Down-regulation of XBP1s alleviates the senescence of renal tubular epithelial cells induced by hypoxia/reoxygenation through Sirt3/SOD2/mtROS signaling pathway / 器官移植

Objective To investigate the role and mechanism of spliced X-box binding protein 1 (XBP1s) in the senescence of primary renal tubular epithelial cells induced by hypoxia/reoxygenation (H/R). Methods Primary renal tubular epithelial cells were divided into the normal control group (NC group), H/R group, empty adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), empty adenovirus+H/R treatment group (Ad-shNC+H/R group) and targeted silencing XBP1s adenovirus+H/R treatment group (Ad-shXBP1s +H/R group), respectively. The expression levels of XBP1s in the NC, H/R, Ad-shNC and Ad-shXBP1s groups were measured. The number of cells stained with β-galactosidase, the expression levels of cell aging markers including p53, p21 and γH2AX, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined in the Ad-shNC, Ad-shNC+H/R and Ad-shXBP1s+H/R groups. Chromatin immunoprecipitation was employed to verify Sirtuin 3 (Sirt3) of XBP1s transcription regulation, and the expression levels of Sirt3 and downstream SOD2 after down-regulation of XBP1s were detected. Mitochondrial reactive oxygen species (mtROS) were detected by flow cytometry. Results Compared with the NC group, the expression level of XBP1s was up-regulated in the H/R group. Compared with the Ad-shNC group, the expression level of XBP1s was down-regulated in the Ad-shXBP1s group (both P<0.001). Compared with the Ad-shNC group, the number of cells stained with β-galactosidase was increased, the expression levels of p53, p21 and γH2AX were up-regulated, the levels of ROS, MDA and mtROS were increased, the SOD activity was decreased, the expression level of Sirt3 was down-regulated, and the ratio of Ac-SOD2/SOD2 was increased in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the number of cells stained with β-galactosidase was decreased, the expression levels of p53, p21 and γH2AX were down-regulated, the levels of ROS, MDA and mtROS were decreased, the SOD activity was increased, the expression level of Sirt3 was up-regulated and the ratio of Ac-SOD2/SOD2 was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Conclusions Down-regulation of XBP1s may ameliorate the senescence of primary renal tubular epithelial cells induced by H/R, which probably plays a role through the Sirt3/SOD2/mtROS signaling pathway.

Crosstalk between activating transcription factor 6 and the inositol-requiring enzyme 1 - X-box binding protein 1 pathway in oxygen-glucose deprivation/reoxygenation-injured HT22 cells / 中华危重病急救医学

