D-半乳糖诱导大鼠内耳氧化性应激、线粒体损伤和凋亡及长期高脂饮食对D-半乳糖诱导的老化大鼠内耳的影响

发布时间：2018-01-26 00:25

本文关键词： 年龄相关性听力损失线粒体 NADPH氧化酶3 氧化性应激激活型caspase-3 凋亡高脂饮食 D-半乳糖听力损伤氧化性应激线粒体凋亡　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：第一部分D-半乳糖诱导大鼠内耳氧化性应激、线粒体损伤和凋亡目的：线粒体DNA (mitochondrial DNA,mtDNA)的氧化性损伤和凋亡是老化的重要特征。我们之前利用连续8周注射D-半乳糖(D-galactose, D-gal)的方法建立了大鼠老化模型,并且发现在D-gal诱导的老化大鼠内耳中氧化性应激的增加和mtDNA常见缺失(common deletion, CD)的增加。但是,在D-gal诱导的老化大鼠内耳中,目前没有直接的证据表明mtDNA CD的累积是由氧化性损伤引起的；同时此模型内耳中活性氧(reactive oxygen species, ROS)的来源以及凋亡情况仍不清楚。方法：听力正常,无中耳疾患的40只5周龄雄性Spragua-Dawley大鼠随机分成2组(各20只)：①D-半乳糖组(D-gal group):每日颈背部皮下注射D-gal (500mg/kg),连续8周；②对照组(control group):每日颈背部皮下注射同体积的生理盐水,连续8周。听性脑干反应(auditory brainstem response, ABR)检测每组大鼠听功能。比色法检测大鼠血清H202,总超氧化物歧化酶(total superoxide dismutase, T-SOD)活性以及丙二醛(malondialdehyde, MDA)含量。实时定量PCR检测大鼠内耳mtDNA CD的累积以及NADPH氧化酶3(NOX3)和P22phox]mRNA水平的变化。透射电子显微镜检术(Transmission Electron Microscopy, TEM)观察大鼠内耳组织细胞中线粒体超微结构的改变。免疫组织化学法检测大鼠耳蜗8-羟基-2-脱氧鸟苷(8-hydroxy-2-deoxyguanosine,8-OHdG)以及NOX3和P22phox蛋白水平的变化。免疫印迹法检测大鼠内耳组织中激活型caspase-3(cleaved caspase-3)的表达。原位末端转移酶标记(terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling, TUNEL染色检测大鼠耳蜗细胞凋亡发生的情况。结果：D-半乳糖组大鼠血清H202和MDA含量比对照组明显增加,T-SOD水平比对照组明显降低,差异有统计学意义(P0.01)。D-半乳糖组大鼠内耳组织中mtDNACD累积比对照组明显增加,差异有统计学意义(P0.01)。TEM发现D-半乳糖组大鼠耳蜗组织细胞中线粒体表现为基质密度降低,甚至发生严重变性。D-半乳糖组大鼠耳蜗组织中8-OHdG、NOX3、P22phox和cleaved caspase-3的表达比对照组明显升高,差异有统计学意义(P0.01)。TUNEL染色发现少量凋亡细胞局限于D-半乳糖组大鼠耳蜗血管纹,而对照组大鼠耳蜗未发现凋亡细胞。两组大鼠ABR阈值差异无统计学意义(P0.05)。结论：在内耳老化过程中mtDNACD的积累可能是NADPH氧化酶相关的氧化性应激造成的,并且caspase-3介导的细胞凋亡可能参与了内耳的老化过程。第二部分长期高脂饮食加重D-半乳糖诱导的老化大鼠内耳氧化性应激,线粒体损伤和凋亡目的：研究12个月的高脂饮食对Sprague-Dawley大鼠内耳及D-半乳糖诱导的老化大鼠内耳的老化过程的作用。方法：104只5周龄雄性Spragua-Dawley大鼠随机分成4组(各26只)：①对照组(control group):饲喂基础饮食12个月,前8周每日颈背部皮下注射和D-半乳糖组同体积的生理盐水；②D-半乳糖组(D-gal group):饲喂基础饮食12个月,前8周每日颈背部皮下注射D-gal (500mg/kg);③高脂饮食组(HFD group):饲喂高脂饮食12个月,前8周每日颈背部皮下注射和D-半乳糖组同体积的生理盐水；④D-半乳糖+高脂饮食组(D-gal+HFD group):饲喂高脂饮食12个月,前8周每日颈背部皮下注射D-gal (500mg/kg)。造模结束后,听性脑干反应(auditory brainstem response, ABR)检测每组大鼠听功能；每组大鼠称重,比色法检测大鼠血清甘油三脂(triglycerides,TG),总胆固醇(total cholesterol, TC),游离脂肪酸(nonesterified fatty acids, NEFA)和H202含量；实时定量PCR检测大鼠内耳mtDNACD的累积以及NADPH氧化酶3(NOX3), P22phox, uncoupling protein2(UCP2)和uncoupling protein3(UCP3) mRNA水平的变化；免疫组织化学法检测大鼠耳蜗NOX3蛋白水平的表达；免疫印迹法检测大鼠内耳组织中NOX3,P22phox,UCP2,UCP3和激活型caspase-3(cleaved caspase-3)的蛋白水平的表达；透射电子显微镜检术(Transmission Electron Microscopy, TEM)观察大鼠耳蜗血管纹边缘细胞线粒体超微结构的改变；原位末端转移酶标记(terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling, TUNEL染色检测大鼠耳蜗细胞凋亡情况。结果：D-半乳糖组和D-半乳糖+高脂饮食组大鼠ABR阈值在4,8,16和32kHz比对照组升高,差异有统计学意义(P0.05)；高脂饮食组大鼠ABR阈值数值上在上述4个频率均比对照组升高,但仅在32kHz差异有统计学意义(P0.05)；同时,D-半乳糖+高脂饮食组大鼠ABR阈值在16和32kHz比D-半乳糖组升高,差异有统计学意义(P0.05)。