An Exploratory Study On Gut Microbiota Of Patients With Atopic Dermatitis And The Effect Of PTQX Formulae On Intestinal Flora

Objective1.To investigate the characteristics of gut microbiota of patients with atopic dermatitis(AD)by comparing with healthy controls based on the 16 S rDNA gene sequence analyses.2.To evaluate the clinical efficacy and safety of PTQX formulae on patients with AD and explore the effects of PTQX formulae on intestinal flora.Methods1.Clinical investigations:45 patients with AD were recruited for this research.The dignosis of AD was made according to Hanifin&Rajka criteria.The clinical severity of the eczema was evaluated by SCORAD(SCORing Atopic Dermatitis),IGA(ivestigator's global assessment),and DLQI(dermatology life quality index),or CDLQI(children's dermatology life quality index).A questionnaire was completed.Based on the expert consensus of TCM diagnosis and treatment program of AD in 2013,the syndrome differentiation of AD patients was performed.A total of 39 healthy controls without atopic diseases were enrolled,and the intestinal flora questionnaires were filled in.Fecal samples were collected from patients with AD and healthy controls.2.Clinical trials: We started a double-blind,randomized,controlled study,26 cases of AD patients were randomly divided into treatment group(basic treatment + PTQX formulae)and control group(basic treatment + placebo),13 cases in each group.The patients were assigned to undergo a 12-week treatment,and clinical follow-ups were done at 0 weeks,4 weeks,8 weeks and 12 weeks of the intervention.On each occasion,the socres of IGA,SCORAD and DLQI/CDLQI and fecal samples collections were needed.3.Gut microbiota analysis: all collected fecal samples were sent to Wuhan high-throughput sequencing lab for the detection of bacterias.Combined with clinical data,the characteristics of gut microbiota were in analysis.Results1.Comparison of intestinal flora between patients with AD and healthy controls:(1)Baseline: the 45 cases of AD patients and 39 cases of healthy controls were all residents of Guangdong Province,and there were no statistical differences in gender and age between the two groups(P>0.05).(2)Assessment of the AD patients: the severity of the disease was mainly moderate,the IGA was 3(3,4),SCORAD was(46.63±13.99),and DLQI/CDLQI was(11.79±5.22).The TCM syndrome differentiations of patients were mainly the heart fire and spleen deficiency syndrome,which accounted for75.56%,followed by the spleen deficiency and wetness syndrome(20.0%),and the blood deficiency and wind-dryness syndrome(4.44%).(3)Gut microbiota analysis: 83 samples of qualified feces were collected,and divided into AD patients group(n=44)and healthy controls group(n=39).Comparison results:in terms of Alpha diversity index,there were no significant differences in observed species,chao index and ace index between the two groups(P>0.05);but the shannon index was higher and simpson index was lower in AD group than those in the healthy controls(P<0.05).At the Phylum level,Bacteroidetes was the most abundant and Firmicutes was secondary: in healthy controls,the relative abundance of Bacteroidetes was 62.40%,and Firmicutes was 26.73%;In AD group,the relative abundance of Bacteroidetes was 59.19%,and Firmicutes was 29.49%.There were no statistically significant differences between the two groups(P>0.05).The proportion of Lentisphaerae was higher in AD group than those in the healthy controls(P=0.023).At the Class level,the relative abundance of Alphaproteobacteria and Deltaproteobacteria in AD group were higher(P<0.05).At the Order level,the relative abundance of Desulfovibrionales in AD group was higher(P<0.05).At the Family level,Desulfovibrionaceae and Porphyromonadaceae were more in AD group than those in the healthy controls(P<0.05).At the Genus level,the proportions of Bilophila,Oscillospira,Coprococcus and Parabacteroides in AD group were higher(P<0.05);Faecalibacterium was more in AD group(P=0.063);important probiotics such as Bifidobacterium,Lactobacillus and Akkermansia had no significant differences between the two groups(P>0.05).At the Species level,the relative abundances of Bacteroides_ovatus,Bacteroides_uniformis,Clostridium_citroniae,and Ruminococcus torques were higher in AD group(P<0.05);Prevotella_stercorea were less in AD group(P<0.05).In addition,the relative abundance of faecalium