Expression of N-methyl-D-aspartic acid receptor and endoplasmic reticulum stress related pathway proteins in brain tissue of fluorosis rats / 中国组织工程研究

BACKGROUND:Previous studies have shown that N-methyl-D-aspartic acid receptor(NMDA)receptors are associated with fluorine,but the role in fluoride-induced endoplasmic reticulum stress remains unclear. OBJECTIVE:To observe the changes of excitatory neurotransmitter NMDA receptor and endoplasmic reticulum stress IRE1α-ASK1-JNK pathway protein expression in brain tissue of rats with experimental fluorosis,and to investigate the pathogenesis of neurological injury in fluorosis by giving NMDA receptor inhibitor to SH-SY5Y cells. METHODS:(1)Animal model:18 1-month-old SD rats were randomly divided into control group(drinking water fluoride content<0.5 mg/L),low fluoride group(drinking water fluoride content 10.0 mg/L)and high fluoride group(drinking water fluoride content 100.0 mg/L),with 6 rats in each group,half of each sex.After 6 months of fluoride intake,the rats were observed for the occurrence of dental fluorosis,and the 24-hour urinary fluoride content was measured.After anesthesia and euthanasia,the brain tissue of rats was taken to observe the pathological changes.Western blot assay was used to detect NMDA receptors and IRE1α,ASK1 and JNK protein expression in the brain tissue.(2)Cell model:SH-SY5Y cells were cultured in vitro and treated with sodium fluoride at final concentrations of 0.3 mmol/L and 3 mmol/L.The fluoride-stained cells were interfered with 10 μmol/L NMDA receptor antagonists Ifenprodil and MK-801 to observe the relevant protein changes. RESULTS AND CONCLUSION:(1)The incidence of dental fluorosis and urinary fluoride level in rats in the high fluoride group were significantly higher than that in the control and low fluoride groups(P<0.05).(2)Compared with the control group,the cytoplasm of neuronal cells in the CA3 area of the hippocampus in the low fluoride group was slightly more basophilic,while the neuronal cells in the CA3 area of the high fluoride group were disorganized,with increased basophilicity and some of the nuclei solidified.(3)In rat brain tissue,the expressions of NR2A in the high fluoride group and NR2B in the low fluoride group were significantly higher compared with the control group(P<0.05),and NR2B,IRE1,ASK1,and p-JNK protein expression levels were increased in the high fluoride group compared with the control and low fluoride groups(P<0.05).(4)In SH-SY5Y cells,NR1,NR2A and NR2B protein expressions were significantly increased in the high fluoride group compared to the control group(P<0.05).The protein levels of NR1 and NR2A were significantly reduced in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group(P<0.05).NR2B protein expression was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group(P<0.05).(5)In SH-SY5Y cells,IRE1,ASK1,and p-JNK protein expression was significantly higher in the high fluoride group compared with the control group(P<0.05),while ASK1 and p-JNK protein expressions were significantly decreased in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group(P<0.05).IRE1 protein level was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group(P<0.05).(6)It is concluded that excessive fluorine intake activates NMDA receptors in the central nervous system,causing increased expression of endoplasmic reticulum stress IRE1α,ASK1,and p-JNK proteins,and the use of NMDA receptor inhibitors has a mitigating effect on endoplasmic reticulum stress caused by fluorosis.

Effects of Ziziphi Spinosae Semen-Albiziae Flos on the IRE1α/ASK1/JNK Pathway of Endoplasmic Reticulum Stress in Hippocampus of Depression Model Rats / 中药新药与临床药理

Objective Based on the endoplasmic reticulum stress(ERS)inositol-requiring enzyme-1α(IRE1α)/apoptosis signal-regulated kinase 1(ASK1)/c-Jun N-terminal kinase(JNK)pathway to investigate the intervention effect of Ziziphi Spinosae Semen(ZSS)-Albiziae Flos(AF)on the depression model of rats,which were prepared by solitary rearing combined with chronic unpredictable mild stress(CUMS).Methods A total of 144 rats were randomly divided into normal group,model group,high-,medium-and low-dose groups of ZSS-AF,and Venlafaxine group.In addition to the normal group,the rats in other groups were isolated for 21 days combined with CUMS to prepare the depression model.The behavioral changes of rats were observed by open field test and Morris water maze test.The ultrastructural changes of hippocampal neurons were observed by transmission electron microscope.TUNEL staining was used to observe the apoptosis of nerve cells in hippocampus.The protein expression levels of IRE1α,phosphorylated(P)-IRE1α,ASK1,P-ASK1,JNK,P-JNK,B cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cysteme aspartate specific protease-3(Caspase-3)in hippocampus were detected by Western Blot.The mRNA expression levels of IRE1α,ASK1,JNK,Bax,Bcl-2 and Caspase-3 in hippocampus were detected by Real-Time PCR.Results Compared with the normal group,the scores of horizontal movement and vertical movement in the open field test of rats in the model group were decreased(P＜0.01).In the water maze test,the escape latency was increased and the number of crossing the original platform was decreased(P＜0.01).The number of hippocampal apoptosis was increased(P＜0.01).The protein expression levels of P-IRE1 α/IRE1 α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 and mRNA expressions of IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were increased,while the protein and mRNA expression levels of Bcl-2 were decreased(P＜0.01).Compared with model group,the scores of horizontal movement and vertical movement in the open field test of rats in each dose ZSS-AF groups and Venlafaxine group were increased(P＜0.01).In the water maze test,the escape latency was decreased and the times of crossing the original platform was increased(P＜0.01).The number of hippocampal apoptosis was decreased(P＜0.01).The mRNA expression levels of P-IRE1α/IRE1α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 protein and IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were decreased,while the protein and mRNA expression levels of Bcl-2 were increased(P＜0.05,P＜0.01).The effect of medium-dose ZSS-AF group was better than that of high-and low-dose groups.Conclusion ZSS-AF may play an antidepressant role by regulating IRE1α/ASK1/JNK pathway of endoplasmic reticulum stress.

