Tripterine promotes pyroptosis of liver cancer cells by regulating ATF4/caspase-3/GSDME signaling pathway / 中国药理学通报

Aim To explore the new mechanism of triptolide (TRI) inhibiting the progression of hepatocellular carcinoma (HCC) . Methods Different concentrations (0, 0 . 5, 2, and 8 jjunol • L~) of TRI were administered to act on liver cancer cells, and then the cell phenotypes and possible mechanisms were explored using experimental methods such as CCK-8, cell cloning, Transwell, and protein immunoblotting; siRNA was used to interfere with the target gene GSDME and its role was determined. Finally, the mechanism of TRI inhibiting the growth of HCC cells in vivo was validated using a transplanted tumor model. Results TRI could inhibit the proliferation, cloning, and invasion of HCC cells, and promote cell apoptosis. Immunoblotting results showed that the expression of GSDME was significantly upregulated in HepG2 or He-pal-6 hepatocellular carcinoma after TRI treatment, while the expression of cleaved caspase-3 and PARP also significantly increased. Knocking out GSDME could partially reverse TRI-induced cell apoptosis. At the same time, cells knocked down by GSDME had stronger cloning and migration abilities, and the apoptosis rate was reduced compared to the TRI treatment group alone. In vivo experiments showed that TRI inhibited HCC tumor growth, and the TRI + siGSDME group had a faster tumor growth rate than the TRI treatment group alone did. In addition, after TRI stimulation, p-eIF2a and ATF4 in HepG2 and Hepal-6 cells significantly increased. The immunofluorescence results showed a dose-dependent increase in the number of ATF4 positive cells in HepG2 and Hepal-6 cells after TRI stimulation. Conclusion The inhibitory effect of TRI on the growth and invasion of liver cancer cells may be related to its regulation of the ATF4/caspase-3/GSDME signaling pathway and promotion of liver cancer cell apoptosis.

Research Progress of Endoplasmic Reticulum Stress PERK-eIF2α-AFT4-CHOP Signaling Pathway in Hematological Malignancies / 肿瘤防治研究

The biosynthesis and maturation of proteins are primarily regulated by the endoplasmic reticulum in its physiological state. Thus, the disruption of physiological homeostasis initiates the buildup of unfolded and misfolded proteins in the endoplasmic reticulum, resulting in endoplasmic reticulum stress (ERS) and unfolded protein response (UPR). One of the important pathways by which UPR maintains intracellular homeostasis under ERS is activating protein kinase R-like endoplasmic reticulum kinase (PERK). The activation of the PERK pathway stimulates eukaryotic translation initiation factor 2 subunit-α (eIF2α) phosphorylation and the selective translation of active transcription factor 4 (ATF4), and PERK induces cell apoptosis by directly binding to the promoter of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). This signaling pathway is also one of the important mechanisms by which UPR participates in the regulation of hematological malignancies and immune cells in a tumor microenvironment. This article provides an overview of advancements in research into the PERK-eIF2α-ATF4-CHOP signaling pathway in hematological malignancies and the potential therapeutic benefits of targeting this signaling pathway.

