【作者】 邹云锋；

【导师】 陈学敏；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2007， 博士

【摘要】 硒是一种具有重要生物功能的必需微量元素,在生命活动中发挥着重要作用。硒在环境中的分布存在有明显的地域差异,一般热带以及亚热带含硒较高,而温带以及森林或草原地带含硒较低。国外有研究表明,一些肿瘤如胃癌、食道癌、直肠癌等的发病率和硒的地理分布呈负相关,低硒地区肿瘤的发病率和死亡率比较高。国内也有研究通过分析不同肝癌高发区的粮食硒的水平后,发现硒的分布有明显的地区差异,并且与肝癌的流行负相关。一些流行病学调查和动物实验也表明高浓度的硒具有化学防癌作用。硒的抗肿瘤作用的机制有不少报道,但是总的来说,其防癌、抗癌作用机理目前还不是十分清楚。认为可能主要有以下几个方面:1.抑制肿瘤细胞增殖,促进肿瘤细胞凋亡。各种形式的硒均可以抑制CDC2+和PKC(蛋白激酶C)的活性,从而抑制细胞增殖;硒可以和还原性谷胱甘肽(GSH)反应产生活性氧(ROS),启动一些凋亡信号途径而促使肿瘤细胞凋亡。2.降低一些诱癌因子的诱变性。硒可以降低某些能激活致癌原的羟化酶如芳基羟化酶的活性;硒还可以和一些致癌性的金属离子相互作用,从而拮抗它们的活性。3.调节机体免疫系统,增强人体免疫系统的抗癌能力。4.调节一些生物体内抗氧化酶如谷胱甘肽过氧化酶的活性,防止脂质过氧化,保护生物膜不受损伤,防止突变。另一方面,硒的生理需要量范围比较窄,有人认为硒的摄入量超过生理需要量10倍就可能达到中毒阈剂量水平,30～50倍可导致中毒。目前有关硒化合物的毒性作用机制也不是十分清楚,主要认为可能有以下两个方面:1.硒化合物的毒性和其催化产生的ROS有关。硒在较高浓度下产生ROS,它们可以和一些生物大分子包括蛋白质、DNA、脂质发生反应,从而使其受损。2.硒可使体内的一些代谢酶如琥珀酸脱氢酶等失活,从而使机体受损。总而言之,无论是硒的抗肿瘤作用还是硒的毒性作用可能都和ROS有关。ROS和一些信号转导途径也密切相关,无论是内源性还是外源性的ROS都可以调节丝裂原活化蛋白激酶(MAPKs)的活性。不同的信号途径通过MAPKs调节细胞生理过程的很多方面,包括细胞增殖、分化和凋亡。本研究用硒处理HepG2细胞以及给Wistar大鼠灌胃后,利用彗星实验、流式细胞术、Western-blot等方法观察细胞的DNA损伤和凋亡情况以及ROS和MAPK的表达水平,探讨硒染毒产生ROS在MAPKs信号转导通路中的调节作用。本研究共分两部分。第一部分硒诱导HepG2细胞凋亡的作用机制的研究首先为了了解不同浓度硒对细胞活性的影响,本研究应用MTT试验检测了不同剂量(0、2.5、5、10、20μmol/L)处理HepG2 (12、24、48 h)不同时间后的细胞活性。结果表明随着浓度以及作用时间的增加,亚硒酸钠对HepG2的抑制增殖作用越明显。10μmol/L亚硒酸钠作用12 h和5μmol/L亚硒酸钠作用24 h后细胞活性与对照组比较明显降低,差异有显著性(P<0.05)。10、20μmol/L的亚硒酸钠处理细胞后,在三个观察时间点(12、24、48 h)都可见细胞活性明显降低。为了了解硒处理细胞后ROS的变化情况,本研究用流式细胞仪测定不同浓度硒(0、2.5、5、10、20μmol/L)处理细胞不同时间(0.5、1、2 h)后细胞内ROS的水平。ROS的测定是采用DCFH-DA,它本身不发荧光,进入细胞内后水解生成非荧光的DCFH,在ROS的存在下氧化成发荧光的DCF。结果显示5、10、20μmol/L亚硒酸钠作用于HepG2 lh,随着浓度增加,产生的ROS也增加。与对照组相比,当亚硒酸钠浓度≥5μmol/L时,流式峰明显右移,平均荧光强度增加(P<0.05)。10μmol/L亚硒酸钠处理HepG2细胞0.5 h后,即可以见到到荧光强度明显增加(P<0.05)。为了阐明不同浓度硒处理细胞后其DNA损伤以及凋亡情况,我们通过彗星试验(单细胞琼脂糖凝胶电泳)、流式细胞术来检测细胞损伤以及凋亡情况。结果发现0、2.5、5、10、20μmol/L亚硒酸钠作用HepG2 24小时后,当亚硒酸钠浓度≥5μmol/L时,Olive尾矩明显增加,DNA损伤加重,与对照组相比有统计学意义(P<0.05)。流式细胞术检测结果表明0、2.5、5、10、20μmol/L亚硒酸钠处理HepG2 24 h后, 5、10、20μmol/L组早期凋亡细胞以及晚期凋亡细胞/坏死细胞均比对照组高((P<0.05)。随亚硒酸钠剂量从0μmol/L增加到20μmol/L,早期凋亡率和晚期凋亡/坏死细胞率分别从1.11%、2.60%增加到16.6%、10.15%。10μmol/L亚硒酸钠处理HepG2 0、12、24、48 h后,早期凋亡和晚期凋亡/坏死细胞分别从1.11%、2.60%增加到23.52%、14.8%。为了了解MAPKs在亚硒酸钠导致细胞凋亡中的可能作用,我们用不同浓度(0、2.5、5、10、20μmol/L)亚硒酸钠处理HepG2细胞4 h后,使用Western-blot技术检测c-Jun氨基末端激酶(JNK)、细胞外信号调节激酶(ERK)以及p38的表达水平。结果表明各处理组p38、磷酸化p38、ERK1/2和磷酸化ERK1/2水平和对照组无明显差异,而磷酸化JNK1/2的水平随着亚硒酸钠的剂量的增加而增加。10μmol/L亚硒酸钠处理HepG2细胞0、1、2、4、8 h后,用Western-blot技术检测磷酸化的JNK1/2水平。结果1 h后磷酸化的JNK1和JNK2分别为对照的1.58和1.80倍;4 h后磷酸化的JNK1和JNK2分别为对照的2.48和5.03倍;8 h后,和亚硒酸钠处理4 h相比,磷酸化的JNK1/2水平下降。最后为了进一步阐明ROS以及JNK1/2在细胞凋亡中的可能作用以及ROS对JNK1/2的影响,我们使用ROS清除剂N-乙酰半胱氨酸(NAC)后来看亚硒酸钠处理细胞对细胞活性、DNA损伤程度、凋亡情况以及JNK1/2表达水平的影响,以及使用JNK1/2抑制剂sp600125后来看凋亡情况以及JNK1/2表达水平的变化。结果表明NAC增加了细胞活性并降低了细胞凋亡率以及DNA损伤程度,并且NAC处理的亚硒酸钠组ROS水平下降,Western-blot结果表明NAC抑制了JNK1的磷酸化。和10μmol/L亚硒酸钠单独作用组比较,Sp600125预处理的10μmol/L亚硒酸钠组磷酸化的JNI1/2水平降低(P<0.05),并且与对照组比较,细胞早期凋亡率也下降(P<0.05)。第二部分硒亚急性染毒对wistar大鼠的影响为了阐明硒的毒作用表现及氧化应激在其中所起的作用,我们将36只雄性wistar大鼠随机分成6组,各组剂量分别为0、0.125、0.25、0.5、1、2mg/kg体重,经口灌胃7周,每天同一时间灌胃一次.每周记录一次大鼠体重以及饲料消耗量,染毒结束后断头取血检测临床生化指标,取肝、肾等组织称重、做病理切变并用彗星实验分析其DNA损伤情况.结果表明,染毒七周后,和对照组相比,0.125mg/kg组大鼠的平均体重增加,但是经检验无统计学意义; 2mg/kg亚硒酸钠组,7周后体重降低(P<0.05)。脏器系数分析结果表明,与对照组相比,1mg/kg和2mg/k亚硒酸钠组,肝脏的相对重量增加(P<0.05);2mg/kg亚硒酸钠组,肾脏和脾脏的相对重量增加(P<0.01)。