Identification of a natural PLA2 inhibitor from the marine fungus Aspergillus sp. c1 for MAFLD treatment that suppressed lipotoxicity by inhibiting the IRE-1α/XBP-1s axis and JNK signaling

Lipotoxicity is a pivotal factor that initiates and exacerbates liver injury and is involved in the development of metabolic-associated fatty liver disease (MAFLD). However, there are few reported lipotoxicity inhibitors. Here, we identified a natural anti-lipotoxicity candidate, HN-001, from the marine fungus Aspergillus sp. C1. HN-001 dose- and time- dependently reversed palmitic acid (PA)-induced hepatocyte death. This protection was associated with IRE-1α-mediated XBP-1 splicing inhibition, which resulted in suppression of XBP-1s nuclear translocation and transcriptional regulation. Knockdown of XBP-1s attenuated lipotoxicity, but no additional ameliorative effect of HN-001 on lipotoxicity was observed in XBP-1s knockdown hepatocytes. Notably, the ER stress and lipotoxicity amelioration was associated with PLA2. Both HN-001 and the PLA2 inhibitor MAFP inhibited PLA2 activity, reduced lysophosphatidylcholine (LPC) level, subsequently ameliorated lipotoxicity. In contrast, overexpression of PLA2 caused exacerbation of lipotoxicity and weakened the anti-lipotoxic effects of HN-001. Additionally, HN-001 treatment suppressed the downstream pro-apoptotic JNK pathway. In vivo, chronic administration of HN-001 (i.p.) in mice alleviated all manifestations of MAFLD, including hepatic steatosis, liver injury, inflammation, and fibrogenesis. These effects were correlated with PLA2/IRE-1α/XBP-1s axis and JNK signaling suppression. These data indicate that HN-001 has therapeutic potential for MAFLD because it suppresses lipotoxicity, and provide a natural structural basis for developing anti-MAFLD candidates.

Effects of Angelica sinensis polysaccharide on the apoptosis of cardiomyocytes in diabetic KK-Ay mice / 中国药房

OBJECTIVE To investigate the effects of Angelica sinensis polysaccharide on the apoptosis of cardiomyocytes in diabetic KK-Ay mice. METHODS KK-Ay mice were randomly divided into model group， metformin group （200 mg/kg） and A. sinensis polysaccharide high-dose， medium-dose and low-dose groups （400， 200 and 100 mg/kg）； C57BL/6J mice were included in blank group， with 8 mice in each group. Each group was given relevant medicine intragastrically or normal saline， once a day， for consecutive 4 weeks. After the final administration， the levels of fasting glucose， total cholesterol （TC）， low-density lipoprotein cholesterol （LDL-C） and insulin （INS） were detected； the protein expressions of B-cell lymphoma 2 （Bcl-2）， cleaved- caspase-3， apoptosis signal-regulated kinase 1 （ASK1）， phosphorylated c-Jun N-terminal kinase （p-JNK）， phosphorylated inositol- requiring enzyme 1α （p-IRE1α） in myocardium， and apoptosis in cardiomyocytes were also detected. RESULTS Compared with model group， the fasting glucose， TC and LDL-C content， apoptotic rate of cardiomyocyte， protein expressions of p-JNK and p- IRE1α， ASK1， cleaved-caspase-3 were significantly lower in the metformin group and A. sinensis polysaccharide medium-dose， high-dose groups； INS level and relative expression of Bcl-2 protein were significantly increased （P＜0.05 or P＜0.01）. CONCLUSIONS A. sinensis polysaccharide can improve the levels of blood glucose and blood lipid and inhibit cardiomyocyte apoptosis in diabetic KK-Ay mice， and the mechanism may be related to the inhibition of IRE1/ASK1/JNK signaling pathway.

