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Objective To detect the expressions of X-box binding protein 1 unspliced （xbp1u） and X-box binding protein 1 spliced （xbp1s） mRNA in peripheral blood CD19+B cells of patients with lupus nephritis （LN） and healthy subjects and explore the clinical significance of XBP1 in LN patients. Methods The expression levels of xbp1u and xbp1s in peripheral blood CD19+B cells of 65 LN patients and 30 healthy controls were detected by real-time polymerase chain reaction （RT-PCR）. And the ratio of CD19−CD138+plasma cells/CD19+B cells in peripheral blood was detected by flow cytometry. Results The expression level of xbp1u mRNA in CD19+B cells of active LN group （0.078±0.034） was higher than that in control group（0.035±0.011）（P＜0.05） and in stable group（0.049±0.016）（P＜0.05）. The expression level of xbp1s mRNA in CD19+B cells of active LN group（0.076±0.037）was higher than that in control group（0.022±0.010）（P＜0.05） and stable group（0.043±0.017）（P＜0.05）. The ratio of CD19−CD138+ plasma cells /CD19+B cells in LN group（0.059±0.024）was significantly higher than that in control group （P＜0.01） and stable group （0.073±0.036）（P＜0.05）; XBP1 expression in LN patients was correlated positively with the ratio of CD19−CD138+ plasma cells/CD19+B cells （r value = 0.138 & 0.036）, positively with anti-ds-DNA antibody and systemic lupus erythematosus disease activity index（SLEDAI）score （r value = 0.417 & 0.058） and negatively with hemoglobin and complement C3 （r value = −0.559 & −0.219）. Conclusion The expressions of xbp1u and xbp1s mRNA in peripheral blood mononuclear cells of LN patients in active group are higher than those in healthy group. Both are correlated positively with SLEDAI score and anti-DS DNA antibody. The expression of XBP1 is up-regulated in B cells of LN patients and it is significantly correlated with plasma cells. It implies that XBP1 may participate in the pathogenesis of LN through promoting the differentiation of B cells.

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Objective: To investigate the mechanism and clinical value of nicotinamide phosphoribosyltransferase (NAMPT) in multiple myeloma (MM)., Methods: RT-qPCR and Western blot were used to detect the expression of NAMPT in MM cells and normal bone marrow mononuclear cells. The biological function of NAMPT was analyzed by cell proliferation and apoptosis assay, small interfering RNA silencing, overexpression assay and chromatin immunoprecipitation assay., Results: The mRNA and protein expression levels of NAMPT in MM cell lines (MM1R, MM1S, U266 and RPMI-8226) were significantly higher than those in normal bone marrow mononuclear cells ( P < 0.001), and were most obvious in U266 cells. Compared with Si-NC group, the proliferation of U266 cells in Si- NAMPT group was significantly inhibited at 24, 48 and 72 h after transfection ( P =0.006, P < 0.001, P =0.001), and the apoptosis rate of U266 cells was significantly increased at 48 h after transfection ( P < 0.001). Compared with Flag-NC group, U266 cell proliferation in Flag- NAMPT group was significantly increased ( P =0.003, P =0.002, P < 0.001), while the apoptosis rate decreased significantly at 48 h after transfection. The expression of NAMPT in U266 cells was regulated by XBP1 at transcriptional level. The proliferation rate of U266 cells with XBP1 or NAMPT stable knockout or MKC3946 pretreated with bortezomib was significantly decreased, the levels of BCL-2 mRNA and protein were also significantly decreased, while the levels of BAX mRNA and protein were significantly increased, moreover, the cleavage degree of caspase-3 significantly decreased, while caspase-3/7 activity increased dramatically ( P < 0.05)., Conclusions: The high expression of NAMPT in MM cell line can promote MM cell proliferation and inhibit apoptosis. NAMPT is regulated by IRE1α-XBP1 signaling pathway in U266 cells. Stable knockdown of NAMPT or blocking of IRE1α-XBP1 pathway can significantly increase the sensitivity of U266 cells to bortezomib.

