Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava 84005, Slovakia;

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1 Int. J. Mol. Sci. 215, 16, ; doi:1.339/ijms Article OPEN ACCESS Interntionl Journl of Moleculr Sciences ISSN Quercetin Improves Postischemic Recovery of Hert Function in Doxorubicin-Treted Rts nd Prevents Doxorubicin-Induced Mtrix Metlloproteinse-2 Activtion nd Apoptosis Induction Monik Brteková 1, Petr Šimončíková 1, Mári Fogrssyová 1, Monik Ivnová 1, Ľudmil Okruhlicová 1, Nrcis Tribulová 1, Im Dovinová 2 nd Miroslv Brnčík 1, * 1 Institute for Hert Reserch, Slovk Acdemy of Sciences, Brtislv 845, Slovki; E-Mils: Monik.Brtekov@svb.sk (M.B.); Petr.Simoncikov@svb.sk (P.S.); mri.fogrssyov@zet.sk (M.F.); Monik.Strniskov@svb.sk (M.I.); Ludmil.Okruhlicov@svb.sk (L.O.); Nrcis.Tribulov@svb.sk (N.T.) 2 Institute of Norml nd Pthologicl Physiology, Slovk Acdemy of Sciences, Brtislv 845, Slovki; E-Mil: Im.Dovinov@svb.sk * Author to whom correspondence should be ddressed; E-Mil: Miroslv.Brncik@svb.sk; Tel.: ; Fx: Acdemic Editor: Chng Won Choi Received: 27 December 214 / Accepted: 24 Mrch 215 / Published: 13 April 215 Abstrct: Quercetin (QCT) is flvonoid tht possesses vrious biologicl functions including nti-oxidtive nd rdicl-scvenging ctivities. Moreover, QCT exerts some preventive ctions in tretment of crdiovsculr diseses. The im of present study ws to explore effects of prolonged dministrtion of QCT on chnges induced by repeted ppliction of doxorubicin (DOX) in rt herts. We focused on the ultrstructure of myocrdium, mtrix metlloproteinses (MMPs), biometric prmeters, nd poptosis induction. Our im ws lso to exmine effects of QCT on ischemic tolernce in herts exposed to chronic effects of DOX, nd to determine possible mechnisms underlying effects of QCT. Our results showed tht QCT prevented severl negtive chronic effects of DOX: (I) reversed DOX-induced blood pressure increse; (II) medited improvement of deleterious effects of DOX on ultrstructure of left ventricle; (III) prevented DOX-induced effects on tissue MMP-2 ctivtion; nd (iv) reversed effects of DOX on poptosis induction nd superoxide dismutse inhibition. Moreover, we showed tht rt herts exposed to effects of QCT were more resistnt to ischemi/reperfusion injury. Effects of QCT on modultion

2 Int. J. Mol. Sci. 215, of ischemic tolernce were linked to Akt kinse ctivtion nd connexin-43 up-regultion. Tken together, these results demonstrte tht prolonged tretment with QCT prevented negtive chronic effects of DOX on blood pressure, cellulr dmge, MMP-2 ctivtion, nd poptosis induction. Moreover, QCT influenced myocrdil responses to cute ischemic stress. These fcts bring new insights into mechnisms of QCT ction on rt herts exposed to the chronic effects of DOX. Keywords: ; doxorubicin; hert; ischemic tolernce; mtrix metlloproteinses; cell signling 1. Introduction Quercetin (QCT; 3,5,7,3',4'-penthydroxyflvone) is polyphenolic compound present in vrious foods including vegetbles, fruit nd wine [1]. This flvonoid possesses vrious biologicl functions including nti-oxidtive, nti-inflmmtory, nti-cogultion, nd oxygen rdicl-scvenging ctivities [2 4]. Recently, QCT hs been found to hve certin preventive ctions in the tretment of crdiovsculr diseses [5 11]. Animl studies demonstrted tht QCT exerts vsodilting nd blood pressure-lowering effects in spontneous hypertensive rts [5,6] nd in rts fed high-ft high sucrose diet [7]. An cute ppliction of QCT before ischemi or during reperfusion hs been found to protect the myocrdium from ischemi/reperfusion injury [8,9]. In nother study, significnt reduction of the myocrdil infrct size in both norml nd dibetic nimls by QCT hs been reported [1]. This modultion of ischemic tolernce in dibetic herts suggests tht QCT cn influence ischemic tolernce in herts ffected by nother prolonged pthologicl stress stimuli. Quercetin hs lso been found to hve beneficil effects in combting cdmium-induced oxidtive crdiotoxicity nd dyslipidemi in rts [11]. An ppliction of nthrcycline doxorubicin (DOX), known stress fctor, cn induce chronic pthologicl chnges in myocrdium. DOX is used in tretment of vrious cncers such s solid tumours, leukemi, lymphoms nd soft tissue srcom [12], however, its clinicl utility hs been hmpered by its dverse crdic effects. The mechnisms of its crdiotoxicity my be multifctoril [13], including the impirment of mitochondril energetics by increse in the mitochondril clcium nd rective oxygen species (ROS) leding to oxidtive stress, cell necrosis nd induction of pro-poptotic signling pthwys [12,14]. An ppliction of DOX ws lso found to influence sensitivity of herts to ischemic injury. A recent study demonstrted tht cute dministrtion of DOX upon induction of ischemi significntly incresed the infrct size [15]. On the other hnd, prolonged exposure of rts to DOX induced dptive responses of myocrdium ssocited with modultion of myocrdil resistnce to cute ischemic insult [16]. The im of this study ws to explore the effects of prolonged dministrtion of on the chnges induced by repeted ppliction of doxorubicin in rt herts. We focused on chnges in the ultrstructure of myocrdium, mtrix metlloproteinses, biometric prmeters, nd poptosis induction. Furthermore, we investigted the effects of QCT on ischemic tolernce in rt herts exposed to the chronic effects of DOX nd we lso imed to ssess the moleculr mechnisms underlying the effects of QCT.

