Maslinic Acid Protects against Streptozotocin-Induced Diabetic Retinopathy by Activating Nrf2 and Suppressing NF- κ B

Abstract

This study tested the protective effect of maslinic acid (MA) against diabetic retinopathy (DR) in rats with type 1 diabetes mellitus (T1DM) and investigated possible mechanisms of action. DM was introduced by streptozotocin (STZ) (65 mg/kg, i.p.). Control and STZ (T1DM) were divided into 2 subgroups, which received either the vehicle or MA (80 mg/kg). Serum, pancreases, and retinas were collected for further use. MA significantly reduced fasting glucose levels in the control and T1DM rats but enhanced fasting insulin levels and partially increased the size of the islets of Langerhans and the number of β-cells in T1DM rats. In addition, MA significantly improved the retina structure by preventing the reduction in the area between the inner and outer limiting membranes (ILM and OLM, respectively) and increasing the number of cells forming the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Associated with these effects, MA significantly reduced the total levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), as well as the nuclear levels of NF-κB p65, mRNA levels of Bax, and protein levels of cleaved caspase-3 in the retinas of T1DM rats. However, MA significantly lowered levels of reactive oxygen species (ROS) and malondialdehyde (MDA) but significantly increased the nuclear levels of Nrf2, protein levels of Bcl2, and total levels of superoxide dismutase (SOD) and reduced glutathione (GSH) in the retinas of the control and T1DM rats. In conclusion, MA prevents DR by antioxidant potential mediated by the activation of Nrf2.

Figure 1

Photomicrographs of the pancreases from all groups of rats. A & B represent control and MA-treated rats. In both images, the islets of Langerhans (thick arrow) appeared circular and were of large size. The exocrine (α)-cells were located at the periphery and had normally small cells with dark nuclei (long thin arrow), whereas the endocrine β-cells (short arrow) are located centrally with larger with light nuclei. C represents a T1DM rat in which the islets of Langerhans appeared smaller (thick arrow) with a severe reduction in the number of the β-cells (short arrow). Not the dilated blood vessel with increased inflammatory cells (arrowhead). D represents T1DM + MA-treated rats and showed an increase in the size of the islet of the Langerhans and the number of β-cells (short arrow) (200X).

Figure 2

Histopathological photomicrographs of the retina of all groups of rats as stained with hematoxylin and eosin (H & E) staining. 200X. All layers of the retina including the inner limiting membrane (ILM), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), outer limiting membrane (OLM), and photoreceptors (rods and cones) were identified in all sections. A and B were taken from control and MA-treated rats and showed a normal thickness of all layers with an abundant cell number in the GCL (long arrow) and INL (short arrow) and intact photoreceptor layer (thick arrow) and ONL. Not the thickness between the ILM and OLM layers (black double arrow), ONL (yellow double arrow), and INL (red double arrow). (C) was taken from T1DM-induced rats and showed a significant reduction in the thickness. Besides, severe damage in the photoreceptor layer (Thick arrow) with severe loss of the majority of the cells in the GCL (long arrow) and INL (short arrow) was seen. (D) was taken from a T1DM + MA-treated rat and showed an almost normal retina structure with a preserved thickness of all layers and rods that were associated with an increased number of cells composing the INL (200X).

Figure 3

Levels of inflammatory markers in the retina of all groups of rats. Data were analyzed by 1-way ANOVA, and all results were presented as mean ± SD (n = 8). Values are considered significantly different at P < 0.05. ^a: significantly different as compared to control rats. ^b: significantly different as compared to MA-treated rats. ^c: significantly different as compared to T1DM-induced rats.

Figure 4

Levels of markers of oxidative stress in the retina of all groups of rats. Data were analyzed by 1-way ANOVA, and all results were presented as mean ± SD (n = 8). Values are considered significantly different at P < 0.05. ^a: significantly different as compared to control rats. ^b: significantly different as compared to MA-treated rats. ^c: significantly different as compared to T1DM-induced rats.

Figure 5

The nuclear levels of Nrf2 as detected by ELISA and by western blotting in the retina of all groups of rats. Data were analyzed by 1-way ANOVA, and all results were presented as mean ± SD (n = 8). Values are considered significantly different at P < 0.05. ^a: significantly different as compared to control rats. ^b: significantly different as compared to MA-treated rats. ^c: significantly different as compared to T1DM-induced rats.

Figure 6

mRNA level of Bax and Bcl2 and their ratio, as well as the total protein levels of cleaved caspase-3 as detected by western blotting in the retina of all groups of rats. Data were analyzed by 1-way ANOVA and all results were presented as mean ± SD (n = 8). Values are considered significantly different at P < 0.05. ^a: significantly different as compared to control rats. ^b: Significantly different as compared to MA-treated rats. ^c: Significantly different as compared to T1DM-induced rats.