Renoprotective effect of berberine via intonation on apoptosis and mitochondrial-dependent pathway in renal ischemia reperfusion-induced mutilation

Figures & data

Table 1. Primer sequences for KIM-1, NGAL, TNF-α, caspase-3, Bax, Bcl-2 and β-actin.

Gene	Primer sequences (5′–3′)	Length (bp)
KIM-1	5′-ACTCCTGCAGACTGGAATGG-3′ 5′-CAAAGCTCAGAGAGCCCATC-3′	212
NGAL	5′-GATGTTGTTATCCTTGAGGCCC-3′ 5′-CACTGACTCACGACCAGTTTGCC-3′	230
TNF-α	5′-AAGCCTGTAGCCCATGTTGT-3′ 5′-CAGATAGATGGGCTCATACC-3′	295
Caspase-3	5′-CTCGGTCTGGTACAGATGTCGATG-3′ 5′-GGTTAACCCGGGTAAGAATGTGCA-3′	238
Bax	5′-GGGAATTCTGGAGCTGCAGAGGATGATT-3′ 5′-GCGGATCCAAGTTGCCATCAGCAAACAT-3′	96
Bcl-2	5′-CAGATGCACCTGACGCCCTT-3′ 5′-CCCAGCCTCCGTTATCCTGGA-3′	195
β-Actin	5′-AGGCATCCTGACCCTGAAGTAC-3′ 5′-TCTTCATGAGGTAGTCTGTCAG-3′	764

Table 2. Effect of berberine on RIR induced alteration in kidney function.

Treatment	Kidney weight (mg)	Urine flow (ml)	Serum creatinine (mg/dL)	BUN (mg/dL)
Sham	722.7 ± 26.74	33.66 ± 1.15	1.31 ± 0.18	21.77 ± 2.04
RIR	926.3 ± 36.61###	64.58 ± 2.78###	5.43 ± 0.68###	75.49 ± 5.04###
B (10)	832.5 ± 27.17	56.60 ± 4.498	4.110 ± 0.6721	70.39 ± 7.87
B (20)	831.5 ± 27.06	50.10 ± 4.575*	2.988 ± 0.5094*	50.81 ± 5.66*
B (40)	795.8 ± 16.30**	47.92 ± 4.054*	2.033 ± 0.35**	33.57 ± 1.58***

Notes: Data are expressed as mean ± SEM (n = 6) and analyzed by one-way ANOVA followed by Dunnett’s test multiple range test. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the renal ischemia reperfusion group. ###p < 0.001 as compared to sham control group. RIR: Renal ischemia reperfusion group; B (10): berberine (10 mg/kg, p.o.) treated group; B (20): berberine (20 mg/kg, p.o.) treated group; B (40): berberine (40 mg/kg, p.o.) treated group; BUN: blood urea nitrogen.

Figure 1. Effect of berberine on RIR induced alteration in hemodynamic changes. Data are expressed as mean ± SEM (n = 6) and analyzed by one-way ANOVA followed by Dunnett’s test multiple range test. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the renal ischemia reperfusion group. ###p < 0.001 as compared to sham control group. RIR: Renal ischemia reperfusion group; B (10): berberine (10 mg/kg, p.o.) treated group; B (20): berberine (20 mg/kg, p.o.) treated group; B (40): berberine (40 mg/kg, p.o.) treated group.

Table 3. Effect of berberine on RIR induced alteration in left ventricular function.

Treatment	EDP (mmHg)	dp/dtmax (mmHg/s)	dp/dtmin (mmHg/s)	Pressure time index	Contractility index	Tau (ms)
Sham	3.79 ± 0.40	1370 ± 186.4	−1257 ± 154.6	4.627 ± 0.44	18.50 ± 1.410	17.17 ± 1.27
RIR	9.69 ± 0.82###	2959 ± 169.1###	−3225 ± 305.7###	12.38 ± 0.99###	7.837 ± 0.8042###	26.85 ± 1.54###
B (10)	8.66 ± 0.87	2494 ± 145.8	−2282 ± 527.0	11.09 ± 1.16	12.47 ± 1.720	24.06 ± 1.63
B (20)	6.41 ± 0.71*	1994 ± 147.8**	−1803 ± 292.1**	7.79 ± 1.14*	11.48 ± 1.779	19.35 ± 1.93**
B (40)	5.20 ± 0.74***	1577 ± 208.0***	−1726 ± 400.5**	6.05 ± 1.12***	15.60 ± 1.707**	19.61 ± 1.22**

Notes: Data are expressed as mean ± SEM (n = 6) and analyzed by one-way ANOVA followed by Dunnett’s test multiple range test. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the renal ischemia reperfusion group. ###p < 0.001 as compared to sham control group. RIR: Renal ischemia reperfusion group; B (10): berberine (10 mg/kg, p.o.) treated group; B (20): berberine (20 mg/kg, p.o.) treated group; B (40): berberine (40 mg/kg, p.o.) treated group; EDP: end diastolic pressure.

