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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.
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Objective:To investigate the expression and significance of endoplasmic reticulum stress apoptosis pathway related proteins in renal cortex of rats with chronic fluorosis.Methods:Twenty four healthy SD rats were divided into 4 groups (6 rats/group, half male and half female) according to their body mass (100 - 120 g) by random number table method, rats in control group drank tap water (fluoride content < 0.5 mg/L), and in low, medium and high fluoride groups drank tap water with fluoride content (sodium fluoride) of 10, 50 and 100 mg/L, respectively. After 180 days of feeding, dental fluorosis was examined, 24-hour urine sample was collected and the content of fluoride in urine was detected by fluoride ion selective electrode method. Renal tissue was taken after anesthesia, and the pathological changes of renal cortex were observed by hematoxylin-eosin (HE) staining. The expressions of endoplasmic reticulum stress apoptosis pathway related proteins [inositol-requiring enzyme 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), C-Jun N-terminal kinase (JNK) and phosphorylated JNK (P-JNK)] were determined by immunohistochemical staining in rat renal cortex.Results:No dental fluorosis was found in control group. The incidence of dental fluorosis in low, medium and high fluoride groups were 2/6, 5/6 and 6/6, respectively. Compared with control group [(5.707 ± 1.190) mg/L], the urinary fluoride in low, medium and high fluoride groups [(17.028 ± 3.006), (34.378 ± 12.045), (94.759 ± 31.773) mg/L] was significantly higher ( P < 0.05), and the urinary fluoride in high fluoride group was higher than that in low and medium fluoride groups ( P < 0.05). HE staining showed that, compared with control group, the cell volume of renal tubules and glomeruli in medium and high fluoride groups increased, the cells arranged closely, and the eosinophilia of the cytoplasm increased. The immunohistochemical staining results showed that there was no significant difference in the expression of JNK protein in rat renal cortex between control group and low, medium and high fluoride groups ( F = 0.07, P > 0.05). The expressions of IRE1α, ASK1 and P-JNK proteins in rat renal cortex in high fluoride group were higher than those of control, low and medium fluoride groups ( P < 0.05), and the expressions of IRE1α and ASK1 proteins in medium fluoride group were significantly higher than those in control and low fluoride groups ( P < 0.05). Conclusion:Long-term excessive fluoride intake can lead to renal cortex injury in rats, and the mechanism of injury may be related to the activation of IRE1α-ASK1-JNK endoplasmic reticulum stress apoptosis pathway.
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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.
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Objective To investigate the regulatory effects of activated endoplasmic reticulum stress（ERS） and unfolded protein response（UPR） on the immune behavior of the stressed muscle fibers in inflammatory environments induced by interferon-γ（IFN-γ）. Methods The myogenic precursor cells（MPCs） of C57 BL/6 mice cultured in vitro were differentiated into multinucleated myogenic tubes by horse serum and then to set up: 1. Control group; 2. IFN-γ group; 3. Tunicamycin group; 4. Thapsigargin group; 5. IFN-γ and 4-phenylbutyrate（4-PBA） combined treatment group; 6. IFN-γ, TG and 4-PBA combined treatment group; 7. IFN-γ and 4μ8 c combined treatment group; 8. IFN-γ, TG and 4μ8 c combined treatment group; 9. IFN-γ and GSK2606414 combined treatment group; 10. IFN-γ, TG and GSK2606414 combined treatment group. The level of myokines gene was detected by Real-time PCR. The expression of UPR key molecules including eukaryotic intiatio factor 2α（eIF2α）, inositol requrring enzyme 1α（IRE1α） and activating transcription factor 6（ATF6） in muscle fibers was observed by immunofluorescence. Western blotting was used to detect immune molecules related to muscle cells, myokines and key molecules of UPR. Luminex analyzed the levels of pro-inflammatory myokines in muscle fibers. Results The expression of H-2 Kb, H2-Ea, Toll like receptor 3（TLR3）, p-eIF2α and p-IRE1α were up-regulated in IFN-γ induced inflammatory environment. The expression of H-2 Kb, H2-Ea, TLR3 and myokines in the group with UPR inhibitor 4-PBA was down-regulated compared with IFN-γ group, and the expression of these molecules in the group with IRE1α specific inhibitor 4μ8 c was down-regulated compared with the IFN-γ group. The addition of protein kinase R-like endoplasmic eticulum（PERK） specific inhibitor GSK2606414 showed no significant change. Conclusion In IFN-γ induced inflammatory environment, the UPR-IRE1α pathway activates and inhibits the synthesis of muscle fiber immune-related molecules, which further inhibits the muscle fiber mediated immune response and facilitates muscle regeneration.