3 Int. J. Mol. Sci. 215, Results 2.1. Effects of Quercetin on Biometric Prmeters The ppliction of QCT lone did not influence registered prmeters significntly. There ws significnt decrese of body weight nd weight gin of rts in the DOX-treted group in comprison to sline-treted nimls (Tble 1). Appliction of QCT further potentited the DOX-induced effects on modultion of BW nd weight gin; however, the chnges (DOX QCT vs. DOX) were not sttisticlly significnt. The ppliction of DOX or QCT lone did not influence the weight of the whole hert nd weight of the left ventricle in comprison to conditions, nd comprisons between DOX nd DOX QCT groups lso did not show sttisticlly significnt chnges. Eight weeks fter the end of the DOX tretment, the systolic blood pressure (SBP) nd hert rte were significntly incresed in comprison with nimls. The tretment with QCT ttenuted the DOX-induced effects nd reversed the blood pressure nd hert rte increse in DOX-treted rts (Tble 1). Tble 1. Effects of on biometric prmeters in norml nd doxorubicin-treted rts. BW, body weight; HW, hert weight; LV, left ventricle; SBP, systolic blood pressure; HR, hert rte; C, rts; QCT, -treted rts; DOX, doxorubicin-treted rts; DOX QCT, rts treted with both doxorubicin nd. Dt re presented s the men ± SEM (n = 12 per group). Sttisticl significnce ws reveled by one wy ANOVA with Bonferroni post-hoc test nd sttisticl differences were lwys determined mong groups C nd DOX (or QCT) () s well s DOX nd DOX QCT (b), p <.5 vs. C, b p <.5 vs. DOX. Sttisticlly significnt chnges re in Tble mrked in bold. Prmeter C QCT DOX DOX QCT BW (g) ± ± ± ± 17.1 Weight gin (g) ± ± ± ± 16.3 HW (g) 1.43 ± ± ± ±.6 HW/BW ± ± ± ±.174 LV (mg) 87.6 ± ± ± ± 34. SBP (mm Hg) 113 ± 4 12 ± ± ± 5 b HR (bets/min) 369 ± ± ± ± 9 b 2.2. Electron Microscopic Anlysis of Quercetin Effects on Ultrstructurl Chnges Induced by Doxorubicin An electron microscopic exmintion of the herts of rts showed intct ultrstructure of the myocytes, without significnt bnormlities in the extrcellulr spce (Figure 1A). The tretment of rts with QCT did not hve deleterious effects on ultrstructure of the tissue of left ventricle (Figure 1B). On the other hnd, the ppliction of DOX resulted in more serious heterogeneous subcellulr bnormlities of crdiomyocytes s well s extrcellulr spce (Figure 1C). The ltter ws mnifested by n incresed density of extrcellulr mtrix proteins, the ccumultion of perivsculr collgen, the presence of lrge nd/or smll vcuoles nd fibroblsts with their long projections. Electron microscopy of the left ventricle of the rts exposed to the effects of both DOX nd QCT

4 Int. J. Mol. Sci. 215, (Figure 1D) showed tht ppliction of QCT medited improvement of severl deleterious subcellulr ltertions (of extrcellulr mtrix) induced by DOX. A c mc B c mc cp vc mit ECS C col vc cp D c mc fib ECS vc c mc c mc c mc cp ECS cp Figure 1. Electron microscopic imges showing qulittive chnges in ultrstructure of the left ventricle of rt herts. (A) Electron microgrph of rt hert showing norml rchitecture of crdiomyocytes nd without chnges in ECS; (B) Electron microgrph of the myocrdium fter tretment with QCT; (C) Electron microgrph of the myocrdium ffected with DOX demonstrting subcellulr ltertions of crdiomyocytes nd extrcellulr spce (rrows); (D) Ultrstructure of the myocrdium of rts treted with both QCT nd DOX. Arrows indicte improvement of some deleterious subcellulr ltertions induced by DOX. ECS: extrcellulr spce; cmc: crdiomyocytes; cp: cpillry; col: collgen; mit: mitochondri; vc: vcuole; fib: fibroblst. (A) originl mgnifiction 8; (B) originl mgnifiction 8; (C) originl mgnifiction 6; (D) originl mgnifiction Quercetin Tretment Modultes the Doxorubicin-Induced Effects on Mtrix Metlloproteinse-2 The MMPs ctivities in hert tissue s well s in blood plsm smples were nlyzed by zymogrphy using geltin s substrte. The positions of 63- nd 72-kD forms of MMP-2 were identified using corresponding positive s. In tissue of the left ventricle tretment with DOX induced up-regultion of 72-kD MMP-2 ctivities but the ppliction of QCT prevented these DOX-induced effects on tissue MMP-2 ctivtion (Figure 2A,B). The observed effects of QCT nd DOX on MMP-2 ctivities were not ssocited with modultion of the protein levels of this enzyme.

