mTOR inhibisyonu, arka bacak iskemisi/reperfüzyon zedelenmelerinde apoptozu ve oksidatif stresi düzenler

Yıl 2023, Cilt: 48 Sayı: 4, 1322 - 1332, 29.12.2023

Zarife Pire Demet Sinem Güden Meryem Temiz Reşitoğlu Sefika Pınar Şenol Özden Vezir Nehir Sucu Bahar Tunçtan Kafait U. Malik Seyhan Şahan Fırat

https://doi.org/10.17826/cumj.1353689

Öz

Amaç: İskemi/reperfüzyon (İ/R)’nun neden olduğu zedelenmeler, oksidan/antioksidan dengenin bozulması ve apopitoz nedeniyle hedef organlarda ve takip eden uzak organlarda gelişebilecek komplikasyonlar ile ilgili ciddi klinik olayları temsil eder. İ/R zedelenmeleriyle ilgili yapılan son çalışmalarda farklı organlarda gelişebilecek İ/R kaynaklı zedelenmelerde rapamisinin memeli hedefi (mTOR)’un etkileri halen tartışılmaya devam etmektedir. Bu çalışmada arka bacak İ/R zedelenmesinde mTOR inhibisyonunun olası koruyucu etkisi ve bu etkisine aracılık edebilecek antiapoptotik ve antioksidan etki mekanizmalarının araştırılması amaçlandı.
Gereç ve yöntem: Wistar sıçanlar dört gruba ayrıldı. Sıçanlarda arka bacak İ/R zedelenmesini oluşturmak için anestezi altında her iki arka ekstremitelerine turnike uygulanarak 4 saat iskemi ve ardından turnikeler açılarak 4 saat reperfüzyon uygulandı. Reperfüzyondan 1 saat önce rapamisin (1 mg/kg) veya salin (4 mL/kg) periton içine uygulandı. Deneylerin sonunda gastroknemius kası, böbrek ve kanları alındı. Kas ve böbrek dokularında İ/R’de gelişebilecek zedelenmeler ribozomal protein S6 (rpS6), ökaryotik başlatma faktörü 4E-bağlayıcı protein 1 (4EBP1), kaspaz-3, Bcl-2 ile ilişkili X proteini (Bax) ve B-hücreli lenfoma (Bcl)-2’nin protein ekspresyonu ve/veya fosforilasyonu ile NADPH oksidaz seviyesi ve toplam antioksidan kapasitesi ölçülerek değerlendirildi.
Bulgular: İ/R'nin neden olduğu organ zedelemeleri artan rpS6, 4EBP1, kaspaz-3, Bax ve azalan Bcl-2 ekspresyon ve/veya fosforilasyonu ile buna eşlik eden toplam antioksidan kapasitede azalma ve NADPH oksidaz seviyesinde artma ile gösterildi. Bir mTOR inhibitörü olan rapamisin uygulaması, İ/R zedelenmesine aracılık eden etkileri ortadan kaldırdı.
Sonuç: Bulgularımız, mTOR sinyal iletisinin etkinleşmesinin oksidan/antioksidan dengedeki bozulmanın sonucu olarak apopitozun etkinleşmesi aracılığıyla arka bacaklarda İ/R’nin neden olduğu doku ve organ zedelenmelerinde önemli bir rol oynadığını göstermektedir.

Anahtar Kelimeler

Apopitoz, iskemi/reperfüzyon zedelenmesi, mTOR, oksidatif stres

Proje Numarası

2018-2-TP2-2984

mTOR inhibition modulates apoptosis and oxidative stress in hindlimb ischemia/reperfusion injury

