叔丁基对苯二酚对亚砷酸钠致Chang liver细胞毒性的影响

目的 探讨叔丁基对苯二酚(tert-butylhydroquinone,tBHQ)对亚砷酸钠(NaAsO_2)致Chang liver细胞毒性的影响.方法 Chang liver细胞培养48 h后分别以20、40、60和80 μmo/L的NaAsO_2染毒24和48 h,作为NaAsO_2单独作用组.以5和20μmol/L的tBHQ预处理Chang liver细胞24h,以40和60μmol/L的NaAsO_2染毒24和48h,作为tBHQ预处理组;对照组处理同NaAsO_2:单独作用组.每个浓度设3个复孔.用Alamar Blue法检测细胞活力.结果 NaAsO_2单独作用24和48 h组Alamar Blue还原率显著下降,与对照组比较,差异有统计学意义(P<0.05);且NaAsO_2单独作用48 h组的Alamar Blue还原率均显著低于24 h组,差异有统计学意义(P＜0.01).5 μmol/L的tBHQ预处理组与对应的60 μmol/L NaAsO_2单独作用24h组相比,显著提高了Alamar Blue还原率(P＜0.01);20 μmol/L的tBHQ预处理组的Alamar Blue还原率均显著高于相对应NaAsO_2单独作用24 h组(P＜0.05).5 μmol/L的tBHQ预处理组的Alaraar Blue还原率均显著高于相对应NaAsO_2单独作用48h组(P＜0.01);20μmol/L的tBHQ预处理组与对应的40μmol/L NaAsO_2单独作用48h组相比,显著提高了Alamar Blue还原率(P<0.01).结论 tBHQ能够降低NaAsO_2致Chang liver细胞的毒性,增强细胞对NaAsO_2毒性的抵抗能力.

Abstract：

Objective To study the antagonism of text-butylhydroquinone (tBHQ)to NaAsO_2 induced cytotoxicity in Chang liver cells in vitro.Methods Chang liver cells were exposed to NaAsO_2(0,20,40,60 and 80μmol/L)for 24 and 48 hours,or Chang liver cells were treated with tBHQ(5 and 20 μmol/L),then exposed to NaAsO_2(40 and 60 μmol/L)for 24 and 48 hours.The conditions of control group was the same as NaAsO_2 group.Alamar Blue was used to evaluate the viabifity of cells.Results Chang liver cells were exposed to NaAsO_2 for 24 and 48 hours,Alamar Blue reduction rates decreased significantly and Alamar Blue reduction rates of 48 hours group were lower than 24 hours group (P<0.01).Alamar Blue reduction rate of 5 μmol/L tBHQ pretreatment group was higher than 60 μmol/L NaAsO_2 group for 24 h(P<0.01);Alamar Blue reduction rate of 20μmol/L tBHQ pretreatment group were higher than respective NaAsO_2 group for 24 h(P＜0.05).Alamar Blue reduction rate of 5 μmol/L tBHQ pretreatment group were higher than respective NaAsO_2 group for 48 h(P＜0.01);Alamar Blue reduction rate of 20 μmol/L tBHQ pretreatment group was higher than 40 μmol/L NaAsO_2 group for 48 h(P<0.01).Conclusion tBHQ can decrease the cytotoxieity induced by NaAsO_2 in Chang liver cells,increase the resistance to the cytotoxieity induced by NaAsO_2 in Chang liver cells.     

叶酸对亚砷酸钠致Chang肝细胞毒性和氧化应激的影响

目的 观察不同浓度叶酸对无机砷所致肝细胞毒性和氧化应激状态的影响.方法 分别以无叶酸(0μmol/L)、正常叶酸(2.3 μmol/L)、高叶酸(10 μmol/L)培养液和20 μmol/L亚砷酸钠(NaAsO_2)对Chang肝细胞进行处理.染砷24 h后,以3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测细胞活力,以流式细胞术Annexin V/PI双染法检测细胞凋亡情况,以2',7'-二乙酰二氯荧光素(DCFH-DA)测定细胞内活性氧(ROS)水平,分别用DTNB法和硫代巴比妥酸比色法测定细胞内还原型谷胱甘肽(GSH)和丙二醛(MDA)含量.结果 NaAsO_2所致细胞活力降低和细胞内ROS、MDA相对水平的升高在无叶酸培养条件下显著加剧(P<0.05),在高叶酸条件下显著减轻(P<0.05);NaAsO_2诱导的细胞内GSH相对含量升高在无叶酸条件下显著减弱(P<0.05),在高叶酸环境下显著增强(P<0.05);NaAsO_2诱导的凋亡细胞在无叶酸条件下显著增多(P<0.05).结论 叶酸可改善无机砷所致氧化应激,减轻无机砷的肝细胞毒性.

