PCB118引言部分.doc

Polychlorinated biphenyls 118 (PCB118) induced oxidative stress and MAPK signaling mediates its pro-apoptotic effects in islet beta cellsIntroductionWith the increasing incidence of diabetes and the growing awareness of the medical complication, the study on the pathogenesis of diabetes became a valuable topic. Plenty of research1 showed that insulin resistance and dysfunction of pancreatic -cells were the main mechanism of type 2 diabetes mellitus (T2DM). The insulin resistance is initial factor, while the dysfunction of pancreatic -cells is decisive factor. All of the diabetic patients were associated with dysfunctional -cells. The apoptosis of -cells result from internal and external factors directly lead to seriously damage of pancreas. Afterwards deficiency of insulin secretion played an important role in the process of diabetes. However the specific mechanism of -cells apoptosis is not fully elucidated. Oxidative stress and endoplasmic reticulum stress were two probable theories in the present study stage.Owing to the serious environmental pollution, some nonbiodegradable and bioaccumulative toxic substances such as polychlorinated biphenyls(PCBs) might have a bad influence in endocrine system and immune system. PCBs was synthetic organic compounds which was insulative and antiflaming. The feature of stable chemical property and the ability to resist pyrolysis were principal reason why it could widely used in industrial production. Recent epidemiological studies 2showed that exposure to environmental toxins was an important factor for the development of T2DM and there was close relationship between diabetes and environmental toxins such as PCBs. The mechanism may be that PCBs induced islet cells apoptosis and insulin secretion capacity was weakened, then diabetes was induced or aggravated. In order to explore the specific mechanism on islet cell apoptosis, our research use rat INS-1 cells as observation subject and treated with PCB118. The results suggest that PCB118 might induce apoptosis in INS-1 cells by activating oxidative stress. 1Yoshimoto K, Ozawa S, Ishida H. Pathogenic mechanism of type 2 diabetes mellitus from impaired glucose tolerance /borderline type and its reversibilityJ. Nihon Rinsho, 2005( 63) : 95-992 Gray SL, Shaw AC, Gagne AX, Chan HM. Chronic exposure to PCBs (Aroclor 1254) exacerbates obesity-induced insulin resistance and hyperinsulinemia in miceJ. J Toxicol Environ Health A. 2013;76(12):701-15.近年来，随着糖尿病发病率的升高以及人们对其并发症危害性认识的提高，对糖尿病发病机制的研究已成为极具研究价值的热点课题。多年的研究表明，胰岛素抵抗和细胞功能障碍是2型糖尿病发病的两大机制。其中，胰岛素抵抗是2型糖尿病发生的始动因素，而细胞功能障碍是2型糖尿病发生的决定因素，所有2型糖尿病患者均有细胞功能受损。多种原因导致的细胞凋亡可直接导致细胞功能严重受损，但凋亡的具体机制目前并未完全阐明，目前，氧化应激和内质网应激是两种重要的学说。由于环境污染问题日益严重，自然界中一些难以降解的、具有生物蓄积性和高毒性的物质如多氯联苯（PCBs）会对人体的内分泌系统和免疫系统造成重大影响。多氯联苯（PCBs）是一类化学性质稳定，绝缘、阻燃、抗热解能力较好的人工合成有机化合物，被广泛应用于工业生产的众多环节。近年来有研究显示，多氯联苯与糖尿病之间存在密切联系。（参考文献）其机制可能是由于PCBs能诱导胰岛细胞凋亡，胰岛素分泌能力减弱，从而诱发或加重糖尿病。为了探讨PCB118诱导胰岛细胞凋亡的具体机制，本文选择大鼠胰岛细胞株（INS-1）作为观察对象，用PCB118对其干预，研究结果显示，PCB118可能通过激活INS-1细胞内的氧化应激从而诱导凋亡的发生。(第一次写不打算用)Polychlorinated biphenyls (PCBs) are toxic synthetic chemicals which are refractory in nature. Because of the widely using between the 1930s and 1970s, the morbidity of many related diseases was rapidly increased. Recent epidemiological studies showed that exposure to environmental toxicants was an important factor for the development of type 2 diabetes (T2D) (Gray SL et al,2013). In addition, there are reports of associations between PCB, obesity, and diabetes ( Lyche et al., 2010;Airaksinen et al., 2011).The pathogenesis was related to the bioaccumulation of persistent organic