sirt1 regulates the inflammatory response of vascular adventitial fibroblasts through autophagy and related signaling pathway

Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 DOI: 10.1159/000457878 © 2017 The Author(s) online:February February 2017 ZZZNDUJHUFRPFSE Published online: 03,03, 2017 Published by S Karger AG, Basel and Biochemistry Published www.karger.com/cpb 569 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ Accepted: December 07, 2016 This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) (http://www.karger.com/Services/OpenAccessLicense) Usage and distribution IRUFRPPHUFLDOSXUSRVHVDVZHOODVDQ\GLVWULEXWLRQRIPRGLÀHGPDWHULDOUHTXLUHVZULWWHQSHUPLVVLRQ Original Paper 6,575HJXODWHVWKH,QÁDPPDWRU\ 5HVSRQVHRI9DVFXODU$GYHQWLWLDO )LEUREODVWVWKURXJK$XWRSKDJ\DQG 5HODWHG6LJQDOLQJ3DWKZD\ Wei-Rong Wanga,b Ting-Ting Lic Ting Jingd Yan-Xiang Lid Xiao-Feng Yangd Yan-Hao Hed Wei Zhangd Ji-Ye Zhange Rong Lind a Research Institute of Atherosclerotic Disease, Xi’an Jiaotong University Cardiovascular Research Center, Xi’an, bLaboratory Animal Center, Xi’an Jiaotong University Health Science Center, Xi’an, cDepartment of Pharmacy, Xi'an Chest Hospital, Xi'an, dDepartment of Pharmacology, Xi’an Jiaotong University Health Science Center, Xi’an, eSchool of Pharmacy, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, China H\:RUGV 6,57‡9DVFXODUDGYHQWLWLDOÀEUREODVWV‡$XWRSKDJ\‡,QÁDPPDWLRQ‡$NWP725 Dr Rong Lin, PhD Department of Pharmacology, Xi’an Jiaotong University Health Science Center No.76 Yanta West Road, Xi’an, Shaanxi 710061 (China) Tel +86 29 82657691; Fax +86 29 82657833 E-Mail linrong@mail.xjtu.edu.cn Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM $EVWUDFW Background/Aims: Autophagy is a lysosomal degradation pathway that is essential for cellular survival, differentiation, and homeostasis Sirtuin (SIRT1), a NAD+-dependent deacetylase, plays a pivotal role in modulation of autophagy Recent studies found that autophagy was LQYROYHG LQ WKH UHJXODWLRQ RI LQÁDPPDWRU\ UHVSRQVH ,Q WKLV VWXG\ ZH DLPHG WR GHWHUPLQH WKHHIIHFWRI6,57RQDXWRSKDJ\DQGLQÁDPPDWLRQDQGZKHWKHUDXWRSKDJ\FDQUHJXODWHWKH LQÁDPPDWRU\ UHVSRQVH LQ YDVFXODU DGYHQWLWLDO ÀEUREODVWV 9$)V Methods: Cell autophagy ZDV HYDOXDWHG E\ ÁXRUHVFHQFH PLFURVFRSH DQG WUDQVPLVVLRQ HOHFWURQ PLFURVFRS\ 7KH expression of protein and mRNA were determined by Western blot analysis and real time-PCR The production of cytokine was detected by ELISA Results: 71)įLQGXFHGDXWRSKDJ\DQG LQFUHDVHG6,57H[SUHVVLRQLQ9$)V6,57DFWLYDWRUUHVYHUDWUROHQKDQFHG71)įLQGXFHG9$) autophagy In contrast, SIRT1 knockdown attenuated VAF autophagy Both the Akt inhibitor 0. DQG P725 LQKLELWRU UDSDP\FLQ IXUWKHU LQFUHDVHG 71)įLQGXFHG 9$) DXWRSKDJ\ Furthermore, SIRT1 knockdown increased Akt phosphorylation and inhibited the autophagy in VAFs However, MK2206 attenuated the effect of SIRT1 knockdown on VAF autophagy In addition, ingenuity pathway analysis showed that there is a relationship between cell DXWRSKDJ\DQGLQÁDPPDWLRQ:HIRXQGWKDW6,57NQRFNGRZQLQFUHDVHGWKHH[SUHVVLRQRI 1/53 DQG LQWHUOHXNLQ ,/ DQG SURPRWHG WKH SURGXFWLRQ RI ,/DŽ LQ 9$)V )XUWKHU VWXG\ showed that autophagy activation decreased the expression of NLRP3 and IL-6 and inhibited WKHSURGXFWLRQRI,/DŽZKHUHDVDXWRSKDJ\LQKLELWLRQLQFUHDVHGWKHLQÁDPPDWRU\UHVSRQVHRI VAFs More importantly, our study showed that autophagy was involved in the degradation of NLRP3 through the autophagy-lysosome pathway Conclusion: SIRT1 not only regulates VAF DXWRSKDJ\WKURXJKWKH$NWP725VLJQDOLQJSDWKZD\EXWDOVRVXSSUHVVHVWKHLQÁDPPDWRU\ © 2017 The Author(s) response of VAFs through autophagy Published by S Karger AG, Basel Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 570 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ ,QWURGXFWLRQ Autophagy is an evolutionarily ancient process of intracellular catabolism necessary to preserve cellular homeostasis in response to a wide variety of stress [1] Accumulating evidence suggests that autophagy principally serves an adaptive role to protect organisms against various pathologies, including cancer, aging, and cardiovascular diseases [2-4] A growing number of studies found that the NAD+-dependent class III histone/protein deacetylase sirtuin (SIRT1) was involved in the regulation of autophagy in cardiovascular diseases Overexpression of SIRT1 increased the autophagy of vascular endothelial cells ȋ•Ȍǡ™Š‡”‡ƒ•‘…†‘™‘ˆͳ”‡•—Ž–‡†‹ƒ•‹‰‹ϐ‹…ƒ–†‡…”‡ƒ•‡‹–Š‡ƒ—–‘’Šƒ‰›‘ˆ VECs [5] In addition, SIRT1 also promoted the autophagy of cardiomyocytes [6] The vascular adventitia, a supporting