限制蛋氨酸摄入通过内源性硫化氢延缓肾脏衰老及衰老相关分泌表型

发布时间：2018-07-21 10:52

【摘要】：目的:衰老广义上被认为是时间依赖性引起的功能下降。肾脏也随着衰老发生相关改变,如肾皮质的变薄、肾小球硬化、肾间质纤维化、肾小管萎缩以及肾血管硬化。同时衰老细胞对自身微环境的影响,如衰老相关分泌表型(senescence associated secretory phenotype, SASP)也对加速衰老进程具有重要意义。限制饮食摄入(dietaryrestriction,DR)已被证明是一种延缓衰老的有效干预方式。机制涉及增加自噬、减轻炎症和氧化应激、增加胰岛素敏感性、上调SIRT1等。同时,降低特定营养素的摄入量,而非整体的热量,也发挥着保护作用,尤其以限制蛋白质和特定的氨基酸发挥作用更为突出。限制饮食中蛋氨酸(methionine restriction,MR)摄取可通过提高健康代谢标志物、限制脂肪堆积、增强胰岛素敏感性,从而对机体产生保护作用。已有研究表明MR可以延长果蝇、小鼠等动物模型的寿命,但具体分子机制尚不明确。其他学者的研究显示,DR引起的内源性硫化氢(H2S)增多是发挥保护作用的关键分子,而以蛋氨酸为主的含硫氨基酸恰恰是H2S在体内合成的重要来源。在本研究中,我们通过观察20月龄C57雄性小鼠自由饮食(Ad libitum,AL)及限蛋氨酸(MR)饮食后肾脏衰老标志物及SASP (IL-1、IL-6、IL-8)的改变,明确限制饮食中蛋氨酸摄取能否通过提高内源性H2S表达发挥延缓肾脏衰老的作用。并对机制进行深入研究,检测了 SASP的相关信号通路AMPK/mTOR的表达水平,探究H2S对该通路的调控作用。本研究对于探索延缓衰老相关肾脏疾病新的治疗方案具有重要的意义。方法:将12只SPF级C57BL/6雄性小鼠,正常条件喂养至20月龄后随机分为2组:①老年限蛋氨酸组(old methionine restriction,OMR):蛋氨酸限制至0.15%,胱氨酸缺乏(n=6);②老年对照组(old ad libitum, OAL):正常自由饮食(n=6);两组间其他条件相同且单位重量的饲料所含总热量一致。取6只2月龄SPF级C57BL/6雄性小鼠作为青年对照组(young ad libitum, YAL)。2个月后处死小鼠并留取小鼠肾脏病理及分生标本,检测动物生化指标。Western blot及ELISA检测SASP相关蛋白含量。体外实验中,用硫酸吲哚酚(Indoxyl sulfate,IS)诱导人肾小管上皮细胞衰老,将细胞分为3组:①对照组(DMEM)、②IS组(DMEM+250μM IS)、③IS+MR组(250uMIS+蛋氨酸6mg/L、缺乏胱氨酸的DMEM),刺激48小时后收集细胞及其上清液,Western blot及ELISA检测相关蛋白含量。免疫荧光检测SASP成分IL-1β、IL-8。体内外实验中我们均对各组AMPK/mTOR信号通路的表达情况进行了检测。随后我们为了验证AMPK/mTOR通路的关键作用,在培养基中加入AMPK抑制剂Compound C,刺激48小时后收集细胞及其上清液,Western blot及ELISA检测相关蛋白含量。结果:1)体内动物实验显示,OAL组小鼠肾脏出现明显肾小球硬化、肾小管间质损伤、肾小管萎缩及空泡变性、炎症细胞浸润等病理改变,而OMR组改变较轻,说明MR可在一定程度上延缓肾脏衰老相关改变。Western blot结果显示,与OMR组相比,OAL组小鼠肾脏中的H2S合成相关酶CGL降低,衰老相关标志物p16、p53、p21显著升高,磷酸化AMPK下降、mTOR及下游4E-BP1磷酸化水平明显升高,说明OMR可降低衰老肾脏中衰老标志物表达并激活AMPK通路。2)ELISA显示OMR组较OAL组血液中H2S含量升高,肾脏组织中IL-1β、IL-6水平降低。3)生化结果显示白蛋白、肌酐及相关指标组间无统计学差异,但OMR组血糖(7.13± 0.48mmol/L)及胆固醇(2.47± 0.36 mmol/L)较 OAL 组的血糖(11.36±1.67mmol/L)与胆固醇(3.61±0.36mmol/L)下降明显。4)体外实验显示,与对照组相比,IS组衰老标志物p53、p21的表达增加且β半乳糖苷酶染色阳性细胞增多,证实了IS对于衰老的诱导作用。而IS+MR组比IS组H2S合成相关酶CGL显著升高,衰老标志物p53、p21显著降低,磷酸化AMPK增多、mTOR及4E-BP1磷酸化显著降低。5) ELISA显示IS+MR组细胞中H2S含量显著升高,细胞培养上清中IL-1 β、IL-6显著降低。6)免疫荧光显示IL-1 β与IL-8在IS+MR组明显下降。7)在加入AMPK抑制剂Compound C后,IS+MR+CC组较IS组比较,虽H2S合成相关酶CGL依旧有所上升,但两组间衰老标志物及AMPK/mTOR关键蛋白表达量以及IL-1 β、IL-6表达均无明显差异,MR的保护作用减弱,提示AMPK/mTOR可能是H2S发挥作用的关键通路之一,H2S位于AMPK上游进行调控。结论:肾脏衰老过程中伴随着衰老标志物及SASP成分(IL-1β、IL-6、IL-8)表达增加。AMPK/mTOR在SASP的调节中具有重要作用,且H2S可对AMPK发挥调控作用。限蛋氨酸饮食可通过提高内源性H2S进而上调AMPK,抑制mTOR及下游4E-BP-1的磷酸化、减少肾脏衰老相关分泌表型的蛋白合成。本研究为今后延缓肾脏衰老可提供新的研究思路与并对肾脏衰老的防治具有重要意义。
[Abstract]:Objective: senescence is generally considered to be a time dependent decline in function. Kidney also changes with aging, such as the thinning of the renal cortex, glomerulosclerosis, renal interstitial fibrosis, renal tubule atrophy, and renal vascular hardening. And the effects of aging cells on their microenvironment, such as the aging related secretory phenotype (senescence associate) D secretory phenotype, SASP) also plays an important role in accelerating the aging process. Restriction of dietary intake (dietaryrestriction, DR) has been