Objective: To observe the effect of moxibustion at Governor Vessel acupoints on inositol requiring enzyme 1 （IRE1） / X-box binding protein 1 （XBP1） pathway in hippocampal CA1 region of rats with vascular dementia （VD）, so as to explore its mechanisms in the treatment of VD., Methods: Male SD rats were randomly divided into normal, sham operation, model, moxibustion （Moxi） and medication groups （n=12）. The VD model was established by permanent ligation of bilateral common carotid arteries. For rats of the Moxi group, mild moxibustion was given to "Baihui" （GV20）, "Dazhui" （GV14） and "Fengfu" （GV16） for 20 min each point, once a day for consecutive 6 days per week, for a total of 4 weeks. For rats of the medication group, intragastric perfusion of nimodipine was given 3 times each day with total dose of 2 mg•kg -1 •d -1 for 4 weeks. Morris water maze test was used to detect the learning and memory ability of rats before and after modeling as well as after intervention. The apoptosis rate of nerve cells in hippocampal CA1 region was detected by TUNEL staining. The proteins and mRNA expression levels of IRE1, XBP1, B-cell lymphoma/leukemia-2 （Bcl-2）, Bcl-2 associated X protein （Bax） in hippocampal CA1 region were detected by Western blot and real-time quantitative PCR, respectively., Results: Compared with the sham operation group, the average escape latency was significantly prolonged （ P <0.01）, the number of times crossing the original platform was significantly reduced （ P <0.01）, the apoptosis rate of nerve cells in hippocampal CA1 region was significantly increased （ P <0.01）, the proteins and mRNA expression levels of IRE1, XBP1 and Bax were significantly increased （ P <0.01）, and the expression levels of Bcl-2 protein and mRNA were significantly decreased （ P <0.01） in rats of the model group. After treatment, compared with the model group, the average escape latency was significantly shortened （ P <0.01）, the number of times crossing the original platform was increased （ P <0.05）, the apoptosis rate of nerve cells in hippocampal CA1 region was significantly decreased （ P <0.01）, the protein and mRNA expression levels of IRE1, XBP1 and Bax were decreased （ P <0.05, P <0.01）, and the expression levels of Bcl-2 protein and mRNA were increased （ P <0.05, P <0.01） in rats of the Moxi group and medication group. There was no significant difference in the above indexes between the Moxi group and the medication group., Conclusion: Moxibustion at the acupoints of Governor Vessel can improve the cognitive function of VD rats, and its mechanism may be related to regulating IRE1/XBP1 pathway, inhibiting the release of pro-apoptotic protein Bax, increasing the expression of anti-apoptotic protein Bcl-2, and thus inhibiting the apoptosis of hippocampal nerve cells.

Objectives: This study aims to investigate the effect of X-box binding protein 1 (XBP1), a key signal molecule of ERS, on the insulin signaling pathway in adipocytes stimulated by Porphyromonas gingivalis ( P. gingivalis )-lipopolysaccharide (LPS), a pathogenic bacterium of periodontitis., Methods: Primary cultured rat adipocytes were stimulated by P. gingivalis -LPS (100 ng·mL -1 ) for 4, 8, 12, and 24 h. The protein expression levels of insulin receptor substrate-1 (IRS-1), phosphoinositide dependent protein kinase (p-PDK-1), and protein kinase B (p-AKT-1) in the insulin signaling pathway were detected by Western blot analysis. pLVX-NC1, pLVX-XBP1, pLVX-NC2, and pLVX-XBP1-RNAi were transfected into adipocytes, respectively. The transfected rat adipocytes were stimulated by P. gingivalis -LPS, and the protein expression of the insulin signaling pathway was detected by Western blot., Results: The Western Blot showed decreased protein expression of the insulin signaling pathway in rat adipocytes stimulated with P. gingivalis -LPS compared with the control, and the difference was statistically significant ( P <0.05). The protein expression levels of IRS-1, p-PDK-1, and p-AKT in the rat adipocytes of pLVX-XBP1 were significantly higher than those in pLVX-NC1 at 8 and 12 h after P. gingivalis -LPS stimulation ( P <0.05). The protein expression levels of IRS-1, p-PDK-1, and p-AKT in the rat adipocytes of pLVX-XBP1-RNAi were significantly lower than those in pLVX-NC2 at 4, 8, 12, and 24 h after P. gingivalis -LPS stimulation ( P <0.05)., Conclusions: P. gingivalis -LPS regulates the insulin signaling pathway in adipocytes th-rough XBP1.

