| Objective: SD rats model were induced for investigating the relation between Helicobacter pylori gastritis and NF-kappa B signaling pathway,and specific blockers of different sites of NF-kappa B signaling pathway were used for finding the effective point of Polygonum capitatum,which can offord experimental basis for the treatment of Helicobacter pylori gastritis and the development of new drugs.Methods:(1)Helicobacter pylori gastritis rats model were induced by gavage method,and all rats were randomly divided into blank control group(n=16)and experimental group(n=32).The experimental group was given intragastric administration of 1.5mL of H.pylori of 1×109cfu/mL.The blank group was replaced by the same amount of sterile brain heart infusion broth.At the end of time after gavage of 8w,two groups were randomly selected from 3 SD rats,which were sacrificed,then to observe the colonization of H.pylori,by gastric urease test in rats,microaerophilic culture,Warthin-Starry silver staining method and HE staining of gastric mucosal inflammation,according to Lausanne standards to identify the animal model of Helicobacter pylori gastritis.(2)dividing the experimental group SD rats into model group and Polygonum capitatum gruop.Polygonum capitatum group According to the weight of intragastric 1.723g/kg·d,model group and blank group were treated with Sterile deionized water,lasting gavage 2w.After the last time,feeding 1mon normally.(3)Using Real-Time PCR assay the effect of Polygonum capitatum on gene expression of IκKβ,IκBα,NF-kappa B/p65 and TNF-alpha of NF-kappa B signaling pathway in Helicobacter pylori gastritis rats,and using Western Blotting assay the effect of Polygonum capitatum on protein of IκKβ,p-IκBα,IκBα,cytoplasmic and nuclear NF-kappa B/p65 and TNF-alpha.(4)Cells model of Helicobacter pylori infection were induced by H.pylori SS2000 and immortalized human gastric epithelial cells(GES-1)to culture of 12 h with a ratio of 100:1,then taking the supernatant to do micro aerobic culture and test proinflammatory factor TNF-alpha,and observing the changes of ultrastructure of GES-1 cells by electron microscope.(5)MTT assay was used to observe the effect of Polygonum capitatum on proliferation of Helicobacter pylori gastritis cells,setting 0-640 μg/mL concentration(7 concentration)of Polygonum capitatum,which were used to cultured with Helicobacter pylori gastritis cells 48 h respectively,the highest concentration had no significant inhibitory effect on the cells for subsequent experiments.(6)The GES-1 cells were divided into control group,Polygonum capitatum blank group and experimental group.The experimental group includes model group,NF-kappa B inhibitor group(parthenolide,Bay11-7082,PDTC,SN50 four inhibitor control group),Polygonum capitatum + inhibitor group,four inhibitor combined group,Polygonum capitatum + four inhibitor combination group.The experimental group was infected with H.pylori,then each group was given corresponding drug intervention with 48 h.Western Blotting,Real-Time PCR were taken to detect expression of IκKβ,p-IκBα,IκBα,cytoplasmic and nuclear NF-kappa B/p65 and downstream factor TNF-alpha,which to explore the mechanism of Polygonum capitatum effect on NF-B signaling pathway.Results:(1)After 8W with H.pylori infection,all the tests were positive.These results showed that Helicobacter pylori gastritis animal model was successful.(2)HE staining showed that compared with the model group,inflammatory cell infiltration was significantly reduced in Polygonum capitatum group,which suggesting that Polygonum capitatum can improve gastric mucosal inflammation caused by H.pylori infection.(3)qRT-PCR experiment showed that SD rats after H.pylori infection,gastric mucosa IκKβ,TNF-α mRNA relative expression than blank group were raised 9.346 ±0.927 and 4.579±0.480 times(P<0.05).Making treatment by Polygonum capitatum,IκKβ and TNF-α mRNA relative expression then model group were reduced 13.333±0.721 and 1.906±0.067times(P<0.05).(4)Western Blot experiment showed that after H.pylori infection,rat gastric mucosal protein IκKβ,p-IκBα,Cytoplasmic NF-κB/p65,TNF-α and nuclear protein NF-κB/p65 than blank group were raised 1.868±0.037,3.256±0.026,2.127±0.020,21.306±0.175 and 45.089±0.030 times(P<0.05).Making treatment by Polygonum capitatum,cytoplasmic NF-κB/p65 than model group was increased 1.740±0.025 times(P<0.05),nuclear NF-κB/p65 and TNF-α were decreased 10.213±0.001 and 2.791±0.013 times(P<0.05).These suggested that Polygonum capitatum could inhibit the NF-κB/p65 nuclear transfer,in order to reduce inflammation.(5)After12 hours H.pylori infection on GES-1 cells,the level of TNF-α was 1.980±0.594 times higher than that in the blank group(P<0.05).The morphology of cells was observed by optical microscope,which showed that cells in the blank group were spindle shaped,the surface was smooth and the particles were few.However when they were infected by H.pylori,most of the cells were elongated,the surface was not smooth,and the particulate matter increased significantly.Under the scanning electron microscope,it was found that the cells in the model group were long and thin.It was found that the H.pylori adhered to the cell surface.Transmission electron microscopy showed H.pylori infected cell surface villi lower than that in the blank group,many pseudopodia in cytoplasm swelling,autop Helicobacter pylori gastritis osomes were lower than that in the blank group.(6)The results of MTT colorimetric assay showed the proliferation of GES-1 cells infected with H.pylori could be significantly inhibited when the concentration was higher than 80 g/mL.(7)qRT-PCR showed that IκKβ and TNF-α of model group were 3.953±0.069 and 2.114±0.159 times higher than that of the blank group(P<0.05);Compared with the model group,the IκKβ,TNF-α mRNA in Polygonum capitatum group were down 3.831±0.159,1.757±0.062 times(P<0.05),which in PDTC group and SN50 group had no obvious change,but which in Polygonum capitatum+PDTC group were down 4.167±0.001、1.923±0.028 times(P<0.05),and in Polygonum capitatum+SN50 group wer e down 4.255±0.005、2.611±0.055 times.(8)Western Blot results showed that that p-IκBα,cytoplasmic NF-κB/p65,nuclear NF-κB/p65 and TNF-α protein in model group were higher 3.953±0.069 and 2.114±0.159 times than in blank group(P<0.05);Compared with the model group,p-IκBα was down1.983±0.096 times,IκBα and cytoplasmic NF-κB/p65 was up 1.747±0.092 and 2.199±0.153 times,however nuclear NF-κB/p65 and TNF-α were down 2.295±0.007 and 13.216±0.992 times in Polygonum capitatum group(P<0.05);p-IκBαand TNF-αin Polygonum capitatum +Bay11-7082 group was lower 1.547±0.040 and 8.947±0.015 times than in Bay11-7082 group(P < 0.05),but there were no significant difference than in Polygonum capitatum group。IκBαin Polygonum capitatum + Parthenolide group was higher 2.618±0.132 times than Parthenolide group(P<0.05),but there were no significant difference than in Polygonum capitatum group。p-IκBαin Polygonum capitatum+SN50 group was lower 2.010±0.051 times than in SN50 group(P<0.05),but there were no significant difference than in Polygonum capitatum group。Conclusion:(1)H.pylori infection can cause cell damage,decreasing villi and swelling of the body.(2)Polygonum capitatum down-regulation expression of IκKβ and TNF-α mRNA,and inhibit the phosphorylation of IκBα,reduce the degradation of IκBα,which inhibit the dissociation of IκBα and NF-κB,thus inhibit production of inflammatory factor effectively,and ease inflammation... |
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