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Objective To observe the effects of different folate levels on inorganic arsenic-induced cytotoxicity and oxidative stress status. Methods Chang human hepatocytes were cultured with 20 μmol/L sodium arsenite (NaAsO_2) in medium containing 0 μmol/L, 2.3 μmol/L and 10 μmol/L of folate, respectively. After 24 h of treatment with NaAsO_2, cell viability was analyzed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, apoptosis was assessed by Annexin V/PI double staining, levels of ROS were detected by 2' ,7' -dichlorodihydrofluorescein diacetate (DCFH-DA) probe, and intracellular reduced glutathione (GSH) and malondialdehyde (MDA) content were determined by DTNB method and formation of thiobarbituric acid reactive substances, respectively. Results The decrease of cell viability and the increase of intracellular ROS and MDA induced by NaAsO_2 were significantly exacerbated by folate deficiency but attenuated by folate supplement (P<0.05). Moreover, the NaAsO_2-induced elevation of intracellular GSH was attenuated by folate deficiency but aggravated by folate supplement (P<0.05). Apoptosis cells induced by NaAsO_2 were significantly increased by folate deficiency (P<0.05). Conclusion Folate can attenuate the oxidative stress and cytotoxicity induced by inorganic arsenic in hepatocytes.     

兴奋性氨基酸递质系统在乐果诱导星形胶质细胞活化中的作用

Objective To study the involvement of excitatory amino acid system in astrocytes activation caused by dimethoate. Methods Pure-cultured astrocytes were gained by three passages from primary cultured rat nerve cells, then treated with 10-6,10-5,10-4 mol/L dimethoate for 48 h, 50 μmol/L and 100μmol/L MK801, a NMDA receptor blocker, was used to intervene the effects induced by 10-4 mol/L dimethoate.HPLC-FLD was utilized to measure the concentrations of excitatory amino acid (EAA), RT-PCR was used to detect the expression levels of NR2B, GLT-1, GLAST, GFAP and S100β mRNA, and immunofluresence staining method was applied to measure the expression levels of GFAP and S100β proteins. Results The expression levels of GLAST mRNA in all exposure groups were 67.8% ,68.6% and 76.2% of control level,respectively, which were significantly lower than that of control group (P＜0.05); The concentrations of EAA significantly decreased in 10-4 mol/L dimethoate group, as compared with control group (P＜0.01); the expression levels of GFAP mRNA in 10-4 mol/L dimethoate group, of S100β mRNA in 10-5 mol/L dimethoate group, of GFAP protein in 10-4 mol/L and 10-5 mol/L dimethoate groups and S100β protein in 10-4 mol/L dimethoate group were significantly higher than those in control group (P＜0.01). The expression levels of GLT-1 and GLAST mRNA in 10-4 mol/L dimethoate plus 50 μ mol/L or 100 μ mol/L MK801 groups increased significantly, as compared with 10-4 mol/L dimethoate group (P＜0.01), the expression levels of NR2B mRNA in 10-4 mol/L dimethoate plus 50 μ mol/L or 100 μmol/L MK801 groups increased significantly, as compared with control group (P＜0.05 or P＜0.01); the concentration of Glu in 10-4 mol/L dimethoate plus 100 μ mol/L MK801 group increased significantly, as compared with 10-4 mol/L dimethoate group (P＜0.01); the expression levels of GFAP mRNA and protein in10-4 mol/L dimethoate plus 50 μ mol/L or 100 μ mol/L MK801 groups decreased significantly (P＜0.01); S100β protein expression level in 50 μ mol/L MK801 intervention group was significantly higher than thatl in control group (P＜0.01). Conclusion Excitatory amino acid system involved in astrocytes activation caused by dimethoate. MK801 was useful to control astrocytes gliosis.     

1,4-苯醌暴露下小鼠骨髓细胞p15基因表达和甲基化状态

目的 探讨1,4-苯醌(1,4-BQ)暴露下,体外原代培养的小鼠骨髓细胞中抑癌基因p15的mRNA表达及启动子区的甲基化状态.方法 体外分离小鼠骨髓细胞,测定0、0.1、1.0、5.0、10.0、20.0、40.0 μmol/L的1,4-BQ对小鼠骨髓细胞活力的影响,最终确定以0、0.1、1.0、10.0 μmol/L的1,4-BQ染毒骨髓细胞24 h;实时荧光定量聚合酶链反应(PCR)检测p15基因mRNA的表达变化;重亚硫酸盐测序法(BSP)检测启动子区域CpG岛的甲基化状态.结果 1,4-BQ对小鼠骨髓细胞具有剂量依赖的毒性作用,其LC50为8.3 μmol/L(95%CI:4.6～10.6 μmol/L).与对照组(100.0%)比较,10.0、20.0、40.0μmol/L 1,4-BQ染毒组小鼠的骨髓细胞生存率(35.9%、35.8%、30.5%)均明显降低,差异有统计学意义(P＜0.01).10.0μmol/L染毒组p15 mRNA表达为对照组的43%,与对照组比较,1.0和10.0 μmol/L染毒组p15基因mRNA表达量明显下降,差异有统计学意义(P＜0.05或P＜0.01).BSP测序结果显示,染毒组和对照组相同,p15基因启动子区域CpG岛上56个甲基化位点仍保持非甲基化状态.结论 1,4-BQ暴露可导致原代培养的小鼠骨髓细胞中p15基因的mRNA表达量下降,但启动子区域CpG岛的甲基化状态未受影响,提示甲基化不参与1,4-BQ介导的p15基因表达下调.