Mechanism of Yishen Tongluo Prescription in Inhibiting Endoplasmic Reticulum Stress and Improving Apoptosis of Renal Tubular Epithelial Cells Based on PERK/ATF4/CHOP / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Yishen Tongluo prescription （YSTLP） on apoptosis of renal tubular epithelial cells and explore the mechanism based on endoplasmic reticulum stress pathway of protein kinase R-like endoplasmic reticulum kinase （PERK）/activating transcription factor 4 （ATF4）/transcription factor C/EBP homologous protein （CHOP）. MethodThe db/db mice were randomly divided into model group， valsartan group （10 mg·kg-1）， and low， middle， high-dose YSTLP groups （1， 2.5， 5 g·kg-1）. Samples were collected after eight weeks of drug intervention. In addition， db/m mice in the same litter served as the control group. Human renal tubular epithelial cells （HK-2） were cultured in vitro and divided into the control group， advanced glycated end-product （AGE） group， and AGE + low， middle， and high-dose YSTLP groups （100， 200， 400 mg·L-1）. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect the apoptosis rate of HK-2 cells. Methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay was conducted to detect the viability of HK-2 cells. Calcium fluorescence probe staining and luciferase reporter gene method were adopted to detect the luciferase activity of folded protein response element （UPRE） and endoplasmic reticulum stress. Immunohistochemical （IHC） analysis was carried out to measure the protein expressions of phosphorylated PKR （p-PERK）， CHOP， and ATF4. Real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of CHOP and X-box binding protein 1 （XBP1） in mouse kidney and HK-2 cells. Western blot was used to detect the protein expression level of p-PERK， PERK， CHOP， ATF4， B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， and cleaved Caspase-3 in mouse kidney and HK-2 cells. ResultIn the cellular assay， HK-2 cell viability was significantly reduced， and the apoptosis rate was elevated in the AGE group compared with the control group （P<0.01）. The mRNA and protein expression levels of apoptosis-related factor Bcl-2 were significantly reduced （P<0.01）， and those of Bax were significantly increased （P<0.01）. The protein expression level of cleaved Caspase-3 was significantly increased （P<0.01）. Compared with the AGE group， YSTLP administration treatment resulted in elevated cell viability and reduced apoptosis rate （P<0.01）. The mRNA and protein expression levels of Bcl-2 were significantly elevated in a time- and dose-dependent manner （P<0.01）， and those of Bax were significantly reduced in a time- and dose-dependent manner. The protein expression level of cleaved Caspase-3 was significantly reduced in a time- and dose-dependent manner （P<0.01）. The intracellular Ca2+ imbalance and UPRE luciferase fluorescence intensity were increased in the AGE group compared with the control group （P<0.01）. The mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 were significantly increased （P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the AGE group， YSTLP effectively improved intracellular Ca2+ imbalance in HK-2 cells and decreased UPRE luciferase fluorescence intensity in a dose-dependent manner （P<0.01）. It reduced the mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 （P<0.01） and the protein expression levels of intracellular p-PERK， CHOP， and ATF4 in a dose- and time-dependent manner （P<0.01）. In animal experiments， the protein expression level of Bcl-2 was significantly reduced（P<0.01）， and that of cleaved Caspase-3 and Bax was significantly increased in the model group compared with the control group （P<0.05）. The protein expression level of Bcl-2 was dose-dependently elevated， and that of cleaved Caspase-3 and Bax was dose-dependently decreased in the YSTLP groups compared with the model group （P<0.01）. Compared with the control group， the mRNA expression levels of CHOP and XBP1 were significantly elevated in the model group （P<0.05， P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the model group， YSTLP significantly decreased the mRNA expression levels of CHOP and XBP1 （P<0.01） and the protein expression levels of p-PERK， CHOP， and ATF4 （P<0.01）. ConclusionYSTLP can effectively inhibit endoplasmic reticulum stress and improve apoptosis of renal tubular epithelial cells， and its mechanism may be related to the regulation of the PERK/AFT4/CHOP pathway.

Effects and mechanisms of total flavones of Abelmoschus manihot in attenuating diabetic tubulopathy by targeting endoplasmic reticulum stress-induced cell apoptosis / 中国中药杂志

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.

