Clinical Efficacy Evaluation Of Lianpu Yin Modified Decoction Combined With Quadruple Therapy In The Treatment Of Hp Associated Gastritis And Mechanism Study Of Its Correlation With Intestinal Microecology

Objective1.To investigate the current status of infection and drug resistance of Helicobacter pylori（Hp）in this area.2.To evaluate the clinical efficacy and safety of Lianpu Yin Modified Decoction combined with quadruple therapy in the treatment of Hp associated gastritis.3.To investigate the effects of Hp antibody typing on the tongue flora and analyze the characteristic bacteria;To investigate the effects of Hp antibody typing on intestinal flora and analyze the characteristic bacteria.4.To analyze the effects and characteristics of Hp eradication therapy on intestinal microecology;Based on intestinal microecology,to explore the mechanism of Lianpu Yin Modified Decoction.Methods1.Literature research: Combing and analyzing the status quo of Hp infection and drug resistance,pathogenesis,diagnosis and treatment plan,TCM etiology and pathogenesis,syndrome differentiation and treatment by referring to relevant domestic and foreign literature and the latest Hp diagnosis and treatment guidelines.2 clinical study :（1）A retrospective study was conducted to analyze the Hp infection status and TCM syndrome differentiation characteristics of16,549 patients with spleen and stomach diseases in Wuhan area from January2019 to November 2021.The current status of drug resistance of Hp strains isolated from clinical culture from January 2018 to April 2021 was analyzed and the comparative analysis of four time periods was conducted to dynamically understand the changing trend of Hp resistance in this area,so as to guide the selection of Hp eradication program.（2）76 patients who met the criteria were included and randomly assigned to the bismuth quadruple combination group（control group,n=38）,and Lianpu Yin Modified Decoction combined with bismuth compound group（treatment group,n=38）.The control group was given the standard dose of rabeprazole,amoxicillin,furazolidone and bismuth potassium citrate for 2 weeks;The treatment group was treated with Lianpu Yin Modified Decoction combined with bismuth quadruple therapy for 2 weeks,and then treated with Lianpu Yin Modified Decoction alone for another 2 weeks.The patients were re-examined at 2 weeks of treatment,2 weeks of drug withdrawal and 4 weeks of drug withdrawal.Hp eradication rate,main symptom efficacy,TCM syndrome efficacy and incidence of adverse reactions were observed and analyzed before and after treatment.The levels of Tumor necrosis factor-α（TNF-α）,interleukin-1（IL-1）,interleukin-6（IL-6）,Interleukin-8（IL-8）,interleukin-10（IL-10）,interleukin-17A（IL-17A）,interleukin-23（INTERleukin-23）,Il-23),Gastrin（GAS）,Motilin（MTL）,substance-P（Substance P）were detected by enzyme-linked immunosorbent assay（ELISA）.3.Experimental study :（1）Western blotting was used to detect A variety of Hp Ig G antibodies in serum of patients,including cytotoxin Cag A-116 KD antibody,vacuolating toxin Vac A（95KD antibody and 91 KD antibody）, Ure A-66 KD antibody and UREB-30 KD antibody.According to the antibody expression of Cag A and Vac A,Patients were classified as type 3,they were divided into type I:positive anti-cytotoxin-related gene A（Cag A）antibody and/or anti-vacuolar cytotoxin A（Vac A）antibody,whether positive anti-urease A antibody or anti-urease B antibody).Type II :Cag A negative and Vac A negative,with only Ure B positive and/or Ure A positive;Type III（negative）:with all negative indicators.The intestinal and tongue coating microbial communities of patients with type 3 were detected by 16 Sr DNA high-throughput sequencing.（2）In the clinical study,Feces samples of patients in the two treatment groups were collected before treatment（Week0）,2 weeks of drug withdrawal（Week2）,4 weeks of drug withdrawal（Week4）,8 weeks of drug withdrawal（Week 8）and 11 weeks of drug withdrawal（Week 11）.16 S r DNA sequencing technology was used to detect the changes of intestinal microbial community before and after treatment.Results1 Literature research :（1）Antibiotic resistance is a major clinical problem in Hp eradication therapy.（2）Syndrome differentiation and treatment under the mode of "disease-syndrome" combination is the basic principle of TCM treatment of HP-related diseases.