对心脏和睾丸则未见影响,各组之间未见差异(P>0.05)。为了检测不同亚硒酸钠浓度对肝肾功能的影响,对肝肾组织做了病理切片并检测了一些临床生化指标。病理切片分析结果显示2mg/kg组肝细胞水肿、变性,组织呈大片空泡样细胞,其它各组未见明显变化;肾组织可见充血、水肿,其它与对照组比较无明显差异。生化分析结果表明随亚硒酸钠剂量增加,丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)均增加,与对照组相比,1mg/kg和2mg/kg亚硒酸钠组,谷丙转氨酶(ALT)和谷草转氨酶(AST)均增加明显(P<0.05)。而各组之间血清尿素和肌酐未见明显差异(P>0.05)。为了进一步了解亚硒酸钠染毒产生ROS所造成的氧化应激,我们检测了血清和肝组织的还原性谷胱甘肽(GSH)、超氧化物歧化酶(SOD)酶活性以及脂质过氧化产物丙二醛(MDA)的含量.结果显示血清和肝组织各组间SOD活性无明显差异(P>0.05);血清和肝组织的丙二醛(MDA)含量除了2mg/kg亚硒酸钠组与对照组相比明显升高(P<0.05),其余各组和对照组之间无明显差异(P>0.05);血清GSH在0.5、1、2mg/kg亚硒酸钠组和对照组相比明显降低(P<0.05),且有随剂量增加而降低的趋势,肝组织GSH在1、2mg/kg亚硒酸钠组和对照组相比明显降低(P<0.05)。我们通过彗星实验进一步研究亚硒酸钠对大鼠肝、肾的DNA损伤情况。实验结果显示和对照组相比较,0.5、1、2mg/kg亚硒酸钠组肝、肾细胞OTM值增加,表明DNA损伤增加(P<0.05)。综上所述,本研究结果表明:(1)亚硒酸钠可抑制HepG2细胞活性,且具有剂量和时间依赖性,NAC可以拮抗亚硒酸钠对细胞活性的抑制作用。(2)亚硒酸钠可使HepG2细胞内的ROS增加,而NAC可以使亚硒酸钠诱导的ROS下降.(3)亚硒酸钠可导致HepG2细胞DNA受损,细胞凋亡增加,NAC可以使亚硒酸钠导致的细胞DNA受损程度降低,并减少细胞凋亡。(4)亚硒酸钠可激活JNK1/2,NAC和sp600125可使JNK1/2磷酸化激活受到抑制,并且sp600125降低了亚硒酸钠导致的细胞凋亡。(5) 2 mg亚硒酸钠/kg体重亚急性染毒主要导致大鼠肝、肾受损,尤其是肝脏,血清和肝组织的GSH降低,MDA增加,机体的氧化平衡受到破坏可能在亚硒酸钠的毒性中发挥了重要作用。(6)彗星实验表明DNA损伤可能是亚硒酸钠导致损害比较敏感的指标,可以考虑作为一个亚硒酸钠导致机体损伤的早期评价指标。更多还原

【Abstract】 Selenium is an essential micronutrient with important biological functions. It plays a key role in the organism. The distribution of selenite is different in different area. Generally speaking, there are much selenite in tropical zone and subtroptical zone, but threre are little in temperate zone and prairie. An foreign investigation showed that cancer death rates were inversely correlated with the geographic distribution of Se in forage crops, and cancer of stomach, oesophagus and rectum were found to be particularly high in selenium-poor areas. A domestic investigation showed that the incidences of liver cancer were inversely correlated with Se in crops, and the distribution of Se was different in different area. Some epidemiological studies and experimental models have also indicated that high levels of Se compounds prevent cancer.There are some reports about the mechanism for the inhibited cancer of Se compounds, but it remains largely unclear about the mechanism of the cancer chemoprevention and inhibition. Now the mechanism may be the following reason: 1. Se compounds can inhibit cancer cells proliferation and promote them apoptosis. Various kinds of Se compounds can inhibit cells proliferation by decreasing the activity of CDC2+ and PKC. ROS can be induced by Se compounds reacted with GSH. Some apoptosis pathway is initiated, so cancer cells are induced apoptosis. 2. The mutagenicity of carcinogenic factor can be decreased by Se compounds. Se compounds decrease the activity of hydroxylase that can active carcinogen. Se compounds also can decrease the carcinogenicity of some carcinogenic metal. 3. By modulating immune system, Se compounds can enhance the anticancer ability. 