5 Int. J. Mol. Sci. 215, A B C C Q Q C C Q Q -Dox + Dox 72 kd MMP-2 72 kd MMP-2 63 kd MMP-2 GAPDH Intensity of recrtion (% of ) b Figure 2. Effect of QCT nd DOX tretment on tissue mtrix metlloproteinse-2. (A) At the top is record showing the ctivities of MMP-2 nlyzed using geltin zymogrphy; in the middle, western blot record showing MMP-2 protein levels nlyzed using specific ntibody tht rects with both the 72 nd 63 kd forms of MMP-2, nd t the bottom is documented the protein loding using GAPDH; (B) Quntittive nlysis of the tissue 72-kD MMP-2 ctivities. Dt re expressed s percentge of vlue for corresponding. Ech br represents men ± S.E.M. of seven independent tissue smples per group. Sttisticl significnce is reveled by Student s unpired t-test, p <.5 vs. sline-treted ( DOX) rts; b p <.5 vs. DOX-treted (+DOX) rts. By zymogrphic nlysis of blood plsm smples we identified using positive s the ctivities of 63- nd 72-kD MMP-2. We found significntly incresed ctivities of 72-kD MMP-2 in plsm of rts exposed to the prolonged effects of DOX (Figure 3). The observed increse in 72-kD MMP-2 ctivities fter DOX tretment in plsm correlted with increse of MMP-2 ctivities in the left ventricle. However, tretment with QCT filed to prevent the DOX-induced effects on ctivtion of circulting plsm MMP-2 (Figure 3). A B C C Q Q C C Q Q -Dox + Dox MMP-2 Intensity of recrtion (% of ) Figure 3. Effects of QCT nd DOX tretment on plsm MMP-2 ctivities. (A) Zymogrm showing the ctivities of circulting plsm MMP-2 nlyzed using geltin zymogrphy; (B) Quntittive nlysis of plsm MMP-2 ctivities. Dt re expressed s percentge of vlue for corresponding. Ech br represents men ± S.E.M. of seven independent plsm smples per group. Sttisticl significnce is reveled by Student s unpired t-test, p <.5 vs. sline-treted ( DOX) rts.

6 Int. J. Mol. Sci. 215, Quercetin Prevents the Negtive Effects of Doxorubicin on Apoptosis Induction nd Superoxide Dismutse Inhibition Detection with specific ntibody documented n incresed content of cleved PARP (poly(adenosine Diphosphte-Ribose) Polymerse) in the left ventricle of rts exposed to the prolonged effects of DOX (Figure 4A,B). Apoptosis induction by DOX ws confirmed lso by cspse-3 ctivtion (Figure 4A,C). The observed dt show tht QCT prevented the negtive effects of DOX on poptosis induction nd its ppliction reversed the DOX-induced cspse-3 ctivtion (Figure 4A,C) nd PARP clevge (Figure 4A,B). A cleved PARP cleved cspse 3 GAPDH C C Q Q C C Q Q B -Dox + Dox C cleved PARP/GAPDH protein rtio b cleved cspse-3/gapdh protein rtio , b Figure 4. Effect of QCT nd DOX on mrkers of poptosis induction in the left ventricle. (A) The protein levels of cleved cspse-3 nd cleved PARP were determined by western blot nlysis using specific ntibodies. The protein loding is documented using GAPDH; (B) Quntifiction of cleved cspse-3 content normlized to the GAPDH protein levels; (C) Quntifiction of cleved PARP content normlized to the GAPDH protein levels. Dt were obtined from western blot records nd ech br represents men ± S.E.M. of seven tissue smples per group. Sttisticl significnce is reveled by Student s unpired t-test, p <.5 vs. sline-treted ( DOX) rts; b p <.5 vs. DOX-treted (+DOX) rts. The induction of poptosis s well s ctivtion of the non-cleved, oxidtively ctivted 72-kD form of tissue ventriculr MMP-2 suggested potentil ltertions in ctivities of enzymes involved in rdicl (superoxide) formtion. We found tht the effects of DOX were ssocited with reduction of totl superoxide dismutse (SOD) ctivities. QCT tretment prevented the negtive effects of DOX on SOD inhibition (Figure 5A). Moreover, QCT lone induced significnt stimultion of SOD ctivities

