Clinical And Mechanism Observation Of Recurrent Urinary Tract Infection In Postmenopausal Women Belonging To Liver Depression And Damp-heat Syndrome

Objective:1.Theoretical study on the treatment of rUTI in postmenopausal patients based on the theory of liver depression and damp heat.2.To evaluate the clinical efficacy and safety of Zhuolin Granules in the treatment of rUTI in postmenopausal patients.3.To observe the characteristics of urinary microbiota and fecal microbiota in postmenopausal patients with rUTI,and to invest the efficacy of Zhuolin Granules on urinary microbiota and fecal microbiota in postmenopausal women with rUTI.Methods:1.Clinical research:120 postmenopausal patients with rUTI were randomly divided into the treatment group and the control group,60 patients in each group.The control group were treated with oral antibiotics for 2 weeks.After remission,the control group was treated with low-dose antibiotics every night and continued for 12 weeks.The treatment group took Zhuolin Granules orally twice a day,200ml each time,after 4 weeks of oral administration,withdrawal for 2 weeks,2 weeks of medication,lweek of withdrawal,repeated 3 times.Both groups were treated for 14 weeks totally.The patients were followed up for 6-months after treatment.The clinical efficacy and adverse reactions were observed.2.Experimental study:16 postmenopausal patients with rUTI were randomly divided into the Chinese Medicine group and the Western Medicine group,8 patients in each group.Cases in the Western Medicine group were treated with oral sensitive antibiotics for 2 weeks,after remission,they were treated with small dose of antibiotics every night for 2 weeks.Cases in the Chinese Medicine group were treated with Zhuolin granules twice a day,200ml each time,for 4 weeks.Eight healthy volunteers were selected as the Healthy Control group.16S rDNA sequencing technology was used to detect the urine and fecal microbiota of postmenopausal patients with rUTI and healthy people,in order to explore the characteristics of urine and fecal microbiota between the rUTI group and the Healthy Control group,and also to evaluate the efficacy of Zhuolin Granules on urinary microbiota and fecal microbiota in postmenopausal women with rUTI.Results:1.Clinical research①After 14 weeks of treatment,the total effective rate of the treatment group was 96.77%,and that of the control group was 85%.The short-term clinical efficacy of the treatment group was significant better than that of the control group（P<0.01）.Six months after treatment,the complete cure rate of the treatment group was significantly higher than that of the control group（P<0.01）.During the follow-up period of 6 months after treatment,3 cases were lost in the control group,and 1 case in the treatment group.In the treatment group,5 cases recurred,while 28 cases recurred in the control group.②Tchinese medicine syndrome scores:At different observation points,the difference of the Tchinese medicine syndrome scores of the treatment group was significant statistically（P<0.01）,while the difference of Tchinese medicine syndrome scores in the control group only appeared after 14 weeks treatment.After 14 weeks of treatment and 6 months after treatment,the Tchinese medicine syndrome scores in the treatment group were significantly lower than that of the control group（P<0.01）.③Kaplan-Meier（KM）survival analysis showed that the rate of UTI relapse-free survival of the treatment group was significantly better than that of the control group（P<0.01）.④The incidence of adverse reactions in the treatment group was lower than that in the control group（3.33%vs 20%,P<0.01）.The main adverse reactions in the treatment group were gastrointestinal discomfort（3.33%）.The adverse reactions in the control group were gastrointestinal discomfort（10%）,elevated liver enzymes（1.67%）and leukopenia（8.33%）.There were no serious adverse reactions in both groups.2.Experimental study2.1 Urine microbiota analysis（1）OTU analysis.① The number of OTUs in the urine samples of postmenopausal patients with rUTI was higher than that of healthy controls,but the difference was not statistically significant（P>0.05）.② The number of OTUs in the urine samples of the chinese medicine group after treatment was lower than that before treatment,and the number of OTUs in the urine samples of the western medicine group after treatment was higher than that before treatment,but the differences were not statistically significant（P>0.05）.（2）Principal Component Analysis.① The distribution of the urine samples in the healthy control group was relatively concentrated in PCA diagram,indicating that the similarity of the components of the urine samples in the healthy control group was higher than that in the rUTI group.