Abstract：

Objective To detect the expression and the CpG island methylation status of tumor suppressor gene p15 after exposure to 1,4-benzoquinone (1,4-BQ) in primary cultivated C57BL/6J mouse bone marrow cells in vitro. Methods The mouse bone marrow cells were isolated in vitro. The effect of 0, 0.1, 1, 5,10, 20, and 40 μmol/L 1,4-BQ on cell viability (CKK-8) was detected. 0, 0.1, 1, 10 μmol/L 1,4-BQ were used to intoxicate the mouse bone marrow cells for 24 h; Real-time PCR was employed to analyze the mRNA expression level of p15; The bisulfite sequencing PCR (BSP) was used to look into the methylation status of CpG islands in p15 promoter region. Results 1,4-BQ exhibited dose-dependent toxicity to mouse bone marrow cells, and the LC50 was 8.3 μmol/L (95% CI: 4.6-10.6 μmol/L). The mRNA expression of p15 in 10 μ mol/L group was only equivalent to 43% of control group. Compared with control group, the decrease of p15 mRNA expression in 1 and l0 μ mol/L concentration were obvious, and the differences had statistical significance(P＜0.05 or P＜0.01 ). BSP sequencing results were same between the exposure groups and control group, the 56 CpG sites on CpG islands remained in the state of unmethylated. Conclusion mRNA expression of p15 gene decreases after exposure to 1,4-BQ, but the CpG islands menthylation status in promoter is not affected, suggesting that methvlation does not participate in 1,4-BQ-mediated p15 gene expression decrease, other effect mechanisms still need to be investigated.     

镉对HepG2细胞DNA损伤作用以及对gadd基因表达的影响

目的 探讨氯化镉对HepG2细胞DNA损伤作用以及对gadd153和gadd45β启动子和mRNA表达的影响.方法 应用彗星试验检测氯化镉对HepG2细胞的DNA损伤作用;分别构建含有gadd153和gadd45β启动子和荧光素酶报告基因的载体pGADD153-Luc和pG45-Luc,荧光素酶活性检测反映gadd153和gadd45β启动子的活性,生物发光检测荧光素酶活性;反转录-聚合酶链反应(RT-PCR)检测gadd153和gadd45β基因mRNA的表达.结果 彗星试验结果显示,在100、300 μmol/L氯化镉处理细胞24 h后,Olive尾矩(分别为0.78±0.06、1.10±0.12)明显高于对照组(0.53±0.08),差异有统计学意义(P<0.05),并有良好的剂量-效应关系(r=0.9761,P<0.05);报告基因分析显示,1、5、10 μmol/L氯化镉处理组gadd153启动子表达[分别为(9.45±1.26)、(11.76±1.12)、(21.14±1.47)RIU/μg Pro]均明显高于对照组[(7.02±0.82)RIU/μg Pro]差异均有统计学意义(P<0.05),并有良好的剂量-效应关系(r=0.8755,P<0.05);5、10μmol/L氯化镉处理组gadd45β启动子表达明显高于对照组,差异有统计学意义(P<0.05),并有良好的剂量-效应关系(r=0.8856,P<0.05);RT-PCR结果显示,1、5、10μmol/L氯化镉处理组gadd153 mRNA表达均明显高于对照组,5、10 μmol/L氯化镉处理组gadd45β mRNA表达明显高于对照组,差异有统计学意义(P<0.05).结论 氯化镉可诱导HepG2细胞DNA损伤,较低剂量氯化镉即使未引起明显的DNA损伤,亦可促进HepG2细胞gadd153和gadd45β启动子和mRNA的表达.