Effects of daphresin on synovial fibroblasts in rheumatoid arthritis based on PERK/ATF4/CHOP signaling pathway / 中国药理学通报

Aim To investigate the effects of DAP on human rheumatoid arthritis synovial fibroblasts(RA-FLS)and its relationship with endoplasmic reticulum stress(ERS)PERK/ATF4/CHOP signaling pathway.Methods RA-FLS cells were cultured and identified by immunofluorescence assay for Vimentin and CD68.CCK-8 detected(0,5,10,20,30,40,50,60,70)mg·L-1 DAP on the proliferation activity of RA-FLS cells.According to the proliferation activity,the experiment was divided into blank group(Blank),low dose group(L-DAP),medium dose group(M-DAP),high dose group(H-DAP)and high dose+specific inhibitor 4-PBA group(H-DAP+4-PBA).The levels of TNF-α and IL-6 were detected by ELISA.Cell apoptosis was detected by flow cytometry.The invasion and migration of cells were detected by Transwell and scratches assays,and the expressions of endoplasmic reticulum stress-related proteins GRP78,PERK,P-PERK,ATF4,CHOP,Caspase-12,C-Caspase-12 and Bcl-2 were assessed by Western blot.Results CCK-8 results showed that compared with 0 mg·L-1 DAP group,the proliferation activity of each group in 20-70 mg·L-1 DAP group was significantly different(P<0.05),and the proliferation rate corresponding to 0,20,40 and 60 mg·L-1 DAP group was significantly different(P<0.05).This concentration was used as the basis for blank,low-dose,medium-dose,high-dose groups and high-dose+4-PBA experimental grouping.DAP could inhibit the release of inflammatory cytokines IL-6 and TNF-α from RA-FLS cells in concentration,reduce the invasion ability of cells,promote apoptosis,upregulate the expression of PERK,P-PERK,ATF4,GRP78,CHOP,Caspase-12, C-caspase-12 proteins and decrease the expression level of anti-apoptotic protein Bcl-2.Another set of experiments demonstrated that high dose DAP+4-PBA could up-regulate the expression of IL-6 and TNF-α,as well as the invasion of cells,and inhibit the expression of apoptosis and ERs-related proteins.Conclusions Daphresin regulates the secretion of inflammatory factors by activating the PERK/ATF4/CHOP signaling pathway,inhibits the proliferation and invasion of RA-FLS cells,and induces apoptosis,which is expected to be a potential therapeutic pathway for RA.

A small molecule UPR modulator for diabetes identified by high throughput screening

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

Protective effect of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress / 中国中药杂志

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P<0.05 or P<0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P<0.05 or P<0.01), while Bcl-2 was significantly up-regulated(P<0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P<0.01), whereas Bcl-2 was significantly up-regulated(P<0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

Effect of endoplasmic reticulum stress on the osteogenic differentiation of periodontal ligament cells under continuous static pressure / 口腔疾病防治

Objective@#To study the effect of continuous static pressure on the endoplasmic reticulum of human periodontal ligament cells (hPDLCs) and the mechanism of osteogenic differentiation.@*Methods@#hPDLCs cultured in vitro were subjected to 1 g/cm 2 of continuous compressive pressure (CCP) by custom-made, round, glass panes for 0, 2, 4, and 6 h, respectively. Alkaline phosphatase staining was used to detect osteogenic differentiation, and real-time quantitative PCR was used to detect the expression of protein kinase receptor-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and transcription activation factor 4 (ATF-4). The 0 h loading group was the control group.@*Results@#After CCP treatment, the alkaline phosphatase staining of hPDLCs was blue-violet and significantly stronger than that of cells in the control group. The expression levels of PERK and ATF4 in the hPDLCs after CCP treatment were higher than those of cells in the control group (P < 0.05) and increased over time (P < 0.05). The expression of eIF2α was lower in the experimental groups than in the control group (P < 0.05) and decreased over time (P < 0.05).@*Conclusion @#Mechanical stimulation can activate ERS in hPDLCs, leading to enhanced PERK-eIF2α-ATF4 signaling and inducing osteogenic differentiation.

