Effect of Zuojin Formula on proliferation and apoptosis of human GES-1 cells infected by Helicobacter pylori / 中草药

Objective: To study the effect of Zuojin Formula on proliferation and apoptosis of human normal gastric epithelial cells (GES-1) cells infected by Helicobacter pylori. Methods: GES-1 cells were infected by H. pylori at different multiplicity of infection (1∶1, 50∶1, 100∶1, 200∶1, 300∶1) for 12, 24, 48 h; GES-1 cells infected by H. pylori were treated with different concentrations (0.5, 1.0, 2.0, 4.0 μg/mL) of Zuojin Formula, and the cells were harvested after 12, 24, and 48 h. The proliferation activity of cells was detected by CCK-8, and the apoptosis rate of GES-1 cells was measured by Anexin V-FITC apoptosis detecting kit and flow cytometry. Moreover, the expression of Caspase-3 was detected by Western blot and the morphological changes of apoptotic cells were detected by Hochest staining. Results: After infected by H. pylori, the cell viability showed a descending trend while the apoptotic rate showed tendency to ascend, with the increasing of multiplicity of infection and infection time. GES-1 cells were treated with multiplicity of infection of H. pylori at 100∶1 for 12, 24, 48 h, and the cell viability decreased to (80.57 ± 1.21)%, (70.04 ± 3.21)%, and (67.74 ± 2.91)%, while the apoptotic rate increased to (23.74 ± 1.71)%, (53.60 ± 1.87)%, and (70.67 ± 2.87)%. After co-cultured with 1.0 μg/mL Zuojin Formula for 24 h, the cell viability increased to (97.67 ± 1.04)%, and the apoptotic rate decreased to (31.04 ± 1.02)%, and the difference was statistically significant compared with the corresponding model group (P < 0.01). The results of Western blotting showed that the protein expression of Caspase-3 of all treatment groups was obviously decreased. Based on the morphological point of view, this result was further verified. Conclusion: GES-1 infected by H. pylori NCTC11637 can inhibit cell proliferation and induce apoptosis, while ZuoJinFang could protect GES-1 cells from cell damage.

Study on the proliferation and the expression of S100A8 and S100A9 in Helicobacter pylor-infected human gastric epithelial cells GES-1 / 中华消化杂志

Objective@#To investigate the effects of Helicobacter pylori (H.pylori) on the proliferation of GES-1 cells and the expressions of S100A8 and S100A9 in human gastric epithelial cell line GES-1. @*Methods@#H. pylori were co-cultured with GES-1 cells at different infection plural (muhiplieity of infection (MOI) 50∶1, 100∶1, 200∶1), then the cells and cell culture supernatants were collected. The proliferative activity was detected by cell counting kit 8 (CCK-8) methods. The expression of S100A8 and S100A9 at mRNA level was determined by reverse transcription polymerase chain reaction (RT-PCR). The levels of S100A8 and S100A9 proteins and tumor necrosis factor-alpha (TNF-α) in cell culture supernatants were measured by enzyme linked immunosorbent assay (ELISA). T test and Pearson method were performed for statistical analysis. @*Results@#The negative control group was taken as the baseline, the proliferation rates of GES-1 cells of the H. pylori multiplicity 50∶1 group, 100∶1 group and 200∶1 group were (105.51±4.78)%, (168.97±11.29)% and (64.05±10.11)%, respectively. There was no statistically significant difference in the proliferation rate of GES-1 cells between H. pylori multiplicity 50∶1 group and negative control group (t=0.69, P=0.51). The proliferation rate of GES-1 cells of the H. pylori multiplicity 100∶1 group was higher than that of the negative control group, and the difference was statistically significant (t=10.63, P<0.01). The proliferation rate of GES-1 in the H. pylori multiplicity 200∶1 group was lower than that of the negative control group, and the difference was statistically significant (t=-5.54, P<0.01). The expression of S100A8 and S100A9 at the mRNA level in the H. pylori multiplicity 200∶1 group was 0.31±0.21 and 8.66±4.08, respectively, which were higher than those of the negative control group (0.06±0.05 and 0.08±0.08), and the differences were statistically significant (t=10.20 and 6.89, both P<0.05). The expressions of S100A8 at the protein level of H. pylori multiplicity 50∶1, 100∶1, and 200∶1 groups were (112.21±1.25) ng/mL, (120.39±1.61) ng/mL and (121.28±0.71) ng/mL, respectively; while the expression of S100A9 at the protein level were (179.43±2.44) ng/mL, (191.47±1.98) ng/mL and (201.80±2.06) ng/mL, respectively; and the expression of TNF-α levels were (285.52±3.64) ng/mL, (320.08±2.28) ng/mL and (350.97±2.90) ng/mL, respectively; which were all higher than those of the negative control group ((76.14±1.30) ng/mL, (161.35±1.31) ng/mL and (270.08±2.96) ng/mL, respectively), and the differences were statistically significant (tS100A8=35.09, 43.06, 43.92, tS100A9 = 11.13, 18.54, 24.90, tTNF-α= 6.34, 20.54, 33.23; all P<0.01). The expressions of S100A8 and S100A9 at the protein level were positively correlated with TNF-α (r=0.92 and 0.95, both P<0.01). @*Conclusion@#S100A8 and S100A9 may be involved in the process of H. pylori induced proliferation disorder and inflammation in GES-1 cells.

