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1.
Article in Chinese | WPRIM | ID: wpr-920528

ABSTRACT

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.

2.
Acta Pharmaceutica Sinica B ; (6): 3983-3993, 2021.
Article in English | WPRIM | ID: wpr-922454

ABSTRACT

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and

3.
Acupuncture Research ; (6): 873-877, 2019.
Article in Chinese | WPRIM | ID: wpr-844221

ABSTRACT

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of eukaryotic initiation factor 2α (EIF2α), activating transcription factor 4 (ATF4), glucose regulator protein-78 / immunoglobulin heavy-chain-binding protein (GRP78/Bip) in the substantia nigra (SN) in rats with Parkinson's disease (PD), so as to explore its mechanism underlying improvement of PD. METHODS: Forty-eight male SD rats were randomly divided into control, sham-operation, model and EA groups (n=12 in each group). The PD model was established by 28-day consecutive subcutaneous injection of rotenone (1 mg/kg dissolved in dimethyl sulfoxide and normal saline) at the back shoulder. EA (2 Hz, 1 mA) was applied to "Fengfu" (GV16) and "Taichong" (LR3) for 30 min, once daily for 2 weeks. The behavio-ral changes of rats in each group were measured and scored at 28th day and 44th day, respectively. The expressions of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in the SN were observed by immunohistochemistry, and the expressions of EIF2α, ATF4 and GRP78/Bip were detected by Western blot. RESULTS: Following modeling and compared with the control and sham-operation groups, the behavioral scores of rats in the model group were elevated (P<0.01), which were significantly decreased by EA intervention (P<0.01). The expression of TH decreased whereas the α-syn, EIF2α, ATF4 and GRP78/Bip increased in the rats of model group, and EA intervention reversed these changes (all P<0.01). CONCLUSION: EA at GV16 and LR3 can improve PD rats' behavioral changes, which is probably related with its effects in up-regulating the expression of TH in the SN and down-regulating the expression of α-syn and EIF2α-ATF4-GRP78/Bip signaling.

4.
Article in English | WPRIM | ID: wpr-777129

ABSTRACT

OBJECTIVE@#To test the hypothesis that the inhibition of endoplasmic reticulum (ER) stress-induced apoptosis in oxidized low-density lipoproteins (ox-LDL)-induced human aortic-vascular smooth muscle cells (HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) signaling pathway by Pollen Typhae total flavone (PTF).@*METHODS@#Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an HPTF group (70 μg/mL high ox-LDL+500 μg/mL PTF), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), and a LPTF group (70 μg/mL high ox-LDL+100 μg/mL PTF) in the first part; and a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), a shRNA group (transducted with PERK shRNA lentiviral particles), a scramble shRNA group (transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group (250 μg/mL PTF+70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group (70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their mRNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to test cell viability, and the level of apoptosis was monitored by flow cytometry.@*RESULTS@#The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and shRNA groups. Moreover, the ox-LDL group had increased protein and mRNA levels of binding immunoglobulin protein and ER-associated apoptosis proteins, such as PERK, eIF2α, ATF4 and CHOP, which were attenuated in PTF treatment groups and shRNA groups.@*CONCLUSIONS@#The apoptosis induced by ox-LDL had a strong relation to ER stress. The protective effect of PTF on ER stressinduced apoptosis was associated with inhibition of the PERK-eIF2α-ATF4-CHOP pathway, which might be a potential therapeutic strategy for enhancing the stability of atherosclerotic plaques.

5.
Article in Chinese | WPRIM | ID: wpr-744253

ABSTRACT

AIM:To investigate the effects of Jiawei-Naotai formula (JWNTF) on ATF4/CHOP/Puma pathway in hippocampal neurons of ovariectomized female rats with cerebral ischemia.METHODS:The female rats were randomly divided into sham group, model group, JWNTF group and positive control group.The rats, expect in the sham group, were ovariectomized.The rats in each group were intragastric administration 11 days after ovariectomy.The rats in sham group and model group were given a gavage of 0.9%Na Cl, while the rats in other groups were administrated by corresponding therapy intragastrically for 3 d.The regional cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) suture method 14 days after ovariectomy.The behaviors of the rats were evaluated 24 h after cerebral ischemia.The mRNA levels of Bax, Bcl-2 and caspase-3 were detected by RT-qPCR, and the protein expression of Bax, Bcl-2, caspase-3, ATF4, CHOP and Puma was determined by Western blot.RESULTS:Compared with sham group, the neurobehavioral scores significantly increased in other groups (P<0.05).Compared with model group, the neurobehavioral scores were significantly decreased in positive control group and JWNTF group (P<0.05).The protein expression of Bax, caspase-3, ATF4, CHOP and Puma, and the mRNA expression of Bax and caspase-3 in the hippocampus were much higher, and Bcl-2 was lower in model group than those in sham group (P<0.05).JWNTF significantly reduced the protein expression of Bax, caspase-3, ATF4 and CHOP, and the mRNA expression of Puma, Bax and caspase-3, and markedly increased the expression of Bcl-2 at mRNA and protein levels compared with model group.CONCLUSION:The JWNTF protects against brain damage induced by cerebral ischemia, which may be related to inhibitiing the expression of ATF4/CHOP/Puma pathway-related molecules at mRNA and protein levels.

