Chang TH et al / Acta Pharmacol Sin 2002 Sep; 23 (9): 769-774

Effects of dl-praeruptorin A on interleukin-6 level and Fas, bax, bcl-2 protein expression in ischemia-reperfusion myocardium¹

CHANG Tian-Hui², LIU Xiao-Yang, ZHANG Xin-Hua, WANG Huai-Liang

Department of Clinical Pharmacology, China Medical University, Shenyang 110001, China

¹ Project partly supported by the Key Foundation of the Ministry of Public Health of China, ¡í 88402257.

² Correspondence to Prof CHANG Tian-Hui. Phn 86-24-2325-6666, ext 5130 or 5547. E-mail thchang99@yahoo.com.cn

Received 2001-12-04 Accepted 2002-05-30

KEY WORDS dl-praeruptorin A; myocardium; ischemia; reperfusion injury; apoptosis; interleukin-6

ABSTRACT

AIM: To investigate the effects of dl-praeruptorin (Pd-Ia) on interleukin-6 (IL-6) level and apoptosis-related protein expression in ischemia-reperfusion myocardium. METHODS: Left anterior descending coronary artery was subjected to 30 min ischemia followed by 120 min reperfusion in open-chest anesthetized rats. Serum IL-6 level was measured by radioimmunoassay. Apoptosis-related protein Fas, bax, and bcl-2 expression was detected by immunohistochemistry and computer image analysis system. Infiltration of neutrophils was observed using Hematoxylin-Eosin staining under optical microscope. RESULTS: Pd-Ia 2.0 mg·kg ^-1 iv lowered serum IL-6 level and Fas, bax, bcl-2 expression under conditions with hypotension and without changes on heart rate, but increased the ratio of bcl-2/bax. There existed a close linearity and positive correlation between IL-6 level and Fas, bax, bcl-2 expression. Whereas, the infiltration of neutrophils was mild. CONCLUSION: Pd-Ia elicits a novel target in the therapeutic prevention of postischemic cardiomyocyte death. The reason might be associated with modulating the expression of some immediate-early genes including IL-6, Fas, bax, and bcl-2 in ischemia-reperfusion myocardium.

INTRODUCTION

A number of studies have shown that ischemia-reperfusion induce inflammatory cytokine gene expression such as tumor necrosis factor-a (TNF-a )^[1,2], interleukin (IL)-b, IL-6, IL-8, interferon-g^[3,4], and intercellular adhesion molecule-1^[5] in myocardium. These overexpressed myocardial cytokines may play a critical role in the progress of myocardial dysfunction, including ischemia-reperfusion injury (MIR), vascular wall remodeling^[6], heart failure^[7], and cardiac hypertrophy^[8]. Recent evidence suggested that locally produced TNF could also contribute to postischemic myocardial dysfunction via direct depression of contractility and induction of cardiomyocyte apopto-sis^[9]. However, the molecular mechanism of controlling and regulating the expression of these immediate-early genes during MIR has remained unclear. As cardiomyocyte apoptosis is one of the major contributors to the pathogenesis, accordingly, prevention of it may be a reasonable therapeutic strategy.

In our previous studies, a pyranocoumarin component dl-praeruptorin (Pd-Ia) isolated from Baihua Qianhu, a traditional Chinese medicine (Peucedanum praeruptorum Dunn), as a Ca²⁺-influx blocker^[10-12] and K⁺-channel opener^[13], displays myocardial protection^[14-16]. In the present study, we mainly investigated the effects of Pd-Ia on serum IL-6 level and Fas, bax, bcl-2 protein expression in ischemia-reperfusion myocardium of rats, in order to further explore the activity and mechanism of Pd-Ia.

MATERIALS AND METHODS

Drugs and reagents Pd-Ia was presented friendly by Prof OKUYAMA T (Department of Pharmacognosy and Phytochemistry, Meiji College of Pharmacy, Tokyo 154, Japan); ¹²⁵I-interleukin-6 radioimmunization kit was produced by 301 Radioimmunization Research Center, Beijing, China; Rabbit anti-rat monoclonal antibodies (including Fas, bax, and bcl-2) were prepared by Santa Cruz Biological Technology Co; Instant-type SABC (Strept-Avidin-Biotin-enzyme Complex) immunohistochemistry kit was provided by Wuhan Boster Biological Technology Co.