Jak1/Stat3 Is an Upstream Signaling of NF-kappaB Activation in Helicobacter pylori-Induced IL-8 Production in Gastric Epithelial AGS Cells

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-kappaB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-kappaB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IkappaBalpha were assessed by Western blot analysis, and NF-kappaB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-kappaB, determined by phosphorylation of IkappaBalpha and NF-kappaB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-kappaB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-kappaB is inhibited and inflammatory cytokine expression is suppressed.

Jak1/Stat3 Is an Upstream Signaling of NF-kappaB Activation in Helicobacter pylori-Induced IL-8 Production in Gastric Epithelial AGS Cells

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-kappaB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-kappaB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IkappaBalpha were assessed by Western blot analysis, and NF-kappaB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-kappaB, determined by phosphorylation of IkappaBalpha and NF-kappaB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-kappaB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-kappaB is inhibited and inflammatory cytokine expression is suppressed.

Diphenyleneiodonium Inhibits Apoptotic Cell Death of Gastric Epithelial Cells Infected with Helicobacter pylori in a Korean Isolate

NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacter pylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediate apoptotic cell death, direct involvement of NADPH oxidase on H. pylori-induced apoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reported as cagA+, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelial AGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability, hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the development of gastric inflammation associated with H. pylori infection by suppressing abnormal apoptotic cell death of gastric epithelial cells.

HTRA1 gene expression in gastric epithelial cells

OBJECTIVES@#To explore HtrA1 gene expression and its regulation in human gastric cancers.@*METHODS@#The HtrA1 mRNA levels were examined by QPCR analysis and confirmed its expression with Northern blot analysis. The HtrA1 protein levels in all six gastric epithelial cell lines were investigated by Western blot analysis. Gene copy number was accessed and then sequenced the coding region from each mRNA in all six cell lines. The HtrA1 promoter region DNA methylation status was detected by using bisulfite sequencing analysis. Effect of decitabine and TSA on HTRA1 expression in gastric cancer cell line was determined by RTPCR.@*RESULTS@#HIC analysis indicated that HtrA1 was highly expressed in normal epithelium, but dramatically down-regulated in gastric carcinoma tissues and variably expressed in tumor-adjacent tissues. HtrA1 gene expression was dramatically decreased in gastric carcinoma cells compared to non-tumorigenic counterparts. The HtrA1 gene loss in any of the 4 breast cancer cell lines was not detected. Total 14 CpGs in this region were all methylated in gastric cancer cells, whereas two normal cells, GES-1 and HFI-145, were having several unmethylated cytosines in this region. HtrA1 showed as ~Mr 44,000, Expression of HtrA1 protein was not observed in any of the four gastric cancer cell lines, BGC-823, MKN-45, SGC-7901and MKN-28. HtrA1 expression was observed in the HFI-145and GES-1 cell lines.@*CONCLUSIONS@#The epigenetic silencing for HtrA1 gene expression could provide a possible strategy for re-activating HtrA1 gene expression in gastric cancer cells, thus facilitating further investigation of HtrA1's role in chemotherapy.

The Induction of Matrix Metalloproteinase-9 Expression by Helicobacterium Pylori / 中国医师杂志

Objective To study the pathogenic molecular mechanism of H.pylori through analyzing its effect of on MMP-9 expression. Methods BGC-823 cells were cocultured with ATCC49503(CagA +)and HP 030811 (CagA -) H.pylori strains respectively, then the total cellelar RNA was extracted. The MMP-9 mRNA expression in BGC-823 cells, 9 cases of H.pylori-positive and 9 cases of H.pylori-negative gastric ulcer biopsy tissues was detected by RT-PCR. The MMP-9 protein expression on the paraffin-imbedded tissue sections of 17 cases of H.pylori-positive and 14 cases of H.pylori-negative gastric ulcer tissues was detected with immunohistochemical technique, and was quantified by image quantitative analysis. Results H.pylori induced MMP-9 expression in BGC-823, and the level of MMP-9 expression induced by CagA -postive H.pylori strain was higher than that by CagA -negative one. MMP-9 expression level was higher in the H.pylori-positive gastric ulcer tissues than that in the H.pylori-negative ones. Conclusion H.pylori-induced MMP-9 expression may play an important role in gastric ulcer formation and gastric carcinogenesis.