Effects of Baicalein on Cortical Proinflammatory Cytokines and the Intestinal Microbiome in Senescence Accelerated Mouse Prone 8

Baicalein, a flavonoid derived from the roots of <i>Scutellariae baicalensis</i> Georgi, has shown health benefits for an array of human diseases including dementia. The senescence-accelerated mouse prone 8 (SAMP8) strain is extensively used as a senile dementia model. To further investigate the effects of baicalein in SAMP8 mice, behavioral testing, biochemical detection, and gut microbiota analysis were performed. The results demonstrated that treatment with baicalein ameliorated the senescence status of the SAMP8 mice, as manifested by reducing the grading score of senescence. Additionally, baicalein improved the cognitive functions of the SAMP8 mice, including spatial learning and memory abilities, object recognition memory, and olfactory memory. Furthermore, baicalein significantly inhibited the release of proinflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-α (TNF-α) in the brain cortex of SAMP8 mice. Gut microbiota analysis revealed that treatment with baicalein markedly altered the abundance of six genera in SAMP8 mice. Correlation analysis indicated that the abundances of <i>Mucispirillum</i>, <i>Bacteroides</i>, and <i>Sutterella</i> were negatively correlated with cognitive abilities and that <i>Christensenellaceae</i> was positively correlated with cognition. Furthermore, the abundance of <i>Christensenellaceae</i> was negatively correlated with the levels of IL-6 and TNF-α, while [<i>Prevotella</i>] was positively correlated with the levels of IL-1β and IL-6. In addition, <i>Mucispirillum</i> and <i>Bacteroides</i> were positively correlated with the level of IL-6 in the brain cortex. These data indicated that baicalein ameliorates senescence status and improves cognitive function in SAMP8 mice and that this effect might be attributable to suppression of cortical proinflammatory cytokines and modulation of the intestinal microbiome

PCA scores plots derived from UPLC/Q-TOF MS data for extracts obtained from four different tissues (A); Permutation test with 200 permutations of PLS-DA model (B); PLS-DA loadings plot obtained from the metabolic profiles of four different tissues (C); Peak area intensity of triterpenoid saponins differences between the four tissue groups (D).

<p>^* The stems, leaves, and seeds group compared with the roots group, <i>p</i><0.05.</p

CYP450s and UGTs involved in triterpenoid saponin biosynthesis.

<p>CYP450s and UGTs involved in triterpenoid saponin biosynthesis.</p

Expression levels of candidate reference genes in the different tissues by qRT-PCR (A); Average expression stability values (M) of the candidate reference genes calculated by geNorm (B); Gene expression stability and ranking of reference genes as calculated by NormFinder (C).

<p>Expression levels of candidate reference genes in the different tissues by qRT-PCR (A); Average expression stability values (M) of the candidate reference genes calculated by geNorm (B); Gene expression stability and ranking of reference genes as calculated by NormFinder (C).</p

UPLC/Q-TOF MS-Based Metabolomics and qRT-PCR in Enzyme Gene Screening with Key Role in Triterpenoid Saponin Biosynthesis of <i>Polygala tenuifolia</i>

<div><p>Background</p><p>The dried root of <i>Polygala tenuifolia</i>, named Radix Polygalae, is a well-known traditional Chinese medicine. Triterpenoid saponins are some of the most important components of Radix Polygalae extracts and are widely studied because of their valuable pharmacological properties. However, the relationship between gene expression and triterpenoid saponin biosynthesis in <i>P. tenuifolia</i> is unclear.</p><p>Methodology/Findings</p><p>In this study, ultra-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS)-based metabolomic analysis was performed to identify and quantify the different chemical constituents of the roots, stems, leaves, and seeds of <i>P. tenuifolia</i>. A total of 22 marker compounds (VIP>1) were explored, and significant differences in all 7 triterpenoid saponins among the different tissues were found. We also observed an efficient reference gene GAPDH for different tissues in this plant and determined the expression level of some genes in the triterpenoid saponin biosynthetic pathway. Results showed that MVA pathway has more important functions in the triterpenoid saponin biosynthesis of <i>P. tenuifolia</i>. The expression levels of squalene synthase (SQS), squalene monooxygenase (SQE), and beta-amyrin synthase (β-AS) were highly correlated with the peak area intensity of triterpenoid saponins compared with data from UPLC/Q-TOF MS-based metabolomic analysis.</p><p>Conclusions/Significance</p><p>This finding suggested that a combination of UPLC/Q-TOF MS-based metabolomics and gene expression analysis can effectively elucidate the mechanism of triterpenoid saponin biosynthesis and can provide useful information on gene discovery. These findings can serve as a reference for using the overexpression of genes encoding for SQS, SQE, and/or β-AS to increase the triterpenoid saponin production of <i>P. tenuifolia</i>.</p></div

VIP values of PCA and peak area intensity of tissue metabolites measured by UPLC/Q-TOF MS.

<p>VIP values of PCA and peak area intensity of tissue metabolites measured by UPLC/Q-TOF MS.</p

Expression pattern of genes involved in triterpenoid saponin backbone biosynthesis pathway in the different tissues of <i>P. Tenuifolia</i> by qRT-PCR (A); QRT-PCR analysis of CYP450s and UGTs in the different tissues of P. Tenuifolia (B).

<p>^* The stems, leaves, and seeds group compared with the roots group, <i>p</i><0.05.</p

Primer sequences for qRT-PCR.

<p>Primer sequences for qRT-PCR.</p

Compounds identified in <i>P. Tenuifolia</i> by UPLC/Q-TOF MS.

<p>Compounds identified in <i>P. Tenuifolia</i> by UPLC/Q-TOF MS.</p

MS TIC chromatograms of roots (A), stems (B), leaves (C), and seeds (D) of <i>P. tenuifolia</i> by UPLC/Q-TOF MS in negative ion mode.

<p>MS TIC chromatograms of roots (A), stems (B), leaves (C), and seeds (D) of <i>P. tenuifolia</i> by UPLC/Q-TOF MS in negative ion mode.</p