臺灣博碩士論文加值系統

第一部分：高血壓引起的心臟肥大和心功能減弱是早期心力衰竭的主要特徵。我們之前的研究發現熱休克因子2（HSF2）的SUMO化在自發性高血壓大鼠（SHR）的心臟中被MEL-18嚴重削弱。然而調節MEL-18之機制在高血壓誘導的心臟肥大中的潛在機制的作用仍然難以捉摸。在這項研究中，我們確定了哺乳動物靶向雷帕黴素複合物1（mTORC1）激活MEL-18促進 HSF2去SUMO化使類胰島素生長因子II接受器(IGF-IIR)造成心臟肥大，同時在體外和體內藉由血管收縮素II（ANG II）刺激mTORC1上調MEL-18。總結，白藜蘆醇和雷帕黴素抑制mTORC1可完全消除MEL-18的下游靶點並恢復HSF2的SUMO化以減輕心臟功能障礙。我們的研究結果顯示，不論在體外或體內mTORC1-MEL-18-HSF2-IGFIIR機制是心肌細胞肥大的關鍵調控途徑。另外，雷帕黴素可能是改善由IGF-IIR所造成的心臟肥大達到改善心臟功能障礙和減輕高血壓的潛在治療藥物。
第二部分：龍葵是一種天然的化合物，已經被證實可以降低甘油三酯，低密度膽固醇，還可以增加高密度膽固醇。越來越多的文獻指出蛋白激酶C家族參與高血壓相關的心臟疾病。然而究竟龍葵如何改善了心臟肥大之機制仍然未知。根據我們先前的結果，我們發現雷帕黴素複合物1在血管收縮素II所誘導的心臟肥大中起關鍵作用。有力的文獻指出，蛋白激酶C-ζ調節mTORC1所介導的高血壓相關疾病，這意味著蛋白激酶C-ζ可能是抑制血管收縮素II誘導的心臟肥大的心臟保護治療策略。在本研究，我們確定了蛋白激酶C的信號傳遞路徑在ANG II所誘導的心臟肥大下通過mTORC1信號傳導正調節胰島素樣生長因子II受體。但是，龍葵的施用降低了高血壓，特別是在舒張壓下，並且還顯著恢復了熱休克因子2上的SUMO化以抑制IGF-IIR上調。綜上所述，這些研究結果發現龍葵透過救援熱休因子2的SUMO化來下調類胰島素生長因子II接受器，最終減少BNP表現量和心臟肥大，從而改善體高血壓造成的心臟肥大。

第一部分：Hypertension-induced cardiac hypertrophy to attenuate cardiac function is the major characteristics of early-stage heart failure. Our previous studies found that SUMOylation of the heat shock factor 2 (HSF2) was severely attenuated by MEL-18 in the heart of spontaneously hypertensive rats (SHR). However, the role of underlying mechanism regulating MEL-18 in hypertension-induced cardiac hypertrophy remains elusive. In this study, we identified mammalian of target of Rapamycin compex1 (mTORC1) activated MEL-18 to deSUMOylate HSF2 for insulin-like growth factor II receptor (IGF-IIR)-mediated cardiac hypertrophy. mTORC1 activation by angiotensin II (ANG II) is responsible for MEL-18 upregulation both in vitro and in vivo. Furthermore, inhibition of mTORC1 by resveratrol and Rapamycin completely abrogated downstream targets of MEL-18 and restored the SUMOylation of HSF2 to alleviate the cardiac dysfunction. Our results revealed an unanticipated mTORC1-MEL-18-HSF2-IGFIIR axis was a critical regulatory pathway of cardiomyocyte hypertrophy in vitro and in vivo, suggesting that Rapamycin could be a potential therapeutic candidate to alleviate cardiac dysfunction and attenuate high blood pressure during hypertension-induced cardiac hypertrophy.