Objective:To study the crosstalk between the activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) - X-box binding protein 1 (XBP1) pathway in oxygen-glucose deprivation/reoxygenation (OGD/R)-injured mouse hippocampal neuronal cell line HT22.Methods:The OGD/R-injured HT22 cell model was used to observe the changes of the indicators of endoplasmic reticulum stress (ERS), cell viability, and apoptosis at different OGD/R time points (0, 3, 6, 12, and 24 hours). HT22 cells in the logarithmic growth phase were randomized into blank control group, control+ATF6 activator (AA147) group, control+IRE1 inhibitor (4μ8c) group, OGD/R model group, OGD/R+AA147 group and OGD/R+4μ8c group (10 μmol/L AA147 or 16 μmol/L 4μ8c was given during the whole process in the AA147 group and 4μ8c group). Western blotting was used to detect the expression of ERS-related proteins [glucose-regulated protein 78 (GRP78), phosphorylated-inositol-requiring enzyme 1 (p-IRE1), and phosphorylated-eukaryotic translation initiation factor-2α (p-eIF2α)], and apoptosis-related proteins (Bcl-2, Bax, caspase-3, and cleaved caspase-3). The mRNA of ERS-related genes, and ATF6 [homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (Herpud1), protein disulfide isomerase associated 4 (Pdia4) and Sel-1 suppressor of lin-12-like (Sel1L)] and spliced XBP1 [XBP1s, include DnaJ heat shock protein family member B9 (Erdj4), Sec24 related gene family, member D (Sec24d) and signal sequence receptor, gamma (Ssr3)] induced transcriptional response-related genes were measured by real-time quantitative polymerase chain reaction (RT-qPCR). A cell counting kit-8 (CCK-8) assay was used to detect the viability of HT22 cells. Immunofluorescence was utilized to test the expression of cleaved caspase-3.Results:Compared with the blank control group, the expression of ERS-related proteins p-IRE1 and p-eIF2α were significantly increased at 12 hours and 3 hours following OGD/R, respectively (p-IRE1/β-actin: 2.09±0.10 vs. 1.00±0.00, p-eIF2α/β-actin: 1.39±0.11 vs. 1.00±0.00, both P < 0.01). The mRNA expressions of ERS-related genes [ATF6, XBP1s, unspliced XBP1 (XBP1u), activating transcription factor 4 (ATF4), CCAAT/EBP homologous protein (CHOP)] were also upregulated in different OGD/R timepoint in HT22 cells, which indicated ERS was activated in OGD/R-stimulated HT22 cells. Compared with the OGD/R model group, the expression of protein p-IRE1 was not changed, but the mRNA of XBP1s and XBP1u were obviously downregulated in the OGD/R+AA147 group [XBP1s (2 -ΔΔCt): 0.76 (0.71, 0.92) vs. 1.13 (1.03, 1.29), XBP1u (2 -ΔΔCt): 0.29±0.05 vs. 0.52±0.04, both P < 0.01], whereas the expressions of XBP1s-induced transcriptional response downstream genes did not change significantly. Compared with the OGD/R model group, the protein of short-form ATF6 (sATF6) and GRP78 were not changed after administration of 4μ8c, neither was the mRNA expression of ATF6-induced transcriptional response-related genes. These results showed that the mRNA expression of XBP1s and XBP1u were inhibited by AA147-induced activation of ATF6, but no crosstalk was observed between the transcriptional response induced by ATF6 and XBP1s. Compared with the blank control group, the cell viability decreased significantly at OGD/R 3 hours [(44.64±5.12) % vs. (99.13±5.76) %, P < 0.01], the ratios of apoptosis-related proteins Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly increased at OGD/R 3 hours and OGD 0 hour, respectively (Bax/Bcl-2: 6.15±1.65 vs. 1.00±0.00, cleaved caspase-3/caspase-3: 17.48±2.75 vs. 1.00±0.00, both P < 0.01), which indicated that apoptosis was activated in OGD/R-treated HT22 cells. Compared with the OGD/R model group, the cell viability decreased significantly [(36.52±17.78)% vs. (69.90±9.43)%, P < 0.01], and the ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly upregulated in the OGD/R+AA147 group in HT22 cells (Bax/Bcl-2: 2.06±0.31 vs. 1.10±0.25, cleaved caspase-3/caspase-3: 3.35±0.59 vs. 0.55±0.09, both P < 0.01). Conclusion:Under our experimental conditions, no obvious crosstalk between the transcriptional response induced by ATF6 and XBP1s was observed, while ATF6 activation induced by AA147 suppressed mRNA expression of XBP1s and XBP1u and promoted cell death in OGD/R-treated HT22 cells.

Study of endoplasmic reticulum stress in human dental pulp cells induced by oxygen-glucose deprivation culture in vitro / 口腔疾病防治

