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Objective @#To investigate the role and mechanism of bone formation caused by the ratio of advanced platelet-rich fibrin (A-PRF) and β-tricalcium phosphate (β-TCP) in rabbit femur defect model, which provides a new idea for clinical treatment of bone defect.@*Methods @#Twenty-four New Zealand white rabbits were divided into model group, 1∶1 complex group (A-PRF∶β-TCP=1∶1), 2∶1 complex group (A-PRF∶β- TCP=2∶1) and 4∶1 complex group (A-PRF∶β- TCP=4∶1）, with 6 rabbits in each group. Femoral defect models were constructed in each group. In the composite group, the bone defect was filled with composite material, while in the model group, no material was filled. After 8 weeks, the animals were euthanized and specimens were collected. Bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.SP) and trabecular number (Tb.N) in femoral defect tissue were measured by micro-CT and photographed. Hematoxylin - eosin staining was used to detect the pathological changes of new bone tissue. The morphological changes of the new bone tissue were observed by scanning electron microscopy. Determination of phospho-mitogen activated protein kinase p38 (p-p38MAPK), CCAAT/enhancer binding protein homologous protein (CHOP) and phospho-cysteine aspartic protease-3 (p-Caspase3) in newborn femur by ELISA. The mRNA expressions of osteoprotegerin (OPG), bone morphogenetic protein-2 (BMP-2), receptor activator of nuclear factor kappa-B ligand (RANKL) and p38MAPK were detected by real-time quantitative PCR. The expression of OPG, BMP-2, RANKL, p-p38MAPK and p-Caspase3 protein in the new bone tissue was observed by immunohistochemistry. @*Results @#In the model group, bone formation in the femoral defect area was slow and osteogenic quality was poor. Compared with the model group, the bone formation and neocapillaries of femoral defect area in the complex group was good, BMD, BV.TV, Tb.Th, Tb.N were increased, and Tb.Sp were decreased, the expressions of p-p38MAPK, CHOP and p-Caspase3 were decreased, and the mRNA and protein expressions of OPG and BMP-2 were increased. The mRNA expression of RANKL and p38MAPK was decreased. Apoptosis in new bone tissue of each group showed the lowest apoptosis rate in samples of the 2∶1 complex group (P<0.05); A-PRF: β-TCP=2∶1 ratio has the best osteogenic effect. @*Conclusion@#The complex composed of A-PRF and β-TCP can promote the expression of OPG, inhibit the expression of RANKL and phosphorylation of p38MAPK, reduce the apoptosis of new bone tissue cells, and promote osteogenic differentiation.
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ObjectiveTo investigate the protective effect of modified Huangqi Guizhi Wuwutang （MHGW） on endoplasmic reticulum stress in the sciatic nerve of diabetes rats based on the pathways of inositol-requiring enzyme 1α （IRE1α） and CCAAT/enhancer-binding protein homologous protein （CHOP）. MethodSixty rats were fed on a high-sugar and high-fat diet for six weeks， followed by intraperitoneal injection of streptozotocin at a dose of 35 mg·kg-1. Random blood glucose levels were measured three days later and rats with a sustained blood glucose level ≥ 16.7 mmol·L-1 were included in study （n=48）. The rats were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg-1·d-1）， a high-dose MHGW group （2.5 g·kg-1·d-1）， and a low-dose MHGW group （1.25 g·kg-1·d-1）. Another 10 rats were assigned to the normal group. The intervention lasted for 16 weeks. After 16 weeks， the sciatic nerve structure of the rats in each group was observed under light microscopy using Luxol fast blue （LFB） staining. Transmission electron microscopy was used to observe the ultrastructure of the sciatic nerve. Chemiluminescence method was employed to measure the serum reactive oxygen species （ROS） levels. Western blot and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to evaluate the expression of p-IRE1α protein， IRE1α mRNA， CHOP protein， and CHOP mRNA in the sciatic nerve of the rats. ResultCompared with the normal group， the model group showed elevated serum ROS levels （P<0.01）. In contrast， the serum ROS levels were significantly reduced in the treatment groups compared with those in the model group （P<0.01）. The sciatic nerve of the model group showed pathological changes compared with that in the normal group， while the treatment groups exhibited improvement in sciatic nerve pathology compared with the model group. The protein expression of p-IRE1α and CHOP in the sciatic nerve significantly increased in the model group as compared with that in the normal group （P<0.01）. However， the treatment groups showed a significant decrease in the protein expression of p-IRE1α and CHOP in the sciatic nerve compared with the model group （P<0.05， P<0.01）. Furthermore， compared with the normal group， the model group showed upregulated mRNA expression of IRE1α and CHOP in the sciatic nerve （P<0.01）， while the treatment groups exhibited a significant decrease in the mRNA expression of IRE1α and CHOP compared with the model group （P<0.01）. ConclusionMHGW can alleviate endoplasmic reticulum stress-induced cell apoptosis and improve the structure and function of the sciatic nerve in diabetes rats by inhibiting the expression of IRE1α/CHOP pathway-related proteins and mRNA， thereby preventing and treating peripheral neuropathy in diabetes.