Experimental protocol Forty-seven healthy male Wistar rats of both sexes, weighing 274 g±16 g were randomly divided into 7 subgroups (n=6-7). Group I, sham operation control; Group II, operation control; Group III, solvent control (PEG400); Group IV, positive control (nifedipine); Group V, Pd-Ia 0.5 mg·kg ^-1; Group VI, Pd-Ia 1.0 mg·kg ^_1; Group VII, Pd-Ia 2.0 mg·kg ^-1.

Under urethane-chloralose anaesthesis, open-chest, and artificial ventilation, left anterior descending coronary artery of rat was dissected. A 5-0 silk suture was passed around the vessel and an occlusive snare was placed around it. Thirty minutes ischemia followed by 120 min reperfusion was produced as reported previously^[14]. Mean carotid arterial pressure (BP) and heart rate (HR) were measured using 8-channel recorder (RM-6000, Nihon Kohden).

A bolus of Pd-Ia was injected iv while onset of reperfusion. Blood sample was drawn out from right ventricular chamber at 120 min after reperfusion. Serum IL-6 level was determined by radioimmunoassay. Partial myocardium from area subjected to MIR was scissored snap-frozen, and sliced by freezing micro-tome. Fas, bax, and bcl-2 expression was surveyed by immunohistochemistry and computer image analysis system^[17]. Chromogenic reaction was performed with 3',3-diaminobenzidine 0.5 g/L (25 ¡æ±1 ¡æ, 10 min), counterstained in hematoxylin and observed under light microscopy. Brown staining in epicyte or cytoplasm was evaluated as positive expression, which was examined by calculating the ratio of mean optic density and area in positive stain, namely, positive expressive index (PEI).

Infiltration of neutrophil was examined by Hematoxylin-Eosin staining under optical microscope.

Statistical analysis All data was expressed as mean±SD. Unpaired student's t-test was used to assess statistical significance of differences between

Pd-Ia and various related control groups. Comparison of parameters before and after administration of drugs was performed using one-way ANOVA analysis of variance. P<0.05 was considered to be significant.

Effects of Pd-Ia on serum IL-6 level Pd-Ia decreased serum IL-6 level at higher dose of 2.0 mg/kg markedly [(116±11) ng/L vs (153±15) ng/L in solvent control, P<0.05]. This decrease was similar to that of nifedipine. IL-6 level in operation control was higher than that of sham control, suggesting that MIR induced the overexpression of IL-6, but no significant change was found in solvent control (Tab 1).

Tab 1. Effects of Pd-Ia on serum IL-6 level and Fas, bax, bcl-2 expression in ischemia-reperfusion myocardium of rats. PEI: positive expressive index; Sham: sham control; Oper: operation control; Sol: solvent control; Nif: nifedipine control. Mean±SD. ^bP<0.05, ^cP<0.01 vs solvent group. ^fP<0.01 vs sham group.

Group	Dose/mg×kg-1	n	IL-6/ng×L-1	PEI/%			bcl-2/bax
				Fas	bax	bcl-2
Sham	-	7	122± 6	1.9±0.5c	1.4±0.3c	1.8±0.5c	1.35±0.11c
Oper	-	6	152±18	7.0±2.4f	5.9±1.4f	5.7±1.0f	0.96±0.11f
Sol	-	7	153±15	6.7±1.5f	6.0±1.0f	5.4±0.9f	0.92±0.09f
Nif	5.0	6	124±19b	3.6±0.6c	1.8±0.4c	3.6±0.6b	2.05±0.22c
Pd-Ia	0.5	7	129±18	3.8±0.7b	3.5±0.4c	4.2±1.0	1.16±0.17
	1.0	7	120±16b	3.5±0.7c	2.8±0.3c	3.4±0.9c	1.28±0.23c
	2.0	7	116±11b	3.4±0.9c	2.1±0.4c	2.9±0.7c	1.42±0.06c