Effects of 1,25-(OH)2D3on endoplasmic reticulum stress and ATG12 expression in pulmonary fibrosis / 医学研究生学报

Objective It is not yet clear whether 1,25-(OH)2D3acts on endoplasmic reticulum stress(ERS)and autoph-agy in pulmonary fibrosis(PF).This study aimed to investigate the roles of ERS and autophagy in the development and progression of pulmonary fibrosis in rats and the effects of 1,25-(OH)2D3on the expressions of ERS-related molecules and autophagy-related gene 12 (ATG12). Methods Ninety male SD rats were randomly divided into a control, a PF model and a treatment group of equal number.Bleomycin was injected into the tracheas of the latter two groups of rats to induce PF.On the second day after modeling, the rats of the treatment group were injected intraperitoneally with 1,25-(OH)2D3at 2 μg/kg,those of the PF model group with 1,25-(OH)2D3solvent at 200 μL,and those of the control group with iso-tonic saline at 200 μL,all once 2 days.Then the mRNA expressions of PERK,ATF4 and ATG12 were measured by real-time PCR and the protein expressions of PERK and ATF4 detected by immunohistochemistry. Results At 14, 21 and 28 days after treat-ment,the expression levels of PERK were significantly higher in the PF model group(2.30±0.19, 3.59±0.27, and 4.63±0.19) and treatment group(1.44±0.34,1.92±0.17,and 2.52±0.15)than in the control(1.01±0.23,1.05±0.09,and 1.04±0.08)(P<0.05), and so were the expression levels of ATF4 in the PF models(2.10±0.12, 3.91±0.14, and 6.20±0.28)and treated rats (1.49±0.27,2.52±0.42, and 4.02±0.31)than in the controls(1.04±0.07,1.05±0.08,and 1.03±0.10)(P<0.05).Compared with the control group,the PF model and treatment groups showed markedly increased expression levels of ATG 12 mRNA at 14 days (P<0.05), but decreased at 21 and 28 days(P<0.05).Both the expressions of PERK and ATF4 proteins were remarkably higher in the model and treatment groups than in the control at 14,21 and 28 days(P<0.05), increasing in a time-dependent manner. Conclusion By suppressing the PERK -eIF2α-ATF4 signaling pathway,1,25-(OH)2D3inhibits the development and progression of pulmonary fibrosis.

Effects of ribosomal protein L41 (RPL41) on the proliferation and apoptosis of human retinoblastoma Y79 cells and its mechanisms / 眼科新进展

Objective To study the effects of ribosomal protein L41 (RPL41) on the proliferation and apoptosis of human retinoblastoma Y79 cells and its underlying mechanisms.Methods Y79 cells were seeded in RPMI 1640 medium containing 10％ fetal bovine serum for passage culture.Then the cells were divided into control group,with cells left untreatment,(40 μmol · L-1,80 μmol · L 1 and 120 μmol · L-1) RPL41 treatment group according to the concentration.Next CellTiter-Glo fluorescence cell viability testing system was used to observe the viability of Y79 cells in all groups,and flow cytometry was applied to measure the cell apoptotic rate in 100 μmol · L 1 RPL41 treatment group,with Hoechst staining for the observation of nuclear morphometry of apoptotic cells,and finally,Western blot was used to determine the expression of activating transcription factor 4 (ATF4) of each group.Results Compared with the control group,the viability of Y79 cells in the 40 μmol · L-1 RPL41 treatment group was (97.9 ± 1.5) ％,with no significant difference (P =0.055);and the viability in the 80 μmol · L-1 and 120 μmol · L-1 RPL41 treatment group was (87.6 ± 1.8)％ and (63.9 ± 2.0) ％,respectively,both of which were significantly different from the control group (both P ＜ 0.05),so RPL41 inhibited the viability of Y79 cells,and 100 μmol · L-1 RPL41 promoted the apoptosis of Y79 cells,with the apoptotic rate of (17.33 ± 2.47)％.Compared with normal cells,the apoptotic cells in the 100 μmol · L 1 RPL41 treatment group showed bright color and smaller cell volume by Hoechst staining.Western blot showed that PRL41 significantly decreased the expression of ATF4 protein and the expression of ATF4 protein in the 40 μmol · L 1,80 μmol · L-1 and 120 μmol · L 1 treatment group were 0.76 ± 0.04,0.29 ± 0.04,0.29 ± 0.05,respectively,all of which were significantly different from the control group (all P ＜ 0.01).Conclusion RPL41 can inhibit the proliferation and promote the apoptosis of human retinoblastoma Y79 cell,and its mechanism may be related to the expression of ATF4.