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Objective:To investigate the effect of Ziziphi Spinosae Semen （ZSS） and Albiziae Flos （AF） on behavior and endoplasmic reticulum stress endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase （PERK）/activated transcription factor 4 （ATF4）/CCAAT enhancer binding protein （CHOP） pathway in depression model rats， and to explore its antidepressant mechanism. Method:The male SD rats were divided into normal group， model group， ZSS-AF high dose， middle dose and low dose groups （16， 8， 4 g·kg<sup>-1</sup>） and Venlafaxine group （0.008 g·kg<sup>-1</sup>）， <italic>n</italic>=15 in each group. Except the normal group， the depression model was established in the rats of other 5 groups by the method of chronic unpredictable mild stress （CUMS） combined with isolated feeding. The normal group and model group were given with distilled water by gavage when modeling， while other groups received corresponding drug by intragastric administration for 21 days. Behavior changes of rats in each group were observed by the open field test and sugar water consumption test on 1<sup>th </sup>and 21<sup>th</sup>day of the experiment. The protein expressions of PERK， CHOP， B-cell lymphoma-2 associated X protein （Bax） and cysteine-containing aspartate-specific proteases-3（Caspase-3） were detected by Western blot（WB）， the ultrastructural changes of the hippocampus were observed by transmission electron microscope， the apoptosis of hippocampal neurons was observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL） method. Result:Compared with the normal group， the scores of open field test and sugar water consumption rate in model group rats decreased （<italic>P</italic><0.01）. Compared with the model group， the scores of open field test and water consumption rate increased （<italic>P</italic><0.01） in ZSS-AF groups and Venlafaxine group. Transmission electron microscope showed that the changes of neuronal damage in hippocampal were revealed in the model group， whereas those neuronal damages were relieved in ZSS-AF groups and Venlafaxine group. TUNEL method showed that the number of apoptotic neurons in hippocampal increased in the model group （<italic>P</italic><0.01）， but decreased in ZSS-AFgroups and Venlafaxine group （<italic>P</italic><0.01）. WB results showed that as compared with the normal group， protein expressions of PERK， CHOP， Bax and Caspase-3 were up-regulated significantly in the model group （<italic>P</italic><0.01）， whereas those were down-regulated in ZSS-AF groups and Venlafaxine group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:The antidepressant effect of ZSS-AF herbal pair may be correlated with the regulation of endoplasmic reticulum stress PERK/ATF4/-CHOP pathway.
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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.
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Objective To investigate the effects of homocysteine(Hcy) on myocardial injury and its possible mechanisms.Methods The selected H9C2 cardiomyocytes were intervened with various concentrations of Hcy and 4-phenyl butyric acid(4-PBA).The H9C2 cells were divided into the control group,H400 group and H400P2 group.The control group used the common medium,the H400 group was added with 400 μmol/L Hcy,the H400P2 group was added with 2 mmol/L 4-PBBA on the basis of H400 group.The cell livability was detected by using cell counting kit-8 (CCK-8).Apoptosis was evaluated by using the terminal deoxynucleotidyl transferase mediated nick-end labelling(TUNEL) staining.The ERO1α expression was determined by using immunocytochemistry,and the protein expression difference was determined by using Western blot.Results The injury of Hey on H9C2 cardiomyocytes showed a concentration-dependent manner(F=2 039.958,P＜0.01).Compared with the control group,the apoptosis percentages and expression levels of PERK,p-PERK,CHOP and ERO1α in the H400 group were increased(P＜0.01);while which in the H400P2 group were decreased,the difference was statistically significant(P＜0.05).Conclusion Hcy mediates myocardial apoptosis through endoplasmic reticulum stress mechanism.
