ABSTRACT
Objective To evaluate the effect of spliced X-box binding protein 1 (XBP1s) on hypoxia/reoxygenation (H/R) injury of mouse renal tubular epithelial cells and unravel underlying mechanism. Methods Mouse renal tubular epithelial cells were divided into adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), Ad-shNC+H/R group and Ad-shXBP1s+H/R group. The apoptosis level, mitochondrial reactive oxygen activity, mitochondrial membrane potential and mitochondrial calcium ion level were detected in each group. Chromatin immunocoprecipitation followed by sequencing (ChIP-seq) was employed to analyze the binding sites of XBP1s in regulating the inositol 1,4,5-trisphosphate receptor (ITPR) family. The expression levels of XBP1s and ITPR family messenger RNA (mRNA) and protein were determined in each group. Results Compared with the Ad-shNC group, the apoptosis level was higher, mitochondrial reactive oxygen species level was increased, mitochondrial membrane potential was decreased and mitochondrial calcium ion level was elevated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the apoptosis level was lower, mitochondrial reactive oxygen species level was decreased, mitochondrial membrane potential was elevated, and mitochondrial calcium ion level was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 mRNAs and proteins were down-regulated in the Ad-shXBP1s group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 proteins were up-regulated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 were down-regulated in the Ad-shXBP1s+H/R group (all P<0.05). ChIP-seq results showed that XBP1s could bind to the promoter and exon of ITPR1, the exon of ITPR2, and the exon of ITPR3. Conclusions XBP1s may affect mitochondria-associated endoplasmic reticulum membrane structure and function by directly regulating ITPR transcription and translation. Down-regulating XBP1s may inhibit ITPR expression and mitigate mitochondrial damage.
ABSTRACT
Objective:To evaluate the role of 1, 4, 5-inositol triphosphate receptor (IP3R) in necroptosis of hippocampal neurons induced by sevoflurane anesthesia in aged rats.Methods:Sixty healthy male Sprague-Dawley rats, aged 18 months, weighing 500-600 g, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), sevoflurane anesthesia group (group S) and sevoflurane anesthesia + IP3R antagonist group (group S+ I). S and S+ I groups inhaled 2% sevoflurane for 5 h. In group S+ I, IP3 receptor antagonist 2-APB 3 mg/kg was intraperitoneally injected at 10 min before sevoflurane inhalation, and the equal volume of dimethyl sulfoxide was intraperitoneally injected in group C and group S. Morris water maze test was used to test the cognitive function on the day after the end of sevoflurane anesthesia.Then the animals were sacrificed and the brain tissues were obtained for microscopic examination of the pathological changes after HE staining and Nissl staining (with a light microscope) and for determination of the free calcium concentration ([Ca 2+ ] i) and rate of necroptosis of hippocampal neurons (by flow cytometry) and expression of IP3R, receptor-interacting protein kinase-1 (RIPK1), receptor-interacting protein kinase-3 (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) (by Western blot). Results:Compared with group C, the escape latency was significantly prolonged, the times of crossing the platform were reduced, the time of staying at the target quadrant was shortened, the [Ca 2+ ] i and necroptosis rate of hippocampal neurons were increased, and the expression of IP3R, RIPK1, RIPK3 and p-MLKL in hippocampal neurons was up-regulated in group S and group S+ I ( P<0.05). Compared with group S, the escape latency was significantly shortened, the times of crossing the platform were increased, the time of staying at the target quadrant was prolonged, the [Ca 2+ ] i and necroptosis rate of hippocampal neurons were decreased, and the expression of IP3R, RIPK1, RIPK3 and p-MLKL in hippocampal neurons was down-regulated in group S+ I ( P<0.05). Conclusions:The mechanism by which sevoflurane induces cognitive dysfunction may be related to the imbalance of calcium homeostasis caused by activation of IP3R and thus inducing programmed necrosis in aged rats.