（3）The diagnosis and treatment plan of TCM and Western medicine is consistent with China’s national conditions,and the implementation of individual-holistic treatment is the key to improve the eradication rate of Hp.2 Clinical research:（1）Part three and part four show that among 16549 patients,5544 were Hp positive and the infection rate was 33.50%.The Hp infection rates were 35.30%（1542/4368）,34.66%（1439/4152）and31.92%（2563/8029）in 2019,2020 and 2021,respectively.There was statistical difference between 2021（31.92%）and 2019 and 2020（P < 0.05）.The infection rate of Hp increased with age（P < 0.05）,and there was no significant difference between male and female Hp infection rates in different age groups（P > 0.05）.In Hp positive patients,the proportion of dampness-heat syndrome of spleen and stomach was 41.21%,the proportion of disharmony between liver and stomach was 21.4%,and the proportion of weakness of spleen and stomach was 12.17%.The drug resistance rates of Hp to clarithromycin,levofloxacin,amoxicillin,furazolidone,metronidazole and tetracycline were 35.78%,29.97%,4.89%,1.07%,73.39% and 0.76%,respectively.Hp strains were mainly resistant to single and double drug resistance,accounting for 67.43% of the total drug resistance,and metronidazole-clarithromycin resistance rate was 14.83%.From 2018 to 2020,the drug resistance rate of Hp to clarithromycin,levofloxacin and metronidazole increased year by year,the level in 2021 was lower than that in2020（P < 0.05）,and the drug resistance rate to levofloxacin alone increased year by year,from 0.88% to 6.98%（P < 0.05）.Triple drug resistance rate of Hp to metronidazole clarithromycin levofloxacin showed an increasing trend from 2018 to 2020（P < 0.05）.（2）Part five shows that comparison of Hp eradication rate between the two groups,Hp eradication rate in the control group was 84.85%（28/33）by PP analysis and 73.68%（28/38）by ITT analysis;Hp eradication rate was89.19%（33/37）in the treatment group（PP analysis）,and 86.84%（33/38）in the treatment group（ITT analysis）.There was no significant difference between the two groups（P >0.05）.After 2 weeks of treatment,2 weeks of drug withdrawal and 4 weeks of drug withdrawal,the total effective rate of Lianpu Yin modified Decoction group was significantly higher than that of bismuth quadruple combination group（P< 0.05）.The TCM syndrome score in Lianpu Yin modified Decoction group was significantly lower than that in bismuth quadruple combination group（P<0.05）.With the extension of time,Lianpu Yin modified Decoction group was better than bismuth quadruple combination group in alleviating clinical symptoms（P<0.05）.Lianpu Yin modified Decoction group was significantly better than bismuth quadruple combination group in improving the single symptoms of "sticky mouth and bitter","heavy limbs","bad breath" and "abdominal fullness"（P<0.05）.After treatment,the serum levels of IL-6,IL-8,IL-1β,TNF-A and IL-17 A were decreased（P<0.05）,while the levels of IL-10 and IL-23 were increased（P<0.05）,SP and GAS levels decreased（P<0.05）,while MLT levels increased（P<0.05）.The incidence of adverse reactions was 9.1% in the control group and 5.4% in the treatment group.There was no significant difference between the two groups（P>0.05）.3 Experimental study :（1）The results of 16 Sr DNA high-throughput sequencing showed that the value of type II index was the lowest and that of type III index was the highest on Chao index.On Shannon index,the value of type I index is the smallest,type III index is the largest,and type II is in the middle.β diversity analysis and species composition analysis showed that there were differences in the composition of liverwort community among different types of tongue.There was no difference in the relative abundance of the three types of tongue coatings at class level,order level,family level and genus level（P > 0.05）.At phylum level,the relative abundance of Firmicutes III was higher than that of Firmicutes I and II（P<0.05）.The relative abundance of Proteobacteria of type I was higher than that of Type II and III（P<0.05）.Analysis of three types of differential bacteria,Proteobacteria,Gammaproteobacteria,Burkholderiales,Neisseriaceae,Neisseria,Ultured bacterium and Prevotella_shahii were found in type I community and the abundance of them increased,and the abundance of Firmicutes in type III community increased（P<0.05）.The results of functional prediction showed that the relative enrichment of terpenoids in amino acid metabolism,carbohydrate metabolism,energy metabolism,cofactor metabolism and vitamin metabolism increased among the three types.The results of intestinal flora detection by 16 Sr DNA high-throughput sequencing showed that compared with the diversity of α,on Chao index,the value of type II index was the smallest and that of type III index was the largest;on Shannon index,the value of type I index was the largest and that of type II index was the smallest.β diversity analysis and species composition analysis showed that there were differences in the composition of liverwort community among different types of tongue.At phylum level,there was no difference between type I and type II microorganisms（P > 0.05）.The relative abundance of Patescibacteria in II was higher than that in III（P < 0.05）.At the genus level,there were no differences among the three types of intestinal microbial species（P>0.05）.The relative abundance of Bacteroidia,Bacteroidales and Eubacterium-nodatum-group in type III was higher than that in type I（P < 0.05）.The results of functional prediction showed that the relative enrichment of the three types was increased with the pathways of amino acid metabolism,sugar metabolism,lipid metabolism,terpenoid metabolism,and biosynthesis and metabolism of polysaccharides（P < 0.05）.