Anat Cell Biol.  2021 Dec;54(4):465-478. 10.5115/acb.21.105.

The role of hesperidin in ameliorating retinal changes in rats with experimentally induced type 1 diabetes mellitus and the active role of vascular endothelial growth factor and glial fibrillary acidic protein

• Shehata AS
• Mohamed DA
• Hagras SM
• El-Beah SM
• Elnegris HM

• KMID: 2523577
• DOI: http://doi.org/10.5115/acb.21.105

Abstract

Patients with type 1 diabetes mellitus (T 1 DM) are vulnerable to developing diabetic retinopathy even under insulin therapy. Thus, this study was designed to evaluate the efficacy of hesperidin and insulin in rats with T 1 DM compared with insulin alone in improving diabetic retinal changes. Eighty rats were divided into four equal groups: group I, control rats without diabetes; group II, untreated rats with diabetes; group III, rats with diabetes treated daily with subcutaneous (SC) doses of long-acting insulin; and group IV, a rat with diabetes in which hesperidin was orally administered with SC insulin. The animals were assessed histologically, morphometrically, and biochemically. In group II, the thickness of all retinal layers decreased histologically. Ultrastructurally, degenerated retinal neurons and congested blood vessels were observed. Immunostaining detected elevated gene expression of advanced glycation end products. Gene expression of vascular endothelial growth factor, and glial fibrillary acidic protein were elevated. In this study, hesperidin supplementation with insulin significantly improved the retinal histological changes, supported by morphometric findings, compared with insulin alone. Moreover, treatment with hesperidin significantly reduced malondialdehyde and elevated serum antioxidant markers, including superoxide dismutase and catalase; furthermore, glutathione peroxidase decreased. Hesperidin might be an effective supplement for improving diabetic retinal complications occurring even with insulin treatment.

Keyword

Hesperidin, Retina; Type I diabetes mellitus; Vascular endothelial growth factor; Glial fibrillary acidic protein

Figure

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  Fig. 1 Photomicrograph, paraffin sections stained with H&E, scale bar=20 µm (A–D: ×400), scale bar=10 µm (E–H: ×1,000). (A, E) Retina of control group: showing layers of the retina consisting of photoreceptor outer segment (Ph), outer nuclear (ON), outer plexiform (OP), inner nuclear (IN), inner plexiform (IP), and ganglion cell layers (arrow). (B, F) T1DM group: showing observable decreased thickness of all retinal layers. Wide spaces appear between few and dark nuclei of outer and IN layers (arrow head). Nerve fibers of IP layer are widely separated (curved arrow). Ganglion cells are few with pyknotic nuclei (n). Congested B.V also seen. (C, G) Sections in retina of (T1DM+insulin) group: showing decreased thickness of all retinal layers. Wide spaces appear between few and dark nuclei of outer and IN layers (arrow head). Nerve fibers of IP layer are widely separated (arrow). Ganglion cells are few and small n. Congested B.V also seen. (D, H) Retina of (T1DM+insulin+hesp) group: showing layers of the retina with normal structure. Consisting of Ph, ON, OP, IN, IP, and ganglion cell layers (arrow). T1DM, type 1 diabetes mellitus; hesp, hesperetin 7-rhamnoglucoside; B.V, blood vessel.

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  Fig. 2 (A–D) Photomicrograph, advanced glycation end products (AGE)-immunostained paraffin sections, scale bar=20 µm; Anti-AGE ×400. (A) Control group: showing negative cytoplasmic immunoreactivity in all retinal layers. (B) T1DM group: showing strong positive cytoplasmic immunoreactivity in photoreceptor (black arrows), nuclear layers (arrowheads), plexiform layers (stars) and ganglion cell layer (curved arrows). (C) T1DM+insulin group: showing mild positive cytoplasmic immunoreactivity in photoreceptor (arrow), nuclear layers (arrowhead), plexiform layers (stars) and ganglion cell layer (curved arrow). (D) T1DM+insulin+hesp group: showing negative cytoplasmic immunoreactivity in all retinal layers. T1DM, type 1 diabetes mellitus; hesp, hesperetin 7-rhamnoglucoside.

