Streptozotocin-induced diabetic cardiomyopathy in rats: ameliorative effect of PIPERINE via Bcl2, Bax/Bcl2, and caspase-3 pathways

Figures & data

Table 1. Effect of piperine on STZ-induced alterations in body weight, heart weight, heart weight body weight ratio, blood glucose level, food intake, water intake, urine output, and serum insulin in diabetic rats

Treatment	Body Weight (g)	Heart Weight (mg)	Heart Weight Body Weight Ratio (mg/g)	Blood glucose (mg/dL)	Food intake (g)	Water Intake (mL)	Urine output (mL)	Serum Insulin (mg/L)
Normal (ND)	277.30 ± 6.97	140.30 ± 2.44	0.51 ± 0.02	142.30 ± 14.68	29.65 ± 2.68	45.94 ± 6.05	8.25 ± 2.77	2.47 ± 0.15
STZ control	197.20 ± 6.78^#	199.00 ± 3.98^#	1.02 ± 0.04^#	414.00 ± 9.94^#	78.94 ± 3.38^#	126.50 ± 5.21^#	51.62 ± 2.84^#	0.17 ± 0.13^#
STZ+ I (10)	261.80 ± 6.69^*$	153.00 ± 3.80^*$	0.58 ± 0.02^*$	166.90 ± 16.40^*$	35.60 ± 2.87^*$	49.75 ± 6.47^*$	21.96 ± 1.40^*$	2.34 ± 0.13
STZ+ P (10)	201.00 ± 6.40	184.10 ± 4.25	0.92 ± 0.04	365.20 ± 17.03	79.62 ± 1.65	111.20 ± 6.77	47.91 ± 2.75	0.42 ± 0.12
STZ+ P (20)	225.30 ± 4.66^*$	176.20 ± 4.51^*$	0.81 ± 0.03^*$	311.30 ± 12.37^*$	57.49 ± 2.01^*$	82.28 ± 5.63^*$	33.82 ± 2.12^*$	1.02 ± 0.14^*$
STZ+ P (40)	234.30 ± 4.67^*$	162.70 ± 5.12^*$	0.70 ± 0.03^*$	214.80 ± 14.36^*$	47.71 ± 2.33^*$	62.70 ± 6.17^*$	29.95 ± 1.99^*$	1.45 ± 0.14^*$
STZ+P (40)+I (10)	266.20 ± 3.73*	151.60 ± 4.86^*$	0.58 ± 0.01^*$	157.30 ± 13.89*	33.19 ± 2.26*	45.95 ± 7.43^*$	16.21 ± 2.22*	2.35 ± 0.10*

Data are expressed as mean±S.E.M. (n = 6) and analyzed by one-way ANOVA followed by Tukey’s multiple range tests. ⁎p < 0.05 as compared to the diabetic control group, ^#p < 0.05 as compared to the normal group,^$p < 0.05 as compared to one another (Piperine and insulin) and ^&p < 0.05 as compared to P (40). ND: non-diabetic rats; STZ: diabetic (STZ) control rats; P (10): piperine (10 mg/kg, p.o.) treated rats; P (20): piperine(20 mg/kg, p.o.) treated rats; P (40): piperine (40 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats. ANOVA: analysis of variance; I: insulin; P: piperine; SEM: standard error mean; STZ: Streptozotocin.

Figure 1. Effect of piperine on STZ induced increased heart volume in diabetic rats

[a] Normal: ND Non-diabetic rats; [b] STZ control: STZ control diabetic rats; [c]STZ+I(10): Insulin (10 mg/kg, p.o.) treated rats; [d] STZ+P(10): piperine (10 mg/kg, p.o.) treated rats; [e] STZ+P(20): piperine (20 mg/kg, p.o.) treated rats; [f]STZ+P(40): piperine (40 mg/kg, p.o.) treated rats; [g] STZ+ I(10)+ P(40): insulin (10 mg/kg, s.c.) and piperine (40 mg/kg, p.o.) treated rats. I: insulin P: piperine; STZ: Streptozotocin.