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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.
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OBJECTIVE To investigate the effect of total flavonoids of Epimedium (TFE) on the decline of secretion function of senile testis-supporting cells (Sertoli cells), and to explore their molecular mechanism from inositol-requiring enzyme 1α(IRE1α)/X-box- binding protein 1(XBP1) signaling pathway. METHODS Thirty-six SPF 18-month-old SD male rats were randomly divided into natural aging group, low and high dose TFE groups, with 12 rats in each group. Another ten 2-month-old rats were used as a youth control group. TFE was ig given once a day, and the ig administration was continued every five days after a two-day interval, and the administration lasted for 4 months. Then, the testicular index was calculated. The testicular histomorphology was observed by HE staining; while quantitative-PCR (qPCR) and Western blotting were used to detect the mRNA and protein expression levels of glial cell derived neurotrophic factor (GDNF), bone morphogenetic protein 4 (BMP4) and stem cell factor (SCF), and Western blotting protein expression levels of p-IRE1α and XBP1 in testes. The number of Sertoli cells in the testis and the localization of p-IRE1α in the testis were detected by immunofluorescence. RESULTS Compared with the young control group, the testicular index decreased significantly in the natural aging group (P<0.01), but was significantly up-regulated after TFE 40 m g · k g- 1 (P<0.05). The results of HE showed that the morphological structure of the testicular seminiferous tubules in the natural aging group were disorderly, and some spermatogenic cells were shed. The TFE group could improve the morphological structure of the seminiferous tubules of the testis. qPCR and Western blotting results showed that compared with the young control group, the mRNA and protein expression levels of GDNF, BMP4 and SCF in Sertoli cells in the natural aging group were significantly down-regulated (P< 0.01). However, the expression of secretory factors in TFE group was significantly up-regulated (P< 0.05, P<0.01). Western blotting results showed that compared with the young control group, the expression of p-IRE1α and XBP1 protein in the testis of the natural aging group was significantly down-regulated (P<0.01), but the expression of p-IRE1α and XBP1 protein in the TFE group was significantly up-regulated (P<0.01). The results of immunofluorescence also showed that there was no significant difference in the number of Sertoli cells between the natural aging group and the TFE group compared with the young control group, while the expression of p-IRE1α was localized both in cytoplasm of Sertoli cells and spermatogenic cells. CONCLUSION TFE can significantly improve the decline of secretion function of testicular Sertoli cells induced by aging, and the mechanism may be related to the regulation of IRE1α/XBP1 signaling pathway.
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Objective To investigate the effect of Xuebijing injection on lipopolysaccharide (LPS)-mediated the procoagulant activity of tissue factor (TF) in abdominal aortic endothelial cells from rats.Methods Abdominal aortic endothelial cells from rats were randomLy(random number) divided into the control group,LPS group (500 ng/mL),Xuebijing group (1,5,25 μL/mL),and LPS+Xuebijing group (1,5,25 μL/mL),respectively.Cell proliferation was measured by CCK8 and lactate dehydrogenase (LDH) level in supematants was determined at 24,48,and 72 h;Expressions ofinositol-requiring enzyme-1α (IRE 1α),unspliced-box binding protein-1 (uXBP-1),spliced-box binding protein 1 sXBP-1),and protein disulfide isomerase (PDI) were determined by Western blotting at 72 h.Procoagulant activity of TF was measured as the ability of monolayer to support activation of factor with the addition of a and Ca2+ by chromogenic substrate method.Results Compared with the control group,the cell proliferation was decreased and LDH level was increased in the LPS group (P＜0.05),and there were markedly up-regulated in the expression of IRE1 α,uXBP1,sXBP1,and PDI (P＜0.05).Compared with the control group,treatment with Xuebijing injection could promote cell proliferation and reduce the release of LDH (P＜0.05 or P＜0.01),which were gradually enhanced along with the observational intervals.Compared with the LPS group,the LPS+Xuebijing group showed obviously higher cell proliferation and lower release of LDH (P＜0.05 or P＜0.01),expressions of IRE 1 α,uXBP 1,sXBP 1,and PDI were significantly reduced (P＜0.01);meanwhile,F Ⅹ a activity was decreased in the LPS+Xuebijing group,and 5 μ L/mL Xuebijing was the optimal dose in down-regulation of F Ⅹ a.Conclusions These results suggest that treatment with Xuebijing injection can markedly down-regulate the expression of PDI by inhibiting the IRE1α-XBP1 signaling pathway to suppress the procoagulant activity of TF in abdominal aortic endothelial cells from rats.
