[Effect and mechanism of Yueju Pills on breast cancer induced by DMBA in rats].

This paper aims to explore the inhibitory effect of Yueju Pills on breast cancer and decipher the underlying mechanism. A total of 92 SPF-grade SD female rats were involved in this study, and 14 of them were randomly selected into control group. The remaining 78 rats were administrated with 7,12-dimethylbenzanthracene(DMBA) by gavage to establish the breast cancer model. The modeled rats were randomized into model, tamoxifen(1.9 mg·kg~(-1)·d~(-1)), and low-and high-dose(17, 34 g·kg~(-1)·d~(-1)) Yueju Pills groups. The mental state, food intake, and activities of the rats were observed daily, and the body weight was measured on alternate days. After 12 weeks of administration, the rats were sacrificed and the tumor weight was measured. The serum estrogen and progeste-rone levels were determined by enzyme-linked immunosorbent assay. The histopathological changes of the breast and tumor were observed by hematoxylin-eosin staining. Western blot was employed to measure the protein levels of glucose transporter 1(GLUT1), lactate dehydrogenase A(LDHA), phosphofructokinase muscle(PFKM), pyruvate kinase isozyme type M2(PKM2), hexokinase 2(HK2), nuclear factor-kappaB(NF-κB), and phosphorylated NF-κB. The intestinal microbiome was examined by 16S rRNA high-throughput sequencing. The results showed that compared with the model group, high and low-dose Yueju Pills showed the tumor inhibition rate of 15.8% and 64.5%, respectively, and the low dose group had stronger inhibitory effect. Compared with the control group, the model group presented elevated the levels of estrogen and progesterone in serum. The administration of Yueju Pills lowered such ele-vation, and the low-dose group showed stronger lowering effect(P<0.05). Compared with the model group, Yueju Pills reduced the glands with increased breast tissue, the degree of breast duct expansion, the number and area of acinar cavity, the secretions, and the layers of mammary epithelial cells. Furthermore, Yueju Pills down-regulated the expression of GLUT1, LDHA, PFKM, PKM2, HK2, and NF-κB(P<0.05) and altered the diversity, composition, structure, and abundance of intestinal flora. The results showed that Yueju Pills could inhibit breast cancer by regulating the secretion of estrogen and progesterone, glycolysis, inflammatory cytokines, and intestinal flora.

Pediatric body mass index trajectories and the risk of hypertension among adolescents in China: a retrospective cohort study.

BACKGROUND: The impact of pediatric body mass index (BMI) trajectories on the risk of adolescent hypertension (HTN) determined by three separate visits remains unclear. This longitudinal study aims to identify potential pediatric sex-specific BMI trajectories and to assess their associations with HTN and HTN subtypes. METHODS: Based on the Health Promotion Program for Children and Adolescents (HPPCA) in Suzhou, China, a total of 24,426 participants who had initial normal blood pressure (BP) and had at least four BMI measurements during 2012-2020 were included. HTN was defined as simultaneously having three separate visits of elevated BP in 2020. Latent class growth models were used to explore sex-specific BMI trajectories, whose associations with HTN and HTN subtypes were further examined by logistic regression. RESULTS: The incidence of HTN determined through three separate visits was 3.34%. Four trajectories were identified for both sexes: low BMI increasing, medium BMI increasing, high BMI increasing, and highest BMI increasing. Compared to the medium BMI increasing group, the odds ratio (95% confidential interval) for developing adolescent HTN of the low, high, and highest BMI increasing groups among boys were 0.54 (0.39, 0.75), 1.90 (1.44, 2.51), and 2.89 (1.90, 4.39), respectively; and the corresponding values for girls were 0.66 (0.48, 0.90), 2.30 (1.72, 3.09), and 4.71 (3.06, 7.26). Similar gradually elevated associations between different trajectories with isolated systolic hypertension, systolic and diastolic hypertension were observed. CONCLUSION: Current results emphasized the adverse effects of stable high BMI on HTN and the benefits of maintaining normal weight throughout childhood.

Targeting tryptophan metabolism reveals Clematichinenoside AR alleviates triptolide-induced hepatotoxicity.

Liver toxicity induced by Triptolide (TP) has limited its clinical application on rheumatoid arthritis (RA). Saponins have been proved as an efficacious remedy to mitigate hepatotoxicity. However, the mechanism of reducing hepatotoxicity by saponins intervention remains incompletely characterized. Tryptophan (Trp) metabolites activate transcriptional regulators to mediate host detoxification responses. Our study aimed to investigate whether Clematichinenoside AR (C-AR) could attenuate TP-induced liver damage by regulating Trp metabolism. We used targeted metabolomics to quantify Trp metabolites in the serum and liver samples of collagen-induced arthritis rats treated by TP. Multiple comparison analyses helped the evaluation of promising biomarkers. The pronounced changed levels of Trp, indole acetic acid, and indole-3-carboxaldehyde in the serum and indole acetic acid, indole, and tryptamine in the liver are relevant to TP-induced liver injury. Intervention with C-AR could relieve TP-induced hepatotoxicity evidenced by ameliorative serum parameters and hepatic histology. In addition, C-AR regulated the levels of these indoles biomarker candidates to normal. Therapeutic modulation with natural compounds might be a useful clinical strategy to ameliorate toxicity induced by TP. Deciphering Trp metabolism will facilitate a better understanding of the pathogenesis of diseases and drug responding.

