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Objective@#To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells (hDPCs) in vitro.@*Methods@#This study was reviewed and approved by the Ethics Committee. hDPCs were cultured using a modified tissue explant technique in vitro. The effects of different concentrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium (MTT) assay. Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium, and the cells were induced in vitro. Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells, and the mRNA expression of the key differentiation factors was detected by RT-qPCR. Moreover, the expression of key molecules of endoplasmic reticulum stress (ERS) was detected by RT-qPCR and Western blot. The data were analyzed with the SPSS 18.0 software package.@*Results@#Low concentration of NaF (0.1 mmol/L) could stimulate cell proliferation in vitro, while a high concentration (5-10 mmol/L) could inhibit cell proliferation (P<0.05). According to the literature and the experimental data, 0.1 mmol/L NaF was selected as the following experimental concentration. The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vitro. The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) and osteocalcin (OCN), were increased (P<0.05). The mRNA levels of ERS markers (splicing x-box binding protein-1 (sXBP1), glucose-regulated protein 78 (GRP78) and activating transcription Factor 4 (ATF4) were increased in NaF-treated cells. The protein expression levels of key ER stress molecules (phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α) and ATF4) were higher in NaF-treated cells.@*Conclusion@#A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.
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Mucormycosis is an infectious disease characterized by rapidly progressive vascular invasiveness, thrombosis and tissue necrosis, which could be potentially responsible for blocking the exudation of leukocytes to infection sites and affecting drug distribution. Glucose-regulated protein 78 (GRP78) is a key protein involved in regulating the invasiveness of Mucorales. Endoplasmic reticulum GRP78 is overexpressed under various stress conditions and transported to the cell membrane to become a cell surface receptor for Mucorales entering into vascular endothelial cells. This article reviewed the mechanisms and pathogenesis of GRP78-mediated host cell invasion and summarized the progress in related targeted drugs, aiming to provide reference for developing multi-target intervention against mucormycosis.
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Objective:To investigate the significance and level of serum GRP78 and GRP94 in the early stage ofacute pulmonary embolism.Methods:Ninety APE patients (APE group) and forty healthy controls (control group) in Affiliated Zhongshan Hospital of Dalian University from January 2018 to March 2020 were recruited. The level of serum D-Dimer, GRP78 and GRP94 were compared between the two groups. The pulmonary embolism group was divided into three groups according to guidelines for the diagnosis and treatment of pulmonary embolism in ESC in 2019: low risk, medium risk, high risk, The level of serum D-Dimer, GRP78, GRP94, blood gas, pulmonary arterial pressure, PESI mark of three groups were compared.Results:Two groups had no significant difference in sex, age, body mass index ( P>0.05), but the level of serum D-Dimer, GRP78 and GRP94 in control group and APE group were higher than those in the control group: (3.86 ± 1.82) mg/L vs. (0.31 ± 0.15) mg/L, (2.68 ± 0.71) μg/L vs. (1.64 ± 0.38) μg/L, (1.31 ± 0.29) μg/L vs. (0.98 ± 0.13) μg/L ( P<0.05). The levels of serum D-Dimer, GRP78, GRP94, pulmonary arterial pressure, PO2, PESI score of different degree group (low risk, medium risk, high risk) were higher than those of low risk group and medium risk group: (5.63 ± 1.75) mg/L vs. (2.29 ± 0.51) and (3.64 ± 1.02) mg/L, (3.24 ± 0.76) μg/L vs. (2.17 ± 0.41) μg/L and (2.64 ± 0.47) μg/L, (1.57 ± 0.33) μg/L vs. (1.12 ± 0.13) and (1.26 ± 0.14) μg/L, (47.75 ± 6.98) mmHg (1 mmHg = 0.133 kPa) vs. (26.15 ± 4.63) and (35.21 ± 5.85) mmHg, (123.5 ± 20.59) scores vs. (85.5 ± 14.31) and (102.5 ± 13.32) scores ( P<0.05), and that of medium risk group were higher than those of low risk group ( P<0.05). But PO 2 of high risk group was lower than that of low risk group and medium risk group ( P<0.05), and PO 2 of medium risk group was lower than that of low risk group ( P< 0.05). pH of three group had no significant difference ( P>0.05). PCO 2 of high risk group was lower than that of low risk group and medium risk group ( P<0.05), and PCO 2 of medium risk group and low risk group had no significant difference ( P>0.05). The level of serum D-Dimer, GRP78, GRP94 were positively correlated withPESI score ( r = 0.610, 0.622, 0.627, P<0.01). After treatment, the levels of D-Dimer, GRP78 and GRP94 were significantly decreased ( P<0.05). Conclusions:The level of serum GRP78 and GRP94 are connected with acute pulmonary embolism, and it can reflect the severity of the disease.