4. The activity of some anti-oxidized enzyme can be modulated by Se compounds. Se compounds prevent lipid being oxidized and biomembrane being impaired, so the incidence of mutation can be decreased.The nutritional requirement of Se is small. The reports showed that toxic threshold level may be ten fold of nutritional requirement of Se, and human could be poisoned for ingesting thirty to fifty fold of nutritional requirement of Se. Now the mechanism of toxic effect is still not very clear. The two aspects are being considered as below: 1. ROS is thought being related with the toxicity of Se compounds. ROS can be induced by higher concentration of Se compounds, and it can react with biomacromolecule, including protein, DNA and lipid. Thus biomacromolecule are impaired. 2. Some metabolic enzyme can be inactive by Se compounds, so organism is damaged.In a word, no matter the anticancer ability or the toxicity of Se compounds is related with ROS. ROS is also closely related with some signal transduction pathway. Both exogenous and endogenous ROS can modulate the activity of MAPKs. MAPKs are regulated by distinct signal transduction pathways that control many aspects of mammalian cellular physiology, including cell growth, differentiation and apoptosis.In the present study, after Hepg2 cells were treated with selenite and male Wistar rat were administered by selenite, the DNA damage was measured by comet essays. The apoptosis rate and the levels of ROS were detected by flow cytometry. The levels of MAPK were determined by western-blot. Our study investigate how MAPKs are modulated by ROS.Part I The Investigation of Mechanisms about HepG2 Apoptosis Induced by SeleniteIn order to know how the cell viability is affected by selenite, after HepG2 was treated by selenite (0, 2.5, 5, 10, 20μmol/L) for 12, 24, 48 h, then the cell viability was detected. The results showed the cell viability decreases with the increase of time exposed to selentie and the concentration of selenite. Comparing with the control, the cell viability was decreased after HepG2 cells were treated with 10μmol/L selenite for 12 h or 5μmol/L selenite for 24 h (P<0.05). The cell viability was decreased after they are exposed to 10 or 20μmol/L selenite for 12, 24 and 48 h.For exploring how the level of ROS changed after HepG2 cells were treated by selenite, the level of ROS was measured by flow cytometry after the cells were treated by selenite (0, 2.5, 5, 10, 20μmol/L) for 0.5, 1, 2 h. 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) readily diffuses through the cell membrane and is deacetylated by esterases to non-fluorescent 2′, 7′-dichoorofluorescin (DCFH). Further, DCFH could be rapidly oxidized to highly fluorescent DCF in the presence of ROS. The results showed that the increase in ROS was dose-dependent, and ROS increased significantly after cells exposed to 5, 10, or 20μmol/L for one hour. The level of ROS was increased in time-dependent manner, when cells were exposed to 10μmol/L of Se, and a significant increase in ROS was observed 30 min after the treatment with Se.The comet assays and flow cytometry were used to detect the DNA damage and apoptosis rate of cells after they are exposed to selenite. Chromosomal DNA strand breaks as measured by OTM value were increased in HepG2 cells exposed to different dose of Se (i.e., 5, 10, or 20μmol/L), compared with the controls of untreated HepG2 cells. No difference was observed in the