Objective@#Oxygen-glucose deprivation (OGD) is used to mimic ischemia in vitro to observe whether endoplasmic reticulum (ER) stress is involved in human dental pulp cells (hDPCs) after OGD and to better understand the regulatory mechanism of hDPCs in ischemia.@*Methods@# hDPCs were cultured in glucose-free DMEM and hypoxia (volume fraction 2% O2) to establish an hDPCs OGD model in vitro, which mimics hDPCs ischemia in vitro. hDPCs were divided into a control group (normal culture) and an experimental group (OGD 0 h, 2 h, 4 h and 8 h groups). After pretreatment with OGD for 0, 2, 4 and 8 h, hDPC viability was measured by methylthiazol tetrazolium (MTT) assay. qRT-PCR was used to detect the mRNA expression of ER stress markers [splicing x-box binding protein1 (sXBP1), activating transcription Factor 4 (ATF4) and C/EBP homologous protein (chop)]. Western blot was used to detect the protein expression of ER stress markers [phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-perk) and phosphorylated eukaryotic initiation factor-2α (p-eIF2α)]. @*Results@#Compared with OGD 0 h group, cell viability of hDPCs decreased when exposed to OGD treatment for 2 h, 4 h and 8 h. Compared with the control group, mRNA expressions of ER stress makers (sXBP1, ATF4 and chop) and the protein expressions of ER stress protein markers (p-perk andp-eIF2α) increased in OGD treatment cells after 4 h were higher in OGD cells. The differences were statistically significant (P<0.05).@*Conclusion@#The results indicate that ER stress response is involved in hDPCs in OGD treatment.

Silencing XBP1 expression enhances the sensitivity of human osteosarcoma HOS cells to MPPα-PDT / 肿瘤

Objective: To investigate the role of X-box binding protein 1 (XBP1) in the treatment of human osteosarcoma HOS cells by pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPα-PDT), and to explore the possible mechanism. Methods: HOS cells were treated by MPPα-PDT for 6, 12 and 24 h, and the expression levels of inositol-requiring enzyme 1 alpha (IRE1α)-XBP1 pathway-related protein IRE1α and XBP1 were detected by Western blotting. The specific siRNA targeting XBP1 gene was transfected into HOS cells by lipofection, and treated by MPPα-PDT. The cells were divided into the blank group, siRNA-negative control (NC) group, siRNA-XBP1 group, MPPα-PDT group and MPPα-PDT+siRNA-XBP1 group. The expressions of XBP1 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The proliferation of HOS cells was detected by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry (FCM). The expression levels of cleaved-caspase 3 and cleaved-poly ADP-ribose polymerase (cleaved-PARP) were determined by Western blotting. The intracellular level of reactive oxygen species (ROS) was detected by DCFH-DA probe staining, then the expression levels of Catalase and superoxide dismutase 1 (SOD1) proteins were detected by Western blotting. Results: The expression levels of IRE1α-XBP1 pathway-related proteins IRE1α and XBP1 were increased after the treatment of MPPα-PDT (both P < 0.05 ). siRNA-XBP1 inhibited the expression levels of XBP1 mRNA and protein (both P < 0.01). Silencing siRNA-XBP1 expression inhibited the proliferation activity of HOS cells (P < 0.01), up-regulated the apoptosis rate and the expression level of apoptosis-related protein cleaved-caspase 3 (both P < 0.05), and down-regulated the expression levels of antioxidant enzyme-related proteins Catalase and SOD1 (both P < 0.05). Compared with the MPPα-PDT group, the proliferation activity of HOS cells treated with MPPα-PDT+siRNA-XBP1 was decreased (P < 0.01), the apoptosis rate and the expression levels of cleaved-caspase 3 and cleaved-PARP were increased (all P < 0.05), the intracellular ROS level was up-regulated (P < 0.01), and the expression levels of Catalase and SOD1 were decreased (both P < 0.01). Conclusion: MPPα-PDT can induce the activation of IRE1α-XBP1 pathway in HOS cells. Silencing XBP1 can inhibit the proliferation activity of HOS cells, up-regulate the apoptosis rate, and increase the sensitivity of HOS cells to MPPα-PDT. The mechanism may be related to the up-regulation of intracellular ROS level and the down-regulation of anti-oxidation molecules.

Flavonoids from the aerial parts of Lespedeza cuneata / 药学学报

The chemical constituents of the aerial parts of Lespedeza cuneata (Dum. Cour.) G. Don were investigated using chromatographic techniques, such as silica gel, reversed phase MPLC and preparative HPLC. Five compounds were isolated and their structures were elucidated by spectroscopic data and physicochemical properties, which were identified as 7-O-glucosyllaburnetin (1), kaempferol-3-O-β-D-galactopyranoside (2), kaempferol-3-O-α-L-rhamnoside (3), vitexin (4), and isovitexin (5). Among those, compound 1 is a new compound, compounds 2-3 were isolated from this plant for the first time. Compounds 1-5 were tested for their anti-ulcerative colitis activity by dual luciferase report gene assay targeting xbp1. Compared with control group, compound 1 showed a certain activity on activating the transcription of xbp1, with its relative activating ratio being 1.80 times.