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miR-340 can promote the proliferation and invasion of cancer cells, but how miR-340 regulates the occurrence and development of cancer in colon cancer is rarely reported. This study aims to explore the biological function and target gene regulation mechanism of miR-340 in colorectal cancer cells. Firstly, RT-qPCR was used to detect the expression level of miR-340 in different colorectal cancer cell lines, and then miR-340 was overexpressed or inhibited in COLO-205 cells. CCK-8, Transwell migration and invasion assay, and flow cytometry were performed to analyze the cell ability of proliferation, migration and invasion, as well as cell apoptosis and cell cycle. Finally, after bioinformatics prediction of miR-340 target genes, luciferase reporter gene and Western blot experiment were applied to verify those target genes. The results showed that miR-340 was downregulated in COLO-205 cells. Compared with the control group, cell proliferation, migration and invasion were significantly inhibited in the miR-340 overexpression group, but were promoted in the miR-340 suppression group (P<0. 01). The results of flow cytometry showed that the percentage of apoptosis in the miR-340 overexpression group was significantly increased, while the percentage of apoptosis in the miR-340 inhibition group was decreased (P<0. 01). The bioinformatics analysis of the overexpression miR-340 transfection group showed that the 3′UTR of glucose regulated protein 78 kD (GRP78) had a miR-340-5p binding site, and the luciferase activity was significantly reduced in the overexpression miR-340 group (P<0. 01); Western blot results also showed that overexpression of miR-340 can inhibit the expression of GRP78, while inhibiting miR-340 expression, the expression of GRP78 is relieved. In summary, miR-340 can directly target GRP78 to promote the apoptosis of COLO-205 cells and inhibit their proliferation, migration and invasion.
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Aim To investigate whether endoplasmic reticulum stress is involved in the neurotoxicity of sodi¬um arsenite and clarify whether over-expression of 3-mercaptopyruvate sulfurtransferase (MPST) regulates endoplasmic reticulum stress induced by arsenic. Methods The SH-SY5Y cell line stably expressing the exogenous MPST gene was obtained by constructing the lentiviral vector of MPST gene. The SH-SY5Y cells were randomly divided into six groups, the SR-MPST over-expression group stably expressing the exogenous MPST gene, SH-PEB control group transfected with empty vector, the arsenite treatment group ( NaAs02 group ), TUDC A treatment group ( blocker of endoplasmic reticulum stress ) and TUDC A + NaAs02 group. Western blot was used to examine the protein expression of GRP78 and CHOP after different treatment. Results Although MPST overexpression had no significant effects on the expression of GRP78 and CHOP proteins, NaAs02 could significantly increased the protein levels of GRP78 and CHOP ( P < 0. 01 ) and the up-regulation of GRP78 and CHOP proteins caused by NaAs02 could be blocked by the treatment of TUDC A. In addition, the inhibition by MPST overexpression on the arsenic-induced increase of GRP78 and CHOP proteins (P <0. 01 ) could also be reversed by the TUDC A treatment significantly. Conclusions The GRP78/ CHOP endoplasmic reticulum stress pathway is involved in the neurotoxic damage induced by arsenic; MPST overexpression may decrease arsenic-induced endoplasmic reticulum stress.
