Antioxidants, Vol. 11, Pages 2398: 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

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 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.

Antioxidants 11 02398 g001

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 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.

Antioxidants 11 02398 g002

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 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.

Antioxidants 11 02398 g003

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 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.

Antioxidants 11 02398 g004 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, “ Antioxidants 11 02398 i001”—Necrotic cell, “ Antioxidants 11 02398 i002”—Hydropic degeneration (HD); IC—Islets of β cells, Aci—Acinar cell. 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, “ Antioxidants 11 02398 i001”—Necrotic cell, “ Antioxidants 11 02398 i002”—Hydropic degeneration (HD); IC—Islets of β cells, Aci—Acinar cell. Antioxidants 11 02398 g005

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 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.

Antioxidants 11 02398 g006

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 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.

Antioxidants 11 02398 g007

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 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.

Antioxidants 11 02398 g008

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 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.

Antioxidants 11 02398 g009

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.

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.

Antioxidants 11 02398 g010

Table 1. Name and sequences of the primers used for qRT-PCR.

Table 1. Name and sequences of the primers used for qRT-PCR.

Gene
SymbolGene
DescriptionPrimerSequences (5′→3′)Gene Bank
Accession No.β-ACTINβ-actin proteinFGGCATCCTGACCCTGAAGTANM_031144.3RGGGGTGTTGAAGGTCTCAAACATCatalaseFACGAGATGGCACACTTTGACAGNM_012520.2RTGGGTTTCTCTTCTGGCTATGGSOD2Superoxide dismutase-2FAGCTGCACCACAGCAAGCACNM_017051.2RTCCACCACCCTTAGGGCTCAGPX1Glutathione peroxidase-1FAAGGTGCTGCTCATTGAGAATGNM_030826.4RCGTCTGGACCTACCAGGAACTGAPDHGlyceraldehyde-3-phosphate dehydrogenaseFGGTGAAGTTCGGAGTCAACGGANM_017008.4RGAGGGATCTCGCTCCTGGAAGAPON-1Paraoxonase -1FTGCTGGCTCACAAGATTCACXM_039108462.1RTCAAAGCTGAGGACCTTCAATPFK-1Phosphofructokinase-lFTTACCGATCACCCTCGTTCCTXM_008772798.3RTTCCCCTTAGTGCTGGGATCT

Table 2. Effects of different doses of LSML extract on the changes of organs weight pancreas, kidney, liver, and spleen in rats with STZ-induced diabetic and control groups.

Table 2. Effects of different doses of LSML extract on the changes of organs weight pancreas, kidney, liver, and spleen in rats with STZ-induced diabetic and control groups.

GroupsPancreas Weight
(g)Relative
Pancreas Weight (g)Kidney Weight
(g)Relative
Kidney Weight
(g)Liver
Weight
(g)Relative Liver Weight
(g)Spleen Weight
(g)Relative Spleen Weight
(g)Normal
Control3.67 ± 0.290.80 ± 0.060.93 ± 0.070.18 ± 0.036.86 ± 1.061.49 ± 0.220.65 ± 0.06 0.14 ± 0.01 STZ +
Silymarin3.44 ± 0.70 ns1.57 ± 0.35 a1.01 ± 0.12 ns0.55 ± 0.07 a7.15 ± 0.65 ns3.26 ± 0.38 b0.61 ± 0.08 ns0.27 ± 0.03 aSTZ1.99 ± 0.13 a1.20 ± 0.04 c1.56 ± 0.06 a0.94 ± 0.06 a11.96 ± 1.91 a7.24 ± 1.15 a0.40 ± 0.07 b0.24 ±0.05 bSTZ + LSML652.556 ± 0.26 c1.25 ± 0.14 b1.45 ± 0.25 a0.71 ± 0.12 a10.74 ± 2.13 c5.23 ± 1.03 a0.42 ± 0.12 c0.21 ± 0.05 nsSTZ + LSML1252.99 ± 0.31 ns0.92 ± 0.07 ns1.35 ± 0.09 c0.42 ± 0.04 a8.69 ± 1.47 ns2.67 ± 0.47 ns0.52 ± 0.12 ns0.16 ± 0.03 nsSTZ + LSML2503.22 ± 0.39 ns0.90 ± 0.12 ns1.18 ± 0.09 ns0.33 ± 0.03 ns8.11 ± 2.01 ns2.27 ± 0.56 ns0.58 ± 0.16 ns0.16 ± 0.05 ns

Table 3. Effects of Lasia spinosa extracts on histopathological changes in the morphology of pancreas tissue sections.

Table 3. Effects of Lasia spinosa extracts on histopathological changes in the morphology of pancreas tissue sections.

GroupDegenerated CellNecrotic CellDiameter of Islet of Langerhans (μm)Area Occupied by β-Cell/Islet of ± Langerhans (μm2)Normal control (NC)--215 ± 17.324600 ± 7447Standard (STD)++170 ± 11.5428,800 ± 3925Diabetic control (DC)++++Islets are extensively disrupted to countLSML 65 (T1)++195 ± 28.8637,400 ± 9750LSML 125 (T2)--285 ± 05.7781,200 ± 3290LSML 250 (T3)++250 ± 34.6461,600 ± 1732

留言 (0)

沒有登入
gif