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.

Objective: To study the inhibition and mechanism on lung cancer A549 cells xenografts in BALB / c nude mice., Methods: 40 BALB / c mice were selected to establish lung cancer xenografts models with human lung adenocarcinoma A549 cells,which were randomized into model group,cisplatin group( 0. 002 g / kg), Yiqi Chutan Formula low-dose group( 3. 0 g / kg),Yiqi Chutan Formula high-dose group( 6. 0 g / kg) and combination group[Yiqi Chutan Formula 6. 0( g / kg) / cisplatin 0. 002( g / kg) ], with eight mice in each group. Yiqi Chutan Formula was given once a day with 0. 2 m L from 8th day after modeling,and successive medication for14 days. After given cisplatin with 0. 2 m L by intraperitoneal injection once a day in 17 th day,and modeling for 5 days. The tumor weight and tumor volume were detected. And the tumor inhibitory rate were calculated. The expression of CNX and XBP1 was detected by immunohistochemistry,Western blotting and and real-time quantitative PCR., Results: Compared with the model group, the tumor weight and the tumor volume were lower in cisplatin group, Yiqi Chutan Formula high-dose group and combination group( P < 0. 01);and the combination group was better than the cisplatin group( P < 0. 01). The results of immunohistochemistry, Western blotting and real-time quantitative PCR showed that, compared with the model group, the expression of CNX and XBP1 were lower in cisplatin group, Yiqi Chutan Formula high-dose group and combination group( P < 0. 01),and the combination group was better than the cisplatin group( P < 0. 05 or P < 0. 01)., Conclusion: Yiqi Chutan Formula can inhibit A549 lung cancer cells in BALB/c nude mice. Combined with chemotherapy drugs, the killing effect on lung cancer can be enhanceed. Its mechanism may be related to down-regulating CNX and XBP1 to inhibit the unfolded protein response to lead to apoptosis.

The saturated free fatty acid (FFA), palmitate, could induce apoptosis in various cell types, but little is known about its effects on human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). Here, we investigated whether palmitate induced apoptosis and endoplasmic reticulum (ER) stress in hUC-MSCs. hUC-MSCs were stained by labeled antibodies and identified by flow cytometry. After administration with palmitate, apoptotic cell was assessed by flow cytometry using the Annexin V-FITC/7-AAD apoptosis detection kit. Relative spliced XBP1 levels were analyzed using semi-quantitative RT-PCR. The mRNA of BiP, GRP94, ATF4 and CHOP were analyzed by real-time PCR. Relative BiP and CHOP protein were analyzed using Western blot analysis. The results showed that hUC-MSCs were homogeneously positive for MSC markers; palmitate increased apoptosis of hUC-MSCs and activated XBP1 splicing, BiP, GRP94, ATF4 and CHOP transcription. These findings suggest that palmitate induces apoptosis and ER stress in hUC-MSCs.

Objective: To explore the effects of GRP78 suppression on the sensitivity to cisplatin and elucidate the role and mechanism of GRP78 in ovarian cancer cisplatin resistance., Methods: The GRP78 siRNA expression plasmid was constructed to suppress GRP78 expression. Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. Endoplasmic reticulum stress-related apoptosis related protein expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. And cell apoptosis was detected by flow cytometry., Results: The expressions of GRP78, CHOP and cleaved-caspase 4 were induced significantly by cisplatin (6 mg/L) in SKOV3 cells. And the expression of GRP78 was induced significantly by cisplatin in SKOV3/DDP cells. But the expressions of CHOP and cleaved-caspase 4 showed no significant difference. Inhibition of GRP78 expression and cisplatin combined treatment significantly increased the expressions of cleaved-caspase 4 and cleaved-caspase 3 in SKOV3/DDP cells. Inhibition of GRP78 expression reduced the cisplatin-induced up-regulations of p-Akt and p-mTOR and induced XBP1 mRNA shear expression and CHOP mRNA expression., Conclusion: Inhibition of GRP78 expression reverses cisplatin resistance in SKOV3/DDP cells. The mechanism may be through the activity of Akt/mTOR signaling pathway, CHOP expression levels and caspase activity.