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Purpose: Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease, which could even induce severe brain damage in some patients. As the underlying mechanism of the invasion and replication process still remains largely unknown, we investigated the role of candidate proteins expressed during EV71 invasion in human brain microvascular endothelial cells (HBMECs) to delineate the pathophysiological mechanism of EV-71 infection. Materials and Methods: Ninety-one candidate EV71-associated proteins which could bind the major capsid protein (viral protein 1 [VP1]) of EV71 on the HBMEC were identified by applying an analysis of glutathione-S-transferase pull-down coupling with liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). Seventy-eight kDa glucose-regulated protein 78 (GRP78) binding to the VP1 protein was further validated by co-immunoprecipitation, immunofluorescence and western blot analysis. To explore the role of GRP78 in EV71 infection, GRP78 was knocked down and overexpressed in HBMEC and was verified by TCID50 assay. Results: LC-ESI-MS/MS-identified 91 proteins were subjected to gene ontology analysis, and on molecular and biological function analysis revealed GRP78 act as an important binding protein in mediating EV71 infection. In addition, immunofluorescence demonstrated the co-localisation of GRP78 and VP1 in cytoplasm of the infected HBMEC. The TCID50 assay showed that knockdown of GRP78 could attenuate the replication capacity of EV71 in HBMEC, and the overexpression could increase the virus titre in HBEMC at 24 h post-infection suggesting that GRP78 was associated with the replication capacity of EV71 in HBMEC. Conclusion: These findings provided evidence that GRP78 plays an important role during the progression of EV71 infection as a mediator in HBMEC.
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Objective: To investigate the effects and mechanisms of iridoid glycosides of Scrophulariae Radix (IGRS) via endoplasmic reticulum stress-mediated apoptosis pathway on the primary cortical neurons induced by oxygen glucose deprivation/reperfusion (OGD/R). Methods: Newborn SD rats were performed primary cortical neurons culture. And the primary cortical neurons were pretreated with IGRS (50, 100, and 200 μg/mL) for 24 h, and the in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was applied. The neurons purity and morphology were observed under inverted microscope, the cell viability was detected by MTT assay; the intracellular lactate dehydrogenase (LDH) level and superoxide dismutase (SOD) activity were detected by commercial kit. The apoptotic rate was detected by flow cytometry. The expression of C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78) and Caspase-12 protein were detected by western blotting. Results: The cultured primary cortical neurons were plump with high purity in good condition. Compared with the control group, the primary cortical neurons were retracted and rounded after OGD/R treatment, and the surface of the neurons became rough; The cell viability and SOD activity were significantly decreased; The LDH level and apoptotic rate were evidently increased; The expression of CHOP, Caspase-12, and GRP78 were significantly increased. Compared with the model group, IGRS could relieve neurons damage, increase cell viability and SOD activity, decrease LDH level and apoptotic rate, and down-regulate the expression of CHOP, Caspase-12, and GRP78. Conclusion: IGRS can antagonize the neuronal damage induced by OGD/R in primary cortical neurons, and its mechanism is related to the inhibition of endoplasmic reticulum stress-mediated apoptosis.
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@#Glucose-regulated protein 94(Grp94), an endoplasmic reticulum resident Hsp90 paralog, has a limited set of client proteins. Selective inhibition of Grp94 has emerged as a new direction for the development of drugs targeting the Hsp90 chaperone system. Now Grp94-Probe, an affinity-based probe of Grp94, was designed and synthesized based on DDO-5813, a most potent Grp94-selective inhibitor we found previously. Using fluorescence polarization(FP)assay and double staining assay with ER-Red in cells, we confirmed the binding of Grp94-Probe with ER Grp94. The FR results showed that the probe exhibited high affinity for Grp94N(EC50=117. 9 nmol/L)without exhibiting obvious Hsp90α inhibition, Moreover, as a fluorescence probe molecule, Grp94-Probe could better distinguish the inhibitory activity of compounds for Grp94N. The results of fluorescence analysis in cells showed that Grp94-Probe could co-stain with ER-Red in the endoplasmic reticulum, and the fluorescence did not decay rapidly with time after 4 h of staining, which further indicated the binding of Grp94-Probe with Grp94 in cells. This Grp94 selective probe can be further used for biology evaluation of Grp94 inhibitor and exploration of Grp94 biological functions.