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Objective: To detect the expressions of inositol-requiring enzyme-1α (IRE1α) and X box-binding protein-1 (XBP1) mRNA and proteins in mouse testis cells, and to explore the role of IRE1-XBP1 pathway activation in the endoplasmic reticulum stress induced by low dose radiation (LDR) in the mouse testis cells. Methods: A total of 50 heatly male ICR mice were randomly divided into 10 groups. After the mice were irradiated with 75 mGy X-ray in whole body at different time (0, 3, 6, 12, and 24 h) or with different doses (0, 50, 75, 100, and 200 mGy) of X-ray fori2 h, the expression levels of IRElα, T-XBP1 and S-XBP1 mRNA and proteins were measured by real-time quantitative PCR and Western blotting method, respectively. Results: The expression levels of IRElα, T-XBP1 and S-XBP1 mRNA (except T-XBP1 and S-XBP1 mRNA at 3 h after irradiation) in the testis cells of the mice after irradiated with 75 mGy X-ray for 0-24 h were increased with the time prolongation, and reached to the maximum value at 6 h after irradiation, then were gradually decreased. Compared with 0 h group, the expression levels of IRElα mRNA (6, 12, and 24 h after irradiation), T-XBP1 mRNA and S-XBP1 mRNA (6 and 12 h after irradiation) were significantly increased (P<0. 05 or P<0. 01); the expression intensities of IRElα and S-XBP1 proteins were increased; the expression intensities of IRE1α (6 and 12 h after irradiation) and S-XBP1 (24 h after irradiation) proteins were increased most significantly, but the expression intensity of T-XBP1 protein was slightly decreased. The expression levels of IRE1α, T-XBP1 and S-XBP1 mRNA in the testis cells of the mice after irradiated with 0-200 mGy for 12 h were increased, and reached to the maximum values after 75 mGy irradiation, then they were gradually decreased, even below 0 mGy. Compared with 0 mGy group, the expression levels of IRE1α mRNA in the testis cells of the mice in 75 and 200 mGy groups and the expression levels of T-XBP1 mRNA and S-XBP1 mRNA in 75 mGy group were significantly increased (P<0. 05 or P<0. 01); the expression intensities of IREla protein in 75 mGy group and S-XBP1 protein in 75 and 200 mGy groups were increased mostly, while the expression intensity of T-XBP1 protein had no obvious change. Conclusion: LDR can induce the increase of IRE1α and S-XBP1 mRNAs and proteins, and activate the IRE1-XBP1 pathway in endoplasmic reticulum stress.
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PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.