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Objective To investigate the effects of the cadmium chloride on the DNA damage and the expression of the gadd153 and gadd45β promoter and mRNA in HepG2 cells.Methods DNA damage induced by cadmium chloride was detected by comet assay.The plasmids (pGADD153-Luc and pG45-Luc ) containing DNA damage and repair inducible gene 153 and 45 (gadd153 and gadd45β) promoter and luciferase and gadd45β reporter gene were constructed.The activity of gadd 153 and gadd45β promoter were represented by the luciferase activity,the inducible luciferase activities was detected by bioluminescence.The expression of gaddl53 and gadd45β mRNA was detected by RT-PCR.Results The results of comet assay indicated that Olive Tail Moment induced by the cadmium chloride increased significantly at the dose of 100,300 μmol/L,compared with the control (P<0.05).The luciferase activity analysis showed that the expression levels of gaddl53 promoter increased significantly in 1,5,10 μmol/L treatment group,compared with the control (P<0.05).The expression levels of gadd45β promoter in 5,10μmol/L treatment group were significantly higher than that in control group (P<0.05).The expression levels of gaddl53 mRNA induced by cadmium chloride at the doses of 1,5,10 μ-mol/L and the expression levels of gadd45β mRNA induced at the doses of 5,10μmol/L were significantly higher than thoae in control group (P<0.05).Conclusion The cadmium chloride can induce the DNA damage and increase the expression levels of the gaddl53 and gadd45β promoters in HepG2 cells.     

脱氧雪腐镰刀菌烯醇对人胚胎膝关节软骨细胞的毒性及作用机制

目的 探讨脱氧雪腐镰刀菌烯醇(DON)对人胚胎膝关节软骨细胞的毒性及作用机制.方法 取原代培养人胚胎膝关节软骨,用DMEM/F12培养基进行培养和传代,在培养基中加入DON,建立以下DON染毒浓度:0.1、0.2、0.4、1.0 μg/ml组和空白对照组.在处理72 h后进行相关研究指标的检测:分别用ELISA法检测培养上清基质金属蛋白酶13(MMP-13)、前列腺素E2(PGE2)含量,硝酸还原酶法检测上清液的一氧化氮(NO)含量,流式细胞术法检测软骨细胞凋亡情况、软骨细胞内诱导型一氧化氮合酶(iNOS)和Ⅱ型胶原表达的情况,RT-PCR法分析iNOS mRNA和Ⅱ型胶原mRNA的表达.结果 DON组凋亡率为6.78%～19.05%,高于对照组的1.20%,凋亡率随DON浓度增高而增加(F=174.761,P<0.05).DON组上清液NO含量为20.8～40.7 μmol/L,高于对照组的10.2 μmol/L(F=91.966,P<0.05);MMP-13含量为0.25～0.56 μmol/L,高于对照组的0 μmol/L(F=78.420,P<0.05);PGE2含量为3.2～20.6 μmol/L,高于对照组的1.6 μmol/L(F=276.453,P<0.05).DON组软骨细胞iNOS表达强度为14.8%～56.8%,高于对照组7.1%(F=214.614,P<0.05).高剂量DON组(0.4 μg/ml和1.0 μg/ml)Ⅱ型胶原表达强度分别为56.7%和52.7%,低于对照组的62.2%(F=5.134,P<0.05).DON组软骨细胞内iNOS mRNA表达的吸光度值(A值)为1.07～1.33,高于对照组的0.62(F=8.358,P<0.05).高剂量DON组(0.4、1.0 μg/ml)软骨细胞内Ⅱ型胶原mRNA表达量分别为0.83和0.82,低于对照组的1.14(F=7.887,P<0.05).结论 DON导致人软骨细胞合成NO增加,并通过NO调节MMP-13和PGE2等促进Ⅱ型胶原等软骨基质的降解和软骨毒性损伤;DON可促进软骨细胞的凋亡.

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Objective This study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol(DON)on articular chondrocytes in human embryo.Methods Articular cartilage cells were isolated from knees of human embryo and cultured in DMEM/F12 medium.The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings: control group and group with DON of 0.1,0.2,0.4,1.0 μg/ml.The effects of DON were observed 72 hours after incubation.Cell apoptosis was assayed by flow cytometry(FCM) with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits;NO was measured by Griess assay with spectrophotometer.Inducible nitric oxide synthase(iNOS) and collagen Ⅱ in cells were detected by FCM.The expression levels of iNOS,mRNA and collagen Ⅱ mRNA were measured with RT-PCR.Results The rates of cell apoptosis in DON groups were 6.78%-19.05%,which were significantly higher than that in control(1.20%,F=174.761,P<0.05).The levels of NO in DON groups were 20.8-40.7 μmol/L,which were significantly higher than that in control(10.2 μmol/L,F=91.966,P<0.05).The levels of MMP-13 in DON groups were 0.25-0.56 μmol/L,which were significantly higher than that in control(0 μmol/L,F=78.420,P<0.05).The levels of PGE2 in DON groups were 3.2-20.6 μmol/L,which were significantly higher than that in control(11.6 μmol/L,F=276.453,P<0.05).The proportions of cells with positive iNOS in DON groups were 14.8%-56.8% which were significantly higher than that in controls (7.1%,F=214.614,P<0.05).The proportions of cells with positive collagen Ⅱ in groups with DON of 0.4 μg/ml and 1.0 μg/ml were 56.7% and 52.7%,which were significantly lower than that in control(62.2%,F=5.134,P<0.05).The relative absorbance values of iNOS mRNA in DON groups were 1.07-1.33,which were significantly higher than that in control(0.62,F=8.358,P<0.05).The levels of collagen Ⅱ mRNA in groups with DON of 0.4 μg/ml and 1.0 μg/ml were 0.83 and 0.82,which were significantly lower than that in control (1.14,F=7.887,P<0.05).Conclusion DON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13,which both promoted resolution of articular cartilage matrix such as collagen Ⅱ.DON induced apoptosis in articular cartilage cells.     