Endoplasmic Reticulum Stress Induces MUC5AC and MUC5B Expression in Human Nasal Airway Epithelial Cells

OBJECTIVES: Endoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated. METHODS: In primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptasepolymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved. RESULTS: Tunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B. CONCLUSION.: These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.

Effect of exogenous insulin on IRE1α-XBP1 signaling pathway in pancreatic tissues during cardiopulmonary bypass-caused insulin resistance in rabbits / 中华麻醉学杂志

Objective To evaluate the effect of exogenous insulin on inositol-requiring protein 1α (IRE1α)-X-box-binding protein 1 (XBP1) signaling pathways in pancreatic tissues during cardiopulmonary bypass (CPB)-caused insulin resistance in rabbits.Methods Forty healthy adult New Zealand white rabbits of both sexes,weighing 2.5-3.0 kg,were divided into 4 groups (n =10 each) using a random number table method:control group (group C),group CPB,insulin group (group I),and normal saline control group (group NS).CPB was established in group CPB.Insulin was intravenously infused in a dose of 1.2 ml/h from establishing CPB to 1 day after operation,and the infusion rate of insulin was regulated according to the blood glucose (maintaining at 7.2-8.3 mmol/L) in group I.CPB was established,and normal saline was intravenously infused from the beginning of operation to 1 day after operation in group NS.Before CPB (T1) and at 15,30 and 60 min after aortic opening (T2-4),blood samples were collected from the left femoral artery,the plasma was separated,the blood glucose level was detected by oxidase method,the level of glucagon was detected by the radioimmunoassay method,and the insulin resistance index was calculated.Animals were sacrificed at T4,and pancreatic tissues were obtained for determination of the expression of IRE1α,XBP1,c-Jun N-terminal kinase (JNK) and caspase-12 protein and mRNA (by Western blot or fluorescent quantitative real-time polymerase chain reaction) and for examination of the pathological changes (by haematoxylin and eosin staining).Results Compared with group C,blood glucose and glucagon concentrations and insulin resistance index were significantly increased at T2-4,and the expression of IRE1α,XBP1,JNK and caspase-12 was up-regulated at T4 in CPB,I and NS groups (P＜0.05).Compared with group CPB or group NS,blood glucose and glucagon concentrations and insulin resistance index were significantly decreased at T2-4,the expression of IRE1α,XBP1,JNK and caspase-12 was down-regulated at T4 (P＜0.05),and the pathological changes of pancreatic tissues were significantly attenuated in group I.There was no significant difference in the parameters mentioned above between group CPB and group NS (P＞0.05).Conclusion The mechanism by which exogenous insulin reduces CPB-caused insulin resistance may be related to inhibiting IRE1α-XBP1 signaling pathways in pancreatic tissues of rabbits.

Effect of Epimedium flavonoids on decreasing secretion function of Sertoli cells in testis of natural aging rats / 中国药理学与毒理学杂志

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.