proved to be an effective intervention to delay aging. The mechanism involves increasing autophagy, alleviating inflammation and oxidative stress, increasing insulin sensitivity, up-regulated SIRT1, etc., and reducing the intake of specific nutrients. Quantity, not the whole heat, also plays a protective role, especially in limiting protein and specific amino acids. Restriction of dietary methionine (methionine restriction, MR) uptake can be protected by improving health metabolic markers, limiting fat accumulation, increasing insulin sensitivity, and thus protecting the body. Studies have shown that MR can prolong the life of Drosophila, mice and other animal models, but the specific molecular mechanism is not clear. Other scholars have shown that the increase of endogenous hydrogen sulfide (H2S) caused by DR is the key molecule to play a protective role, and methionine based sulfur containing amino acid is the important source of H2S in the body. By observing the changes in renal senescence markers and SASP (IL-1, IL-6, IL-8) in the 20 month old C57 male mice free diet (Ad libitum, AL) and methionine (MR) diet, we clearly restrict the effect of methionine uptake in diet by improving endogenous H2S expression to postpone renal senescence. The expression level of the related signal pathway of SASP, AMPK/mTOR, was used to explore the role of H2S in the regulation of the pathway. This study was of great significance for exploring new treatment schemes for postponing aging related renal diseases. Methods: 12 SPF grade C57BL/6 male mice were fed to 2 groups after the normal conditions were fed to 20 month old groups: (OL) D methionine restriction, OMR): methionine was limited to 0.15%, cystine deficiency (n=6); (2) the old control group (old ad libitum, OAL): normal free diet (n=6); the total calories of the two groups with the same conditions and unit weight were the same. 6 2 month old SPF C57BL/6 male mice were taken as the young control group. After month, the mice were killed and the Kidney Pathological and pariaplastic specimens were left, and the biochemical indexes of.Western blot and ELISA were detected. In vitro, the renal tubular epithelial cell senescence was induced by Indoxyl sulfate (IS), and the cells were divided into 3 groups: (1) control group (DMEM) and IS group (DMEM+250 mu M IS). Group MR (250uMIS+ methionine 6mg/L, lack of DMEM of cystine), collected cells and their supernatant after 48 hours of stimulation, Western blot and ELISA detection related protein content. Immunofluorescence detection SASP component IL-1 beta, IL-8. in vivo and in vivo, we all detected the expression of AMPK/mTOR signaling pathway in each group. Then we used to verify AMPK. The key role of /mTOR pathway was the addition of AMPK inhibitor Compound C in the medium. After 48 hours of stimulation, the cells and their supernatants were collected and the content of