um_prausnitzii in AD patients was higher,but the difference was not statistically significant(P=0.063).Important probiotics such as Akkermansia_muciniphila showed no significant difference between two groups(P=0.922).2.Clinical study results: 24 AD patients were statistically analyzed,13 in the treatment group and 11 in the control group.There were no statistically differences in age,gender and severity of disease between the two groups before the intervention(P>0.05).The results showed that the IGA,SCORAD and DLQI/CDLQI scores of patients were all reduced after the intervention in both groups.In the treatment group,the scores of patients were all reduced at the 4-week,8-week or 12-week points,and the differences were statistically significant(P<0.05).In the control group,The IGA scores were reduced after treatment,but at the 8-week point,the difference was not statistically significant(P>0.05);the scores of SCORAD were reduced after 4-week and 12-week treatment in control group(P<0.05),but at the 8-week point,the difference was not statistically significant(P>0.05);The DLQI/CDLQI scores of the control group were reduced after treatment,but there were no statistical significances(P>0.05).Comparison between groups: there were no statistically significant differences in IGA,SCORAD or DLQI/CDLQI scores at all the observation points between the two groups(P>0.05).3.Comparison of intestinal microflora of AD patients before and after treatment of placebo and PTQX formulae:The study included 23 fecal samples before the treatment,18 fecal samples at 4 weeks,6 fecal samples at 8 weeks,and 15 fecal samples at 12 weeks.Due to the small sample size at 8 weeks,the individual influence factors were obvious,so we just compared the samples at the points of 0,4,and 12 weeks.Results: in terms of Alpha diversity index,there were no statistically significant changes at each points in both groups(P>0.05).At the level of Phylum,there were no statistically significant changes in both groups(P>0.05);however,in the placebo group,the relative abundance of Bacteroidetes decreased with time and Firmicutes was on the rise after treatment.The trend in the treatment group was reversed.The two groups showed the opposite trend in the change of Bacteroidetes/Firmicutes ratio.At the level of Class,there were no statistically significant changes in the both groups(P>0.05).At the level of Order,there were no statistically significant changes in placebo group(P>0.05);but in the treatment group,the relative abundance of Actinomycetales decreased significantly at 12 weeks(P<0.05).The proportion of Campylobacterales showed no significant difference between the treatment group and healthy controls before the intervention,but it decreased to 0% after a 12-week treatment,which was different from those in healthy controls(P<0.05).At the level of Family,the proportions of Micrococcaceae were reduced in both groups(P<0.05).At the level of Genus,the relative abundance of Rothia in the placebo group decreased to 0% at 12 weeks(P<0.05).Actinomyces,Enterobacter,Lactococcus and Rothia were less abundant in the treatment group at 12 weeks(P<0.05).At the Species level,Rothia_mucilaginosa became less abundant in both groups at 12 weeks(P<0.05).In other species,there were no statistical differences between the two groups(P>0.05).Conclusion1.The Alpha diversity of gut microbiota in patients with AD is higher than those of healthy controls and the structure of intestinal flora of AD patients is basically consistent with healthy controls,but there are differences on the abundance of some floras:At the Phylum level,Lentisphaerae is more abundant in AD patients;At the Class level,the proportions of Alphaproteobacteria and Deltaproteobacteria are higher in AD patients.At the Order level,Desulfovibrionales is more in AD patients.At the Family level,the relatvie abundances of Desulfovibrionaceae and Porphyromonadaceae are higher in AD patients.At the Genus level,the proportions of Bilophila,Oscillospira,Coprococcus and Parabacteroides are higher in AD patients.At the Species level,Bacteroides_ovatus,Bacteroides_uniformis,Clostridium_citroniae and Ruminococcus_torques are more abundant in AD patients;Prevotella stercorea are less abundant in AD patients.2.PTQX formulae may reduce Alpha diversity of gut microbiota in AD patients and have a moderating effect on Bacteroidetes/Firmicutes ratio.