| Objective:according to the preliminary experimental results show that Polygonum capitatum to chronic gastritis and gastric mucosal repair effect more obvious observed Polygonum capitatum can regulate gastrointestinal hormone secretion, maintain the normal gastrointestinal immune system function, suggesting that Polygonum capitatum in vivo in addition to the direct inhibition of H.pylori, and innate immune regulation and restoration has a close relationship. This experiment through the detection of H.pylori infection and after treatment of rats with the innate immune response is an important pattern recognition molecules of TLR4 and DC-SIGN protein levels, and immune response, gastrointestinal hormones(gas, SS mRNA expression changes to understand the effect of Polygonum capitatum Buch of H.pylori infection caused by immune response; conducive to further interpretation of the pyloric helicobacter pylori gastric mucosal injury of innate immune regulation mechanism, and then discusses mechanism of Polygonum capitatum anti H.pylori and adjust and repair, clinical research and treatment of H.pylori infection chronic gastritis and new drug to provide valuable experimental evidence.Methods:(1) H.pylori SS2000 culture: the H.pylori SS2000 evenly coated on the Columbia blood agar, 37℃three gas incubator culture 72 h, observation of bacterial growth, stable passage 3times after H.pylori identification.(2) Water ban fasting after liquid bacteria strains of H.pylori 109cfu/mL to blank feeding adaptability of H.pylori infection in SD rats of chronic gastritis animal model of SD rats after 1w, 96 male SPF SD mice were randomly divided into model group(70) and control group(16 rats) and drug group(16), all SD rats fasted for 12 h water, first of all, 5g / L NaHCO3 and indomethacin mixture according to the weight of male rats, female rats of 0.5ml pre treatment 1times; intragastric administration to fasted for water, 6 h after the model group wasgiven 1 x standard SS2000 1.5mL gavage for 4 hours the next day gavage, a total of five times, the blank group and drug control group was injected with the same amount of sterile brain heart infusion broth instead.(3) Infection animal model identification:at the end of the 8th week after the end of the gastric irrigation, fasting for 24 h,model group and blank control group were randomly selected from 6 SD rats were put to death, according to the standard of Lausanne, antral mucosa were stained by HE staining, observe the chronic inflammation of gastric mucosa in SD rats changed; on gastric mucosa in SD rats for rapid urease test, bacterial microaerophilic culture and improved W-S silver staining method for the detection of gastric mucosa H.pylori colonization. Model group SD rat gastric mucosa showed chronic inflammation and pathological changes and H.pylori colonization, confirmed the establishment of chronic gastritis animal model;(4) after drug treatment: the animal model was successfully made module is divided into 4 groups(16 rats / group) were Polygonum capitatum high, medium and low dose continuous treatment for 2 weeks, the model group not treated. The drug control group to high doses of Polygonum capitatum to normal SD rats, normal feeding group. Last gavage for 2 weeks were SD rats, remove the gastric mucosa of gastric antrum part, which is divided into 4 parts, a load in brain heart infusion broth save liquid, H.pylori microaerophilic culture; a rapid urease test;a sample into a protective liquid, frozen in the refrigerator –80℃ for RNA extraction;and one in tissue sections for HE staining and immunohistochemistry.(5) Polygonum capitatum effect evaluation: the clearance rate of H.pylori; HE staining was used to observe the gastric mucosal lymphocyte infiltration;(6) the best dosage as effect of Polygonum capitatum as treatment group, should be a important pattern recognition molecules DC-SIGN and TLR4 transcription and translation expression by immunohistochemistry and real-time PCR method for the detection of Polygonum capitatum treatment before and after rat innate immune;(7) using in situ hybridization method for the detection of GAS and SS m RNA expression changes.Results:(1)Identification of urease, catalase, oxidase, Gram staining confirmed that for H.pylori;(2)After inoculation with H.pylori 8W, the model identification, HE staining showed that the mice model of gastric mucosa showed chronic inflammatory changeof gastric mucosa, gastric mucosa H.pylori culture rapid urease positive Giemsa staining, H.pylori colonization, confirmed that the animal model of H.pylori infection of SD in chronic gastritis rats was successfully constructed;(3) The treatment effect of Polygonum capitatum: the gastric mucosa of H.pylori rats culture showed that after different dosages of Polygonum capitatum, H.pylori colonization density decreased to different extent, the high dose of Polygonum capitatum group(172.29 mg extract /mL)clearance rate was 91.573%, the dose group in Polygonum capitatum(57.43 mg extract /m L) clearance rate was 78.2000%, the low dose group(19.14 Polygonum capitatum Mg extract /mL) clearance rate was 76.327%; inflammation of Polygonum capitatum in different doses on the gastric mucosa to improve the situation is different.(4)The results of immunohistochemistry showed that TLR4 and DC-SIGN mainly expressed in epithelial cells of gastric mucosa in cytoplasm in the blank group.Epithelial cells constitute rules of glands, no expression in the model group, TLR4,DC-SIGN strong positive expression of H.pylori upregulated the expression of TLR4 and DC-SIGN, respectively, increased by 1.6 times and 3.2 times(P <0.01), by Polygonum capitatum after treatment, positive expression decreased, were down 1.33 times and 2.6 times(P < 0.01), Polygonum capitatum blank group compared with the control group, no statistical significance, P > 0.05;(5)real-time PCR showed that after H.pylori stimulation, the model group of TLR4 mRNA and DC-SIGN expression was significantly upregulated, were 2.7 times and 3 times(P<0.05), the head Polygonum capitatum role, TLR4 mRNA and DC-SIGN mRNA expression levels have obvious downward trend(P<0.05), 1.97 times, respectively, and 1.76 times.Blank group and drug control group no statistical difference;(6)The ISH showed that:gastrin is secreted mainly by antral G cells, somatostatin secreted by the gastric antral D cells, H.pylori infection after gas expression was significantly increased, the positive expression than the blank group, by scanning the gray value calculation of gas optical density values showed upregulation of 1.26 times(P<0.01), SS expression was significantly decreased, optical density values were significantly down regulated1.19 times(P<0.01), effect of Polygonum capitatum gas cut 1.12 times(P<0.05); SS expression slightly increased the level of, but not significant(P>0.05).Conclusion:(1)Polygonum capitatum high dose of H.pylori scavenging effect is the best, and the improvement of gastric mucosal inflammation effect is good;(2) Polygonum capitatum by pattern recognition receptors TLR regulation, DC-SIGN to balance the innate immunity;(3) Polygonum capitatum may regulate the function of gastrointestinal hormone balance. |
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