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Objective To investigate the effect of electroacupuncture on the expressions of endoplasmic reticulum stress-related proteins in the substantia nigra in a rat model of rotenone-induced Parkinson disease.Methods One hundred and twenty healthy male SD rats were randomized to normal, sham operation, model, electroacupuncture pretreatment, and 7-day, 14-day, 21-day and 28-day electroacupuncture treatment groups, 15 rats each. A model of Parkinson disease was made by subcutaneous injection of rotenone in the nape. The sham operation group received an injection of equal volume of sunflower oil emulsion. The normal, sham operation and model groups were not treated. The electroacupuncture treatment group received electroacupuncture at points Fengfu (GV 16)and Taichong (LR 3) after model making. The electroacupuncture pretreatment group received model making after 7 days of electroacupuncture treatment. TH and α-syn positive cells expressions in rat substantia nigra were determined by immunohistochemical method. BiP and CHOP expressions in rat substantia nigra were examined by Western blotting. Results The model group rats had obvious syndrome characteristic of PD and rat behavior score decreased significantly after electroacupuncture intervention (P<0.01).α-syn positive cells expression and BiP and CHOP expressions in rat substantia nigra increased significantly and TH positive cells expression in rat substantia nigra decreased significantly in the model group compare with the normal and sham operation groups (P<0.01).α-syn positive cells expression and BiP and CHOP expressions in rat substantia nigra decreased significantly and TH positive cells expression in rat substantia nigra increased significantly in every electroacupuncture treatment group compare with the model group (P<0.01).Conclusions The mechanism of electroacupuncture prevention and treatment of Parkinson disease may be related to its significant inhibition of the expressions of endoplasmic reticulum stress-related proteins and protection of dopaminergic neurons.
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AIM:To investigate whether cigarette smoke(CS)promotes the expression of endoplasmic reticu-lum-associated apoptosis protein CCAAT/enhancer-binding protein homologous protein(CHOP)in rat lung tissues. METHODS:Adult male Wistar rats(n=40)were randomly divided into 4 groups with 10 rats in each group: control group,CS-2 group(exposed to CS for 2 months),CS-4 group(exposed to CS for 4 months)and ex-smoking(Ex-S)group (exposed to CS for 4 months and then quit smoking for 1 month).The percentage of forced expiratory volume in 0.3 second to forced vital capacity(FEV0.3/FVC)and peak expiratory flow(PEF)were measured.TUNEL assay was used to detect the apoptotic cells.In situ hybridization and RT-PCR were used to determine the mRNA expression of CHOP.The methods of immunohistochemistry and Western blot were used to determine the protein expression of CHOP.Western blot was also used to determine the protein levels of protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK,eukaryotic initiation factor(eIF)2αand p-eIF2α.RESULTS:The pulmonary function greatly decreased in the rats exposed to CS for 2 months in comparison with control group(P<0.05),markedly decreased in the rats exposed to CS for 4 months as com-pared with the rats after exposure to CS for 2 months(P<0.05),and was improved little in ex-smoking rats(P>0.05). The structural destruction of the lung was observed in the rats exposed to CS for 2 months,and more obvious changes were found in the rats exposed to CS for 4 months.However,the structural destruction of the lung remained obvious in ex-smok-ing rats.The apoptotic cells were markedly increased in the rats exposed to CS for 2 months and were even more in the rats exposed to CS for 4 months.The apoptotic cells were alveolar epithelial cell I(ACE I),ACE II,vascular endothelial cells and bronchial epithelial cells.The protein levels of p-PERK,p-eIF2αand CHOP were remarkably increased in the rats af-ter exposure to CS for 2 months compared with the control rats(P<0.05),significantly elevated in the rats exposed to CS for 4 months compared with the rats exposed to CS for 2 months(P<0.05),and slightly decreased in ex-smoking rats in comparison with the rats after exposure to CS for 4 months(P>0.05).The total protein levels of PERK and eIF2αdid not change between the control rats and those exposed to CS.CONCLUSION: CS promotes the development of chronic ob-structive pulmonary disease(COPD)by inducing the expression of endoplasmic reticulum-associated apoptosis protein CHOP via PERK/eIF2α/CHOP signaling pathway.