6.
Article in Chinese | WPRIM | ID: wpr-699713

ABSTRACT

Objective To investigate the regulating effects of Krüppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group,and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds,The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid,respectively,and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group;pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6 + pSilencer group;pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4 + pEGFP-C2 group;pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6 + siATF4 group,and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay.Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis,karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group,the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ATF4 transfected group and UVB+pEGFP-C2 plasmid group,respectively,with a significant difference between them (t =23.13,P<0.01).The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group,and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P<0.01).ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37± 0.11,which was significantly higher than 0.31 ±0.11 in the normal control group (t =8.034,P =0.001);the apoptotic rate of the cells was increased in the KLF6+pSilencer group and decreased in the siATF4+pEGFP-C2 group in comparison with the empty plasmid group (P<0.01,P=0.02).In addition,the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6 + pSilencer group (P< 0.01).Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating oathwav of KLF6 to apoptosis.

7.
Tianjin Medical Journal ; (12): 620-624,前插2, 2018.
Article in Chinese | WPRIM | ID: wpr-698079

ABSTRACT

Objective To investigate the therapeutic effect of mechanical loading on obesity and non-alcoholic fatty liver disease. Methods Thirty 6-week-old female C57BL/6 mice (body weight 18 g) were randomly assigned into three groups: normal control group (NC group, n=10), high-fat diet group (HF group, n=10) and high-fat diet with mechanical loading treatment group (HF+L group, n=10). All mice except for NC group were fed with high-fat diet for 12 weeks. After 6 weeks of high-fat diet, mice of HF+L group received 6-week mechanical loading. The whole body composition was analyzed to detect the total body fat content. The mesenteric fat, perirenal fat, inguinal fat, periuterine fat and the liver were collected and weighed. A portion of the liver sample was isolated for histological analysis (Oil red O staining and HE staining) to observe pathologic changes, while the other was used for Western blot assay to detect the expression of eIF2α, p-eIF2α and ATF4, which were the marker proteins of endoplasmic reticulum stress. Results Compared with the NC group, high-fat diet resulted in a significant increase in body weight and body fat (P<0.05). After mechanical loading treatment, the body weight and body fat were significantly decreased in the HF+L group compared with those of HF group (P<0.05). Hepatic histological analysis showed that high-fat diet induced hepatic steatosis, which was effectively alleviated by mechanical loading treatment (P<0.05). Western blot analysis indicated that high-fat diet led to higher expression levels of p-eIF2α and ATF4 in liver, and mechanical loading was effective in inhibiting the increased expressions of p-eIF2α and ATF4. Conclusion Mechanical loading can effectively alleviate obesity and non-alcoholic fatty liver disease caused by high-fat diet, and its effects may be associated with endoplasmic reticulum stress in liver.

8.
The Journal of Practical Medicine ; (24): 1424-1427,1434, 2018.
Article in Chinese | WPRIM | ID: wpr-697791

ABSTRACT

Objective To investigate he intervention effect of knockdown of activating transcription factor 4(ATF-4)on fructose-induced lipid accumulation in liver cells. Methods HepG2 cells were divided into the control group(C),high fructose group(F),high-fructose+negative control group(F+NC)and high-fructose+ATF-4 siRNA group(F+ATF-4-). The mRNA level of gens of the upstream transcriptional factors and ERS markers was detected. The protein level of ACC,FAS and SCD-1 was also detected. Results Compared with group C,the mRNA expression of SREBP-1c,ChREBP,GRP78 and CHOP was increased(P < 0.01),while ATF-4 knock-down decreased the expression of the above genes(P < 0.01 ,respectively). Compared with group C ,the protein expression of ACC,FAS and SCD-1 was increased in group F(P<0.01,respectively). While ATF-4 knockdown decreased the protein expression of ACC ,FAS and SCD-1. Conclusions ATF-4 knockdown can improve the lipid steatosis induced by fructose through down-regulating lipid lipogenesis ,indicating ATF-4 possesses a regulatory effect on lipogenesis.