group treated with 2.5μmol/L of Se and control group. After HepG2 cells were exposed to different doses (5, 10 and 20μmol/L) of Se for 24 hour, significant differences were observed between them and 0μmol/L Se. The percentage of early apoptosis and late apoptosis/necrosis increased from 1.11% to 16.60% and from 2.60% to 10.15%. After HepG2 were treated with 10μmol/L Se for 0 h, 12 h, 24 h and 48 h, the percentage of early apoptosis and late apoptosis/necrosis increased from 1.11% to 23.52% and from 2.60% to 14.80.In order to know the role of MAPKs in the apoptosis induced by selenite, the level of c-JUN N-terminal kinase, extracellular signal-regulated kinase and p38 were detected by western-blot after HepG2 were exposed to selenite (0, 2.5, 5, 10, 20μmol/L) for 4 h. Western blot analyses showed than the amounts of p38, phospho-p38, ERK1/2 and phospho-ERK1/2 were the same as in untreated cells, whereas the levels of phospho-JNK1/2 increased in a dose-dependent after HepG2 cells were exposed to Se for 4 h. Interestingly, the amounts of JNK2 increased with the doses of Se increasing, whereas levels of JNK1 were no difference between cells exposed to Se and untreated cells. The results showed changes in phospho-JNK1/2 in HepG2 cells with exposure to 10μmol/L Se for 0, 1, 2, 4 and 8 h. After an hour exposure to 10μmol/L Se, it caused increases in phospho-JNK1 about 1.58-fold and phospho-JNK2 about 1.80-fold above the control. After 4 h the increase in phospho-JNK1 was about 2.48-fold and phospho-JNK2 about 5.03-fold above the control. After 8 h, compared with cells treated with Se for 4 h, the levels of phospho-JNK1/2 decreased.Finally for further illuminating the role of ROS and JNK1/2 in the apoptosis induced by selenite, the viability, DNA damage, apoptosis rate and the level of JNK1/2 were detected after N-acetylcysteine (NAC), one of antioxidants, and sp600125, one special inhibitor of JNK, were treated with selenite. NAC increased cell viability and inhibited apoptosis and DNA damage in the cells exposed to Se. To confirm that the inhibition of apoptosis was associated with the antioxidant effects of NAC, the effects of NAC on the generation of ROS were examined. Our data showed that NAC indeed decreased DCF fluorescence, suggesting the increase of ROS was inhibited by NAC. Furthermore, western blot results show that NAC inhibited JNK1 phosphorylation induced by Se. Sp600125 decreased the rates of early apoptotic cell induction by 10μmol/L Se (P<0.05). Furthermore, sp600125 significantly diminished the activation of JNK1/2 when HepG2 exposed to 10μmol/L Se for 4 h (P<0.05).Part II A subchronic toxicity study of selenite in wistar ratIn order to explore the subchronic toxicity of selenite and the role of oxidative stress, 36 male wistar rats were randomly divided into 6 groups with 6 rats each group. The rats were administered saline or selenite at the dose of 0.125, 0.25, 0.5, 1, 2 mg/kg bw Se orally each day for 7 consecutive weeks. Body weights and feed consumption were measured. At the end of the test period, the rats were decapitated to obtain blood, l for clinical chemistry, selected