Xiaojianzhong decoction prevents gastric precancerous lesions in rats by inhibiting autophagy and glycolysis in gastric mucosal cells

Figure 1 Effects of Xiaojianzhong decoction on body weight, gastric mucosal pathological conditions and liver kidney toxicity in N-methyl-N’-nitro-N-nitrosoguanidine-induced gastric precancerous lesions rats. A: Schematic diagram of model construction and treatment; B: Schematic diagram of weight change in rats; C: Representative images of hematoxylin-eosin and alcian blue-periodic acid-Schiff staining of gastric tissues; D-G: Ki-67 and caudal-type homeobox protein 2 (CDX-2) expression was determined using immunohistochemistry (n = 5); Ki-67 and CDX-2 positive score was determined using Image J; H: Superoxide dismutase and malondialdehyde levels in serum (n = 3); I: Alanine aminotransferase and creatinine levels in serum (n = 3). Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group. XJZ-L: Low dose of Xiaojianzhong decoction; XJZ-M: Middle dose of Xiaojianzhong decoction; XJZ-H: High dose of Xiaojianzhong decoction; SOD: Superoxide dismutase; MDA: Malondialdehyde; ALT: Alanine aminotransferase; Cr: Creatinine.

Figure 2 Result of the high-performance liquid chromatography method coupled with triple-quadrupole tandem mass spectrometry assays of the chemical composition of Xiaojianzhong decoction. A: High-performance liquid chromatography chromatogram; B-F: Compounds contained in Xiaojianzhong decoction.

Figure 3 Effects of Xiaojianzhong decoction on autophagy of gastric mucosal epithelial cells in N-methyl-N’-nitro-N-nitrosoguanidine-induced gastric precancerous lesions rats. A: Observation of autophagy in rat gastric epithelial cells by transmission electron microscope (the red arrows in the figure are autophagosomes or autophagolysosomes); B and C: Western blot analysis was performed to detect BCL2-interacting protein 1 (Beclin-1), microtubule associated protein 1 light chain 3 (LC-3) II protein expression (n = 3); D and E: Beclin-1, LC-3II mRNA expression was determined using real-time polymerase chain reaction analysis (n = 3); F and G: p62 expression was determined using immunohistochemistry (n = 5); p62 positive score was determined using Image J; H-J: B cell lymphoma/leukemia-2 and adenovirus E1B19000 interacting protein 3 (Bnip-3), Beclin-1 proteins expression was determined using immunofluorescence (n = 5); Bnip-3, Beclin-1 porteins positive score was determined using ImageJ. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group. XJZ-L: Low dose of Xiaojianzhong decoction; XJZ-M: Middle dose of Xiaojianzhong decoction; XJZ-H: High dose of Xiaojianzhong decoction; Beclin-1: BCL2-interacting protein 1; LC-3: Microtubule associated protein 1 light chain 3.

Figure 4 Xiaojianzhong decoction up-regulates phosphatidylinositol 3-kimase/protein kinase B/mammalian target of rapamycin signaling pathway in N-methyl-N’-nitro-N-nitrosoguanidine-induced gastric precancerous lesions rats. A-C: Western blot analysis was performed to detect phosphorylated-phosphatidylinositol 3-kimase (p-PI3K), phosphorylated-protein kinase B (p-AKT), and phosphorylated-mammalian target of rapamycin (p-mTOR) protein expression (n = 3); D-F: PI3K, AKT, and mTOR mRNA expression was determined using real-time polymerase chain reaction analysis (n = 3). Data are expressed as mean ± standard deviation. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group. XJZ-L: Low dose of Xiaojianzhong decoction; XJZ-M: Middle dose of Xiaojianzhong decoction; XJZ-H: High dose of Xiaojianzhong decoction.