第二部分：Solanum Nigrum is a natural compound that can decrease triglycerides, cholesterol, VLDL, and LDL cholesterol, and also increase HDL cholesterol. Growing evidences shows Protein Kinase C (PKC) family is involved in hypertension-related cardiac disease. However, the underlying mechanism of Solanum Nigrum ameliorated the cardiac hypertrophy remains unknown. According to our results, we found that mammalian target of Rapamycin complex 1 (mTORC1) plays a critical role in angiotensin II (ANG II) induced cardiac hypertrophy. Growing evidences suggest that PKC-zeta regulates mTORC1 activation-mediated hypertension-related disease, implying PKC-zeta may be a cardioprotective therapeutic strategy to suppress ANG II-induced cardiac hypertrophy. Here, we identified PKC signaling pathways modulates insulin-like growth factor II receptor (IGF-IIR) expression by mTORC1 signaling under ANG II-induced cardiac hypertrophy. But, Administration of Solanum Nigrum reduces high blood pressure , particularly in diastolic pressure and also markedly restored SUMOylation on HSF2 to inhibit IGF-IIR upregulation. Taken together, these findings demonstrated that Solanum Nigrum improves hypertension-related hypertrophy in vivo and in vitro, by rescuing SUMOylation of HSF2 to downregulate IGF-IIR, eventually reduce BNP expression and cardiac hypertrophy.

中文摘要 1
Abstract 2
Abbreviation 3
Introduction 5
Motivation 8
Materials and Methods 9
1. Experimental animals and the administration of Rapamycin 9
2. Cell culture and transient transfection 9
3. Western blot analysis and immunoprecipitation 10
4. Antibodies and reagents 11
5. Cell viability assay 11
6. Co-immunoprecipitation (Co-IP) 12
7. Luciferase and fluorescence reporter assay 13
8. Assessment of cardiomyocyte size in vitro 13
9. Immunohistochemistry 14
10. Neonatal rat ventricular myocyte primary culture 15
11. Statistical analysis 15
Results 16
Discussion 21
References 24
Figures 27

Part II
中文摘要 36
Abstract 37
Introduction 40
Motivation 44
Materials and Methods 45
1. Experimental animals and the administration of Solanum Nigrum (S.N) 45
2. Cell culture and transient transfection 45
3. Western blot analysis and immunoprecipitation 46
4. Antibodies and reagents 47
5. Cell viability assay 48
6. Luciferase reporter assay 48
7. Assessment of cardiomyocyte size in vitro 49
8. Statistical analysis 49
Results 50
Discussion 52
References 55
Figures 58

第一部分：1.Gray, M.O., et al., Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from fibroblasts. Cardiovasc Res, 1998. 40(2): p. 352-63.
2.Mazzolai, L., et al., Increased cardiac angiotensin II levels induce right and left ventricular hypertrophy in normotensive mice. Hypertension, 2000. 35(4): p. 985-91.
3.Ichihara, S., et al., Angiotensin II type 2 receptor is essential for left ventricular hypertrophy and cardiac fibrosis in chronic angiotensin II-induced hypertension. Circulation, 2001. 104(3): p. 346-51.
4.Huang, C.Y., et al., p53-mediated miR-18 repression activates HSF2 for IGF-IIR-dependent myocyte hypertrophy in hypertension-induced heart failure. Cell Death Dis, 2017. 8(8): p. e2990-.
5.Huang, C.Y., et al., HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy. J Cell Physiol, 2018. 233(2): p. 979-989.
6.Kjeldsen, S.E., et al., Mechanism of angiotensin II type 1 receptor blocker action in the regression of left ventricular hypertrophy. J Clin Hypertens (Greenwich), 2006. 8(7): p. 487-92.
7.Crowley, S.D., et al., Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proc Natl Acad Sci U S A, 2006. 103(47): p. 17985-90.
8.Hafizi, S., et al., ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells. Am J Physiol Heart Circ Physiol, 2004. 287(3): p. H1232-8.
