【摘要】：目的:衰老廣義上被認(rèn)為是時(shí)間依賴性引起的功能下降。腎臟也隨著衰老發(fā)生相關(guān)改變,如腎皮質(zhì)的變薄、腎小球硬化、腎間質(zhì)纖維化、腎小管萎縮以及腎血管硬化。同時(shí)衰老細(xì)胞對(duì)自身微環(huán)境的影響,如衰老相關(guān)分泌表型(senescence associated secretory phenotype, SASP)也對(duì)加速衰老進(jìn)程具有重要意義。限制飲食攝入(dietaryrestriction,DR)已被證明是一種延緩衰老的有效干預(yù)方式。機(jī)制涉及增加自噬、減輕炎癥和氧化應(yīng)激、增加胰島素敏感性、上調(diào)SIRT1等。同時(shí),降低特定營(yíng)養(yǎng)素的攝入量,而非整體的熱量,也發(fā)揮著保護(hù)作用,尤其以限制蛋白質(zhì)和特定的氨基酸發(fā)揮作用更為突出。限制飲食中蛋氨酸(methionine restriction,MR)攝取可通過提高健康代謝標(biāo)志物、限制脂肪堆積、增強(qiáng)胰島素敏感性,從而對(duì)機(jī)體產(chǎn)生保護(hù)作用。已有研究表明MR可以延長(zhǎng)果蠅、小鼠等動(dòng)物模型的壽命,但具體分子機(jī)制尚不明確。其他學(xué)者的研究顯示,DR引起的內(nèi)源性硫化氫(H2S)增多是發(fā)揮保護(hù)作用的關(guān)鍵分子,而以蛋氨酸為主的含硫氨基酸恰恰是H2S在體內(nèi)合成的重要來源。在本研究中,我們通過觀察20月齡C57雄性小鼠自由飲食(Ad libitum,AL)及限蛋氨酸(MR)飲食后腎臟衰老標(biāo)志物及SASP (IL-1、IL-6、IL-8)的改變,明確限制飲食中蛋氨酸攝取能否通過提高內(nèi)源性H2S表達(dá)發(fā)揮延緩腎臟衰老的作用。并對(duì)機(jī)制進(jìn)行深入研究,檢測(cè)了 SASP的相關(guān)信號(hào)通路AMPK/mTOR的表達(dá)水平,探究H2S對(duì)該通路的調(diào)控作用。本研究對(duì)于探索延緩衰老相關(guān)腎臟疾病新的治療方案具有重要的意義。方法:將12只SPF級(jí)C57BL/6雄性小鼠,正常條件喂養(yǎng)至20月齡后隨機(jī)分為2組:①老年限蛋氨酸組(old methionine restriction,OMR):蛋氨酸限制至0.15%,胱氨酸缺乏(n=6);②老年對(duì)照組(old ad libitum, OAL):正常自由飲食(n=6);兩組間其他條件相同且單位重量的飼料所含總熱量一致。取6只2月齡SPF級(jí)C57BL/6雄性小鼠作為青年對(duì)照組(young ad libitum, YAL)。2個(gè)月后處死小鼠并留取小鼠腎臟病理及分生標(biāo)本,檢測(cè)動(dòng)物生化指標(biāo)。Western blot及ELISA檢測(cè)SASP相關(guān)蛋白含量。體外實(shí)驗(yàn)中,用硫酸吲哚酚(Indoxyl sulfate,IS)誘導(dǎo)人腎小管上皮細(xì)胞衰老,將細(xì)胞分為3組:①對(duì)照組(DMEM)、②IS組(DMEM+250μM IS)、③IS+MR組(250uMIS+蛋氨酸6mg/L、缺乏胱氨酸的DMEM),刺激48小時(shí)后收集細(xì)胞及其上清液,Western blot及ELISA檢測(cè)相關(guān)蛋白含量。免疫熒光檢測(cè)SASP成分IL-1β、IL-8。