To investigate the effect of Huangqi injection combined with puerarin injection on the myocardium of the mice with type 2 diabetes. METHODS:Diabetic KKAy mice were randomly divided into model group and treatment group (Huangqi injection combined with puerarin injection). The male KKAy mice of the same age were used as control group. All mice were sacrificed at 21, 24 and 28 weeks. Morphological changes of the myocardium were observed by HE staining. Apoptosis of the cardiomyocytes was measured by TUNEL staining. The mRNA levels of glucose-regulated protein 78(GRP78), C/EBP hoinologous protein (CHOP) and p53 up-regulated modulator of apoptosis (PUMA) were detected by real-time PCR, and the protein levels of GRP78, CHOP, PUMA, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, poly(ADP-ribose) polymerase (PARP) and cleaved PARP were determined by Western blot. RESULTS:Cardiomyocyte hypertrophy, partly dissolved sarcoplasm and necrosis were observed in model group, and these lesion were alleviated in treatment group. Obvious increased apoptosis in model group and significantly decreased apoptosis of cardiomyocytes in treatment group was observed (P<0.05). At 21, 24 and 28 weeks, the mRNA and protein levels of GRP78, CHOP and PUMA and the protein levels of cleaved caspase-3, cleaved caspase-9 and cleaved PARP in model group were increased significantly as compared with control group (P<0.01), and these in treated group were decreased compared with model group. CONCLUSION:Huangqi injection combined with puerarin injection has cardioprotective effects on type 2 diabetes mice and its mechanism of the action was implemented via inhibiting the activation of endoplasmic reticulum stress and caspase pathway, thus resulting in suppressed apoptosis.     

Effects of capsaicin on cognitive impairment and mitochondria-associated endoplasmic reticulum membranes in rats with chronic cerebral hypoperfusion

AIM:To observe the effects of capsaicin on cognitive impairment and mitochondria-associated endoplasmic reticulum membranes (MAMs) of hippocampal CA1 area in the rats with chronic cerebral hypoperfusion (CCH), and to investigate the underlying molecule mechanism of cognitive defects induced by ischemia. METHODS:Healthy male Sprague-Dawley (SD) rats(n=48) were randomly divided into sham operation (sham) group,CCH model (CCH) group, capsaicin group,and solvent group, 12 rats in each group. Capsaicin at 2.5 mg/kg was intraperitoneally injected twice a week for 4 weeks, starting on the 7th day after surgery. The rats in solvent group were given the same amount of solvent at the same time and under the same conditions. Morris water maze, object recognition test and open field test were conducted to analyze the cognitive related behavior performance on the 4th week after surgery. The changes of MAMs in the hippocampal CA1 region were observed under transmission electron microscope, the co-localization of the MAMs was observed by immunofluorescence double-labeling, and the expression of mitofusin 2 (Mfn2) in the hippocampal tissue was determined by Western blot.RESULTS:Four weeks after the operation, the behavior tests showed that the cognitive function of CCH rats was impaired compared with sham operation group. Compared with solvent group, spatial learning and memory in capsaicin group was improved significantly. The results of transmission electron microscope and confocal microscope showed that the distance of MAMs in the hippocampal CA1 area of CCH rats was increased compared with sham operation group, and the co-localization of the contacts was decreased (P<0.05). Compared with solvent group, the correlation between the mitochondria and ER in capsaicin group was increased (P<0.05). The protein level of Mfn2 in CCH group was significantly lower than that in sham group (P<0.05). Compared with solvent group, the protein level of Mfn2 in capsaicin group was higher (P<0.05). CONCLUSION:CCH rats showed decreased cognitive function and loosen MAMs. Capsaicin improves the cognitive behavior of CCH rats by up-regulation of MAMs.     