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Aim: To explore the role of apoptosis stimulating protein 2 of p53 (ASPP2) on L-NAME induced apoptosis of placental trophoblast cells by regulating glucose-regulated protein78(GRP78), and provide a theoretical basis for the study of clinical pregnancy-induced hypertension. Methods: The HTR-8/SVneo human placental trophoblast cells were cultured in vitro, and in the absence (control group) or presence of 100 μmol · L-1 L-NAME (L-NAME group) for 48 h. The effects of L-NAME on placental trophoblast cell apoptosis were tested using flow cytometry and AO/EB assay. The expressions of caspase-12, GRP78 and ASPP2 were detected by Western blot. The ASPP2 interference with adenovirus was used to transfect the cells, and the mRNA expression level of ASPP2 and the protein expression level of GRP78 were detected by qRT-PCR and Western blot, respectively. After treated with 100 (xrnol · L-1 L-NAME for 48 h, the protein expression of caspase-12 and GRP78 was detected by Western blot and immunofluorescence. Results: Compared with control group, the placental trophoblast cell apoptosis significantly increased in L-NAME group (P < 0. 05). AO/EB staining showed that compared with control group, the majority of cells in L-NAME group showed bright orange and the number of late apoptotic cells increased significantly. At the same time, caspase-12, GRP78 and ASPP2 protein expression increased (P < 0. 05, P < 0. 01). After interfering with ASPP2, caspase-12 and GRP78 protein expressions decreased (P < 0. 05). Conclusions: Down-regulation of ASPP2 could decrease GRP78 expression and inhibit L-NAME-induced apoptosis in placental trophoblast cells.
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Objective: To investigate the relationship between the expression of glucose-regulated protein 78 (GRP78) and gemcitabine chemotherapy in the patients with non-small cell lung cancer (NSCLC) and the effects of GRP78 on the viability of lung adenocarcinoma SPCA1 cells and the chemosensitivity of gemcitabine, and to elucidate its mechanisms. Methods: The positive expression rates of GRP78 in 32 cases of cancer tissue of the NSCLC patients were detected by immunohistochemical staining. The SPCA1 cells with high expression of GRP78 were selected as the subjects. RNA interference technique was used to down-regulate the expression of GRP78 in SPCA1 cells (interference group) and the cells treated with shNC were used as control group. MTT assay was used to detect the viabilities of SPCA1 cells in various groups. Negative control group, interference group, control + gemcitabine group, and interference+ gemcitabine group were set up; colone formation assay was used to detect the colone formation rates of SPCA1 cells in various groups; Western blotting method was used to detect the expression amount of Akt, p-Akt, PI3K, and p-PI3K in the SPCA1 cells in various groups. Results: The immunohistochemical staining results showed that the positive expression rate of GRP78 in cancer tissue in the remission NSCLC patients was significantly higher than that in the no-remission NSCLC patients after treated with gemcitabine (P<0. 05). The MTT assay results showed that compared with negative control group, the viability of SPCA1 cells in interference group was decreased significantly (P<0. 05), and the viability of SPCA1 cells in interference + gemcitabine group was significantly decresed (P<0. 05). The Western blotting results showed that compared with negative control group, the expression amounts of p-Akt and p-PI3K in the SPCA1 cells in interference group were decreased, and the expression amounts of p-Akt and p-PI3K in the SPCA1 cells in interference+gemcitabine group were decreased significantly. Conclusion: Interference of GRP78 may increase the sensitivity of gemcitabine to chemotherapy, and GRP78 may reduce the sensitivity of NSCLC patients to gemcitabine through PI3K/Akt pathway.
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Glucose-regulated protein 78 (GRP78) plays an important role in the development of drug resistance in cancer, and GRP78 targeted therapy has become a hotspot of cancer research. In this article, we briefly introduce GRP78, review the research progress on the roles of GRP78 in drug resistance to cancer therapies, including chemotherapy, endocrine therapy and targeted therapy, and the mechanisms of resistance. The progress on GRP78 targeted therapy for cancer is also summarized.