In silico production of relative correction factor for the quantitative analysis of multi-components by single marker method.

INTRODUCTION: Traditional methods to derive experimentally-generated relative correction factors (RCFs) for the quantitative analysis of herbal multi-components by single marker (QAMS) method require reference standards and multiple validations with different instruments and columns, which hampers high throughput implementation. OBJECTIVES: To effectively reduce the application amounts of raw material and provide higher and more stable accuracy, this study aimed to develop a method to computationally generate RCFs of herbal components. MATERIALS AND METHODS: This strategy included the published data collection, calibration curves screening, computer algorithm-based RCFs generation and accuracy validation. RESULTS: Using the in silico approach, we have successfully produced 133 RCFs for the multi-component quantitative analysis of 63 widely used herbs. CONCLUSION: Compared with conventional RCFs, this in silico method would be a low cost and highly efficient way to produce practical RCFs for the QAMS method.

Orthogonal label and label-free dual pretreatment for targeted profiling of neurotransmitters in enteric nervous system.

Neurotransmitter (NT) abnormalities in the enteric nervous system have been reported as crucial roles to regulate the intestinal inflammation and gut immune homeostasis. Capturing quantitative changes at the NT metabolome provides an opportunity to develop an understanding of neuroimmune-mediated inflammation. Given the wide diversity of chemical characterizations in the NTs, only partial coverage of the NT metabolome can be simultaneously quantified in a single-run analysis. Herein, we summarized the distribution of functional groups of compound entries in the NT metabolome. Based on this information, an orthogonal dansyl-labeling and label-free dual pretreatment approach was separately designed to target phenol and amine NTs and tertiary amine and choline NTs. By combining the dansyl-labeled and unlabeled NTs within a single vial, a comprehensive and practical approach was optimized for quantifying high coverage of NT metabolome in a single-run analysis on the reversed-phase C18 column. Method validation indicated good linearity with correlation coefficients (R2) > 0.99, intra- and interday accuracy with relative error < ±20%, and precision with relative standard deviations of ≤15%. With this method, we could simultaneously monitor the alterations of cholines, amines, amino acids, tryptophan and phenylalanine biological pathways in dextran sulphate sodium-induced colitis mice. The measured levels of NT metabolome ranged from 0.0007 to 3.540 µg/mg in intestinal contents and 0.013-154.54 µg/mL in serum samples. The NT metabolism was disrupted by colitis, characterized by the changed NT levels in serum and excessive amino acid NTs accumulation in the intestinal contents. We envisage that the orthogonal approach is of great significance for the comprehensive determination of targeted metabolomics. NTs have the potential to be biomarkers for clinical metabolomics.

Saponins from Clematis mandshurica Rupr. regulates gut microbiota and its metabolites during alleviation of collagen-induced arthritis in rats.

Gut microbiota and their metabolites (short-chain fatty acids, SCFAs) are associated with the pathogenesis of rheumatoid arthritis (RA). Total Clematis triterpenoid saponins (CTSs) prepared from Clematis mandshurica Rupr. possess therapeutic benefits for arthritic diseases. However, the poor pharmacokinetic properties of CTSs have obstructed the translation of these natural agents to drugs. Here, we examined the effects of CTSs on arthritis symptoms, gut microbiota and SCFAs in rats with collagen-induced arthritis (CIA). Our results showed that the arthritis index scores of CIA rats treated with CTSs were significantly lower than those of the model group. Most importantly, CTSs moderated gut microbial dysbiosis and significantly downregulated the total SCFA concentration in CIA rats. Compared to the control group, CTSs treatment have no significant side effects on the gut microbiota and SCFA metabolism in normal rats. Two differential analyses (LEfSe and DESeq2) were combined to study the details of the changes in gut microbiome, and twenty-four marker taxa at the genus level were identified via a comparison among control, model and CIA rats treated with high doses of CTSs. In particular, the mostly significantly increased gram-negative (G-) and decreased gram-positive (G+) genera in CIA rats were well restored by CTSs. The observed SCFA concentrations demonstrated that CTSs tend to maintain the balance of the gut microbiota. The data presented herein suggest that CTSs could ameliorate arthritis-associated gut microbial dysbiosis and may be potential adjuvant drugs that could provide relief from the gastrointestinal damage caused as a side effect of commonly used drugs.

