Characteristics and Functional Differences of Intestinal Flora in Type 2 Diabetes Mellitus Patients with Dampness Heat Trapping Spleen Syndrome and Qi-Yin Deficiency Syndrome： An Analysis Based on High-throughput Sequencing Technology / 中国实验方剂学杂志

ObjectiveTo explore the structural characteristics and functional differences of intestinal flora in patients with type 2 diabetes mellitus （T2DM） of dampness heat trapping spleen（DHTS） syndrome and Qi-Yin deficiency（QYD） syndrome. MethodFrom June 2018 to January 2020，62 T2DM patients with DHTS syndrome and 60 with QYD syndrome were selected from Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine. Serum and fecal samples were collected to compare body mass index（BMI），glucose and lipid metabolism，fasting insulin （FINS） and fasting C-peptide （FCP） levels，and homeostasis model assessment of insulin resistance（HOMA-IR） of the two syndrome types. Fecal samples were extracted for DNA database construction，and 16S rDNA high-throughput sequencing was used to analyze and compare the intestinal flora and metabolic pathways. Result① The BMI，fasting plasma glucose（FPG），2-hour postprandial blood glucose （2 h PBG），total cholesterol（TC），triglyceride（TG），low density lipoprotein（LDL），FINS，FCP，and HOMA-IR were higher in patients with DHTS syndrome than in patients with QYD syndrome，and the high density lipoprotein（HDL） of the former was lower than that of the latter，（P<0.05，P<0.01）. ② In terms of species composition and differences，Bacteroidetes， Clostridia and Gammaproteobacteria were dominant at the class level，and the relative abundance of Clostridia，Mollicutes and Verrucomicrobiae in QYD syndrome group was higher than that in DHTS syndrome group. At the order level，Bacteroidales，Clostridiales and Enterobacteriales were mainly found. The relative abundance of Clostridiales，Erysipelotrichales and Verrucomicrobiales in QYD syndrome group was obviously higher than that in DHTS syndrome group，while Aeromonadales in the former was lower than that in the latter （P<0.05）. At the family level，Bacteroidaceae，Prevotellaceae and Ruminococcaceae were predominant. The relative abundance of Ruminococcaceae，Porphyromonadaceae and Erysipelotrichaceae in QYD syndrome group was higher than that in DHTS syndrome group（P<0.05）. At the genus level，Bacteroides，Prevotella and Parabacteroides were mainly found. The relative abundance of Parabacteroides，Butyrivibrio and Ruminiclostridium in QYD syndrome group was higher than that in DHTS syndrome group，while that of Klebsiella and Megasphaera in DHTS syndrome group was higher than that in QYD syndrome group（P<0.05）. ③ Through Venn analysis of operational taxonomic units（OTU），it was found that there were 49 OTUs in patients with DHTS syndrome patients and 47 OTUs in QYD syndrome patients. ④ The results of OTU β diversity and α analysis showed that Shannon and Simpson indexes had statistical differences，while Ace and Chao indexes had no statistical differences. The intestinal microbial diversity of patients with QYD syndrome was higher than that of patients with DHTS syndrome（P<0.05）. The analysis of similarities （ANOSIM） showed that the difference of β diversity between the two groups was significant（P<0.05）. ⑤ Linear discriminant analysis Effect Size（LEfSe） results demonstrated that Klebsiella，Megasphaera and Aeromonadales could be selected as the key biomarkers for DHTS syndrome； 14 bacteria such as Ruminiclostridium，Burkholderiaceae，Lautropia，Butyrivibrio，Erysipelotrichales can be selected as the key biomarkers for QYD syndrome. ⑥Functional annotation and analysis showed that the DHTS syndrome involved 9 metabolic pathways，including arginine and proline metabolism，lipopolysaccharide biosynthesis，nicotinic acid and nicotinamide metabolism，while the QYD syndrome involved 10 metabolic pathways，including acarbose and valinomycin biosynthesis，glucagon signaling pathway and NOD-like receptor signaling pathway. ConclusionThere are obvious differences in intestinal flora and functions in T2DM patients of DHTS syndrome and QYD syndrome，which can be used as reference for traditional Chinese medicine （TCM） syndrome differentiation and the target of TCM treatment.

Study on tanshinones regulating root-associated microbiomes of Salvia miltiorrhiza / 中国中药杂志

The plant root-associated microbiomes include root microbiome and rhizosphere microbiome, which are closely related to plant life activities. Nearly 30% of photosynthesis products of plants are used to synthesize root compounds, there is evidence that root compounds regulate and significantly affect the root microbiome Tanshinones are the main hydrophobic components in Salvia miltiorrhiza. In order to study whether these compounds can regulate the root-associated microbiomes of S. miltiorrhiza, our study first identified a white root S. miltiorrhiza(BG) which contains little tanshinones. Retain of the fifth intron of tanshinones synthesis key enzyme gene SmCPS1 leading to the early termination of the SmCPS1 gene, and a stable white root phenotype. Further, wild type(WT) and BG were planted in greenhouse with nutrient soil(Pindstrup, Denmark) and Shandong soil(collected from the S. miltiorrhiza base in Weifang, Shandong), then high-throughput sequencing was used to analyze the root-associated microbiomes. The results showed that the tanshinones significantly affected the root-associated microbiomes of S. miltiorrhiza, and the impact on root microbiomes was more significant. There are significant differences between WT and BG root microbiomes in species richness, dominant strains and co-occurrence network. Tanshinones have a certain repelling effect on Bacilli which belongs to Gram-positive, while specifically attract some Gram-negative bacteria such as Betaproteobacteria and some specific genus of Alphaproteobacteria. This study determined the important role of tanshinones in regulating the structure of root-associated microbiomes from multiple angles, and shed a light for further improving the quality and yield of S. miltiorrhiza through microenvironment regulation.

