姬松茸多糖在镉致鸡外周血液淋巴细胞和肾脏损伤中的作用研究

发布时间：2018-06-06 02:46

本文选题：镉 + 姬松茸多糖　；参考：《东北农业大学》2017年硕士论文

【摘要】：镉是一种有害重金属物质,可造成人和动物机体外周血液淋巴细胞(Peripheral blood lymphocytes,PBLs)和肾脏毒性损伤,破坏自身免疫系统。姬松茸多糖(Agaricus blazei Murill Polysaccharides,ABP)能够治疗镉带来的毒性损伤。本试验从细胞凋亡水平、模式识别受体TLR4信号通路及抗氧化角度研究ABP对镉中毒鸡PBLs和肾脏的影响,以明确ABP对镉中毒鸡的保护作用。将7日龄生长状态良好的雏鸡随机分为四组,每组20只。镉组饲喂含氯化镉140 mg/kg的全价饲料,多糖组饮食为全价饲料并给予0.2 m L ABP,糖镉组饲喂含氯化镉140 mg/kg的全价饲料,ABP 0.2 m L,对照组鸡以正常饲养方式使用全价饲料饲喂。分别在第20、40、60试验日时,心脏采取鸡外周血液,分离、收集外周血液淋巴细胞,细胞培养24 h后收集细胞上清,并采取鸡肾脏,通过电感耦合等离子-质谱法(IPC-MS)检测鸡PBLs及肾脏镉含量;流式细胞术测定并计算鸡PBLs凋亡率;实时荧光定量PCR法对鸡PBLs凋亡基因Caspase-3、Bax和Bcl-2 m RNA,PBLs和肾脏TLR4及TLR4信号通路相关因子(My D88、TRIF、NF-κB、IRF3)mRNA水平进行检测;Western blot方法分析TLR4蛋白变化;双抗体夹心ELISA法检测PBLs和肾脏炎症因子IL-1β、IL-6、TNF-α的含量;通过MDA、T-SOD、GSH-p X测试盒对鸡肾脏中相应因子进行检测。结果发现,镉组鸡PBLs和肾脏组织中的镉含量显著高于对照组,ABP治疗后其镉含量明显下降。镉组鸡PBLs细胞凋亡率最高,ABP治疗后凋亡率显著抑制,同时多糖组细胞凋亡率明显低于对照组。同对照组相比,镉组鸡PBLs中Caspase-3、Bax的mRNA水平升高,Bcl-2 mRNA水平降低,Bax/Bcl-2明显升高,ABP干预后,上述变化得到显著抑制。ABP能显著抑制镉诱导的鸡PBLs和肾脏TLR4表达以及My D88、TRIF、NF-κB、IRF3 m RNA水平。降低镉组鸡炎症因子IL-1β、IL-6、TNF-α含量。镉组鸡MDA含量显著高于对照组,而T-SOD、GSH-p X的活性明显降低,姬松茸多糖进行治疗后得到明显改善,且多糖组MDA的含量与对照组相比显著降低,T-SOD、GSH-p X的活性显著升高。上述结果表明,镉能够诱导鸡外周血液淋巴细胞凋亡,活化鸡PBLs和肾脏组织TLR4信号通路,增加炎症因子的表达释放,损害鸡肾脏氧化-抗氧化功能,造成鸡PBLs和肾脏损伤,ABP对镉引起的上述损伤均有明显的保护作用。综上所述,本试验通过建立鸡镉中毒模型,研究镉对鸡PBLs和肾脏毒性损伤作用以及姬松茸多糖对镉毒性的影响。为今后进一步研究镉毒性及ABP的治疗机制提供理论依据。
[Abstract]:Cadmium is a harmful heavy metal substance, which can damage peripheral blood lymphocytes PBLs and kidney toxicity in human and animal peripheral blood, and destroy the autoimmune system. Agaricus blazei Murill polysaccharide ABP can treat the toxic injury caused by cadmium. The effects of ABP on PBLs and kidney of cadmium poisoned chickens were studied in terms of cell apoptosis level, pattern recognition receptor TLR4 signaling pathway and antioxidation angle, in order to clarify the protective effect of ABP on cadmium intoxicated chickens. The 7-day-old chicks were randomly divided into four groups with 20 chickens in each group. The cadmium group was fed with the full price diet containing cadmium chloride 140 mg/kg, the polysaccharide group was fed the full price diet and the 0.2 m L ABP, glucose cadmium group was fed with ABP 0.2 mL containing cadmium chloride 140 mg/kg, while the control group was fed with the full price diet in the normal way. On the 20th day of the experiment, chicken peripheral blood was isolated, lymphocytes were collected, supernatants were collected after 24 hours of cell culture, and chicken kidneys were taken. The contents of cadmium in PBLs and kidney were detected by inductively coupled plasma-mass spectrometry (ICP-MS), and the apoptosis rate of chicken PBLs was measured and calculated by flow cytometry. The changes of TLR4 protein in chicken PBLs apoptosis gene Caspase-3 Bax and Bcl-2 m RNA-PBLs and renal TLR4 and TLR4 signal transduction related factors were detected by real-time fluorescence quantitative PCR. The changes of TLR4 protein were analyzed by Western blot, and the contents of PBLs and the renal inflammatory factor IL-1 尾 -IL-6TNF- 伪 were detected by double antibody sandwich ELISA method. The corresponding factors in chicken kidney were detected by MDA-T-SODX GSH-p X test box. The results showed that the content of cadmium in PBLs and renal tissue in cadmium group was significantly higher than that in control group after treatment. The apoptosis rate of PBLs cells in cadmium group was the highest and the apoptosis rate in polysaccharide group was significantly lower than that in control group. Compared with the control group, the mRNA level of Caspase-3 and Bax in PBLs of cadmium group was increased and the level of Bcl-2 mRNA was decreased. The above changes were significantly inhibited. ABP could significantly inhibit the expression of PBLs and renal TLR4 induced by cadmium, and the level of NF- 魏 IRF3 m RNA of my D88 trifion (NF- 魏 BIF-IRF3). The content of IL-1 尾 -IL-6 TNF- 伪 was decreased in cadmium group. The content of MDA in cadmium group was significantly higher than that in control group, while the activity of T-SODN GSH-pX was decreased significantly, and the content of MDA in polysaccharide group was significantly improved after treatment, and the activity of GSH-pX in polysaccharide group was significantly lower than that in control group. These results suggest that cadmium can induce apoptosis of lymphocytes in chicken peripheral blood, activate TLR4 signaling pathway in chicken PBLs and kidney tissue, increase the expression and release of inflammatory factors, and damage the oxidation-antioxidation function of chicken kidney. Both PBLs and ABP have obvious protective effects on cadmium-induced injury. To sum up, the effects of cadmium on PBLs and kidney toxicity and the effects of polysaccharides of Agaricus blazei Murrill on cadmium toxicity were studied. It provides a theoretical basis for the further study of cadmium toxicity and the therapeutic mechanism of ABP.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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