Effect of Modified Huangqi Guizhi Wuwutang on Stress-related IRE1α/CHOP Pathway of Sciatic Nerve Endoplasmic Reticulum in Diabetes Rats / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of modified Huangqi Guizhi Wuwutang （MHGW） on endoplasmic reticulum stress in the sciatic nerve of diabetes rats based on the pathways of inositol-requiring enzyme 1α （IRE1α） and CCAAT/enhancer-binding protein homologous protein （CHOP）. MethodSixty rats were fed on a high-sugar and high-fat diet for six weeks， followed by intraperitoneal injection of streptozotocin at a dose of 35 mg·kg-1. Random blood glucose levels were measured three days later and rats with a sustained blood glucose level ≥ 16.7 mmol·L-1 were included in study （n=48）. The rats were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg-1·d-1）， a high-dose MHGW group （2.5 g·kg-1·d-1）， and a low-dose MHGW group （1.25 g·kg-1·d-1）. Another 10 rats were assigned to the normal group. The intervention lasted for 16 weeks. After 16 weeks， the sciatic nerve structure of the rats in each group was observed under light microscopy using Luxol fast blue （LFB） staining. Transmission electron microscopy was used to observe the ultrastructure of the sciatic nerve. Chemiluminescence method was employed to measure the serum reactive oxygen species （ROS） levels. Western blot and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to evaluate the expression of p-IRE1α protein， IRE1α mRNA， CHOP protein， and CHOP mRNA in the sciatic nerve of the rats. ResultCompared with the normal group， the model group showed elevated serum ROS levels （P<0.01）. In contrast， the serum ROS levels were significantly reduced in the treatment groups compared with those in the model group （P<0.01）. The sciatic nerve of the model group showed pathological changes compared with that in the normal group， while the treatment groups exhibited improvement in sciatic nerve pathology compared with the model group. The protein expression of p-IRE1α and CHOP in the sciatic nerve significantly increased in the model group as compared with that in the normal group （P<0.01）. However， the treatment groups showed a significant decrease in the protein expression of p-IRE1α and CHOP in the sciatic nerve compared with the model group （P<0.05， P<0.01）. Furthermore， compared with the normal group， the model group showed upregulated mRNA expression of IRE1α and CHOP in the sciatic nerve （P<0.01）， while the treatment groups exhibited a significant decrease in the mRNA expression of IRE1α and CHOP compared with the model group （P<0.01）. ConclusionMHGW can alleviate endoplasmic reticulum stress-induced cell apoptosis and improve the structure and function of the sciatic nerve in diabetes rats by inhibiting the expression of IRE1α/CHOP pathway-related proteins and mRNA， thereby preventing and treating peripheral neuropathy in diabetes.

Effect of fritinib on angiogenesis, tumor growth and IRE1-ASK1-JNK pathway in triple negative breast cancer / 国际肿瘤学杂志

Objective:To investigate the effects of fritinib on angiogenesis, tumor growth and inositol requiring enzyme 1 (IRE1) -apoptosis signal regulating kinase 1 (ASK1) -c-Jun N-terminal kinase (JNK) pathway in triple negative breast cancer.Methods:Triple negative breast cancer cells MDA-MB-231 were taken and divided into normal saline (NS) group, low-dose fritinib (LD) group, medium-dose fritinib (MD) group and high-dose fritinib (HD) group. NS group was added with 100 μmol/L normal saline. LD group, MD group and HD group were added with 25, 50 and 100 μmol/L fritinib, respectively. Cell proliferation was detected by MTT assay, cell apoptosis was detected by flow cytometry, the ability of cells to form mimicry vessels was observed by three-dimensional cell culture, and the related indexes of angiogenesis and IRE1-ASK1-JNK pathway were detected by Western blotting. Twenty triple-negative breast cancer rat models were divided into control group and experimental group by random number table method, with 10 rats in each group. The control group was given normal saline gavage and the experimental group was given 100 μmol/L fritinib gavage. The tumor growth of rats in the two groups was observed and recorded.Results:The 48 h cell proliferation rates of NS group, LD group, MD group and HD group were (85.44±5.58) %, (73.24±4.95) %, (61.53±4.07) % and (50.23±2.97) %, respectively ( F=4.01, P=0.002). Compared with the NS group, the cell proliferation rate in LD, MD and HD groups was significantly decreased in a dose-dependent manner (all P<0.05). The apoptosis rates of NS group, LD group, MD group and HD group were (3.41±0.39) %, (18.75±1.94) %, (24.97±2.58) % and (38.62±3.27) %, respectively ( F=18.99, P<0.001). Compared with the NS group, the apoptosis rate of LD, MD and HD groups was significantly increased in a dose-dependent manner (all P<0.05). The number of mimicry vessels in NS group, LD group, MD group and HD group was 19.58±2.11, 15.67±2.02, 11.57±1.73 and 5.20±1.23, respectively ( F=3.28, P=0.008). Compared with the NS group, the number of mimicry vessels in LD group, MD group and HD group was significantly reduced. The results were dose-dependent (all P<0.05). vascular endothelial growth factor (VEGF) protein expression in NS group, LD group, MD group and HD group was 2.36±0.21, 1.79±0.17, 1.48±0.14 and 0.94±0.10, respectively ( F=5.17, P<0.001). The expression of IRE1 protein was 1.18±0.12, 1.67±0.18, 2.03±0.24 and 2.39±0.28, respectively ( F=5.55, P<0.001). The expression of ASK1 protein was 1.09±0.11, 1.46±0.13, 1.81±0.18, 2.33±0.21 ( F=5.32, P<0.001), respectively. JNK protein expression was 1.01±0.09, 1.48±0.14, 1.86±0.21 and 2.28±0.24, respectively ( F=6.92, P<0.001). Compared with the NS group, VEGF protein expression in LD, MD and HD groups was significantly decreased, and the expressions of IRE1, ASK1 and JNK were significantly increased in a dose-dependent manner (all P<0.05). Compared with the control group, the tumor weight and volume of the experimental group were significantly decreased [ (0.55±0.10) g vs. (1.37±0.15) g, t=14.38, P<0.001; (77.39±3.21) mm 3vs. (118.26±5.34) mm 3, t=20.74, P<0.001], tumor inhibition rate was significantly increased [ (71.23±3.85) % vs. (32.56±3.08) %, t=24.80, P<0.001]. Conclusion:Fritinib has an inhibitory effect on the activity of triple negative breast cancer cells, which can significantly reduce their angiogenesis and inhibit tumor growth. Moreover, it is related to the activation of IRE1-ASK1-JNK pathway.

