Inhibition of N-methyl-N-nitrosourea-induced gastric tumorigenesis by Liuwei Dihuang Pill in db/db mice

Figure 1 Hematoxylin and eosin staining of stomach tissue. A: Gastric dysplasia in non-diabetic control group (db/m); B: Gastric dysplasia in diabetic control group (db/db); C: Gastric cancer in diabetic control group (db/db); D: Gastric dysplasia in metformin-treated group (db/db); E: Gastric dysplasia in LDP-treated group (db/db). The black arrows indicate cell dysplasia with irregular and hyperchromatic cell nuclei. The red arrows show invasive gastric cancer cells in the submucosa. Bar represents 50 μm. LDP: Liuwei Dihuang Pill; MNU: N-methyl-N-nitrosourea.

Figure 2 Cell proliferation assay with immunohistological staining of Ki67 in gastric dysplasia. A: Ki67 expression in non-diabetic control group (n = 2); B: Ki67 expression in diabetic control group (n = 6); C: Ki67 expression in metformin-treated group (n = 1); D: Ki67 expression in LDP-treated group (n = 1); E: Ki67-positive cells in each group. Results are presented as mean ± SE. ^bP < 0.01 vs control. Bar represents 25 μm. LDP: Liuwei Dihuang Pill; MNU: N-methyl-N-nitrosourea.

Figure 3 Effects of Liuwei Dihuang Pill on fasting blood glucose, insulin, insulin-like growth factor and insulin resistance. The fasting blood glucose at baseline (weeks 1 and 20) and week 30 was measured. The levels of insulin and IGF-1 were measured with ELISA kits. HOMA-IR was calculated for insulin resistance. Data represent the mean ± SE (n = 9). ^aP < 0.05, ^bP < 0.01 vs control. ELISA: Enzyme-linked immunosorbent assay; HOMA-IR: Homeostatic model assessment of insulin resistance; IGF-1: Insulin-like growth factor-1.

Figure 4 Effects of Liuwei Dihuang Pill on gene expression in gastric tissues. mRNA expression of IGF-1, IGF-1R, IGF-2, IGF-2R and insulin receptor was determined in stomach tissues by qRT-PCR. mRNA expression was normalized to that of GAPDH. Data represent the mean ± SE. ^aP < 0.05, ^bP < 0.01 vs control. IGF-1/2: Insulin-like growth factor-1 or 2; IGF-1/2R: Insulin-like growth factor-1 or 2 receptor; LDP: Liuwei Dihuang Pill; qRT-PCR: Quantitative reverse transcription-polymerase chain reaction.

Figure 5 Effects of Liuwei Dihuang Pill on insulin and insulin-like growth factor signaling pathways. A: A representative blot is presented. IGF-1R, AKT and their phosphorylated status were determined by western blotting. β-actin served as a loading control; B: Intensity of protein bands was determined with Image-J 1.46r software. The expression ratios of target proteins relative to β-actin were calculated. Data represent the mean ± SE. ^aP < 0.05, ^bP < 0.01 vs control. IGF-1R: Insulin-like growth factor-1 receptor.

Figure 6 Effects of Liuwei Dihuang Pill on serum adiponectin and leptin. Serum levels of adiponectin and leptin were examined with ELISA kits. Values are the mean ± SE. ^aP < 0.05, ^bP < 0.01 vs control. ELISA: Enzyme-linked immunosorbent assay; IGF-1: Insulin-like growth factor; LDP: Liuwei Dihuang Pill.