| Objective: The anti-inflammatory effects and the mechanisms of Polygonum capitatum(P.capitatum)to the clinical therapy of Helicobacter pylori(H.pylori)gastritis were investigated to provide theoretical and experimental basis.The animal model of H.pylori gastritis were established with SD rats.Investigations were made on gastritis inflammatory cytokine TNF-?,and the proteins and mRNA expression related to p38 mitogen-activated protein kinase(p38MAPK)signaling pathway.Methods:(1)A total of 48 specific-pathogen-free(SPF)SD rats were randomly divided into three groups: blank group,model group and P.capitatum group.(2)The rats in each group were pretreated with 2g/L NaHCO3.The rats in the model group and the P.capitatum group were treated with H.pylori SS2000 solution(1 × 109 cfu / mL),1.5mL/rat.The blank group received sterile Brain Heart Infusion Broth once every other day for total of 5 times,then they were fed normally for 8 weeks.Three rats were randomly selected from each group,and their gastric mucosa tissues were used for model identification and specimen extraction.(3)With reference of the Lausanne standards,the rapid urease test,gastric mucosal tissue micro-aerobic culture test,paraffin section with improved Warthin-Starry silver staining and HE staining were positive.These results have determined that the H.pylori gastritis model was constructed successfully.(4)After proven animal model was constructed successfully,the rats in the P.capitatum group were treated with P.capitatum solution with a concentration of 172.29 mg/mL.The administration of P.capitatum solution were made according to the clinical intragastric administration standards and based on the average body weight of the rats.The female rats received 3 mL while the male rats received 3.6 mL,once a day for 2 weeks.The rates blank group and model group were given equal volume of sterile ddH2 O.Four weeks after the last intragastric treatment,gastric mucosa tissues were extracted from all the rats and stored.(5)The techniques described in step 3 were used to determine the eradication rate of H.pylori gastritis and the improvement of gastric mucosa inflammation in SD rats.(6)Real Time PCR and Western-Blot were used to detect the expression of inflammatory cytokines(MKK3/6,p38,CREB,and TNF-? mRNA)and the respective phosphorylated proteins in p38 MAPK signaling pathway in the gastric mucosa.(7)The effects of P.capitatum on the localization of phosphorylated p38 protein and p38 protein in gastric mucosal cells were studied by using the Laser Scanning Confocal Microscopy.Results:(1)H.pylori gastritis SD rats were successfully constructed: the model group was positive for H.pylori rapid urease test.In the micro-aerobic culture,the model group showed a lot of moist,colorless,semi-transparent,and needle-like H.pylori small colony growth.The modified Warthin-Starry silver staining of the gastric mucosa sections showed distinct black rod-shaped,short rod and spherical H.pylori.The HE staining showed the gastric mucosa of the model group was very irregular;the tissues had obvious hyperemia and edema,and a lot of inflamed cells.(2)In the P.capitatum treatment group,the gastric mucosa of SD rats did not show erosion of the cells and the inflammation was improved,hyperemia was observed only in few areas,the amount of the inflammatory cells was significantly less than that of the model group,H.pylori was not found in the gastric mucosa.(3)Compare to the blank group,the model group showed an increase in MKK3,MKK6,p38 and TNF-? mRNA,by 1.392±0.121 times,2.211±0.520 times,2.152±0.384 times and 4.579±0.476 times(P<0.05),respectively.Compare to the model group,the treatment group showed a decrease in P-MKK3,MKK6,P-p38 and P-CREB,by 1.564±0.122 times,3.133±0.076 times 1.892±0.111 times and 1.906±0.062 times(P<0.05),respectively.Compared with the blank group,the CREB mRNA of the model group was 1.794 ± 0.141 times lower than that of the blank group.Compared with the model group,CREB mRNA of the treatment group was 1.245 ± 0.097 times higher than that of the model group.(4)Compared with the blank group,the model group showed an increase in phosphorylated MKK3/6,p38,CREB and TNF-? proteins by 1.859±0.024 times,2.532±0.042 times,3.695±0.034 times and 21.306±0.175 times(P<0.05),respectively.Compared with the model group,the treatment group showed a decrease in phosphorylated MKK3/6,p38,CREB and TNF-? proteins by 1.670±0.011 times,1.498±0.036 times,1.821±0.006 times and 2.791±0.013 times(P<0.05),respectively.(5)In the laser confocal microscopy,it was found that the phosphorylated p38 protein in the gastric mucosa of the model group was significantly higher than that in the blank group,and the red fluorescent signal of the p38 proteins closely match with the blue fluorescent of the nucleus.The treatment group displayed a significantly less amount of fluorescent signals of the p38 proteins than the blank group.In the model group,the p38 proteins with red fluorescence signal were distributed in patches-like pattern in the cytoplasm and nucleus.In the treatment group,the red fluorescent signals of the p38 proteins were significantly weakened and were not in the nucleus.Conclusion:(1)P.capitatum showed inhibition of MKK3/6,p38,CREB protein phosphorylation activation in the p38 MAPK signaling pathway.P.capitatum showed its ability of anti-H.pylori gastritis.(2)The phosphorylated p38 protein maybe the main target for the P.capitatum to act against H.pylori gastritis. |
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