The purpose of the present study is to explore the effect of oxidized low density lipoprotein (ox-LDL) on the induction of endoplasmic reticulum stress (ERS) and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process. RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum, and then treated with ox-LDL (25, 50 and 100 mg/L), anti-CD36 monoclonal antibody+ox-LDL and tunicamycin (TM), respectively. After incubation for 24 h, the cells were collected. The cellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymatic colorimetry. The expression of glucose-regulated protein 94 (GRP94), a molecular marker of ERS, was determined by immunocytochemistry assay. The levels of GRP94 protein, phosphorylated inositol-requiring enzyme 1 (p-IRE1) and X box binding protein 1 (XBP1) in RAW264.7 cells were detected by Western blotting. The results indicated that after incubation with ox-LDL (25, 50 and 100 mg/L) for 24 h, a large amount of lipid droplets were found in the cytoplasm, and the contents of cellular total cholesterol were increased by 2.1, 2.8 and 3.1 folds compared with the control, respectively. Anti-CD36 antibody decreased markedly the cellular lipid accumulation induced by ox-LDL at 100 mg/L. Both ox-LDL and TM, a specific ERS inducer, could up-regulate the protein expression of GRP94 in a dose-dependent manner. Furthermore, p-IRE1 and XBP1, two key components of the unfolded protein response, were also significantly induced by the treatment with ox-LDL. The up-regulations of the three proteins induced by ox-LDL were inhibited significantly when the macrophages were pre-incubated with anti-CD36 antibody. These results suggest that ox-LDL may induce ERS in a dose-dependent way and subsequently activate the unfolded protein response signaling pathway in RAW264.7 macrophages, which is potentially mediated by scavenger receptor CD36.

The aim of this study was to explore the effect of 2 different spliceosomes of X-box binding protein 1 (XBP-1), the spliced form XBP-1s and unspliced form XBP-1u, on myeloma cell differentiation and its mechanism. The overexpression plasmids pcDNA3.1-C-XBP1u and pcDNA3.1-C-XBP1s were constructed and transfected into myeloma cell line U266, RPMI8226. The morphology of U266 and RPMI 8226 cells was observed by means of light microscope, the expression rate of CD49e on cell surface was detected by flow cytometry, the ELISA was used to determine the changes of light chain protein level in supernatants of cell culture, the Western blot was used to assay the expression changes of XBP1u and XBP1s. The results showed that the overexpression of XBP1u could promote the myeloma cell differentiation morphologically displaying the maturation of plasmocytes, the CD49e positive expression rates on surface of U266 and RPMI8226 cells were obviously up-regulated from 9.02±0.3% and 5.17±0.92% in control group to 27.7±1.14% and 13.97±1.79% respectively (p<0.01), the levels of light chain protein in supernatants of U266 and RPMI 8226 cell cultures increased from 474.75±19.52 ng/ml and 289.44±6.19 ng/ml in control group to 692.34±21.17 ng/ml and 401.55±13.7 ng/ml respectively (p<0.01, p<0.05), while the above-mentioned parameters in the overexpressed XBP-1s showed no significant changes, which indicated no promotive effect of overexpressed XBP1s on myeloma cell differentiation. It is concluded that the up-regulation of XBP-1u expression plays an important role in the differentiation of myeloma cells.

This study was purposed to investigate the effect of xbp-1 gene silencing on bortezomib-induced apoptosis in multiple myeloma cell line NCI-H929 (H929). After xbp-1 gene expression was interfered by small hairpin RNA, the cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression level of XBP-1 protein was detected by Western blot. The results showed that XBP-1 protein level of H929 cells was inhibited effectively by the PLL3.7 lentiviral vector mediated expression xbp-1 shRNA. The apoptosis rate was significantly higher in xbp-1 shRNA-expressing cells than in untreated control group [(10.13±0.61)% vs (2.5±0.2)%, p<0.05]. After treatment with bortezomib, the apoptosis rate of XBP-1 protein functionally deficient H929 cells was significantly higher than those in vector control group [(45.07±1)% vs (19.53±0.8)%, p<0.05]. It is concluded that xbp-1 gene silencing can significantly enhance the pro-apoptotic activity of bortezomib in multiple myeloma cells.