tissue surrounding the blood vessels, is recently found to participate in the initiation and development of atherosclerosis [7, 8] Our previous study showed that ͳ‹Š‹„‹–‡†–Š‡ƒ’‘’–‘•‹•‘ˆ˜ƒ•…—Žƒ”ƒ†˜‡–‹–‹ƒŽϐ‹„”‘„Žƒ•–•ȋ •ȌȏͻȐǤ‘™‡˜‡”ǡ–Š‡”‘Ž‡ ‘ˆ ƒ—–‘’Šƒ‰›‹…ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡•ǡƒ†™Š‡–Š‡”ͳ…ƒ”‡‰—Žƒ–‡–Š‡ƒ—–‘’Šƒ‰› ‘ˆ •”‡ƒ‹—…Ž‡ƒ”Ǥ Recent studies found that autophagy plays an important role in the modulation of ‹ϐŽƒƒ–‘”›”‡•’‘•‡ǡƒ†—†‡”•–ƒ†‹‰–Š‡„ƒŽƒ…‡„‡–™‡‡–Š‡•‡–™‘’”‘…‡••‡•ƒ› point to possible therapeutic targets for diseases [10] The SIRT1 activator resveratrol (RSV) ƒ––‡—ƒ–‡†˜ƒ•…—Žƒ”‡†‘–Š‡Ž‹ƒŽ‹ϐŽƒƒ–‹‘ƒ†‹’”‘˜‡†˜ƒ•…—Žƒ”ˆ—…–‹‘„›‹†—…‹‰ ƒ—–‘’Šƒ‰› ȏͳͳǡ ͳʹȐǡ ™Š‹Ž‡ ͳ ‹Š‹„‹–‘” •‹”–‹‘Ž ’”‘‘–‡† –Š‡ ‹ϐŽƒƒ–‘”› ”‡•’‘•‡ ‘ˆ Š—ƒ Ǧͳ …‡ŽŽ• –Š”‘—‰Š ‹Š‹„‹–‹‘ ‘ˆ ƒ—–‘’Šƒ‰› ȏͳ͵ȐǤ  ”‡…‡– •–—†› •Š‘™‡† –Šƒ– ˆ‡‘ϐ‹„”ƒ–‡’”‡˜‡–‡†Š‹‰Š‰Ž—…‘•‡Ǧ‹†—…‡†‹ϐŽƒƒ–‘”›”‡•’‘•‡‹…ƒ”†‹ƒ…›‘„Žƒ•–•„› upregulating SIRT1-mediated autophagy [14] So we suppose that SIRT1 is involved in the ”‡‰—Žƒ–‹‘‘ˆ‹ϐŽƒƒ–‘”›”‡•’‘•‡‹ •–Š”‘—‰Šƒ—–‘’Šƒ‰›Ǥ–‹•‰‡‡”ƒŽŽ›ƒ……‡’–‡†–Šƒ– the Akt/mTOR signaling pathway is a key regulator of autophagy [15] It has been reported that both hyperphosphatemia and silica nanoparticles could induce the autophagy of VECs through the inhibition of the Akt/mTOR pathway [16, 17] Another study also showed that selectively inhibition of Akt/mTOR pathway activated the autophagy of macrophages and inhibited the atherosclerosis progression [18] Thus, in the present study, we investigated –Š‡‡ˆˆ‡…–•‘ˆͳ‘ƒ—–‘’Šƒ‰›ƒ†‹ϐŽƒƒ–‹‘ǡƒ†™Š‡–Š‡”ƒ—–‘’Šƒ‰›…ƒ”‡‰—Žƒ–‡ –Š‡‹ϐŽƒƒ–‘”›”‡•’‘•‡‹ •Ǥ 0DWHULDOVDQG0HWKRGV Cell culture ƒŽ‡ ’”ƒ‰—‡Ǧƒ™Ž‡› ”ƒ–• ™‡”‡ ƒƒ‡•–Š‡–‹œ‡† ™‹–Š —”‡–Šƒ‡Ǥ ƒ•…—Žƒ” ƒ†˜‡–‹–‹ƒŽ ϐ‹„”‘„Žƒ•–• ™‡”‡ ‹•‘Žƒ–‡†ƒ•†‡•…”‹„‡†’”‡˜‹‘—•Ž›ȏͻȐǤ ”‹‡ϐŽ›ǡthe thoracic aorta were removed and stripped of perivascular …‘‡…–‹˜‡–‹••—‡ǡ–Š‡™‡”‡…Ž‡ƒ‡†™‹–Š’Š‘•’Šƒ–‡„—ˆˆ‡”‡†•ƒŽ‹‡ȋȌ—†‡”•–‡”‹Ž‡…‘†‹–‹‘•ǤŠ‡ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM Materials  ǦȽ™ƒ•’—”…Šƒ•‡†ˆ”‘‡’”‘‡…Šȋ‘…›‹ŽŽǡ ǡȌǤǡ•‹”–‹‘Žƒ†‘‘†ƒ•›Ž…ƒ†ƒ˜‡”‹‡ ȋȌ ™‡”‡ ‘„–ƒ‹‡† ˆ”‘ ‹‰ƒǦŽ†”‹…Š ȋƒ‹– ‘—‹•ǡ ǡ ȌǤ Š‡ ʹʹͲ͸ǡ ”ƒ’ƒ›…‹ǡ ͵Ǧǡ „ƒϐ‹Ž‘›…‹ ͳ ƒ†  Ǧͳ͵ʹ ™‡”‡ ’—”…Šƒ•‡† ˆ”‘ ‡ŽŽ‡……Š‡ ȋ‘—•–‘ǡ ǡ ȌǤ –‹„‘†‹‡• ƒ‰ƒ‹•– ‡…Ž‹ͳǡ͵ƒ†͵™‡”‡‘„–ƒ‹‡†ˆ”‘„…ƒȋƒ„”‹†‰‡ǡǡȌǤŠ‡ͳƒ–‹„‘†›™ƒ•‘”†‡”‡† ˆ”‘ƒ–ƒ”—œ‹‘–‡…Š‘Ž‘‰›ȋƒ–ƒ”—œǡǡȌǤ–‹„‘†‹‡•ƒ‰ƒ‹•––ǡ’Ǧ–ƒ†ȾǦƒ…–‹ƒ•™‡ŽŽƒ• ‰‘ƒ–ƒ–‹Ǧ”ƒ„„‹–‰ Ǧ™‡”‡‘”†‡”‡†ˆ”‘‡ŽŽ‹‰ƒŽ‹‰‡…Š‘Ž‘‰›ȋ‡˜‡”Ž›ǡǡȌǤ‹’‘ˆ‡…–ƒ‹‡ ʹͲͲͲ–”ƒ•ˆ‡…–‹‘”‡ƒ‰‡–™ƒ•’—”…Šƒ•‡†ˆ”‘˜‹–”‘‰‡ȋƒ”Ž•„ƒ†ǡǡȌǤ‹•‘Ž—•ǡ”‹‡…”‹’– ƒ•–‡”‹šƒ†”‡‹ššƒ“ ™‡”‡’—”…Šƒ•‡†ˆ”‘ƒƒ”ƒ‹‘‘’ƒ›ȋƒƒ”ƒǡŠ‹‰ƒǡ ƒ’ƒȌǤ  ”ƒ– ‹–‡”Ž‡—‹ ȋȌǦͳȾ ‡œ›‡ǦŽ‹‡† ‹—‘•‘”„‡– ƒ••ƒ› ȋȌ ‹– ™ƒ• ‘„–ƒ‹‡† ˆ”‘ œ‘ ‹ˆ‡ …‹‡…‡•ȋ ƒ”‹‰†ƒŽ‡ǡǡȌǤŠ‡–ƒ†‡ϐŽ—‘”‡•…‡–Ǧ–ƒ‰‰‡†͵ȋ Ǧ Ǧ͵Ȍ™ƒ•’—”…Šƒ•‡†ˆ”‘ ƒŠ‡‰‹‘–‡…Š‘Ž‘‰›ǡ…ǤȋŠƒ‰Šƒ‹ǡŠ‹ƒȌǤŽŽ‘–Š‡”…Š‡‹…ƒŽ•™‡”‡‘ˆŠ‹‰Š’—”‹–›ƒ†™‡”‡‘„–ƒ‹‡† from commercial sources Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 571 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ media and endothelium were separated from the adventitia and the isolated adventitia was cut into pieces ƒ†’Žƒ…‡†‹—Ž„‡……‘ǯ•‘†‹ϐ‹‡†ƒ‰Ž‡ǯ•‡†‹—ȋȌ•—’’Ž‡‡–‡†™‹–ŠʹͲΨˆ‡–ƒŽ…ƒŽˆ•‡”—ǡͳͲͲ Ȁ’‡‹…‹ŽŽ‹ƒ†ͳͲͲɊ‰Ȁ•–”‡’–‘›…‹‹ƒŠ—‹†‹ϐ‹‡†ƒ–‘•’Š‡”‡™‹–ŠͷΨ2ƒ–͵͹ιǤŠ‡…‡ŽŽ• were characterized by immunocytochemistry MDC labeling The cells were seeded in 24-well plates at 1×105 cells per well After treatment with increasing …‘…‡–”ƒ–‹‘• ‘ˆ  ǦȽ ˆ‘” ʹͶ Šǡ  • ™‡”‡ ‹…—„ƒ–‡† ™‹–Š ͷͲ Ɋ‘ŽȀ  ƒ– ͵͹ι ˆ‘” ʹͲ ‹ ƒ† –Š‡™‡”‡™ƒ•Š‡†™‹–Š–Š”‡‡–‹‡•ƒ†ϐ‹š‡†™‹–ŠͶΨ’ƒ”ƒˆ‘”ƒŽ†‡Š›†‡Ǥ ‹ƒŽŽ›ǡ‹–”ƒ…‡ŽŽ—Žƒ” ϐŽ—‘”‡•…‡…‡Ž‡˜‡Ž•™‡”‡‘„•‡”˜‡†—†‡”ƒŽ›’—•ϐŽ—‘”‡•…‡…‡‹…”‘•…‘’‡Ǥ mRFP-GFP-LC3 assay Š‡…‡ŽŽ•™‡”‡–”ƒ•†—…‡†™‹–Šƒ†‡‘˜‹”—•Šƒ”„‘”‹‰ Ǧ Ǧ͵ƒ–ͷͲͲǤ™‡Ž˜‡Š‘—”•ƒˆ–‡” ƒ†‡‘˜‹”—• –”ƒ•†—…–‹‘ǡ –Š‡ …‡ŽŽ• ™‡”‡ –”‡ƒ–‡†  ǦȽ ˆ‘” ʹͶ ŠǤ Š‡ ”‡•—Ž–• ™‡”‡ ˜‹•—ƒŽ‹œ‡† —•‹‰ Žƒ•‡” •…ƒ‹‰…‘ˆ‘…ƒŽ‹…”‘•…‘’‡ȋ‹‘ǡ‘›‘ǡ ƒ’ƒȌǤ Western blot analysis Š‡…‡ŽŽ•™‡”‡™ƒ•Š‡†‹ƒ†–‘–ƒŽ’”‘–‡‹™ƒ•‡š–”ƒ…–‡†™‹–ŠŽ›•‹•„—ˆˆ‡”…‘–ƒ‹‹‰ʹΨ ’”‘–‡ƒ•‡‹Š‹„‹–‘”…‘…–ƒ‹ŽǤ”‘–‡‹•ƒ’Ž‡•™‡”‡•‡’ƒ”ƒ–‡†„›Ǧ ƒ†–”ƒ•ˆ‡””‡†–‘’‘Ž›˜‹›Ž‹†‡‡ †‹ϐŽ—‘”‹†‡ ‡„”ƒ‡•Ǥ ˆ–‡” „Ž‘…‹‰ ™‹–Š ͷΨ ‘ˆƒ– †”› ‹Žǡ –Š‡ ‡„”ƒ‡• ™‡”‡ ‹…—„ƒ–‡† ™‹–Š •’‡…‹ϐ‹… ƒ–‹„‘†‹‡• ‘˜‡”‹‰Š– ƒ– ͶιǤ Š‡ ’”‹ƒ”› ƒ–‹„‘†‹‡• —•‡† ™‡”‡ ƒ–‹Ǧ͵ ȋ†‹Ž—–‡† ͳǣͷͲͲȌǡ ƒ–‹Ǧ ‡…Ž‹ͳȋ†‹Ž—–‡†ͳǣͳͲͲͲȌǡƒ–‹Ǧͳȋ†‹Ž—–‡†ͳǣͳͲͲȌǡƒ–‹Ǧ–ȋ†‹Ž—–‡†ͳǣͳͲͲͲȌǡƒ–‹Ǧ’Ǧ–ȋ†‹Ž—–‡†ͳǣͳͲͲͲȌ ƒ†ƒ–‹ǦȾǦƒ…–‹ȋ†‹Ž—–‡†ͳǣͶͲͲȌǤŠ‡‡„”ƒ‡•™‡”‡–Š‡‹…—„ƒ–‡†ˆ‘”ʹŠƒ–”‘‘–‡’‡”ƒ–—”‡™‹–Š Ǧ…‘Œ—‰ƒ–‡† ƒ–‹Ǧ”ƒ„„‹– •‡…‘†ƒ”› ‰  ȋ†‹Ž—–‡† ͳǣͳͲǡͲͲͲ ‘” ͳǣͷͲͲͲȌǤ –‹„‘†› „‹†‹‰ ™ƒ• †‡–‡…–‡† ™‹–Š–Š‡‡Šƒ…‡††‡–‡…–‹‘•›•–‡ǤŠ‡”‡Žƒ–‹˜‡‹–‡•‹–›‘ˆ’”‘–‡‹„ƒ†•™ƒ•“—ƒ–‹ϐ‹‡†„›—ƒ–‹–› ‡ƒŽ›•‹•‘ˆ–™ƒ”‡ȋ‹‘ƒ†ǡ‡”…—Ž‡•ǡȌƒ†ȾǦƒ…–‹™ƒ•—•‡†ƒ•ƒ‹–‡”ƒŽ…‘–”‘ŽǤ Quantitative real-time PCR Total RNA was extracted from the cells using the TRIzol reagent cDNA was synthesized using the ”‹‡…”‹’–‡ƒ‰‡–‹–ˆ‘ŽŽ‘™‹‰–Š‡ƒ—ˆƒ…–—”‡”ǯ•‹•–”—…–‹‘•ǤŠ‡’”‹‡”•—•‡†‹”‡ƒŽǦ–‹‡ ™‡”‡†‡•‹‰‡†™‹–Š”‹‡””‡‹‡”ͷǤͲȋ•‡‡ƒ„Ž‡ͳȌǤ…™ƒ•ƒ’Ž‹ϐ‹‡†—•‹‰–Š‡”‡‹ššƒ“ ‹–Ǥ ™ƒ•—•‡†ƒ•ƒ‡†‘‰‡‘—•…‘–”‘Žƒ†”‡Žƒ–‹˜‡‰‡‡‡š’”‡••‹‘™ƒ•“—ƒ–‹–ƒ–‹˜‡Ž›ƒƒŽ›œ‡†„› the comparative Ct method (2-ᇞᇞCTȌǤƒ–ƒ™‡”‡‘”ƒŽ‹œ‡†–‘ Ž‡˜‡Ž•Ǥ Transmission electron microscope Š‡ …‡ŽŽ• ™‡”‡ …‘ŽŽ‡…–‡† „› –”›’•‹‹œƒ–‹‘ ƒ† …‡–”‹ˆ—‰ƒ–‹‘ǡ ƒ† –Š‡ ™‡”‡ ϐ‹š‡† ™‹–Š ʹǤͷΨ ‰Ž—–ƒ”ƒŽ†‡Š›†‡ ƒ† ͳΨ ‘•‹— –‡–”‘š‹†‡ ˆ‘ŽŽ‘™‡† „› †‡Š›†”ƒ–‹‘ ‹ ƒ ƒ•…‡†‹‰ •‡”‹‡• ‘ˆ ‡–Šƒ‘ŽǤ The samples were embedded in Durcopan ACM for h, and ultrathin sections were cut using a Leica Ž–”ƒ‹…”‘–‘‡͸ǡŠ‡•‡…–‹‘•™‡”‡–Š‡•–ƒ‹‡†™‹–Š—”ƒ›Žƒ…‡–ƒ–‡ƒ†Ž‡ƒ†…‹–”ƒ–‡ǡƒ†‡šƒ‹‡† ™‹–Šƒ‡…ƒ‹ 2ͳʹ–”ƒ•‹••‹‘‡Ž‡…–”‘‹…”‘•…‘’‡ȋ ‘’ƒ›ǡ‘ŽŽƒ†ȌǤ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM Table ”‹‡”•‡“—‡…‡—•‡†ˆ‘””‡ƒŽǦ–‹‡ƒƒŽ›•‹• Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 572 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ RNA interference Cells were transfected with SIRT1 siRNA or a negative control (NC siRNA) chemically synthesized by Šƒ‰Šƒ‹ ‡‡Šƒ”ƒ‘”’‘”ƒ–‹‘ȋ ǡŠ‹ƒȌǤŠ‡•‹ƒ†‹’‘ˆ‡…–ƒ‹‡ʹͲͲͲ™‡”‡•‡’ƒ”ƒ–‡Ž›†‹Ž—–‡† in serum-free DMEM and incubated for at room temperature The two solutions were gently mixed ƒ†‹…—„ƒ–‡†ˆ‘”ʹͲ‹ƒ†–Š‡™‡”‡ƒ††‡†–‘–Š‡…‡ŽŽ•Ǥ •™‡”‡–”ƒ•ˆ‡…–‡†™‹–Š•‹Ǧ‹’‘ˆ‡…–ƒ‹‡ …‘’Ž‡š‡•ƒ†‹…—„ƒ–‡†ˆ‘”ͶͺŠƒ–͵͹鋐ƒ2 incubator prior to experimental use Ingenuity pathway analysis ‰‡—‹–› ’ƒ–Š™ƒ› ƒƒŽ›•‹• ȋȌ ™ƒ• ’‡”ˆ‘”‡† „› Šƒ‰Šƒ‹ Ž‘—† …‹‡–‹ϐ‹… ‡…Š‘Ž‘‰› ‘Ǥǡ –† (Shanghai, China) Enzyme-linked immunosorbent assay Š‡…‘…‡–”ƒ–‹‘‘ˆǦͳȾ‹…‡ŽŽ…—Ž–—”‡•—’‡”ƒ–ƒ–•™ƒ•‡ƒ•—”‡†„›ƒ••ƒ›ǤŠ‡ƒ••ƒ›™ƒ• performed according to the manufacturer’s instructions Statistical analysis ƒ–ƒƒ”‡’”‡•‡–‡†ƒ•–Š‡‡ƒάˆ”‘–Š”‡‡‹†‡’‡†‡–‡š’‡”‹‡–•Ǥ–ƒ–‹•–‹…ƒŽ•‹‰‹ϐ‹…ƒ…‡™ƒ• determined using one-way analysis of variance P δͲǤͲͷ™ƒ•…‘•‹†‡”‡†•–ƒ–‹•–‹…ƒŽŽ›•‹‰‹ϐ‹…ƒ–Ǥ  ǦȽ‹†—…‡•ƒ—–‘’Šƒ‰›‹ •  •™‡”‡–”‡ƒ–‡†™‹–Š†‹ˆˆ‡”‡–…‘…‡–”ƒ–‹‘•‘ˆ ǦȽȋʹǤͷǡͷǡͳͲƒ†ʹͲ‰ȀȌˆ‘” ʹͶŠǡƒ†…‡ŽŽƒ—–‘’Šƒ‰›™ƒ•‡˜ƒŽ—ƒ–‡†—•‹‰ϐŽ—‘”‡•…‡…‡‹…”‘•…‘’‡ǡ‡•–‡”„Ž‘–ƒƒŽ›•‹•ǡ ”‡ƒŽ –‹‡Ǧǡ ƒ† –”ƒ•‹••‹‘ ‡Ž‡…–”‘ ‹…”‘•…‘’›Ǥ ‡ ˆ‘—† –Šƒ–  ǦȽ ‹…”‡ƒ•‡† –Š‡ ƒ……——Žƒ–‹‘ ‘ˆ ƒ—–‘’Šƒ‰‹… ˜ƒ…—‘Ž‡• ‹  • …‘’ƒ”‡† ™‹–Š –Š‡ …‘–”‘Ž ‰”‘—’ „›  •–ƒ‹‹‰Ǥ Š‡”‡ ‹• ƒ •‹‰‹ϐ‹…ƒ– ‹…”‡ƒ•‡ ‹ –Š‡ ƒ……——Žƒ–‹‘ ‘ˆ ǦŽƒ„‡Ž‡† ϐŽ—‘”‡•…‡– ’ƒ”–‹…Ž‡• ‹  • •–‹—Žƒ–‡† ™‹–Š  ǦȽ ȋͷ ‰ȀȌ ȋ ‹‰Ǥ ͳȌǤ Š‡ …‘˜‡”•‹‘ ‘ˆ ͵Ϩto ͵ϩ–Š”‘—‰Š’”‘–‡‘Ž›–‹……Ž‡ƒ˜ƒ‰‡‹•”‡‰ƒ”†‡†ƒ•ƒŠƒŽŽƒ”‘ˆƒƒŽ‹ƒƒ—–‘’Šƒ‰›Ǥ‡ –Š‡”‡ˆ‘”‡‹˜‡•–‹‰ƒ–‡†–Š‡”ƒ–‹‘‘ˆ͵Ϩ–‘͵ϩ„›‡•–‡”„Ž‘–ƒƒŽ›•‹•Ǥ••Š‘™‹ ‹‰Ǥ ͳǡ–Š‡”ƒ–‹‘‘ˆ͵ϩȀ͵Ϩ™ƒ•‹…”‡ƒ•‡†™Š‡–Š‡ •™‡”‡•–‹—Žƒ–‡†™‹–Š ǦȽȋͷ ng/mL, PδͲǤͲͳȌǤ ǦȽȋͷ‰ȀȌƒŽ•‘‹…”‡ƒ•‡†–Š‡‡š’”‡••‹‘‘ˆ͵‹ •ȋ ‹‰Ǥ 1C) In addition, we found that Beclin1 exhibited increased levels of protein and mRNA in  ••–‹—Žƒ–‡†™‹–Š ǦȽȋͷƒ†ͳͲ‰ȀȌȋ ‹‰Ǥͳƒ†ȌǤ‡‡š–’‡”ˆ‘”‡†–Š‡ Ǧ Ǧ͵ƒ••ƒ›–‘‹•‹‰Š–‹–‘–Š‡†›ƒ‹…ƒ–—”‡‘ˆƒ—–‘’Šƒ‰‹…ϐŽ—š‹ •Ǥ‡‘„•‡”˜‡†ƒ ƒ……——Žƒ–‹‘‘ˆ›‡ŽŽ‘™’—…–ƒ‹–Š‡…‘–”‘Ž •ǤŠ‡ƒ—–‘’Šƒ‰›™ƒ•‹†—…‡†„› ǦȽ ȋͷ ‰ȀȌǡ –Š‡ ”‡† ’—…–ƒ ™ƒ• ˆ‘—† –‘ ƒ……——Žƒ–‡ ‹  • …‘’ƒ”‡† ™‹–Š –Š‡ …‘–”‘Ž group (PδͲǤͲͳȌǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ– ǦȽƒ…–‹˜ƒ–‡†–Š‡ƒ—–‘’Šƒ‰‹…ϐŽ—š‹ •ȋ ‹‰Ǥ ͳ ǡ ƒ†ȌǤ‘ˆ—”–Š‡”…‘ϐ‹”–Š‡•‡”‡•—Ž–•ǡ–Š‡ƒ—–‘’Šƒ‰‘•‘‡ˆ‘”ƒ–‹‘™ƒ•ƒ••‡••‡† „›–”ƒ•‹••‹‘‡Ž‡…–”‘‹…”‘•…‘’›ǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ– ǦȽȋͷ‰ȀȌƒ”‡†Ž› ‹…”‡ƒ•‡†–Š‡ƒ—–‘’Šƒ‰‘•‘‡ˆ‘”ƒ–‹‘‹ •…‘’ƒ”‡†™‹–Š–Š‡…‘–”‘Ž‰”‘—’ȋ ‹‰ǤͳȌǤ Š‡•‡”‡•—Ž–•‹†‹…ƒ–‡–Šƒ– ǦȽ‹†—…‡• ƒ—–‘’Šƒ‰›„›‹…”‡ƒ•‹‰–Š‡ƒ—–‘’Šƒ‰‹…ϐŽ—š ƒ†–Š‡…‘˜‡”•‹‘ƒ†‡š’”‡••‹‘‘ˆ͵ƒ†‡…Ž‹ͳǡƒ†ƒ—–‘’Šƒ‰‘•‘‡ˆ‘”ƒ–‹‘Ǥ ‘ †‡–‡”‹‡ ™Š‡–Š‡”  ǦȽǦ‹†—…‡† ƒ—–‘’Šƒ‰› ‹• ”‡Žƒ–‡† ™‹–Š ‹…”‡ƒ•‡† ƒ—–‘’Šƒ‰‘•‘‡ ˆ‘”ƒ–‹‘ ‘” †‡…”‡ƒ•‡† ƒ—–‘’Šƒ‰‘•‘ƒŽ ϐŽ—šǡ –Š‡  • ™‡”‡ ’”‡–”‡ƒ–‡† ™‹–Š Ž›•‘•‘‡ ‹Š‹„‹–‘” „ƒϐ‹Ž‘›…‹ ͳ ȋʹͲ Ȍ ‘” Ž›•‘•‘ƒŽ ’”‘–‡ƒ•‡ ‹Š‹„‹–‘”  Ǧͳ͵ʹ ȋͳͲ—Ȍˆ‘”ͳŠƒ†™‡”‡‡š’‘•‡†–‘ ǦȽȋͷ‰ȀȌˆ‘”ƒƒ††‹–‹‘ƒŽʹͶŠǡ”‡•’‡…–‹˜‡Ž›Ǥ Š‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩƒ†–Š‡‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ‹ •™‡”‡†‡–‡…–‡†„› ‡•–‡”„Ž‘–ƒƒŽ›•‹•ǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ–„ƒϐ‹Ž‘›…‹ͳ‹…”‡ƒ•‡†–Š‡…‘˜‡”•‹‘‘ˆ ͵Ϩ–‘ ͵ϩ ƒ† –Š‡ ‡š’”‡••‹‘ ‘ˆ ‡…Ž‹ͳ …‘’ƒ”‡† ™‹–Š –Š‡  ǦȽ ‰”‘—’Ǥ ‘™‡˜‡”ǡ  Ǧͳ͵ʹ Šƒ• ‘ ‡ˆˆ‡…– ‘ –Š‡ …‘˜‡”•‹‘ ‘ˆ ͵Ϩ–‘ ͵ϩand the expression of Beclin1 ȋ ‹‰Ǥͳ ƒ†ȌǤŠ‡•‡”‡•—Ž–••—‰‰‡•––Šƒ– ǦȽ‹†—…‡•–Š‡ˆ‘”ƒ–‹‘ƒ†ƒ–—”ƒ–‹‘‘ˆ ƒ—–‘’Šƒ‰‘•‘‡•‹ •Ǥ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM Results Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 573 Fig  ǦȽ‹†—…‡•ƒ—–‘’Šƒ‰›‹ •Ǥ •™‡”‡–”‡ƒ–‡†™‹–Š ǦȽƒ–…‘…‡–”ƒ–‹‘•‘ˆʹǤͷǡͷǡͳͲƒ† ʹͲ‰Ȁˆ‘”ʹͶŠǤȋȌŠ‡ƒ……——Žƒ–‹‘‘ˆƒ—–‘’Šƒ‰‹…˜ƒ…—‘Ž‡•™ƒ•‡˜ƒŽ—ƒ–‡†„›•–ƒ‹‹‰ȋ•‹œ‡„ƒ”ǣ ʹͲɊȌǤȋƒ†ȌŠ‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩƒ†–Š‡‡š’”‡••‹‘‘ˆ͵™‡”‡ƒ••‡••‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹•ƒ†”‡ƒŽ–‹‡Ǧǡ”‡•’‡…–‹˜‡Ž›Ǥȋƒ†ȌŠ‡‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ’”‘–‡‹ƒ† ™‡”‡ ƒ••‡••‡† „› ‡•–‡” „Ž‘– ƒƒŽ›•‹• ƒ† ”‡ƒŽ –‹‡Ǧǡ ”‡•’‡…–‹˜‡Ž›Ǥ ȋ Ȍ ‡ŽŽ• ™‡”‡ –”ƒ•†—…‡† ™‹–Š  Ǧ Ǧ͵ˆ‘”ͳʹŠǤŠ‡–Š‡…‡ŽŽ•™‡”‡–”‡ƒ–‡†™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǤŠ‡‡”‰‡†‹ƒ‰‡• ȋ›‡ŽŽ‘™Ȍ•Š‘™‘˜‡”Žƒ›‘ˆ Ǧ͵ȋ‰”‡‡Ȍƒ† Ǧ͵ȋ”‡†Ȍȋ•‹œ‡„ƒ”ǣͺɊȌǤȋ Ȍ‡ƒ—„‡”‘ˆ  ƒ† †‘–•’‡”…‡ŽŽǤȋȌ‡ƒ—„‡”‘ˆƒ—–‘’Šƒ‰‘•‘‡•”‡’”‡•‡–‡†„››‡ŽŽ‘™†‘–•‹‡”‰‡†‹ƒ‰‡• ƒ†ƒ—–‘Ž›•‘•‘‡•”‡’”‡•‡–‡†„›”‡††‘–•‹‡”‰‡†‹ƒ‰‡•’‡”…‡ŽŽȋα͵Ͳ…‡ŽŽ•’‡”…‘†‹–‹‘Ȍˆ”‘–Š”‡‡ ‹†‡’‡†‡–‡š’‡”‹‡–•ǤȋȌ •™‡”‡–”‡ƒ–‡†™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǡƒ†–Š‡ƒ—–‘’Šƒ‰‘•‘‡ ˆ‘”ƒ–‹‘™ƒ•ƒ••‡••‡†„›–”ƒ•‹••‹‘‡Ž‡…–”‘‹…”‘•…‘’›ƒ–έͶͲͲͲͲƒ‰‹ϐ‹…ƒ–‹‘Ǥȋ ƒ†Ȍ •™‡”‡ ’”‡–”‡ƒ–‡†™‹–ŠŽ›•‘•‘‡‹Š‹„‹–‘”„ƒϐ‹Ž‘›…‹ͳȋʹͲȌƒ†Ž›•‘•‘ƒŽ’”‘–‡ƒ•‡‹Š‹„‹–‘” Ǧͳ͵ʹȋͳͲ —Ȍˆ‘”ͳŠƒ†™‡”‡‡š’‘•‡†–‘ ǦȽˆ‘”ƒƒ††‹–‹‘ƒŽʹͶŠǡ”‡•’‡…–‹˜‡Ž›ǤŠ‡…‘˜‡”•‹‘‘ˆ͵–‘͵  ƒ†–Š‡‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ‹ •™‡”‡†‡–‡…–‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹•ǤŠ‡”‡•—Ž–•ƒ”‡‡š’”‡••‡†ƒ• –Š‡‡ƒάǡα͵ǤȗPδͲǤͲͷƒ†ȗȗP < 0.01 vs the control group #P < 0.05 vs.–Š‡ ǦȽ‰”‘—’Ǥ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 574 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ ͷ’”‘‘–‡• ǦȽǦ‹†—…‡† ƒ—–‘’Šƒ‰› ‘‹˜‡•–‹‰ƒ–‡–Š‡‡ˆˆ‡…–‘ˆ ǦȽ‘SIRT1‡š’”‡••‹‘ǡ •™‡”‡–”‡ƒ–‡†™‹–Š ǦȽ (2.5, 5, 10, 20 ng/mL) for 24 h, and the expression of SIRT1™‡”‡ƒ••‡••‡†„›‡•–‡”„Ž‘– ƒƒŽ›•‹•ƒ†”‡ƒŽ–‹‡ǦǤ‡ˆ‘—†–Šƒ–Ž‘™…‘…‡–”ƒ–‹‘•‘ˆ ǦȽȋʹǤͷƒ†ͷ‰ȀȌ increased the expression of SIRT1 protein, whereas SIRT1 ‡š’”‡••‹‘™ƒ•†‡…”‡ƒ•‡†‹ • •–‹—Žƒ–‡†™‹–Š ǦȽȋʹͲ‰ȀȌȋ ‹‰ǤʹȌǤŠ‡‡š’”‡••‹‘‘ˆSIRT1 mRNA was increased ™Š‡–Š‡ •™‡”‡•–‹—Žƒ–‡†™‹–Š ǦȽȋͷƒ†ͳͲ‰ȀǡP < 0.05) compared with the …‘–”‘Ž‰”‘—’ȋ ‹‰ǤʹȌǤ To assess the role of SIRT1‹ ƒ—–‘’Šƒ‰›ǡ–Š‡…‡ŽŽ•™‡”‡’”‡–”‡ƒ–‡†™‹–Š–Š‡SIRT1 ƒ…–‹˜ƒ–‘”ȋʹͲɊȌ‘”–Š‡‹Š‹„‹–‘”•‹”–‹‘ŽȋͳͷɊȌˆ‘”ͳŠƒ†™‡”‡‡š’‘•‡†–‘ ǦȽȋͷ ‰ȀȌˆ‘”ƒƒ††‹–‹‘ƒŽʹͶŠǤ‡ˆ‘—†–Šƒ–ˆ—”–Š‡”‹…”‡ƒ•‡†–Š‡‡š’”‡••‹‘‘ˆͳ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM Fig The effects of ͳƒ…–‹˜ƒ–‘”ƒ†‹Š‹„‹–‘”‘ ǦȽǦ‹†—…‡† ƒ—–‘’Šƒ‰›Ǥȋƒ†Ȍ •™‡”‡ –”‡ƒ–‡†™‹–Š ǦȽƒ–…‘…‡–”ƒ–‹‘•‘ˆʹǤͷǡͷǡͳͲƒ†ʹͲ‰Ȁˆ‘”ʹͶŠǡƒ†–Š‡‡š’”‡••‹‘‘ˆͳ’”‘–‡‹ƒ†™‡”‡ƒ••‡••‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹•ƒ†”‡ƒŽ–‹‡Ǧǡ”‡•’‡…–‹˜‡Ž›Ǥ •™‡”‡’”‡–”‡ƒted with the SIRTͳƒ…–‹˜ƒ–‘”ȋʹͲɊȌ‘”–Š‡‹Š‹„‹–‘”•‹”–‹‘ŽȋͳͷɊȌˆ‘”ͳŠƒ†–Š‡‡š’‘•‡†–‘ ǦȽ (5 ng/mL) for an additional 24 h (C and D) The expression of SIRT1 protein and mRNA were determined (E ƒ† ȌŠ‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩƒ†–Š‡‡š’”‡••‹‘‘ˆ͵™‡”‡†‡–‡”‹‡†Ǥȋ ƒ†ȌŠ‡ ‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ’”‘–‡‹ƒ†™‡”‡†‡–‡”‹‡†ǤŠ‡”‡•—Ž–•ƒ”‡‡š’”‡••‡†ƒ•–Š‡‡ƒάǡα͵Ǥ ȗPδͲǤͲͷƒ†ȗȗP < 0.01 vs the control group #P < 0.05 and ##P < 0.01 vs.–Š‡ ǦȽ‰”‘—’Ǥ Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 575 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ Fig ͳ ‘…†‘™ ‹Š‹„‹–•  ǦȽǦ‹†—…‡†   ƒ—–‘’Šƒ‰›Ǥ ȋ ƒ† Ȍ  • ™‡”‡ –”ƒ•ˆ‡…–‡† ™‹–Š ͳ•‹‘”•‹ˆ‘”ͶͺŠǡ–Š‡‡š’”‡••‹‘‘ˆͳ™‡”‡ƒ••‡••‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹•ƒ†”‡ƒŽ –‹‡ǦǤ •™‡”‡–”ƒ•ˆ‡…–‡†™‹–Šͳ•‹ƒ†™‡”‡–Š‡–”‡ƒ–‡†™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǡȋ ƒ†ȌŠ‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩƒ†–Š‡‡š’”‡••‹‘‘ˆ͵™‡”‡†‡–‡”‹‡†Ǥȋƒ† ȌŠ‡ ‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ’”‘–‡‹ƒ†™‡”‡†‡–‡”‹‡†ǤŠ‡”‡•—Ž–•ƒ”‡‡š’”‡••‡†ƒ•–Š‡‡ƒάǡα͵Ǥ ȗPδͲǤͲͷƒ†ȗȗP < 0.01 vs the NC siRNA group ##P < 0.01 vs.–Š‡•‹Ϊ ǦȽ‰”‘—’Ǥ Š‡–Ȁ‹•‹˜‘Ž˜‡†‹–Š‡ ǦȽǦ‹†—…‡†ƒ—–‘’Šƒ‰›‹ • ‘ ‹˜‡•–‹‰ƒ–‡ ™Š‡–Š‡” –Š‡ –Ȁ •‹‰ƒŽ‹‰ ’ƒ–Š™ƒ› ‹• ‹˜‘Ž˜‡† ‹ –Š‡  ǦȽǦ ‹†—…‡† ƒ—–‘’Šƒ‰›ǡ–Š‡ •™‡”‡’”‡–”‡ƒ–‡†™‹–Š ǦȽƒ–†‹ˆˆ‡”‡–…‘…‡–”ƒ–‹‘• ȋʹǤͷǡͷǡͳͲǡʹͲ‰ȀȌˆ‘”ʹͶŠǤŠ‡’Š‘•’Š‘”›Žƒ–‹‘‘ˆ–™ƒ•–Š‡ƒ••‡••‡†„›‡•–‡” „Ž‘–ƒƒŽ›•‹•ǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ– ǦȽƒ”‡†Ž›†‡…”‡ƒ•‡†–Š‡’Š‘•’Š‘”›Žƒ–‹‘‘ˆ– ‹ƒ…‘…‡–”ƒ–‹‘Ǧ†‡’‡†‡–ƒ‡”‹ •ǤŠ‡”‡™ƒ•ƒ•–ƒ–‹•–‹…ƒŽŽ›•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡ ‹–Š‡’Š‘•’Š‘”›Žƒ–‹‘‘ˆ–„‡–™‡‡–Š‡ ǦȽ‰”‘—’•ƒ†–Š‡…‘–”‘Ž‰”‘—’ȋͷƒ†ͳͲ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM ȋ ‹‰Ǥʹƒ†Ȍǡƒ†–Š‡’”‘‘–‡†–Š‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩand Beclin1 expression …‘’ƒ”‡† ™‹–Š –Š‡  ǦȽ ‰”‘—’ ȋ ‹‰Ǥ ʹǡ ǡ  ƒ† ȌǤ  …‘–”ƒ•–ǡ •‹”–‹‘Ž †‡…”‡ƒ•‡† –Š‡ ‡š’”‡••‹‘‘ˆͳǡƒ†ƒ––‡—ƒ–‡†–Š‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩand Beclin1 expression ‹ •ȋ ‹‰Ǥʹǡǡǡ ǡ ƒ†ȌǤ ‘ˆ—”–Š‡”…‘ϐ‹”–Š‡‡ˆˆ‡…–‘ˆͳ‘ ƒ—–‘’Šƒ‰›ǡ •™‡”‡–”ƒ•ˆ‡…–‡†™‹–Š SIRT1 siRNA or NC siRNA for 48 h, and then the expression of SIRT1 protein and mRNA ™‡”‡‡ƒ•—”‡†ǤŠ‡‘…†‘™ ‡ˆϐ‹…‹‡…›‘ˆͳ™ƒ• ƒ’’”‘š‹ƒ–‡Ž› ͹ͷΨǡƒ• ƒ••‡••‡† „› ‡•–‡” „Ž‘– ƒƒŽ›•‹• ƒ† ”‡ƒŽ –‹‡Ǧ ȋ ‹‰Ǥ ͵ ƒ† ȌǤ  • –”ƒ•ˆ‡…–‡† ™‹–Š ͳ •‹™‡”‡–Š‡–”‡ƒ–‡†™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǡ–Š‡‡š’”‡••‹‘‘ˆ͵ƒ†‡…Ž‹ͳ were determined The results showed that SIRT1 knockdown decreased the conversion of ͵Ϩ–‘͵ϩƒ†–Š‡‡š’”‡••‹‘‘ˆ͵‹ •…‘’ƒ”‡†™‹–Š–Š‡ ǦȽ‰”‘—’ȋP δͲǤͲͳǡ ‹‰Ǥ͵ƒ†ȌǤƒ††‹–‹‘ǡͳ‘…†‘™ƒŽ•‘‹Š‹„‹–‡†–Š‡‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ ’”‘–‡‹ƒ†‹ •ȋPδͲǤͲͳǡ ‹‰Ǥ͵ƒ† ȌǤƒ‡–‘‰‡–Š‡”ǡ–Š‡•‡”‡•—Ž–•‹†‹…ƒ–‡ –Šƒ–ͳ’”‘‘–‡• ǦȽǦ‹†—…‡† ƒ—–‘’Šƒ‰›Ǥ Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 576 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ Fig Š‡ –Ȁ ‹• ‹˜‘Ž˜‡† ‹ –Š‡  ǦȽǦ‹†—…‡† ƒ—–‘’Šƒ‰› ‹  •Ǥ ȋȌ  • ™‡”‡ –”‡ƒ–‡† ™‹–Š  ǦȽƒ–…‘…‡–”ƒ–‹‘•‘ˆʹǤͷǡͷǡͳͲƒ†ʹͲ‰Ȁˆ‘”ʹͶŠǡ–Š‡–Š‡’”‘–‡‹‡š’”‡••‹‘‘ˆ’Ǧ–ƒ†– ™‡”‡ ƒ••‡••‡†„› ‡•–‡”„Ž‘– ƒƒŽ›•‹•Ǥ • ™‡”‡ ’”‡–”‡ƒ–‡†™‹–Š ʹʹͲ͸ȋͷɊȌ ˆ‘”ͳ Š ’”‹‘”–‘ •–‹—Žƒ–‹‘™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǡȋȌ–Š‡’”‘–‡‹‡š’”‡••‹‘‘ˆ’Ǧ–ƒ†–™‡”‡†‡–‡”‹‡†Ǥȋ ƒ†ȌŠ‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩƒ†–Š‡‡š’”‡••‹‘‘ˆ͵™‡”‡†‡–‡”‹‡†Ǥȋƒ† ȌŠ‡ ‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ’”‘–‡‹ƒ†™‡”‡†‡–‡”‹‡†Ǥ •™‡”‡’”‡–”‡ƒ–‡†™‹–Š–Š‡‹Š‹„‹–‘” ”ƒ’ƒ›…‹ȋͳͲͲȌˆ‘”ͳŠƒ†–Š‡‡š’‘•‡†–‘ ǦȽȋͷ‰ȀȌˆ‘”ƒƒ††‹–‹‘ƒŽʹͶŠǤȋ ƒ†ȌŠ‡ …‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩƒ†–Š‡‡š’”‡••‹‘‘ˆ͵™‡”‡†‡–‡”‹‡†Ǥȋƒ† ȌŠ‡‡š’”‡••‹‘‘ˆ ‡…Ž‹ͳ’”‘–‡‹ƒ†™‡”‡†‡–‡”‹‡†ǤŠ‡”‡•—Ž–•ƒ”‡‡š’”‡••‡†ƒ•–Š‡‡ƒάǡα͵ǤȗP < 0.05 and ȗȗP < 0.01 vs the control group #P < 0.05 and ##P < 0.01 vs –Š‡ ǦȽ‰”‘—’Ǥ ͷ”‡‰—Žƒ–‡• ƒ—–‘’Šƒ‰›–Š”‘—‰Š–Š‡–Ȁ•‹‰ƒŽ‹‰’ƒ–Š™ƒ› ‹˜‡–Š‡•‡”‡•—Ž–•ǡ‹–‹•‡…‡••ƒ”›–‘‡š’Ž‘”‡™Š‡–Š‡”ͳ”‡‰—Žƒ–‡• ƒ—–‘’Šƒ‰› –Š”‘—‰Š–Š‡–Ȁ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›Ǥ •™‡”‡–”ƒ•ˆ‡…–‡†™‹–Šͳ•‹‘” Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM ng/mL, P < 0.05; 20 ng/mL, PδͲǤͲͳǢ ‹‰ǤͶȌǤŠ‡ǡ •™‡”‡’”‡–”‡ƒ–‡†™‹–Š–‹Š‹„‹–‘” ʹʹͲ͸ˆ‘”ͳŠ’”‹‘”–‘•–‹—Žƒ–‹‘™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ– ʹʹͲ͸‹Š‹„‹–‡†–Š‡’Š‘•’Š‘”›Žƒ–‹‘‘ˆ–ȋ ‹‰ǤͶȌƒ†‹…”‡ƒ•‡†–Š‡’”‘–‡‹ƒ† ‡š’”‡••‹‘‘ˆ͵ƒ†‡…Ž‹ͳ…‘’ƒ”‡†™‹–Š–Š‡ ǦȽ‰”‘—’ȋPδͲǤͲͷǡ ‹‰ǤͶǡǡƒ† ȌǤ Š‡•‡”‡•—Ž–•‹†‹…ƒ–‡–Šƒ––‹Š‹„‹–• ǦȽǦ‹†—…‡†ƒ—–‘’Šƒ‰›‹ •Ǥ ‘†‡–‡”‹‡™Š‡–Š‡”‹•ƒŽ•‘‹˜‘Ž˜‡†‹–Š‡”‡‰—Žƒ–‹‘‘ˆ ƒ—–‘’Šƒ‰›ǡ–Š‡ mTOR inhibitor rapamycin (100 nM) was used The results showed that rapamycin further ‹…”‡ƒ•‡† ƒ—–‘’Šƒ‰›‹†—…‡†„› ǦȽȋPδͲǤͲͷǡ ‹‰ǤͶ ǡǡƒ† ȌǤŠ‡•‡ƒ„‘˜‡”‡•—Ž–• ‹†‹…ƒ–‡–Šƒ––Š‡–Ȁ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›‹Š‹„‹–• ǦȽǦ‹†—…‡†ƒ—–‘’Šƒ‰›‹ •Ǥ Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 577 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ Fig ͳ”‡‰—Žƒ–‡• ƒ—–‘’Šƒ‰›–Š”‘—‰Š–Š‡–Ȁ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›Ǥ •™‡”‡–”ƒ•ˆ‡…–‡† ™‹–Šͳ•‹‘”•‹ˆ‘”ͶͺŠ’”‹‘”–‘•–‹—Žƒ–‹‘™‹–Š ǦȽˆ‘”ʹͶŠǡȋȌ–Š‡’Š‘•’Š‘”›Žƒ–‹‘‘ˆ –™ƒ•ƒ••‡••‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹•ǤȗPδͲǤͲͷƒ†ȗȗP < 0.01 vs the NC siRNA group #P < 0.05 vs the •‹Ϊ ǦȽ‰”‘—’Ǥ •–”ƒ•ˆ‡…–‡†™‹–Šͳ•‹™‡”‡’”‡–”‡ƒ–‡†™‹–ŠʹʹͲ͸ˆ‘”ͳŠ’”‹‘”–‘ •–‹—Žƒ–‹‘™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǡƒ†ȋƒ†Ȍ–Š‡…‘˜‡”•‹‘‘ˆ͵Ϩ–‘͵ϩ and the expressi‘‘ˆ͵™‡”‡†‡–‡”‹‡†Ǥȋƒ†ȌŠ‡‡š’”‡••‹‘‘ˆ‡…Ž‹ͳ’”‘–‡‹ƒ†™‡”‡†‡–‡”‹‡†Ǥ ȗPδͲǤͲͷƒ†ȗȗP < 0.01 vs.–Š‡•‹Ϊ ǦȽ‰”‘—’Ǥ#P < 0.05 and ##P < 0.01 vs.–Š‡ͳ•‹Ϊ ǦȽ ‰”‘—’Ǥ‡•—Ž–•ƒ”‡‡š’”‡••‡†ƒ•–Š‡‡ƒάǡα͵Ǥ ͷ•—’’”‡••‡•–Š‡‹ϔŽƒƒ–‘”›”‡•’‘•‡‘ˆ •–Š”‘—‰Šƒ—–‘’Šƒ‰› •‘ˆ–™ƒ”‡Ž‹••’‡…‹ϐ‹…‰‡‡•–‘ƒ†ƒ–ƒ„ƒ•‡‘ˆ‰‡‡ˆ—…–‹‘•‰Ž‡ƒ‡†ˆ”‘„‹‘‡†‹…ƒŽ ”‡•‡ƒ”…ŠŽ‹–‡”ƒ–—”‡Ǥ”‹‡ϐŽ›ǡƒ•‡–‘ˆ‰‡‡•‹†‡–‹ϐ‹‡†„›ƒ—„‡†•‡ƒ”…Š™ƒ•—’Ž‘ƒ†‡†‹–‘ ƒ ™‡„Ǧ„ƒ•‡† ƒ’’Ž‹…ƒ–‹‘ ƒ† ‡ƒ…Š ‰‡‡ ‹†‡–‹ϐ‹‡” ™ƒ• ƒ’’‡† –‘ ‹–• …‘””‡•’‘†‹‰ ‰‡‡ ‘„Œ‡…–‹–Š‡‰‡—‹–›‘™Ž‡†‰‡ƒ•‡ǤŠ‡‹–‡”ƒ…–‹‘•„‡–™‡‡ƒ—–‘’Šƒ‰›ƒ†•‹”–—‹• ‹…ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡•‹–Š‡‰‡—‹–›‘™Ž‡†‰‡ƒ•‡™‡”‡…‘•‹†‡”‡†ǤŠ‡”‡•—Ž–• indicated that SIRT1 played a regulatory role in cell autophagy, which was consistent with our ƒ„‘˜‡”‡•—Ž–•Ǥ‡ƒ™Š‹Ž‡ǡ™‡ˆ‘—†–Šƒ–ͳ‘†—Žƒ–‡†–Š‡’”‘†—…–‹‘‘ˆ‹ϐŽƒƒ–‘”› Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM •‹ͶͺŠ’”‹‘”–‘•–‹—Žƒ–‹‘™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǤŠ‡”‡•—Ž–•”‡˜‡ƒŽ‡†–Šƒ– ͳ ‘…†‘™ ƒ—‰‡–‡† –Š‡ ’Š‘•’Š‘”›Žƒ–‹‘ ‘ˆ – ‹  • •–‹—Žƒ–‡† ™‹–Š  ǦȽ ȋ ‹‰Ǥ ͷȌǤ Š‡ǡ  • –”ƒ•ˆ‡…–‡† ™‹–Š ͳ •‹ ™‡”‡ ’”‡–”‡ƒ–‡† ™‹–Š ʹʹͲ͸ ˆ‘” ͳ Š ’”‹‘” –‘ •–‹—Žƒ–‹‘ ™‹–Š  ǦȽ ˆ‘” ʹͶ ŠǤ Š‡ ”‡•—Ž–• ‹†‹…ƒ–‡† –Šƒ– ͳ ‘…†‘™ ‹Š‹„‹–‡† –Š‡  ǦȽǦ‹†—…‡† ‡š’”‡••‹‘ ‘ˆ ͵ ƒ† ‡…Ž‹ͳ ‹  •Ǥ ‘™‡˜‡”ǡ ʹʹͲ͸ ƒ––‡—ƒ–‡†–Š‡‡ˆˆ‡…–‘ˆͳ‘…†‘™‘–Š‡‡š’”‡••‹‘‘ˆ͵ƒ†‡…Ž‹ͳȋ ‹‰Ǥͷǡǡ ƒ†ȌǤŠ‡•‡”‡•—Ž–••—‰‰‡•––Šƒ–ͳ”‡‰—Žƒ–‡• ƒ—–‘’Šƒ‰›–Š”‘—‰Š–Š‡‹Š‹„‹–‹‘ of Akt/mTOR signaling pathway Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 578 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ …›–‘‹‡Ǧ͸ƒ†ǦͳȾ„›ƒƒŽ›•‹•ȋ ‹‰Ǥ͸ȌǤ‡–Š‡”‡ˆ‘”‡ˆ‘…—•‡†‘—”•–—†›‘–Š‡”‘Ž‡ ‘ˆͳ‹–Š‡‡š’”‡••‹‘‘ˆ͵ƒ†Ǧ͸ƒ†–Š‡’”‘†—…–‹‘‘ˆǦͳȾ‹ •Ǥ—””‡•—Ž–• •Š‘™‡†–Šƒ–ͳ‘…†‘™‹…”‡ƒ•‡†–Š‡‡š’”‡••‹‘‘ˆ͵’”‘–‡‹ƒ†Ǧ͸ ƒ†’”‘‘–‡†–Š‡’”‘†—…–‹‘‘ˆǦͳȾ‹ ••–‹—Žƒ–‡†™‹–Š ǦȽȋ ‹‰Ǥ͸ǡƒ†ȌǤ  ƒ††‹–‹‘ǡ –Š‡  ƒƒŽ›•‹• ”‡˜‡ƒŽ‡† –Šƒ– –Š‡”‡ ™ƒ• ƒ ”‡Žƒ–‹‘•Š‹’ „‡–™‡‡ …‡ŽŽ ƒ—–‘’Šƒ‰› ƒ† ‹ϐŽƒƒ–‘”› …›–‘‹‡ ǦͳȾ ƒ† Ǧ͸ ȋ ‹‰Ǥ ͸ȌǤ ‘ ‹˜‡•–‹‰ƒ–‡ ™Š‡–Š‡” ƒ—–‘’Šƒ‰›…ƒ”‡‰—Žƒ–‡–Š‡‹ϐŽƒƒ–‘”›”‡•’‘•‡‘ˆ •ǡ–Š‡ •™‡”‡’”‡–”‡ƒ–‡†™‹–Š ƒ—–‘’Šƒ‰›ƒ…–‹˜ƒ–‘””ƒ’ƒ›…‹ȋͳͲͲȌ‘”ƒ—–‘’Šƒ‰›‹Š‹„‹–‘”͵ǦȋͷȌˆ‘”ͳŠ’”‹‘” Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM Fig ͳ•—’’”‡••‡•–Š‡‹ϐŽƒƒ–‘”›”‡•’‘•‡‘ˆ •–Š”‘—‰Šƒ—–‘’Šƒ‰›ǤȋȌ‰‡—‹–›’ƒ–Š™ƒ›ƒƒŽ›•‹•™ƒ•—•‡†–‘‹†‡–‹ˆ›–Š‡”‡Žƒ–‹‘•Š‹’•ƒ‘‰•‹”–—‹•ǡƒ—–‘’Šƒ‰›ƒ†‹ϐŽƒƒ–‹‘‹…ƒ”†‹‘˜ƒ•…—Žƒ” †‹•‡ƒ•‡•Ǥȋǡƒ†Ȍ •™‡”‡–”ƒ•ˆ‡…–‡†™‹–Šͳ•‹‘”•‹ˆ‘”ͶͺŠ’”‹‘”–‘•–‹—Žƒ–‹‘ ™‹–Š ǦȽˆ‘”ʹͶŠǡ–Š‡‡š’”‡••‹‘‘ˆ͵’”‘–‡‹ƒ†Ǧ͸™ƒ•ƒ••‡••‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹• ƒ†”‡ƒŽ–‹‡Ǧǡ”‡•’‡…–‹˜‡Ž›ǤŠ‡ǦͳȾ’”‘†—…–‹‘™ƒ•ƒ••‡••‡†„›ǤȗPδͲǤͲͷƒ†ȗȗP < 0.01 vs the NC siRNA group #P < 0.05 and ##P < 0.01 vs.