organs were weighed and specified tissues from all animals were subsequent histopathological examination. The livers and kidneys were taken to prepare the single-cell suspensions for comet assay. Oliver tail moment (OTM) was used to evaluate DNA damage of cells from the rats.After 7 weeks, comparing with the control, the average body of the rats in 0.125mg/kg bw group increased, but the increase in not significant; the average body of the rats in 2 mg/kg bw group decreased (P<0.05). Comparing with the control, the relative liver in 1 and 2 mg/kg bw group increased (P<0.05), and the relative kidney and spleen in 2 mg/kg bw group increased (P<0.05), but no difference was seen in the relative heart and testis between different group.The results of pathological section showed hepatic cells presented hydropic degeneration, and a lot of vacuolation could be observed in 2mg/kg bw group. Hyperemia and hydropic change kidney could be observed in 2mg/kg bw group. With the increasing of doses of selenite, the level of alanine aminotransferase and aspartate amino transferase increased. Furthermore, the level of alanine aminotransferase and aspartate amino transferase is higher than the control (P<0.05), but no difference was observed in urea and creatinine of blood serum among different groups.We detected the level of glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) in blood serum and livers to explore the oxidative stress induced by selenite. Among different groups, no difference was observed in the level of SOD in blood serum and livers (P>0.05). The level of MDA in blood serum and livers in 2 mg/kg bw group was higher than in the control (P<0.05). The level of GSH in blood serum among 0.5, 1, 2 mg/kg bw groups is lower than in the control, however, only The level of GSH in liver in 1, 2 mg/kg bw groups is lower than the control.Comet assays was used to detect the DNA damage of liver and kidney cells induced by selenite. Comparing with the control, the results showed that the OTM of 0.5, 1, 2 mg/kg bw groups increased (P<0.05), and it indicated that selenite induced liver and kidney cells DNA damage.Conclusions:1) Se inhibited cells viability in a dose- and time-dependent manner. NAC could increase cell viability.2) HepG2 cells exposed to Se result in intracellular ROS increasing, and NAC can decrease the level of ROS induced by selenite.3) The dose of Se higher than 5μmol/L can induce apoptosis and DNA damage in HepG2, and NAC can inhibit the effects induced by selenite.4) The amounts of phospho-JNK1/2 increased with the doses of Se increasing. NAC inhibits JNK1 phosphorylation induced by Selenite. Sp600125 decreases the apoptosis induced by Selenite.5) 2 mg/kg selenite impairs the liver and kidney of the rats. The level of GSH in liver and blood serum decreases, and the level of MDA in liver and blood serum increases. Oxidative stress plays a key role in the damage induced by selenite.6) The results of comet assay show that DNA damage may be sensitive to the impairment induced by selenite. DNA damage may be the early evaluation indexes of the impairment induced by selenite.更多还原