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<p><b>Objective</b>To investigate the correlation of the single nucleotide polymorphisms (SNPs) rs12009, rs1140763 and rs16927997 in the 3'-untranslated region (3'UTR) of the glucose-regulated protein 78 (GRP78) gene with the risk of male asthenozoospermia (AZS).</p><p><b>METHODS</b>We included 400 AZS patients in the AZS group and another 400 fertile men as normal controls. Using the SNaPshot technique, we genotyped the rs12009, rs1140763 and rs16927997 polymorphisms in the 3'UTR of the GRP78 gene in all the male subjects and analyzed the association of the three SNPs with AZS.</p><p><b>RESULTS</b>The percentage of progressively motile sperm was significantly lower in the AZS group than in the normal controls (［20.09 ± 8.18］ % vs ［57.16 ± 13.45］ %, P <0.01). Three genotypes of CC, CT and TT and 2 alleles of C and T were found in rs12009 and rs1140763 of the GRP78 gene, and another three genotypes of GG, GA and AA and two alleles of G and A were observed in rs16927997. There were no statistically significant differences between the control and AZS groups in the frequencies of the C and T alleles in rs12009 (44.3% vs 47.3% and 55.7% vs 52.7%, P >0.05) or rs1140763 (50.0% vs 52.0% and 50.0% vs 48.0%, P >0.05) or those of the G and A alleles in rs16927997 (6.0% vs 4.4% and 94.0% vs 95.6%, P >0.05), nor in the genotypes and allele frequencies of the 3 polymorphisms (P >0.05). Furthermore, three haplotypes of C-C-A, T-C-G and T-T-A were observed in the male subjects but showed no evident correlation between the AZS and normal control groups.</p><p><b>CONCLUSIONS</b>The polymorphisms in the 3'UTR of the GRP78 gene are not correlated with the risk of male asthenozoospermia.</p>
Subject(s)
Female , Humans , Male , 3' Untranslated Regions , Genetics , Alleles , Asthenozoospermia , Genetics , Gene Frequency , Genetic Predisposition to Disease , Genotype , Haplotypes , Heat-Shock Proteins , Genetics , Polymorphism, Single Nucleotide , RiskABSTRACT
Objective To study the expression of protein 94 (GRP94) and C/EBP-homologous protein (CHOP) in myocardial tissue of hypertensive rats and to investigate the effects of amlodipine on endoplasm retieulum stress ( ERS) and ventricular hypertrophy in abdominal aortic banded rats .Methods One hundred and twenty adult male SD rats with criteria were divided randomly into three groups:sham-operated group , abdominal aortic banding ( AAB) and AAB treated with amlodipine (AAB+Aml)groups(n=40).In sham-operated rats, the abdominal aorta was isola-ted but not constricted , while the abdominal aorta were constricted in AAB rats .The AAB+Aml group was treated by abdominal aortic constriction and treated with amlodipine [10 mg/(kg· d)].According to the time of surgery, each group was further divided into 2, 4 and 8-week postoperative subgroups ( n=6 ) .Mean arterial pressure (MAP), the 1eft ventricular mass (LVM) and body mass (BM) were measured and (LVM/BM) was calculated. The morphology of cardiomyocytes was observed by HE staining .The protein level of GRP 94 and CHOP was ana-lyzedbyimmunohistochemistryandwesternblot.Results 1)Withtimeaftersurgery,MAPandLVM/BWof AAB group increased gradually , and they were obviously higher than the sham operation group [ MAP: 2 weeks (144 ±10)vs(118 ±9), 4 weeks (163±8)vs(120±7), 8 weeks(177±10)vs(120±6)mmHg;LVW/BW:2 weeks (2.21±0.17) vs (1.91±0.12), 4 weeks (2.45±0.16) vs (2.01±0.14), 8 weeks (2.68±0.15) vs ( 2.05 ±0.09 ) mmHg;( P<0.05 ) ] .The cardiomyocytes in AAB group were hypertrophic as compared to the sham group.The expression of GRP94 in AAB group increased significantly at 2 weeks post-operation, and reached peak level at 4 weeks after the surgery and was on the decline thereafter .The expression of CHOP and GRP94 in AAB rats were significantly higher than sham group, and reached the peak at the 8 weeks after surger-y.2)Treatment with amlodipine significantly reduced MAP and LVM/BW in AAB rats[(MAP:2 weeks (126± 6) vs (144±10), 4 weeks (125±8) vs (163±8), 8 weeks (128±5) vs (177±10)mmHg;LVM/BW:2 weeks ( 1.94 ±0.15 ) vs ( 2.21 ±0.17 ) , 4 weeks ( 2.13 ±0.08 ) vs ( 2.45 ±0.16 ) , 8 weeks ( 2.18 ±0.10 ) vs ( 2.68 ± 0.15)mg/g;(P<0.05)].The myocardial hypertrophic was alleviated in AAB+Aml group.The level of GRP94 and CHOP in AAB+Aml group was lower than those in AAB rats ( all P<0.05 ) .Conclusions Endoplasmic reticulum stress is involved in the ventricular remodeling caused by hypertension , and use of amlodipine can reduce ventricu-lar hypertrophy in hypertension models possibly by inhibiting endoplasmic reticulum stress via down -regulation of GRP94 and CHOP .