Hypoxia activation IRE1a/JNK pathway regulates the proliferation and apoptosis of pulmonary artery smooth muscle cells in mice / 中华急诊医学杂志

Objective:To investigate whether hypoxia induces endoplasmic reticulum stress (ERS) through inositol-dependent enzyme 1α (IRE1α) and activates JNK pathway to participate in the proliferation and apoptosis of pulmonary artery smooth muscle cells in mice.Methods:Mouse pulmonary artery smooth muscle cell line (MPASMCs) was cultured in vitro to establish the hypoxic MPASMCs model. The expression of hypoxia-inducible factor-1α (HIF-1α), glucose-regulated protein 78 (GRP78) and JNK pathway genes were detected. The expression of IRE1α was knocked down by siRNA transfection, JNK pathway specific inhibitor 1, 9-pyrazoxanolone and pyrazoxanolone (SP600125) was used to inhibit JNK pathway, and XBP-1s plasmid was transfected to increase the expression of XBP-1s. CCK8 assay and proliferating cell nuclear antigen (PCNA) protein detection were used to observe the cell proliferation. Cell apoptosis was detected by flow cytometry and the expression of B-cell lymphoma-2 (BCL-2) and BCL-2 associated X protein (BAX) protein.Results:HIF-1αexpression was significantly up-regulated in MPASMCs cultured under hypoxia. The expression of GRP78, phosphorylated IRE1α (P-IRE1α) and phosphorylated JNK (P-JNK) increased after hypoxia, indicating that ERS and JNK pathways mediated by IRE1α were activated. When IRE1α expression was inhibited by si-IRE1a, the expression of P-JNK decreased, indicating that JNK pathway was inhibited. The expression of PCNA protein was up-regulated in the hypoxia group, and CCK8 assay indicated that the cell proliferation was up-regulated. The expression of BAX and BCL-2 protein were down-regulated in the hypoxia group, and the level of apoptosis was down-regulated. The above changes were delayed after SiIRE1a inhibited the expression of IRE1α. Treatment with SP600125 could also partially delay the pro-proliferation and anti-apoptosis changes induced by hypoxia. Overexpression of XBP-1s under normoxia activated the JNK pathway, accompanied by hypoxia-like changes.Conclusions:Hypoxia activates IRE1α-mediated endoplasmic reticulum stress, which promotes the proliferation and inhibits apoptosis of pulmonary artery smooth muscle cells through JNK pathway in mice.

Triptolide induces apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1/c-Jun N-terminal kinase signaling pathway / 中华皮肤科杂志

Objective:To investigate the effect of triptolide on the apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1 (IRE1) /c-Jun N-terminal kinase (JNK) signaling pathway, and to explore its possible mechanisms.Methods:Cultured A375 cells were treated with triptolide at different concentrations of 0, 50, 100, 200 nmol/L (experimental control group, 50, 100, 200 nmol/L triptolide groups, respectively), and a blank control group (DMEM high-glucose medium without cells) was set up. Methyl thiazol tetrazolium (MTT) assay was performed to evaluate the cell viability at 24, 48, and 72 hours after the start of treatment, flow cytometry to detect cell apoptosis at 24 hours after the start of treatment, and real-time fluorescence-based quantitative PCR (RT-qPCR) and Western blot analysis were conducted to determine mRNA and protein expression of IRE1, JNK, and c-Jun, respectively. After pretreatment with the JNK inhibitor SP600125 for 72 hours, some A375 cells were then treated with 100 nmol/L triptolide for 24 hours (SP600125 + 100 nmol/L triptolide group), and the A375 cells only treated with 100 nmol/L triptolide served as control group (100 nmol/L triptolide group). Effects of triptolide on the mRNA expression of IRE1, JNK, and c-Jun in A375 cells, as well as on cell apoptosis, were investigated. Statistical analysis was performed using two-way analysis of variance, one-way analysis of variance, and Dunnett′s test.Results:After the treatment with different concentrations of triptolide for different durations, the cell viability was significantly lower in all triptolide groups than in the experimental control group ( Ftriptolide concentration = 18.36, P = 0.002), and gradually decreased over time ( F time = 8.54, P = 0.018). After 24-hour treatment, the apoptosis rate of A375 cells significantly differed among the 4 groups treated with different concentrations of triptolide ( F = 5 234.97, P < 0.001) ; additionally, the apoptosis rate was significantly higher in the 50, 100, and 200 nmol/L triptolide groups (16.99% ± 0.33%, 30.78% ± 0.40%, 38.91% ± 0.51%, respectively) than in the experimental control group (4.33% ± 0.02%, all P < 0.05), and gradually increased with the rising concentrations of triptolide. The mRNA expression levels of IRE1, JNK, and c-Jun were all significantly higher in the 50, 100, and 200 nmol/L triptolide groups than in the experimental control group (all P < 0.05), and gradually increased with the increase of triptolide concentration. Moreover, the protein expression levels of IRE1, JNK, c-Jun, p-JNK, and p-c-Jun in A375 cells in the triptolide groups also showed the same trend. After pretreatment with the JNK inhibitor SP600125 for 72 hours, the apoptosis rate was significantly lower in the SP600125 + 100 nmol/L triptolide group (21.88% ± 0.55%) than in the 100 nmol/L triptolide group without SP600125 pretreatment (30.78% ± 0.40%, t = -22.51, P < 0.001), and the mRNA expression levels of IRE1, JNK, and c-Jun were also significantly decreased in the SP600125 + 100 nmol/L triptolide group compared with the 100 nmol/L triptolide group (all P < 0.05) . Conclusion:Triptolide may induce apoptosis of human melanoma A375 cells by activating the IRE1/JNK signaling pathway.

