Effect Of β1-integrin In Autophagy Of Gastric Epithelial Cells Induced By Helicobacter Pylori Infection

ObjectiveThe role and mechanism of β1-integrin in autophagy of gastric epithelial cells induced by Helicobacter pylori.Methods1.The expression of β1-integrin and LC3 Ⅱ protein in gastric mucosa of H.pylori positive and negative human were detected by immunofluorescence technique;H.pylori was co-cultured with GES-1 cells,the expression of β1-integrin and LC3 II was detected by Western blot.2.The cells were divided into three experimental groups,and the normal GES-1 cells were blank groups.GES-1 cells transfected with Negative-siRNA were negative control groups.GES-1 cells transfected with β1-integrin-siRNA were the silencingβ1-integrin group.H.pyori was co-cultured with GES-1 cells,and the expression changes of autophagy-related proteins Beclin-1,LC3II,Atg5,Atg12,Atg16L1,Atg7 and Atg3 were detected by Western blot.The formation of autophagosomes was observed by transmission electron microscopy.The distribution of cytosolic mRFP-GFP-LC3 protein was observed by laser confocal microscopy.The cells were treated with the autophagic flow inhibitor bafilomycin A1(bafA1),H.pylori was co-cultured with GES-1 cells,and the expression of P62 and LC3Ⅱ was detected by Western blot.3.H.pylori was co-cultured with GES-1 cells,and the apoptosis rate was detected by flow cytometry.The cells were treated with autophagy inhibitor 3-methyladenosine(3-MA).Then,H.pylori was co-cultured with GES-1 cells,the apoptosis rate was detected by flow cytometry.The ratio of cleaved-caspase3/pro-caspase3 and cleaved-PARP/pro-PARP was detected by Western blot.Results1.The expression of LC3 Ⅱ was significantly increased in H.pylori positive gastric mucosa tissue samples,the expression of β1-integrin was significantly decreased,and the expression of LC3 II was negatively correlated with the expression of β1-integrin.The fluorescence intensity of LC3 Ⅱ in H.pylori positive gastric mucosa specimens was 24.5±6.4,and the fluorescence intensity in negative samples was 9.6±3.5.The fluorescence intensity of LC3 Ⅱ in positive samples was significantly higher than that in negative samples(P<0.001);H.pylori positive The fluorescence intensity of β1-integrin in gastric mucosa tissue specimens was 20.6±10.7,and the fluorescence intensity in negative samples was 31.3± 12.5.The fluorescence intensity of β1-integrin in positive samples was significantly lower than that in negative samples(P<0.001).2.After infection with H.pylori,the expression of LC3 Ⅱ in the cells increased and the expression of β1-integrin decreased.After 12 hours of co-culture with H.pylori,the results of western blot showed that the expression of LC3 Ⅱ was significantly increased(P<0.01),and the expression of LC3 Ⅱ was significantly increased(P<0.01).3.β1-integrin reduced the expression of LC3 Ⅱ,Atg5-Atg12,Atg16L1 and inhibited the formation of autophagosomes.After co-culture of H.pylori with different experimental groups of GES-1 for 12 h,Western blot showed that the expression of LC3 Ⅱ,Atg5-Atg12 and Atg16L1 in silencing β1-integrin group was significantly higher than that in the negative control group(P<0.05).The expression of Beclin-1,Atg7 and Atg3,was not statistically significant compared with the negative control group;the expression of LC3 Ⅱ,Atg5-Atg12 and Atg16L1 in the blank group was significantly increased(P<0.05),and the expression levels of Atg7,Atg3 and Beclin-1 were not statistically significant.Transmission electron microscopy showed that the number of autophagosomes in the silencing β1-integrin group was significantly higher than that in the negative control group(P<0.01);the number of autophagosomes in the blank group increased.The number of autophagosomes in the blank group was 1.73±1.05/cell,2.09±1.16/cell in the blank with H.pylori group,2.7±1.27/cell in the negative control group,3.88±2.15/cell in the negative with H.pylori group,and 3.45±1.83/cell in the silencedβ1-integrin group.The silenced β1-integrin group was 6.55 ± 3.06/cell.The results of laser confocal microscopy showed that the number of autophagosomes and autophagosomes in silencing β1-integrin group increased;the number of autophagosomes in the blank group increased.4.β1-integrin inhibits P62 degradation.After co-culture of H.pylorii with different experimental groups of GES-1 for 12 h,the results of Western blot showed that the expression of P62 in the silencedβ1-integrin group was significantly lower than that in the negative control group(P<0.01),and the expression of LC3 Ⅱ was significantly increased compared with the negative control group.(P<0.05).The expression of P62 in the blank group was significantly increased(P<0.05),and the expression level of LC3 Ⅱ was significantly increased(P<0.05).After treatment with autophagic flow inhibitor batroxine(bafA1),the expression of P62 and LC3 Ⅱ in the silenced β1-integrin group was significantly increased(P<0.05).5.