高脂饮食组和D-半乳糖+高脂饮食组大鼠血清TG、TC和NEFA水平比对照组和D-半乳糖组升高,差异有统计学意义(P0.01)；D-半乳糖组、高脂饮食组和D-半乳糖+高脂饮食组大鼠血清H202水平比对照组升高,差异有统计学意义(P0.01),其中D-半乳糖+高脂饮食组大鼠血清H202水平最高,差异有统计学意义(P0.01)。D-半乳糖组、高脂饮食组和D-半乳糖+高脂饮食组大鼠内耳组织NOX3、P22phox、UCP2、UCP3和cleaved caspase-3的表达比对照组升高,其中D-半乳糖+高脂饮食组最高,差异有统计学意义(P0.01)。D-半乳糖组、高脂饮食组和D-半乳糖+高脂饮食组大鼠内耳组织mtDNA CD的累积比对照组增加,差异有统计学意义(P0.05)。D-半乳糖组、高脂饮食组和D-半乳糖+高脂饮食组大鼠耳蜗血管纹边缘细胞线粒体有不同程度的肿胀,伴有线粒体基质密度降低。TUNEL染色发现D-半乳糖组、高脂饮食组和D-半乳糖+高脂饮食组凋亡细胞比对照组明显增多,且凋亡细胞主要局限于血管纹；同时,在D-半乳糖+高脂饮食组大鼠耳蜗Corti器发现少量凋亡细胞。结论：长期高脂饮食可诱导内耳氧化性应激、线粒体损伤和凋亡。长期高脂饮食可加速年龄相关性听力损失的进展。
[Abstract]:The first part of D- galactose induced oxidative stress, mitochondrial damage and apoptosis in the inner ear of rats
Objective: mitochondrial DNA (mitochondrial DNA mtDNA) on the oxidative damage and apoptosis is an important feature of aging. We use before 8 weeks of continuous injection of D- galactose (D-galactose, D-gal) method to establish the model of aging rats, and found that in D-gal induced aging increased oxidative stress in the inner ear of rats and mtDNA common deletion (common deletion, CD) increased. However, in aging rats induced by D-gal in the inner ear, there is no direct evidence that the accumulation of mtDNA CD caused by oxidative damage; active oxygen at the same time, this model in the inner ear (reactive oxygen species, ROS) as well as the source of apoptosis remains unclear.
Methods: normal hearing, no middle ear diseases of the 40 5 week old male Spragua-Dawley rats were randomly divided into 2 groups (20 each): D- group (D-gal group) galactose: daily subcutaneous injection of D-gal (500mg/kg), for 8 weeks; the control group (control group): saline daily journal subcutaneous injection of the same volume for 8 consecutive weeks. Auditory brainstem response (auditory brainstem, response, ABR) function to detect the rats. Detect rats serum H202 assay, total superoxide dismutase (total superoxide, dismutase, T-SOD) activity and malondialdehyde (malondialdehyde, MDA). Real time quantitative content detection of PCR accumulation in the rat inner ear and mtDNA CD NADPH (NOX3) oxidase 3 changes and P22phox]mRNA level. Microscopy transmission electron microscopy (Transmission Electron, Microscopy, TEM) to observe the mitochondrial ultrastructure of cells of the inner ear tissues of rats without change. Immunohistochemical method was used to detect the rat cochlea 8- -2- hydroxy deoxyguanosine (8-hydroxy-2-deoxyguanosine, 8-OHdG) and the changes of NOX3 and P22phox protein levels. Caspase-3 activation in rats were detected by Western blotting in inner ear tissues (cleaved caspase-3). The expression of TDT mediated dUTP nick end labeling (terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP nick-end-labelling), TUNEL staining of rat cochlear cell apoptosis detection occurs.