Effects of Pd-Ia on Fas, bax, and bcl-2 expression Pd-Ia inhibited Fas, bax, and bcl-2 expression (Fig 1). At the dose of Pd-Ia 2.0 mg·kg ^_1, PEI of Fas, bas, and bcl-2 were decreased significantly (3.4 %±0.9 %, 2.1 %±0.4 %, and 2.9 %±0.7 % vs 6.7 %±1.5 %, 6.0 %±1.0 %, and 5.4 %±0.9 % in solvent control, P<0.01), in contrast, it increased the ratio of bcl-2/bax (1.42±0.06 vs 0.92±0.09 in solvent control, P<0.01). The attenuation was a little similar but weak to that of nifedipine. Fas, bax, bcl-2 expression in operation control was markedly higher than sham control (P<0.01), whereas the ratio of bcl-2/bax was lower (P<0.05, Tab 1), indicating that MIR assuredly lead to apoptosis-related protein overexpression.

Fig 1. Effects of Pd-Ia on Fas (b), bax (c), and bcl-2 (d) protein expression in left ventricular myocardium subjected to 30 min ischemia followed by 120 min reperfusion in open-chest anesthetized rats treated by a bolus injection intravenously of solvent (PEG 400) solution 1 mL/kg (a) or dl-praeruptorin A 2.0 mg/kg (b, c, and d) immediatedly before reperfusion. Epicyte or cytoplasma brown staining indicates positive expression. ×200.

Correlation analysis between serum IL-6 level and Fas, bax, and bcl-2 expression There existed a close linearity and positive correlation between serum IL-6 level and Fas, bax, bcl-2 expression (Fig 2). The correlation coefficients (r) were 0.8324, 0.7429, and 0.7423 (P<0.01), respectively.

Fig 2. Correlativity analysis between serum IL-6 level and Fas, bax, bcl-2 protein expression in ischemia-reperfusion myocardium of rats treated iv with Pd-Ia 2.0 mg/kg. PEI: positive expression index.

Effects of Pd-Ia on infiltration of inflammatory cells The infiltration of neutrophils in Pd-Ia group displayed mild, and was not seen in sham control, whereas most appeared in operation and solvent controls (Fig 3).

Fig 3. Micrograph demonstrating inflammatory cell infiltration using Hematoxylin-Eosin staining in left ventricular myocardium subjected to a 30 min ischemia followed by a 120 min reperfusion in open-chest anesthetized rats treated by a bolus injection intravenously of solvent (PEG 400) solution 1 mL/kg (upper) or dl-praeruptorin A 2.0
mg/kg (down) immediately before reperfusion. Most nucleolus of neutrophils staining in blue or violet, or more seen intraluminally indicates inflammatory cell infiltration. ×200.

Effects of Pd-Ia on BP and HR Pd-Ia decreased BP significantly, and the duration sustained over 60 min (Tab 2). The BP at maximal dose reached from 12.6 kPa±0.7 kPa to 9.4 kPa±1.2 kPa at 30 min after administration (25 %±6 % decreased, P<0.01 vs solvent group), but the potency was weaker than that of nifedipine (41 %±11 % decreased, P<0.05 vs nifedipine group). Furthermore, Pd-Ia had no influence on HR.

Tab 2. Effects of Pd-Ia on BP in ischemia-reperfusion myocardium of rats. Sham: sham control; Oper: operation control; Sol: solvent control; Nif: nifedipine control. T₀: time before drug; T₃₀: 30 min after drug; T₆₀: 60 min after drug. Mean±SD. ^cP<0.01 vs solvent group. ^fP<0.01 vs sham group. ^hP<0.05 vs nifedipine group.