β1-integrin promotes apoptosis induced by H.pylori.After H.pylorii and different experimental groups of GES-1 cells were co-cultured for 12 h,The apoptosis rate of AnnexinV-FITC/PI double staining method was 8.87±0.97%in the blank group,11.93±1.87%in the blank with H.pylori group,9.67± 1.49%in the negative control group,and 16.87± 1.79%in the negative control with H.pylori group.The silenced β1-integrin group was 9.13±0.94%,and the silenced β1-integrin with H.pylori group was 12.33±1.11%.The mitochondrial membrane potential(△Ψ)detected by JC-1 showed that the△Ψ in the silenced β1-integrin group was higher than that in the negative control group(P<0.05),and the △Ψ in the blank group was decreased(P<0.05),that is,the level of apoptosis in the silenced β1-integrin group was lower,and the level of apoptosis in the blank group was increased.The results of JC-1 detection of mitochondrial membrane potential showed that the blank group △Ψ was 3.80±1.2,the blank with H.pylori group was 1.66±0.27,the negative control group was 2.10±0.42,and the negative control with H.pyori group was 0.88±0.11.The silenced △Ψ1-integrin group was 2.44 ± 0.41,and the silenced β1-integrin with H.pylori group was 1.25±0.1.Western blot results showed that the ratio of cleaved-caspase3/pro-caspase3 and cleaved-PARP/PARP in the silenced β1-integrin group was lower than that in the negative control group(P<0.01),and in the blank group,cleaved-caspase3/pro-caspase3 and cleaved-PARP/PARP,the ratio was increased(p<0.05).6.After inhibition of autophagy,the level of apoptosis induced by H.pylori is increased.After autophagy inhibitor 3-methyladenosine(3-MA)was used to treat GES-1 cells in the control groups and H.pylori was co-cultured with GES-1 cells for 12 h.The results of Annexin V-FITC/PI double staining showed that the apoptotic rate of the treated with 3-MA group was higher than that of the non-treated 3-MA group.The apoptotic rate of the silenced β1-integrin group was lower than that of the negative control group(P<0.05),and the apoptosis rate of the blank group was increased(P<0.05).The apoptotic rate was 11.43±0.66%in the blank group,25.50±2.42%in the blank with H.pylori group,12.13±2.09%in the negative control group,39.27±4.20%in the negative control with H.pylori group,and 12.37±2.08%in the silencedβ1-integrin group.The silenced β1-integrin group with H.pylori group was 25.33±1.45%.The mitochondrial membrane potential(△Ψ)detected by JC-1 showed that the△Ψ of the 3-MA treatment group was lower than that of the 3-MA non-treatment group,and the apoptosis level was increased.The △Ψ in the silenced β1-integrin group was higher than that in the negative control group(P<0.05),and the △Ψ in the blank group was decreased(P<0.05).The apoptosis level of the silenced β1-integrin group was lower than that of the negative control group,and the apoptosis level of the blank group was increased.The results of JC-1 detection of mitochondrial membrane potential(△Ψ)showed that the blank group △Ψ was 2.04±0.56,the blank with H.pylori group was 0.45±0.19,the negative control group was 1.74±0.58,and the negative control with H.pylori group was 0.30±0.06.The silenced β1-integrin group was 1.15±0.71,and the silenced β1-integrin with H.pylori group was 0.50±0.02.7.After inhibit of autophagy,integrin β1 promotes H.pylori-induced apoptosis.The autophagy inhibitor 3-methyladenosine(3-MA)was treated with silencingβ1-integrin and negative control cells,and co-cultured with H.pylori for 12 h.Annexin V-FITC/PI double staining showed that apoptosis rate of β1-integrin group was lower than that of the negative control group(P<0.05).The negative control group was 12.37±2.08%,the negative control co-treated with H.p.ylori group was 25.5±2.42%,the silenced β1-integrin group was 39.27±4.2%,and the silencedβ1-integrin group with H.pylori group was 25.33±1.45%.The mitochondrial membrane potential(△Ψ)detected by JC-1 showed that the△Ψ in the silencing β1-integrin group was higher than that in the negative control group(P<0.05),that is,the level of apoptosis in the silencing β1-integrin group was lower than that in the negative control group.The mitochondrial membrane potential(△Ψ)detected by JC-1 showed 1.74±0.58 in the negative control group,0.3±0.06 in the negative control with H.pylori group,1.15±0.71 in the silencing β1-integrin group,and 0.5 ± 0.02 in the silent β1-integrin with H.pylori group.Western blot analysis showed that the ratio of cleaved-caspase3/pro-caspase3 and cleaved-PARP/pro-PARP in the silenced β1-integrin group was lower than that in the negative group(P<0.01),the level of apoptosis in the silenced β1-integrin group was lower than that in the negative group.ConclusionsHelicobacter pylori attenuates the inhibition of ATG5-ATG12 and ATG16L1 by reducing the expression of β1-integrin in gastric epithelial cells,promoting autophagy and inhibiting apoptosis.