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Oective: To investigate the expression of glucose regulating protein 78 (GRP78) in the sciatic nerve cells of the rats with diabetic peripheral neuropathy (DPN), and to elucidate the clinical significance and mechanism of Adjust Zang Dredge Meridian electroacupuncture in the treatment of DPN. Methods: A total of 86 adult male SD rats were divided into normal control group (n=5) and model group (n=81). The diabetic rat models were built through one-time injection of streptozocin (STZ), and then the diabetic rats were randomly divided into model control group (n=27), medicine intervention group (n=26), and electroacupuncture intervention group (n= 26). The rats in normal control group and model control group were fed with the maintenance feed. During the course of experiment, no special treatment was done for the rats in normal control group and model control group. The rats in drug intervention group were treated with intragastric administration of mecobalamin, 20 mg · kg-1 · d-1, once per day. The rats in electroacupuncture intervention group were treated with Adjust Zang Dredge Meridian electroacupuncture in the bilateral Feishu, Pishu, Shenshu, Hegu, Zusanli, Sanyinjiao and Taichong, once per day, 30 min per time. The thermal sensitivity value, motor nerve conduction velocity (MCV) and sensory nerve conduction velocity (SCV) of the tibial nerve of the rats in various groups were measured, the ultrastructures of sciatic nerve of the rats in various groups were observed under transmission electron microscope, the apoptotic rates of sciatic nerve cells of the rats in various groups were detected by TUNEL method, the expressions of GRP78 in sciatic nerve cells of the rats in various groups were detected by immumofluorescence, and the expression levels of GRP78 in sciatic nerve of the rats in various groups were detected by Western blotting method. Results: Compared with medicine intervention group, the thermal sensitivity value of the rats in electroacupuncture intervention group was significantly decresed (P<0. 01). Compared with model control group, the MCV and SCV of tibial nerve of the rats in medicine intervention group and electroacupuncture intervention group were increased after 12-week-intervention (P<0. 01); compared with before intervention, the MCV and SCV of tibial nerve of the rats in various groups were decreased after 12-week-intervention (P<0. 01). Under transmission electron microscope, some of the myelin sheath lamellae of sciatic nerve of the rats in model control group showed irregular membrane-like mass, and most of the thicker myelinated nerve fibers were seriously involved; the above changes were alleviated in medicine intervention group and electroacupuncture intervention group. The TUNEL assay results showed that compared with model control group, the apoptotic rates of sciatic nerve cells of the rats in medicine intervention group and electroacupuncture intervention group were significantly decreased (P < 0. 01). The Western blotting results showed that compared with model control group, the expression levels of GRP78 in sciatic nerve cells of the rats in medicine intervention group and electroacupuncture intervention group were significantly decreased (P < 0.05). Conclusion: Adjust Zang Dredge Meridian electroacupuncture can reduce the expression level of GRP78 in sciatic nerve cells of the DPN rats, inhibit the occurrence of endoplasmic reticulum stress (ERS), decrease the apoptotic rate of cells, and protect the nerves.
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OBJECTIVE: To investigate the role and mechanism of the endoplasmic reticulum stress(ERS) pathway of apoptosis mediated by inositol-requiring enzyme-1(IRE1) in the intervention of silicosis fibrosis in rats using polyguanylic acid(PolyG).METHODS: The specific pathogen free adult male SD rats were randomly divided into control group(24rats),silicosis model group(24 rats),PolyG intervention group(16 rats) and PolyG treatment group(16 rats).The silicosis fibrosis rat model was constructed using the single inhalable intratracheal instillation method.The rats in the control group were injected with 1 mL of 0.9% sodium chloride solution.The other 3 groups were given 1 mL of silica suspension at 50.0 g/L mass concentration.The rats in PolyG intervention group on the day of model construction and rats in PolyG treatment group on the 28 th day after model construction were all given PolyG with 2.5 mg/kg body weight by one time tail vein intravenous injection.Eight rats in the PolyG intervention group and PolyG treatment group were sacrificed respectively on day 28 and day 56 after injection.The pathological changes of lung tissue in each group were observed.The expression of glucose regulated protein-78(GRP78),IRE1,CCAAT/enhancer-binding protein homologous protein(CHOP),Casepase-3,Casepase-12,type Ⅰ collagen and type Ⅲ collagen in lung tissue was detected by the Western blot.RESULTS: The histopathology examination results showed that the structure of lung tissue in control group was normal.The alveolar structure of the lung tissue of the silicosis model group was severe,and the fibrous nodules and a large amount of collagen deposition appeared.The silicosis nodules and collagen deposition in PolyG intervention group and PolyG treatment group were less than those in silicosis model group.The expression of GRP78,IRE1,CHOP,Casepase-3,Casepase-12,type Ⅰ collagen and type Ⅲ collagen in silicosis model group was higher than that of control group(P <0.05).The expression of the above 7 proteins in the PolyG intervention group and PolyG treatment group was lower than that of silicosis model group(P<0.05),higher than that of control group(P<0.05),except IRE1 and CHOP in PolyG intervention group.On day 56 after model construction,the expression of GRP78,IRE1,Casepase-3,Casepase-12,typeⅠ collagen and type Ⅲ collagen in PolyG intervention group were lower than that of PolyG treatment group(P<0.05).CONCLUSION: The unfolded protein response of ERS mediated by IRE1 may participate in the process of PolyG the intervention on silicosis fibrosis in rats.PolyG can effectively prevent and treat silicosis fibrosis.Prophylactic administration is recommended.