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Objective By establishing multivariate necrotizing enterocolitis (NEC) model in newborn rats to study the expression of the related molecules protein kinase R-like ER kinase(PERK),pho-protein kinase R-like ER kinase(p-PERK),eukaryotic translation initiation factor 2 alpha(eIF2a),pho-eukaryotic translation initiation factor 2 alpha (p-eIF2a),CCAAT/enhancer-binding protein-homologous protein (CHOP) in the PERK/eIF2a/ CHOP signaling pathway to explore the pathogenesis of NEC,and in order to provide a theoretical basis for clinical pre-vention and treatment of NEC.Methods One hundred and fifty neonatal Sprague-Dawley (SD) rats were divided into 3 groups according to random number table.NEC group:fed with formula,experienced hypoxia and cold stress,and lipopolysaccharide(LPS) fed via orogastric gavage to induce NEC model.Salubrinal(SAL) group:estabhshing NEC model and intraperitoneal injection of SAL (1 mg/kg).Control group:the same amount of 9 g/L saline was injected intraperitoneally.Ten rats selected from each group randomly were decapitated at 0,12,24,48 and 72 h,respectively,and intestinal tissues were obtained,intestinal general situation and made pathology scores observed.Expressions of PERK,p-PERK,eIF2a,p-eIF2a,CHOP proteins were detected by Western blot technique and the expression levels of CHOP mRNA gene were detected by real-time polymerase chain reaction.Results The control group had no NEC.The incidence of NEC in NEC group was 90％ (9/10 cases).The incidence of NEC in SAL group was 40％ (4/10 cases).The p-PERK and p-eIF2a protein expressions in NEC and SAL groups were increased with the modeling time.The p-PERK and p-eIF2a protein expressions of NEC and SAL groups were up-regulated compared with the control group (p-PERK:1.528 ± 0.264,1.402 ± 0.233 vs.0.303 ± 0.036;p-eIF2a:0.969 ± 0.076,1.173 ± 0.066 vs.0.209 ±0.045;P ＜0.05).The p-eIF2a protein expressions of SAL group were higher than those of the NEC group (P ＜ 0.05).The CHOP protein and CHOP mRNA expressions in the NEC and the SAL groups were increased with the modeling time.The CHOP protein and CHOP mRNA expressions of NEC and SAL groups were up-regulated than those of the control group (CHOP protein:1.456 ± 0.223,0.929 ± 0.064 vs.0.165 ± 0.026;CHOP mRNA:0.343 ±0.035,0.198 ± 0.044 vs.0.017 ± 0.010;P ＜ 0.05).The CHOP protein and CHOP mRNA expressions of SAL group were lower than that in the NEC group (P ＜ O.05).Conclusions The PERK/eIF2a/CHOP signal pathway may participate in NEC,and salubrinal probably by inhibiting p-eIF2a dephosphorylation,and inhibiting CHOP gene and protein expression to work.
