Graviola (Annona muricata) attenuates behavioural alterations and testicular oxidative stress induced by streptozotocin in diabetic rats

Abstract

Oxidative stresses intensify the progression of diabetes-related behavioural changes and testicular injuries. Graviola (Annona muricata), a small tree of the Annonaceae family, has been investigated for its protective effects against diabetic complications, oxidative stress, and neuropathies. This study was planned to investigate the effects of graviola on behavioural alterations and testicular oxidative status of streptozotocin (STZ; 65 mg/kg)-induced diabetic rats. Forty adult male Wistar rats were equally allocated into four groups: control (received normal saline 8 ml/kg orally once daily), diabetic (received normal saline orally once daily), graviola (GR; received 100 mg/kg/day; orally once daily), and diabetic with graviola (Diabetic+GR; received 100 mg/kg/day; once daily). Behavioural functions were assessed using standard behavioural paradigms. Also, oxidative statuses of testis were evaluated. Results of behavioural observations showed that diabetes induced depression-like behaviours, reduction of exploratory and locomotor activities, decreased memory performance, and increased stress-linked behaviours. These variations in diabetic rats were happened due to oxidative stress. Interestingly, treatment of diabetic rats with graviola for four weeks alleviated all behavioural changes due to diabetes. Also, rats in graviola-treated groups had greater testicular testosterone and estradiol levels compared with diabetic rats due to significant rise in testicular acetyl-CoA acetyltransferase 2 expression. In the same context, graviola enhanced the antioxidant status of testicular tissues by significantly restoring the testicular glutathione and total superoxide dismutase that fell during diabetes. In addition, Graviola significantly decreased the expression of apoptotic (Bax) and inflammatory (interleukin-1β) testicular genes. In conclusion, these data propose that both the hypoglycemic and antioxidative potential of graviola are possible mechanisms that improve behavioural alterations and protect testis in diabetic animals. Concomitantly, further clinical studies in human are required to validate the current study.

Fig 1. Open field test in diabetic and graviola-treated rats.

(A) number of crossing, (B) number of grooming, (C) number of rearing, and (D) number of defecations. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. Control. ⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001 vs. Diabetic. ^xP < 0.05 and ^xxxP < 0.001 vs. Diabetic+GR. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons. GR, graviola.

Fig 2. Elevated plus maze in diabetic and graviola-treated rats.

(A) frequencies of entries to the open arms, (B) frequencies of entries to the closed arms, (C) times of open arms entry, (D) number of grooming, (E) number of rearing, and (F) number of defecations. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. Control. ⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001 vs. Diabetic. ^xP < 0.05 and ^xxxP < 0.001 vs. Diabetic+GR. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons. GR, graviola.

Fig 3

Body weight (A) and serum glucose (B). The data represented that the body weights of the Diabetic group were significantly decreased compared with control, while rats in GR and Diabetic+GR groups had significant increases in their body weights in comparison with Diabetics. ^*P < 0.05 and ^***P < 0.001 vs. Control. ⁺⁺⁺P < 0.001 vs. Diabetic. ^xxxP < 0.001 vs. Diabetic+GR. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons. GR, graviola.

Fig 4

Testicular (A) ACP, (B) ALP, (C) testosterone, and (D) estradiol levels. Represented a restoration in testicular function that monitored by significant increases in testicular testosterone and estradiol levels in graviola-treated groups compared with diabetic rats. Samples (n = 5). ^***P < 0.001 vs. Control. ⁺⁺⁺P < 0.001 vs. Diabetic. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons. ACP, acid phosphatase. ALP, alkaline phosphatase. GR, graviola.

Fig 5

Testicular (A) MDA, (B) NO, (C) GSH, and (D) T.SOD levels. Graviola supplementation to diabetic rats significantly restored the testicular GSH levels and T.SOD activities that depleted due to diabetes. Samples (n = 5). ^**P < 0.01 and ^***P < 0.001 vs. Control. ⁺⁺⁺P < 0.001 vs. Diabetic. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons. MDA, malondialdehyde. NO, nitric oxide. GSH, reduced glutathione. T.SOD, Total superoxide dismutase. GR, graviola.

Fig 6

RT-PCR validation of (A) CYP17A1 and (B) ACAT2. Graviola significantly increased the CYP17A1 gene expression levels in graviola-treated groups that led to improvement of steroidogenesis in diabetic rats. In addition, graviola downregulated ACAT2 that let to spare more free cholesterol for steroidogenesis. Samples (n = 5). ^**P < 0.01 and ^***P < 0.001 vs. Control. ⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001 vs. Diabetic. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons. CYP17A1, cytochrome P450 17A1. ACAT2, acetyl-CoA acetyltransferase 2. GR, graviola.

Fig 7

RT-PCR validation of (A) Bax, (B) Bcl2, and (C) IL-1β genes. graviola-treated rats possessed improvement in testicular tissues that evidenced by downregulations in apoptotic and inflammatory genes. Samples (n = 5). ^**P < 0.01 and ^***P < 0.001 vs. Control. ⁺⁺⁺P < 0.001 vs. Diabetic. ^xP < 0.05 and ^xxP < 0.01 vs. Diabetic+GR. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons. Bax, Bcl-2-associated X. Bcl2, B-cell lymphoma 2. IL-1β, interleukin 1 beta. GR, graviola.