Clinical Observation On The Effect Of Hp Infection On Gastric Microbiota And Mechanism Research Of Lian-Pu-Yin On Hp-Related Gastritis With Spleen And Stomach Damp-Heat Syndrome Model Rats

Objective1 To explore the current research status of Helicobacter pylori(Hp)-related gastritis.2 To explore the relationship between the pathological state of the body,the changes of gastric mucosal flora,and the inflammatory microenvironment after Hp infection,to reveal the mechanism of the formation of Hp-related gastritis with spleen and stomach damp-heat syndrome.3 To explore the mechanism of Lianpu Yin(LPY)in the treatment of Hp-related gastritis with spleen and stomach damp-heat syndrome.(1)To establish a rat model of Hp-related gastritis with spleen and stomach damp-heat syndrome,and evaluate the model.(2)To observe the effects of LPY on the apoptosis of gastric mucosa,immune cell differentiation,and related cytokine expression in model rats,confirm that LPY can regulate immune cell differentiation and related cytokine,improve cell apoptosis and play a protective role in mucosal membranes.Methods1 Theoretical discussion:By consulting relevant literature,analyzing the pathological mechanism of Hp-related gastritis,the etiology and pathogenesis of Chinese medicine,the distribution of syndromes,and the current status of treatment.2 Clinical research:Divide chronic gastritis patients into Hp infection with spleen and stomach damp-heat syndrome group(Hp⁺PWSR,n=17),non-Hp infection with spleen and stomach damp-heat syndrome group(Hp^-PWSR,n=11),and non-Hp infection with spleen and stomach weakness syndrome group(Hp^-PWXR,n=14).Endoscopic observation of gastric mucosa-related pathology in each group.Hematoxylin-eosin(HE)staining was used to observe the morphological characteristics of gastric mucosa.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of interferon-?(IFN-?),tumor necrosis factor-?(TNF-?),interleukin1?(IL-1?),interleukin 4(IL-4),interleukin 6(IL-6),interleukin8(IL-8),interleukin 10(IL-10),interleukin 12(IL-12),interleukin 17a(IL-17a),interleukin 18(IL-18),transforming growth factor-?(TGF-?),interleukin 21(IL-21),CXC chemokine ligand 12(CXCL12)in the serum of each group.Immunohistochemistry(IHC)was used to detect the expression of CD4⁺T cells and CD8⁺T cells in gastric mucosal tissue.16S r DNA high-throughput sequencing was used to detect gastric mucosal flora.3 Experimental study:(1)High-sugar and high-fat diet,56°ethanol gavage,artificial climate box to create damp and heat external environment,combined with Hp bacterial liquid gavage,construct a rat model of Hp-related gastritis with spleen and stomach damp-heat syndrome.The model is evaluated by the general conditions of rats,the colonization of Hp in the gastric mucosa,and gastric mucosal tissue morphology of rats in each group.(2)60 Wistar rats were randomly divided into normal control group(NC,n=10),Hp-related gastritis spleen and stomach damp-heat syndrome model group(MD,n=10),and LPY low-dose group(LPYL,n=10),LPY medium-dose group(LPYM,n=10),LPY high-dose group(LPYH,n=10),and quadruple therapy group(n=10).LPYH,LPYM and LPYL groups were given 15,7.5,3 g·kg^-1·d^-1 by gavage,and quadruple therapy group was given omeprazole(2 mg·kg^-1)+amoxicillin(100 mg·kg^-1)+clarithromycin(50 mg·kg^-1)+colloidal pectin capsule(35 mg·kg^-1)by gavage,2 times/d.NC was given the equal volume of normal saline for 21 consecutive days.Western blot was used to detect Caspase-3,B-cell lymphoma-2 associated X(Bax),B-cell lymphoma-2(Bcl-2)protein expression in the gastric mucosa of rats in each group.IHC was used to detect Bax protein expression.Flow cytometry was used to detect helper T cell 1(Th1),Th2,Th17,and regulatory T cell(Treg)differentiation.ELISA was used to detect the expression of IFN-?,TNF-?,IL-4,IL-10,IL-17a,TGF-?in the serum of each group.Results1 Theoretical discussion:(1)After Hp infection,the imbalance of the flora and immunity lead to gastric diseases.(2)The Chinese medicine syndrome types of Hp-related gastritis are mainly divided into spleen and stomach damp-heat syndrome,spleen and stomach weakness(cold)syndrome,and cold-heat complex syndromes,of which spleen-stomach damp-heat syndrome is a common syndrome type.(3)LPY can improve clinical symptoms,increase Hp eradication rate,and reduce adverse reactions through multiple channels and multiple targets.2 Clinical research:(1)Observation under endoscopy showed that The proportion of Hp⁺PWSR with gastric mucosal congestion and edema,erythema,bleeding spots,rough mucosa,erosion,and increased mucus were significantly higher than that of Hp^-PWXR(P<0.05),gastric mucosal rough was significantly higher than that of Hp^-PWSR(P<0.05).The proportion of Hp^-PWSR with gastric mucosal congestion and edema,erythema,erosion,and increased mucus was significantly higher than that of Hp^-PWXR(P<0.05).(2)HE staining showed that gastric mucosal tissue damage,inflammatory cell infiltration,and inflammatory activity were the heaviest in the Hp+PWSR group.(3)IHC results showed that the expressions of CD4⁺T cells and CD8⁺T cells in gastric mucosa of Hp⁺PWSR patients were significantly higher than those of Hp^-PWXR(P<0.05).(4)The results of ELISA showed that the expression of IFN-??TNF-??IL-1??IL-8?IL-12?IL-21?IL-17a levels of Hp⁺PWSR patients were significantly higher than those of Hp^-PWXR(P<0.05),the expression of IL-4 and IL-10 were significantly lower than those of Hp^-PWXR(P<0.05).(5)16S r DNA high-throughput sequencing results showed that the Richness index,ACE index,Chao1 index,Shannon index of Hp⁺PWSR were significantly lower than those of Hp^-PWSR and Hp^-PWXR(P<0.05).?diversity analysis and species composition analysis showed that there are differences in the composition of the flora of each group.The PICRUSt function prediction results showed that the gastric mucosal flora of patients with Hp infection is involved in the metabolism of cofactors and vitamins,lipid metabolism,ubiquinone and other terpenoids-quinone biosythesis,adipocytokine signaling,xylene degradation,mineral absorption,retinol metabolism are weaker than those of non-Hp infection patients(P<0.05),replication,recombination and repair proteins,glutamatergic synapse,cytochrome P450,novobiocin biosynthesis,bacterial secretion system,fatty acid biosynthesis were stronger than those of non-Hp infection patients(P<0.05).3 Experimental study:(1)Rats in MD group gained slower body weight,reduced mobility,liked to curl up,had damp coats,prolapsed anus,slowed reactions,sluggish eyesight,thin stools,and smelly odor.The results of rapid urease test and Giemsa staining showed that the colonization rate of Hp in gastric mucosa was 90%.HE staining showed a large number of inflammatory cell infiltration and tissue damage in the gastric mucosal tissue of the MD group.(2)(1)HE staining results showed that the congestion and swelling,gland arrangement and inflammatory cell infiltration of the gastric mucosa of rats in the LPYH,LPYM and LPYL groups were improved,and the improvement was most obvious in the LPYH group.(2)Western Blot results showed that,compared with the NC group,the expression of Caspase-3 and Bax protein in the gastric mucosa tissue of the MD group were significantly increased(P<0.05),and the expression of Bcl-2 protein was significantly decreased(P<0.05).Compared with the MD group,the expression of Caspase-3 and Bax protein in the quadruple therapy group and LPYH group were significantly reduced(P<0.05),and the expression of Bcl-2 protein was significantly increased(P<0.05);the expression of Bcl-2 protein in the LPYL and LPYM groups was significantly increased(P<0.05).(3)IHC results showed that Bax protein is expressed in the cell cytoplasm,and the protein expression trend is consistent with Western Blot.(4)The results of flow cell technique showed that compared with the NC group,the expression of Th1,Th2,Th17,Treg,and ratio of Th1/Th2,Th17/Treg were increased significantly in the MD group(P<0.05).Compared with the MD group,the expression of Th1,Th2,Th17,and the ratio of Th1/Th2,Th17/Treg were decreased in the quadruple therapy and LPYH groups(P<0.05).(5)ELISA results showed that,compared with the NC group,the levels of IFN-?,TNF-?,and IL-17a of the MD group were increased(P<0.05),and the levels of IL-4,IL-10,and TGF-?were decreased(P<0.05).Compared with the MD group,the levels of IFN-?,TNF-?,and IL-17a in the quadruple therapy and LPYH groups were significantly reduced(P<0.05),the levels of IL-4,IL-10 and TGF-?were increased(P<0.05);the levels of IFN-?,TNF-?,and IL-17a in the LPYM group were significantly reduced(P<0.05),IL-4 and IL-10 levels were increased significantly(P<0.05);the levels of TNF-?and IL-17a in the gastric mucosa of rats in LPYL group were significantly reduced(P<0.05),IL-4,IL-10 levels were significantly increased(P<0.05).Conclusions1 Spleen and stomach damp-heat syndrome is the most common Chinese medicine syndrome of Hp-related gastritis.LPY has a definite treatment effect.In-depth study of the mechanism of LPY will help provide new ideas for the treatment of Hp-related gastritis.2 The appearance of pathological changes of Hp-related gastritis with spleen and stomach damp-heat syndrome is related to the structure and function of the host gastric mucosal flora change,the body's immune response is unbalanced,the inflammatory state is enhanced,and the mucosal pathological damage is aggravated.3 Compound factor intervention can successfully construct a rat model of Hp-related gastritis with spleen and stomach damp-heat syndrome.LPY can improve cell apoptosis by regulating the differentiation of immune cells and the secretion of related cytokines,and exert a protective effect on the gastric mucosa of model rats.