ABSTRACT
OBJECTIVE: To investigate the effect of beryllium sulfate( BeSO_4) on apoptosis of human embryonic lung fibroblast( MRC-5 cell). METHODS: MRC-5 cells were cultured in vitro and randomly divided into 6 groups: a control group,a low-,medium- and high-dose BeSO_4 group,an antagonist group,and an activator group. The former 4 groups were given final concentrations of 0,1,10 and 100 μmol / L of BeSO_4,respectively. The combined treatment of BeSO_4and2-aminoethoxydiphenyl borate( 10 μmol / L final concentration) was used in the antagonist group. The combined treatment of BeSO_4 and inositol triphosphate( IP3)( 10 μmol / L final concentration) was used in the activator group. After 24 and48 hours of culture,the cells were harvested. The apoptosis of MRC-5 cells was detected by flow cytometry. The intracellular calcium ion( Ca~(2+)) was detected using laser scanning confocal microscope. Quantitative real-time polymerase chain reaction was used to detect the relative expression of IP_3RⅢ and B-cell lymphoma-2( BCL-2) mRNA and the protein expression of IP_3RⅢ and IP3 were detected by enzyme-linked immunosorbent assay. RESULTS: The apoptosis rates of cells in the 3 BeSO_4 dose groups at the time points of 24 and 48 hours were lower than those in the control group at the same time points( P < 0. 05). The apoptosis rate of the antagonist group was lower than those in medium-dose BeSO_4 group and control group at the same time points( P < 0. 05). At the time point of 48 hours,the apoptosis rate of the activator group was lower than that of control group( P < 0. 05) and higher than that of the medium-dose BeSO_4group( P < 0. 05). As for the Ca~(2+)concentration at time point of 24 hours,the low-dose BeSO_4 group was lower than the control group( P < 0. 05),and the high-dose BeSO_4 group was higher than the control group( P < 0. 05). The Ca~(2+)concentrations at time point of 48 hours in the medium- and high-dose BeSO_4 groups were lower than that in the control group( P < 0. 05). Compared with the medium-dose BeSO_4 group and control group at time points of 24 and 48 hours,the Ca~(2+)concentrations in the antagonist group decreased( P < 0. 05),while thoes of the activator group increased( P < 0. 05). The expression of BCL-2and IP_3RⅢmRNA in the 3 BeSO_4 groups,the activator and antagonist group were higher than those of the control group( P <0. 05). The expression of IP3 R Ⅲ protein at the time point of 24 hours in the medium-dose BeSO_4 group,the activator group and the antagonist group were lower than that of control group( P < 0. 05). The expression of IP_3RⅢ protein at the time point of 48 hours,the high-dose BeSO_4 group was lower than the control group( P < 0. 05); the activator and antagonist groups were higher than the medium-dose BeSO_4group( P < 0. 05). The expression of IP3 protein in the lowand medium-dose BeSO_4 groups and the activator group were higher than that in the control group( P < 0. 05). The expression of IP3 protein in activator group was higher than the medium-dose group( P < 0. 05). CONCLUSION: BeSO_4 might change the Ca~(2+)concentration and inhibite the apoptosis of MRC-5 cell through regulating the IP3 R / Ca~(2+)pathway,IP3 can improve the decrease of Ca~(2+)concentration in MRC-5 cells induced by BeSO_4.
ABSTRACT
Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca2+ release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca2+ homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca2+ metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (gamma)-irradiation. In irradiated RKO cells, Ca2+ influx via activation of NCX reverse mode was enhanced and a decline of [Ca2+]i via forward mode was accelerated. The amount of Ca2+ released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca2+]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that gamma-irradiation elicits enhancement of cellular Ca2+ metabolism in radiation-sensitive RKO cells yielding programmed cell death.
Subject(s)
Humans , Calcium , Cell Death , Colorectal Neoplasms , Cytochromes c , DNA Damage , Homeostasis , Inositol 1,4,5-Trisphosphate Receptors , Metabolism , MitochondriaABSTRACT
Objective To investigate the changes of inositol 1,4,5-triphosphate receptor typeⅠ(IP3RⅠ)expression in the myocardial tissue of rats with endotoxic shock and probe the possible mechanisms of myocardial depression.Methods Thirty-three male Wistar rats were divided into four groups:control group (n=6) were injected normal saline(4ml/kg),and the other 3 groups (n=9) were injected endotoxin via femoral vein with a different dose of 10mg/kg,5mg/kg,and the artificial sacrificed group(10mg/kg,the rats were sacrificed when the mean arterial pressure was first rapidly decreased,then gradually increased to the normal level).IP3RⅠexpression in the myocardial tissue of rats was detected by immunohistochemistry,Western blot and colloidal gold immunoelectron microscopy technique.Results IP3RⅠexpressions were increased significantly in each group of rats with endotoxic shock than in the control group (P<0.01),and most obviously enhanced in the the artificial sacrificed group.The expression of IP3RⅠwas related to the level of mean arterial pressure.Ectopic expression of IP3RⅠwas observed around the cell membrane of the cardiac myocytes.Conclusion (1)The expression of IP3RⅠenhances in the myocardial tissue of rats with endotoxic shock,and exists ectopic expression.(2) The expression of IP3RⅠwas related to the level of mean arterial pressure.(3) IP3RⅠ may be related to the cardiac contractility and involved in the regulation of mean arterial pressure.
