Inhibitory effect of astragalus membranaceus injection on apoptosis and autophagy induced by enterovirus 71 / 中国生物制品学杂志

@#Objective To investigate the effect of astragalus membranaceus（AM）injection on apoptosis and autophagy of human gastric epithelial cell line（GES⁃1）induced by enterovirus 71（EV71）. Methods GES⁃1 cells were cultured in vitro and divided into infected group（EV71 infected at a MOI of 3 and control group（no virus infected）. The morpho⁃logical changes of EV71 infected cells were observed by inverted microscope. The level of VP1 in GES⁃1 cells infected with EV71 was detected by Western blot；CCK⁃8 assay was used to detect the viability of GES⁃1 cells infected with EV71；Nuclear staining with DAPI was used to observe the morphological changes of nuclear apoptosis infected with EV71. GES⁃1 cells were divided into control group（without virus infection），infection group and AM intervention group with final concentration of 1，2. 5，5 and 10 μg／mL，respectively. Western blot was used to detect the effect of AM intervention on the expression of apoptosis⁃related proteins Caspase⁃3，PARP and autophagy⁃related proteins LC3 and P62 in GES⁃1 cells infected withEV71. CCK⁃8 method was used to detect the effect of AM intervention on the viability of GES⁃1 cells infected with EV71. Results GES⁃1 cells were round，shrunken with nuclear pyknosis and uneven size；VP1 level increased（t = 41. 56，P < 0. 01），cell viability decreased（t = 19. 07，P < 0. 01），Caspase⁃3 and PARP proteins were cut off（t = 35. 29 and 3. 648， P < 0. 01 and 0. 021 8，respectively），LC3Ⅱ／LC3Ⅰ ratio increased（t = 10. 16，P = 0. 000 5）and P62 protein was degraded（t = 68. 68，P < 0. 01）；AM inhibited the degradation of Caspase⁃3，PARP and P62 proteins induced by EV71 （t = 52. 66，59. 60 and 40. 22，respectively，each P < 0. 01）and increased the ratio of LC3Ⅱ／LC3Ⅰ（t = 5. 521，P = 0. 005 3），andreducedtheinhibitoryeffectofEV71ontheviabilityofGES⁃1cells（t =4. 420，P =0. 0115）. Conclusion EV71 infection induced apoptosis of GES⁃ 1 cells and AM intervention inhibited EV71 induced apoptosis by inhibiting EV71 induced autophagy.

Role and mechanism of IGF2BP3 in malignant transformation of human gastric epithelial cells induced by MNNG / 环境与职业医学

Background N6-methyladenosine (m6A) RNA methylation may play an important role in the process of malignant transformation of cells induced by environmental carcinogens. However, the specific roles and mechanisms need to be further explored. Objective To explore the role and mechanism of m6A binding protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the malignant transformation of human gastric mucosal epithelial cells GES-1 induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Methods Based on the GES-1 malignant transformation cells MC-30, a stable knockdown IGF2BP3 MC-30 cell line (MC30-shIGF2BP3, abbreviated as MC30-shI3) was constructed by lentiviral transfection technology, and a negative control group (MC30-NC) was also prepared. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were applied to detect the mRNA expression and protein levels of IGF2BP3. RNA binding protein immunoprecipitation (RIP-qPCR) was used to examine the combination between IGF2BP3 protein and MYC mRNA in malignant cells MC-30. Furthermore, the stability of MYC mRNA was detected by actinomycin D assay. CCK-8 and Transwell respectively were employed to detect cell proliferation, migration, and invasion. Western blotting was applied to detect the expression of EMT markers (N-cadherin, Vimentin, α-SMA, and Snail). The role of the downstream target gene MYC was further elucidated by a rescue assay in MC30-shI3 cells transfected with a plasmid overexpressing MYC to observe changes in cellular phenotypes (proliferation, migration, invasion) and expression of key EMT proteins. Results Compared with the control group, the expression of IGF2BP3 mRNA was up-regulated after 5, 10, 20, and 40 μmol·L−1 MNNG infection of GES-1 cells (P<0.05). After 20 μmol·L−1 MNNG infection, the expression level of IGF2BP3 mRNA increased with prolongation of exposure time (P<0.05). Compared with the control group, the mRNA and protein expression levels of IGF2BP3 were up-regulated in the 10th, 20th, and 30th generations of 5 μmol·L−1 MNNG malignant transformation (P<0.05). The results of qRT-PCR and Western blotting showed that, compared with the MC30-NC group, the IGF2BP3 and MYC mRNA expression and protein expression decreased in the MC30-shI3 group (P<0.01). The CCK8 and transwell assay results showed that, compared with the MC30-NC group, the cell proliferation, migration, and invasion abilities significantly reduced in the MC30-shI3 group (P<0.01). The results of the Western blotting showed that, compared with the MC30-NC group, the protein levels of EMT markers N-cadherin, Vimentin, α-SMA, and Snail decreased in the MC30-shI3 group (P<0.01). The results of RIP-qPCR showed that, compared with the IgG group, the mRNA level was higher for the enriched MYC in the IGF2BP3 group (P<0.01); the results of the actinomycin D assay showed that, compared with the MC30-NC group, the stability of MYC mRNA significantly reduced in the MC30-shI3 group (P<0.01). While the rescue experiment showed that, compared with the IGF2BP3 knock-down+vector group, the MYC protein level significantly increased in the IGF2BP3 knock-down + MYC over-expression group (P<0.01), the proliferation, migration, and invasion abilities significantly enhanced (P<0.01), and the EMT key proteins (N-cadherin, Vimentin, α-SMA, Snail) increased in the MC30-shI3+MYC group (P<0.01). Conclusion Exposure to MNNG could result in up-regulation of IGF2BP3 expression in GES-1 cells. IGF2BP3 may enhance the proliferation, migration, and invasion of malignantly transformed human gastric epithelial cells by binding to MYC mRNA and increasing its stability and expression level and thus promoting the EMT process, which in turn affects the progression of malignant transformation.