• 

  Fig. 3 Electron micrograph of control group, scale bar=2 µm (A: ×2,000; B–D: ×1,500). (A) Showing small highly condensed heterochromatic rod nuclei (N) with rim of cytoplasm (arrow), inner nuclear layer (INL) containing, bipolar neuron with large ovoid euchromatic nucleus (B), few mitochondria (arrowheads) and ribosomes (serrated arrows). Outer plexiform (OP) layer is seen. (B, C) Muller cells with oval electron dens nucleus (M) and cytoplasmic processes (curved arrow). Amacrine cell with indented nuclei is seen (Am), also retinal blood vessel (B.V) is seen. (D) Inner plexiform layer (stars) containing thick nerves fibers.

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  Fig. 4 Electron micrograph of type 1 diabetes mellitus (T1DM) group, scale bar =2 µm (A–C: ×1,500; D: ×1,200). (A) Showing nuclei (N) of rods widely separated and surrounded with cytoplasm with multiple vacuoles (v). (B, C) Inner nuclear layer containing, degenerated neuron with rarified cytoplasm (arrowheads) and dark nuclei. Another neuron has apoptotic nucleus (star) and multiple cytoplasmic vacuoles (v), neuronal nuclei with clumped chromatin (curved arrow). Many Muller (M) cells also seen with its cytoplasmic processes (arrow), dilated congested retinal blood vessel (B.V) with thick basement membrane is present. (D) Microglia with dense nuclei are seen (N) and inner plexiform layer (IPL) are seen.

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  Fig. 5 Electron micrograph of (T1DM+insulin) group, scale bar= 2 µm (A, D: ×1,000; B, C: ×1,500). (A) Showing rods with small electron dense nuclei (N) and narrow outer plexiform (OPL) (arrow). (B, C) Inner nuclear layer containing some neuron with rarified vacuolated cytoplasm (arrowhead) and remnants of nuclei (curved arrow). Another neuron appeared normal (stars). Muller cells (M) are also seen. (D) Retinal blood vessel with normal dimeter and intact basement membrane (B.V) and inner plexiform layer (IPL) are seen. T1DM, type 1 diabetes mellitus.

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  Fig. 6 Electron micrograph of (T1DM+ insulin+hesp) group, scale bar=2 µm (A: ×1,500; B: ×2,000; C, D: ×1,200). (A) Showing preserved ultrastructure of most layers of retina. Rods with electron dense nuclei (N) and outer plexiform layer (OPL) with normal thickness an organization (arrow). (B–D) Inner nuclear layer containing many neurons with electron lucent cytoplasm (arrowheads) and euchromatic nuclei (curved arrows). Other neurons appeared with heterochromatic nuclei (star). Microglia (g), retinal blood vessel with normal basement membrane (B.V), Muller cells and inner plexiform layer (IPL) are seen. T1DM, type 1 diabetes mellitus; hesp, hesperetin 7-rhamnoglucoside.

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  Fig. 7 Statistical analysis of thickness of the retinal layers (µm) in different studied groups. Values are presented as mean±SD. Significant with control (*P<0.05). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer; IPL, inner plexiform layer; T1DM, type 1 diabetes mellitus; hesp, hesperetin 7-rhamnoglucoside; NS, non-significant with control.

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  Fig. 8 Statistical analysis of retinal content of antioxidant enzymes SOD, CAT, GSH-Px, and oxidant marker MDA in all studied groups. Values are presented as mean±SD. Significant with control (*P<0.05). SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; NS, non-significant with control; T1DM, type 1 diabetes mellitus; hesp, hesperetin 7-rhamnoglucoside.

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  Fig. 9 Statistical analysis of relative gene expression of GFAP and VEGF in rat retina in different studied groups. Significant with control (***P≤0.001). VEGF, vascular endothelial growth factor; GFAP, glial fibrillary acidic protein; T1DM, type 1 diabetes mellitus; hesp, hesperetin 7-rhamnoglucoside; NS, non-significant with control.

Reference

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