Table 2. Effect of piperine on STZ-induced alterations in echocardiogram parameters in diabetic rats

Treatment	Heart Rate (BPM)	QRS interval (ms)	QT Interval (ms)	QTc Interval (ms)	RR interval (ms)
Normal (ND)	354.70 ± 10.75	11.34 ± 1.32	49.89 ± 3.43	138.6 ± 8.14	126.10 ± 8.12
STZ control	257.30 ± 13.89^#	28.00 ± 0.93^#	83.36 ± 3.14^#	226.70 ± 4.97^#	234.70 ± 8.84^#
STZ + I (10)	327.80 ± 11.47^*$	16.20 ± 1.35^*$	55.11 ± 3.79^*$	156.10 ± 11.13^*$	156.50 ± 11.28^*$
STZ +P (10)	268.90 ± 14.52	26.13 ± 1.48	71.61 ± 1.66	194.30 ± 13.68	200.00 ± 7.27
STZ +P (20)	318.30 ± 9.37^*$	21.59 ± 1.02^*$	69.11 ± 2.61^*$	183.90 ± 5.51^*$	189.90 ± 7.23^*$
STZ + P (40)	324.40 ± 8.47^*$	19.62 ± 1.34^*$	67.41 ± 2.64^*$	177.80 ± 7.06^*$	185.70 ± 7.66^*$
STZ + I (10) + P (10)	334.30 ± 11.75^*$	12.17 ± 0.90*	51.15 ± 2.37*	134.10 ± 9.27*	141.30 ± 13.09*

Data are expressed as mean±S.E.M. (n = 6) and analyzed by one-way ANOVA followed by Tukey’s multiple range tests. ⁎p < 0.05 as compared to the diabetic control group, ^#p < 0.05 as compared to the normal group,^$p < 0.05 as compared to one another (Piperine and insulin), and ^&p < 0.05 as compared to P (40). ND: non-diabetic rats; STZ: diabetic (STZ) control rats; P (10): piperine (10 mg/kg, p.o.) treated rats; P (20): piperine(20 mg/kg, p.o.) treated rats; P (40): piperine (40 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats. ANOVA: analysis of variance; I: insulin; P: piperine; SEM: standard error mean; STZ: Streptozotocin.

Table 3. Effect of piperine on STZ-induced alterations hemodynamic parameters in diabetic rats

Treatment	SBP (mmHg)	DBP (mmHg)	LVEDP	MABP (mmHg)	Max dp/dt	Min dp/dt	Pressure time index	Contractility index	Tau(ms)
Normal (ND)	111.20 ± 5.63	83.58 ± 3.78	5.44 ± 0.71	124.40 ± 6.17	3517.00 ± 155.10	−2739.00 ± 193.20	18.83 ± 1.76	59.97 ± 5.16	5.46 ± 0.44
STZ control	161.00 ± 5.07^#	148.80 ± 3.85^#	17.91 ± 0.80^#	68.39 ± 7.12^#	2291.00 ± 176.60^#	−1028.00 ± 106.10^#	34.96 ± 1.54^#	24.28 ± 2.10^#	12.77 ± 0.71^#
STZ + I (10)	114.30 ± 5.04^*$	91.68 ± 3.81^*$	8.92 ± 0.45^*$	112.5 ± 7.70^*$	3479.00 ± 130.30^*$	−2342.00 ± 165.40^*$	25.70 ± 0.89^*$	50.97 ± 3.87^*$	5.67 ± 0.99^*$
STZ +P (10)	140.40 ± 3.80	135.80 ± 4.49	15.16 ± 0.66	81.47 ± 6.88	2719.00 ± 191.40	−1710.00 ± 191.30	31.43 ± 1.04	40.91 ± 4.17	11.83 ± 0.53
STZ +P (20)	136.60 ± 3.41^*$	113.90 ± 4.81^*$	13.84 ± 0.64^*$	102.80 ± 7.43^*$	3035.00 ± 81.94^*$	−1951.00 ± 178.90^*$	28.41 ± 0.91^*$	50.70 ± 4.13^*$	9.11 ± 0.95^*$
STZ + P (40)	133.40 ± 4.52^*$	106.70 ± 3.12^*$	12.58 ± 0.69^*$	104.5 ± 4.18^*$	3260.00 ± 180.90^*$	−1821.00 ± 214.60^*$	27.28 ± 1.58^*$	52.89 ± 3.95^*$	8.99 ± 0.80^*$
STZ + I (10) + P (10)	109.70 ± 3.96*	87.40 ± 4.12*	8.46 ± 0.82*	118.3 ± 6.03^*$	3490.00 ± 160.80^*$	−2799.00 ± 63.75*	21.03 ± 1.24*	54.25 ± 5.95^*$	5.33 ± 0.94^*$