9.Zhang, J., et al., MEL-18 interacts with HSF2 and the SUMO E2 UBC9 to inhibit HSF2 sumoylation. J Biol Chem, 2008. 283(12): p. 7464-9.
10.Zhang, J. and K.D. Sarge, Mel-18 interacts with RanGAP1 and inhibits its sumoylation. Biochem Biophys Res Commun, 2008. 375(2): p. 252-5.
11.Lee, J.Y., et al., MEL-18 loss mediates estrogen receptor-alpha downregulation and hormone independence. J Clin Invest, 2015. 125(5): p. 1801-14.
12.Huang, C.Y., et al., Inhibition of HSF2 SUMOylation via MEL18 upregulates IGF-IIR and leads to hypertension-induced cardiac hypertrophy. Int J Cardiol, 2018. 257: p. 283-290.
13.Sciarretta, S., M. Volpe, and J. Sadoshima, Mammalian target of rapamycin signaling in cardiac physiology and disease. Circ Res, 2014. 114(3): p. 549-64.
14.Laplante, M. and D.M. Sabatini, mTOR signaling in growth control and disease. Cell, 2012. 149(2): p. 274-93.
15.Zhang, D., et al., MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice. J Clin Invest, 2010. 120(8): p. 2805-16.
16.Huang, C.Y., et al., Mitochondrial ROS-induced ERK1/2 activation and HSF2-mediated AT1 R upregulation are required for doxorubicin-induced cardiotoxicity. J Cell Physiol, 2018. 233(1): p. 463-475.
17.Anckar, J., et al., Inhibition of DNA Binding by Differential Sumoylation of Heat Shock Factors. Mol Cell Biol, 2006. 26(3): p. 955-64.
18.Sadoshima, J. and S. Izumo, Rapamycin selectively inhibits angiotensin II-induced increase in protein synthesis in cardiac myocytes in vitro. Potential role of 70-kD S6 kinase in angiotensin II-induced cardiac hypertrophy. Circ Res, 1995. 77(6): p. 1040-52.
19.Nikolaeva, I., et al., Differential roles for Akt and mTORC1 in the hypertrophy of Pten mutant neurons, a cellular model of brain overgrowth disorders. Neuroscience, 2017. 354: p. 196-207.
20.Xu, L. and M. Brink, mTOR, cardiomyocytes and inflammation in cardiac hypertrophy. Biochim Biophys Acta, 2016. 1863(7 Pt B): p. 1894-903.
21.Sciarretta, S., et al., New Insights Into the Role of mTOR Signaling in the Cardiovascular System. Circ Res, 2018. 122(3): p. 489-505.
22.Child, D.D., et al., Cardiac mTORC1 Dysregulation Impacts Stress Adaptation and Survival in Huntington''s Disease. Cell Rep, 2018. 23(4): p. 1020-1033.
23.Huang, C.Y., et al., ANG II promotes IGF-IIR expression and cardiomyocyte apoptosis by inhibiting HSF1 via JNK activation and SIRT1 degradation. Cell Death Differ, 2014. 21(8): p. 1262-74.
24.Huang, C.Y., et al., Data supporting the angiotensin II activates MEL18 to deSUMOylate HSF2 for hypertension-related heart failure. Data Brief, 2018. 16: p. 521-526.
25.Zheng, B., et al., mTORC1 and mTORC2 play different roles in regulating cardiomyocyte differentiation from embryonic stem cells. Int J Dev Biol, 2017. 61(1-2): p. 65-72.
26.Gonzalez-Teran, B., et al., p38gamma and delta promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation. Nat Commun, 2016. 7: p. 10477.
27.Volkers, M., et al., Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1. Proc Natl Acad Sci U S A, 2013. 110(31): p. 12661-6.
28.Liu, D., R.P. Ceddia, and S. Collins, Cardiac natriuretic peptides promote adipose ''browning'' through mTOR complex-1. Mol Metab, 2018. 9: p. 192-198.