7 Int. J. Mol. Sci. 215, in nimls. The observed chnges in SOD ctivities in DOX-treted rts were in positive correltion with protein levels of SOD-2 isoform. DOX induced down-regultion of SOD-2 protein nd reversed these DOX-induced effects on SOD-2 (Figure 5B). On the other hnd, QCT nd DOX did not influence the protein expression of SOD-1. A B SOD ctivity (U/mg protein) b SOD-2 protein levels (% of ) b Figure 5. Effects of QCT nd DOX tretment on superoxide dismutse (SOD) totl ctivities nd protein levels. (A) The SOD ctivities were nlyzed using the SOD Assy kit in tissue smples of the left ventricle. The specific ctivities re expressed in units per mg of proteins nd re presented s men ± S.E.M.; (B) Quntittive nlysis of SOD-2 protein levels. Dt re expressed s percentge of vlue for corresponding. Ech br represents men ± S.E.M. of seven independent tissue smples per group. Sttisticl significnce is reveled by Student s unpired t-test, p <.5 vs. sline-treted ( DOX) rts; b p <.5 vs. DOX-treted (+DOX) rts Prolonged Appliction of Quercetin Improves Postischemic Recovery of Crdic Function of Isolted Rt Herts Left ventriculr developed pressure (LVDP, systolic minus distolic pressure), mximl rtes of pressure development +(dp/dt)mx nd fll (dp/dt)mx (s the indexes of contrction nd relxtion), s well s coronry flow (CF) were used to ssess crdic function. There were no significnt differences in the bseline vlues of ll these prmeters between ll four groups. After ischemi/reperfusion, the recovery of these prmeters ws determined nd expressed s percentge of pre-ischemic bseline vlues. In the 4th minute of reperfusion, the recovery of severl functionl prmeters of the herts ws significntly improved in both QCT-treted groups ginst QCT-non-treted nimls ( or DOX-treted, respectively). Recovery of LVDP ws significntly higher in QCT ginst s well s in DOX QCT ginst DOX (Figure 6A). Similrly, the mximl postischemic recovery of (dp/dt)mx ws significntly incresed fter ppliction of QCT nd tht in both sline (QCT) nd DOX-treted (DOX QCT) rts (Figure 6B). A tendency of n improved recovery fter the tretment with ws lso observed for +(dp/dt)mx but the chnges were significnt only in rts exposed to the effects of both QCT nd DOX (Figure 6C). During the entire experiment there were no significnt differences in the coronry flow. Figure 6D indictes the vlues in the 4th minute of reperfusion.

8 Int. J. Mol. Sci. 215, A B LVDP recovery (% of bseline vlue) , b -dp/dtmx recovery (% of bseline vlue) , b C D +dp/dtmx recovery (% of bseline vlue) , b coronry flow (% of bseline vlue) Figure 6. Effects of on ischemic tolernce of herts isolted from norml nd doxorubicin-treted rts. (A) Postischemic recovery of the left ventriculr developed pressure LVDP; (B) Postischemic recovery of the mximl rte of pressure development +(dp/dt)mx; (C) Postischemic recovery of the mximl rtes of pressure fll (dp/dt)mx; (D) Postischemic recovery of the coronry flow. The recovery of prmeters ws determined fter 3 min lsting globl ischemi followed by 4-min reperfusion. The recovery of prmeters ws expressed s percentge of pre-ischemic bseline vlues. Ech br represents men ± S.E.M. of eight independent mesurements. p <.5 vs. sline-treted ( DOX) rts; b p <.5 vs. DOX-treted (+DOX) rts Crdioprotective Effects of Quercetin Are Associted with Akt Kinse Activtion nd Connexin-43 Up-Regultion To chrcterize the possible mechnisms involved in crdioprotective effects of QCT, we investigted the chnges in Akt kinse. The obtined dt showed tht there re no differences in the levels of totl Akt kinse between the experimentl groups (Figure 7A). However, detection with phospho-specific ntibody reveled n incresed phosphoryltion of Akt kinse specificlly on Ser473 in the left ventricle of rt herts exposed to the effects of QCT either in sline- or doxorubicin-treted groups of rts (Figure 7A). Prolonged effects of DOX were lso ssocited with significntly incresed specific phosphoryltion of Akt kinse. From the point of the modultion of ischemic tolernce it is importnt tht ppliction of QCT further potentited Akt kinse ctivtion. The observed levels of ctive Ser473 phosphorylted Akt kinse (fter QCT nd/or QCT+DOX tretment) were lso incresed in reltion to totl Akt kinse levels (Figure 7B).

9 Int. J. Mol. Sci. 215, A B C Q C Q -Dox + Dox Akt P-Akt phospho-akt / Akt rtio C D C C Q Q C C Q Q -Dox + Dox Cx-43 Intensity of recrtion (% of ) , b Figure 7. Effect of QCT nd DOX on Akt kinse nd connexin-43. (A) Western blot records showing the chnges in protein levels nd specific phosphoryltion of Akt kinse. The chnges in ctivtion of Akt kinse were determined using n ntibody which rects with Akt kinse phosphorylted specificlly on Ser473; (B) The quntifiction of Akt kinse phosphoryltion (ctivtion). Dt were obtined from western blot records nd re expressed s rtio of content of phosphorylted Akt kinse to totl Akt kinse. Ech br represents men ± S.E.M. of seven tissue smples per group. Sttisticl significnce is reveled by Student s unpired t-test, p <.5 vs. sline-treted ( DOX) rts; b p <.5 vs. DOX-treted (+DOX) rts; (C) Protein levels of Connexin-43 (Cx-43) were determined by western blot nlysis using specific ntibody. The upper Cx-43 bnd represents the expression of highly phosphorylted Cx-43, while the lower bnd corresponds to Cx-43 phosphorylted to lower extent (degree); (D) Quntifiction of content of the lower phosphorylted form of Cx-43. Brs represent men ± S.E.M. of mesurements of seven independent tissue smples per group. Sttisticl significnce is reveled by Student s unpired t-test; p <.5 vs. sline-treted ( DOX) rts; b p <.5 vs. DOX-treted (+DOX) rts. Connexin-43 (Cx-43) protein expression ws nlyzed using specific ntibody detecting both phosphorylted nd unphosphorylted Cx-43. Doxorubicin tretment induced reduction of Cx-43 protein, especilly of low phosphorylted form of Cx-43, but tretment reversed the DOX-induced reduction of Cx-43 (Figure 7C,D). The levels of Cx-43 fter ppliction of QCT even exceed the vlues observed in nimls.

