Upregulation of Antioxidative Gene Expression by Lasia spinosa Organic Extract Improves the Predisposing Biomarkers and Tissue Architectures in Streptozotocin-Induced Diabetic Models of Long Evans Rats

Abstract

Plants are an entity essential to the function of the biosphere as well as human health. In the context of human health, this research investigated the effect of Lasia spinosa (Lour) leaf methanolic extracts (LSML) on antioxidative enzymes and gene expression as well as biochemical and histological markers in a streptozotocin (STZ)-induced diabetes model. Fructose-fed streptozotocin (STZ)-induced diabetic animals were subjected to a four-week intervention followed by the assessment of the animal’s blood and tissues for enzymatic, biochemical, histological, and genetic changes. LSML-treated groups were shown to decrease plasma glucose levels and improve body and organ weights compared to the untreated group in a dose-dependent manner. At the doses of 125 and 250 mg/kg b.w., LSML were able to normalize serum, hepatic, and renal biochemical parameters and restore the pancreas, kidney, liver, and spleen tissue architectures to their native state. A considerable increase (p < 0.01) of liver antioxidant enzymes CAT, SOD, GSH, and a decrease of MDA level in LSML-treated groups were found at higher doses. The improved mRNA expression level of antioxidant genes CAT, SOD2, PON1, and PFK1 was also found at the doses of 125 mg/kg and 250 mg/kg BW when compared to untreated control groups. The results demonstrate that LSML impacts the upregulation of antioxidative gene expressions, thus improving the diabetic complications in animal models which need to be affirmed by compound-based antioxidative actions for therapeutic development.

Keywords: Lasia spinosa; MDA; PON1; SOD2; antioxidant; β-ACTIN.

Figure 1

Effect of LSML extract on the changes of (a) food consumption, (b) fluid intake, (c) Fasting Blood Glucose level; (d) oral glucose tolerance test (OGTT) and (e) body weight in STZ-induced diabetic rat model (n = 5). Data are expressed as Mean ± SD. Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by Tukey’s Multiple Comparison Test using GraphPad Prism 8.0 Software. The a–d superscript letters over the lines of the figure indicate the significant differences between and among the treatment groups at p < 0.05. Different letters indicate that the differences are significant while same letters do nonsignificant. Here, * = p < 0.05; ** = p < 0.01; *** = p < 0.001, **** = p < 0.0001 and ns as not significant when compared with normal control group. #### = p < 0.0001 and ns as not significant when compared with diabetic control group.

Figure 2

Effect of different doses of LSML extract on the changes of serum (a) ALP, (b) AST, (c) ALT, and (d) TB level assessed against STZ-induced diabetes in Long Evan rats. Results are represented as the mean ± SD, where n = 5. ### = p < 0.001; #### = p < 0.0001 and ns = not significant, when compared with diabetic control group. **** = p < 0.0001, ** = p < 0.01, * = p < 0.05 and ns = not significant, when compared with normal control group.

Figure 3

Effect of different doses of LSML on the changes of serum (a) triglycerides (TG), (b) total cholesterol (TC), (c) high density lipoprotein (HDL-C), (d) low density lipoprotein (HDL-C) and (e) very low-density lipoprotein (VLDL-C) level assessed against streptozotocin-induced diabetes in Long Evan rats. Results are represented as the mean ± SD, where n = 5. #### = p < 0.0001, ### = p < 0.001, ## = p < 0.01, and ns = not significant when compared with the diabetic control group. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant, when compared with the normal control group.

Figure 4

Effect of different doses of LSML on the changes of serum (a) creatinine level; (b) total protein concentration; (c) uric acid concentration assessed against streptozotocin-induced diabetes in Long Evan rats. Results are represented as the mean ± SD, where n = 5. #### = p < 0.0001, ### = p < 0.001, ## = p < 0.01, # = p < 0.05 and ns = not significant when compared with the diabetic control group. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant, when compared with the normal control group.