Effects of isoflurane on learning and memory and the expression of GRP78,ATF4 and CHOP in frontal lobe neurons of aged rats / 中华行为医学与脑科学杂志

Objective To observe the changes of learning and memory ability and the expression of GRP8,ATF4 and CHOP in the frontal lobe neurons of rats after isoflurane anesthesia.Methods Male SD aged rats were randomly divided into 4 groups with 15 in each group.Rats in ISO group received 1.5% isoflurane 2 h,SAL group received intraperitoneal injection Salubrinal(1 mg/kg),ISO+SAL group received 1.5% 2 h after intraperitoneal injection Salubrinal(1 mg/kg),C group only inhaled 30% air and oxygen mixture.Morris water maze (MWM) test was performed 24 hours after isoflurane anesthesia,and then the left frontal lobe of rats was collected,gene transcription and protein expression changes of 78-kDa glucose-regulated protein(GRP78),activating transcription factor 4(ATF4),C/EBP homologous protein(CHOP) were evaluated by real-time fluorescence quantitative PCR and immunofluorescence.Results Compared with the C group,the latent time of ISO group was significantly prolonged(ISO group(19.10±2.98)s vs C group (10.54±2.05)s,P<0.05);the number of times passing through the target platform of ISO group was decreased significantly(ISO group (6.78±1.47) vs Cgroup (9.03±1.69),P<0.05);protein expression level of GRP78 was significantly increased in group ISO (ISO group (965.8±86.5) vs C group(247.5±46.3),P<0.05);protein expression level of ATF4 was significantly increased in group ISO(ISO group (470±69.4) vs C group (275.4±56.3),P<0.05) protein expression level of CHOP was significantly increased in group ISO(ISO group (618.7±83.3) vs C group(174.5±71.2),P<0.05).The transcription trends of GRP78,ATF4,CHOP were consistent with their protein expression.Conclusion The decrease of short-term memory ability after isoflurane anesthesia may be related to the activation of endoplasmic reticulum stress pathway in frontal lobe neurons.

Effects of Different Exercises on cAMP/CREB/ATF4 Pathway and Bone Formation in Bone of T2DM Mice / 中国运动医学杂志