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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 role of endoplasmic reticulum stress (ERS) pathway involving protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) in apoptosis in lungs of rats with bronchopulmonary dysplasis (BPD).Methods Forty eight premature SD rats were divided into BPD group and control group according to random number table.Rats in BPD group were continually exposed to O2 with volumetric concentration factor of 850 mL/L,while rats in control group were exposed to air.Lung tissues in each group were obtained in 7,14 and 21 days respectively.The apoptosis in lung cells was evaluated by terminal dexynucleotifyl transferase-mediated dUTP nick end labeling (TUNEL) assay.The mRNA levels of glucose regulated protein 78 (GRP78),PERK,ATF4 and CHOP were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).The protein levels of GRP78,phosphorylated PERK (pho-PERK),ATF4 and CHOP were detected by using Western blot.Results Compared with control group,the lung cells of the rats in BPD group developed more serious apoptosis.Furthermore,the apoptosis index (AI) in lung cells of the rats increased rapidly with the hyperoxia exposure time.This had been statistically verified by comparison with the control group at different timing(7 d:15.50 ± 0.58 vs 1.25 ± 0.50,14 d:27.75 ± 1.71 vs 3.25 ± 0.96,21 d:50.50 ±3.70 vs 4.00 ± 1.15 ;t =57.00,20.58,25.16,all P ＜0.01).The mRNA levels of GRP78,PERK,ATF4 and CHOP in BPD group increased significantly compared to the control group [GRP78:7 d (33.88 ± 3.73) vs (11.65 ± 1.00),14 d (54.50 ±2.18)vs(12.84 ± 1.41),21 d (95.34 ± 7.61)vs(12.43 ±0.59) ;PERK:7 d (5.23 ±0.92)vs (1.45 ±0.46),14 d (7.60 ± 1.56)vs(2.18 ±0.97),21 d (16.55 ±0.50)vs(2.90 ± 1.18) ;ATF4:7 d (23.04 ± 2.45)vs(12.56 ±2.81),14 d (28.66 ±2.66)vs(15.18 ±2.92),21 d (36.63 ±2.99)vs(15.14 ±2.09) ;CHOP:7 d (2.21 ±0.19)vs(0.81 ±0.02),14 d (4.19 ±0.17)vs(0.90 ±0.08),21 d (6.08 ±0.38)vs(0.88 ±0.10) ;all P ＜ 0.05].The protein levels of GRP78,pho-PERK,ATF4 and CHOP in BPD group increased significantly as well [GRP78:7 d (1.33 ±0.03)vs(0.85 ±0.04),14 d (1.31 ±0.02)vs(0.92 ±0.01),21 d (1.82 ±0.28)vs(0.87 ± 0.01);pho-PERK:7 d (0.68±0.02)vs(0.54±0.01),14 d (1.04±0.01)vs(0.65±0.01),21 d (1.29± 0.02)vs(0.73 ±0.01) ;ATF4:7 d (1.26 ±0.01) vs(0.83 ±0.01),14 d (1.39 ±0.02) vs (0.87 ±0.02),21 d (1.67 ±0.02)vs(0.94 ±0.02) ;CHOP:7 d (1.37 ±0.01)vs(0.47 ±0.06),14 d (1.50 ±0.04)vs(0.74 ±0.05),21 d (1.61 ± 0.03) vs (0.55 ± 0.02) ; all P ＜ 0.05].Positive correlation was demonstrated between the expression levels of CHOP protein and AI,PERK,ATF4 in the BPD group (r =0.87,0,92,0.93 respectively,all P ＜ 0.05).Conclusion PERK-ATF4-CHOP mediated ERS may participate in and contribute to the apoptosis mechanism in lungs of rats with BPD.
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Objective To investigate the effects of dexmedetomidine pre-treatment on pneumonocyte apoptosis and CCAAT/enhancer binding protein homologous protein (CHOP) in acute lung injury (ALI) induced by ischemia/reperfusion (I/R) during orthotopic liver transplantation in rats.Methods Forty adult male Sprague-Dawley (SD) rats were randomly divided into four groups by random number table method: sham operation group, I/R model group, dexmedetomidine low dose group and dexmedetomidine high dose group, 10 rats per group. Hepatic artery was ligated and cut off by two cuff method, and the portal vein was completely opened after donor liver transplanted into the recipient, thus, a hepatic I/R model was established. The perihepatic ligaments of rats were just separated after laparotomy in sham operation group and no other special treatment was performed. One hour prior to I/R, dexmedetomidine at a dose of 2.5μg·kg-1·h-1 and 5.0μg·kg-1·h-1, respectively, were pumped intravenously and finished within 1 hour in the rats of low dose group and high dose group. After experiment, the lung tissue was taken, and the lung wet/dry weight (W/D) ratio was determined. Pathological changes of lung tissue were observed and alveolar damage index of quantitative assessment (IQA) was tested by light microscope, and changes of ultrastructure of lung tissue were observed by transmission electron microscope. The mRNA and protein expressions of CHOP were detected respectively by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. The apoptosis in lung tissue was determined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) method and apoptosis index (AI) was calculated.Results Compared to sham operation group, the lung W/D ratio (4.94±0.84 vs. 2.29±0.54), IQA [(40.52±5.15)% vs. (4.55±1.85)%] and AI [(36.57±5.85)% vs. (2.85±0.95)%] in I/R model group were significantly higher (allP < 0.01); remarkable injury of lung tissue was confirmed by light microscope and transmission electron microscope in the I/R model group. Compared to I/R model group, the W/D ratio (3.29±0.85, 2.68±0.78 vs. 4.94±0.84), IQA [(23.69±2.62)%, (15.86±3.61)% vs. (40.52±5.15)%] and AI [(25.73±3.71)%, (14.66±2.61)% vs. (36.57±5.85)%] in dexmedetomidine low and high dose groups were markedly lower (allP < 0.01); under light and transmission electron microscopes, the injury of lung tissue in these two dose groups was notably alleviated. There was a large amount of apoptotic cells of pulmonary vascular endothelium and alveolar epithelium in I/R model group, while the cell apoptosis was distinctly decreased in dexmedetomidine low and high dose groups compared to that in model group. Compared to sham operation group, the expressions of CHOP mRNA [absorbance (A) value: 0.96±0.18 vs. 0.43±0.08] and protein (gray scale: 2.79±0.74 vs. 1.02±0.27) were significantly higher in I/R model group (bothP < 0.01). Compared to I/R model group, the expressions of CHOP mRNA (A value: 0.69±0.13, 0.56±0.12 vs. 0.96±0.18) and protein (gray scale: 1.96±0.58, 1.34±0.49 vs. 2.79±0.74) were significantly lower in dexmedetomidine low and high dose groups, the decrease in dexmedetomidine high dose group being more marked (allP < 0.01).Conclusion The pretreatment of dexmedetomidine can protect lung tissue against I/R injury during liver transplantation in rats, and the mechanism may be related to the suppression of CHOP activation and alleviation of lung tissue cell apoptosis.
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Objective To investigate the function of melatonin (MEL)on liver transplantation by detecting the effect of MEL on glucose regulated protein-78 (GRP-78)and CCAAT enhancer-binding protein homologous protein (CHOP)involved in the endoplasmic reticulum stress (ERS)pathways in livers of rats with liver transplantation.Methods The rat orthotopic liver transplantation model was established by using‘magnetic-loop method’.Male SD rats were randomized into 3 groups:the sham operation group (Sham group),the orthotopic liver transplantation group (OLT group)and the orthotopic liver transplantation +MEL treatment group (OLT +MEL group),8 rats in each group.Serum sample and liver sample were collected at 24 h after operation.Serum alanine aminotransferase (ALT)and aspartate aminotransferase (AST)levels were detected.Liver tissues were stained with hematoxylin-eosin (HE)and pathological changes of liver tissues of each group were observed.The messenger ribonucleic acid (mRNA)and protein expression levels of GRP-78 and CHOP were detected by polymerase chain reaction and Western blot.Results Compared with the Sham group,the serum ALT and AST of the OLT group increased significantly (both in P <0.01 ),liver tissues injury was serious,and mRNA and protein expression levels of GRP-78 and CHOP increased significantly (all in P <0.01).Compared with the OLT group,ALT and AST of the OLT +MEL group decreased significantly (both in P <0.05),liver tissues injury was alleviated,and mRNA and protein expression levels of GRP-78 and CHOP decreased significantly (all in P <0.05 ).Conclusions MEL may reduce mRNA and protein expression levels of GRP-78 and CHOP involved in the ERS pathways of transplant liver,which may be one of its mechanisms to alleviate liver injury after liver transplantation.
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Objective To explore the neuronal apoptosis,endoplasmic reticulum stress(ERS)-related factors glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein-homologous protein (CHOP) expressions in hippocampus and possible mechanism of brain protection by 2-deoxy-D-glucose(2-DG) on their expressions in epileptic brain damage rats induced by pentetrazole.Methods Rats aged 21 d (n =120) were randomly divided into 3 groups:control group,status epilepticus (SE) group and SE + DG group,with 40 rats in each group.On a SE rat model,the neuronal apoptosis in the hippocampus was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling method.And the expression of GRP78 and CHOP were detected by Western blot and Real-time PCR.Results Apoptotic cells in the hippocampus were increased remarkably at 12 h,24 h,and 48 h after the seizures,as compared with those of control rats.In SE group,chromatin condensation and breakdown of the nucleus in neurons in hippocampus were also easily observed by Hoechst staining.Meanwhile,the ERS was induced by SE,as witness by up-regulating GRP78 expression at both protein and mRNA level.The expression of GRP78 protein in the hippocampus was elevated at 3 h,6 h and 12 h,with the maximum elevation noted at 6 h after SE.The GRP78 transcript expression was increased at 3 h,6 h,and 12 h after SE.DG treatment enhanced the changes of GRP78 protein and transcript at 3 h,and abrogated the increase of GRP78 at 6 h and 12 h,as compared with those in SE group.CHOP activation occurred at 12 h,24 h and 48 h in SE group.And the CHOP-mRNA expression was the same as the CHOP protein expression.The expression of CHOP protein and mRNA was decreased at 12 h,24 h,and 48 h after using DG.Conclusion The endoplasmic reticulum stress response mediated by CHOP seems to be involved in the neuronal apoptosis caused by status epilepticus.