Table 4. Effect of berberine on RIR induced alteration in oxido-nitrosative stress, membrane bound enzymes, and cytochrome oxidase.

Treatment	SOD (U/mg of PRT)	GSH (µg/mg PRT)	MDA (nmol/l/mg of protein)	MPO (U/mg of protein)	Na-K-ATPase (μmoles/mg of protein)	Ca-K-ATPase (μmoles/mg of protein)	Cytochrome oxidase (nmol cyto-C oxidized/min/mg protein)
Sham	12.47 ± 0.73	30.92 ± 1.21	3.16 ± 0.55	3.123 ± 0.5289	6.94 ± 0.55	4.39 ± 0.3041	245.50 ± 16.24
RIR	5.42 ± 0.42###	22.22 ± 1.30###	9.52 ± 0.67###	10.63 ± 1.14###	1.938 ± 0.35###	10.98 ± 0.76###	78.62 ± 8.55###
B (10)	7.26 ± 0.48	22.20 ± 1.147	8.03 ± 0.51	7.39 ± 0.66*	3.53 ± 0.41	8.66 ± 1.12	221.52 ± 10.85
B (20)	8.96 ± 0.74**	27.60 ± 1.026*	6.133 ± 0.61**	5.458 ± 0.83**	4.060 ± 0.66*	5.92 ± 1.187**	165.84 ± 12.02**
B (40)	10.97 ± 0.59***	28.44 ± 0.70**	5.09 ± 0.29***	4.05 ± 0.58***	4.773 ± 0.45**	4.71 ± 0.57***	98.75 ± 8.55***

Notes: Data are expressed as mean ± SEM (n = 4) and analyzed by one-way ANOVA followed by Dunnett’s test multiple range test. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the renal ischemia reperfusion group. ###p < 0.001 as compared to sham control group. RIR: Renal ischemia reperfusion group; B (10): berberine (10 mg/kg, p.o.) treated group; B (20): berberine (20 mg/kg, p.o.) treated group; B (40): berberine (40 mg/kg, p.o.) treated group.

Figure 2. Effect of berberine on RIR induced alteration in NGAL, KIM-1 and TNF-α mRNA expression (A), quantitative representation of mRNA expression of NGAL (B), KIM-1 (C), and TNF-α (D). Data are expressed as mean ± SEM (n = 4) and analyzed by one-way ANOVA followed by Dunnett’s test multiple range test. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the renal ischemia reperfusion group. ###p < 0.001 as compared to sham control group. RIR: Renal ischemia reperfusion group; S: Sham control group; B (10): berberine (10 mg/kg, p.o.) treated group; B (20): berberine (20 mg/kg, p.o.) treated group; B (40): berberine (40 mg/kg, p.o.) treated group.

Figure 3. Effect of berberine on RIR induced alteration in caspase-3, Bax, and Bcl-2 mRNA expression (A), quantitative representation of mRNA expression of caspase-3 (B), Bax (C), and Bcl-2 (D). Data are expressed as mean ± SEM (n = 4) and analyzed by one-way ANOVA followed by Dunnett’s test multiple range test. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with the renal ischemia reperfusion group. ###p < 0.001 as compared to sham control group. RIR: Renal ischemia reperfusion group; S: Sham control group; B (10): berberine (10 mg/kg, p.o.) treated group; B (20): berberine (20 mg/kg, p.o.) treated group; B (40): berberine (40 mg/kg, p.o.) treated group.

Figure 4. Effect of berberine on RIR induced alteration in kidney histology. Photomicrograph of sections of kidney of (A) normal, (B) RIR control rats, (C) Berberine (20 mg/kg) treated rats, and (D) Berberine (40 mg/kg) treated rats. H & E staining at 100×.

Table 5. Effect of berberine on RIR induced alteration in kidney histology.

Parameter	Glomerular hypertrophy	Inflammatory infiltration	Congestion	Edema	Necrosis
Sham	0	0	0	0	0
RIR	++	+++	++	++	++
B (20)	++	+	+	+	++
B (40)	+	+	+	++	+

Notes: 0: No abnormality detected, +: damage/active changes up to less than 25%, ++: damage/active changes up to less than 50%, +++: damage/active changes up to less than 75%.