Protective effect of against ethanol-induced gastric ulcer and its mechanism / 浙江大学学报·医学版

: To investigate the protective effect of (FD) against ethanol-induced gastric ulcer and its mechanism. : Human gastric epithelial GES-1 cells were divided into normal control group, model control group, FD 95% alcohol extract group, FD 50% alcohol extract group and FD decoction extract group. Gastric ulcer was induced by treatment with 1% ethanol in GES-1 cells. The cell proliferation was detected with MTT method in each group. Sixty SD rats were randomly divided into normal control group, model control group, ranitidine group and low-dose, medium-dose, high-dose FD 95% alcohol extract groups (150, 300, 600 mg/kg). The corresponding drugs were administrated by gavage for The gastric ulcer model was induced by intragastric administration of anhydrous ethanol. The gastric ulcer area and ulcer inhibition rate of rats were measured in each group; the degree of gastricmucosal damage was observed by scanning electron microscopy; the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β in serum and the content of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT) in gastric tissues were detected by ELISA method. : 95% alcohol extract of FD had the strongest protective effect on proliferation of GES-1 cells. In animal experiments, compared with the normal control group, a large area of ulcers appeared on the gastric mucosa in the model control group, while the ulcer areas of the FD groups and ranitidine group were significantly smaller than that of the model control group (all <0.05). Compared with the model control group, FD groups and ranitidine group significantly reduced the levels of TNF-α, IL-1β, IL-6 in serum and the MDA content in the gastric tissues, and increased the activity of SOD, CAT and GSH in gastric tissues (all <0.05). : The 95% alcohol extract of FD can reduce the levels of TNF-α, IL-1β and IL-6 in serum and the content of MDA in gastric tissues, and increase the activity of SOD, CAT and GSH in gastric tissues to achieve the protective effect against gastric ulcer.

Helicobacter pylori virulence factor vacuolating cytotoxin A ( VacA) promotes homologous recombi-nation repair in GES-1 cells / 中华微生物学和免疫学杂志

Objective To analyze the impact of Helicobacter pylori standard strain (Hp P12) and its virulence factor vacuolating cytotoxin A ( VacA) on DNA damage and homologous recombination ( HR) repair in a human gastric epithelial cell line (GES-1). Methods Strains of Hp P12 and vacA gene knock-out Hp P12 ( Hp P12 ΔvacA) were respectively used to infect GES-1 cells at a multiplicity of infection of 100. GES-1 cells treated with etoposide (50μmol/L) or mitomycin (0. 5μg/ml) for 2 h were used as posi-tive control. Western blot and immunofluorescence were performed to detect the expression of DNA damage marker protein γH2AX and key HR repair proteins (Rad51, pMRE11, CtIP and pCtIP) and the recruitment of them at DNA damage sites. Human embryonic kidney HEK-293 ( DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA strains to verify the impact of VacA on HR repair efficiency. Results The expres-sion and recruitment of γH2AX and key HR repair proteins ( Rad51, pMRE11, CtIP and pCtIP) were in-creased in Hp P12-infected cells as compared with that in uninfected and Hp P12 ΔvacA-infected cells ( all P<0. 05). To evaluate the HR repair efficiency, I-SceⅠ plasmid-transfected HEK-293 (DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA and the results showed that green fluorescent protein ( GFP)-positive cells were decreased after infection, especially in Hp P12 ΔvacA-infected cells (both P<0. 05). Conclusions Hp P12 infection could cause DNA damage and promote HR repair in GES-1 cells, in which the virulence factor VacA played an important role.