（2）The results showed that the α diversity index（Chao,Shannon,Simpson index）of bismuth quadruple combination group decreased significantly at W2 compared with W0.The α diversity index（Chao,Shannon and Simpson index）of intestinal flora at W4,W8 and W11 was higher than that at W2.Diversity analysis and species composition analysis showed that the composition of intestinal flora was different in each group.Compared with the differences before treatment,At genus level,compared with W0,The abundance of beneficial bacteria Ruminococcus,Dorea and Eubacterium decreased 2 weeks after drug withdrawal（P < 0.05）,4 weeks after drug withdrawal,The abundance of beneficial bacteria Eubacterium,Agathobacter,Fusicatenibacter,Erysipelotrichace AE-UCG-003 and Neisseria were lower than before treatment（P<0.05）.The abundance of beneficial bacteria Roseburia and Lachnospiraceae increased at W8（P < 0.05）,and the abundance of Monoglobus increased at W11（P < 0.05）.The Chao index of Lianpu Yin Modified Decoction group was higher at 2and 4 weeks after eradication than before（P <0.05）.The β diversity analysis and species composition analysis of showed that there were differences in the composition of intestinal flora in each group.At phylum level,the relative abundance of Firmicutes decreased at 2 weeks after eradication compared with W0 before eradication.The relative abundance of Bacteroidetes increased（P < 0.05）,and the ratio of B: F increased gradually.In addition,beneficial bacteria Clostridium,Megasphaera,Dorea and Megamonas were significantly increased compared with those before eradication（P<0.05）.The value of Subdoligranulum decreased at W2 and was significantly lower than W0 at W11（P<0.05）.Comparative analysis of changes in intestinal flora of the two sets of scheme at the same time node,At the genus level,2 weeks after drug withdrawal,The relative abundance of beneficial bacteria,such as Lachnospira,Eubacterium and Clostridium,in Lianpu Yin modified Decoction group was significantly higher than that in bismuth quadruple combination group（P < 0.05）.At 4 weeks after drug withdrawal,the relative abundance of beneficial pili spironella Lachnospiraceae-ND3007-group and Lachnospiraceae-NK4A136 group was higher than that of bismuth quadruple（P<0.05）.The contents of Subdoligranulum,Collinsella and Coprococcus in bismuth quadruple combination group were significantly higher than those in Lianpu Yin modified Decoction group at W8（P < 0.05）.Comparative analysis of B: F value before and after the eradication of the two sets of scheme showed that B: F value after the bismuth quadruple treatment was higher than that before the eradication,but the range was smaller,and B: F value decreases at W8.The B: F value of Lianpu Yin modified Decoction group increased gradually with the extension of time,and was higher than that of bismuth quadruple combination group at W4.Conclusions1.Antibiotic resistance is a major clinical problem in Hp eradication therapy.Syndrome differentiation and treatment under the mode of "disease-syndrome" combination is the basic principle of TCM treatment of Hp-related diseases.2.The infection rate of Hp in Wuhan showed a decreasing trend in recent 3years and was lower than the national average.Dampness-heat syndrome of spleen and stomach was the primary syndrome type of Hp infection.The infection rate of Hp increased with the increase of age,but there was no correlation between Hp infection and sex.The drug resistance rates of Hp to clarithromycin,levofloxacin and metronidazole were high in this area.The drug resistance of amoxicillin,furazolidone and tetracycline was low.3.Lianpu Yin Modified Decoction combined with quadruple therapy to eradicate Hp and significantly improve the clinical symptoms of Hp infection.In the improvement of "sticky and bitter mouth","limbs sleepy heavy","halitosis","abdominal fullness" single symptom efficacy and with the extension of time,its relief of clinical symptoms highlighted advantages.4.Different antibody types of Hp affect the diversity of tongue and intestinal flora,which was correlated with virulence.The diversity of liverwort community of type III was significantly higher than that of type I and type II.Type II had the lowest diversity of intestinal flora,which might be related to Patescibacteria.Type III intestinal flora is more beneficial bacteria,and type I intestinal flora is less beneficial bacteria.5.The potential characteristic bacteria of type I are Proteobacteria,Gammaproteobacteria,Burkholderiales,Neisseriaceae and Neisseria uncultured-bacterium,Prevotella-shahii;Type III was Firmicutes.No characteristic bacteria of tongue coating were found in type II.The characteristic bacteria of type III intestinal flora were Bacteroidia,Bacteroidales and Eubacterium-nodatum-group.6.Lianpu Yin Modified Decoction has certain advantages in maintaining the diversity of intestinal flora.may promote the growth of beneficial bacteria,reduce harmful bacteria,adjust the proportion of Bacteroidetes and Firmicutes,improve B: F value to adjust the imbalance of intestinal flora,significantly relieve gastrointestinal symptoms,and play its dominant role.