Öz

Purpose: Ischemia/reperfusion (I/R)-induced injuries represent serious clinical events regarding profound target organ destructions followed by remote organ complications due to the loss of oxidant/antioxidant balance and apoptosis. Recent studies examining the mammalian target of rapamycin (mTOR) during I/R injury in different organs have remained a matter of debate. The current study aimed to explore further the protective and underlying antiapoptotic and antioxidant mechanisms of mammalian target of rapamycin (mTOR) inhibition in hindlimb (HL) schemia/reperfusion (I/R)injury.
Materials and Methods: Occlusion of bilateral hindlimbs for 4 h with tourniquets was carried out under anesthesia to induce I/R for 4 h in rats. Rapamycin (1 mg/kg) or saline (4 mL/kg) was injected intraperitoneally 1 h before reperfusion. Gastrocnemius muscle, kidney, and blood were collected at the end of the experiments for analysis. Muscle and kidney damages were evaluated by measuring protein expression and/or phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 (rpS6), B-cell lymphoma 2 (Bcl-2), caspase-3, and Bcl-2-associated X protein (Bax) with NADPH oxidase level and total antioxidant capacity in tissues or sera.
Results: I/R-induced organ damages were demonstrated by enhanced phosphorylation and/or expression of rpS6, 4EBP1, caspase-3, and Bax with a significant reduction in Bcl-2 accompanied by a decreased total antioxidant capacity and increased level of NADPH oxidase. Administration of rapamycin, an inhibitor mTOR, protected against I/R-mediated injuries.
Conclusion: Our findings suggest that the activation of mTOR signaling plays a crucial role in HL I/R-triggered organ damages presumably through the activation of apoptosis as a result of oxidant/antioxidant imbalance.