10 Int. J. Mol. Sci. 215, Discussion The present study clerly shows tht prolonged tretment with QCT possesses beneficil effects ginst DOX-induced chnges in rt herts s evident from lowered systolic blood pressure, decresed cellulr dmge, ttenuted tissue MMP-2 ctivtion, nd decresed poptosis induction. Moreover, we showed tht QCT enhnced function of isolted herts of sline- nd doxorubicin-treted rts nd these crdioprotective effects of QCT were relized through incresed recovery of contrctile function fter ischemi/reperfusion. Our dt demonstrte tht QCT protects by reversing DOX-induced increse of systolic blood pressure. Interestingly, the prolonged ppliction of QCT decresed blood pressure in DOX-treted (hypertensive) nimls, but not in rts with norml blood pressure (, sline-treted). Similr ntihypertensive effects of QCT were found in severl niml models of incresed blood pressure: this flvonoid hs been shown to decrese blood pressure in spontneously hypertensive rts [5], Dhl slt-sensitive rts [17], nd rts fed with high-ft, high-sucrose diet [7]. There exist severl mechnisms potentilly involved in ntihypertensive effects of QCT tht cn include decrese in oxidtive stress s well s improved vsculr function, nd this is supported by ntioxidnt effects of QCT, nd its bility to hve vsodiltor effects on rt rteries [18]. It hs been previously demonstrted tht chronic effects of nthrcyclines re ssocited with profound chnges in left ventriculr morphology nd function s well s with ltertions in the collgen network [19]. In the present study we observed heterogeneous subcellulr ltertions in the left ventricle fter DOX tretment nd we found tht ppliction of QCT resulted in prevention of some deleterious subcellulr nd extrcellulr spce chnges induced by DOX. The positive effects of QCT on ultrstructure of the left ventricle were ssocited with prevention of MMP-2 ctivtion. MMP-2 is n enzyme tht plys n importnt role in processes of extrcellulr mtrix remodeling nd its ctivtion could be relted to structurl disorgniztions of the crdic extrcellulr spce. The ctivtion of myocrdil nd circulting MMPs, especilly MMP-2, is closely ssocited with the progression of the toxic effects of DOX [2] nd these enzymes were lso found to be mrkers of cute DOX-induced crdiotoxicity in mice [21]. We found modultion of ctivity of the 72-kD MMP-2, the form of MMP-2 tht my be oxidtively ctivted through conformtionl chnges induced by rdicls. This suggested potentil ltertions in enzyme ctivities of enzymes involved in rdicl formtion. We found tht the effects of DOX on 72-kD MMP-2 ctivtion in the left ventricle were ssocited with reduction of totl superoxide dismutse (SOD) ctivities, nd QCT tretment prevented the negtive effects of DOX on both 72-kD MMP-2 ctivtion nd SOD inhibition. Thus, the effects of QCT on DOX-induced chnges in tissue 72-kD MMP-2 ctivities cn lso be explined through the observed effects on recovery of SOD ctivities. MMP-2 is sensitive to oxidtive stress nd rective oxygen species (ROS) cn ctivte pro-mmp-2 (72-kD MMP-2) by oxidtion of the sulfide bond in the prodomin of the MMP, nd peroxynitrite ( rection product of superoxide nd nitric oxide) ctivtes pro-mmp vi interction with glutthione [22 24]. The connection between superoxide levels (SOD) nd MMP-2 ws lso demonstrted in study showing incresed superoxide production nd MMP-2 ctivtion induced by high glucose in bovine retinl endothelil cells [25]. Addition of the MnSOD mimic MnTBAP, prevented the high glucose-induced increse of superoxide levels nd