The inhibitory effect of Averrhoa carambola DMDD on high glucose-induced endoplasmic reticulum stress IRE1α pathway and inflammation in renal tubular epithelial cell HK-2 / 中国药理学通报

Aim To investigate the inhibition effect of 2-dodecyl-6-methoxycyclohexa-2, 5-diene-l, 4-dione ( DMDD) on renal tubular epithelial cell HK-2 endo¬plasmic reticulum stress and inflammatory responses induced by high glucose. Methods HK-2 cells were cultured in vitro and divided into normal group, high glucose group, endoplasmic reticulum stress inhibitor 4-PBA group (5 mmoL • L ) , DMDD high, medium and low dose groups (8,4,2 μmol • L

Research progress of IRE1α inhibitors / 药学学报

Inositol requiring enzyme 1 alpha (IRE1α), a widespread transmembrane protein in mammals, is an endoplasmic reticulum stress (ER stress) receptor. Among the three signaling pathways of the unfolded protein response (UPR), the IRE1α pathway is the most conservative. And there is a growing body of evidence that the occurrence and development of tumors is closely related to the over-expression of IRE1α. Therefore, the study of the IRE1α inhibitors is of great significance to the discovery of new anti-tumor drugs and has been attracting more and more attention. In the hope of providing ideas for the research of targeting IRE1α for cancer therapy, this paper reviewed the data of representative IRE1α inhibitors, including inhibitory activity, the mechanism of action, structural characteristics, and so on.

Effect of Huangqi Guizhi Wuwutang on Diabetic Peripheral Neuropathy in MKR Mice via Regulating Endoplasmic Reticulum Stress / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Huangqi Guizhi Wuwutang （HQGZWWT） in the treatment of diabetic peripheral neuropathy （DPN） in MKR mice via regulating endoplasmic reticulum （ER） stress. MethodThirty-two 8-week-old MKR mice （half were male and half were female） were fed with a high-fat diet for four weeks， and then 1% streptozotocin （STZ） was injected intraperitoneally for five days. After the blood glucose was stabilized， the mice were housed in the cage covered with ice bags for another one hour stimulation per day for four weeks. Mice with fasting blood glucose （FBG） value ≥11.1 mmol·L-1 were randomly divided into model group ， Huangqi Guizhi Wuwutang in original dosage group （30 g·kg-1·d-1）， Huangqi Guizhi Wuwutang in formula dosage group （6.25 g·kg-1·d-1）， and positive drug group （mecobalamin tablets， 0.17 mg·kg-1·d-1）. Another eight MKR mice of the same age were set as blank group and eight FVB mice were normal group. After four weeks of intragastric administration in each group， the change in FBG was tested， and hematoxylin and eosin （HE） staining and transmission electron microscope were used for observing the morphology of sciatic nerve tissue. In addition， the expression of c-Jun N-terminal kinase （JNK）， phosphorylated c-Jun N-terminal kinase （p-JNK） and inositol requiring enzyme 1α （IRE1α） proteins was determined by immunohistochemical test and Western blot （WB）. ResultCompared with the conditions in the normal group and blank group， the time of paw withdrawal， paw licking and tail flick in the model group was shortened （P<0.01）， and the conduction velocity of sciatic nerve was decreased （P<0.01）. Compared with the conditions in the model group， the behavioral and functional indicators were improved by HQGZWWT （P<0.05，P<0.01）. The immunohistochemical test revealed the JNK expression was elevated in the model group compared with the conditions in the normal group and blank group （P<0.05）， while that was lowered by HQGZWWT compared with the condition in the model group （P<0.05）. However， there was no difference among the treatment groups. According to the WB， the expression of IRE1α and p-JNK in the model group was enhanced compared with the conditions in the normal group and blank group （P<0.05，P<0.01）， while that was decreased by HQGZWWT compared with the condition in the model group （P<0.05，P<0.01）. No difference was observed between the HQGZWWTO and HQGZWWTF groups. ConclusionHQGZWWT can improve the neurophysiological function and pathological damage of sciatic nerve， which may be related to its delaying the ER stress response of sciatic nerve.