Inconsistent effect of chloroquine on apoptosis of normal gastric epithelial cells GES-1 and gastric cancer cell line HGC-27 / 中国免疫学杂志

Objective:To compare the effect of chloroquine on apoptosis of normal gastric epithelial cells and gastric cancer cell line HGC-27. Methods:Change of these two kinds of cells were observed by inverted microscope after treating with CQ. HGC-27 cells were detected on the effect of apoptosis by DAPI nuclear staining after treating with CQ. The proliferation of cells were measured by CCK-8. Changes of mitochondrial membrane potential were investigated by JC-1 after treating with CQ. The expression of apoptosis protein effector enzyme Caspase-3 and substrate PARP in these two kinds of cells were tested by Western blot after using chloroquine (CQ) and rapamycin ( rapamycin, RAP ) to treat cells 72 h. Results: After treated with 10 μmol/L CQ 72 h, morphological characteristics of GES-1 cells and HGC-27 cells could be visible under the microscope,CQ induced apoptosis of GES-1 cells,on the contrary,it could make the HGC-27 cell get widened,the number of apoptotic cells gradually increased,the cell density decreased,cell atrophy and gradually turned round,cytoplasm reduced,at last,lose normal cell morphology. After two kinds of cells treated with CQ 72 h,as for GES-1 cell nuclei stained light,nuclear size and shape were not changed,however,HGC-27 nuclei showed pyknosis or granular fluorescence dense concentrated form. CCK-8 results showed that comparing with normal gastric epithelial cells GES-1,the pro-liferation of gastric cancer HGC-27 cells activity could be inhibited by CQ. JC-1 results showed that the change of the red fluorescence to green fluorescence in HGC-27 cells treated by CQ. Western blot showed that after being treated with CQ and RAP in normal gastric epithelial cells and HGC-27 cell line 72 h,the expression of apoptosis protein Caspase-3 and PARP in gastric cancer cell HGC-27 decreased significantly,comparing to that in GES-1 cells. Conclusion:Compared to normal gastric epithelial cells,CQ can inhibit human gastric cancer HGC-27 cell viability and induce apoptosis.

Distribution of the bla OXA, bla VEB-1, and bla GES-1 genes and resistance patterns of ESBL-producing Pseudomonas aeruginosa isolated from hospitals in Tehran and Qazvin, Iran

Abstract INTRODUCTION: Pseudomonas aeruginosa is one of the most common nosocomial pathogens. The emergence of extended spectrum β-lactamases (ESBLs) has been increasingly reported as a major clinical concern worldwide. The main aim of the present study was to determine the distribution of bla OXA, bla PER-1, bla VEB-1, and bla GES-1 genes among ESBL-producing P. aeruginosa isolated from two distinct provinces in Iran. METHODS: In this study, a total of 75 (27.5%) ESBL-producing isolates were identified from 273 P. aeruginosa isolates collected from patients in Qazvin and Tehran. Phenotypic detection of ESBLs and antimicrobial susceptibility testing were performed according to the Clinical and Laboratory Standards Institute guidelines. PCR and sequencing were employed to detect bla OXA-1, bla OXA, bla GES-1, bla PER-1, and bla VEB-1 genes. Isolate genetic relationships were evaluated by repetitive extragenic palindromic sequence-based PCR (REP-PCR). RESULTS: In total, 59 (78.7%) of the ESBL-producing isolates showed multidrug resistance. The highest rates of susceptibility were observed against colistin (75 isolates, 100%) and polymyxin B (75, 100%) followed by amikacin (44, 58.7%), and piperacillin-tazobactam (40, 53.3%). The bla OXA-1 (37.3%) gene was the most common of the genes investigated, followed by bla OXA-4 (32%), bla GES-1 (16%), and bla VEB-1 (13.3%). REP-PCR identified three different genotypes: types A (89.3%), B (6.7%), and C (4%). CONCLUSIONS: We found a significant presence of bla OXA-1, bla OXA-4, bla GES-1, and bla VEB-1 genes among P. aeruginosa isolates, highlighting the need for suitable infection control strategies to effectively treat patients and prevent the further distribution of these resistant organisms.

