| Background: Alpiniae officinarum rhizome(AO),the dried rhizomes of the Alpinia officinarum Hance(Zingiberaceae),was first reported in the "Mingyi Bielu" and was listed as a Chinese herbal medicine with medium quality.It is recorded in the Chinese Pharmacopoeia.Its medicinal properties and flavour are pungent and hot.Its channel orientation belongs to the spleen and stomach.It has the traditional functions such as warming stomach and vomiting,reliefing cold and pain and has been used for treating heart and spleen cold pain,spleen deficiency and cold malaria.Modern pharmacological research showed that AO possess various bioactivities such as anti-inflammatory,anti-oxidation,anti-bacterial,anti-ulcer and protection of the gastric mucosa.Chinese herbal medicines such as Jiang Baiweiyan tablets,Liangfuxiangsu powder and Weiyanfang using AO as a monarch drug are widely used in clinical for treatment of chronic gastritis.Chronic gastritis is a non-erosive inflammatory change in the gastric mucosa.Histologically,it is characterized by significant inflammatory cell infiltration,abnormal epithelial proliferation,gastric atrophy,and scar formation.Helicobacter pylori(H.pylori)is the strongest known risk factor for chronic gastritis.Clinically,AO is often used for treatment of H.pylori-positive chronic gastritis,but its mechanism of action has not yet been elucidated.In this thesis,the multi-disciplinary research methods were used to screen the anti-H.pylori-associated gastritis active fraction of different solvent extracts of AO.In addition,the mechanism of anti-H.pylori-associated gastritis of the active fracition was investigated.Objective: To screen the active fraction of AO against H.pylori-associated gastritis,explore its mechanism of action,and provide a modern scientific basis for explaining the traditional efficacy of AO against gastric diseases and for clarifying the theory of traditional Chinese medicine.Methods:(1)Using the HPLC method recorded in Chinese Pharmacopoeia under item of AO(C18 column,mobile phase: methanol-0.2%phosphoric acid(55:45),detection wavelength: 266 nm,column temperature:25 °C)with the content of galangin as the index to selected the high-quality source AO medicinal materials.The systematic solvent extraction method was used to obtain the different solvent extractions of AO.(2)The cytotoxicity of different extractions on human gastric mucosal cells(GES-1)was assayed by MTT assay.The effect of different solvent extracts of AO on the IL-8 level of GES-1 gastritis cells induced by H.pylori was detected by ELISA to screen the active fraction of AO against H.pylori-associated gastritis.(3)An H.pylori associated mouse gastritis model was constructed firstly.The effects of active fraction on the behaviors and gastric acid environment of mice with H.pylori-associated gastritis were observed.The inhibitory effect of active fraction on H.pylori was tested by urease assay.The morphology of H.pylori-associated gastritis mice was observed by HE staining.The effects of active fraction on the expression of IL-8 in gastric tissue of mice with H.pylori-associated gastritis was studied by immunohistochemical technique.Through above studies to investigate the pharmacodynamics of AO against H.pylori-associated gastritis.(4)The expression levels of inflammatory cytokines of IL-1?,IL-1ra,IL-17 and TNF-? in serum were detected by ELISA.The expression of p65 protein in gastric tissue of mice withH.pylori-associated gastritis was detected by immunohistochemistry.Western blot analysis was used to detect the expression levels of NF-?B p65,IKK?,IKK?,I?B? and MAPK p38,ERK1/2 and JNK in gastric tissue of mice.By doing above assay,to explore the mechanism of anti-H.pylori-associated gastritis of active fraction.Results: The HPLC quantitative results showed that the material from Guangdong with batch number of B505071 has the highest galangin content of14.17 mg/g and it was extracted by systematic solvent method to obtain total extract,petroleum ether extracts,dichloromethane extracts,ethyl acetate extracts,n-butanol extracts and water soluble extracts.The results of MTT assay showed that all six extract fractions had no cytotoxicity on GES-1 cells with the concentration less than 10 ?g/m L,and when the concentration was increased to 20 ?g/m L,the petroleum ether extracts and dichloromethane extracts showed cytotoxicity against GES-1cells,while the ethyl acetate extraction(EAE)and n-butanol extracts are still no cytotoxic with concentration up to 160 ?g/m L.The results of ELISA showed that H.pylori induced a significant increase in the expression of IL-8in GES-1 cells,which was significantly different from the control group(P <0.01).GES-1 cells was were treated with different solvent extracts of AO at 4?g/m L for 12 h and the results showed that the release of IL-8 from GES-1cells induced by H.pylori infection was significantly inhibited by the petroleum ether extraction,dichloromethane extraction and EAE.pharmacodynamic studies in vivo showed that H.pylori infection significantly inhibited the weight of mice with gastritis,there was a significant difference compared with the control group(P < 0.01);after oral administration of EAE for 21 days,the mice gained weight and there was a significant difference compared with the model group(P < 0.05).H.pylori infection significantly reduced the gastric weight coefficient of mice to 0.591,compared with the control group,there was a significant difference(P < 0.05).After intragastric administration of high does of EAE,the gastric weight coefficient of mice increased to 0.613,there was a significant difference(P <0.05)compared with the model group.H.pylori infection increased the p H of the gastric juice in mice to 3.4,compared with the control group,there was a significant difference(P < 0.01).After given medium and high doses of EAE,the p H of the gastric juice of mice decreased to 2.5 and 2.2,respectively,compared with the model group,there were significant difference(P < 0.05,P< 0.01).The H.pylori infection rate in the stomach of mice in the model group was 100%.Compared with the control group,there was a significant difference(P < 0.01).The medium and high doses of EAE reduce the H.pylori infection rate of mice to 50% and 30%,respectively,compared with the model group,there was a significant difference(P < 0.05,P < 0.01).H.pylori infection caused inflammatory lesions in the stomach tissue of mice,and significantly up-regulated the expression of IL-8 in gastric tissues.The AO EAE reduced the inflammatory lesions of gastric tissue and inhibited the expression of IL-8 in gastric tissues in a dose-dependent manner.The mechanism studies on anti-H.pylori-associated gastritis showed that the EAE significantly reduced the expression of inflammatory factors of IL-1?,TNF-? and IL-17 in the serum of H.pylori-associated gastritis mice and promoted the expression of IL-1ra in a dose-dependent manner,compared with the model group,there was a significant difference(P < 0.05,P < 0.01.The expression of NF-?B pathway and MAPK pathway-related proteins was detected by Western blot.The EAE significantly down-regulated p65,p-p65,p-I?B?,IKK?,p-IKK?/?,p-p38,p-ERK1/2 and the expression level of p-JNK protein,compared with the model group,there was a significant difference(P< 0.05,P < 0.01).Conclusion: Ethyl acetate extraction of AO can effectively improve the weight loss and gastric p H rise of H.pylori-induced gastritis mice,restore gastric weight coefficient,reduce H.pylori infection rate,reduce gastric inflammatory lesions,and exerted the effect of anti-H.pylori-associated gastritis.The mechanism of action maybe as follows: 1.Inhibition of IKK-mediated I?B? phosphorylation by inhibiting H.pylori-induced IKK activation,thus inhibiting NF-?B p65 activation,finally inhibiting the production of downstream proteins of IL-1?,IL-8,IL-17,TNF-? and promoting the production of IL-1ra so that inhibits H.pylori-associated gastritis;2.The catalytic effect of activated MAPK on NF-?B phosphorylation is inhibited by inhibiting the activation of MAPK and thus exerted the effect of anti-H.pylori-associated gastritis. |
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