Data are expressed as mean±S.E.M. (n = 6) and analyzed by one-way ANOVA followed by Tukey’s multiple range tests. ⁎p < 0.05 as compared to the diabetic control group, ^#p < 0.05 as compared to the normal group,^$p < 0.05 as compared to one another (Piperine and insulin), and ^&p < 0.05 as compared to P (40). ND: non-diabetic rats; STZ: diabetic (STZ) control rats; P (10): piperine (10 mg/kg, p.o.) treated rats; P (20): piperine(20 mg/kg, p.o.) treated rats; P (40): piperine (40 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats. ANOVA: analysis of variance; DBP: diastolic blood pressure; I: insulin; LVEDP: left ventricular end-diastolic pressure; MABP: mean arterial blood pressure; P: piperine; SBP: systolic blood pressure; SEM: standard error mean; STZ: Streptozotocin.

Table 4. Effect of piperine on STZ-induced alterations SOD, GSH, MDA, NO, Na-K-ATPase, serum CK-MB and serum LDH in diabetic rats

Treatment	SOD (U/mg of protein)	GSH (μg/mg protein)	MDA (nmol/l/mg of protein)	NO (μg/mL)	Na-K-ATPase (μmoles/mg of protein)	Serum CK-MB (IU/I)	Serum LDH (IU/I)
Normal (ND)	14.90 ± 0.49	47.33 ± 2.72	2.05 ± 0.53	130.20 ± 18.26	10.5 ± 0.83	969.60 ± 85.24	1120.00 ± 108.00
STZ control	5.27 ± 0.62^#	17.15 ± 1.14^#	7.33 ± 0.54^#	312.40 ± 10.71^#	3.15 ± 0.43^#	1910.00 ± 95.82^#	2316.00 ± 92.94^#
STZ + I (10)	12.61 ± 1.40^*$	39.08 ± 3.13^*$	3.04 ± 0.57^*$	177.90 ± 17.44^*$	9.08 ± 0.35^*$	1199.00 ± 92.84^*$	1371.00 ± 119.60^*$
STZ + P (10)	8.71 ± 0.68	17.41 ± 1.16	6.85 ± 0.43	286.90 ± 14.14	4.38 ± 0.42	1822.00 ± 118.10	2099.00 ± 104.50
STZ + P (20)	9.94 ± 0.78^*$	28.48 ± 1.62^*$	4.76 ± 0.39^*$	231.30 ± 23.56^*$	6.18 ± 0.62^*$	1429.00 ± 52.10^*$	1771.00 ± 109.00^*$
STZ + P (40)	9.67 ± 0.57^*$	29.21 ± 2.79^*$	4.65 ± 0.64^*$	227.50 ± 20.85^*$	6.47 ± 0.44^*$	1416.00 ± 68.71^*$	1773.00 ± 89.11^*$
STZ + I (10) + P (40)	14.87 ± 1.24*	41.83 ± 2.05*	2.32 ± 0.30*	132.80 ± 10.29*	9.93 ± 0.62*	1011.00 ± 54.93*	1159.00 ± 131.70*

Data are expressed as mean±S.E.M. (n = 6) and analyzed by one-way ANOVA followed by Tukey’s multiple range tests. ⁎p < 0.05 as compared to the diabetic control group, ^#p < 0.05 as compared to the normal group,^$p < 0.05 as compared to one another (Piperine and insulin), and ^&p < 0.05 as compared to P (40). ND: non-diabetic rats; STZ: diabetic (STZ) control rats; P (10): piperine (10 mg/kg, p.o.) treated rats; P (20): piperine(20 mg/kg, p.o.) treated rats; P (40): piperine (40 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats. ANOVA: analysis of variance;CK-MB: Creatine Kinase–MB; GSH: reduced glutathione; I: insulin; LDH: lactate dehydrogenase; MDA: lipid peroxidation; NO: nitric oxide;P: piperine; SEM: standard error mean; SOD: superoxide dismutase; STZ: Streptozotocin.