Preconditioning of ulinastatin alleviates GES-1 cell injury induced by oxygen and glucose deprivation / 实用医学杂志

Objective To observe the effects of the preconditioning of ulinastatin on GES-1 cell injury induced by oxygen and glucose deprivation (OGD). Methods GES-1 cells were cultured in vitro and divided into three groups: normal control group (group N), oxygen and glucose deprivation group (group O), and ulinastatin preconditioning group (group U). The OGD model of GES-1 cells were established by glucose-free medium and three-gas incubator for 6h. Ulinastatin was added to group U 12h before the deprivation of oxygen and glucose. The cell viability and apoptosis were determined by cck-8 and flow cytometry respectively. Western Blot was used to examine the protein expression of Caspase-3 and Cleaved Caspase-3. The TRPV1 mRNA expression was measured by quantitative real-time PCR. Results As compared with group N, the viability of GES-1 was decreased, the apoptotic rate and the expression of Caspase-3 and Cleaved Caspase-3 were increased, and the TRPV1 mRNA expression decreased greatly in group O (P < 0.05). As compared with group O, the aforementioned changes were significantly inhibited in group U. Conclusions Ulinastatin preconditioning could effectively inhibit GES-1 cell injury induced by OGD, which may be related to the inhibition of apoptosis and the upregulation of TRPV1 mRNA expression.

Stress-related hormone norepinephrine induces interleukin-6 expression in GES-1 cells

In the current literature, there is evidence that psychological factors can affect the incidence and progression of some cancers. Interleukin 6 (IL-6) is known to be elevated in individuals experiencing chronic stress and is also involved in oncogenesis and cancer progression. However, the precise mechanism of IL-6 induction by the stress-related hormone norepinephrine (NE) is not clear, and, furthermore, there are no reports about the effect of NE on IL-6 expression in gastric epithelial cells. In this study, we examined the effect of NE on IL-6 expression in immortalized human gastric epithelial cells (GES-1 cells). Using real-time PCR and enzyme-linked immunoassay, we demonstrated that NE can induce IL-6 mRNA and protein expression in GES-1 cells. The induction is through the β-adrenergic receptor-cAMP-protein kinase A pathway and mainly at the transcriptional level. Progressive 5′-deletions and site-directed mutagenesis of the parental construct show that, although activating-protein-1 (AP-1), cAMP-responsive element binding protein (CREB), CCAAT-enhancer binding protein-β (C/EBP-β), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding sites are all required in the basal transcription of IL-6, only AP-1 and CREB binding sites in the IL-6 promoter are required in NE-induced IL-6 expression. The results suggest that chronic stress may increase IL-6 secretion of human gastric epithelial cells, at least in part, by the stress-associated hormone norepinephrine, and provides basic data on stress and gastric cancer progression.

Study on the effects of intestinal trefoil factor on gastric mucosal epithelial cell proliferation and its signal transduction mechanism / 医学研究生学报

Objective To explore the effects of intestinal trefoil factor ( ITF) on gastric mucosal epithelial cell proliferation and its possible molecular mechanism . Methods The cultured GES-1 cells were treated with ITF in the concentration of 100 ng/mL and 500 ng/mL in vitro, and then were observed using microscope for the morphological analysis .The Cell Counting Kit-8 ( CCK-8) was used to detect the proliferation activity of GES-1.The cultured GES-1 cells were treated with 100 ng/mL ITF and the specific inhibitor of PI3K/Akt signaling pathway LY294002 (15μmol/L) in vitro, and then were observed using microscope for the morphological analysis . The proliferation activity of treated GES-1cells was detected using CCK-8 and the expressions of p-Akt and Akt of PI3K/Akt signaling pathway were determined by Western blot . Results Compared with the control group , the proliferation activity of GES-1 cells in-creased after being treated with ITF and the higher concentration of ITF induced the higher proliferation activity .LY294002 inhibited the increased proliferation activity of GES-1induced by ITF.The data of Western blot indicated that ITF induced the expression of p -Akt and activated the P3IK/Akt signaling pathway to modulate the proliferation activity of GES -1 cells.However, LY294002 inhibited the PI3K/Akt signaling pathway and then decreased the proliferation activity of GES -1 cells. Conclusion ITF could promote the proliferation ac-tivity of GES-1 cells by activating PI3K/Akt signaling pathway .

Expression and clinical significance of Wnt5a in gastric carcinoma / 实用医学杂志

Objective To study the significance of Wnt5a gene expression in gastric carcinoma (GC). Methods The Wnt5a mRNA expressions in SGC7901 cell line and normal gastric mucosa GES-1 cell line were detected by RT-PCR. The Wnt5a protein expressions in 60 GC and 30 matched tumor-adjacent normal tissue samples were examined by immunohistochemistry using the streptavidin-peroxidase method.χ2 test was used to analyze Wnt5a expression on the clinical pathologic features of gastric carcinoma. Results Both Wnt5a mRNA and Wnt5a protein were significantly higher in SGC7901 cells than those in GES-1 cells. The positive rate of Wnt5a protein expression was also significantly higher in GC than that in tumor-adjacent normal tissue (P < 0.05). Positive expression of Wnt5a was associated with higher degree of regional lymph node metastasis and the later TNM stage (P < 0.05). Conclusion Higher expression of Wnt5a seems to promote invasion and metastasis of gastric cancer, and Wnt5a might play an oncogene-like role in the development of gastric carcinoma.