體內(nèi)外實(shí)驗(yàn)中我們均對(duì)各組AMPK/mTOR信號(hào)通路的表達(dá)情況進(jìn)行了檢測(cè)。隨后我們?yōu)榱蓑?yàn)證AMPK/mTOR通路的關(guān)鍵作用,在培養(yǎng)基中加入AMPK抑制劑Compound C,刺激48小時(shí)后收集細(xì)胞及其上清液,Western blot及ELISA檢測(cè)相關(guān)蛋白含量。結(jié)果:1)體內(nèi)動(dòng)物實(shí)驗(yàn)顯示,OAL組小鼠腎臟出現(xiàn)明顯腎小球硬化、腎小管間質(zhì)損傷、腎小管萎縮及空泡變性、炎癥細(xì)胞浸潤(rùn)等病理改變,而OMR組改變較輕,說明MR可在一定程度上延緩腎臟衰老相關(guān)改變。Western blot結(jié)果顯示,與OMR組相比,OAL組小鼠腎臟中的H2S合成相關(guān)酶CGL降低,衰老相關(guān)標(biāo)志物p16、p53、p21顯著升高,磷酸化AMPK下降、mTOR及下游4E-BP1磷酸化水平明顯升高,說明OMR可降低衰老腎臟中衰老標(biāo)志物表達(dá)并激活A(yù)MPK通路。2)ELISA顯示OMR組較OAL組血液中H2S含量升高,腎臟組織中IL-1β、IL-6水平降低。3)生化結(jié)果顯示白蛋白、肌酐及相關(guān)指標(biāo)組間無統(tǒng)計(jì)學(xué)差異,但OMR組血糖(7.13± 0.48mmol/L)及膽固醇(2.47± 0.36 mmol/L)較 OAL 組的血糖(11.36±1.67mmol/L)與膽固醇(3.61±0.36mmol/L)下降明顯。4)體外實(shí)驗(yàn)顯示,與對(duì)照組相比,IS組衰老標(biāo)志物p53、p21的表達(dá)增加且β半乳糖苷酶染色陽性細(xì)胞增多,證實(shí)了IS對(duì)于衰老的誘導(dǎo)作用。而IS+MR組比IS組H2S合成相關(guān)酶CGL顯著升高,衰老標(biāo)志物p53、p21顯著降低,磷酸化AMPK增多、mTOR及4E-BP1磷酸化顯著降低。5) ELISA顯示IS+MR組細(xì)胞中H2S含量顯著升高,細(xì)胞培養(yǎng)上清中IL-1 β、IL-6顯著降低。6)免疫熒光顯示IL-1 β與IL-8在IS+MR組明顯下降。7)在加入AMPK抑制劑Compound C后,IS+MR+CC組較IS組比較,雖H2S合成相關(guān)酶CGL依舊有所上升,但兩組間衰老標(biāo)志物及AMPK/mTOR關(guān)鍵蛋白表達(dá)量以及IL-1 β、IL-6表達(dá)均無明顯差異,MR的保護(hù)作用減弱,提示AMPK/mTOR可能是H2S發(fā)揮作用的關(guān)鍵通路之一,H2S位于AMPK上游進(jìn)行調(diào)控。結(jié)論:腎臟衰老過程中伴隨著衰老標(biāo)志物及SASP成分(IL-1β、IL-6、IL-8)表達(dá)增加。AMPK/mTOR在SASP的調(diào)節(jié)中具有重要作用,且H2S可對(duì)AMPK發(fā)揮調(diào)控作用。限蛋氨酸飲食可通過提高內(nèi)源性H2S進(jìn)而上調(diào)AMPK,抑制mTOR及下游4E-BP-1的磷酸化、減少腎臟衰老相關(guān)分泌表型的蛋白合成。本研究為今后延緩腎臟衰老可提供新的研究思路與并對(duì)腎臟衰老的防治具有重要意義。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692

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