Anahtar Kelimeler

apoptosis, reperfusion injury, mTOR, oxidants, antioxidants

Proje Numarası

2018-2-TP2-2984

Kaynakça

• Blaisdell WF. The pathophysiology of skeletal muscle ischemia and the reperfusion syndrome: a review. Cardiovasc Surg. 2002;10:620-30.
• Drysch M, Wallner C, Schmidt SV, Reinkemeier F, Wagner JM, Lehnhardt M et al. An optimized low-pressure tourniquet murine hind limb ischemia reperfusion model: inducing acute ischemia reperfusion injury in C57BL/6 wild type mice. PLoS One. 2019;14:e0210961.
• Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention of ischemia–reperfusion injury basic pathophysiology of ischemia–reperfusion injury. Anesthesiology. 2001;94:1133-8.
• Yassin MMI, Harkin DW, Barros D’Sa AAB, Halliday MI, Rowlands BJ. Lower limb ischemia-reperfusion injury triggers a systemic inflammatory response and multiple organ dysfunction. World J Surg. 2002;26:115-21.
• Bayrak S, Yurekli I, Gokalp O, Kiray M, Bademci MS, Ozcem B et al. Assessment of protective effects of methylprednisolone and pheniramine maleate on reperfusion injury in kidney after distant organ ischemia: a rat model. Ann Vasc Surg. 2012;26:559-65.
• Takhtfooladi MA, Jahanshahi A, Jahanshahi G, Sotoudeh A, Takhtfooladi HA, Khansari M. Protective effect of N-acetylcysteine on kidney as a remote organ after skeletal muscle ischemia-reperfusion. Acta Cir Bras. 2012;27:611-5.
• Takhtfooladi MA, Takhtfooladi HA, Moayer F, Karimi P, Asl HA. Effect of otostegia persica extraction on renal injury induced by hindlimb ischemia-reperfusion: a rat model. Int J Surg. 2015;13:124-30.
• Collino M, Aragno M, Mastrocola R, Gallicchio M, Rosa AC, Dianzani C et al. Modulation of the oxidative stress and inflammatory response by PPAR-γ agonists in the hippocampus of rats exposed to cerebral ischemia/reperfusion. Eur J Pharmacol. 2006;530:70-80.
• Teruya R, Fagundes DJ, Oshima CTF, Brasileiro JL, Marks G, Ynouye CM et al. The effects of pentoxifylline into the kidneys of rats in a model of unilateral hindlimb ischemia/reperfusion injury. Acta Cir Bras. 2008;23:29-35.
• Liu S, Yang Y, Gao H, Zhou N, Wang P, Zhang Y et al. Trehalose attenuates renal ischemia-reperfusion injury by enhancing autophagy and inhibiting oxidative stress and inflammation. Am J Physiol Renal Physiol. 2020;318:994-1005.
• Akcay A, Nguyen Q, Edelstein CL. Mediators of inflammation in acute kidney injury. Mediators Inflamm. 2009;2009:137072.
• Havasi A, Borkan SC. Apoptosis and acute kidney injury. Kidney Int. 2011;80:29-40.
• Kezic A, Spasojevic I, Lezaic V, Bajcetic M. Mitochondria-targeted antioxidants: future perspectives in kidney ischemia reperfusion injury. Oxid Med Cell Longev. 2016;2016:2950503.
• Candilio L, Malik A, Hausenloy DJ. Protection of organs other than the heart by remote ischemic conditioning. J Cardiovasc Med. 2013;14:193-205.
• Clair D, Shah S, Weber J. Current state of diagnosis and management of critical limb ischemia. Curr Cardiol Rep. 2012;14:160-70.
• Liang S, Xu Z, Ruan Y, Niu T, Guo W, Jiang W et al. Isoquercitrin attenuates renal ischemia/reperfusion injury through antioxidation, anti-inflammation, and antiapoptosis in mice. Transplant Proc. 2020;52:1014-9.
• Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004;18:1926-45.
• Moschetta M, Reale A, Marasco C, Vacca A, Carratu MR. Therapeutic targeting of the mTOR-signalling pathway in cancer: benefits and limitations. Br J Pharmacol. 2014;171:3801-13.
• Zhang T, Guo J, Gu J, Chen K, Li H, Wang J. Protective role of mTOR in liver ischemia/reperfusion injury: involvement of inflammation and autophagy. Oxid Med Cell Longev. 2019;2019:7861290.
• Weichhart T, Costantino G, Poglitsch M, Rosner M, Zeyda M, Stuhlmeier KM et al. The TSC-mTOR signaling pathway regulates the innate inflammatory response. Immunity. 2008;29:565-77.
• Khan S, Salloum F, Das A, Xi L, Vetrovec GW, Kukreja RC. Rapamycin confers preconditioning-like protection against ischemia–reperfusion injury in isolated mouse heart and cardiomyocytes. J Mol Cell Cardiol. 2006;41:256-64.
• Jiang M, Liu K, Luo J, Dong Z. Autophagy is a renoprotective mechanism during in vitro hypoxia and in vivo ischemia-reperfusion injury. Am J Pathol. 2010;176:1181-92.
• Alshaman R, Truong L, Oyekan A. Role of mechanistic target of rapamycin (mTOR) in renal function and ischaemia-reperfusion induced kidney injury. Clin Exp Pharmacol Physiol. 2016;43:1087-96.
• Aoyagi T, Kusakari Y, Xiao CY, Inouye BT, Takahashi M, Scherrer-Crosbie M et al. Cardiac mTOR protects the heart against ischemia-reperfusion injury. Am J Physiol Heart Circul Physiol. 2012;303:75-85.
• Kocak Z, Temiz-Resitoglu M, Guden DS, Vezir O, Sucu N, Balcı S et al. Modulation of oxidative–nitrosative stress and inflammatory response by rapamycin in target and distant organs in rats exposed to hindlimb ischemia–reperfusion: the role of mammalian target of rapamycin. Can J Physiol Pharmacol. 2019;97:1193-203.