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Objective:To investigate the effect of mH2A and Butein on mitogen-activation protein kinase (MAPK) signaling pathway through targeting glucose regulated protein of 78 (GRP78) in regulating the biological behavior of melanoma.Methods:Immunohistochemistry was used to detect the expressions of mH2A and GRP78 in melanoma and adjacent normal tissues.The relationship between mH2A and GRP78,and the dinicopathological data was analyzed.The relationship between GRP78 and mH2A protein was detected by immunoprecipitation assay.The protein expressions of mH2A and GRP78 was detected by Western blot.The invasion ability of melanoma after sliencing mH2A was detected by Transwell invasion assay.The migration ability of melanoma after sliencing mH2A was detected by wound healing assays.The protein expressions of MEK and ERK1 after sliencing mH2A was detected by Westem blot.Results:The expressions of mH2A and GRP78 in melanoma tissues was significantly lower than that in adjacent normal tissues (P＜0.05).Butein enhanced the expression of mH2A (P＜0.05).The mH2A regulated the GRP78 protein (P＜0.05).Silencing mH2A promoted the invasion and migration of melanoma A375 cells.The MEK and ERK1 protein expressions were up-regulated after silencing Butein.Conclusion:Butein promotes the role of mH2A in regulating GRP78 and modulating the biological behavior of melanoma cells through MAPK signaling pathway.
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Objective:To observe the effect of Toudingyikezhu extract on the morphology of liver tissue and endoplasmic reticulum stress(ERS) of the rats with nonalcoholic fatty liver disease(NAFLD),and to clarify the possible mechanism of Toudingyikezhu of intervention of NAFLD.Methods:The NAFLD rat models were established by intraperitoneal injection of carbon tetrachloride solution combined with high-fat diet.A total 60 SD rats were randomly divided into control group,model group,low,middle and high doses of Toudingyikezhu extract groups,phosphatidylcholine group(n=10).The doses of Toudingyikezhu extracts were 0.50,1.00 and 2.00 g·mL-1,and the dose of phosphatidylcholine was 0.20 g·kg-1,4 weeks after administration,the body weights and liver wet weights were detected,and the liver indexes were calculated.The texture,elasticity,color and morphology of liver tissue of the rats were observed by HE staning.The fatty degeneration,inflammation and necrosis scores of liver tissue of the rats were calculated.The levels of serum tumor necrosis factor-α(TNF-α),interleukin-1 (IL-1) and interleukin-6(IL-6) of the rats were detected by ELISA method.Immunohistochemical SP method was used to determine the expression levels of GRP78 protein,and PCR method was used to determine the expression levels of GRP78 mRNA.Results:Compared with control group,the morphology of liver tissue of the rats in model group were abnormal under light microscope;the liver index,fatty degeneration,inflammation and necrosis scores of liver tissue of the rats in model group were increased(P<0.05),and the expression levels of GRP78 protein and mRNA were decreased(P<0.01).Compared with model group,the liver tissue pathology of the rats had different degrees of improvement;the fatty degeneration,inflammation and necrosis scores of liver tissue and the serum TNF-α,IL-1,IL-6 levels of the rats in different doses of Toudingyikezhu extract groups were decreased(P<0.05 or P<0.01);the expression levels of GRP78 protein and mRNA were decreased (P <0.05 or P<0.01).Conclusion:Toudingyikezhu extract may inhibit or block the ERS,reduce the synthesis of lipids and accelerate its decomposition in order to resist the injury of liver cells of the NAFLD rats by down-regulating the GRP78 expression.