ABSTRACT
Objective To inquire into T-type calcium channel effects on RyR and IP 3R of sarcoplasmic reticulum in atrial myocytes during atrial fibrillation.Methods Ten dogs underwent continuous rapid atrial pacing (500 beats/min) to create persistent atrial fibrillation.In five rapidly-paced dogs,mibefradil dihydrochloride was given beginning 2 days after pacemaker implantation,continuing until the twenty-fourth week.A group of size-matched dogs (n=5) without given mibefradil was used as a pure atrial fibrillation group.Another group of size-matched dogs (n=5) without pacemaker implantation was used as a control group.Canine atrial myocytes isolated by enzymatic dissociation were used to study T-type Ca 2+ channel blocker effects on expression and function changes of RyR and IP 3R in atrial myocytes by confocal microscopy.Results RyR/IP 3R ratio (0.2965?0.01812) was markedly lower than that of control group (2.7043?0.2293),but there was no difference compared with that of atrial fibrillation group (0.2472?0.1355).Ca 2+transient of atrial myocytes was nremarkably changed (1.3031?0.1056)(P
ABSTRACT
Objective:To investigate the effect of TGF-? on the expression of type Ⅰ inositol 1,4,5-triphosphate receptor in WB rat liver epithelial cells.Methods:The expression of type Ⅰ inositol 1,4,5-triphosphate receptor protein and mRNA were detected by Western blot and RT-PCR in WB rat liver epithelial cells in vitro stimulating with TGF-?.Results:The expression level of type Ⅰ inositol 1,4,5-triphosphate receptor protein and mRNA in WB rat liver epithelial cells were both increased after stimulating with TGF-? in several time points and reached the highest at 8 h stimulated, and then decreased.Conclusion:TGF-? enhance the IP_3R1 protein and mRNA expression in WB rat liver epithelial cells.
ABSTRACT
AIM: To investigate the role of phosphoinositide pathway in the formation of pressure-overload cardiac hypertrophy. METHODS: Cardiac hypertrophy was induced in male Sprague-Dawley rats with coarctation of abdominal aorta, whole heart weight/body weight ratio was tested after 10 or 30 days of operation. Content of G?q/11 protein in left ventricle was detected by immunoblot analysis and concentration of IP 3 was measured by radioimmunoassay. RESULTS: At 10 and 30 days, whole heart weight/body weight ratio of coarctation aorta (CA) group was higher than that of sham-operated (SO) rats ( P 0.05). At 10 days, the level of IP 3 significantly increased in left ventricle of CA rats compared with the control animals ( P
ABSTRACT
AIM:To investigate the role of 1,4,5-trisphosphate inositol(IP3)and Fas gene expression in apoptosis of HepG2 cells induced by quercetin.METHODS:HepG2 cells were treated with quercetin at different concentrations(including 20,40,60,80 ?mol/L)for 72 h and treated with 60 ?mol/L quercetin for 6 h,12 h,24 h,48 h and 72 h.IP3,Fas mRNA,Fas protein and apoptosis rate were assayed by IP3-3H Birtrak assay,RT-PCR,Western blotting and flow cytometry,respectively.RESULTS:When HepG2 cells were incubated with different concentrations of quercetin for 72 h,the IP3 content was lower than those in control.Fas mRNA expression,Fas protein expression and the apoptosis rate were higher than those in control.When HepG2 cells were incubated with quercetin for 6 h,12 h,24 h,48 h,72 h,the IP3 contents were lower than those in control incubated with 60 ?mol/L quercetin for 12 h.Fas mRNA expression was higher than that in control incubated with 60 ?mol/L quercetin for 12 h.Fas protein expression was higher than that in control.The apoptosis rate was significantly higher than that in control incubated with 60 ?mol/L quercetin for 24 h(P
ABSTRACT
Objective To detect the expression of inositol 1,4,5-triphosphate receptor (IP 3R) subtypes in normal rat airway smooth muscle cells (ASMCs) and changes during chronic asthma formation. Methods ASMCs were cultured by collagen enzyme digestion method. The expressions of subtypes of IP 3R were detected by RT-PCR and the purified PCR products were linked with pGEM-T vector for DNA sequencing. Chronic asthma model was established with egg albumin. The changes of IP 3Rs were detected by RT-PCR method. Results All subtypes of IP 3R were expressed in airway smooth muscle cells of normal rats. The expression of IP 3R1 in asthma groups increased obviously as compared with that in the control group (P