• Sucu N, Unlu A, Tamer L, Aytacoglu B, Coskun B, Bilgin R et al. Effects of trimetazidine on tissue damage in kidney after hindlimb ischemia-reperfusion. Pharmacol Res. 2002;46:345-9.
• Sari AN, Kacan M, Unsal D, Sahan-Firat S, Buharalioglu CK, Vezir O et al. Contribution of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway to ischemia/reperfusion-induced oxidative/nitrosative stress and inflammation leading to distant and target organ injury in rats. Eur J Pharmacol. 2014;723:234-45.
• Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54.
• Tunctan B, Korkmaz B, Sari AN, Kacan M, Unsal D, Serin MS et al. Contribution of iNOS/sGC/PKG pathway, COX-2, CYP4A1, and gp91phox to the protective effect of 5,14-HEDGE, a 20-HETE mimetic, against vasodilation, hypotension, tachycardia, and inflammation in a rat model of septic shock. Nitric Oxide. 2013;33:18-41.
• Temiz-Resitoglu M, Kucukkavruk SP, Guden DS, Cecen P, Sari AN, Tunctan B et al. Activation of mTOR/IκB-α/NF-κB pathway contributes to LPS-induced hypotension and inflammation in rats. Eur J Pharmacol. 2017;802:7-19.
• Sahan-Firat S, Temiz-Resitoglu M, Guden DS, Kucukkavruk SP, Tunctan B, Sari AN et al. Protection by mTOR inhibition on zymosan-induced systemic inflammatory response and oxidative/nitrosative stress: contribution of mTOR/MEK1/ERK1/2/IKKβ/IκB-α/NF-κB signalling pathway. Inflammation. 2018;41:276-98.
• Wu M, Zhang H, Kai J, Zhu F, Dong J, Xu Z et al. Rapamycin prevents cerebral stroke by modulating apoptosis and autophagy in penumbra in rats. Ann Clin Transl Neurol. 2017;5:138-46.
• Fan W, Cheng K, Qin X, Narsinh KH, Wang S, Hu S et al. mTORC1 and mTORC2 play different roles in the functional survival of transplanted adipose-derived stromal cells in hind limb ischemic mice via regulating inflammation in vivo. Stem Cells. 2013;31:203-14.
• Liu C, Peng M, Zheng L, Zhao Y, Wang R, Su Q et al. Enhanced autophagy alleviates injury during hindlimb ischemia/reperfusion in mice. Exp Ther Med. 2019;18:1669-76.
• Liu D, Xu L, Zhang X, Shi C, Qiao S, Ma Z et al. Snapshot: Implications for mTOR in aging-related ischemia/reperfusion injury. Aging Dis. 2019;10:116-33.
• Fan W, Han D, Sun Z, Ma S, Gao L, Chen J et al. Endothelial deletion of mTORC1 protects against hindlimb ischemia in diabetic mice via activation of autophagy, attenuation of oxidative stress and alleviation of inflammation. Free Radic Biol Med. 2017;108:725-40.
• Ayuso MI, Hernández-Jiménez M, Martín ME, Salinas M, Alcázar A. New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress. J Biol Chem. 2010;285:34355-63.
• Song Y, Liu W, Ding Y, Jia Y, Zhao J, Wang F et al. Salvianolic acid A ameliorates renal ischemia/reperfusion injury by activating Akt/mTOR/4EBP1 signaling pathway. Am J Physiol Renal Physiol. 2018;315:254-62.
• Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T et al. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res. 2007;100:914-22.
• Sciarretta S, Volpe M, Sadoshima J. Mammalian target of rapamycin signaling in cardiac physiology and disease. Circ Res. 2014;114:549-64.
• Zong H, Li X, Lin H, Hou C, Ma F. Lipoxin A4 pretreatment mitigates skeletal muscle ischemia-reperfusion injury in rats. Am J Transl Res. 2017;9:1139-50.
• Huang TL, Wang WC, Tu C, Yang ZY, Bramwell D, Sun XJ. Hydrogen-rich saline attenuates ischemia-reperfusion injury in skeletal muscle. J Surg Res. 2015;194:471-80.
• Das A, Salloum F, Durrant D, Ockaili R, Kukreja R. Rapamycin protects against myocardial ischemia-reperfusion injury through JAK2-STAT3 signaling pathway. J Mol Cell Cardiol. 2012;53:858-69.
• Filippone SM, Samidurai A, Roh SK, Cain CK, He J, Salloum FN et al. Reperfusion therapy with rapamycin attenuates myocardial infarction through activation of AKT and ERK. Oxid Med Cell Longev. 2017;2017:4619720.
• Li Q, Gao S, Kang Z, Zhang M, Zhao X, Zhai Y et al. Rapamycin enhances mitophagy and attenuates apoptosis after spinal ischemia-reperfusion injury. Front Neurosci. 2018;12:865.
• Yin L, Ye S, Chen Z, Zeng Y. Rapamycin preconditioning attenuates transient focal cerebral ischemia/reperfusion injury in mice. Int J Neurosci. 2012;122:748-56.
• Kim J, Kil IS, Seok YM, Yang ES, Kim DK, Lim DG et al. Orchiectomy attenuates post-ischemic oxidative stress and ischemia/reperfusion injury in mice. A role for manganese superoxide dismutase. J Biol Chem. 2006;281:20349-56.
• Chen Q, Parker CW, Devine I, Ondrasik R, Habtamu T, Bartol KD et al. Apocynin exerts dose-dependent cardioprotective effects by attenuating reactive oxygen species in ischemia/reperfusion. Cardiovasc Pharm Open Access. 2006;5:2.
• Ma LL, Ma X, Kong FJ, Guo JJ, Shi HT, Zhu JB et al. Mammalian target of rapamycin inhibition attenuates myocardial ischaemia-reperfusion injury in hypertrophic heart. J Cell Mol Med. 2018;22:1708-19.
• Ghasemnejad-Berenji M, Ghazi-Khansari M, Yazdani I, Saravi SSS, Nobakht M, Abdollahi A et al. Rapamycin protects testes against germ cell apoptosis and oxidative stress induced by testicular ischemia-reperfusion. Iran J Basic Med Sci. 2017;20:905-11.