9.
Chinese Journal of Diabetes ; (12): 856-858, 2015.
Article in Chinese | WPRIM | ID: wpr-482406

ABSTRACT

[Summary] Activating transcription factor 4 (ATF4) is an alkaline leucine zipper transcriptional factor ,which is involved in many physiological metabolism processes such as stress response ,inflammation and tumor growth. Moreover ,recent studies have shown that ATF4 also plays a key role in regulating lipid and glucose metabolism ,insulin secretion and insulin sensitivity ,which may related to pathways such as endoplasmic reticulum stress (ERS ) ,peroxisome proliferator‐activated receptor gamma coactivator (PGC1α) and target of rapamycin (TOR). This article summarized the recent research progress of ATF4 in regulating glucose and lipid metabolism.

10.
Article in Chinese | WPRIM | ID: wpr-443582

ABSTRACT

BACKGROUND:Activating transcription factor 4 is found as an activating factor that can regulate osteogenic differentiation and function, and plays a critical role in the osteogenic differentiation. OBJECTIVE:To investigate the expression and significance of activating transcription factor 4 in the osteogenic differentiation of bone marrow mesenchymal stem cells from Sprague-Dawley rats. METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured using the whole bone marrow adherence method, and ossification revulsant was added to induce passage 3 cells. cells with no osteogenic induction served as controls. RT-PCR and western blot assay were employed to dynamical y monitor expression of activating transcription factor 4. RESULTS AND CONCLUSION:RT-PCR results showed that activating transcription factor 4 mRNA expression increased with the increasing osteogenic differentiation, and peaked at day 16. Western blot analysis showed that the protein expression of activating transcription factor 4 tended to increase with the increasing osteogenic differentiation, peaked at day 16 and stil maintained at a higher level at day 19. Compared with the uninduced cells, activating transcription factor 4 in the induced cells exhibited a higher expression at mRNA and protein levels (P<0.05). These findings indicate that activating transcription factor 4 expression is elevated during osteogenic differentiation, showing a positive correlation with osteogenic ability of bone marrow mesenchymal stem cells.

11.
Chinese Journal of Nephrology ; (12): 920-925, 2013.
Article in Chinese | WPRIM | ID: wpr-439387

ABSTRACT

Objective To investigate the effects of the rat serum with chronic renal failure (CRF) on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4) of rat arterial vascular smooth muscle cells(VSMCs) cultured in vitro,and explore the possible mechanism.Methods To establish the rat model of CRF by 5/6 nephrectomy,VSMCs were incubated in the media with the 10% of CRF serum or control serum in vitro.The mRNA expressions of ubiquitin(Ub),ubiquitin activating enzyme(E1),ubiquitin ligases enzymes (β-transducin repeat containing protein 1,β-TrCP1),p300 and ATF4 in the rat VSMCs were examined by using realtime PCR.Expressions of E1,β-TrCP1,p300 and ATF4 proteins in response to the CRF serum in VSMCs were determined by Western blotting analysis.The enzyme activities of 20S proteasomes in the total protein were examined by using three special fluorogenic peptide substrates.Results The CRF serum significantly promoted the mRNA expressions of Ub,E1,β-TrCP1,p300 and ATF4 in VSMCs in a time dependent manner.Compared with that in control serum group,the mRNA levels of Ub,E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P < 0.01).The CRF serum also increased the protein expressions of E1,β-TrCP1 and p300 in a time dependent manner.The expression of ATF4 was decreased,but the difference was not significant (P > 0.05).Compared with that in control serum group,the protein expressions of E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P < 0.01).The activities of 20S proteasomes in the CRF serum group were significantly increased in a time dependent manner.Compared with that in control serum group,the activities of 20S proteasomes in the CRF serum group increased significantly (P < 0.01).Conclusions The serum of CRF rat can effectively active the ubiquitin-proteasome pathway,but ATF4 ubiquitinylated degradation is blocked.The latter may be associated with increased expression of p300.

12.
Chinese Journal of Nephrology ; (12): 706-710, 2009.
Article in Chinese | WPRIM | ID: wpr-380388

ABSTRACT

Objective To investigate the effects of the serum from chronic renal failure (CRF) rats on endoplasmic reticulum stress (ERS) and activation of activating transcription factor 4 (ATF4) of rat arterial smooth muscle cells (ASMCs) cultured in vitro and explore the possible mechanism. Methods The rat model of CRF was established by 5/6 nephrectomy. ASMCs were incubated in the media with 10% or 20% serum of CRF rats cultured in vitro. ATF4 nuclear translocation were analyzed by immunofluorescence. GRP78 (glucose regulated protein 78), p-PERK (pancreatic ER kinase) and ATF4 proteins in response to the CRF serum in ASMCs were determined by Western blot. The ATF4-DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA). Results The CRF serum could significantly promote the expression of GRP78 of ASMCs in dose-dependent manner. Under the stimulation of 20% CRF serum, nuclear translocation of ATF4 in ASMCs was found. Compared with control serum group, the expression of ATF4 and p-PERK of CRF serum group was increased significantly (P<0.01). The CRF serum could increase the ATF4-DNA binding activity. Conclusion The CRF rat serum can effectively induce ERS of ASMCs and activate the pathway of PERK-ATF4.

13.
Article in Chinese | WPRIM | ID: wpr-567995

ABSTRACT

Objective To investigate the expression and significance of activating transcription factor 4 (ATF4) in the hepatocyte steatosis models induced by oleic acid,and to explore the relationship between the expression of ATF4 and nonalcoholic fatty liver disease (NAFLD). Methods L02 cells were treated by 20 mg/ml oleic acid for 0,24,48 and 72 h respectively to induce the hepatocytes steatosis,and then the cells were collected and the total RNA and protein were extracted. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot analysis were used to detect the expressions of ATF4 gene at different time points. Results With the development of deterioration of steatosis,the expression levels of ATF4 mRNA were 1.01?0.12,1.87?0.24,2.01?0.13,and 1.79?0.19 respectively correspondingly from 0 h to 72 h. The peak of ATF4 mRNA expression was reached at the 48th hour,and then showed the tendency of down-regulation. At the same time points,the protein expression of ATF4 were 0.31?0.16,0.57?0.14,0.91?0.20,and 0.89?0.17 separately,and the protein expression was maintained at a high level up to the 72nd hour. There were significantly differences compared with the control group(P

14.
Article in Chinese | WPRIM | ID: wpr-566465

ABSTRACT

Leukoencephalopathy with vanishing white matter(VWM) is one of the most prevalent inherited white matter disorders in childhood,and it′s the only known hereditary human disease due to the direct defects in protein synthesis process,with the gene defects in EIF2B1-5,encoding the five subunits of eukaryotic translation initiation factor(eIF2B ?,?,?,? and ?) respectively.eIF2B is essential for the protein translation initiation process,and its action is realized via eukaryotic translation initiation factor2(eIF2).Phosphorylation of eIF2? and eIF2B? is an important way to regulate eIF2B function,and thus play a key role in control of the protein translation level under physiological condition.Mutant eIF2B results in functional defects and decrease of the overall protein translation in cells,but in increase the translation of proteins with multiple upstream open reading frames,such as activating transcription factor 4(AFT4),which leads to the susceptibility to un-folded protein response under stress,and the following apoptosis.The exact pathogenic mechanisms of VWM are far from well understood.It′s suggested that level of AFT4 in cells with eIF2B mutations is higher than in wild type cells under physiological condition,which makes the mutant cells more susceptible to endoplasmic reticulum(ER) stress and unfolded protein response(UPR).Under stress,the defect eIF2B leads to a vicious cycle of UPR activation,which may underlie the neurological aggravation in VWM patients after minor stress,a specific cli-nical feature of VWM.Elucidating the pathogenesis of VWM will be helpful to further understand the protein translation process in eukaryotic cells,and provide a clue for possible therapeutic targets and treatment strategies in the future.Abstract:SUMM ARY Leukoencephalopathy with vanishing white matter(VWM) is one of the most prevalent in-herited white matter d isorders in childhood,and i′ts the only known hered itary human d isease due to the d irect defects in protein synthesis process,with the gene defects inEIF2B1-5,encod ing the five sub-units of eukaryotic translation initiation factor(eIF2B?,?,?,?and?) respectively.eIF2B is essential for the protein translation initiation process,and its action is realized via eukaryotic translation initiation factor2(eIF2).Phosphorylation of eIF2?and eIF2B?is an important way to regulate eIF2B function,and thus play a key role in control of the protein translation level under physiological cond ition.Mutant eIF2B results in functional defects and decrease of the overall protein translation in cells,but in increase the translation of proteins with multiple upstream open read ing frames,such as activating transcription factor 4(AFT4),which leads to the susceptibility to un-folded protein response under stress,and the following apoptosis.The exact pathogenic mechanisms ofVWM are far from well understood.I′ts sugges-ted that level ofAFT4 in cells with eIF2B mutations is higher than in wild type cells under physiological cond ition,which makes the mutant cellsmore susceptible to endoplasm ic reticulum(ER) stress and un-folded protein response(UPR).Under stress,the defect eIF2B leads to a vicious cycle ofUPR activa-tion,which may underlie the neurological aggravation in VWM patients afterm inor stress,a specific cli-nical feature ofVWM.E lucidating the pathogenesis ofVWM will be helpful to further understand the pro-tein translation process in eukaryotic cells,and provide a clue for possible therapeutic targets and treat-ment strategies in the future.

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