三甲基氯化锡对亚慢性染毒大鼠肾氢钾ATP酶活力及蛋白与基因表达的研究

Objective To study the activity、protein and gene expression of renal HK-ATPase (HKA) in rats subchronicly exposed to trimethyltin chloride (TMT). Methods In subchronic toxic test (14-week), 55 female SD rats (age, 6 weeks) were divided randomly into 5 groups: control, low, medium, high and super high dosage, respectively, which drank water with TMT of 0, 8.20, 32.81, 131.25 and 262.50 μg·kg-1 ·d-1 for 14 weeks.Then serum K+ levels were measured; the activities of H K-ATPase (HKA) in kidneys were detected by the method of determinenate phosphorus content;Western Blot assay and real-time PCR were used to exam the protein and mRNA expression levels of HKA in kidneys, respectively. Results The serum K + level in super-high dosage group was (5.6±0.4) mmol/L, which was significantly lower than that [(6.9±0.3) mmol/L] in control group (P＜0.01).The HKA enzymatic activity of kidneys in low and super high dosage groups was 4.50±1.45 and 4.55±0.72 μmolPi·mg prot-1h-1, respectively, which were significantly lower than that (6.55±0.77 μmol Pi ·mg prot-1h-1) in control group (P＜0.05). Conclusion When rats were exposed subchronicly to TMT, the renal HKA activity could reduce, but the expression levels of HKA protein and mRNA did not decrease.     

叔丁基对苯二酚对百草枯神经细胞毒性和氧化应激的拮抗作用

Objective To investigate the protective effects of the tert-butylhydroquinone (tBHQ)pretreatment on neurotoxicity and oxidative stress induced by paraquat (PQ) in PC12 cells. Methods Cytoyoxicity of PC12 cells was measured by MTT assay, following the PC12 cells treatment with different concentrations of 100, 300 μmol/L PQ for 24 h and 48 h. PC12 cells were pretreated with or without 40 μmol/L tBHQ for 4 h, PC 12 cells were exposed to PQ at the doses of 0, 100, 300 μmol/L for 24 h and 48 h, respectively.The viability of PC 12 cells was measured by MTT assay, the apoptosis rates of PC 12 cells were detected by flow cytometry (FCM) and the malondialdehyde (MDA) levels of PC 12 cells were examine by thiobarbituric acid (TBA) method. Results When the exposure doses of PQ were 100 and 300 μmol/L for 24 h, the viability of PC 12 cells pretreated with tBHQ was significantly higher than that of PC 12 cells only exposed to PQ (P＜0.05 or P＜0.01). When the exposure dose of PQ was 100μmol/L for 48 h, the viability of PC12 cells pretreated with tBHQ was significantly higher than that of PC12 cells only exposed to PQ (P＜0.01). When the exposure doses of PQ were 100 and 300 μmol/L for 24 h, the apoptosis rates and MDA levels of PC12 cells pretreated with tBHQ were significantly lower than those of PC12 cells only exposed to PQ (P＜0.05 or P＜0.01). Conclusions tBHQ preteatment can reduce the cytotoxicity, apoptosis and oxidative stress induced by PQ in PC12 cells.     