IRE1α deficiency impairs autophagy in chondrocytes by upregulating calcium homeostasis endoplasmic reticulum protein / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).@*METHODS@#Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay.@*RESULTS@#Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.@*CONCLUSION@#IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.

Fufang Danshen dripping pills protect vascular smooth muscle cells by reducing endoplasmic reticulum stress and autophagy through regulating IRE1-TRAF2-ASK1 -JNK pathway / 中国药理学通报

Aim To investigate the molecular mechanism of the protection of vascular smooth muscle cells by the regulation of endoplasmic reticulum stress and autophagy by Fufang Danshen dripping pills.Methods Fifty patients with atherosclerosis were randomly divided into two groups; one group received normal treatment,while the other group was added Fufang Danshen dripping pills,and the clinical efficacy was observed.Vascular smooth muscle cells were divided into control,ox LDL model,Fufang Danshen dripping pill group,Fufang Danshen dripping pill+endoplasmic reticulum stress inhibitor group,and Fufang Danshen dripping pill+endoplasmic reticulum stress inducer group.Proliferation was detected,and vasodilator function factors and oxidative stress were measured in each group,ER stress marker proteins,autophagy marker proteins and apoptosis related protein expression were detected,and activation of the IRE1-TRAF2-ASK1-JNK signaling pathway was detected.Results Compared with control group,various indicators of cells in ox-LDL model group showed that they were under ER stress,high oxidative stress,high autophagy status,and the IRE1-TRAF2-ASK1-JNK pathway was found to be over activated.However,compared with ox LDL model group,the above indicators in Fufang Danshen dripping pill group and Fufang Danshen dripping pill+endoplasmic reticulum stress inhibitor group were significantly better,the IRE1-TRAF2-ASK1-JNK pathway was over activated,and the endoplasmic reticulum stress inhibitor was more obvious,and Fufang Danshen dripping pill+endoplasmic reticulum stress inducer group significantly reversed the improved effects of Fufang Danshen dripping pills.Conclusions Fufang Danshen dripping pills protect vascular smooth muscle cells by inhibiting excessive activation of the IRE1-TRAF2-ASK1-JNK pathway,decreasing endoplasmic reticulum stress,maintaining proper autophagy,and inhibiting abnormal cell proliferation and apoptosis.

miRNA-101a inhibits liver fibrosis induced by activated hepatic stellate cell through upregulating IRE1α signaling pathway / 中华肝胆外科杂志

Objective:To investigate the role of microRNA (miRNA)-101a in the liver fibrosis induced by activated hepatic stellate cell (HSC), through upregulating IRE1α signaling pathway.Methods:Carbon tetrachloride (CCl 4) induced liver fibrosis model of mice was established. RT-PCR was used to measure the mRNA level of miRNA-101a in liver tissue of mice. Protein level of α smooth muscle actin(αSMA), collagen I and IRE1α were investigated by Western blot. It was divide into Vehicle, TGFβ1, TGFβ1+ miRNA-NC and TGFβ1+ miRNA-M groups. TGFβ1+ miRNA-NC and TGFβ1+ miRNA-M group were transfected with miRNA-101a mimic negative control and miRNA-101a mimic, respectively. After the corresponding treatments, mRNA level of miRNA-101a was detected by RT-PCR, the protein level of αSMA、collagen I and IRE1α were measured by Western blot. Results:Compared to normal mice, the fibrotic deposition in liver tissue of CCl 4 group was increased significantly [(0.17±0.06) vs. (2.09±0.39), P<0.001)]. Protein level of αSMA, collagen I and IRE1α was increased significantly in the model group [(1.00±0.23) vs. (4.09±0.80), (1.00±0.21) vs. (4.98±1.19), (1.00±0.24) vs. (3.27±0.65), all P<0.001)]. While the mRNA level of miRNA-101a was decreased (1.00±0.05) vs. (0.43±0.05), P<0.001). In vitro study, we found that TGFβ1 could inhibit the mRNA expression of miRNA-101a, induced HSC-T6 activation and then up-regulated protein expression of αSMA, collagen I and IRE1α. Compared to TGFβ1+ miRNA-NC group, the expression of miRNA-101a in TGFβ1+ miRNA-M group increased significantly [(0.59±0.19) vs. (1.89±0.20), P<0.001)]. The protein levels of αSMA, collagen I were reduced by over expression of miRNA-101a [(2.65±0.69) vs. (0.84±0.13), (3.15±0.59) vs. (1.31±0.25), all P<0.05)], and the protein content of IRE1α was down-regulated [ (2.63±0.47) vs. (1.03±0.15), P<0.001)]. Conclusion:miRNA-101a may play a critical role in the inhibition of HSC activation and liver fibrogenesis by blocking IRE1α signaling pathway.