Figure 5

Histopathological interpretation of pancreas tissue sections from different groups of the experimental diabetic animals. Light microscopic image of hematoxylin and Eosin-stained rat pancreas (microscopic resolution: 10 × 40). Here, the different groups are denoted by NC—Normal control, STD—Standard, DC—Diabetic control, T1—LSML65, T2—LSML125, and T3—LSML250. The symbols indicate that “★”—Cellular degeneration, “”—Necrotic cell, “”—Hydropic degeneration (HD); IC—Islets of β cells, Aci—Acinar cell.

Figure 6

Histopathological interpretation of kidney tissue sections of different groups of the experimental diabetic animals. Light micrographs of hematoxylin and eosin staining of rat kidney (microscopic resolution: 10 × 40). Here, the different groups are denoted by NC—Normal control, STD—Standard, DC—Diabetic control, T1—LSML65, T2—LSML125, and T3—LSML250. The arrow shows that BC—Bowman’s capsule, BS—Bowman’s space, G—Glomerulus, DCT—Distal convoluted tubule, PCT—Proximal convoluted tubule.

Figure 7

Histopathological interpretation of liver tissue sections from different groups of the experimental diabetic animals. Light microscopic image of hematoxylin and eosin-stained rat liver (microscopic resolution: 10 × 40). Here, the different groups are denoted by NC—Normal control, STD—Standard, DC—Diabetic control, T1—LSML65, T2—LSML125, and T3—LSML250. The arrow indicates that CV– Central vein, N—Necrosis, KC—Kupffer cell, SD—sinusoidal dilution, H—Hemorrhage, SS—sinusoidal space, AC—Apoptotic cell, IC—Inflammatory cell, ∆—Cellular degeneration, PV—Portal vein, BD—Bile duct.

Figure 8

Histopathological interpretation of spleen tissue sections from different groups of the experimental diabetic animals. Light microscopic image of hematoxylin and eosin-stained rat spleen (microscopic resolution: 10 × 40). Here, the different groups are denoted by NC—Normal control, STD—Standard, DC—Diabetic control, T1—LSML65, T2—LSML125, and T3—LSML250. The arrow indicates that CA—Central arteriole, WP—White pulp, RP—Red pulp, SS—Splenic sinuses.

Figure 9

The effect of different doses of LSML extract on the antioxidant activities of (a) catalase (CAT), (b) superoxide dismutase (SOD), (c) reduced glutathione peroxidase (GPX1), (d) lipid peroxidation (LPO) was assessed against streptozotocin-induced diabetes in Long Evan rats. Results are represented as the mean ± SD, where n = 5. # = p < 0/05, ## = p < 0.01, ### = p <0.001, #### = p < 0.0001 and ns = not significant compared to the hepatic control group. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05 and ns = not significant when compared with the normal control group.

Figure 10

Effect of different doses of LSML extract on (a) CAT (Catalase), (b) SOD2 (Superoxide dismutase 2), (c) GPX1 (Glutathione peroxidase 1), (d) GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), (e) PFK1 (Phosphofructokinase 1), (f) PON1 (Paraoxonase1) mRNA levels in the effect of different doses of LSML extract on liver CAT (Catalase) mRNA expression levels in a diabetic rat model. Total RNAs were isolated from rat hepatocytes. RNA-derived cDNA was used for the qRT-PCR analysis using 45 cycles of real-time PCR program. The relative ratios of mRNA levels were calculated using the 2^−ΔΔCT method normalized with β-ACTIN (Beta actin protein) CT value as the internal control and the control as the calibrator. Values are means (n = 3) and values at the same time point with different lower-case letters displayed above the columns of the figure indicating significant difference or not. Here, a = p < 0.0001, b = p < 0.001, c = p < 0.01, d = p < 0.05 when compared with NC; α = p < 0.0001, γ = p < 0.01 when compared with DC; and ns = not significant when compared with Normal Control and Diabetic Control.