Electroacupuncture improved locomotor function by regulating expression of tyrosine hydroxylase and α-synuclein proteins and transcription activating factor 6 and transcription factor X box binding protein 1 mRNAs in substantia nigra of rats with Parkinson's disease / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on behavioral changes, and expression of tyrosine hydroxylase (TH), α-synuclein(α-syn), transcription activating factor 6 (ATF6) and transcription factor X box binding protein 1 (XBP-1) in the substantia nigra of Parkinson's disease (PD) rats, so as to explore its mechanisms underlying improvement of motor function. METHODS: Thirty-six male SD rats were randomly divided into control, model and EA groups (n=12 rats in each group). The PD model was established by subcutaneous injection of rotenone (2 mg/kg) at the neck and back, once a day for 28 days. EA (2 Hz, 1 mA) was applied to "Fengfu" (GV16) and bilateral "Taichong" (LR3) for 20 min, once a day for 14 successive days. The voluntary motor behavioral changes (total distance, average speed, total movement time, total rest time in 8 min) were detected by open field tests. The immuno-activity of TH and α-syn in the substantia nigra was detected by immunohistochemistry, and the expression of ATF6 mRNA and XBP-1 mRNA detected by fluorescence real-time quantitative PCR. RESULTS: Following modeling and compared with the control group, the total distance, average speed and total movement time of voluntary movement were significantly decreased (P<0.01), and the total rest time was significantly increased (P<0.01). Moreover, the expression of TH was significantly decreased (P<0.01), and that of α-syn protein, ATF6 mRNA and XBP-1 mRNA significantly increased in the model group in comparison with the control group (P<0.01). After the intervention, the total distance, average speed, and total movement time of voluntary movement in the EA group were considerably higher than those in the model group (P<0.01), and the total rest time was obviously decreased in the EA group (P<0.01). The expression level of TH was significantly increased (P<0.01), and those of α-syn, ATF6 mRNA and XBP-1 mRNA were notably decreased in the EA group compared with the model group (P<0.01). CONCLUSION: EA intervention can improve the locomotor function in PD model rats, which is associated with its functions in up-regulating the expression of TH protein and down-regulating the expression of α-syn protein, and ATF6 mRNA and XBP-1 mRNA in the substantia nigra of mesencephalon.

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.

Role of X-Box Binding Protein-1 in Fructose-Induced Lipogenesis in HepG2 Cells / 中华医学杂志(英文版)

<p><b>Background</b>A high consumption of fructose leads to hepatic steatosis. About 20-30% of triglycerides are synthesized via de novo lipogenesis. Some studies showed that endoplasmic reticulum stress (ERS) is involved in this process, while others showed that a lipotoxic environment directly influences ER homeostasis. Here, our aim was to investigate the causal relationship between ERS and fatty acid synthesis and the effect of X-box binding protein-1 (XBP-1), one marker of ERS, on hepatic lipid accumulation stimulated by high fructose.</p><p><b>Methods</b>HepG2 cells were incubated with different concentrations of fructose. Upstream regulators of de novo lipogenesis (i.e., carbohydrate response element-binding protein [ChREBP] and sterol regulatory element-binding protein 1c [SREBP-1c]) were measured by polymerase chain reaction and key lipogenic enzymes (acetyl-CoA carboxylase [ACC], fatty acid synthase [FAS], and stearoyl-CoA desaturase-1 [SCD-1]) by Western blotting. The same lipogenesis-associated factors were then evaluated after exposure of HepG2 cells to high fructose followed by the ERS inhibitor tauroursodeoxycholic acid (TUDCA) or the ERS inducer thapsigargin. Finally, the same lipogenesis-associated factors were evaluated in HepG2 cells after XBP-1 upregulation or downregulation through cell transfection.</p><p><b>Results</b>Exposure to high fructose increased triglyceride levels in a dose- and time-dependent manner and significantly increased mRNA levels of SREBP-1c and ChREBP and protein levels of FAS, ACC, and SCD-1, concomitant with XBP-1 conversion to an active spliced form. Lipogenesis-associated factors induced by high fructose were inhibited by TUDCA and induced by thapsigargin. Triglyceride level in XBP-1-deficient group decreased significantly compared with high-fructose group (4.41 ± 0.54 μmol/g vs. 6.52 ± 0.38 μmol/g, P < 0.001), as mRNA expressions of SREBP-1c (2.92 ± 0.46 vs. 5.08 ± 0.41, P < 0.01) and protein levels of FAS (0.53 ± 0.06 vs. 0.85 ± 0.05, P = 0.01), SCD-1 (0.65 ± 0.06 vs. 0.90 ± 0.04, P = 0.04), and ACC (0.38 ± 0.03 vs. 0.95 ± 0.06, P < 0.01) decreased. Conversely, levels of triglyceride (4.22 ± 0.54 μmol/g vs. 2.41 ± 0.35 μmol/g, P < 0.001), mRNA expression of SREBP-1c (2.70 ± 0.33 vs. 1.00 ± 0.00, P < 0.01), and protein expression of SCD-1 (0.93 ± 0.06 vs. 0.26 ± 0.05, P < 0.01), ACC (0.98 ± 0.09 vs. 0.43 ± 0.03, P < 0.01), and FAS (0.90 ± 0.33 vs. 0.71 ± 0.02, P = 0.04) in XBP-1s-upregulated group increased compared with the untransfected group.</p><p><b>Conclusions</b>ERS is associated with de novo lipogenesis, and XBP-1 partially mediates high-fructose-induced lipid accumulation in HepG2 cells through augmentation of de novo lipogenesis.</p>