Role of PERK pathway in morphine-induced myocardial protection of H9c2 cells

AIM: To explore whether morphine protects oxidative stress-damaged myocardial cells by inhibiting the PERK pathway to reduce endoplasmic reticulum stress and prevent mitochondrial permeability transition pore (mPTP) opening. METHODS: Rat myocardial H9c2 cells were cultured to establish an oxidative stress model, and then randomly divided into control group, H₂O₂ group, H₂O₂+morphine group, H₂O₂+morphine+PERK pathway inhibitor GSK2656157 group, morphine group and GSK2656157 group. Immunohistochemical method was used to detect the effects of morphine on expression of glucose-regulated protein (GRP) 78 and GRP94 induced by oxidative stress. The protein levels of PERK signaling pathway-related molecules were determined by Western blot. Confocal microscopy was used to observe the effects of morphine on mPTP opening and endoplasmic reticulum induced by oxidative stress. Cellular toxicity was detected by lactate dehydrogenase (LDH) kit and cell viability was measured by MTT assay. RESULTS: Compared with control group, GRP78 and GRP94 proteins in H₂O₂ group were strongly expressed, and the brown-yellow particles were significantly increased, but morphine significantly inhibited this process. Compared with control group, the phosphorylation of PERK was significantly reduced with GSK2656157 treatment at different concentrations, among which 2 μmol/L had the most significant effect (P < 0.05). Oxidative stress significantly increased the protein levels of GRP78, GRP94, p-PERK and CHOP, but significantly decreased p-GSK-3β level. These changes were inhibited by morphine, and the effects of morphine were further enhanced by GSK2656157 (P < 0.05). Compared with control group, oxidative stress significantly reduced the fluorescence intensity of mitochondrial TMRE and ER-Tracker Red. Morphine significantly inhibited this effect even when mitochondrial membrane potential was reduced, mPTP was open, and endoplasmic reticulum was damaged, while GSK2656157 further enhanced the effect of morphine (P < 0.05). Compared with control group, H₂O₂ significantly increased cellular toxicity and decreased the cell viability. Morphine inhibited this effect and GSK2656157 significantly enhanced the effect of morphine (P < 0.05). CONCLUSION: Morphine protects cardiac H9c2 cells under oxidative condition by inhibiting endoplasmic reticulum stress through PERK pathway and preventing the mPTP opening via GSK-3β inactivation.     

Effects of calpain-2 and autophagy-related protein 5 on hepatocyte apoptosis induced by endoplasmic reticulum stress

AIMTo investigate the effects of calpain-2 and autophagy-related protein 5 (Atg5) on apoptosis of BRL-3A rat normal liver cells during endoplasmic reticulum stress (ERS) induced by dithiothreitol (DTT). METH?ODS: BRL-3A cells were treated with DTT at 2.0 mmol/L for 0, 6, 12 and 24 h to induce ERS. Real-time cell analysis (RTCA) was used to measure the effect of DTT on BRL-3A cell proliferation. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mRNA expression of calpain-2 and Atg5 was detected by real-time PCR. The protein levels of calpain-2, Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3) were determined by Western blot. The interaction between calpain-2 and Atg5 was investigated by co-immunoprecipitation (Co-IP). RESULTSThe proliferation of BRL-3A cells treated with DTT was significantly inhibited. The apoptosis of BRL-3A cells was significantly increased after DTT treatment for 6, 12 and 24 h as compared with 0 h group (P<0.05). The cell cycle was arrested in G₁ phase after DTT treatment (P<0.05). After DTT treatment for 6, 12 and 24 h, the mRNA expression of calpain-2 and Atg5 in the BRL-3A cells was significantly increased as compared with 0 h group (P<0.05). The protein levels of calpain-2, Atg12 and Atg7 in the cells treated with DTT for 6, 12 and 24 h were significantly higher than those in 0 h group, and the ratio of LC3-II/LC3-I was also significantly higher than that in 0 h group, while Atg5 expression was significantly lower than that in 0 h group (P<0.05). The results of Co-IP found that the anti-calpain-2 antibody precipitated Atg5 protein from the cell lysates, and the anti-Atg5 antibody also precipitated calpain-2 from the cell lysates, which confirmed the interaction between calpain-2 and Atg5. CONCLUSION Calpain-2 may participate in ERS-induced hepatocyte apoptosis by interacting with Atg5.     