29.Urbanska, M., et al., Mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) control the dendritic arbor morphology of hippocampal neurons. J Biol Chem, 2012. 287(36): p. 30240-56.
30.Soesanto, W., et al., Mammalian target of rapamycin is a critical regulator of cardiac hypertrophy in spontaneously hypertensive rats. Hypertension, 2009. 54(6): p. 1321-7.
31.Gu, J., et al., Rapamycin Inhibits Cardiac Hypertrophy by Promoting Autophagy via the MEK/ERK/Beclin-1 Pathway. Front Physiol, 2016. 7.
32.Johnson, S.C., P.S. Rabinovitch, and M. Kaeberlein, mTOR is a key modulator of ageing and age-related disease. Nature, 2013. 493(7432): p. 338-45.
33.Franz, D.N. and J.K. Capal, mTOR inhibitors in the pharmacologic management of tuberous sclerosis complex and their potential role in other rare neurodevelopmental disorders. Orphanet J Rare Dis, 2017. 12.
34.Sciarretta, S., M. Volpe, and J. Sadoshima, mTOR Signaling in Cardiac Physiology and Disease: Sciarretta et al. mTOR signaling in the cardiovascular system. Circ Res, 2014. 114(3): p. 549-64.
35.Bishu, K., et al., Anti-remodeling effects of rapamycin in experimental heart failure: dose response and interaction with angiotensin receptor blockade. PLoS One, 2013. 8(12): p. e81325.
36.Chiao, Y.A., et al., Rapamycin transiently induces mitochondrial remodeling to reprogra
第二部分：1.Oh, S.S., et al., Acute interstitial nephritis induced by Solanum nigrum. Kidney Res Clin Pract, 2016. 35(4): p. 252-4.
2.Jain, R., et al., Solanum nigrum: current perspectives on therapeutic properties. Altern Med Rev, 2011. 16(1): p. 78-85.
3.Wang, Y., et al., Potential Anti-inflammatory Steroidal Saponins from the Berries of Solanum nigrum L. (European Black Nightshade). J Agric Food Chem, 2017. 65(21): p. 4262-4272.
4.Sohrabipour, S., et al., Effect of the administration of Solanum nigrum fruit on blood glucose, lipid profiles, and sensitivity of the vascular mesenteric bed to phenylephrine in streptozotocin-induced diabetic rats. Med Sci Monit Basic Res, 2013. 19: p. 133-40.
5.Sohrabipour, S., et al., Biphasic effect of Solanum nigrum fruit aqueous extract on vascular mesenteric beds in non-diabetic and streptozotocin-induced diabetic rats. Pharmacognosy Res, 2014. 6(2): p. 148-52.
6.Gray, M.O., et al., Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from fibroblasts. Cardiovasc Res, 1998. 40(2): p. 352-63.
7.Mazzolai, L., et al., Increased cardiac angiotensin II levels induce right and left ventricular hypertrophy in normotensive mice. Hypertension, 2000. 35(4): p. 985-91.
8.Ichihara, S., et al., Angiotensin II type 2 receptor is essential for left ventricular hypertrophy and cardiac fibrosis in chronic angiotensin II-induced hypertension. Circulation, 2001. 104(3): p. 346-51.
9.Huang, C.Y., et al., p53-mediated miR-18 repression activates HSF2 for IGF-IIR-dependent myocyte hypertrophy in hypertension-induced heart failure. Cell Death Dis, 2017. 8(8): p. e2990-.
10.Huang, C.Y., et al., HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy. J Cell Physiol, 2018. 233(2): p. 979-989.
11.Kjeldsen, S.E., et al., Mechanism of angiotensin II type 1 receptor blocker action in the regression of left ventricular hypertrophy. J Clin Hypertens (Greenwich), 2006. 8(7): p. 487-92.
12.Crowley, S.D., et al., Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney. Proc Natl Acad Sci U S A, 2006. 103(47): p. 17985-90.
13.Hafizi, S., et al., ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells. Am J Physiol Heart Circ Physiol, 2004. 287(3): p. H1232-8.