pollutants (POP)(Jones et al,2008). These studies indicated that bioaccumulation of POP that coming from exposure to PCB118 may be a new environmental risk factor contributing to the development of T2D in the industrialized world.With the high morbidity of T2D in resent years, hundreds of millions of patients suffered from the complications. Many epidemiological studies has demonstrated the relationship of exposure to environmental toxins and the development of T2D, while the experimental studies that exploring the mechanisms of PCBs effect on Islet cells are few. The present study aimed to determine whether Islet cells will apoptosis under the effect of PCBs and which signal pathway participated in this process. 1. Gray SL, Shaw AC, Gagne AX, Chan HM. Chronic exposure to PCBs (Aroclor 1254) exacerbates obesity-induced insulin resistance and hyperinsulinemia in miceJ. J Toxicol Environ Health A. 2013;76(12):701-15.2. Lyche, J. L., Nourizadeh-Lillabadi, R., Almaas, C., Stavik, B., Berg, V., Skaare, J. U., Alestrom,P. and Ropstad, E. 2010. Nartural mixtures of persistent organic pollutants increase weight gain, advance puberty and induce gene expression associated withy steroid hormones and obesity in female zebrafish. J. Toxicol. Environ. Health A 73: 10321057.3. Airaksinen, R., Rantakokko, P., Eriksson, J. G.,Blomstedt, P., Kajantie, E., and Kivirant, H.2011. Association between type 2 diabetes and exposure to persistent organic pollutants. Diabetes Care 34: 19721979.4,. Jones, O. A., Maguire, M. L., and Griffin, J.L. 2008. Environmental pollution and diabetes: Aneglected association. Lancet 371:287288.2.Materials and methods2.1 ChemicalsPCB118(CAS Number:31508-00-6) was purchased from AccuStandard. Inc(USA). N-Acetyl-L-cysteine(NAC, A9165-25G) was purchased from Sigma Aldrich(USA). 4-PBA没有说明书2.2 Cell culture and treatments INS-1 cells were cultured in RPMI-1640s medium containing 0.05Mm - mercaptoethanol (Sigma), supplemented with10% (v/v) heat-inactivated foetal bovine serum(FBS, Science), 100 unites/ml penicillin-streptomycin solution. Cells were incubated at 37 in 5% CO2 and digested by 1 ml 0.25% parenzyme (Gibco) for 5 minutes when they grown to approximately 80% confluence, then 5 ml fresh medium was used to terminate digestion. INS-1 cells were gently blew and made into single-cell suspension prior to centrifuged for 4 minutes at 1000 RPM. The supernatant was sucked out and appropriate fresh medium was added to disperse the cell aggregate. Cells in logarithmic phase were planked used for experimental research.2.3 Cell morphological observationCells were grown in 12-well tissue culture plates. After treatment, cell morphology was observed by inverted microscope and photos were taken at different time points and magnification times.2.4 Cell viability assayCells were grown in 96-well tissue culture plates. After treatment, medium was sucked out prior to 50l fresh medium and 5l CCK-8 solution were added into culture plates. After gently shaking, INS-1 cells were incubated in 37 for 0.5-1h until the color of part wells turn orange. The absorbance value of each well was determined by ELIASA. Four wells were repeated for each group.2.5 SDS-PAGE and Western blottingSDS-PAGE and Western blotting were performed as described previously 1. Caspase 3, Cleaved Caspase 3, c-Fos, Bcl-xL, CHOP, -actin were purchased from Cell Signaling Technologies(USA).2.6 Reactive oxygen species level detectionBefore treatment, 10mol/L DHE was added into the culture plates and cells were incubated for 20 minutes in 37.Red fluorescence was observed by fluorescence microsco