The IRE1α-miRNA Signaling Pathway Mediates Exercise Intervention in AD / 中国生物化学与分子生物学报

A common pathological features of Alzheimer's disease (AD) is the excessive activation of inositol-requiring enzyme-1α (IRE1α) downstream of endoplasmic reticulum stress (ERS) and the abnormal expression of microRNA (miRNA) with a variety of cellular physiological functions. Studies have found that IRE1α and miRNA are not alone in the AD pathologenesis. IRE1α can regulate the expression of miR-200, miR-7, miR-17 and miR-34, and participate in most AD brain diseases such as Aβ deposition, Tau protein hyperphosphorylation and neuronal apoptosis. It is suggested that IRE1α-miRNA signal abnormality is one of the pathological mechanisms of AD. Exercise can prevent and delay AD, but its mechanism is unclear. Studies have found that exercise could interfere with AD by regulating the IRE1αmiRNA signaling pathway. The specific mechanisms of action include: (1) Exercise improves the adaptation of AD brain energy metabolism and alleviates the excessive activation of brain IRE1α signals. (2) Exercise regulates the expression of miRNA in the brain, exerts epigenetic effects, and reduces pathologies such as Aβ and Tau protein aggregation. (3) The IRE1α-miRNA pathway and its downstream protein changes can mediate exercise to resist the development of AD. This article will review the relationship between IRE1α-miRNA and AD pathology and its exercise feedback mechanism, aiming to provide evidence and ideas for AD diagnosis and treatment strategies.

Role of inositol-requiring kinase 1α/X-box binding protein 1 in airway mucus secretion induced by neutrophil elastase / 上海交通大学学报（医学版）

Objective: To explore the effect of endoplasmic reticulum stress (ERS) on neutrophil elastase (NE) induced mucin 5AC (MUC5AC) production in human airway epithelial cells. Methods: HBE16 airway epithelial cells were cultured and pretreated with reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) or ERS inhibitor 4-phenylbutyrate (4-PBA), or transfected with small interfering RNA (siRNA) against inositolrequiring kinase 1α (IRE-1α) or X-box binding protein 1 (XBP-1), respectively before incubation with NE. NE group and blank control group were also set up. ROS production was assayed by detection kit; expression of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK), activating transcription factor 6 (ATF6), phosphorylated IRE-1α (pIRE-1α), and XBP-1 protein was detected by Western blotting; spliced XBP-1 (XBP-1s) mRNA was measured by real-time PCR; levels of MUC5AC protein in culture supernatant and cytoplasm were assayed by ELISA and immunofluorescence. Results: There was an obvious increase of ROS production with strong elevation of GRP78, ATF6, pPERK, and pIRE-1α protein in NE group cells after 24 h, compared with blank control group (P<0.05). The protein and mRNA of XBP-1s, and MUC5AC production also increased obviously (P<0.05). NAC and 4-PBA reduced ERS-related protein expression and MUC5AC production and secretion (P<0.05). Further studies showed that MUC5AC secretion was also blunted by IRE-1α siRNA or XBP-1 siRNA, accompanied with decreased expression of XBP-1s mRNA and protein (P<0.05). Conclusion: NE induces ERS by producing ROS, and increases MUC5AC protein production and secretion; IRE-1α/XBP-1 play a certain role in this process.

Effect of curcumol on the apoptosis inhibition of traf2 and xbp1 controled by ire1 in rats with obstructive nephropathy / 中国药学杂志

OBJECTIVE: To investigate the effect of curcumol on the apoptosis inhibition of TRAF2 and XBP1 controled by IRE1 in rats with obstructive nephropathy. METHODS: Rats were randomly divided into sham control group, model control group and enalapril group as well as high, medium and low formononetin groups. Animal model of RIF was established by unilateral ureteral obstruction (UUO). The rats were sacrificed at 14 d after UUO, and blood samples were collected. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were examined. Renal tubular damage index was determined by H&E staining. The area of RIF was determined by Masson staining. Expressions of GRP78,IRE1,p-IRE1,TRAF2 and XBP1 in kidney were determined by Western blotting analysis. Apoptosis of tubular epithelial cells was detected by TUNEL assay. RESULTS: Levels of Scr and BUN, tubulointerstitial injury index, RIF and apoptotic index as well as expressions of GRP78,IRE1,p-IRE1,TRAF2 and XBP1 were different between the model control and treatment groups (P<0.05, P<0.01). Compared with the enalapril group, tubulointerstitial injury index and RIF as well as the levels of Scr and BUN were decreased (P < 0.05) in the high curcumol group. CONCLUSION: The curcumol can block the apoptosis of renal tubular epithelial cells through interfering IRE1-XBP1 signaling pathway by inhibiting TRAF2 protein expression mediated by IRE1 phosphorylation. Ultimately, development of RIF was postponed.