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ObjectiveTo investigate the effects and molecular mechanism of endoplasmic reticalam stress (ERS) on albumin-induced apoptosis in renal proximal tubular cells (HKCs). MethodsWestern blot was performed to detect the relationship of the expression of glucose-regulated protein 78 (GRF78) and CCAAT/enhancer-binding protein-homologous protein (CHOP) with the action time and concentration of haman serum albumin (HSA). Expression levels of CHPO mRNA and protein in HKCs after CHOP siRNA transfection were examined by real-time fluorescence quantitative PCR and Western blot respectively. Annexin-V-FITC and PI doable staining cytometry was used to detect the apoptosis of HKCs induced by HSA and influenced by CHOP siRNA. Results(1)After HKCs were stimulatde by 0, 5, 10, 20 g/L albumin for 24 hours respectively, the expression of GRP78, CHOP and HKCs apoptosis were increased with the albumin concentration (P<0.01). After HKCs were stimulated by 20 g/L albumin for 0, 6, 12, 24, 36 hours respectively, the expression of GRP78 was up-regulated at 6-hour, while CHOP and HKCs apoptosis were increased at 12-hour, and significant differences were found among groups (P<0.01). (2) CHOP siRNA significantly inhibited albumin-induced HKC CHOP mRNA and protein expression, as well as HKC apoptosis (P<0.01). ConclusionsRenal tubular cells exposed to high protein load result in EBS. ERS may subsequently lead to tubular damage by activation of pro-apoptosis factor CHOP.
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Objective To explore the inhibitory effects of Zibu Piyin Recipe(ZBPYR)serum on neuron apoptosis induced by tunieamyein(Tm,5 μg/ml)and its mechamsm in vitro by using sero-pharmacological method.Method Totally 12 healthy adult male SD rats(220～250 g)(SPF)were divided randomly into control group and ZBPYR group,6 in each group,then the blank and ZBPYR serum were prepared.The mouse.neuroblastoma cell line Neum2a cells were treated with Tunicamycin(Tin,an inhibitor of N-glycoslytion)to establish the endoplasmic reticulum(ER)stress model.The cells treated by ZBPYR aerum of different concentrations were interventional groups,and the cells treated by blank serum were control group.The viability of Neuro2a cells was meusurcdd by MTT assay.Flow cytometry wus applied to observe the apoptosis of Neuro2a cells.Western blotting was utilized to detect the protein expressions of two molecules,ER molecular chaperone-ucose regulated protein 78(CRP78)and transcriptional factor CCAAT/enhancer-binding protein-homologous protein(CHOP).The results were analyzed by sNK-q test.Results Compared to Tm group(cell viability 0.1673±0.0213,apoptotic rate 62.7050±1.4056),The cell viability of interventional groups(5%0.5295±0.0373,10%0.5843±0.0428,15%0.6274±0.0324)increased significantly(P<0.05);and the apoptotic rate(5%47.8733±2.8166,10%46.3366±1.2748,15%39.8833±1.0524)reduced significantly(P<0.05).The protein expressions of GRP 78(5%2.1228±0.2251,10%1.3293±0.9443,15%;15%0.0931±0.1168)and CHOP(5%1.1776±0.2927,10%0.7290±0.1708,15%0.6577±0.1883)of interventional groups reduced significantly compared with Tm group(GRP78 2.9149±0.5355;CHOP 1.6611±0.2913)P<0.05.Condusions ZBPYR serurn could increase the cell viability of Neuro2a cells treated with Tm and inhibit cell apoptosis.Thereby it may have neuroprotective effects,and the mechanism may be associated with the inhibition of ER stress and apoptosis pathway.