OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8^+T cells an...OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8^+T cells and investigate the curative effect of the combined use.METHODS CD8^+T cells were isolated from normal mice spleen by negative selection using magnetic cell separation.The isolated CD8^+T cells were cultured in RPMI 1640 medium containing 10%FBS and 100 U·mL^(-1)IL-2 and activated by the addition of anti-CD3 and anti-CD28(1 g·L^(-1) each mabs).CD8^+T cells were pretreated for 48 h with drug and the fluo-3 as a marker of intracellular calcium concentration was detected by flow cytometry.The calcineurin(Ca N)levels were assayed with ELISA in CD8^+T cells after 48 h incubation with 10μm CAI.The nuclear translocations of NFAT and AHR were detected by immunofluorescent staining after 48 h of drug treatment.The expression of PD-1 in CD8^+T cells was analyzed by flow cytometry.RESULTS Intracellular fluorescent intensity was markedly debase due to CAI treatment(P<0.01).Meanwhile,the changes of CaN content had a resembled correlation(P<0.01).Immunofluorescence experiment showed that after combination therapy the transfer of NFAT and AHR in nuclear substantially reduced.Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8^+T cells.CONCLUSION CAI and 1-MT could inhibit markedly the expression of PD-1 in CD8^+T cells by inhibiting the nuclear translocation of NFAT and AHR,respectively and the combination of them has synergetic effect.展开更多
目的探讨脑和肌肉芳香烃受体核转运样蛋白1(brain and muscle ARNT-like protein 1,BMAL1)在大鼠牙周炎诱导肝损伤模型中的作用。方法根据随机数字表法将12只Wistar雄性大鼠随机分为对照组和牙周炎组,每组6只。对照组大鼠不做处理。牙...目的探讨脑和肌肉芳香烃受体核转运样蛋白1(brain and muscle ARNT-like protein 1,BMAL1)在大鼠牙周炎诱导肝损伤模型中的作用。方法根据随机数字表法将12只Wistar雄性大鼠随机分为对照组和牙周炎组,每组6只。对照组大鼠不做处理。牙周炎组大鼠通过结扎双侧上颌第一磨牙颈部建立牙周炎模型。建模8周后检测两组大鼠牙周临床指标并处死。显微CT(micro-CT)扫描大鼠上颌骨并分析牙槽骨吸收情况。HE及油红O染色分析两组大鼠牙周组织和肝组织的病理变化。生化试剂盒检测血清中谷草转氨酶(glutamic-oxaloacetic transaminase,GOT)、谷丙转氨酶(glutamic-pyruvic transaminase,GPT)、总胆固醇(total cholesterol,TC)和甘油三酯(triglyceride,TG)等肝功能相关指标。实时荧光定量PCR(real time fluorescent quantitative-PCR,qRT-PCR)、免疫组化和蛋白质印迹法检测肝组织中BMAL1、核因子-κB(nuclear factor kappa-B,NF-κB)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)基因及蛋白的表达水平。原位末端转移酶标记(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL)试剂盒染色法检测肝组织中的细胞凋亡。结果上颌第一磨牙HE染色和micro-CT结果显示,牙周炎组大鼠牙槽骨吸收明显。肝组织病理学结果显示,与对照组相比,牙周炎组肝组织内炎症细胞浸润、肝索结构紊乱且肝细胞中可见大量脂滴形成。大鼠血清生化检测结果显示,牙周炎组大鼠血清中GOT[(62.77±2.59)U/L]、GPT[(47.54±1.04)U/L]、TC[(3.19±0.23)mmol/L]和TG[(1.11±0.09)mmol/L]均较对照组GOT[(38.66±2.47)U/L]、GPT[(31.48±1.57)U/L]、TC[(1.60±0.05)mmol/L]和TG[(0.61±0.09)mmol/L]含量显著升高(P=0.003,P=0.001,P=0.002,P=0.038)。qRT-PCR结果显示,牙周炎组大鼠肝组织中BMAL1 mRNA的表达[(0.60±0.04)%]较对照组[(1.01±0.07)%]显著下调(t=4.80,P=0.009),NF-κB、TNF-αmRNA的表达[(1.62±0.12)%、(2.69±0.16)%]均较对照组[(1.00±0.03)%、(1.03±0.16)%]显著上调(P=0.008,P=0.002)。免疫组化结果显示:牙周炎组大鼠肝组织中BMAL1蛋白表达(平均光密度值)(11.58±2.15)较对照组(22.66±1.67)显著下调(P=0.015),而NF-κB、TNF-α表达(31.77±2.69、24.31±2.32)均较对照组(19.40±1.82、11.92±0.94)显著上调(P=0.019,P=0.008)。蛋白质印迹法结果显示:牙周炎组大鼠肝组织中BMAL1蛋白表达[(0.63±0.10)%]较对照组[(1.00±0.06)%]显著下调(t=3.19,P=0.033),NF-κB、TNF-α表达[(1.61±0.12)%、(2.82±0.23)%]均较对照组[(1.00±0.12)%、(1.00±0.11)%]显著上调(P=0.022,P=0.002)。TUNEL染色结果显示牙周炎组大鼠肝组织中凋亡细胞较对照组增多。结论牙周炎可能通过下调大鼠肝组织中BMAL1的表达激活NF-κB信号分子,引起大鼠肝脏组织中炎症水平和凋亡水平升高,最终诱导肝损伤。展开更多