Expressions of ire1α and xbp1 mRNA and proteins in mouse testis cells induced by low dose ionizing radiation / 吉林大学学报(医学版)

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.

Role of X-box Binding Protein 1 in Acrolein-induced Oxidative Injury of Retinal Pigment Epithelium / 中山大学学报(医学科学版)

[Objective]The oxidative injury of retinal pigment epithelium(RPE)plays a key role in the pathogenesis of age-related macular degeneration(ARMD). This study is to investigate the effects of endoplasmic reticulum stress and the vital transcriptional factor X-box binding protein 1(XBP1)in acrolein-induced oxidative damage of RPE.[Methods]RPE cells were treated with acrolein (75μmol/L)for 2~24 h,expression of glucose regulated protein 78(GRP78)and XBP1 was determined by Western blot analysis. After being transfected with XBP1 siRNA with 24 h,the expression of XBP1 was knocked-down in RPE cells. Protein level of Nrf2 and SOD2 was then determined by Western blot analysis and intracellular Reactive Oxygen Species(ROS)generation was determined by DCF staining. Acrolein was added for 8 h after being transfected with XBP1 siRNA or control siRNA for 24 h. Apoptosis was detected by TUNEL assay before and after the treatment. Subretinal injection of Cre or GFP adenovirus was performed in XBP 1flox mice. After 1 week the mice were sacrificed. Total RNA was extracted from mice eyecups using TRIzol and real-time RT-PCR was performed to determine the two XBP1 down-stream genes,ERdj4 and p58IPK. Cryosectioning and immunofluorescent staining were performed to look at the expression of XBP1,Nrf2 and SOD2 in mice RPE.[Results]Protein level of GRP78 was significantly un-regulated after exposure to acrolein for 2 and 4 h. XBP1 was activated after acrolein treatment for 6 h. Knock-down of XBP1 by siRNA down-regu?lates anti-oxidant genes expression and increased ROS generation in RPE cells. Loss of XBP1 exacerbates acrolein-induced cell apoptosis. XBP1 was knocked-down in the RPE of XBP1flox mice after subretinal injection of Cre adenovirus. Decreased mRNA level of ERdj4 and p58IPK,and decreased Nrf2 and SOD2 expression were seen in the Cre-injected group.[Conclusions]Acrolein induces ER stress and activates XBP1 in RPE cells. Knock-down of XBP1 down-regulates anti-oxidant genes expression ,increases ROS generation,and exacerbates acrolein-induced cell apoptosis. XBP1 plays a role in the anti-oxidant defense in the RPE cells.

Expression of unfolded protein response gene glucose regulated protein 78 and X-box binding protein 1 in esophageal squamous cell carcinoma and their clinical significance / 肿瘤研究与临床