Objective To observe the changes of bone formation in type 2 diabetes mellitus (T2DM) mice and the effect of different exercises on their cAMP/CREB/Atf4 pathway and bone formation.Methods Forty four-week-old C57BL/6 male mice were randomly divided into a normal control group (ZC) of 10 and a T2DM group of 30.T2DM was induced using the high-fat diet and injection of streptozotocin.Then,the T2DM mice were randomly divided into a T2DM control group (TC),a T2DM swimming group (TS) and a T2DM downhill running group (TD),each of 10.The TS and TD groups were trained for eight weeks as their group names indicated.Then the concentration of cAMP in serum was tested using the enzyme-linked immunosorbent assay (ELISA).The reverse transcription-polymerase chain reaction (RT-PCR) was used to test the mRNA expression of the cAMP-response element binding protein (CREB),activate transcription factor 4 (ATF4),osteocalcin (OC),bone gla-protein (OCN)and bone sialoprotein (BSP) in the left tibia,and Western blotting was employed to test the protein expression of CREB in the right femur.The bone marrow mesenchymal stem cells (BMSCs) were taken and induced to differentiate into osteoblasts (OBs) and dyed using the alkaline phosphatase (ALP) solution.The left side hindlimb bone was taken and scanned the bone mineral density (BMD) of the distal end using the Skyscan Micro-CT.Results Compared with group ZC,the concentration of cAMP declined in group TC.Moreover,the mRNA expression of CREB,ATF4,OC,OCN and BSP as well as the protein expression of CREB of group TC were significantly down-regulated(P＜0.01 or P＜0.05),together with the OB osteogenic capacity and BMD (P＜0.01) compared to group ZC.Compared with group TC,significant increase was observed in the mRNA expression of OC and OCN (P＜0.01 or P＜0.05),as well as the OB osteogenic capacity of group TS.The concentration of cAMP of group TD decreased,the mRNA expression of CREB,ATF4,OC and OCN,as well as the protein expression of CREB were all significantly up-regulated (P＜0.01 or P＜0.05) compared with group TD.The OB osteogenic capacity and BMD(P＜0.05) of group TD also increased significantly.Compared with group TS,the concentration of cAMP(P＜0.05) and the OB osteogenic capacity increased,and the mRNA expression of CREB,ATF4 and OC of group TD increased significantly(P＜0.01 or P＜0.05).Conclusion The bone formation metabolism of type 2 diabetic mice is inhibited.The downhill running is superior to swimming in promoting the osteoblast differentiation and bone formation,as well as the bone mineral density through activating the cAMP/CREB/Atf4 pathway in the bone of T2DM mice.

Effects of liraglutide on ATF4/CHOP pathway of pancreatic beta cells in rats fed with high-fat diet / 重庆医学

Objective To observe the effects of liraglutide on GRP78,ATF4,CHOP and TRB3 protein and mRNA expression in rats which fed with high fat diet,and to explore the effect of liraglutide on ATF4/CHOP pathway of pancreas in rats fed with high fat diets.Methods Forty-four male Wistar rats were randomly divided into the control group,high fat group,intervention group 1 and intervention group 2,11 cases in each group.The control group was fed with common food,other 3 groups were fed with high fat diet for 8 weeks.Then the intervention group 1 was given with liraglutide (100 μg · kg-1 · d-1) by subcutaneous injection;the intervention group 2 was given liraglutide (200 μg · kg-1 · d-1) by subcutaneous injection.After 2 weeks medication intervention,the rats were killed.Five rats were taken from each group and performed the hyperinsulinemic englycemic clamp experiment under waking state for calculating the glucose infusion rate (GIR).The fasting blood glucose (FBG),fasting insulin (FINS),serum free fatty acid (FFA),total cholesterol (TC),triglyeeride (TG),low density lipoprotein cholesterol (LDL-C) and high density lipoprotein(HDL) were examined,and islet beta cell function index(HOMA-β) were calculated.PCR and Western blot method were used to detect the expression of pancreas GRP78,ATF4,CHOP and TRB3 protein and mRNA.Results Compared with the control group,the levels of FBG,FFA,TC,FINS,TG and LDL-C in the high fat group were significantly increased,the levels of HDL-C,GIR and HOMA-β were significantly decreased(P＜0.05 or P＜0.01);compared with the high fat group,the levels of HDL-C,GIR and HOMA-β in the intervention group 2 were increased(P＜0.05 or P＜0.01),the other indicators were significantly decreased;compared with the intervention group 1,blood FGB,FFA,TC in the intervention group 2 were decreased,while the levels of GIR and HOMA-β were increased(P＜0.05).Compared with the control group,expression of GRP78,ATF4,CHOP and TRB3 protein and mRNA in the high fat group were significantly increased;compared with the high fat group,the expression of GRP78,ATF4,CHOP and TRB3 protein and mRNA in the intervention group 1 and 2 were gradually decreased with the liraglutide concentration increase.Conclusion Liraglutide can improve insulin resistance and protect pancreatic beta cells in a concentration-dependent manner,its mechanisms may involve in the pancreatic endoplasmic reticulum ATF4/CHOP pathway.