Figure 5 Effects of Xiaojianzhong decoction on gastric mucosal hypoxia in N-methyl-N’-nitro-N-nitrosoguanidine-induced gastric precancerous lesions rats. A and B: Western blot analysis was performed to detect hypoxia-inducible factor 1α (HIF-1α), E1B19000 interacting protein 3 (Bnip-3) protein expression (n = 3); C and D: HIF-1α, Bnip-3 mRNA expression was determined using real-time polymerase chain reaction analysis (n = 3); E-G: Hypoxia-induced autophagy-related protein HIF-1α, Bnip-3 expression was determined using immunofluorescence (n = 5); HIF-1α, Bnip-3 proteins positive score was determined using Image J. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group. XJZ-L: Low dose of Xiaojianzhong decoction; XJZ-M: Middle dose of Xiaojianzhong decoction; XJZ-H: High dose of Xiaojianzhong decoction; HIF-1α: Hypoxia-inducible factor 1α; Bnip-3: B cell lymphoma/Leukemia-2 and adenovirus E1B19000 interacting protein 3.

Figure 6 Effects of Xiaojianzhong decoction on hypoxia-induced glycolysis in gastric mucosal epithelial cells. A-C: Hypoxia-induced glycolysis-related protein hypoxia-inducible factor 1α (HIF-1α), CD147 expression was determined using immunofluorescence (n = 5); HIF-1α, CD147 proteins positive score was determined using Image J; D-F: CD147, monocarboxylate transporter (MCT1), and MCT4 mRNA expression was determined using real-time polymerase chain reaction analysis (n = 3); G and H: Sirtuin 6 (SIRT6) expression was determined using immunohistochemistry (n = 5); SIRT-6 positive score was determined using Image J. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group. XJZ-L: Low dose of Xiaojianzhong decoction; XJZ-M: Middle dose of Xiaojianzhong decoction; XJZ-H: High dose of Xiaojianzhong decoction; HIF-1α: Hypoxia-inducible factor 1α; MCT: Monocarborxylat transporter.

Figure 7 Xiaojianzhong decoction inhibited Unc-51 Like kinase 1 and p53/AMP-activated protein kinase pathway in N-methyl-N’-nitro-N-nitrosoguanidine-induced gastric precancerous lesions rats. A and B: Unc-51 like kinase 1 (ULK1) and p53 protein expression was determined using immunofluorescence (n = 5); ULK1, p53 proteins positive score was determined using Image J; C: ULK1 mRNA expression was determined using real-time polymerase chain reaction analysis (n = 3); D-G: Western blot analysis was performed to detect p-p53, p-AMP-activated protein kinase, p-ULK1 (Ser555), and p-ULK1 (Ser317) protein expression (n = 3); H and I: ULK1 and p53 protein expression was determined using immunofluorescence (n = 5). Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs model group. XJZ-L: Low dose of Xiaojianzhong decoction; XJZ-M: Middle dose of Xiaojianzhong decoction; XJZ-H: High dose of Xiaojianzhong decoction; ULK1: Unc-51 like kinase 1.

Figure 8 Scheme summarizing the protective effects of Xiaojianzhong decoction on N-methyl-N’-nitro-N-nitrosoguanidine-induced gastric precancerous lesions rats via regulation of the p53/AMP-activated protein kinase/Unc-51 like kinase 1, phosphatidylinositol 3-kimase/protein kinase B/mammalian target of rapamycin axis and hypoxia state. AKT: Protein kinase B; AMPK: AMP-activated protein kinase; Bnip-3: B cell lymphoma/Leukemia-2 and adenovirus E1B19000 interacting protein 3; Dys: Dysplasia; GPL: Gastric precancerous lesions; HIF-1α: Hypoxia-inducible factor-1α; IM: Intestinal metaplasia; MCT: Monocarboxylate transporter; MNNG: N-methyl-N’-nitro-N-nitrosoguanidine; PI3K: Phosphatidylinositol 3-kimase; mTOR: Mammalian target of rapamycin signaling pathway; SIRT6: Sirtuin 6; ULK1: Unc-51 like kinase 1.