② The distribution of the urine samples in the chinese medicine group and the western medicine group after treatment were more concentrated than that before treatment,similar to that in the healthy control group,indicating that the urine microbiota of rUTI patients after treatment were close to that of the healthy control group.（3）Alpha diversity analysis.① The Chao index,ACE index and sobs index of the rUTI group were higher than those of the healthy control group,but there were no statistically significant differences（P>0.05）.The Shannon index and Simpson index of the rUTI group were lower than those of the healthy control group,but the differences were not statistically significant（P>0.05）.②The Chao index,ACE index,sobs index,Shannon index,and Simpson index were increased after treatment in the chinese medicine group.The Chao index,ACE index,sobs index,Shannon index,and Simpson index were decreased in the western medicine group.The differences were not statistically significant（P>0.05）.（4）Beta diversity analysis.① The urine microbiota of healthy people overlapped in the urine microbiota of rUTI patients on the PCoA diagram.Based on the measurement of weighted_UniFrac and unweighted_UniFrac distance,it was found that there were differences in the urine microbiota composition between the rUTI group and the healthy control group,with statistical significance（P<0.01）.② After treatment,the distributions of urine microbiota in the chinese medicine group and the western medicine group were more concentrated than that before treatment.（5）Species annotation and analysis.① On the level of phylum classification,the main composition of urine microbiota were Firmicutes,Proteobacteria,Bacteroidetes and Planctomycetes.The abundance of Spirochaetes in the rUTI group was significantly higher than that in the healthy control group（P<0.05）.The abundance of Firmicutes,Proteobacteria and Bacteroidetes increased,while the abundance of Planctomycetes,and the ratio of Firmicutes/Bacteroidetes decreased in the chinese medicine group.The abundance of Firmicutes,Proteobacteria and Planctomycetes increased,while the abundance of Bacteroidetes and the ratio of Firmicutes/Bacteroidetes decreased in the western medicine group.② On the level of class classification,the abundance of Spirochaetia was significantly higher than that in the healthy control group（P<0.05）.The abundance of Bacteroidia,Clostridia and Gammaproteobacteria increased,while the abundance of Planctomycetacia decreased in the chinese medicine group.The abundance of Coriobacteriia was significantly higher in the chinese medicine group after treatment than before treatment（P<0.05）.The abundance of Clostridia and Bacilli increased,while the abundance of Bacteroidia and Gammaproteobacteria decreased in the western medicine group.③ On the level of order classification,the abundance of Spirochaetales and Pasteurellales in the rUTI group were significantly higher than that in the healthy control group（P<0.05）.Compared with before treatment,the proportion of Bacteroidales,Clostridiales and Lactobacillales increased and the proportion of Enterobacteriales decreased in the chinese medicine group after treatment.The proportions of Bacteroidales and Enterobacteriales decreased,and the proportions of Clostridiales and Lactobacillales increased in the western medicine group after treatment compared before treatment.④On the level of family classification,the main composition of urine microbiota were Muribaculaceae,Ruminococcaceae,Lachnospiraceae and Enterobacteriaceae.The abundance of Spirochaetaceae,Veillonellaceae and Pasteurellaceae were significantly higher in the rUTI group than those in the healthy control group（P<0.05）.The abundance of Muribaculaceae and Ruminococcaceae increased,while the abundance of Enterobacteriaceae,Spirochaetaceae and Prevotellaceae decreased in the chinese medicine group after treatment.The abundance of Muribaculaceae,Enterobacteriaceae and Prevotellaceae decreased,while the abundance of Ruminococcaceae and Spirochaetaceae increased in the western medicine group after treatment.⑤On the level of genus classification,the main composition of urine microbiota were Klebsiella,Treponema₂ and Lactobacillus in the healthy control group,while in the rUTI group,the main composition were Klebsiella,Treponema₂ and p-1088-a5_gut_group.The abundance of Treponema₂,Ruminiclostridium₆ and Veillonellaceae_UCG-001 were significantly higher in the rUTI group than that in the healthy control group（P<0.05）.The abundance of Klebsiella,Treponema₂,p-1088-a5_gut_group and Ruminococcaceae_UCG-010 decreased after treatment both in the chinese medicine group and the western medicine group.The abundance of Lactobacillus decreased,while the abundance of Bifidobacterium increased after treatment in the chinese medicine group.The abundance of Lactobacillus increased,while the abundance of Bifidobacterium decreased after treatment in the western medicine group.The differences were not significantly statistically.