Objective To investigate the expression of unfolded protein response (UPR) gene glucose regulated protein 78(GRP78) and X-box binding protein 1(XBP1) in esophageal squamous cell carcinoma, its effect on activation of the endoplasmic reticulum stress (ERS) and its mechanism in the growth of esophageal squamous cell carcinoma. Methods The expressions of GRP78 and XBP1 were detected by immunohistochemistry in 30 samples of esophageal squamous cell carcinoma and 30 samples of normal esophageal squamous epithelium. The correlation between expressions of both proteins and prognosis was analyzed. Results GRP78 positive rate was 83.3 %(25/30) in esophageal carcinoma, while the proportion was 20.0 %(6/30) in normal esophageal (χ2=25.833, P<0.05). XBP1 positive rate was 70.0 % (21/30) in esophageal carcinoma, while the proportion was 26.7%(8/30) in normal esophageal(χ2=20.872, P<0.05). The positive rates of GRP78 and XBP1 in invasive muscular layer of esophageal squamous cell carcinoma were significantly higher than those in invasive mucous layer. Conclusion GRP78 and XBP1 are highly expressed in esophageal squamous cell carcinoma, which may involve the occurrence and development of the esophageal carcinoma.

Valsartan Inhibits Myocardial Apoptosis by Down-regulating Myocardial X-box Binding Protein 1 Expression in Experimental Diabetic Cardiomyopathy Rat’s Model / 中国循环杂志

Objective: To study the relationship between myocardial X-box binding protein 1 (XBP1) expression and myocardial apoptosis in experimental diabetic cardiomyopathy (DCM) rat’s model and to clarify the mechanism of valsartan inhibiting myocardial apoptosis. Methods: A total of 50 Wistar rats were divided into 2 groups: Control group, the rats received intraperitoneal citrate buffer at 65mg/kg,n=10 and Streptozotocin group, the rats received intraperitoneal streptozotocin at 65mg/kg,n=40, all animals were treated for 7 days. DCM model was established in 37 rats (fasting blood glucose ≥ 16.7mmole/L) and they were further divided into 2 groups: DCM group, the rats received intragastric normal saline,n=20 and DCM + valsartan group, the rats received intragastric valsartan at 30mg/kg·day,n=17. The rats were treated for 16 weeks. The body weight, tail blood pressure, glucose and cardiac function were compared among 3 groups. Myocardial apoptosis was detected by TUNEL staining, RNA and protein expressions of myocardial cytochrome C, cleaved caspase 3, glucose regulation protein 78 (GRP78) and XBP1-s were examined by immunolfuorescence, real time RT-PCR and Western blot analysis. Results: Compared with Control group, DCM group showed disordered cardiac structure, more collagen content and myocardial apoptosis,P<0.05; increased RNA and protein expressions of GRP78, XBP1-s, cleaved caspase 3 and cytochrome C,P<0.05. Compared with DCM group, DCM + valsartan group had rather regularly arranged myocardiocytes, less interstitial ifbrosis and myocardial apoptosis,P<0.05; decreased RNA and protein expressions of GRP78, XBP1-s, cleaved caspase 3 and cytochrome C,P<0.05. Conclusion: Valsartan may inhibit myocardial XBP1 activation and therefore, reduce the myocardial apoptosis in experimental DCM rat’s model.

Expression of spliced XBP-1 s and ADRP in kidney of diabetic rats / 临床与实验病理学杂志

Purpose To investigate the expression of XBP-1s and ADRP in kidney of diabetic rats. Methods Diabetic rat models were successfully established by intraperitoneal injection of STZ. After two months rats were sacrificed and XBP-1s and ADRP were de-tected by immunohistochemistry and Western blot. Results XBP-1s and ADRP were located in renal tubular cells and increased by a-bout 2. 017 times and 1. 544 times in comparison with normal control rats (P<0. 05). Moreover, it was shown that high expression of XBP-1s was commonly accompanied with increased ADRP by Pearson correlation analysis and the correlation coefficient was 0. 723 (P<0. 05). Conclusion The increased XBP-1s may cause the up-regulation of ADRP in the kidney of diabetic rats.