Objective: To study the molecular mechanism of differentiation induction by 2-methoxyestradiol (2ME2) of myeloma cell lines., Methods: Differentiation induction effect on myeloma cell lines LP-1, CZ-1 and NCI-H929 which were incubated with 2ME2 and XBP-1, Blimp-1, pax-5 phosphorothioate antisense oligodeoxynucleotide (ASODN) was evaluated by cell morphology, CD49e expression, quantitation of light chain secretion, and the level of pax-5 and XBP-1 mRNA expression., Results: 2ME2 caused morphological, immunophenotypic and the supernatant light chain secretion changes typical of differentiation in all the three myeloma cell lines. 2ME2 up-regulated the XBP-1 mRNA expression. XBP-1 and Blimp-1 ASODNs partially inhibited the differentiation of LP-1, CZ-1, NCI-H929 cells induced by 2ME2; whereas pax-5 ASODN did the contrary. After incubated with pax-5 ASODN for 72 hours, LP-1, CZ-1, NCI-H929 cells exhibited characteristic morphologic feature of differentiation. The expression of CD49e was increased statistically (P < 0.05). Light chain secretion in the supernatant was also increased statistically (P < 0.05). After incubation with Blimp-1 ASODN, the level of XBP-1 mRNA was declined, while the level of pax-5 mRNA increased., Conclusion: 2ME2 could induce cell differentiation and up-regulate XBP-1 mRNA expression in myeloma cell lines. Blimp-1 could help 2ME2 with inducing differentiation of myeloma cells through downregulating pax-5 mRNA and upregulating XBP-1 mRNA.

Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.

Estrogen receptor (ERalpha) plays an important role in the development and progression of breast cancer. Several recent studies have demonstrated that expression of human X-box binding protein 1 (XBP-1) is associated with ERalpha status in breast tumors and overexpressed in a subset of breast tumors. XBP-1 has two splicing variants, which were designated as XBP-1S and XBP-1U, respectively. However, little is known about the expression pattern of XBP-1S and XBP-1U in breast cancer cells and about their roles in ERalpha signaling. In this study, the expression of two splicing forms of XBP-1 was detected in breast cancer cell lines with RT-PCR. Estrogen response element (ERE) -containing luciferase reporter assay was used to determine the effects of XBP-1S and XBP-1U on the transcription activity of ERalpha in MDA-MB-435 breast cancer cells. The result showed that both XBP-1S and XBP-1U enhanced the transcription activity of ERalpha in a hormone-independent and dose-dependent manner and the activity of of XBP-1S is higher than that of XBP-1U. Enhancement of ERE-containing luciferase reporter gene expression by XBP-1S and XBP-1U was dependent on ERalpha. These data suggest that XBP-1S and XBP-1U may play important roles in breast cancer growth and progression through ERalpha signaling.

The estrogen receptor (ERalpha) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that human X-box binding protein 1 (XBP-1) mRNA expression is associated with ERalpha status in breast tumors. More recently, two forms of XBP-1 were identified due to their unique splicing. The two splicing variants of XBP-1 were designated XBP-1S and XBP-1U, respectively. In this study, the coding sequences of XBP-1S and XBP-1U were cloned respectively into the expression vector pcDNA3 harboring FLAG epitope, generating the recombinant plasmids pcDNA3-FLAG-XBP-1S and pcDNA3-FLAG-XBP-1U. Western blot analysis showed that both XBP-1S and XBP-1U were expressed in mammalian cells. To determine the effects of XBP-1S and XBP-1U on the transcriptional activity of ERalpha, MDA-MB-231 breast cancer cells were cotransfected with the expression vectors for ERalpha and either pcDNA3-FLAG-XBP-1S or pcDNA3-FLAG-XBP-1U. The results indicated that XBP-1S and XBP-1U enhanced ERalpha-mediated transcriptional activities in a hormone-independent manner. GST pull-down assay showed that both XBP-1S and XBP-1U interacted with ERalpha. These data suggest that XBP-1S and XBP-1U may play an important role in breast cancer growth and progression through ERalpha signaling.