11 Int. J. Mol. Sci. 215, meliorted the elevtion in MMP-2 ctivities. Moreover, both superoxide production nd MMP-2 ctivtion were prevented through n inhibitor of MMP-2 [25]. We found tht tretment with QCT prevented the ctivtion of pro-poptotic signling pthwy induced in consequence of DOX ppliction. QCT reduced the negtive effects of DOX on poptosis induction nd reversed the DOX-induced cspse-3 ctivtion nd PARP clevge. The observed nti-poptotic effects of QCT in herts of DOX-treted rts my lso be relted to the observed ltertions in SOD ctivities (superoxide formtion) nd re in greement with described nti-oxidtive, oxygen rdicl-scvenging, nd nti-poptotic functions of QCT [2]. We found tht the prolonged ppliction of QCT did not hve positive effects only on hert responses fter chronic stress induced by DOX but medited lso the myocrdil responses to dditionl cute ischemic stress. In the present study we showed tht enhnced postischemic tolernce of isolted herts of both doxorubicin- nd sline-treted rts. The protective effects of QCT were relized through n incresed recovery of contrctile function fter ischemi/reperfusion. The obtined dt suggest tht the QCT-induced protection ginst ischemi/reperfusion injury cn be due to n observed endogenous bseline ctivtion of Akt kinse pthwy. Totl levels of Akt kinse were not modified in herts either by QCT or DOX. In contrst, significnt increse in specific phosphoryltion of Akt kinse ws pprent fter the QCT ppliction. As consequence, the rtio phosphorylted/totl for Akt incresed in nimls exposed to prolonged effects of QCT. The finding tht ppliction of QCT potentited Akt kinse ctivtion not only in sline but lso in DOX-treted rt herts ws importnt, nd Akt kinse ctivtion nd improvement of ischemic tolernce were mximl in nimls co-treted with both QCT nd DOX. Some studies showed tht n cute ppliction of QCT before ischemi (preconditioning) or during reperfusion (postconditioning) protected the myocrdium from ischemi/reperfusion injury [8,9]. In our study we observed protective effects of QCT fter its chronic ppliction nd the crdioprotective effects were relized severl weeks fter the end of the QCT ppliction. Also interesting ws the finding tht in rt herts, the crdioprotective ctions of QCT were even more potentited in rts fter DOX tretment. Appliction of DOX hd negtive effects on the ultrstructure of hert tissue, stimultion of poptosis, nd incresed superoxide production but did not hve negtive effects on modultion of ischemic tolernce. We suggest tht possible explntion of this discrepncy nd bsence of negtive effects of DOX on ischemic tolernce is the observed increse of P-Akt. PI3K/Akt signling pthwy ws found to hve n importnt role in the mechnisms of hert dpttion to pthologicl situtions [26,27] nd its ctivtion ws lso found to ply role in infrct size-limiting mechnisms in the rt hert [28] nd by prevention of injury fter trnsient crdic ischemi in vivo [29]. Severl studies documented tht mechnisms of the incresed myocrdil tolernce ginst injury, induced by ischemi/reperfusion, involve the PI3K/Akt/GSK-3β pthwy with phosphoryltion nd inhibition of the glycogen synthse kinse-3β (GSK-3β) [3 32]. Moreover, phosphoryltion of GSK-3β through PI3K/Akt pthwy cn led to GSK-3β inctivtion with subsequent ccumultion/ctivtion of β-ctenin [33]. Another study showed tht n increse in Akt kinse ctivity is crucil for the incresed levels of heme oxygense-1 (HO-1) induced protection ginst hypoxi-induced injury. Stimultion of Akt kinse resulted in phosphoryltion of GSK-3β t Ser 9. Phosphoryltion t this site inhibited the ctivtion of GSK3-bet, leding to decresed mitochondril permebility trnsition pore (mptp) opening followed by n increse in crdiomyocyte protection [34]. The Akt kinse ctivtion

12 Int. J. Mol. Sci. 215, observed in our study is likely to be protective response to counterct DOX-induced negtive effects on crdic cells. The role of Akt kinse in these effects is lso supported by finding tht Akt kinse ctivtion correltes well with crdioprotection (recovery of crdic function). These two prmeters were mximl in nimls co-treted with both DOX nd QCT. The obtined dt suggest tht crdioprotective effects of QCT could lso be ssocited with observed modultion of Gp junction-ssocited connexin-43 (Cx-43). This protein is criticlly importnt in mny cell processes including intercellulr signl trnsduction, of cell prolifertion, nd coordinted contrction of the hert, nd is suggested to ply n importnt role lso in the pthophysiology of ischemi/reperfusion injury [35,36]. We hve observed tht crdioprotective effects of QCT re ssocited with up-regultion of protein levels of Cx-43. In greement with our dt, the induction of Cx-43 ws lso found in crdioprotection fforded by ischemic preconditioning nd pretretment with dizoxide in isolted rt herts [35]. We hve demonstrted tht exposure of rts to chronic effects of DOX suppressed expression of Cx-43 in the left ventricle nd QCT tretment reversed the DOX-induced effects. An inhibition of crdic expression of Cx43 ws lso demonstrted 14 dys fter DOX dministrtion in mice herts ws observed [37]. 4. Experimentl Section 4.1. Mterils The SOD Assy kit nd ntibody ginst connexin-43 were purchsed from Sigm Aldrich (St. Louis, MO, USA). Primry ntibodies recognizing phosphorylted Akt kinse (Ser473), cleved cspse-3, cleved PARP, nd peroxidse-lbeled nti-rbbit or nti-mouse immunoglobulins were purchsed from Cell Signling Technology (Dnvers, MA, USA). Antibodies ginst totl Akt kinse, MMP-2, SOD-1, SOD-2, nd GAPDH were obtined from Snt Cruz Biotechnology (Snt Cruz, CA, USA) Experimentl Model In the study, 11-weeks old mle Wistr rts were used. All nimls were housed t temperture of C nd fed with regulr pellet diet d libitum. Rts were divided into four experimentl groups: sline-treted (C), doxorubicin-treted (DOX), -treted (QCT), nd doxorubicin+-treted (DOX QCT). In the DOX groups, the rts received DOX in cumultive dose 15 mg/kg for 3 weeks. DOX ws pplied by seven intrperitonel injections, every 3rd dy. The nimls of experimentl groups without ppliction of DOX were treted with sline. Quercetin ws served on piece of bisquit in doses of 2 mg/kg/dy. The dministrtion of QCT strted on the sme dy s the DOX (sline) dministrtion nd continued for further 3 weeks fter the completion of the DOX tretment (totl period of 6 weeks). Eight weeks fter the completion of DOX or sline tretment, the nimls were nesthetized with thiopentl (5 mg kg 1, intrperitonelly); excised herts were either perfused ccording to Lngendorff nd tested on I/R injury or used for seprtion of the ventriculr tissue smples. All niml experiments were performed in ccordnce with the rules issued by the Stte Veterinry Administrtion of the Slovk Republic, legisltion No 289/23 nd with the regultions of the Animl Reserch nd Cre Committee of Institute for Hert Reserch SAS Project 1873/11-221/3, pproved on 3 September 211.