Comparison of Chronologic Change in the Size and Contrast-Enhancement of Ablation Zones on CT Images after Irreversible Electroporation and Radiofrequency Ablation

OBJECTIVE: To compare short-, mid-, and long-term follow-up ablation zone volume alterations as well as imaging features on contrast-enhanced computed tomography (CT) after irreversible electroporation (IRE) of primary and secondary liver tumors with findings subsequent to radiofrequency ablation (RFA). MATERIALS AND METHODS: Volume assessment of 39 ablation zones (19 RFA, 20 IRE) after intervention was performed at four time intervals (day 0 [t1; n = 39], day 1–7 [t2; n = 25], day 8–55 [t3; n = 28], after day 55 [t4; n = 23]) on dual-phase CT. Analysis of peripheral rim enhancement was conducted. Lesion's volume decrease relative to the volume at t1 was calculated and statistically analyzed with respect to patient's sex, age, ablation modality (IRE/RFA), and history of platinum-based chemotherapy (PCT). RESULTS: No influence of patient's sex or age on ablation volume was detected. The decrease in ablation zones' volume was significantly larger (p < 0.05 for all time intervals) after IRE (arterial phase, 7.5%; venous phase, 9.7% of initial volume) compared to RFA (arterial phase, 39.6%; venous phase, 45.3% of initial volume). After RFA, significantly smaller decreases in the ablation volumes, in general, were detected in patients treated with PCT in their history (p = 0.004), which was not detected after IRE (p = 0.288). In the arterial phase, peripheral rim enhancement was frequently detected after both IRE and RFA. In the venous phase, rim-enhancement was depicted significantly more often following IRE at t1 and t2 (pt1 = 0.003, pt2 < 0.001). CONCLUSION: As per our analysis, ablation zone volume decreased significantly in a more rapid and more profound manner after IRE. Lesion's remodeling after RFA but not IRE seems to be influenced by PCT, possibly due to the type of cell death induced by the different ablation modalities.

Anti-lung cancer mechanisms of diterpenoid tanshinone via endoplasmic reticulum stress-mediated apoptosis signal pathway / 中国中药杂志

At present, lung cancer ranks second and first respectively in the incidence and the mortality among malignant tumors. It is urgent to find new effective anti-lung cancer drugs with less side effects and relatively defined mechanisms. Endoplasmic reticulum stress (ERS)-mediated apoptosis pathway is an effective way to promote tumor cell apoptosis; diterpenoid tanshinone (DT), an effective part separated from Salviae Miltiorrhizae Radix et Rhizoma, was found to have an anti-lung cancer effect in previous studies via ERS-induced PERK-EIF2α pathway. In this paper, human lung adenocarcinoma PC9 cell line and nude mouse transplantation tumor model were applied to verify the anti-lung cancer effect of DT in vivo and in vitro, and illuminate the potential mechanism via ERS induced IRE1α/caspase 12 apoptosis pathway. The results showed that in vivo, DT could promote PC9 cell apoptosis in a concentration-dependent manner, up-regulate Bip, IRE1 and TRAF2 protein expressions in tumor tissue, reduce tumor weight and alleviate bodyweight loss. In vitro, DT inhibited the proliferation of PC9 cell line in a concentration-dependent manner, and destroyed the structure of mitochondria in PC9 cell, promoted Bax, IRE1α, Bip, TRAF2 and caspase 12 protein expressions, lower Bcl-2 protein expression in a time-dependent manner. DT shows a good effect on anti-lung cancer both in vivo and in vitro. The mechanism is related to the activation of ERS-induced IRE1α/caspase 12 apoptosis pathway and the promotion of cell apoptosis. ERS-mediated apoptosis pathway may be an important target of DT on anti-lung cancer.

Regulación del metabolismo del hierro: dos sistemas, un mismo objetivo / Iron metabolism regulation: two systems, one goal

La existencia humana está indisolublemente unida al hierro, que es parte de una amplia variedad de enzimas claves como catalasas, aconitasas, ribonucleótido reductasa, peroxidasas y citocromos, que explotan la flexibilidad de su química redox para ejecutar un elevado número de reacciones esenciales para la vida. El cuerpo humano ha evolucionado para conservar el hierro en diferentes formas, incluido su reciclaje después de la ruptura de los eritrocitos y la retención en ausencia de un mecanismo de excreción. El metabolismo del hierro está balanceado por dos sistemas regulatorios: uno sistémico basado en la hormona hepcidina y la proteína exportadora ferroportina, y el otro que controla el metabolismo celular través de las proteínas reguladoras de hierro (IRP) que se unen a los elementos de respuesta al hierro (IRE) de los ARNm regulados. Estos sistemas funcionan de modo coordinado lo que evita, tanto la deficiencia como el exceso del mineral(AU)

Human existence is indissolubly linked to iron, which is part of a wide variety of key enzymes such as catalase, aconitases, ribonucleotide reductase, peroxidases and cytochromes, exploiting the flexibility of its redox chemistry to run a large number of reactions essential for life. Human body has evolved to keep iron in different forms, including recycling after rupture of erythrocytes and the retention without excretion mechanism. Iron metabolism is balanced by two regulatory systems: one based on systemic hormone hepcidin protein export and ferroportin, and the other, which controls cell metabolism through the iron regulatory protein (IRP) binding to the mRNAs regulated iron regulatory elements (IRE). These systems work in a coordinated manner avoiding both deficiency and excess(AU)