兴奋性氨基酸递质系统在乐果诱导星形胶质细胞活化中的作用

目的 探讨兴奋性氨基酸递质系统在乐果染毒后皮层星形胶质细胞活化中的作用.方法 新生大鼠皮层神经细胞传代3次后获得纯化的星形胶质细胞,分别加入终浓度为10-6、10-5、10-4mol/[的乐果,并用50和100 μmol/LN-甲基-N-天门冬氨酸(NMDA)受体非竞争性拮抗剂MK801对10-4mol/L乐果染毒组进行干预.染毒后48 h收获细胞,高效液相色谱-荧光检测系统(HPLC-FLD)方法测定细胞内兴奋性氨基酸递质含量,反转录聚合酶链反应(RT-PCR)法检测NMDA受体NR2B亚基、谷氨酸(Glu)、谷氨酸转运体(GLT-1)、天冬氨酸(Asp)、谷氨酸/天冬氨酸转运体(GLAST)、胶质纤维酸性蛋白(GFAP)及S100β mRNA表达的变化,免疫荧光染色半定量检测GFAP以及S100β的蛋白表达.结果 各剂量染毒组GLASTmRNA表达下降为对照组的67.8%、68.6%和76.2%,差异有统计学意义(P＜0.05);10-4 mol/L染毒组Glu、Asp含量与对照组相比明显下降,差异有统计学意义(P＜0.01);与对照组比较,10-4mol/L染毒组GFAP和10-5mol/L染毒组S100βmRNA表达,10-5、10-4mol/L染毒组GFAP蛋白表达,10-4mol/L染毒组S100β蛋白表达明显上升,差异有统计学意义(P＜0.01),有剂量依赖趋势.对10-4mol/L染毒组给予50和100 μmol/L MK801干预后,GLT-1、GLAST mRNA表达水平较10-4 mol/L染毒组明显上升,差异有统计学意义(P＜0.01),NR2B mRNA表达进一步升高,与未干预前相比,差异无统计学意义(P＞0.05),但均明显高于对照组水平,差异有统计学意义(P＜0.05,P＜0.01);100 μmol/L MK801干预后,Glu的含量升高为10-4mol/L染毒组的1.81倍,差异有统计学意义(P＜0.01);50和100μmol/LMK801干预后,GFAP转录及蛋白水平较未干预前明显下降,差异有统计学意义(P＜0.01),50 μmol/L干预组S100β蛋白表达水平仍然高于对照组,差异有统计学意义(P＜0.01).结论 乐果对兴奋性氨基酸递质系统的影响参与了星形胶质细胞的活化;NMDA受体阻断剂MK801有助于控制星形胶质细胞胶质化.

Abstract：

Objective To study the involvement of excitatory amino acid system in astrocytes activation caused by dimethoate. Methods Pure-cultured astrocytes were gained by three passages from primary cultured rat nerve cells, then treated with 10-6,10-5,10-4 mol/L dimethoate for 48 h, 50 μmol/L and 100μmol/L MK801, a NMDA receptor blocker, was used to intervene the effects induced by 10-4 mol/L dimethoate.HPLC-FLD was utilized to measure the concentrations of excitatory amino acid (EAA), RT-PCR was used to detect the expression levels of NR2B, GLT-1, GLAST, GFAP and S100β mRNA, and immunofluresence staining method was applied to measure the expression levels of GFAP and S100β proteins. Results The expression levels of GLAST mRNA in all exposure groups were 67.8% ,68.6% and 76.2% of control level,respectively, which were significantly lower than that of control group (P＜0.05); The concentrations of EAA significantly decreased in 10-4 mol/L dimethoate group, as compared with control group (P＜0.01); the expression levels of GFAP mRNA in 10-4 mol/L dimethoate group, of S100β mRNA in 10-5 mol/L dimethoate group, of GFAP protein in 10-4 mol/L and 10-5 mol/L dimethoate groups and S100β protein in 10-4 mol/L dimethoate group were significantly higher than those in control group (P＜0.01). The expression levels of GLT-1 and GLAST mRNA in 10-4 mol/L dimethoate plus 50 μ mol/L or 100 μ mol/L MK801 groups increased significantly, as compared with 10-4 mol/L dimethoate group (P＜0.01), the expression levels of NR2B mRNA in 10-4 mol/L dimethoate plus 50 μ mol/L or 100 μmol/L MK801 groups increased significantly, as compared with control group (P＜0.05 or P＜0.01); the concentration of Glu in 10-4 mol/L dimethoate plus 100 μ mol/L MK801 group increased significantly, as compared with 10-4 mol/L dimethoate group (P＜0.01); the expression levels of GFAP mRNA and protein in10-4 mol/L dimethoate plus 50 μ mol/L or 100 μ mol/L MK801 groups decreased significantly (P＜0.01); S100β protein expression level in 50 μ mol/L MK801 intervention group was significantly higher than thatl in control group (P＜0.01). Conclusion Excitatory amino acid system involved in astrocytes activation caused by dimethoate. MK801 was useful to control astrocytes gliosis.     