Table 5. Effect of piperine on STZ-induced alterations in myocardial mitochondrial enzymes activity (complex I, complex II, complex III, and complex IV) in diabetic rats

Treatment	Complex I (nmole of NADH oxidized/min/mg protein)	Complex II (mmole/mg protein)	Complex III (OD at 540 nm)	Complex-IV (nmol cyto-C oxidized/min/mg protein)
Normal (ND)	35.65 ± 2.52	14.9 ± 0.49	0.49 ± 0.03	5092.00 ± 345.80
STZ control	8.08 ± 2.27^#	5.1 ± 0.63^#	0.16 ± 0.01^#	1080.00 ± 157.00^#
STZ + I (10)	29.28 ± 1.96^*$	12.61 ± 1.40^*$	0.41 ± 0.02^*$	3935.00 ± 497.20^*$
STZ + P (10)	14.78 ± 1.29	6.54 ± 0.86	0.25 ± 0.03	2124.00 ± 375.10
STZ + P (20)	18.57 ± 1.98^*$	9.44 ± 0.41^*$	0.28 ± 0.03^*$	2900.00 ± 322.60^*$
STZ + P (40)	20.14 ± 1.59^*$	9.88 ± 0.37^*$	0.30 ± 0.03^*$	3033.00 ± 182.70^*$
STZ + I (10) + P (10)	30.91 ± 2.33*	14.87 ± 1.24*	0.43 ± 0.01*	4830.00 ± 484.30*

Data are expressed as mean±S.E.M. (n = 6) and analyzed by one-way ANOVA followed by Tukey’s multiple range tests. ⁎p < 0.05 as compared to the diabetic control group, ^#p < 0.05 as compared to the normal group,^$p < 0.05 as compared to one another (Piperine and insulin), and ^&p < 0.05 as compared to P (40). ND: non-diabetic rats; STZ: diabetic (STZ) control rats; P (10): piperine (10 mg/kg, p.o.) treated rats; P (20): piperine (20 mg/kg, p.o.) treated rats; P (40): piperine (40 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats. ANOVA: analysis of variance; I: insulin; P: piperine; SEM: standard error mean; STZ: Streptozotocin.

Figure 2. Effect of piperine on STZ-induced altered mRNA protein expression in diabetic rats

Data are expressed as mean±SEM. (n = 4) and analyzed by one-way ANOVA followed by Dunnett’s Multiple Comparison Test.*p < 0.05 as compared to the Diabetic Control group, ^#p < 0.05 as compared to normal group,^$p < 0.05 as compared to one another (Piperine and insulin), and ^&p < 0.05 as compared to P (40). ND: non-diabetic rats; STZ: diabetic (STZ) control rats; P (10): piperine (10 mg/kg, p.o.) treated rats; P (20): piperine (20 mg/kg, p.o.) treated rats; P (40): piperine (40 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats. [a]: ANP/β-Actin ratio; [b]: BNP/β-Actin ratio; [c]: cTn-I/β-Actin ratio; [d]:Bcl2/β-Actin ratio; [e]: Bax/Bcl2 ratio, and [f]: Caspase-3/β-Actin ratio. ANP:Atrial Natriuretic Peptide; ANOVA: analysis of variance; BNP: brain natriuretic peptide; Bcl2:B-cell lymphoma 2; Bax:Bcl-2-associated X protein; cTn-I: cardiac troponin I; I: insulin P: piperine; SEM: standard error mean; STZ: Streptozotocin.

Figure 3. Effect of piperine on STZ-induced alterations in heart histopathology in diabetic rats

Effect of piperine on diabetes-induced alteration in heart histology of rats stained with H&E staining. Photomicrograph of sections of heart of [a] normal, [b] STZ control group, [c] STZ + Insulin (10 mg/kg) treated group, [d] STZ + Piperine (20 mg/kg) treated group, [e] STZ + Piperine (40 mg/kg) treated group, and [f] STZ + Insulin (10 mg/kg) + Piperine (20 mg/kg) treated group. Images at 40X. The quantitative representation of histological score (g). Data are expressed as mean ± SEM (n = 3), and one-way ANOVA followed by Kruskal–Wallis test was applied for post hoc analysis. *p < 0.05 as compared to the Diabetic Control group, ^#p < 0.05 as compared to normal group and ^$p < 0.05 as compared to one another (Piperine and insulin). ANOVA: analysis of variance; H&E: hematoxylin and eosin; I: insulin; P: piperine; SEM: standard error mean; STZ: Streptozotocin.