Molecular mechanisms of the antiglycative and cardioprotective activities of Psidium guajava leaves in the rat diabetic myocardium

Figures & data

Table 1. Primer sequences used for RT-PCR analysis.

Genes	Primer sequences	Amplicon size (bp)
GAPDH (glyceraldehyde-3 phosphate dehydrogenase)	Forward 5′ TGA CAA CTC CCT CAA GAT TGT CA 3′ Reverse 5′ GGC ATG GAC TGT GGT CAT GA 3′	96
RAGE (receptor for advanced glycation end products)	Forward 5′ ACT ACC GAG TCC GAG TCT ACC A 3′ Reverse 5′ GCT CTG ACC GAA GCG TGA 3′	240
TGF β1 (transforming growth factor β1)	Forward 5′ CCG CAA CAA CGC AAT CTA TG 3′ Reverse 5′ AGC CCT GTA TTC CGT CTC CTT 3′	304
CTGF (connective tissue growth factor)	Forward 5′ GGC AGG GCC AAC CAC TGT GC 3′ Reverse 5′ CAG TGC ACT TGC CTG GAT GG 3′	167
BNP (B-type natriuretic peptide)	Forward 5′ GGA CCA AGG CCC TAC AAA AGA 3′ Reverse 5′ CAG AGC TGG GGA AAG AAG AG 3′	245
NFκB (nuclear transcription factor κB)	Forward 5′ CAC CAA AGA CCC ACC TCA CC 3′ Reverse 5′ GGA CCG CAT TCA AGT CAT AGT C 3′	220

Table 2. Blood glucose and serum insulin.

Groups	Blood glucose (mg/dL)	Insulin (IU/L)
C	73.8 ± 6.73^a	14.91 ± 1.43^a
D	433.55 ± 39.47^b	6.43 ± 0.62^b
D + Q	178.35 ± 6.27^c	7.21 ± 0.69^b
D + E	123 ± 11.22^d	11.11 ± 1.06^c

Values expressed as mean ± SD of six rats. Similar alphabets in the same column indicate no significant difference between groups. p < 0.05.

Table 3. Glycated haemoglobin and serum fructosamine.

Groups	Glycated haemoglobin HbA1C (%)	Serum fructosamine (μmol/L)
C	4.71 ± 0.43^a	223.4 ± 20.39^a
D	9.67 ± 0.88^b	279.81 ± 25.53^b
D + Q	7.92 ± 0.73^c	248.05 ± 22.6^a
D + E	5.64 ± 0.52^d	233.7 ± 23.32^a

Values expressed as mean ± SD of six rats. Similar alphabets in the same column indicate no significant difference between groups. p < 0.05.

Figure 1. Equal concentrations of protein aliquots from heart cell lysates were separated using SDS–PAGE and blotted on to nitrocellulose membranes and later probed with anti-AGE antibody. (a) Location of bands ∼60 kDa in the heart extracts of experimental groups. (b) Intensity of bands ∼60 kDa was quantified using Biorad gel doc and plotted. The results presented are average of quadruplicate experiments ± SD statistically significant p ≤ 0.05.

Figure 2. The relative amount of RAGE mRNA was estimated by semi-quantitative RT-PCR. The PCR products were quantified by densitometry and standardized to their respective GAPDH controls. The mean intensity was measured and expressed as INT/mm². Results are expressed as average of quadruplicate experiments ± SD statistically significant p ≤ 0.05.

Figure 3. Equal concentrations of protein aliquots from heart cell lysates were separated using SDS–PAGE and blotted on to nitrocellulose membranes and later probed with anti RAGE antibody. (a) Location of bands ∼45 kDa in the heart extracts of experimental groups. (b) Intensity of bands ∼45 kDa was quantified using Biorad gel doc and plotted. The results presented are average of quadruplicate experiments ± SD statistically significant p ≤ 0.05.

Figure 4. Equal concentrations of protein aliquots (Lowry’s method) from heart cell lysates were separated using SDS–PAGE and blotted on to nitrocellulose membranes and later probed with anti-NFκB p65 antibody. Location of bands ∼65 kDa in the nuclear and cytoplasmic fraction of heart extracts of experimental groups. (b) Intensity of bands ∼65 kDa was quantified using Biorad gel doc and plotted. The results presented are average of quadruplicate experiments ± SD statistically significant p ≤ 0.05.

Figure 5. The relative amount of TGF-β₁ mRNA was estimated by semi-quantitative RT-PCR. The PCR products were quantified by densitometry and standardized to their respective GAPDH controls. The mean intensity was measured and expressed as INT/mm². Results are expressed as average of quadruplicate experiments ± SD statistically significant p ≤ 0.05.

Figure 6. The relative amount of CTGF mRNA was estimated by semi-quantitative RT-PCR. The PCR products were quantified by densitometry and standardized to their respective GAPDH controls. The mean intensity was measured and expressed as INT/mm². Results are expressed as average of quadruplicate experiments ± SD statistically significant p ≤ 0.05.

Figure 7. The relative amount of BNP mRNA was estimated by semi-quantitative RT-PCR. The PCR products were quantified by densitometry and standardized to their respective GAPDH controls. The mean intensity was measured and expressed as INT/mm². Results are expressed as average of quadruplicate experiments ± SD statistically significant p ≤ 0.05.

Figure 8. Light microscopic appearance of the heart sections stained with haematoxylin and eosin (magnification 40×). Group – Control: Heart of control rat showing normal myocardial fibres with normal cell structure. Group – Diabetic: Heart of diabetic rats showing severe necrosis, inflammatory infiltration, oedema with degeneration. Group – Diabetic + Quercetin: Quercetin treatment improved the myocardial degeneration with mild inflammatory infiltration when compared to the heart of diabetic rats. Group – Diabetic + Extract: Administration of PGEt also improved the myocardial degeneration when compared to diabetic control.