Group	Dose/mg¡¤kg-1	n	BP/kPa
			T0	T30	[(T30-T0)/T0]/%	T60	[(T60-T0)/T0]/%
Sham	-	7	16.2¡À1.8	16.2¡À2.6	0¡À5	15¡À4	-8¡À14
Oper	-	6	13.5¡À1.5	13.1¡À1.6	-3¡À6	12.5¡À2.1	-7¡À11
Sol	-	7	10.8¡À2.1	10.7¡À1.9	-1¡À8	9.8¡À1.0	-9¡À10
Nif	5	6	15.1¡À2.3	8.7¡À0.4f	-41¡À11	8.9¡À0.9f	-42¡À10
Pd-Ia	0.5	7	14.2¡À1.5	11.0¡À1.4cfh	-22¡À8	11.3¡À1.7	-20¡À13
	1	7	14.7¡À1.6	11.1¡À0.7cfh	-24¡À7	11.2¡À0.7	-24¡À7
	2	7	12.6¡À0.7	9.4¡À1.2cfh	-25¡À6	9.5¡À1.2	-24¡À14

DISCUSSION

Recent basic experimental and clinical evidence suggest that brief coronary occlusion followed by reperfusion leads to reversible myocardial dysfunction^[18], whereas cardiomyocyte death during MIR is partially mediated by apoptosis^[19,20]. In the present study, we have demonstrated that Pd-Ia reduced serum IL-6 level and Fas, bax, bcl-2 expression during MIR under conditions with hypotension and without changes on HR in rats. Meanwhile, the action of Pd-Ia was strongly similar but weaker to that of nifedipine, a calcium channel blocker. So we hypothesize that the mechanisms of action of Pd-Ia are probably associated with blockade of Ca2+ influx, inhibition of Fas-Fas ligands, and Ca2+-dependence, etc. Those are all capable of abolishing Ca²⁺-overload of cytoplasm evoked by MIR^[21], maintaining balance between intra- and extra-cellular calcium, remaining vasodilatation and stability of mitochondria^[22], finally, leading to lower the occurrence of cardiomyocyte apoptosis. Thus, it is preferable to other vasodilators or calcium antagonists in cardiohemo-dynamic modulation^[23] and cardioprotection during MIR. Secondly, the fact that Pd-Ia reduced Fas, bax, and bcl-2 expression also involved other factors such as fall of IL-6 level, alleviation of neutrophils infiltration, lowering heart load and myocardial oxygen consumption via indirect amelioration of cardio-hemodynamics, etc.

In conclusion, controlling and regulating the expression of some immediate-early genes including IL-6, Fas, bax, and bcl-2, exhibits a relievable or beneficial effect in ischemia-reperfusion myocardium. Pd-Ia elicits a novel target in the therapeutic prevention of postischemic cardiomyocyte death. It might be as a therapeutic agent to open a promising perspectives in prevention of MIR and cardioprotection.

