Zerumbone, a Bioactive Sesquiterpene, Ameliorates Diabetes-Induced Retinal Microvascular Damage through Inhibition of Phospho-p38 Mitogen-Activated Protein Kinase and Nuclear Factor-κB Pathways

Abstract

Zerumbone ameliorates retinal damage by blocking advanced glycation end products and their receptor system in streptozotocin-diabetic rats. Because of the multiple factors involved in diabetic retinopathy (DR) etiology, the mechanisms of zerumbone that are mainly responsible for its ameliorative effect on DR need to be further clarified. In the present study, zerumbone (20 mg or 40 mg/kg) or fenofibric acid (100 mg/kg) was orally administered to diabetic rats by intragastric gavage once daily for three consecutive months. Zerumbone displayed similar characteristics to fenofibric acid in reducing retinal vascular permeability and leukostasis in diabetic rats. Fundus photographs showed that large retinal vessel diameters were decreased in zerumbone-treated diabetic rats. Zerumbone not only down-regulated the gene expression of retinal angiogenic parameters, but also reduced the expression of inflammatory cytokines and chemokines in the retina of diabetic rats. Moreover, zerumbone reduced the p38 MAPK phosphorylation and abrogated the nuclear translocation of NF-κB p65 in the retina of diabetic rats. In conclusion, treatment of diabetic rats with zerumbone attenuates the severity of retinal inflammation and angiogenesis, via inhibition of p38 MAPK and NF-κB signaling pathways. These benefits of zerumbone for DR appear to be linked to its antihyperglycemic and antihyperlipidemic effects.

Keywords: NF-κB; diabetic retinopathy; p38 MAPK; zerumbone.

Figure 1

Changes in body weight (A) and plasma glucose (B), TC (C) and TG (D) in normal or STZ-diabetic rats receiving three months of treatment. STZ-diabetic rats were administered 20 mg/kg/day zerumbone (STZ-ZER 20), 40 mg/kg/day zerumbone (STZ-ZER 40) or 100 mg/kg fenofibric acid (STZ-FA) by oral gavage once daily for three months. Another group of STZ-diabetic rats (STZ-Veh) and normal rats (Normal-Veh) were administered the same volume of vehicle used to prepare the test medication solutions. Data are mean ± SD from eight rats per group. ^a p < 0.05 and ^b p < 0.01 compared to vehicle-treated normal rats, respectively; ^c p < 0.05 and ^d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Figure 2

Fundus photographs (A) and changes in retinal vascular diameters (B) in normal or STZ-diabetic rats receiving three months of treatment. STZ-diabetic rats were administered 20 mg/kg/day zerumbone (STZ-ZER 20), 40 mg/kg/day zerumbone (STZ-ZER 40) or 100 mg/kg fenofibric acid (STZ-FA) by oral gavage once daily for three months. Another group of STZ-diabetic rats (STZ-Veh) and normal rats (Normal-Veh) were administered the same volume of vehicle used to prepare the test medication solutions. Data are mean ± SD from eight rats per group. ^a p < 0.05 and ^b p < 0.01 compared to vehicle-treated normal rats, respectively; ^c p < 0.05 and ^d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Figure 3

Effects on retinal vascular permeability. STZ-diabetic rats were administered 20 mg/kg/day zerumbone (STZ-ZER 20), 40 mg/kg/day zerumbone (STZ-ZER 40) or 100 mg/kg fenofibric acid (STZ-FA) by oral gavage once daily for three months. Another group of STZ-diabetic rats (STZ-Veh) and normal rats (Normal-Veh) were administered the same volume of vehicle used to prepare the test medication solutions. Retinal vascular permeability was measured with EB dye as a tracer. EB was normalized by total protein concentration in the tissue. Permeability is expressed as ng of dye per mg of protein in the tissue. Data are mean ± SD from eight rats per group, and the experiments were repeated independently at least three times with similar results. ^a p < 0.05 and ^b p < 0.01 compared to vehicle-treated normal rats, respectively; ^c p < 0.05 and ^d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Figure 4

Effects on retinal leukostasis. STZ-diabetic rats were administered 20 mg/kg/day zerumbone (STZ-ZER 20), 40 mg/kg/day zerumbone (STZ-ZER 40) or 100 mg/kg fenofibric acid (STZ-FA) by oral gavage once daily for three months. Another group of STZ-diabetic rats (STZ-Veh) and normal rats (Normal-Veh) were administered the same volume of vehicle used to prepare the test medication solutions. The total number of adherent leukocytes per retina was counted. Data are mean ± SD from eight rats per group, and the experiments were repeated independently at least three times with similar results. ^a p < 0.05 and ^b p < 0.01 compared to vehicle-treated normal rats, respectively; ^c p < 0.05 and ^d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Figure 5

Effects on mRNA (left pannels) and protein expression (right pannels) of inflammatory cytokines (A), chemokines (B) and angiogenic parameters (C). STZ-diabetic rats were administered 20 mg/kg/day zerumbone (STZ-ZER 20), 40 mg/kg/day zerumbone (STZ-ZER 40) or 100 mg/kg fenofibric acid (STZ-FA) by oral gavage once daily for three months. Another group of STZ-diabetic rats (STZ-Veh) and normal rats (Normal-Veh) were administered the same volume. Data are mean ± SD from eight rats per group, and the experiments were repeated independently at least three times with similar results. ^a p < 0.05 and ^b p < 0.01 compared to vehicle-treated normal rats, respectively; ^c p < 0.05 and ^d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.

Figure 6

Effects on retinal p38 MAPK (A) and NF-κB (B) activation. STZ-diabetic rats were administered 20 mg/kg/day zerumbone (STZ-ZER 20), 40 mg/kg/day zerumbone (STZ-ZER 40) or 100 mg/kg fenofibric acid (STZ-FA) by oral gavage once daily for three months. Another group of STZ-diabetic rats (STZ-Veh) and normal rats (Normal-Veh) were administered the same volume. The expression and phosporylation of retinal IkB protein have been indicated in left pannel on Figure 6B. NF-κB p65 expression in nuclear and cytosolic fractions of retinas from rats has been shown in right pannel on Figure 6B. Data are mean ± SD from eight rats per group, and the experiments were repeated independently at least three times with similar results. ^a p < 0.05 and ^b p < 0.01 compared to vehicle-treated normal rats, respectively; ^c p < 0.05 and ^d p < 0.01 compared to the values of vehicle-treated STZ-diabetic rats, respectively.