Role of endoplasmic reticulum stress in trimethylamine N-oxide-induced oxidative stress in human umbilical vein endothelial cells

AIM: To investigate whether endoplasmic reticulum stress is involved in trimethylamine N-oxide (TMAO)-mediated oxidative stress in human umbilical vein endothelial cells (HUVECs). METHODS: The cell viability was examined by CCK-8 assay. The cells were stained by DCFH-DA, and the intracellular level of reactive oxygen species (ROS) was observed by phase-contrast microscopy and detected by flow cytometric analysis. The protein levels of phospho-IRE-1α, IRE-1α and GRP78/BiP were detected by Western blot. RESULTS: TMAO exerted no significant effect on the viability of HUVECs. For a long period (>24 h), even a low concentration (10 μmol/L) of TMAO increased the oxidative stress level in the HUVECs (P<0.05). TMAO increased the phosphorylation level of IRE-1α and significantly up-regulated the protein level of GRP78/BiP in HUVECs (P<0.01). Pretreatment with STF-083010, an inhibitor of IRE1α, for 1 h reduced TMAO-induced oxidative stress in HUVECs (P<0.05). CONCLUSION: Endoplasmic reticulum stress is involved in TMAO-induced oxidative stress in HUVECs.     

烟草NbbZIP28突变体的创建及其对病毒侵染胁迫的响应

【目的】bZIP类转录因子参与植物的生长发育、激素信号、抗病性及抗逆性等多种生物胁迫过程。前期研究表明黄瓜花叶病毒(Cucumber mosaic virus,CMV)或烟草花叶病毒(Tobacco mosaic virus,TMV)侵染本氏烟(Nicotiana benthamiana)上调内质网应激（endoplasmic reticulum stress,ERs）因子NbbZIP28;NbbZIP28沉默导致病毒积累量上升,本研究旨在验证NbbZIP28对病毒侵染胁迫的响应机制。【方法】通过CRISPR/Cas9基因编辑技术和烟草遗传转染创建NbbZIP28基因突变植株,以野生型植株为对照,分别浸润接种侵染性克隆TMV-GFP、摩擦接种TMV-GFP或CMV接种液,检测突变体植株对病毒侵染胁迫的敏感性变化,接种CMV后0-48 h,采用qRT-PCR检测内质网应激相关的未折叠蛋白反应（unfolded protein response,UPR)基因的表达。本氏烟接种TMV 24 h、接种CMV 48 h后,在接种叶上浸润融合蛋白NbbZIP28-GFP,瞬时表达48 h后,采用Western blot检测病毒诱导NbbZIP28蛋白的水解激活;采用在线搜索工具PlantCARE分析NbbZIP28启动子区域中参与防卫和应激反应的顺式作用元件。【结果】CRISPR/Cas9定点敲除NbbZIP28后,目的基因靶位点缺失了10个碱基,导致翻译错误、蛋白功能变化。在正常生长条件下,转基因阳性植株与野生型无显著的表型差异。植株接种TMV-GFP后4-8 d,突变体中的病毒浸润斑亮度或初侵染点数目均显著高于野生型,扩展至新叶的速度较快。接种CMV后12-48 h,突变体中UPR相关基因BiP、PDI、CAM及NbbZIP28下游基因NF-YC2的表达量显著低于野生型;5-7 d突变体中的病毒外壳蛋白（coat protein,CP）基因表达量显著高于野生型,花叶及皱缩症状更显著。蛋白质序列比对分析显示NbbZIP28具有与拟南芥AtbZIP28相同的S1P和S2P蛋白酶水解位点。Western blot检测发现,与接种清水对照相比,TMV或CMV侵染显著促进了全长的融合蛋白NbbZIP28-GFP在S1P和S2P位点发生裂解。NbbZIP28启动子序列中包含5个参与热应激反应的顺式作用元件（heat stress response element,HSE）、3个参与低温反应的顺式作用元件（low temperature response element,LTR）、1个参与防卫和应激反应的顺式作用元件（TC-rich repeats）。【结论】病毒侵染促进NbbZIP28的水解激活并上调相关的UPR基因;NbbZIP28敲除导致植株对病毒的敏感性上升,病毒诱导的UPR基因表达被抑制。NbbZIP28为病毒侵染胁迫下的UPR调控因子,在病毒侵染早期通过上调UPR信号和提高寄主基础防卫反应而延缓病毒的侵染和增殖。