Development and validation of a systematic platform for broad-scale profiling of microbial metabolites.

Liquid chromatography-mass spectrometry based profiling of microbial metabolites has been a challenging task due to their diverse physicochemical properties and wide concentration ranges. This study is aimed to develop a systematic platform for the broad-scale profiling of microbial metabolites by integrating aqueous-lipophilic biphasic extractions and chemical derivatizations with a data-dependent automatable metabolite annotation algorithm. This complementary strategy of detection will not only largely expand the metabolite coverage, but also facilitate the drawing out of interested submetabolome using designed chemical derivatizations. Then, the data-dependent metabolite annotation algorithm is able to automatically match the raw MS/MS data with those of compounds in the self-collected databases. The performance of this platform is illustrated through the analysis of two representative bacteria (Escherichia coli and Pseudomonas aeruginosa) and intestinal contents samples from experimental colitis mice. As a result, 292 metabolites corresponding to 875 annotated features distributing over 25 chemical families were putatively annotated in a short time. Of these metabolites, 197 and 218 are respectively from the bacteria and intestinal contents, and 107 are identified in all three biological samples. This systematic platform could be used to accomplete high-coverage detection and high-quality data processing of microbial metabolites. At the same time, chemical derivatization design and the establishment of self-collected databases will facilitate self-driven untargeted analysis.

Identification of human hepatic stimulator substance gene promoter and demonstration of dual regulation of AP1/AP4 cis-acting element in different cell lines.

Human hepatic stimulator substance (hHSS) is a newly identified growth-promoting factor in the liver. HSS is capable of stimulating hepatic regeneration in partial hepatectomized rats, thus, promoting growth of hepatic tumor cells. To understand and elucidate the transcriptional regulation of hHSS gene, the 4890bp of 5'-flanking region of the gene have been isolated and sequenced. The transcriptional start site, located at 248nt upstream from the ATG starting codon, was identified by 5'-rapid amplification cDNA end (5'-RACE). The classical promoter sequences, such as TATA box or GAATT were not identified in the promoter region, instead a GC-rich segment was formed (>70%) by expanding to a longer than 400bp, and immediately upstream from the ATG start codon. The transient transfection assays, using promoter deletion constructs, showed that hHSS promoter was efficiently capable in driving the reporter expression not only in HepG2 cells, but also in Cos7 cells. A region spanning nucleotides in the range of -447 to -358bp revealed a negative regulation on promoter activity in HepG2 cells, but with positive regulation in Cos7 and Hela cells. The promoter activity was obviously influenced by AP1/AP4 (-375/-369nt) mutation in these three cell lines. EMSAs showed that the site was recognized by AP1 in HepG2 cell, and only by an AP4 protein in Cos7 cells. The c-Jun bound to the promoter was further verified by supershift in HepG2 cells and human liver tissue. Chromatin immuno-precipitation (ChIP) demonstrated that there was a direct association of c-Jun with hHSS promoter in HepG2 cells. The c-Jun strongly suppressed hHSS promoter activity in transient expression analyses in HepG2 cells. Mutations in the AP1 binding sites rescued suppression caused by c-Jun, suggesting this was a direct regulation of the hHSS promoter. In contrast, there was no significant effect in c-Jun over-expressed Cos7 and Hela cells. The tissue-specific function of c-Jun in hHSS promoter activity may in part help explain the differences in biology function of hHSS between liver and non-liver cells.

[Correlation analysis between WNK4 gene and essential hypertension].

OBJECTIVE: To investigate association of mutation in WNK4 gene with essential hypertension and to analyze the expression of WNK4 gene. METHODS: cSNP in WNK4 gene in a small samples was detected by sequencing, then PCR-RFLP was performed in 98 patients with essential hypertension and 95 control subjects. The expression profile of WNK4 gene was tested by RT-PCR. RESULTS: A cSNP was detected in WNK4 gene exon7 G1662A, and there were significant differences in the distribution of allele frequency of G1662A between essential hypertension group and control group. WNK4 gene were expressed in the tissues of kidney, brain, lung, heart, spleen and intestine of fetus. CONCLUSION: WNK4 gene is well correlated with essential hypertension.

[Cloning and structural analysis of 5'-flanking sequence of human hepatic stimulator substance (hHSS) gene].

Human hepatic stimulator substance (hHSS) is a newly identified growth-promoting factor in liver. HSS is capable of stimulating the hepatic regeneration in hepaectomized rats and promoting the growth of hepatic tumor cells. To understand and elucidate the regulation of hHSS on hepatocyte growth at genetic level, the 4,890 bp of 5'-flanking region of hHSS gene have been isolated and sequenced. On basis of comparison of the 5'-flanking sequences with GenBank, hHSS gene was localized on human chromosome 16. Transcriptional start site was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA end (5'RACE). The initiation nucleotide was defined as 'C', which located at 254 bp upstream of the reported sequence of exon I.