14.Braz, J.C., et al., PKCα regulates the hypertrophic growth of cardiomyocytes through extracellular signal–regulated kinase1/2 (ERK1/2). J Cell Biol, 2002. 156(5): p. 905-19.
15.Kerkela, R., et al., Identification of PKCalpha isoform-specific effects in cardiac myocytes using antisense phosphorothioate oligonucleotides. Mol Pharmacol, 2002. 62(6): p. 1482-91.
16.Palaniyandi, S.S., et al., Protein kinase C in heart failure: a therapeutic target? Cardiovasc Res, 2009. 82(2): p. 229-39.
17.Konopatskaya, O. and A.W. Poole, Protein kinase Cα: disease regulator and therapeutic target. Trends Pharmacol Sci, 2010. 31(1): p. 8-14.
18.Braz, J.C., et al., PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2). J Cell Biol, 2002. 156(5): p. 905-19.
19.Song, X., et al., Protein kinase C promotes cardiac fibrosis and heart failure by modulating galectin-3 expression. Biochim Biophys Acta, 2015. 1853(2): p. 513-21.
20.Braz, J.C., et al., PKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat Med, 2004. 10(3): p. 248-54.
21.Sil, P., V. Kandaswamy, and S. Sen, Increased protein kinase C activity in myotrophin-induced myocyte growth. Circ Res, 1998. 82(11): p. 1173-88.
22.Dorn, G.W. and T. Force, Protein kinase cascades in the regulation of cardiac hypertrophy. J Clin Invest, 2005. 115(3): p. 527-37.
23.Li, J., et al., PKCzeta interacts with STAT3 and promotes its activation in cardiomyocyte hypertrophy. J Pharmacol Sci, 2016. 132(1): p. 15-23.
24.Garcia-Hoz, C., et al., Protein kinase C (PKC)zeta-mediated Galphaq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts. J Biol Chem, 2012. 287(10): p. 7792-802.
25.Centurione, L., et al., Protein kinase C zeta regulation of hypertrophic and apoptotic events occurring during rat neonatal heart development and growth. Int J Immunopathol Pharmacol, 2005. 18(1): p. 49-58.
26.Heo, K.S., et al., PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation. J Cell Biol, 2011. 193(5): p. 867-84.
27.Takabe, W., N. Alberts-Grill, and H. Jo, Disturbed flow: p53 SUMOylation in the turnover of endothelial cells. J Cell Biol, 2011. 193(5): p. 805-7.
28.Sabri, A., H.H. Hughie, and P.A. Lucchesi, Regulation of hypertrophic and apoptotic signaling pathways by reactive oxygen species in cardiac myocytes. Antioxid Redox Signal, 2003. 5(6): p. 731-40.
29.Naskar, S., et al., Differential and Conditional Activation of PKC-Isoforms Dictates Cardiac Adaptation during Physiological to Pathological Hypertrophy. PLoS One, 2014. 9(8).
30.Geraldes, P. and G.L. King, Activation of Protein Kinase C Isoforms & Its Impact on Diabetic Complications. Circ Res, 2010. 106(8): p. 1319-31.
31.Noh, H. and G.L. King, The role of protein kinase C activation in diabetic nephropathy. Kidney Int Suppl, 2007(106): p. S49-53.
32.Huang, C.Y., et al., Data supporting the angiotensin II activates MEL18 to deSUMOylate HSF2 for hypertension-related heart failure. Data Brief, 2018. 16: p. 521-526.
33.Huang, C.Y., et al., ANG II promotes IGF-IIR expression and cardiomyocyte apoptosis by inhibiting HSF1 via JNK activation and SIRT1 degradation. Cell Death Differ, 2014. 21(8): p. 1262-74.
34.Huang, C.Y., et al., Inhibition of HSF2 SUMOylation via MEL18 upregulates IGF-IIR and leads to hypertension-induced cardiac hypertrophy. Int J Cardiol,