亚砷酸钠联合丁基硫堇亚胺致肝细胞毒性作用

目的研究亚砷酸钠(sodium arsenite,NaAsO2)单独、以及与丁基硫堇亚胺(buthionine sulfoximine,BSO)联合对Chang liver细胞毒性作用。方法常规培养的Chang liver细胞用NaAsO2单独或NaAsO2和BSO联合染毒24h,倒置相差显微镜采集细胞图像;四甲基偶氮噻唑蓝(MTT)法检测细胞活力。结果NaAsO2(0~250μmol/L)明显改变Chang liver细胞的形态并明显降低细胞生存率(P0.01),且呈剂量-反应关系;NaAsO2(5,20μmol/L)和BOS(1 mmol/L)联合作用,其细胞生存率明显低于相应浓度的NaAsO2单独作用组(P0.01)。结论NaAsO2具有明显的细胞毒性;NaAsO2联合BSO能够加重NaAsO2对Chang liver细胞的毒性。     

无机砷对肝细胞转录因子Nrf2及其调控蛋白表达影响

目的探讨不同时间和不同浓度亚砷酸钠(NaAsO2)暴露对Chang肝细胞株NF-E2相关因子2(Nrf2)、及其调控的下游抗氧化酶NAD(P)H:醌氧化还原酶1(NQO1)和血红素单加氧酶-1(HO-1)蛋白表达的影响。方法25μmol/L NaAsO2作用Chang肝细胞2、4、6、12和24 h;或不同浓度的NaAsO2(10、25和50μmol/L)作用Chang肝细胞6 h,免疫印迹法(western blot)检测细胞内Nrf2、NQO1和HO-1的蛋白表达情况。结果 25μmol/L的NaAsO2可以明显诱导Chang肝细胞Nrf2、NQO1和HO-1的蛋白表达(P<0.01);Nrf2蛋白2 h开始明显诱导,4 h表达水平最高,此后随暴露时间的延长虽然表达有所下降,但持续至24 h仍明显高于对照组;NQO1的蛋白表达水平也从4 h开始增加并持续至24 h;HO-1的蛋白表达则从6 h开始明显诱导,且随暴露时间的继续延长表达持续上升,具有明显的时间-效应关系;10、25和50μmol/L NaAsO2染毒6 h,Nrf2、NQO1和HO-1的蛋白表达均随染毒剂量的增加而大量诱导,具有明显的剂量-效应关系(P<0.01)。结论 NaAsO2暴露能够诱导Chang肝细胞Nrf2、NQO1和HO-1蛋白表达增强,且具有一定的剂量-效应关系。     

无机砷对Chang肝细胞核转录因子Nrf2及其下游抗氧化酶mRNA表达的影响

目的观察亚砷酸钠(NaAsO2)对Chang肝细胞株核转录因子Nrf2及Nrf2调控的下游抗氧化酶醌氧化还原酶1(NQO1)和血红素单加氧酶-1(HO-1)mRNA表达的影响。方法分别以不同浓度NaAsO2(5、10、25和50μmol/L)染毒人类Chang肝细胞株6h,采用RT-PCR法测定Nrf2、NQO1和HO-1的mRNA表达水平。结果 5、10、25和50μmol/L NaAsO2暴露6h,Nrf2的mRNA表达分别为对照组的(100.74±3.70)%、(105.96±1.75)%、(101.76±1.01)%和(101.81±6.33)%,与对照组比较,差异未见统计学意义(P>0.05);各暴露组NQO1的mRNA表达均较对照组相比显著增加(P<0.05),分别为对照组的(106.52±3.11)%、(113.27±2.84)%、(111.96±6.96)%和(107.33±2.76)%;NaAsO2暴露还能够显著诱导HO-1的mRNA表达增强,且具有剂量-效应关系,差异具有统计学意义(P<0.01)。5、10、25和50μmol/L NaAsO2暴露,HO-1的mRNA表达分别为对照组的(186.92±1.75)%、(194.08±2.75)%、(196.93±2.55)%和(200.02±0.83)%。结论无机砷暴露未显著增强Chang肝细胞核转录因子Nrf2的转录,却能够显著提高Nrf2调控的下游抗氧化酶NQO1和HO-1的mRNA表达水平。     

无机砷对Chang肝细胞株血红素单加氧酶-1 mRNA和蛋白表达的诱导作用

[目的]观察亚砷酸钠（sodium arsenite,NaAsO2）对Chang肝细胞株血红素单加氧酶-1（heme oxygenase-1,HO-1）mRNA和蛋白表达的诱导作用。[方法]以5μmol／L和10μmol／L的NaAsO2作用Chang肝细胞株2、6、12h和24h,分别用逆转录-聚合酶链反应（RT-PCR）和Western Blot法检测细胞内HO-1的mRNA和蛋白表达情况。[结果]5μmol／L和10μmol／LNaAs02暴露2h开始出现HO-1mRNA的诱导表达;6h的表达水平明显高于对照组和2h暴露组（P〈0．01）。其中,10μmol／LNaAsO2暴露12h和24h的mRNA表达均明显高于该浓度的6h暴露组（P〈0．01）;但24h的HO-1 mRNA表达水平与12h组相比没有明显升高。NaAsO2暴露诱导的HO-1蛋白表达则从6h开始出现,12h组明显高于6h组,24h组明显高于12h组（均P〈0．01）;5μmol／L和10μmol／L NaAsO2分别暴露6、12h和24h的HO-1蛋白表达量分别是对照组的2．80、9．34、18．15和3．97、12．92、23．29倍;此外,10μmol/LNaAsO2暴露12h和24h的HO-1 mRNA和蛋白表达均明显高于对应时间的5μmol／L组（P〈0．01）。[结论]NaAsO2暴露能够有效和持续性诱导Chang肝细胞株HO-1的mRNA和蛋白表达,是无机砷暴露的一种细胞保护性适应性反应。     