To evaluate the protective effects of Glycyrrhiza polysaccharide（GPS） against 2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD）-induced hepatotoxicity in Jian carp,the fish were fed diets containing GPS at doses of 0.1,0....To evaluate the protective effects of Glycyrrhiza polysaccharide（GPS） against 2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD）-induced hepatotoxicity in Jian carp,the fish were fed diets containing GPS at doses of 0.1,0.5 and 1.0 g/kg for 60 days before an intraperitoneal injection of 0.6 μg/kg TCDD at a volume of 0.05 mL/10 g body weight.At 72 hr post-injection,blood and liver samples were taken for biochemical analysis and the fish liver samples were used for the preparation of pathological slices.The results showed that increases in alanine aminotransferase（GPT）,aspartate aminotransferase（GOT）,lactate dehydrogenase（LDH）,and alkaline phosphatase（AKP） in serum induced by TCDD were significantly inhibited by pre-treatment with 1.0 g/kg GPS.Following the 1.0 g/kg GPS pre-treatment,total protein（TP）,albumin（Alb）,catalase（CAT）,glutathione peroxidase（GPx）,total antioxidant capacity（T-AOC） and superoxide dismutase（SOD） activities in liver tissue increased significantly,malondialdehyde（MDA） formation（P ＆lt; 0.05 or P ＆lt; 0.01） was significantly inhibited,and the expression of cytochrome P4501A（CYP1A）,aryl hydrocarbon receptor 2（AHR2） and aryl hydrocarbon receptor nuclear translocator 2（ARNT2） mRNA（P ＆lt; 0.05） was significantly enhanced.Histological observations on fish liver were obtained by preparing paraffin tissue sections via HE staining,and the results showed that histological changes were obviously reduced by 0.5 and 1.0 g/kg GPS.GPS significantly reduced liver tissue damage caused by TCDD.Overall,these results proved the hepatoprotective effect of GPS in protecting against fish liver injury induced by TCDD,and supported the use of GPS（1.0 g/kg） as a hepatoprotective and antioxidant agent in fish.展开更多
基金supported by National Natural Science Foundation of China(81402943)CAMS Major Collaborative Innovation Project(2016-I2M-1-011)PUMC Youth Fund(3332015168)
文摘OBJECTIVE To evaluate whether the IDO1 inhibitor 1-methyl-L-tryptophan(1-MT)combine calcium influx inhibitor carboxyamidotriazole(CAI)could further enhance the suppression of programmed death 1(PD-1)in CD8^+T cells and investigate the curative effect of the combined use.METHODS CD8^+T cells were isolated from normal mice spleen by negative selection using magnetic cell separation.The isolated CD8^+T cells were cultured in RPMI 1640 medium containing 10%FBS and 100 U·mL^(-1)IL-2 and activated by the addition of anti-CD3 and anti-CD28(1 g·L^(-1) each mabs).CD8^+T cells were pretreated for 48 h with drug and the fluo-3 as a marker of intracellular calcium concentration was detected by flow cytometry.The calcineurin(Ca N)levels were assayed with ELISA in CD8^+T cells after 48 h incubation with 10μm CAI.The nuclear translocations of NFAT and AHR were detected by immunofluorescent staining after 48 h of drug treatment.The expression of PD-1 in CD8^+T cells was analyzed by flow cytometry.RESULTS Intracellular fluorescent intensity was markedly debase due to CAI treatment(P<0.01).Meanwhile,the changes of CaN content had a resembled correlation(P<0.01).Immunofluorescence experiment showed that after combination therapy the transfer of NFAT and AHR in nuclear substantially reduced.Flow cytometry revealed that after the combination caused a significant decrease in PD-1 expression in CD8^+T cells.CONCLUSION CAI and 1-MT could inhibit markedly the expression of PD-1 in CD8^+T cells by inhibiting the nuclear translocation of NFAT and AHR,respectively and the combination of them has synergetic effect.