REFERENCES

• 1 Herskowitz A, Choi S, Ansari AA, Wesselingh S. Cytokine mRNA expression in postischemic/reperfusion myocardium. Am J Pathol 1995; 146: 419-28.
• 2 Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, et al. Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-a. Circ Res 1997; 81: 627-35.
• 3 Kukielka GL, Smith CW, LaRosa GJ, Manning AM, Mendoza LH, Daly TJ, et al. Interleukin-8 gene induction in the myocardium after ischemia and reperfusion in vivo. J Clin Invest 1995; 95: 89-103.
• 4 Kukielka GL, Smith CW, Manning AM, Youker KA, Michael LH, Entman ML. Induction of interleukin-6 synthesis in the myocardium: potential role in postreperfusion inflammatory injury. Circulation 1995; 92: 1866-75.
• 5 Yamazaki T, Seko Y, Tamatani T, Miyasaka M, Yagita H, Okumura H, et al. Expression of intercellular adhesion mole-cule-1 in rat heart with ischemia/reperfusion and limitation of infarct size by treatment with antibodies against cell adhesion molecules. Am J Pathol 1993; 143: 410-8.
• 6 Bozkurt B, Kribbs SB, Clubb FJ Jr, Michael LH, Didenko VV, Hormsby PJ, et al. Pathophysiologically relevant concentrations of tumor necrosis factor-a promote progressive left ventricular dysfunction and remodeling in rats. Circulation 1988; 97: 1382-91.
• 7 Bryant D, Becker L, Richardson J, Shelton J, Franco F, Peshock R, et al. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-a. Circulation 1988; 97: 1375-81.
• 8 Kelly RA, Smith TW. Cytokines and cardiac contractile function. Circulation 1997; 95: 778-81.
• 9 Meldrum DR. Tumor necrosis factor in the heart. Am J Physiol 1998; 274: R577-95.
• 10 Chang TH,Li JM, Sun XD, Yu YF, Feng WY, Hao LY, et al. Effects of Pd-Ia, a component of Chinese traditional herb Bai-Hua Qian-Hu, on Ca²⁺-current and action potential duration in single ventricular cells of guinea pig hearts. Jpn J Pharmacognosy 1993; 47: 279-82.
• 11 Li JM, Chang TH, Sun XD, Hao LY, Wang YP, Yu YF, et al. Effect of dl-praeruptorin A on calcium current in ventricular cells of guinea pig. Acta Pharmacol Sin 1994; 15: 525-7.
• 12 Hao LY, Li JM, Chang TH, Sun XD, Wang YP, Zhang KY. Frequency-dependent block of calcium current by (±)-praeruptorin A in single ventricular cell of guinea pig. Chin J Pharmacol Toxicol 1996; 10: 17-20.
• 13 Wang LJ, Li JM, Chang TH, Hao LY, Wang YP, Zhang KY. Effects of Pd-Ia on delayed outward potassium current in single ventricular myocyte of guinea pig. Chin J Pharmacol Toxicol 1995; 9: 192-5.
• 14 Chang TH, Adachi H, Mori N, Saito I, Okuyama T. Effects of Pd-Ia, a pyrano-coumarin isolated from a Chinese medicinal plant, on cardiohemodynamics and myocardial dysfunction after transient ischemia in anesthetized dogs. Eur J Pharmacol 1994; 258:77-84.
• 15 Chang TH, Adachi H, Okuyama T, Zhang KY. Effects of 3'-angeloyloxy-4'-acetoxy-3',4'-dihydroseselin on myocardial dysfunction after a brief ischemia in anesthetized dogs. Acta Pharmacol Sin 1994; 15: 388-91.
• 16 Chang TH, Chen L, Jiang MY, Wang YP. Effects of Peucedanum praeruptorum Dunn, a traditional Chinese medicine on acute myocardial infarction in open-chest anesthetized cats. J China Med Univ 2000; 29: 84-7.
• 17 Leng Y, Feng Y, Cao L, Gu ZP. Effects of droloxifene on apoptosis and Bax, Bcl-2 protein expression of luteal cells in pseudopregnant rats. Acta Pharmacol Sin 2001; 22:155-62.
• 18 Bolli R. Mechanism of myocardial "stunning". Circulation 1990; 82: 723-38.
• 19 Fliss H, Gattinger DA. Apoptosis in ischemia and reperfused rat myocardium. Circ Res 1996; 79: 949-56.
• 20 Williams GT, Smith CA. Molecular regulation of apoptosis: genetic control on cell death. Cell 1993; 74: 777-9.
• 21 Nishida M, Urushidani T, Sakamoto K, Nagao T. L-cis diltiazem attenuates intracellular Ca²⁺ overload by metabolic inhibition in guinea pig myocytes. Eur J Pharmacol 1999; 385: 225-30.
• 22 K Garlid D, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D'Alonzo AJ, et al. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K⁺ channels. Circ Res 1997; 81: 1072-82.
• 23 Chang TH, Adachi H, Okuyama T, Mori N, Saito I, Zhang KY, et al. Effects of 3'-angeloyloxy-4'-acetoxy-3',4'-dihydro-seselin on cardiohemodynamics in anesthetized dogs. Acta Pharmacol Sin 1994; 15: 507-10.