Expression and clinical significance of X-box binding protein 1 in liver cancer / 肿瘤研究与临床

Objective To investigate the possible role of X-box binding protein (XBP1) expression at mRNA level in the hepatocarcinogenesis. Methods The expression of XBP1 at mRNA level was determined with RT-PCR method in liver carcinoma (42 cases), liver tissue adjacent to carcinoma (15 cases) and normal liver tissue (15 cases). XBP1 mRNA relative expression level was obtained by comparing with GAPDH mRNA level. The correlation between XBP1 expression and clinicopathological features of liver carcinoma were respectively analyzed. Results RT-PCR results showed that the expression of XBP1 mRNA in liver carcinoma cases (0.4396 ±0.0241) was significantly higher that that of liver tissues adjacent to carcinoma (0.4152±0.0252) and normal liver tissue (0.4095 ±0.0149), (F =12.79,P =0.001). No significant differences in the expression of XBP1 were found among different age and sex of the patients, different location, histological types, grade and metastatic status in liver carcinoma (P >0.05). Conclusion Increased expression of XBP1 mRNA is found in liver carcinoma but no correlation could be seen between XBP1 expression and clinicopathological features, which XBP1 is involved in the hepatocarcinogenesis.

Role of XBP1 signal transduction pathway in the ischemia/reperfusion injury of graft liver in rats / 中华器官移植杂志

Objective To explore the regulation mechanism of X box binding protein 1 (XBP1)signal transduction pathway for TNF-α and effective approach in ischemia/reperfusion (I/R) injury of liver transplantation for short hairpin RNA (shRNA) interference used to gene therapy in liver graft.Methods Male Sprague-Dawley rats were divided into three groups: the cold ischemia transfection group (CIT), the in vivo transfection group (IVT) and the control group. Experiments of orthotopic liver transplantation were performed by two cuff method. The rats in CIT were perfused with XBP1-shRNA plasmid (pSIXBP1) during cold ischemia phase, those in IVT received the equivalent volume (2 ml) of pSIIRAK 4 after portal vein inoculation, and those in the control group were not subjected to any treatment. Rats were killed at 60 or 180 min after restoring reperfusion of hepatic portal vein.Histopathological damage degree of graft liver was observed by light microscope. The expression levels of XBP1 gene and protein were detected by RT-PCR and Western blotting. The activities of NF-κB and the serum TNF-α level were detected by ELISA. Results All the indexes including the degree of histopathological damage, the expression levels of XBP1 mRNA and protein and the TNF-α level were significantly decreased in CIT as compared with IVT and control group (P＜0. 05). However,there was no significant difference in NF-κB activity among the three groups (P＞0. 05). Conclusion The role of XBP1 pathway in TNF-α gene regulation and that of NF-κB pathway in rat liver I/R injury are two relatively independent aspects, and the depression of XBP1 expression with XBP1 shRNA through portal vein perfusion during cold ischemia phase could effectively alleviate graft hepatic I/R

Screening and Identification of The Proteins Interacting with The Transcription Factor X-box Binding Protein 1 / 生物化学与生物物理进展

X-box binding protein1 (XBP1) is an important transcription factor, which participates in many signal transduction procession.To investigate the biological function of XBP1, yeast two-hybrid system to screen proteins interacting with XBP1 in hepatocytes was performed. The XBP1 coding sequence was amplified by polymerase chain reaction (PCR) method, and was cloned in pGEM-T vector. After the target region was sequenced, it was subcloned into the bait plasmid pGBKT7, then was transformed into yeast AH109(a type). After the expression of bait plasmid pGBKT7-XBP1 in AH109 yeast strains were proved by Western blot. The transformed yeast AH109 was mated with yeast Y187(α type) containing hepatocyte cDNA library plasmids pACT2 in 2 ×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medilium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After sequencing and verifying ORF of positive colonies,7 different kinds of proteins were obtained. In order to further testify the interaction between the screened proteins and XBP1, one of positive colonies MT1E was cloned. The interaction between MT1E and XBP1 in vitro/in vivo were examined successfully with GST pulldown and coimmunoprecipitations. It was shown that MT1E would be a new regulatory protein of XBP1. These screened proteins by yeast two-hybird system were closely correlated with liver fundamental metabolism,protein synthesis and transport,cell proliferation and apoptosis. The results mentioned above contributed to reveal the XBP 1 biological function, and brought some new clues for further exploration of the expressing and regulating mechanism of XBP1.