Comparison of real-time quantitative PCR and 16S rDNA sequencing for detection of female vaginal microbiome / 上海交通大学学报（医学版）

Objective: To compare the differences in the composition of female vaginal flora by real-time quantitative PCR and 16S rDNA sequencing. Methods: Forty-nine healthy reproductive women less than 45 years old were selected. Specimens were collected from posterior fornix. DNA were extracted and the microbiome were ananlyzed by both 16S rDNA V1V2 region sequencing and qPCR of 22 selected genes. The results detected by two methods were compared. Results: According to the classification standard of vaginal community state type (CSTs), qPCR analysis showed that 35 out of 49 samples were dominated by Lactobacillus species, among them, type I (Lactobacillus crispatus, 9), type III (Lactobacillus iners, 24), type IV (no Lactobacillus as the dominant bacteria, 12), type V (Lactobacillus jensenii, 2). 16S rDNA V1V2 region sequence analysis showed that of the 49 samples, 13 belonged to type I, type II (1), type III (23), type IV (8), type V (2). Two methods of vaginal flora classification were consistent for 38 cases, consistent rate was 77.6%. Conclusion: Two methods analysis of vaginal flora showed the different results. If qPCR was used to classify the vaginal microbiome, it was necessary to consider the influence of relevant technical factors on the results.

Comparison of real-time quantitative PCR and 16S rDNA sequencing for detection of female vaginal microbiome / 上海交通大学学报（医学版）

Objective·To compare the differences in the composition of female vaginal flora by real-time quantitative PCR and 16S rDNA sequencing.Methods·Forty-nine healthy reproductive women less than 45 years old were selected.Specimens were collected from posterior fornix.DNA were extracted and the microbiome were ananlyzed by both 16S rDNA V1V2 region sequencing and qPCR of 22 selected genes.The results detected by two methods were compared.Results·According to the classification standard of vaginal community state type (CSTs),qPCR analysis showed that 35 out of 49 samples were dominated by Lactobacillus species,among them,type Ⅰ (Lactobacillus crispatus,9),type Ⅲ (Lactobacillus iners,24),type Ⅳ (no Lactobacillus as the dominant bacteria,12),type Ⅴ (Lactobacillusjensenii,2).16S rDNA V1V2 region sequence analysis showed that of the 49 samples,13 belonged to type Ⅰ,type Ⅱ (1),type Ⅲ (23),type Ⅳ (8),type Ⅴ (2).Two methods of vaginal flora classification were consistent for 38 cases,consistent rate was 77.6％.Conclusion·Two methods analysis of vaginal flora showed the different results.If qPCR was used to classify the vaginal microbiome,it was necessary to consider the influence of relevant technical factors on the results.

Effects of probiotics on alcoholic liver injury in rats and its mechanisms / 中国药理学通报

Aim To observe the protective effects of probiotics on alcoholic liver injury in rats .Methods Male Wistar rats were randomly divided into the follow-ing three groups: control group , normal diet with nor-mal (5 ×108 CFU· kg -1· d -1) treatment group.Ex-cluding the rats in the normal control group , the other animals were initially received intragastric administra-tion with 56%( V/V) ethanol 5.5~11.0 mL· kg -1 · day -1 for 8 weeks.Then the rats’ faeces were collect-ed, and the liver and the small intestine were obtained for pathologic and ultrastructural observation .Serum ALT, AST and ALP was measured by method of bio-chemistry .Serum DAO and D-LA was measured by en-zyme linked immunosorbent assay .The expression of FOXO4 in small intestine was detected by immunohis-tochemistry .The intestinal flora genome DNA was ex-tracted from faeces and the sequence of 16 S rDNA was analyzed by high-throughput sequencing technologies . Results Hepatic steatosis was obviously improved in probiotics treatment groups compared with ethanol-trea-ted group , and the ultrastructural such as mitochondri-al and rough endoplasmic reticulum pathological chan-ges was significantly alleviated . The ultrastructural changes in intestinal were better in probiotics treatment group than in the ethanol-treated group .And ethanol-induced rats ’ serum ALT, AST, ALP, D-LA and DAO levels showed a significant reduction in the probi-otics treatment groups compared with the ethanol-trea-ted group ( P<0.05 ) .The FOXO4 expression was in-creased obviously in the probiotics treatment groups compared with the ethanol-treated group ( P <0.05 ) . And the intestinal flora diversity was impacted after feeding alcohol , and probiotics had a certain regulative action in helping the intestinal flora back to normal state; At phylum level , the Firmicutes quantity was lower and the Bacteroidetes quantity was higher in eth-anol-treated group than those in the control group ( P<0.05 ) , and the conditions were improved after supple-menting probiotics .At genus level , the percent of ge-nus abundance was similar to normal control group in the probiotics treatment groups compared with the etha-nol-treated group .Conclusion Probiotics can relieve liver injury induced by alcohol in rats , and the mecha-nism may be related to the modulation of probiotics on the intestinal flora distribution and intestinal barrier .