⑥ On the level of species classification,in the healthy control group,the main composition of urine microbiota were Klebsiella_variicola,Xanthomonas_campestris and Mycoplasma_pulmonis,while the main composition were Klebsiella_variicola,Mycoplasma_pulmoni and Weissella_paramesenteroides in the rUTI group.After treatment,the proportions of Klebsiella_variicola,Xanthomonas_campestris and Mycoplasma_pulmonis decreased in the chinese medicine group,while the proportions of Klebsiella_variicola and Mycoplasma_pulmonis decreased,and the proportions of Xanthomonas_campestris increased in the western medicine group.2.2 Fecal microbiota analysis（1）OTU analysis.① The number of OTUs of the fecal samples in rUTI patients was less than that in the healthy control group,but the difference was not statistically significant.② After treatment,the number of OTUs of the fecal samples increased,and the shared OTUs with healthy control group decreased in the chinese medicine group.The number of OTUs of the fecal samples increased in the western medicine group,and the shared OTUs with healthy control group increased slightly.（2）Principal Component Analysis.① The distribution of fecal microbiota in the PCA diagram of the healthy control group was relatively concentrated,which showed that the similarity of the components of fecal samples in the healthy control group was higher than that in the rUTI group.② The fecal microbiota in the chinese medicine group was still relatively scattered after treatment,while the fecal microbiota in the western medicine group was more concentrated after treatment,similar to that of the healthy control group,indicated that the fecal microbiota in the western medicine group was close to the healthy control group.（3）Alpha diversity analysis.① The Chao index,ACE index,Shannon index and Simpson index of the rUTI group were less than those of the healthy control group,the sobs index of the rUTI group was significantly lower than that in the healthy control group（P<0.05）.②The Chao index,ACE index,sobs index,Shannon index and Simpson index decreased after treatment in the chinese medicine group,while all the index increased in the western medicine group.The differences were not statistically significant.（4）Beta diversity analysis.① There were cluster and overlap of the fecal microbiota between the rUTI group and the healthy control group.Based on the measurement of weighted_UniFrac distance,it was found that the fecal microbiota composition between the rUTI group and the healthy control group was different,which had statistical significance（P<0.01）.②After treatment,the fecal microbiota distributions of the chinese medicine group and the western medicine group were still different,and were far away from the healthy control group.（5）Species annotation and analysis.① On the level of phylum classification,the main composition of fecal microbiota in the healthy control group were Bacteroidetes,Firmicutes,Verrucomicrobia and Proteobacteria,while the main composition of fecal microbiota in the rUTI group were Bacteroidetes,Firmicutes,and Proteobacteria.The abundance of Bacteroidetes was significantly higher in the rUTI group than that in the healthy control group（P<0.05）.The ratio of F/B and the abundance of Verrucomicrobia were significantly lower in the rUTI group than that in the healthy control group（P<0.05）.After treatment,the abundance of Bacteroidetes decreased,Firmicutes,Proteobacteria and Verrucomicrobia increased,the ratio of F/B increased in the chinese medicine group.While the abundance of Bacteroidetes and Verrucomicrobia increased,Firmicutes and Proteobacteria and ratio of F/B decreased in the western medicine group.② On the level of class classification,the proportion of Bacteroidia was significantly higher in the rUTI group than that in the healthy control group.The proportion of Verrucomicrobiae,Bacilli,Clostridia and Oxyphotobacteria were significantly lower in the rUTI group than that in the healthy control group（P<0.05）.After treatment,the proportions of Bacteroidia,Negativicutes,Gammaproteobacteria increased,while the proportion of Clostridia decreased in the chinese medicine group.The proportions of Bacteroidia and Clostridia increased,while the proportion of Negativicutes decreased in the western medicine group.