不同形态砷化合物对非致瘤性人源性肝细胞的毒性作用

目的探讨不同形态砷化合物对非致瘤性人源性肝(QSG7701)细胞的毒性及氧化应激作用。方法将处于对数生长期的QSG7701细胞分别暴露于亚砷酸钠(砷浓度为1、5、25、100μmol/L)、砷酸钠(砷浓度为10、25、100、500μmol/L)及MMA和DMA(砷浓度均为100、500、1 000、2 000μmol/L)培养24 h,并设对照(含10%胎牛血清的RPMI-1640培养基)。采用四甲基偶氮唑盐(MTT)法测定细胞活性;检测细胞培养液中乳酸脱氢酶(LDH)释放率、谷草转氨酶(AST)活力、总超氧化物歧化酶(T-SOD)活力和丙二醛(MDA)含量。结果一定浓度的As~(Ⅲ)(≥1μmol/L)、As~Ⅴ(≥10μmol/L)、MMA(≥100μmol/L)和DMA(≥2 000μmol/L)均能显著降低QSG7701细胞的存活率,差异有统计学意义(P0.05);且随着As~(Ⅲ)、As~Ⅴ及MMA和DMA染毒浓度的升高,QSG7701细胞的存活率呈逐渐降低的趋势。经计算,QSG7701细胞暴露于As~(Ⅲ)、As~Ⅴ、MMA和DMA 24 h的IC_(50)分别为170.89、863.73、2 235.67、4 045.31μmol/L,对QSG7701细胞生长的抑制作用依次为As~(Ⅲ)As~ⅤMMADMA。在当As~(Ⅲ)浓度为5~100μmol/L、As~Ⅴ浓度为10~500μmol/L及MMA中的砷浓度为1 000、2 000μmol/L和DMA中的砷浓度为2 000μmol/L时,QSG7701细胞的LDH释放率均高于对照组,差异有统计学意义(P0.05);另外,当As~(Ⅲ)浓度为1~100μmol/L、As~Ⅴ浓度为10~500μmol/L及MMA和DMA中的砷浓度为100~2 000μmol/L时,QSG7701细胞培养液中的AST活力均高于对照组;且随着As~(Ⅲ)、As~Ⅴ及MMA和DMA染毒浓度的升高,QSG7701细胞的LDH释放率和细胞培养液中的AST活力呈上升的趋势。与对照组比较,当As~(Ⅲ)浓度为5~100μmol/L、As~Ⅴ浓度为10~500μmol/L时,QSG7701细胞中的MDA含量均较高,而T-SOD活力均较低;与对照组比较,当MMA和DMA中的砷浓度均为500~2 000μmol/L时,QSG7701细胞中的MDA含量均较高;而当MMA中的砷浓度为100~2 000μmol/L和DMA中的砷浓度为100、500μmol/L时,QSG7701细胞中的T-SOD活力均较低,差异有统计学意义(P0.05)。结论在本实验条件下,4种砷化合物均可不同程度地损伤肝细胞膜,破坏肝细胞的氧化平衡状态,产生氧化应激并导致脂质过氧化,对人肝细胞QSG7701的毒性作用依次为As~(Ⅲ)As~ⅤMMADMA。     

亚砷酸钠致SV-HUC-1细胞氧化应激作用的研究

目的 探讨亚砷酸钠(NaAsO_2)对人膀胱上皮永生化细胞SV-HUC-1的氧化应激作用.方法 以不同浓度NaAsO_2(0、0.1、0.2、0.5、1、2、4、6、8、10、20 μmol/L)对SV-HUC-1细胞进行染毒,采用四甲基偶氮唑(MTT)比色法检测细胞活力,利用2',7'-二乙酰二氯荧光素(DCFH-DA)检测细胞内活性氧(ROS)水平,分别应用DTNB比色法、硫代巴比妥酸比色法和黄嘌呤氧化法检测细胞内谷胱甘肽(GSH)含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)活力.结果 与空白对照组比较,全部染毒组细胞活力均下降(P<0.05);染毒24 h后,全部染毒组ROS水平均显著升高(P<0.05);2、4 μmol/LNaAsO_2组细胞GSH含量显著升高(P<0.05);全部染毒组细胞MDA含量均无变化(P>0.05);全部染毒组细胞SOD活力均显著降低(P<0.05).结论 氧化应激可能是NaAsO_2致SV-HUC-1细胞毒性作用的机制之一.