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Objective To explore the endoplasmic reticulum stress (ERS) apoptosis pathway in unilateral ureteral obstruction (UUO) mechanism of renal interstitial fibrosis in rats.Methods The rats were randomly divided into con-trol group and model group,UUO group line ligation of the left ureter,three days 3,7 and 14 days HE and Masson staining were used to observe the renal pathological changes;Take blood retinal venous plexus,separat and determina-tion of serum blood urea nitrogen and creatinine;Western blot was used to find protein 78 (GRP78) glucose regula-tion,endoplasmic reticulum source sex transcription factor (CHOP) and apoptosis related proteins cysteine aspartic acid proteinase 3(caspase-3) and caspase-12 protein expression. Results Compared with control group,the visible UUO model group 1) expansion of renal tubule and renal interstitial fibrosis degree with the extension of ureteral ob-struction time and progressive increase;2) GRP78,CHOP,caspase-3 and caspase-12 protein expression in postoper-ative 3 d have to rise,as the obstruction prolonged,the protein expression more significantly(P<0.01).Conclusions Endoplasmic reticulum stress related trademark protein in UUO rat renal interstitial fibrosis is the increase in expres-sion may promote the early renal interstitial fibrosis and continuous progress.
ABSTRACT
Objective To explore the endoplasmic reticulum stress (ERS) apoptosis pathway in unilateral ureteral obstruction (UUO) mechanism of renal interstitial fibrosis in rats.Methods The rats were randomly divided into con-trol group and model group,UUO group line ligation of the left ureter,three days 3,7 and 14 days HE and Masson staining were used to observe the renal pathological changes;Take blood retinal venous plexus,separat and determina-tion of serum blood urea nitrogen and creatinine;Western blot was used to find protein 78 (GRP78) glucose regula-tion,endoplasmic reticulum source sex transcription factor (CHOP) and apoptosis related proteins cysteine aspartic acid proteinase 3(caspase-3) and caspase-12 protein expression. Results Compared with control group,the visible UUO model group 1) expansion of renal tubule and renal interstitial fibrosis degree with the extension of ureteral ob-struction time and progressive increase;2) GRP78,CHOP,caspase-3 and caspase-12 protein expression in postoper-ative 3 d have to rise,as the obstruction prolonged,the protein expression more significantly(P<0.01).Conclusions Endoplasmic reticulum stress related trademark protein in UUO rat renal interstitial fibrosis is the increase in expres-sion may promote the early renal interstitial fibrosis and continuous progress.
ABSTRACT
Early brain injury (EBI) plays a key role in the pathogenesis of subarachnoid hemorrhage (SAH). This study investigated the role of glucose-regulated protein 78 (GRP78) in EBI after SAH. Male Sprague-Dawley rats (n=108) weighing 260±40 g were divided into control, sham-operated, and operated groups. Blood was injected into the prechiasmatic cistern of rats in the operated group. Neurological scores, ultrastructures of neurons, apoptosis, and GRP78 expression in the hippocampus were examined using Garcia scoring system, transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, and Western blotting at 1, 6, 12, 24, 48, and 72 h after SAH, respectively. The results showed that neurological scores were significantly decreased in the operated group as compared with those in control and sham-operated groups at 12, 24, 48, and 72 h. Metachromatin, chromatin pyknosis at the edge, endoplasmic reticulum swelling, and invagination of nuclear membrane were observed at 24 h in the operated group, indicating the early morphological changes of apoptosis. The number of apoptotic cells was significantly increased in the operated group as compared with that in control and sham-operated groups at 6, 12, 24, 48, and 72 h. The GRP78 protein expression levels in the operated group were significantly elevated at all time points and reached the peak at 12 h. GRP78 expression was positively associated with apoptosis cells and negatively with neurological scores. In conclusion, EBI was demonstrated to occur after SAH and GRP78 was involved in the development of EBI after SAH.