Pomegranate peel extract protects against the development of diabetic cardiomyopathy in rats by inhibiting pyroptosis and downregulating LncRNA-MALAT1

Abstract

Background: Pyroptosis is an inflammatory programmed cell death accompanied by activation of inflammasomes and maturation of pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. Pyroptosis is closely linked to the development of diabetic cardiomyopathy (DC). Pomegranate peel extract (PPE) exhibits a cardioprotective effect due to its antioxidant and anti-inflammatory properties. This study aimed to investigate the underlying mechanisms of the protective effect of PPE on the myocardium in a rat model of DC and determine the underlying molecular mechanism. Methods: Type 1 diabetes (T1DM) was induced in rats by intraperitoneal injection of streptozotocin. The rats in the treated groups received (150 mg/kg) PPE orally and daily for 8 weeks. The effects on the survival rate, lipid profile, serum cardiac troponin-1, lipid peroxidation, and tissue fibrosis were assessed. Additionally, the expression of pyroptosis-related genes (NLRP3 and caspase-1) and lncRNA-MALAT1 in the heart tissue was determined. The PPE was analyzed using UPLC-MS/MS and NMR for characterizing the phytochemical content. Results: Prophylactic treatment with PPE significantly ameliorated cardiac hypertrophy in the diabetic rats and increased the survival rate. Moreover, prophylactic treatment with PPE in the diabetic rats significantly improved the lipid profile, decreased serum cardiac troponin-1, and decreased lipid peroxidation in the myocardial tissue. Histopathological examination of the cardiac tissues showed a marked reduction in fibrosis (decrease in collagen volume and number of TGF-β-positive cells) and preservation of normal myocardial structures in the diabetic rats treated with PPE. There was a significant decrease in the expression of pyroptosis-related genes (NLRP3 and caspase-1) and lncRNA-MALAT1 in the heart tissue of the diabetic rats treated with PPE. In addition, the concentration of IL-1β and caspase-1 significantly decreased in the heart tissue of the same group. The protective effect of PPE on diabetic cardiomyopathy could be due to the inhibition of pyroptosis and downregulation of lncRNA-MALAT1. The phytochemical analysis of the PPE indicated that the major compounds were hexahydroxydiphenic acid glucoside, caffeoylquinic acid, gluconic acid, citric acid, gallic acid, and punicalagin. Conclusion: PPE exhibited a cardioprotective potential in diabetic rats due to its unique antioxidant, anti-inflammatory, and antifibrotic properties and its ability to improve the lipid profile. The protective effect of PPE on DC could be due to the inhibition of the NLRP3/caspase-1/IL-1β signaling pathway and downregulation of lncRNA-MALAT1. PPE could be a promising therapy to protect against the development of DC, but further clinical studies are recommended.

Keywords: IL1β; NLRP3; TGF-β; caspase-1; long non-coding RNA-metastasis-associated lung adenocarcinoma transcript-1; pomegranate peel extract.

FIGURE 1

Total ion chromatogram of the UPLC-PDA-MS/MS analysis of the pomegranate peel extract PPE (top), and the mass spectra of the major constituents (bottom).

FIGURE 2

¹H NMR fingerprint of the pomegranate peel extract PPE (Top) and expanded aromatic protons region (6.30–9.50 ppm) (bottom). The structures of the detected compounds are shown. Red: HHDPG, dark red: caffeoyl quinic acid, dark blue: punicalagin, orange: pedunculagin, black: cyanidin-3-O-glucoside, light blue: ellagic acid derivatives.

FIGURE 3

Effect of the pomegranate peel extract treatment on the studied rat groups (A) heart weight, (B) total body weight, (C) relative heart weight, (D) random blood glucose and fasting serum lipids, and (E) serum cardiac troponin-I concentration. Data are presented as mean ± SD; n = 7. cTn1: cardiac troponin I, Normal: normal rats (negative control group), Diabetic: non-treated diabetic rats (positive control group), N+PPE: normal rats treated with the pomegranate peel extract, Dia+PPE: diabetic rats treated with the pomegranate peel extract. HDLc: high density lipoprotein cholesterol, LDLc: low density lipoprotein cholesterol, Non HDL: non high density lipoprotein, TC: total cholesterol, TG: triglyceride, VLDLc: very low density lipoprotein cholesterol.