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Humans , Anti-Bacterial Agents/pharmacology , Apoptosis , Blotting, Western , C-Reactive Protein/biosynthesis , Cells, Cultured , Endoplasmic Reticulum Stress/drug effects , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Polymerase Chain Reaction , RNA, Messenger/genetics , Retinal Pigment Epithelium/metabolism , Serum Amyloid P-Component/biosynthesis , Tunicamycin/pharmacologyABSTRACT
Objective To explore the expressions of endoplasmic reticulum stress (ERs) related factors including inositol requiring enzyme-1α(IRE1αα),p-IRE1 α,c-jun N-terminal Kinase(JNK),and p-JNK in rats with non-alcoholic fatty liver disease,and to investigate the effect of intervention with glucagon-like peptide 1 (GLP-1) analogue.Methods Forty male Sprague-Dawley rats were divided into normal chow group(n =15) and high-fat diet group(n=25).After 12 weeks,5 rats of each group were used to assess the establishment of rat models with non-alcoholic fatty liver disease.Then the high-fat diet group rats were divided into high-fat diet group (HF,n =10) and GLP-1 group(HG,n=10) and treated with normal saline and GLP-1 analogue for4 weeks respectively.Body weight and biochemical markers in rats were measured.The expressions of IRE1α,p-IRE1α,JNK,and p-JNK were measured by Western blot.Results Compared with the NC group,the levels of body weight,plasma triglyceride (TG),total cholesterol (TC),low density lipoprotein-cholesterol (LDL-C),alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in HF group were significantly higher (all P ＜ 0.01),high density lipoprotein-cholesterol(HDL-C) was decreased(P＜0.01),and p-IRE1 α/IRE1 α and p-JNK/JNK were increased(P＜0.05 and P＜0.01).After GLP-1 treatment,body weight,plasma TG,TC,LDL-C,AST,ALT in HF group were significantly lowered(P＜0.05 or P＜0.01),HDL-C was increased(P＜0.01),p-IRE1 α/IRE1 α and p-JNK/JNK were decreased (P＜0.05 and P＜0.01).Conclusion GLP-1 analogue may improve hepatic steatosis via regulating ERs related IRE1 α-JNK signaling pathway.
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ObjectiveTo investigate the effects of curcumin on type 2 diabetic neuropathic pain (DNP)and expression of inositol-requiring enzyme 1α (IRE1α) in spinal dorsal horn and dorsal root ganglia (DRG) in rats.MethodsType 2 diabetes mellitus was induced by high-fat and high-sucrose diet and intraperitoneal streptozotocin (STZ) 35 mg/kg,and confirmed by fasting blood glucose level ＞ 16.7 mmol/L in male SD rats.Type 2 DNP was confirmed by the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWI.) measured on day 14 after STZ administration ＜ 80％ of the baseline value.The rats were then randomly divided into 3 groups ( n =27 each):DNP group,curcumin group (group Cur) and solvent control group (group SC).Curcumin and corn oil 100 mg/kg (25 mg/ml) were given intraperitoneally once a day for 14 consecutive days starting from 14 days after administration of streptozocin in Cur and SC groups respectively.Another 27 rats were chosen and served as control group (group C) and fed with common fodders.MWT and TWL were measured at 3,7 and 14 day after curcumin administration.The expression of IRE1α in spinal dorsal horn and DRG was detected by Western blot.ResultsCompared with group C,MWT was significantly decreased and TWL was significantly shortened,and the expression of IRE1α was up-regulated in DNP,Cur,and SC groups (P ＜ 0,05),Compared with group DNP,MWT were significantly increased,TWL was significantly prolonged,and the expression of IRE1α in spinal dorsal horn and DRG was down-regulated in group Cur (P ＜ 0.05).There was no significantdifference in the parameters mentioned above between DNP and SC groups (P ＞ 0.05).ConclusionCurcumin can attenuate type 2 1)NP and inhibition of the expression of IRE1α in spinal dorsal horn and DRG is involved in the mechanism.
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Fed with high-fat diet and assessed by hyperinsulinemia-euglycemia clamp technique, rat models with insulin resistance were successfully induced. Compared with normal chow group ( NC ), serum concentrations of free-fatty acids(FFAs) and baseline insulin in high-fat diet group(HF) was higher( P＜0.05 ), the average glucose infusion rate from 60 to 120 min( GIR60-120 ) was lower( P＜0.01 ), and the expression of p-IRE-lα in the liver was higher( P＜0.05 ). Furthermore, the expression of p-IRE-1α in the liver was positvely correlated with the serum concentration of FFAs. All these data indicate that high-fat diet may induce endoplasmic reticulum stress in the liver by elevating serum concentration of FFAs, and may participate in the genesis of insulin resistance via p-IRE-1α.