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Flowchart of PhenoSpD. Simulation of phenotypic correlation estimation First, we simulated the influence of the number of single nucleotide polymorphisms SNPs , sample sizes of two GWASs, and sample overlap between two GWASs on the accuracy of the phenotypic correlation estimation. As shown in Fig. We assumed complex human traits were influenced by both genetic and environmental factors, so we simulated the phenotype data of two correlated human traits phenotype 1 and phenotype 2 with a phenotypic correlation of —0. These genetic and environmental components were further assigned to each of the genetic and environmental factors in the model randomly. We also simulated two extreme cases where either the genetic or environmental components dominate the phenotypic correlation.

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This study aimed to investigate the role of intestinal flora and serum metabolism induced by HD against hypertension. Rats were fed by gavage once a day for 28 days. Rumen bacterium NK4A, Clostridium sp.

MC 40 increased remarkably in M group. Akkermansia, Akkermansia muciniphila, and Lactobacillus intestinalis increased significantly in HD group, which were functionally related to the significant increase of Lachnoclostridium, Faecalibaculum, and Lactobacillus reuteri in W group, which were all probiotics producing butyric acid, lactic acid, and regulating inflammation and other antihypertensive related factors.

HD also changed the serum metabolic pattern of SHRs. The correlation analysis demonstrated that the dominant genius and species in three groups were highly correlated with steroid hormone biosynthesis, arachidonic acid metabolism, tryptophan metabolism, and vitamin B6 metabolism.

Our research indicated that HD had a good antihypertensive effect, which may be driven by the protective intestinal flora and beneficial metabolites induced by it, and the metabolites were closely related to the changes of intestinal flora. It provided new insights for the antihypertensive mechanism of HD. Introduction The intestinal flora as the second genome of the human body was not only directly involved in the normal metabolism but also closely related to the occurrence and development of hypertension [ 1 — 6 ].

It had been found that both spontaneous hypertensive rats SHRs and hypertensive patients had the microecological imbalance of the richness, diversity, and uniformity in intestinal flora and the increased ratio of Firmicutes to Bacteroidetes [ 4 ]. In addition, a meta-analysis of multiple randomized human clinical trials had shown that probiotics could effectively lower blood pressure [ 5 ].

A recent study found that blood pressure was elevated by fecal transplantation from hypertensive human donors to germ-free mice. And their study also confirmed that serum metabolic changes in patients with prehypertension and hypertension were closely related to intestinal flora imbalance [ 6 ]. Metabolomics, as a powerful tool for identifying the changes of endogenous metabolites in pharmacodynamics, had been widely used in exploring the pathological mechanism of cardiovascular and cerebrovascular diseases such as hypertension in recent years [ 7 ].

The metabolic studies of SHRs had found that the changes of metabolites in the pathways of arachidonic acid metabolism, lipid metabolism, fatty acid metabolism, vitamin metabolism, and amino acid metabolism were closely related to the regulation of blood pressure [ 8 — 10 ].

However, the specific mechanism between intestinal flora-host metabolism and blood pressure regulation has not been fully elucidated. Astragalus membranaceus of the Leguminosae family , with Chinese name of Huang qi and the Latin name of Astragalus membranaceus Fisch. Salvia miltiorrhiza of the Lamiaceae family , with Chinese name of Dan shen and the Latin name of Salvia miltiorrhiza Bge.

Both of them have been widely used in the treatment of hypertension in China. It had been found that astragaloside IV, an effective component of Astragalus membranaceus, reduced blood pressure in rats with metabolic syndrome by regulating lipid metabolism and endothelium-dependent vasodilation [ 11 ].

The combination of Astragalus membranaceus and Salvia miltiorrhiza HD could reduce blood lipid and improve insulin resistance, anti-inflammation, and anticoagulation [ 13 — 15 ], all of which were risk factors of hypertension. Our previous studies had found that the combination of HD had better antihypertensive effect than the separated use of herb, but the pharmacological mechanism of blood pressure reduction remained to be further clarified.

Therefore, we speculate that the combination of HD may be involved in the regulation of blood pressure by inducing changes in intestinal flora and serum metabolites.

Materials and Methods 2. According to our previous studies and other reports [ 16 ], HD were prepared in a ratio of , adding fold volume of water to the medicinal material, decocted twice, mixed, and concentrated to 0. The HD group was intragastrically administrated with HD 0. All animals were fed by gavage once a day for 28 days. The supernatants were spun in a vacuum concentrator until they dry. Fecal Collection. HE Staining. All rats were sacrificed on the 28th day and colon tissues were taken and fixed in neutral paraformaldehyde for 48 hours.

The eluents for the positive polarity mode were eluent A 0. Data Processing and Statistical Analysis. After that, peak intensities were normalized to the total spectral intensity.

The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. Specificity and sensitivity of identified potential biomarkers were analyzed by ROC curves, and metabolic pathways involving potential biomarkers were topologically analyzed by the MetaboAnalyst 3.

Beijing, China. PCR products were mixed in equidensity ratios. Library Preparation and Sequencing Analysis. The library quality was assessed on the Qubit 2. The raw reads were spliced and filtered to obtain clean reads, and then all clean reads were clustered by Uparse software Uparse v7. Beta diversity was expressed as PCA. MetaStat and LEfSe methods were used to compare species with significant differences among groups.

Spearman correlation coefficient was calculated to figure out the correlation between genus, species, and metabolites. Results 3. HE staining showed no significant inflammation and dysplasia in intestinal tissue in three groups Figure 1 b. Considering the chronic systemic low-grade inflammatory state in hypertension [ 3 ], pathological changes of intestine were not visually identified in HE staining.

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