–Š‡•‹Ϊ ǦȽ‰”‘—’Ǥȋǡ ƒ† Ȍ •™‡”‡’”‡–”‡ƒ–‡† ™‹–Š–Š‡ƒ—–‘’Šƒ‰›ƒ…–‹˜ƒ–‘””ƒ’ƒ›…‹ȋͳͲͲȌ‘”–Š‡ƒ—–‘’Šƒ‰›‹Š‹„‹–‘”͵ǦȋͷȌˆ‘”ͳŠ’”‹‘”–‘ •–‹—Žƒ–‹‘™‹–Š ǦȽˆ‘”ʹͶŠǡ–Š‡‡š’”‡••‹‘‘ˆ͵’”‘–‡‹ƒ†Ǧ͸™ƒ•ƒ••‡••‡†„›‡•–‡” „Ž‘–ƒƒŽ›•‹•ƒ†”‡ƒŽ–‹‡Ǧǡ”‡•’‡…–‹˜‡Ž›ǤŠ‡ǦͳȾ’”‘†—…–‹‘™ƒ•ƒ••‡••‡†„›ǤȗP < 0.05 and ȗȗP < 0.01 vs the control group #P < 0.05 and ##P < 0.01 vs.–Š‡ ǦȽ‰”‘—’ǤȋȌ •™‡”‡’”‡–”‡ƒ–‡†™‹–Š ’”‘–‡ƒ•‘ƒŽ‹Š‹„‹–‘” Ǧͳ͵ʹ‘”ƒ—–‘Ž›•‘•‘ƒŽ‹Š‹„‹–‘”„ƒϐ‹Ž‘›…‹ͳˆ‘”ͳŠƒ†™‡”‡‡š’‘•‡†–‘ ǦȽ ˆ‘”ƒƒ††‹–‹‘ƒŽʹͶŠǡ–Š‡’”‘–‡‹‡š’”‡••‹‘‘ˆ͵™ƒ•†‡–‡…–‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹•ǤŠ‡”‡•—Ž–• ƒ”‡‡š’”‡••‡†ƒ•–Š‡‡ƒάǡα͵Ǥ Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 579 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ –‘•–‹—Žƒ–‹‘™‹–Š ǦȽȋͷ‰ȀȌˆ‘”ʹͶŠǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ–”ƒ’ƒ›…‹†‡…”‡ƒ•‡† –Š‡ ‡š’”‡••‹‘ ‘ˆ ͵ ’”‘–‡‹ ƒ† Ǧ͸  ƒ† ‹Š‹„‹–‡† –Š‡ ’”‘†—…–‹‘ ‘ˆ ǦͳȾǡ ™Š‡”‡ƒ•͵Ǧ‹…”‡ƒ•‡†–Š‡‡š’”‡••‹‘‘ˆ͵’”‘–‡‹ƒ†Ǧ͸ǡƒ†’”‘‘–‡†–Š‡ ’”‘†—…–‹‘‘ˆǦͳȾ…‘’ƒ”‡†™‹–Š–Š‡ ǦȽ‰”‘—’ȋ ‹‰Ǥ͸ǡ ƒ† ȌǤŠ—•ǡ–Š‡•‡”‡•—Ž–• ‹†‹…ƒ–‡–Šƒ–ͳ•—’’”‡••‡•–Š‡‹ϐŽƒƒ–‘”›”‡•’‘•‡‘ˆ •–Š”‘—‰Šƒ—–‘’Šƒ‰›Ǥ ‘ †‡–‡”‹‡ ™Š‡–Š‡” ƒ—–‘’Šƒ‰› ™ƒ• ‹˜‘Ž˜‡† ‹ †‡‰”ƒ†ƒ–‹‘ ‘ˆ ͵ ‹  •ǡ –Š‡  • ™‡”‡ ’”‡–”‡ƒ–‡† ™‹–Š ’”‘–‡ƒ•‘ƒŽ ‹Š‹„‹–‘”  Ǧͳ͵ʹ ‘” ƒ—–‘Ž›•‘•‘ƒŽ ‹Š‹„‹–‘” „ƒϐ‹Ž‘›…‹ ͳ ˆ‘” ͳ Š ƒ† ™‡”‡ ‡š’‘•‡† –‘  ǦȽ ˆ‘” ƒ ƒ††‹–‹‘ƒŽ ʹͶ Šǡ –Š‡ ’”‘–‡‹ ‡š’”‡••‹‘‘ˆ͵™ƒ•†‡–‡…–‡†„›‡•–‡”„Ž‘–ƒƒŽ›•‹•ǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ– ͳ͵ʹ Šƒ† ‘ ‡ˆˆ‡…– ‘ ͵ ’”‘–‡‹ ‡š’”‡••‹‘ǡ ™Š‡”‡ƒ• „ƒϐ‹Ž‘›…‹ ͳ ‹…”‡ƒ•‡† ƒ”‡†Ž› ͵’”‘–‡‹‡š’”‡••‹‘‹ •…‘’ƒ”‡†™‹–Š–Š‡ ǦȽ‰”‘—’ǤŠ‡•‡”‡•—Ž–••—‰‰‡•– –Šƒ–ƒ—–‘’Šƒ‰›™ƒ•‹˜‘Ž˜‡†‹–Š‡†‡‰”ƒ†ƒ–‹‘‘ˆ͵–Š”‘—‰Š–Š‡ƒ—–‘’Šƒ‰›ǦŽ›•‘•‘‡ ’ƒ–Š™ƒ›ȋ ‹‰Ǥ͸ȌǤ Autophagy is important for maintaining cellular homeostasis and is a survival ‡…Šƒ‹•–Šƒ–‹•—’”‡‰—Žƒ–‡††—”‹‰•–”‡••‹…ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡•ȏͳͻȐǤ‡ˆ‘—†–Šƒ–  ǦȽ‹†—…‡• ƒ—–‘’Šƒ‰›„›’”‘‘–‹‰–Š‡ƒ……——Žƒ–‹‘‘ˆƒ—–‘’Šƒ‰‹…˜ƒ…—‘Ž‡•ƒ† ƒ—–‘’Šƒ‰‹… ϐŽ—šǡ –Š‡ ‹…”‡ƒ•‹‰ –Š‡ ‡š’”‡••‹‘ ‘ˆ ͵ ƒ† ‡…Ž‹ͳ ƒ† ƒ—–‘’Šƒ‰‘•‘‡ ˆ‘”ƒ–‹‘Ǥ–Šƒ•„‡‡”‡’‘”–‡†–Šƒ–„‘–Š ǦȽƒ†‹•—Ž‹ǦŽ‹‡‰”‘™–Šˆƒ…–‘”Ǧͳ‹…”‡ƒ•‡† autophagy by increasing the accumulation of autophagic vacuoles and the expression of ͵ ƒ† ‡…Ž‹ͳ ‹ Š—ƒ ƒ–Š‡”‘•…Ž‡”‘–‹… • ȏʹͲȐǤ  ǦȽ …‘—Ž† †”ƒƒ–‹…ƒŽŽ› ‡Ž‡˜ƒ–‡ ƒ—–‘’Šƒ‰‹…ϐŽ—š‹„‘‡ƒ””‘™Ǧ†‡”‹˜‡†‡•‡…Š›ƒŽ•–‡…‡ŽŽ•ȏʹͳȐǤ–Š‡”•–—†‹‡•ƒŽ•‘ •Š‘™‡†–Šƒ– ǦȽ–”‡ƒ–‡–‹†—…‡†ƒ—–‘’Šƒ‰›‹™‹‰•ƒ”…‘ƒ…‡ŽŽ•ƒ†–”‘’Š‘„Žƒ•–• ȏʹʹǡ ʹ͵ȐǤ ‘”‡‘˜‡”ǡ ‘—” ”‡•—Ž– •Š‘™‡† –Šƒ–  ǦȽ ‹†—…‡• –Š‡ ˆ‘”ƒ–‹‘ ƒ† ƒ–—”ƒ–‹‘ ‘ˆƒ—–‘’Šƒ‰‘•‘‡•‹ •Ǥƒ–ƒˆ”‘–Š‡’”‡•‡–•–—†›•—‰‰‡•––Šƒ– ǦȽ‹†—…‡•  autophagy A growing number of studies support a pivotal role of histone deacetylase SIRT1 in –Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ ƒ—–‘’Šƒ‰› ƒ† …ƒ”†‹‘˜ƒ•…—Žƒ” †‹•‡ƒ•‡• ȏʹͶǡ ʹͷȐǤ ‡ ˆ‘—† –Šƒ– Ž‘™ …‘…‡–”ƒ–‹‘• ‘ˆ  ǦȽ ‹…”‡ƒ•‡† SIRT1 expression and induced autophagy, and SIRT1 ƒ…–‹˜ƒ–‘”ƒ—‰‡–‡†–Š‡ƒ—–‘’Šƒ‰›‘ˆ •‹†—…‡†„› ǦȽǤ–—†‹‡•Šƒ˜‡•Š‘™–Šƒ– RSV upregulated SIRT1 expression and triggered the stimulatory response of shear stress or ‘šǦ‘…‡ŽŽƒ—–‘’Šƒ‰›‹•ȏͷǡʹ͸ȐǤ —”–Š‡”‘”‡ǡ™‡ˆ‘—†–Šƒ–ͳ‹Š‹„‹–‹‘„› •‹”–‹‘Ž‘”ͳ‘…†‘™ƒ––‡—ƒ–‡†…‡ŽŽƒ—–‘’Šƒ‰›‹ ••–‹—Žƒ–‡†™‹–Š ǦȽǤ–Šƒ• „‡‡‘„•‡”˜‡†–Šƒ–ͳ‘…†‘™‘–ƒ„Ž›•—’’”‡••‡††‡Ž’Š‹‹†‹Ǧ͵Ǧ‰Ž—…‘•‹†‡Ǧ‹†—…‡† autophagy in VECs [27] Another study showed that SIRT1 inhibitor or SIRT1 knockdown …‘—Ž† •‹‰‹ϐ‹…ƒ–Ž› ‹Š‹„‹– –Š‡ ’”‘‘–‹˜‡ ‡ˆˆ‡…– ‘ˆ •Š‡ƒ” •–”‡•• ‘ ƒ—–‘’Šƒ‰› ‹ • ȏͷȐǤ Š‡•‡ϐ‹†‹‰••—’’‘”––Š‡‘–‹‘–Šƒ–ͳ’”‘‘–‡• ǦȽǦ‹†—…‡† ƒ—–‘’Šƒ‰›Ǥ It is well accepted that the Akt/mTOR signaling pathway is a key regulator of autophagy The mTOR is a downstream target of the Akt pathway, which can promote cell growth, †‹ˆˆ‡”‡–‹ƒ–‹‘ ƒ† ƒ—–‘’Šƒ‰› ȏʹͺȐǤ ‡ ˆ‘—† –Šƒ– –Š‡ –Ȁ •‹‰ƒŽ‹‰ ’ƒ–Š™ƒ› ‹Š‹„‹–• ǦȽǦ‹†—…‡†ƒ—–‘’Šƒ‰›‹ •Ǥ–Šƒ•„‡‡”‡’‘”–‡†–Šƒ––Š‡‹Š‹„‹–‹‘‘ˆ–Ȁ mTOR pathway activated macrophage autophagy, then suppressed the atherosclerosis and ‡Šƒ…‡• –Š‡ •–ƒ„‹Ž‹–› ‘ˆ ƒ–Š‡”‘•…Ž‡”‘–‹… ’Žƒ“—‡• ȏͳͺȐǤ —”–Š‡” •–—†› •Š‘™‡† –Šƒ– ͳ ‹…”‡ƒ•‡† ƒ—–‘’Šƒ‰›–Š”‘—‰Š–Š‡‹Š‹„‹–‹‘‘ˆ–Ȁ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›Ǥ”‡…‡– study found that overexpression of histone deacetylase SIRT6 could increase autophagy via the attenuation of Akt/mTOR pathway in cigarette smoke extract-induced human bronchial ‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•‡‡•…‡…‡ȏʹͻȐǤƒ‡–‘‰‡–Š‡”ǡ–Š‡•‡”‡•—Ž–••Š‘™–Šƒ–ͳ”‡‰—Žƒ–‡•  autophagy through the Akt/mTOR signaling pathway …‘”‡ƒƒŽ›•‹•ƒŽŽ‘™•—•–‘‹†‡–‹ˆ›‹–‡”ƒ…–‹‘•„‡–™‡‡‰‡‡•ƒ†’”‘–‡‹•ǡ”‡Žƒ–‡† networks, protein function, and signaling pathways in the context of biological processes ȏ͵ͲȐǤ ‹”•–Ž›ǡ–Š‡ƒƒŽ›•‹•”‡˜‡ƒŽ‡†–Šƒ–ͳ…‘—Ž†‘†—Žƒ–‡…‡ŽŽƒ—–‘’Šƒ‰›ǡ™Š‹…Š™ƒ• Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM 'LVFXVVLRQ Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 580 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ Fig Schematic illustration of –Š‡ ‡ˆˆ‡…–• ‘ˆ ͳ ‘ ‹ϐŽƒmation, autophagy and rela–‡† •‹‰ƒŽ‹‰ ’ƒ–Š™ƒ› ‹  •Ǥ ͳ ‘– ‘Ž› ”‡‰—Žƒ–‡•   autophagy through the Akt/ mTOR signaling pathway but ƒŽ•‘ •—’’”‡••‡• –Š‡ ‹ϐŽƒƒ–‘”› ”‡•’‘•‡ ‘ˆ  • –Š”‘—‰Š autophagy ‹ƒ‰”‡‡‡–™‹–Š‘—”ƒ„‘˜‡ϐ‹†‹‰–Šƒ–ͳ”‡‰—Žƒ–‡† ƒ—–‘’Šƒ‰›Ǥƒ††‹–‹‘ǡ™‡ ˆ‘—†–Šƒ––Š‡”‡™ƒ•ƒ‹–‡”ƒ…–‹‘„‡–™‡‡…‡ŽŽƒ—–‘’Šƒ‰›ƒ†–Š‡‹ϐŽƒƒ–‘”›…›–‘‹‡ Ǧ͸ƒ†ǦͳȾ‹…ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡•„›”‡‰—Žƒ–‘”›‡–™‘”ƒƒŽ›•‹•Ǥ–‹•™‡ŽŽ‘™ –Šƒ––Š‡”‡Ž‡ƒ•‡‘ˆǦͳȾ‹•”‡‰—Žƒ–‡†„›–Š‡͵‹ϐŽƒƒ•‘‡ȏ͵ͳȐǤ—”•–—†›•Š‘™‡† –Šƒ–ͳ•—’’”‡••‡†–Š‡‡š’”‡••‹‘‘ˆ–Š‡͵ƒ†Ǧ͸ƒ†–Š‡’”‘†—…–‹‘‘ˆǦͳȾ‹  ǦȽǦ•–‹—Žƒ–‡† •Ǥ”‡…‡–•–—†›•Š‘™‡†–Šƒ–ͳ‡š‡”–‡†ƒ–‹Ǧ‹ϐŽƒƒ–‘”›‡ˆˆ‡…–• „›‹Š‹„‹–‹‰͵‡š’”‡••‹‘‹‡•‡…Š›ƒŽ•–‡…‡ŽŽ•ȏ͵ʹȐǤƒ††‹–‹‘ǡ™‡ˆ‘—†–Šƒ– ƒ—–‘’Šƒ‰›‹Š‹„‹–‡†–Š‡‡š’”‡••‹‘‘ˆ–Š‡͵ƒ†Ǧ͸ƒ†–Š‡’”‘†—…–‹‘‘ˆǦͳȾ‹  ǦȽǦ•–‹—Žƒ–‡† •Ǥ–Šƒ•„‡‡”‡’‘”–‡†–Šƒ–ƒ—–‘’Šƒ‰›•—’’”‡••‡†͵ƒ…–‹˜ƒ–‹‘ ƒ†–Š‡’”‘†—…–‹‘‘ˆǦ͸ƒ†ǦͳȾ‹ƒ–‹‰‡Ǧ’”‡•‡–‹‰…‡ŽŽ••–‹—Žƒ–‡†™‹–Šȏ͵͵ȐǤ Another study also provided some important insights into the roles of the autophagic ’”‘–‡‹•͵ƒ†‡…Ž‹ͳ‹–Š‡”‡‰—Žƒ–‹‘‘ˆ͵Ǧ†‡’‡†‡–‹ϐŽƒƒ•‘‡ƒ…–‹˜ƒ–‹‘ ȏ͵ͶȐǤ Š‡•‡ ”‡•—Ž–• •—‰‰‡•– –Šƒ– ͳ •—’’”‡••‡• –Š‡ ‹ϐŽƒƒ–‘”› ”‡•’‘•‡ ‘ˆ  • –Š”‘—‰Š ƒ—–‘’Šƒ‰›Ǥ —”–Š‡” •–—†› •Š‘™‡† –Šƒ– „ƒϐ‹Ž‘›…‹ ͳ ‹…”‡ƒ•‡† ͵ ’”‘–‡‹ ‡š’”‡••‹‘ǡ ™Š‡”‡ƒ•  Ǧͳ͵ʹ Šƒ† ‘ ‡ˆˆ‡…– ‘ ͵ ’”‘–‡‹ ‡š’”‡••‹‘ ‹  •Ǥ Š‡•‡ ”‡•—Ž–••Š‘™‡†–Šƒ– ǦȽ‡†‹ƒ–‡•͵†‡‰”ƒ†ƒ–‹‘–Š”‘—‰Š–Š‡ƒ—–‘’Šƒ‰›ǦŽ›•‘•‘‡ ’ƒ–Š™ƒ›Ǥ–Šƒ•„‡‡”‡’‘”–‡†–Šƒ–͵‡š’”‡••‹‘™ƒ•‹…”‡ƒ•‡†ƒ”‡†Ž›‹ʹͻ͵ …‡ŽŽ• –”‡ƒ–‡† ™‹–Š „ƒϐ‹Ž‘›…‹ ͳǡ ™Š‡”‡ƒ•  Ǧͳ͵ʹ ’”‘†—…‡† Ž‹––Ž‡ …Šƒ‰‡ ‹ –Š‡ ͵ ’”‘–‡‹ Ž‡˜‡Žǡ •—‰‰‡•–‹‰ –Šƒ– Ǧʹ ‡†‹ƒ–‡• ͵ †‡‰”ƒ†ƒ–‹‘ –Š”‘—‰Š –Š‡ ƒ—–‘’Šƒ‰›Ǧ Ž›•‘•‘‡’ƒ–Š™ƒ›ȏ͵ͷȐǤ  …‘…Ž—•‹‘ǡ ‘—” †ƒ–ƒ ’”‘˜‹†‡ †‹”‡…– ‡˜‹†‡…‡ –Šƒ– ͳ ‘– ‘Ž› ”‡‰—Žƒ–‡•  autophagy through the Akt/mTOR signaling pathway but also suppresses the ‹ϐŽƒƒ–‘”›”‡•’‘•‡‘ˆ •–Š”‘—‰Šƒ—–‘’Šƒ‰›ȋ•—ƒ”‹œ‡†‹ ‹‰͹ȌǤŠ‡•‡ϐ‹†‹‰• ’”‘˜‹†‡ ‡™ ‹•‹‰Š–• ‹–‘ –Š‡ ’‘–‡–‹ƒŽ ”‘Ž‡ ‘ˆ ͳ ‹ ƒ—–‘’Šƒ‰› ƒ† ‹ϐŽƒƒ–‹‘Ǥ Thus, modulating autophagy may represent an attractive therapeutic target for –”‡ƒ–‹‰…ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡•Ǥ —”–Š‡”•–—†‹‡•ƒ”‡‘‰‘‹‰–‘†‡–‡”‹‡–Š‡ in vivo effect ‘ˆͳ‘ƒ—–‘’Šƒ‰›ƒ†‹ϐŽƒƒ–‹‘Ǥ $FNQRZOHGJPHQWV Š‹• •–—†› ™ƒ• •—’’‘”–‡† „› –Š‡ ƒ–‹‘ƒŽ ƒ–—”ƒŽ …‹‡…‡ ‘—†ƒ–‹‘ ‘ˆ Š‹ƒ ȋ‘Ǥ ͺͳʹ͹Ͳ͵Ͷ͹ǡ ‘Ǥ ͺͳͶͲͲʹ͵͵ ƒ† ‘Ǥ ͺͳͶ͹ͲͷͶͻȌǡ –Š‡ ƒ–—”ƒŽ …‹‡…‡ ‘—†ƒ–‹‘ ‘ˆ Šƒƒš‹ ”‘˜‹…‡ ȋʹͲͳ͵Ͳ͵ǦͲʹȌ ƒ† –Š‡ Š‹ƒ ‘•–†‘…–‘”ƒŽ …‹‡…‡ ‘—†ƒ–‹‘ ȋ‘Ǥ ʹͲͳͷͺͳͲ͵ͷȌǤ Š‡ƒ—–Š‘”•†‡…Žƒ”‡–Šƒ––Š‡”‡ƒ”‡‘…‘ϐŽ‹…–•‘ˆ‹–‡”‡•–Ǥ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM 'LVFORVXUH6WDWHPHQW Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 581 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ 10 11 12 13 14 15 16 17 18 19 20 …Š‹ƒ––ƒ”‡ŽŽƒ ǡ‹ŽŽ ǣŠ‡”ƒ’‡—–‹…–ƒ”‰‡–‹‰‘ˆƒ—–‘’Šƒ‰›‹…ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡Ǥ ‘Ž‡ŽŽƒ”†‹‘Ž ʹͲͳͷǢͲͲʹʹǦʹͺʹͺǣ͵Ͳͳʹ͵Ǧ͵ͲͳʹͶǤ ƒƒ‹ǡƒƒ‰—…Š‹ǡƒ‡†ƒǡ‹‰—…Š‹ǡ‹‘•‘ǡƒ‹‹‡ǡ‹›ƒǡ‹œ‘–‡ǡƒ–•——”ƒǡ•ƒŠ‹ǡ ‹•Š‹†ƒǡ‘”‹ǡ‹œ—•Š‹ƒǡ–•—ǣŠ‡”‘Ž‡‘ˆƒ—–‘’Šƒ‰›‹…ƒ”†‹‘›‘…›–‡•‹–Š‡„ƒ•ƒŽ•–ƒ–‡ƒ†‹ ”‡•’‘•‡–‘Š‡‘†›ƒ‹…•–”‡••Ǥƒ–‡†ʹͲͲ͹Ǣͳ͵ǣ͸ͳͻǦ͸ʹͶǤ ‹ƒ‰ǡ‡ǣ—–‘’Šƒ‰›†‡ϐ‹…‹‡…›•–ƒ„‹Ž‹œ‡•ͳ–‘’”‘‘–‡‡’‹–Š‡Ž‹ƒŽǦ‡•‡…Š›ƒŽ–”ƒ•‹–‹‘Ǥ —–‘’Šƒ‰›ʹͲͳͶǢͳͲǣͳͺ͸ͶǦͳͺ͸ͷǤ ‹–Šǡ”‹‡–‘ ǡ‘‰ǡ‡ƒ”•Ǧ”ƒš„‡”‰‡”ǡ‹…‡ ǡ–‡™ƒ”†ǡ‘–ƒǣƒ’ƒ›…‹ƒ† ‹–‡”Ž‡—‹ǦͳȾ‹’ƒ‹”„”ƒ‹Ǧ†‡”‹˜‡†‡—”‘–”‘’Š‹…ˆƒ…–‘”Ǧ†‡’‡†‡–‡—”‘•—”˜‹˜ƒŽ„›‘†—Žƒ–‹‰ ƒ—–‘’Šƒ‰›Ǥ ‹‘ŽŠ‡ʹͲͳͶǢʹͺͻǣʹͲ͸ͳͷǦʹͲ͸ʹͻǤ ‹— ǡ‹ǡŠ‡ǡŠƒ‰ǡƒ‰ǡŠ‹— ǡ‹— ǡŠƒ‰ǡ—ǡ ‹ƒ‰ ǣŠ‡ƒ”•–”‡••”‡‰—Žƒ–‡• ‡†‘–Š‡Ž‹ƒŽ…‡ŽŽƒ—–‘’Šƒ‰›˜‹ƒ”‡†‘š”‡‰—Žƒ–‹‘ƒ†‹”–ͳ‡š’”‡••‹‘Ǥ‡ŽŽ‡ƒ–Š‹•ʹͲͳͷǢ͸ǣ‡ͳͺʹ͹Ǥ ƒ”‹Šƒ”ƒǡƒ‡Œ‹ƒǡƒƒ‡ ǡƒ‹ ǡ‡’‹Š‘ǡƒ†‘•Š‹ƒ ǣ‡ƒ…‡–›Žƒ–‹‘‘ˆ ‘š„›‹”–ͳŽƒ›•ƒ ••‡–‹ƒŽ‘Ž‡‹‡†‹ƒ–‹‰–ƒ”˜ƒ–‹‘Ǧ†—…‡†—–‘’Šƒ‰›‹ƒ”†‹ƒ…›‘…›–‡•Ǥ‹”…‡•ʹͲͳͲǢͳͲ͹ǣͳͶ͹ͲǦ 1482 ƒƒƒǡƒ‰ƒ–ƒǡ‹”ƒ–ƒǡƒ„ƒ–ƒǡƒ–ƒǣ—‰‡–‡†ƒ‰‹‘‰‡‡•‹•‹ƒ†˜‡–‹–‹ƒ’”‘‘–‡•‰”‘™–Š‘ˆ ƒ–Š‡”‘•…Ž‡”‘–‹…’Žƒ“—‡‹ƒ’‘Ž‹’‘’”‘–‡‹Ǧ†‡ϐ‹…‹‡–‹…‡Ǥ–Š‡”‘•…Ž‡”‘•‹•ʹͲͳͳǢʹͳͷǣ͵͸͸Ǧ͵͹͵Ǥ — ǡ ‹ ǡ‹ǡŠ‡ǡ—ǣ†˜‡–‹–‹ƒŽϐ‹„”‘„Žƒ•–•ƒ”‡ƒ…–‹˜ƒ–‡†‹–Š‡‡ƒ”Ž›•–ƒ‰‡•‘ˆƒ–Š‡”‘•…Ž‡”‘•‹•‹ –Š‡ƒ’‘Ž‹’‘’”‘–‡‹‘…‘—–‘—•‡Ǥ‹‘…Š‡‹‘’Š›•‡•‘—ʹͲͲ͹Ǣ͵ͷʹǣ͸ͺͳǦ͸ͺͺǤ ƒ‰ǡƒǡŠƒ‰ ǡ‹ǡ‹ǡƒ‰ǡ‡ ǡŠƒ‰ ǡ ‹ǡ‹ǣͳ‹Š‹„‹–• ǦƒŽ’ŠƒǦ‹†—…‡†               ỗ    ‡†‡”Ǧ‹ˆˆ‡”‡–‡–ƒ„‘Ž‹…ƒ†ƒ•…—Žƒ” ›•ˆ—…–‹‘‹ƒ–•ǣ‘†—Žƒ–‘”›ˆˆ‡…–•‘ˆ‡•˜‡”ƒ–”‘ŽǤ‡ŽŽŠ›•‹‘Ž‹‘…Š‡ʹͲͳͷǢ͵͹ǣͳͶͲ͹ǦͳͶʹͲǤ ƒ‡†ƒǦƒ–ƒƒ„‡ǡ‹–ƒ†ƒǡƒƒ•ƒ‹ǡ‘›ƒǣͳ‹ƒ…–‹˜ƒ–‹‘‹†—…‡•‹ϐŽƒƒ–‹‘–Š”‘—‰Š–Š‡ †›•”‡‰—Žƒ–‹‘‘ˆƒ—–‘’Šƒ‰›‹Š—ƒǦͳ…‡ŽŽ•Ǥ‹‘…Š‡‹‘’Š›•‡•‘—ʹͲͳʹǢͶʹ͹ǣͳͻͳǦͳͻ͸Ǥ Šƒ‰ ǡŠ‡‰ǡ — ǡƒ‰ǡŠ‘—ǡƒ‰ǡƒǡ ‡‰ǡ —ǡ‡ŽŽ‡ǡŠ‡‰ǡƒ ǡŠƒ‰ǡ‹ǡ ƒ‹ǣ ‡‘ϐ‹„”ƒ–‡‹…”‡ƒ•‡•…ƒ”†‹ƒ…ƒ—–‘’Šƒ‰›˜‹ƒ ʹͳȀͳƒ†’”‡˜‡–•ϐ‹„”‘•‹•ƒ†‹ϐŽƒƒ–‹‘‹ –Š‡Š‡ƒ”–•‘ˆ›’‡ͳ†‹ƒ„‡–‹…‹…‡ǤŽ‹…‹ȋ‘†ȌʹͲͳ͸Ǣͳ͵Ͳǣ͸ʹͷǦ͸ͶͳǤ ‡”ƒ•Ǧƒ†‘˜ƒŽǡ±”‡œǦ‘Œƒ• ǡ‡”ž†‡œǦƒ‹ž ǡ‡†”ƒœƒǦŠƒ˜‡””‹ ǣŠ‡”‘Ž‡‘ˆ͵ȀȀ pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration ‡ŽŽ‹‰ƒŽʹͲͳͶǢʹ͸ǣʹ͸ͻͶǦʹ͹ͲͳǤ —ƒ ǡ—ǡ—ǡ‹ǡƒ‰ ǡ ‡‰ǡ ‹ƒ‰ǡ‹ǡŠ‘—ǡ—ǣ‹Ž‹…ƒƒ‘’ƒ”–‹…Ž‡•‹†—…‡ƒ—–‘’Šƒ‰› ƒ†‡†‘–Š‡Ž‹ƒŽ†›•ˆ—…–‹‘˜‹ƒ–Š‡͵Ȁ–Ȁ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›Ǥ– ƒ‘‡†‹…‹‡ʹͲͳͶǢͻǣͷͳ͵ͳǦ 5141 •— ǡ•—ǡ—ƒ‰ǡ‡‡ǡ‹ǡ•ƒ‹ǡŠ‹Šǡ‹ǣ›’‡”’Š‘•’Šƒ–‡‹ƒ‹†—…‡•’”‘–‡…–‹˜‡ ƒ—–‘’Šƒ‰›‹‡†‘–Š‡Ž‹ƒŽ…‡ŽŽ•–Š”‘—‰Š–Š‡‹Š‹„‹–‹‘‘ˆ–Ȁ•‹‰ƒŽ‹‰Ǥ ƒ•…—”‰ʹͲͳͷǢ͸ʹǣʹͳͲǦʹʹͳǤ Šƒ‹ǡŠ‡‰ ǡ—ŒƒŠ‹†ǡƒ‰ǡ‘‰ ǡ‹ǡ‹ ǡŠƒ‰ǡ ‹ǡŠ‡ǣ‡Ž‡…–‹˜‡‹Š‹„‹–‹‘‘ˆ͵Ȁ Akt/mTOR signaling pathway regulates autophagy of macrophage and vulnerability of atherosclerotic ’Žƒ“—‡Ǥ‘‡ʹͲͳͶǢͻǣ‡ͻͲͷ͸͵Ǥ ”‘‰‘ǡ —•–ƒˆ••‘%ǣŠ‡”ƒ’‡—–‹…–ƒ”‰‡–‹‰‘ˆƒ—–‘’Šƒ‰›ǣ’‘–‡–‹ƒŽƒ†…‘…‡”•‹–”‡ƒ–‹‰ …ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡Ǥ‹”…‡•ʹͲͳͷǢͳͳ͸ǣͶͺͻǦͷͲ͵Ǥ ‹ƒ ǡŠ‡‰ ǡ ƒ‰ƒŠƒ”ǡ‰”ƒ™ƒŽǣ•—Ž‹ǦŽ‹‡‰”‘™–Šˆƒ…–‘”Ǧͳƒ† ǦƒŽ’Šƒ”‡‰—Žƒ–‡ƒ—–‘’Šƒ‰› –Š”‘—‰Š…ǦŒ—Ǧ–‡”‹ƒŽ‹ƒ•‡ƒ†–’ƒ–Š™ƒ›•‹Š—ƒƒ–Š‡”‘•…Ž‡”‘–‹…˜ƒ•…—Žƒ”•‘‘–Š…‡ŽŽ•Ǥ —‘Ž‡ŽŽ‹‘ŽʹͲͲ͸ǢͺͶǣͶͶͺǦͶͷͶǤ Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM 5HIHUHQFHV Physiol Biochem 2017;41:569-582 Cellular Physiology Cell Œ7KH$XWKRUV3XEOLVKHGE\6.DUJHU$*%DVHO DOI: 10.1159/000457878 and Biochemistry Published online: February 03, 2017 ZZZNDUJHUFRPFSE 582 :DQJHWDO6,575HJXODWHV,QÁDPPDWLRQWKURXJK$XWRSKDJ\ 22 23 24 25 26 27 28 29 30 31 32 33 34 35 ƒ‰ǡ—›ƒ‰ǡ‹ǡƒ‰ǡ‡‰ǡ‘‰ǡ‘— ǡŠ‡ǡ‹‡ǡ‹—ǡ‹ ǡ‡ǡ—ǡŠ‡ǣ —–‘’Šƒ‰›Žƒ›•ƒ”‘–‡…–‹˜‡‘Ž‡‹—‘”‡…”‘•‹• ƒ…–‘”ǦȽǦ†—…‡†’‘’–‘•‹•‘ˆ‘‡ƒ””‘™Ǧ‡”‹˜‡† ‡•‡…Š›ƒŽ–‡‡ŽŽ•Ǥ–‡‡ŽŽ•‡˜ʹͲͳ͸Ǣʹͷǣ͹ͺͺǦ͹ͻ͹Ǥ Šƒ ǡ™ƒ‰ ǡ‹ǡŠ‘‹ ǡŠǡ‘Šǣ—–‘’Šƒ‰›‹†—…‡†„›–—‘”‡…”‘•‹•ˆƒ…–‘”Ƚ‡†‹ƒ–‡• ‹–”‹•‹…ƒ’‘’–‘•‹•‹–”‘’Š‘„Žƒ•–‹……‡ŽŽ•Ǥ‡’”‘†…‹ʹͲͳͶǢʹͳǣ͸ͳʹǦ͸ʹʹǤ Œƒ˜ƒŠ‡”‹Ǧ‡”‰›ǡ‡Ž‘––‹ǡƒ–Š‹‡— ǡ‡•ƒ­‘ ǡƒ—˜›ǡ‘—“—°”‡ǡ‹‡””‘ ǡ‘†‘‰‘ǣ Ǧ ƒ’’ƒƒ…–‹˜ƒ–‹‘”‡’”‡••‡•–—‘”‡…”‘•‹•ˆƒ…–‘”ǦƒŽ’ŠƒǦ‹†—…‡†ƒ—–‘’Šƒ‰›Ǥ ‹‘ŽŠ‡ʹͲͲ͸Ǣʹͺͳǣ͵Ͳ͵͹͵Ǧ ͵Ͳ͵ͺʹǤ Šƒ‰ǡ‡ ǣƒ”‰‡–‹‰ƒ—–‘’Šƒ‰›ˆ‘”–Š‡–Š‡”ƒ’‡—–‹…ƒ’’Ž‹…ƒ–‹‘‘ˆŠ‹•–‘‡†‡ƒ…‡–›Žƒ•‡‹Š‹„‹–‘”•‹ ‹•…Š‡‹ƒȀ”‡’‡”ˆ—•‹‘Š‡ƒ”–‹Œ—”›Ǥ‹”…—Žƒ–‹‘ʹͲͳͶǢͳʹͻǣͳͲͺͺǦͳͲͻͳǤ —ƒ‰ǡƒǡŠƒ‘ǡŠ‡ ǡ ƒ‘ǡŠƒ‰ǡŠ‡ǡƒǡ‹ǡ ‹ƒ‰ǣͳƒ†  ‡†‹ƒ–‡‘–”ƒ…–‹Ž‡‹ˆˆ‡”‡–‹ƒ–‹‘‘ˆƒ•…—Žƒ”‘‘–Š—•…Ž‡‡ŽŽ•—†‡”›…Ž‹…–”‡–…ŠǤ‡ŽŽŠ›•‹‘Ž ‹‘…Š‡ʹͲͳͷǢ͵͹ǣͳͺͳ͹ǦͳͺʹͻǤ —‘ǡŠ‡ǡ‹ƒ‘ǡ—ǣ‡•˜‡”ƒ–”‘Ž’”‘–‡…–••ˆ”‘‘š‹†‹œ‡†Ǧ‹†—…‡†‘š‹†ƒ–‹˜‡†ƒƒ‰‡„› ƒ—–‘’Šƒ‰›—’”‡‰—Žƒ–‹‘˜‹ƒ–Š‡Ȁͳ’ƒ–Š™ƒ›Ǥƒ”†‹‘˜ƒ•…”—‰•Š‡”ʹͲͳ͵Ǣʹ͹ǣͳͺͻǦͳͻͺǤ ‹ǡŠ‡ǡ‹ǡŠƒ‰ǡŠƒ‰ǡƒ‰ǡƒǡ‡‰ǡŠ‘—ǡ‹ǣ‡Ž’Š‹‹†‹Ǧ͵Ǧ‰Ž—…‘•‹†‡ ’”‘–‡…–•Š—ƒ—„‹Ž‹…ƒŽ˜‡‹‡†‘–Š‡Ž‹ƒŽ…‡ŽŽ•ƒ‰ƒ‹•–‘š‹†‹œ‡†Ž‘™Ǧ†‡•‹–›Ž‹’‘’”‘–‡‹Ǧ‹†—…‡†‹Œ—”›„› ƒ—–‘’Šƒ‰›—’”‡‰—Žƒ–‹‘˜‹ƒ–Š‡Ȁͳ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›Ǥ‘Ž—–” ‘‘†‡•ʹͲͳͶǢͷͺǣͳͻͶͳǦͳͻͷͳǤ ‹ǡ —ƒǣǣƒ’Šƒ”ƒ…‘Ž‘‰‹…–ƒ”‰‡–ˆ‘”ƒ—–‘’Šƒ‰›”‡‰—Žƒ–‹‘Ǥ Ž‹˜‡•–ʹͲͳͷǢͳʹͷǣʹͷǦ͵ʹǤ ƒƒ•ƒƒǡ”ƒ›ƒ ǡƒ”ƒǡ–‘ǡ‘„ƒ›ƒ•Š‹ǡ—”‹–ƒǡƒ—‹ǡ‘•Š‹‹ǡ—‹‘ǡ —Œ‹‹ǡ‹ƒ‰ƒ™ƒ ǡ•—”—•Š‹‰‡ǡ‘Œ‹ƒ ǡ—ƒ–ƒǡŠ‹‹œ—ǡƒ™ƒ‹•Š‹ǡƒ‡‘ǡƒ‹›ƒǡ‹”ƒ‘ ǡ†ƒƒǡ ‘”‹ƒ™ƒǡ‹•Š‹—”ƒǡƒƒ›ƒƒǡ—™ƒ‘ǣ—–‘’Šƒ‰›‹†—…–‹‘„›͸–Š”‘—‰Šƒ––‡—ƒ–‹‘ of insulin-like growth factor signaling is involved in the regulation of human bronchial epithelial cell •‡‡•…‡…‡Ǥ —‘ŽʹͲͳͶǢͳͻʹǣͻͷͺǦͻ͸ͺǤ ”¡‡”ǡ ”‡‡ ǡ‘ŽŽƒ”†  ”ǡ—‰‡†”‡‹…Šǣƒ—•ƒŽƒƒŽ›•‹•ƒ’’”‘ƒ…Š‡•‹‰‡—‹–›ƒ–Š™ƒ›ƒŽ›•‹•Ǥ ‹‘‹ˆ‘”ƒ–‹…•ʹͲͳͶǢ͵Ͳǣͷʹ͵Ǧͷ͵ͲǤ ƒ”–‹‘ ǡ—”•ǡ•…Š‘’’ ǣŠ‡‹ϐŽƒƒ•‘‡ǣ‘Ž‡…—Žƒ”’Žƒ–ˆ‘”–”‹‰‰‡”‹‰ƒ…–‹˜ƒ–‹‘‘ˆ ‹ϐŽƒƒ–‘”›…ƒ•’ƒ•‡•ƒ†’”‘…‡••‹‰‘ˆ’”‘Ǧ„‡–ƒǤ‘Ž‡ŽŽʹͲͲʹǢͳͲǣͶͳ͹ǦͶʹ͸Ǥ —ǡƒ‰ǡ—ǡ—ǡƒ‘ ǡ ƒǡ‹— ǡ—ǡ ƒǡ‹—ǡ ƒ ǣ‡•˜‡”ƒ–”‘Ž‹Š‹„‹–•‹‘‹•‹‰‹””ƒ†‹ƒ–‹‘Ǧ ‹†—…‡†‹ϐŽƒƒ–‹‘‹•„›ƒ…–‹˜ƒ–‹‰ͳƒ†Ž‹‹–‹‰Ǧ͵‹ϐŽƒƒ•‘‡ƒ…–‹˜ƒ–‹‘Ǥ– ‘Ž …‹ʹͲͳ͵ǢͳͶǣͳͶͳͲͷǦͳͶͳͳͺǤ ƒ””‹• ǡƒ”–ƒǡ‘…Š‡ǡ‡‰ ǡ̵Š‡ƒǡŠƒ”’ ǡƒ„‡ǡ”‡ƒ‰Šǡ ‘Ž‡„‘…ǡ•…Š‘’’ ǡ ‘”ˆ‡Ž†ǡ ‹–œ‰‡”ƒŽ†ǡƒ˜‡ŽŽ‡ǣ—–‘’Šƒ‰›…‘–”‘Ž•Ǧͳ„‡–ƒ•‡…”‡–‹‘„›–ƒ”‰‡–‹‰’”‘ǦǦͳ„‡–ƒˆ‘” †‡‰”ƒ†ƒ–‹‘Ǥ ‹‘ŽŠ‡ʹͲͳͳǢʹͺ͸ǣͻͷͺ͹Ǧͻͷͻ͹Ǥ ƒƒŠ‹”ƒǡƒ•’‡Ž ǡƒ–Š‹ƒǡ‡‡ ǡ‘Ž‹ƒ›ǡƒǡ‰Ž‡”– ǡƒ„‹‘˜‹–…Šǡ‡”ƒ†ƒ•ǡ‹ ǡ ‹–œ‰‡”ƒŽ†ǡ›–‡”ǡŠ‘‹ǣ—–‘’Šƒ‰›’”‘–‡‹•”‡‰—Žƒ–‡‹ƒ–‡‹—‡”‡•’‘•‡•„›‹Š‹„‹–‹‰ –Š‡”‡Ž‡ƒ•‡‘ˆ‹–‘…Š‘†”‹ƒŽ‡†‹ƒ–‡†„›–Š‡͵‹ϐŽƒƒ•‘‡Ǥƒ–—‘ŽʹͲͳͳǢͳʹǣʹʹʹǦʹ͵ͲǤ Š—ƒ‰ǡƒ‰ǡŠ‘—ǡŠ‹ƒ‰ǡŠ—ƒ‰ǡ•—ǣǦ‹†—…‡†Ǧʹ‡š’”‡••‹‘•—’’”‡••‡•Ǧ ͳȾ’”‘…‡••‹‰˜‹ƒ‹…”‡ƒ•‹‰ƒ—–‘’Šƒ‰›ƒ†͵†‡‰”ƒ†ƒ–‹‘Ǥ”‘…ƒ–Ž…ƒ†…‹ʹͲͳ͵ǢͳͳͲǣͳ͸Ͳ͹ͻǦ 16084 Downloaded by: Univ of California San Diego 132.239.1.231 - 2/9/2017 5:26:12 AM 21 ... signaling pathway is a key regulator of autophagy [15] It has been reported that both hyperphosphatemia and silica nanoparticles could induce the autophagy of VECs through the inhibition of the Akt/mTOR... pathway [16, 17] Another study also showed that selectively inhibition of Akt/mTOR pathway activated the autophagy of macrophages and inhibited the atherosclerosis progression [18] Thus, in the. .. addition, SIRT1 also promoted the autophagy of cardiomyocytes [6] The vascular adventitia, a supporting tissue surrounding the blood vessels, is recently found to participate in the initiation and