Effects of liraglutide on ATF4/CHOP pathway of pancreatic beta cells in rats fed with high-fat diet / 重庆医学

Objective To observe the effects of liraglutide on GRP78,ATF4,CHOP and TRB3 protein and mRNA expression in rats which fed with high fat diet,and to explore the effect of liraglutide on ATF4/CHOP pathway of pancreas in rats fed with high fat diets.Methods Forty-four male Wistar rats were randomly divided into the control group,high fat group,intervention group 1 and intervention group 2,11 cases in each group.The control group was fed with common food,other 3 groups were fed with high fat diet for 8 weeks.Then the intervention group 1 was given with liraglutide (100 μg · kg-1 · d-1) by subcutaneous injection;the intervention group 2 was given liraglutide (200 μg · kg-1 · d-1) by subcutaneous injection.After 2 weeks medication intervention,the rats were killed.Five rats were taken from each group and performed the hyperinsulinemic englycemic clamp experiment under waking state for calculating the glucose infusion rate (GIR).The fasting blood glucose (FBG),fasting insulin (FINS),serum free fatty acid (FFA),total cholesterol (TC),triglyeeride (TG),low density lipoprotein cholesterol (LDL-C) and high density lipoprotein(HDL) were examined,and islet beta cell function index(HOMA-β) were calculated.PCR and Western blot method were used to detect the expression of pancreas GRP78,ATF4,CHOP and TRB3 protein and mRNA.Results Compared with the control group,the levels of FBG,FFA,TC,FINS,TG and LDL-C in the high fat group were significantly increased,the levels of HDL-C,GIR and HOMA-β were significantly decreased(P＜0.05 or P＜0.01);compared with the high fat group,the levels of HDL-C,GIR and HOMA-β in the intervention group 2 were increased(P＜0.05 or P＜0.01),the other indicators were significantly decreased;compared with the intervention group 1,blood FGB,FFA,TC in the intervention group 2 were decreased,while the levels of GIR and HOMA-β were increased(P＜0.05).Compared with the control group,expression of GRP78,ATF4,CHOP and TRB3 protein and mRNA in the high fat group were significantly increased;compared with the high fat group,the expression of GRP78,ATF4,CHOP and TRB3 protein and mRNA in the intervention group 1 and 2 were gradually decreased with the liraglutide concentration increase.Conclusion Liraglutide can improve insulin resistance and protect pancreatic beta cells in a concentration-dependent manner,its mechanisms may involve in the pancreatic endoplasmic reticulum ATF4/CHOP pathway.

Enhanced activation of PERK-ATF4 pathway by Brefeldin A and cisplatin in human lung cancer GLC-82 ;cells / 实用医学杂志

Objective To investigate the molecular mechanisms of synergistic effects of BFA and CDDP on human lung cancer GLC-82 cells, and to test the levels of PERK-ATF4 pathway. Methods GLC-82 cells were incubated with 50 ng/mL of BFA or/and 2 μg/mL of CDDP for 24 or 48 hours. The levels of PERK, p-PERK and ATF4 in GLC-82 were analyzed by real-time PCRand/or Western Blot. Results The levels of PERK were lowest in CDDP group, but higher in BFA group (P < 0.05), the highest in group of BFA+CDDP (P < 0.05 or P < 0.01). The p-PERK level decreased in group of BFA+CDDP (P < 0.05 or P < 0.01). There was no significant change of ATF4 expression in CDDP group, but ATF4 expression increased slightly in BFA group, and increased further in group of BFA+CDDP (P < 0.05 or P < 0.01)which was also higher than that in BFA group or CDDP group (P < 0.05 or P < 0.01). Conclusions The upregulated levels of PERK and ATF4 by the combination of BFA and CDDP may be one of the mechanisms of synergistic anti-cancer effect of BFA and CDDP on GLC-82 cells.

Research progress of activating transcription factor 4 in glucose and lipid metabolism / 中国糖尿病杂志

[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.