related proteins was detected by Western blot and ELISA. Results: 1) in vivo animal experiments showed that the renal glomerulosclerosis, tubulointerstitial damage, tubuloatrophy and vacuolation of renal tubules in the OAL mice kidney were obvious. Pathological changes such as disease cell infiltration, and OMR group changed slightly, indicating that MR could delay renal aging related changes to.Western blot to a certain extent. Compared with the OMR group, the H2S synthesis related enzyme CGL in the OAL group of mice decreased, the senescence related markers p16, p53, p21 significantly increased, phosphorylation AMPK decreased, and downstream phosphorylation The level obviously increased, indicating that OMR could reduce the expression of senescence markers in the aging kidney and activate the AMPK pathway.2) ELISA showed that the content of H2S in the OMR group was higher than that in the OAL group, and the biochemical results showed that there was no statistical difference between the serum albumin, creatinine and the related indexes in the OAL group, but the OMR group of blood glucose (7.13 + 0.48mmol/L) and in the OMR group had no significant difference. Cholesterol (2.47 + 0.36 mmol/L) was significantly higher than that in group OAL (11.36 + 1.67mmol/L) and cholesterol (3.61 + 0.36mmol/L). In vitro experiments showed that, compared with the control group, the expression of p53, p21, and beta galactosidase staining positive cells increased in the IS group, which confirmed the induction of IS on senescence. The IS+MR group was larger than the IS H2S. Synthesis related enzyme CGL increased significantly, aging markers p53, p21 significantly decreased, phosphorylated AMPK increased, mTOR and 4E-BP1 phosphorylation significantly decreased.5) ELISA showed that H2S content in the IS+MR group cells increased significantly, IL-1 beta in cell culture supernatant and IL-6 significantly decreased After Compound C, the IS+MR+CC group was compared with the IS group, although the H2S synthesis related enzyme CGL still rose, but there was no obvious difference between the two groups of senescence markers and the expression of AMPK/mTOR key proteins and the expression of IL-1 beta and IL-6, and the protective effect of MR was weakened. Regulation. Conclusion: the expression of IL-1 beta, IL-6 (IL-6, IL-8) in the process of renal senescence plays an important role in the regulation of SASP, and H2S can play a regulatory role in AMPK. The methionine diet can increase the endogenous H2S and then up regulate AMPK, inhibit the phosphorylation of mTOR and downstream 4E-BP-1, and reduce the kidney. This study provides new research ideas for postponing renal senescence in the future and is of great significance to the prevention and treatment of renal senescence in the future.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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