文摘目的探讨脑和肌肉芳香烃受体核转运样蛋白1(brain and muscle ARNT-like protein 1,BMAL1)在大鼠牙周炎诱导肝损伤模型中的作用。方法根据随机数字表法将12只Wistar雄性大鼠随机分为对照组和牙周炎组,每组6只。对照组大鼠不做处理。牙周炎组大鼠通过结扎双侧上颌第一磨牙颈部建立牙周炎模型。建模8周后检测两组大鼠牙周临床指标并处死。显微CT(micro-CT)扫描大鼠上颌骨并分析牙槽骨吸收情况。HE及油红O染色分析两组大鼠牙周组织和肝组织的病理变化。生化试剂盒检测血清中谷草转氨酶(glutamic-oxaloacetic transaminase,GOT)、谷丙转氨酶(glutamic-pyruvic transaminase,GPT)、总胆固醇(total cholesterol,TC)和甘油三酯(triglyceride,TG)等肝功能相关指标。实时荧光定量PCR(real time fluorescent quantitative-PCR,qRT-PCR)、免疫组化和蛋白质印迹法检测肝组织中BMAL1、核因子-κB(nuclear factor kappa-B,NF-κB)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)基因及蛋白的表达水平。原位末端转移酶标记(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL)试剂盒染色法检测肝组织中的细胞凋亡。结果上颌第一磨牙HE染色和micro-CT结果显示,牙周炎组大鼠牙槽骨吸收明显。肝组织病理学结果显示,与对照组相比,牙周炎组肝组织内炎症细胞浸润、肝索结构紊乱且肝细胞中可见大量脂滴形成。大鼠血清生化检测结果显示,牙周炎组大鼠血清中GOT[(62.77±2.59)U/L]、GPT[(47.54±1.04)U/L]、TC[(3.19±0.23)mmol/L]和TG[(1.11±0.09)mmol/L]均较对照组GOT[(38.66±2.47)U/L]、GPT[(31.48±1.57)U/L]、TC[(1.60±0.05)mmol/L]和TG[(0.61±0.09)mmol/L]含量显著升高(P=0.003,P=0.001,P=0.002,P=0.038)。qRT-PCR结果显示,牙周炎组大鼠肝组织中BMAL1 mRNA的表达[(0.60±0.04)%]较对照组[(1.01±0.07)%]显著下调(t=4.80,P=0.009),NF-κB、TNF-αmRNA的表达[(1.62±0.12)%、(2.69±0.16)%]均较对照组[(1.00±0.03)%、(1.03±0.16)%]显著上调(P=0.008,P=0.002)。免疫组化结果显示:牙周炎组大鼠肝组织中BMAL1蛋白表达(平均光密度值)(11.58±2.15)较对照组(22.66±1.67)显著下调(P=0.015),而NF-κB、TNF-α表达(31.77±2.69、24.31±2.32)均较对照组(19.40±1.82、11.92±0.94)显著上调(P=0.019,P=0.008)。蛋白质印迹法结果显示:牙周炎组大鼠肝组织中BMAL1蛋白表达[(0.63±0.10)%]较对照组[(1.00±0.06)%]显著下调(t=3.19,P=0.033),NF-κB、TNF-α表达[(1.61±0.12)%、(2.82±0.23)%]均较对照组[(1.00±0.12)%、(1.00±0.11)%]显著上调(P=0.022,P=0.002)。TUNEL染色结果显示牙周炎组大鼠肝组织中凋亡细胞较对照组增多。结论牙周炎可能通过下调大鼠肝组织中BMAL1的表达激活NF-κB信号分子,引起大鼠肝脏组织中炎症水平和凋亡水平升高,最终诱导肝损伤。
基金supported by the National Natural Science Foundation of China(Nos.31202002,31200918)Jiangsu Science and Technology Department(No.BK2012535)the Central Public-Interest Scientific Institution Basal Research Fund(No.2014A08YQ01)
文摘To evaluate the protective effects of Glycyrrhiza polysaccharide（GPS） against 2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD）-induced hepatotoxicity in Jian carp,the fish were fed diets containing GPS at doses of 0.1,0.5 and 1.0 g/kg for 60 days before an intraperitoneal injection of 0.6 μg/kg TCDD at a volume of 0.05 mL/10 g body weight.At 72 hr post-injection,blood and liver samples were taken for biochemical analysis and the fish liver samples were used for the preparation of pathological slices.The results showed that increases in alanine aminotransferase（GPT）,aspartate aminotransferase（GOT）,lactate dehydrogenase（LDH）,and alkaline phosphatase（AKP） in serum induced by TCDD were significantly inhibited by pre-treatment with 1.0 g/kg GPS.Following the 1.0 g/kg GPS pre-treatment,total protein（TP）,albumin（Alb）,catalase（CAT）,glutathione peroxidase（GPx）,total antioxidant capacity（T-AOC） and superoxide dismutase（SOD） activities in liver tissue increased significantly,malondialdehyde（MDA） formation（P ＆lt; 0.05 or P ＆lt; 0.01） was significantly inhibited,and the expression of cytochrome P4501A（CYP1A）,aryl hydrocarbon receptor 2（AHR2） and aryl hydrocarbon receptor nuclear translocator 2（ARNT2） mRNA（P ＆lt; 0.05） was significantly enhanced.Histological observations on fish liver were obtained by preparing paraffin tissue sections via HE staining,and the results showed that histological changes were obviously reduced by 0.5 and 1.0 g/kg GPS.GPS significantly reduced liver tissue damage caused by TCDD.Overall,these results proved the hepatoprotective effect of GPS in protecting against fish liver injury induced by TCDD,and supported the use of GPS（1.0 g/kg） as a hepatoprotective and antioxidant agent in fish.