Results: D- D-galactose rats serum H202 and MDA contents were significantly higher than control group, T-SOD levels were significantly lower than the control group, the difference was statistically significant (P0.01) the inner ear tissue in rats of.D- galactose in mtDNACD accumulation increased significantly than the control group, the difference was statistically significant (P0.01.TEM) found that mitochondrial cochlear tissue group rat D- cells showed galactose matrix density decreased, or even 8-OHdG, serious degeneration of.D- in D-galactose rats cochlea tissues NOX3, expression of P22phox and cleaved caspase-3 were significantly higher than the control group, the difference was statistically significant (P0.01).TUNEL staining showed a few apoptotic cells confined to D- galactose group in rat cochlea the vascular pattern, while the control group rat cochlea no apoptotic cells were found. There was no significant difference between two groups of rats ABR threshold (P0.05).
Conclusion: the accumulation of mtDNACD in the aging process of the inner ear may be caused by NADPH oxidase related oxidative stress, and caspase-3 mediated apoptosis may be involved in the aging process of the inner ear.
The second part of long term high fat diet aggravates the oxidative stress, mitochondrial damage and apoptosis of the aged rats with D- galactose induced aging
Objective: To study the effect of high fat diet for 12 months on the aging process of inner ear of Sprague-Dawley rats and D- galactose induced aging rats.
Methods: 104 5 week old male Spragua-Dawley rats were randomly divided into 4 groups (26 each): control group (control group): fed the basal diet for 12 months, 8 weeks before the daily subcutaneous injection of D- galactose group and the same volume of normal saline; the D- galactose group (D-gal group): fed the basal diet for 12 months, 8 weeks before the daily subcutaneous injection of D-gal (500mg/kg); high fat diet group (HFD group): fed with high fat diet for 12 months, 8 weeks before the daily subcutaneous injection of D- galactose group and the same volume of normal saline; the D- galactose + high fat diet group (D-gal+HFD group): fed with high fat diet for 12 months, 8 weeks before the daily subcutaneous injection of D-gal (500mg/kg). After the modeling, auditory brainstem response (auditory brainstem, response, ABR) function to detect the rats; the rats weighing, than the detection of rat serum glycerol color three fat (triglyceride S, TG), total cholesterol (total, cholesterol, TC), free fatty acids (nonesterified fatty, acids, NEFA) and H202 content accumulated; real time quantitative PCR detection of rat inner ear mtDNACD and NADPH oxidase 3 (NOX3), P22phox uncoupling protein2 (UCP2) and uncoupling protein3 (UCP3) mRNA levels; immunohistochemistry was used to detect the expression level of NOX3 protein in rat cochlea; NOX3 rats were detected by Western blot in the inner ear tissues of P22phox, UCP2, UCP3 and caspase-3 (cleaved caspase-3) activated protein level expression; microscopy transmission electron microscopy (Transmission Electron, Microscopy, TEM) to observe the rat cochlea mitochondria the edge of cell ultrastructure change; TDT mediated dUTP nick end labeling (terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP) nick-end-labelling, TUNEL staining The apoptosis of cochlear cells in rats was detected by color.
Results: D- galactose group and D- galactose + high fat diet group rats ABR threshold in 4,8,16 and 32kHz than the control group increased, the difference was statistically significant (P0.05); numerical threshold of ABR in high fat diet group rats in each of the 4 frequencies were higher than control group, but the difference is only in the 32kHz statistics significance (P0.05); at the same time, D- galactose + high fat diet rats in group ABR and 32kHz than the 16 threshold in D- galactose group increased, the difference was statistically significant (P0.05). Serum TG D- galactose diet group and high-fat + high-fat diet group rats, TC and NEFA levels than the control group galactose and D- group increased, the difference was statistically significant (P0.01); D- galactose group, the serum level of H202 diet group and D- galactose + high fat high-fat diet group rats is higher than that of the control group, the difference was statistically significant (P0.01), the level of serum H202 in D- galactose + high fat diet group were the highest, the difference was statistically significant ( P0.01).D- galactose group, high fat diet group and inner ear tissue D- galactose + high fat diet group rats NOX3, P22phox, UCP2, UCP3 and cleaved expression of Caspase-3 was higher than control, which D- galactose + high fat diet group was the highest, the difference was statistically significant (P0.01).D- galactose group. Cumulative increase in high fat diet group and inner ear tissue D- galactose + high fat diet group rats mtDNA CD than in the control group, the difference was statistically significant (P0.05).D- galactose group, mitochondria in marginal cells of the stria vascularis D- galactose + diet group and high-fat diet group rats with high fat have different degrees of swelling that is associated with decreased mitochondrial matrix density of.TUNEL staining indicated that D- galactose group, high fat diet group and D- galactose + high fat diet group than the control group, the apoptotic cells increased significantly, and the apoptosis was mainly confined to the vascular pattern; at the same time, in D- galactose + high fat diet rats cochlear Corti A small number of apoptotic cells were found.
Conclusion: long term high fat diet can induce oxidative stress in the inner ear, mitochondrial damage and apoptosis. Long term high fat diet can accelerate the progress of age related hearing loss.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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