13 Int. J. Mol. Sci. 215, Systolic Blood Pressure nd Hert Rte Mesurement The systolic blood pressure (SBP) nd hert rte (HR) were mesured by the non-invsive method of til cuff plethysmogrphy (PowerLb 4/3, ADInstruments, Budpest, Hungry) in ll experimentl groups of rts. The mesurements were performed before the first DOX or sline pplictions nd eight weeks fter the end of their ppliction Perfusion Technique nd Determintion of Hert Function Rts were nesthetized (thiopentl, 5 mg/kg, i.p.) nd heprinised (5 IU, i.p.) before hert excision nd perfusion. Herts were rpidly excised, plced in ice-cold perfusion buffer, cnnulted vi the ort nd plced onto the Lngendorff setup for perfusion t constnt perfusion pressure of 73 mm Hg nd temperture of 37 C. Perfusion solution ws modified Krebs Henseleit buffer gssed with 95% O2 nd 5% CO2 (ph 7.4) contining (in mmol/l): NCl 118.; KCl 3.2; MgSO4 1.2; NHCO3 25.; KH2PO4 1.18; CCl2 2.5; glucose 7.. This solution ws filtered through 5 µm porosity filter to remove contminnts. An epicrdil electrogrm ws registered by mens of two stinless steel electrodes ttched to the pex of the hert nd ortic cnnul. Left ventriculr pressure ws mesured by mens of wter-filled blloon inserted into the left ventricle vi the left trium (djusted to obtin end-distolic pressure of 1 8 mm Hg) nd connected to pressure trnsducer. Left ventriculr developed pressure (LVDP, systolic minus distolic pressure), mximl rtes of pressure development nd fll, +(dp/dt)mx nd (dp/dt)mx, s the indexes of contrction nd relxtion, s well s the hert rte (clculted from EG) nd coronry flow were used to ssess crdic function. Recovery of these prmeters fter ischemi/reperfusion ws expressed s percentge of pre-ischemic bseline vlues. Globl ischemi ws mintined for 3 min, followed by 4 min reperfusion. Functionl prmeters of herts were mesured during the whole reperfusion Smples Collection At the end of the experiment, the nimls were nesthetized with thiopentl (5 mg/kg, i.p.) injection, nd were euthnized by thorcotomy nd rpid excision of their herts. Excised herts were weighted nd seprted to the ventricles. The whole hert (HW) nd left ventriculr weights (LVW) were registered. Further processing of the collected left ventriculr tissue smples ws dependent on the following ssy. The tissue left ventriculr smples for biochemicl nlysis were immeditely frozen in liquid nitrogen nd stored t 75 C until use. For the trnsmission electron microscopy studies, smll blocks of trnsmurl left ventriculr tissues were fixed in buffered 2.5% glutrldehyde immeditely fter collection. The plsm smples were prepred from whole rtery blood drwn from the chest of the rts immeditely fter excision of the hert. Citrte ws immeditely dded to the collected blood (resulting concentrtion.76%), followed by centrifugtion of the blood for five min t 12 g to obtin the plsm. The prepred plsm smples were stored t 2 C until further nlysis.