③On the level of order classification,the abundance of Bacteroidales was significantly higher in the rUTI group than that in the healthy control group（P<0.05）,while the abundance of Verrucomicrobiales,Lactobacillales and Clostridiales were significantly lower in the rUTI group than that in the healthy control group（P<0.05）.After treatment,the abundance of Bacteroidales,Selenomonadales,Enterobacteriales and Pasteurellales increased,while the abundance of Clostridiales decreased in the chinese medicine group.The abundance of Bacteroidales and Clostridiales increased,while the abundance of Selenomonadales and Enterobacteriales decreased in the western medicine group after treatment.④ On the level of family classification,the main composition of fecal microbiota were Bacteroidaceae,Ruminococcaceae and Lachnospiraceae both in the rUTI group and the healthy control group.The abundance of Bacteroidaceae and Streptococcaceae were significantly higher in the rUTI group than that in the healthy control group（P<0.01）.The abundance of Akkermansiaceae,Rikenellaceae and Family_XI were significantly higher in the rUTI group than that in the healthy control group（P<0.05）.After treatment,the abundance of Bacteroidaceae and Lachnospiraceae decreased,the abundance of Enterobacteriaceae increased in the chinese medicine group.The abundance of Ruminococcaceae and Rikenellaceae decreased significantly,the abundance of Veillonellaceae increased significantly in the chinese medicine group（P<0.05）.The abundance of Bacteroidaceae,Lachnospiraceae and Ruminococcaceae increased,while the abundance of Veillonellaceae and Enterobacteriaceae decreased after treatment in the western medicine group.⑤On the level of genus classification,the main composition of fecal microbiota in the healthy control group were Bacteroides,Akkermansia,Phascolarctobacterium and Dialister,while the main composition of fecal microbiota in the rUTI group were Bacteroides and Subdoligranulum.The abundance of Bacteroides was significantly higher in the rUTI group than that in the healthy control group（P<0.05）.The abundance of Akkermansia,Alistipes,Dialister and Streptococcus were significantly lower in the rUTI group than that in the healthy control group（P<0.05）.The abundance of Escherichia-Shigella and Bifidobacterium were higher in the rUTI group than that in the healthy control group,and the abundance of Lactobacillus was lower in the rUTI group than that in the healthy control group,but the differences were not statistically significantly.After treatment,the abundance of Bacteroides deseased,and the abundance of Subdoligranulum deseased significantly（P<0.05）in the chinese medicine group.The abundance of Ruminococcaceae_UCG-002 increased significantly after treatment in the western medicine group（P<0.05）.After treatment,the abundance of Lactobacillus and Bifidobacterium increased in the chinese medicine group,while the abundance of Lactobacillus decreased and the abundance of Bifidobacterium increased in the western medicine group,but the differences were not statistically significantly.⑥ On the level of species classification,the main composition of fecal microbiota were Bacteroides_vulgatus and Phascolarctobacterium_faecium both in the rUTI group and the healthy control group.The abundance of Bacteroides_stercoris_ATCC₄3183 was significantly higher in the rUTI group than that in the healthy control group（P<0.05）.After treatment,the abundance of Bacteroides_vulgatus increased,while the abundance of Phascolarctobacterium_faecium and Bacteroides_stercoris_ATCC₄3183 decreased in the chinese medicine group.After treatment,the abundance of Bacteroides_vulgatus decreased,while the abundance of Phascolarctobacterium_faecium and Bacteroides_stercoris_ATCC₄3183 increased in the western medicine group.Conclusion:1.Clinical research:Zhuolin Granules is effective and safe for rUTI in postmenopausal patients.It can improve the cure rate,reduce the recurrence rate,and has a certain long-term effect.2.Experimental study:There were significant differences in the characteristic of fecal microbiota between postmenopausal women with rUTI or without rUTI.The imbalance of fecal microbiota may be one of the pathogenesis of rUTI in postmenopausal women.After Zhuolin Granules and antibiotic treatment,the distribution of urine microbiota in rUTI population was more concentrated than that before treatment,and close to that in healthy population.Zhuolin Granules can increase the abundance of Firmicutes,Verrucomicrobia,and the ratio of Firmicutes/Bacteroidetes of fecal microbiota,increase the proportion of Bifidobacteria in the urine and fecal of rUTI population,increase the proportion of Lactobacillus in the fecal of rUTI population,but the differences were not statistically significant.