FIGURE 4

Effect of the pomegranate peel extract treatment on heart tissue of the studied rat groups. (A): MDA level as marker of lipid peroxidation, (B): IL-1β content detected by ELISA. (C–E): gene expression analysis of lncRNA-MALAT1, NLRP3, and caspase-1 respectively. The gene expression was measured using quantitative RT-PCR. Data are expressed as the mean ± SD. n = 7 for MDA and IL-1β concentration while for gene expression analysis n = 3. IL-1β: interleukin 1 beta; MALAT1: Metastasis associated lung adenocarcinoma transcript 1; MDA: malondialdehyde; NLRP3: nucleotide-binding domain (NOD)-like receptor protein 3, Normal: normal rats (negative control group), Diabetic: non-treated diabetic rats (positive control group), N+PPE: normal rats treated with the pomegranate peel extract, Dia+PPE: diabetic rats treated with the pomegranate peel extract.

FIGURE 5

Microscopic pictures of the cardiac tissue of the studied rat groups stained with H&E. (A) Cardiac wall of the non-diabetic rats in the negative control group showing viable cardiac muscle fibers with distinct cell borders (black arrow), preserved cross striations (blue arrow), and central oval\elongated nuclei (red arrow). (B) Cardiac wall of the non-diabetic rats treated with the pomegranate peel extract showing cardiac muscle fibers with centrally located nuclei (black arrow) and preserved cross striations (red arrow). (C) Cardiac wall of the diabetic rats in the positive control group showing scattered cardiac muscle fibers with small pyknotic nuclei (black arrow), partial loss of cross striations (red arrow), mildly congested intervening blood capillaries (blue arrow), and scattered cytoplasmic vacuoles (yellow arrow). (D) Cardiac wall of the diabetic rats treated with the pomegranate peel extract showing mildly scattered cardiac muscle fibers with small pyknotic nuclei (black arrow), few scattered cytoplasmic vacuoles (blue arrow), and partial loss of cross striations (red arrow).

FIGURE 6

Immunostained cardiac myofibrils against TGF-β. (A) Non-diabetic rats in the negative control group cardiac wall showing negative cell membrane reactivity in cardiac myofibrils. (B) Cardiac wall of non-diabetic rats treated with the pomegranate peel extract showing negative cell membrane reactivity in cardiac myofibrils. (C) Cardiac wall of the diabetic rats in the positive control group showing marked cell membrane reactivity in cardiac myofibrils. (D) Cardiac wall of the diabetic rats treated with the pomegranate peel extract showing weak cell membrane reactivity in cardiac myofibrils. (E) Illustrative figure demonstrating the average number of reactive cells in cardiac myofibrils against TGF-β of the studied rat groups. Data are presented as mean ± SD; n = 6. Normal: normal rats (negative control group). Diabetic: non-treated diabetic rats (positive control group). N + PPE: normal rats treated with the PPE. Dia + PPE: diabetic rats treated with the PPE. a: significant versus negative control group; b: significant versus positive control group, c: significant versus treated normal group; p < 0.05.

FIGURE 7

Masson’s trichome staining of the cardiac tissues of the studied rat groups. (A) Cardiac tissue from the non-diabetic rats in the negative control group showing normal delicate fibrous strand. (B) Cardiac tissue from the non-diabetic rats treated with the pomegranate peel extract showing an appearance similar to that of interstitial collagen as the negative control group. (C) Cardiac tissue from the diabetic rats in the positive control group showing thick fibrous bands. (D) Cardiac tissue from the diabetic rats treated with pomegranate peel extract group illustrating a mild amount of collagen fibers. (E) Illustrative figure demonstrating the quantitative measurement of the optical density of collagen fiber per unit area stained by Masson’s trichome. Data are presented as mean ± SD; n = 6. Normal: normal rats (negative control group). Diabetic: non-treated diabetic rats (positive control group). N + PPE: normal rats treated with the PPE. Dia + PPE: diabetic rats treated with the PPE. a: significant versus negative control group; b: significant versus positive control group, c: significant versus treated normal group; p < 0.05.