14 Int. J. Mol. Sci. 215, Trnsmission Electron Microscopy Smll (1 2 mm 3 ) trnsmurl left ventriculr hert tissue smples were routinely processed for electron microscopy. The smples were fixed in 2.5% glutrldehyde in 1 mmol/l ccodylte buffer t 4 C, wshed, postfixed in 1% OsO4, nd subsequently embedded in Epon 812. Ultrthin sections of the tissue were stined with urnyl cette nd led citrte. The ultrstructure of the myocrdil tissue ws evluted using trnsmission electron microscope Tesl 5 (Tesl, Brno, Czech Republic) Preprtion of Tissue Protein Frctions nd Western Blot Anlysis The tissue smples were resuspended in ice-cold buffer A contining (in mmol/l): 2 Tris-HCl, 25 sucrose, 1. EGTA, 1. dithiothreitol (DTT), 1. phenylmethylsulphonyl fluoride (PMSF) nd.5 sodium orthovndte (ph 7.4) nd homogenized with Teflon homogenizer. The homogentes were centrifuged t 8 g for 5 min t 4 C, the pellets were discrded fter centrifugtion nd the superntnts were centrifuged gin t 16,1 g for 3 min. The superntnts fter the second centrifugtion were used for biochemicl nlysis nd protein concentrtions were estimted by the Brdford method [38]. For western blot nlysis, smples contining equivlent mounts of proteins per lne were seprted by SDS-polycrylmide gel electrophoresis (SDS-PAGE). The proteins fter electrophoretic seprtion were trnsferred onto nitrocellulose membrnes, nd fter blocking of non-specific binding sites, the membrnes were incubted overnight t 4 C with the corresponding specific primry ntibody. The corresponding peroxidse-lbeled nti-rbbit or nti-mouse immunoglobulins were used s secondry ntibodies. Peroxidse rections were detected by the enhnced chemiluminescence (ECL) system Mesurement of MMP-2 Activities by Geltin Zymogrphy The ctivity of MMP-2 ws evluted using zymogrphy in 1% polycrylmide gels contining geltin (2 mg/ml) s substrte for MMP-2. The smples were suspended in Lemmli buffer without 2-mercptoethnol nd loded onto gels without denturtion. After electrophoresis, the gels were wshed twice for 2 min ech with 5 mmol/l Tris-HCl (ph 7.4), contining 2.5% Triton X-1, nd then incubted overnight t 37 C in substrte buffer contining 5 mmol/l Tris-HCl, 1 mmol/l CCl2 nd 1.25% Triton X-1, ph 7.4. After incubtion, the gels were stined with 1% Coomssie Brillint Blue G-25 nd then destined with 4% methnol nd 1% cetic cid. The geltinolytic ctivities of the MMPs were detected s trnsprent bnds ginst drk blue bckground. Recombinnt, ctive MMP-2 ws used s positive to identify the ctive 63 kd MMP-2 form. Seventy-two kd MMP-2 ws identified using fetl bovine serum contining this form of MMP Determintion of Superoxide Dismutse Activity The SOD ctivity ws nlyzed using SOD Assy kit, which ssys SOD ctivity by utilizing highly wter-soluble tetrzolium slt, WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrzolium, monosodium slt), which produces wter-soluble formzn dye upon reduction with superoxide nion. The ctivities were determined following the mnufcturer s instructions. The

15 Int. J. Mol. Sci. 215, chnges in formzn production were nlyzed for 3 min t 37 C using microplte reder (Thermo Scientific Multiscn FC, Vnt, Finlnd). The SOD ctivities were clculted using SOD stndrd curve nd expressed s U mg 1 of protein Sttisticl Evlution Sttisticl differences were lwys determined between two groups; the effects of DOX nd/or QCT were compred to the conditions (DOX or QCT groups vs. C group) nd QCT with DOX to the conditions when only DOX ws pplied (DOX QCT vs. DOX group). Sttisticl significnce ws reveled by one-wy ANOVA with Bonferroni post-hoc test or Student s unpired t-test (Origin softwre). Differences were considered significnt t p <.5 in ll the tests. 5. Conclusions Tken together, our results demonstrte tht prolonged tretment with QCT prevented negtive chronic effects of DOX on blood pressure, cellulr dmge, MMP-2 ctivtion, nd poptosis induction in herts. Tretment with QCT hd positive effects not only on hert responses fter chronic stress induced by DOX, but lso medited myocrdil responses to n dditionl cute ischemic stress. The findings of the current study demonstrte tht prolonged ppliction of QCT improved ischemic tolernce nd tht this improvement ws in concert with triggering of Akt kinse ctivtion nd Cx-43 up-regultion. Acknowledgments This study ws supported by grnts of Scientific Grnt Agency of the Ministry of Eduction of Slovk Republic nd the Slovk Acdemy of Sciences (VEGA SR) No. 2/169/12, 2/14/12, 2/46/12, 2/18/15 nd grnt of Agency for Reserch nd Development APVV Author Contributions Monik Brteková, Petr Šimončíková, Mári Fogrssyová, Monik Ivnová, nd Miroslv Brnčík performed the physiologicl nd biochemicl experiments, Nrcis Tribulová contributed to the connexin-43 determintion, Ľudmil Okruhlicová contributed to the trnsmission electron microscopy, Im Dovinová performed the determintion of SOD ctivities, Monik Brteková, Petr Šimončíková, Nrcis Tribulová contributed to drfting the rticle, Miroslv Brnčík finlized the mnuscript. Conflicts of Interest The uthors declre no conflict of interest. References 1. Hertog, M.G.; Bueno-de-Mesquit, H.B.; Fehily, A.M.; Sweetnm, P.M.; Elwood P.C.; Kromhout, D. Fruit nd vegetble consumption nd cncer mortlity in the Cerphilly Study. Cncer Epidemiol. Biomrk. Prev. 1996, 5,

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