Unraveling the chemical constituents, absorption characteristics, and metabolic profile of Codonopsis Radix based on UPLC-Q- Orbitrap MS.

Codonopsis Radix (CR), a traditional tonic medicinal material in China, has been proven to possess a variety of bioactive functions. However, its chemical composition and in vivo metabolic pattern have not been fully elucidated. In this study, AB-8 macroporous resin column chromatography was employed for the enrichment of small molecular components in CR. Furthermore, a method combining ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry with Acquire X intelligent data acquisition technology software was developed for the preliminary screening and identification of the chemical composition of CR in vitro and their metabolites in vivo. As a result, a total of 116 components were preliminarily characterized in the CR extract, including 28 polyacetylenes, 33 organic acids, 4 amino acids, 23 alkaloids, 9 phenylpropanoids, 6 terpenoids, 2 nucleosides, and 11 others. Additionally, a total of 84 compounds, including 37 prototype components and 47 metabolites, were identified in the plasma, urine, and feces of rats after oral administration of CR. Specifically, 11, 24, 19, 32, and 25 constituents were identified in the heart, liver, spleen, lung, and kidney, respectively. Of note, the lung and spleen are the organs with the highest distribution of CR compounds. These findings will serve as valuable data for future research on the correlation between the chemical composition and pharmacological effects of CR.

UPLC-QTOF-MS based metabolomics unravels the modulatory effect of ginseng water extracts on rats with Qi-deficiency.

Ginseng is commonly used as a nutritional supplement and daily wellness product due to its ability to invigorate qi. As a result, individuals with Qi-deficiency often use ginseng as a health supplement. Ginsenosides and polysaccharides are the primary components of ginseng. However, the therapeutic effects and mechanisms of action of these components in Qi-deficiency remain unclear. This study aimed to determine the modulatory effects and mechanisms of ginseng water extract, ginsenosides, and ginseng polysaccharides in a rat model of Qi-deficiency using metabolomics and network analysis. The rat model of Qi-deficiency was established via swimming fatigue and a restricted diet. Oral administration of different ginseng water extracts for 30 days primarily alleviated oxidative stress and disrupted energy metabolism and immune response dysfunction caused by Qi-deficiency in rats. Ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used for untargeted serum metabolomic analysis. Based on the analysis results, the active constituents of ginseng significantly reversed the changes in serum biomarkers related to Qi-deficiency in rats, particularly energy, amino acid, and unsaturated fatty acid metabolism. Furthermore, analysis of the metabolite-gene network suggested that the anti-Qi-deficiency effects of the ginseng components were mainly associated with toll-like receptor (TLR) signaling and inflammatory response. Additional verification revealed that treatment with the ginseng components effectively reduced the inflammatory response and activation of the myocardial TLR4/NF-κB pathway induced by Qi-deficiency, especially the ginseng water extracts. Therefore, ginseng could be an effective preventive measure against the progression of Qi-deficiency by regulating metabolic and inflammatory responses.

UPLC-Q-TOF/MS based serum and urine metabolomics strategy to analyze the mechanism of nervonic acid in treating Alzheimer's disease.

Nervonic acid is a natural component of breast milk and is frequently used as a food additive due to its excellent neuroprotective effects. Although it has been reported that nervonic acid may play a role in the recovery of human cognitive impairment, its specific mechanism of action is still unclear. In this study, the results of serum biochemical indexes showed that nervonic acid improved inflammation and reduced amyloid ß peptide (Aß) deposition and tau protein phosphorylation in Alzheimer's disease (AD) rats. Subsequently, we further used a metabolomics approach to investigate the potential mechanism of action of nervonic acid in the treatment of AD. The results of serum and urine metabolomics study showed that the intervention of nervonic acid significantly reversed the metabolic profile disorder in AD rats. A total of 52 metabolites were identified. They mainly involved linoleic acid metabolism, alpha-linolenic acid metabolism, phenylalanine metabolism and arachidonic acid metabolism, and all these metabolic pathways were associated with the emergence of inflammation in vivo. It suggests that the therapeutic effect of nervonic acid on AD is likely to be produced by ameliorating inflammation. The results obtained in this study provide new insights into the mechanism of nervonic acid treatment of AD and lay a foundation for the clinical application of nervonic acid in the treatment of Alzheimer's disease.

An integrated approach based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, network pharmacology, and molecular docking to study the key effective compounds and mechanism of action of Platycodi Radix in the treatment of chronic obstructive pulmonary disease.

Platycodi Radix (PR) is a valuable herb that is widely used in the treatment of chronic obstructive pulmonary disease in clinics. However, the mechanism of action for the treatment of chronic obstructive pulmonary disease remains unclear due to the lack of in vivo studies. Our study established a novel integrated strategy based on ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry, network pharmacology, and molecular docking to systematically analyze the tissue distribution and active compounds of PR in vivo and the therapeutic mechanism of chronic obstructive pulmonary disease. First, tissue distribution studies have shown that the lung is the organ with the highest distribution of PR compounds. Subsequently, network pharmacology results showed that the tumor necrosis factor signaling pathway, interleukin-17 signaling pathway, and mitogen-activated protein kinase signaling pathway were the critical mechanisms of PR against chronic obstructive pulmonary disease. Ultimately, molecular docking results showed that the key targets were stably bound to the corresponding active compounds of PR. Our study is of great significance for the screening of the key effective compounds and the study of the mechanism of action in traditional Chinese medicine and provides data to support the further development and utilization of PR.

Farnesoid X receptor represses human sulfotransferase 1A3 expression through direct binding to the promoter.

Human sulfotransferases 1A3 (SULT1A3) has received particular interest, due to their functions of catalyzing the sulfonation of numerous phenolic substrates, including bioactive endogenous molecules and therapeutic agents. However, the regulation of SULT1A3 expression and the underlying mechanism remain unclear. Here, we aimed to investigate the regulation effects of bile acid-activated farnesoid X receptor (FXR) on SULT1A3 expression, and to shed light on the mechanism thereof. Our results demonstrated that FXR agonists (CDCA and GW4064) significantly inhibit the expression of SULT1A3 at mRNA and protein levels. In addition, overexpression of FXR led to decrease in SULT1A3 expression and knockdown of FXR significantly induced the expression of SULT1A3 in protein and mRNA levels, confirming that FXR expression manifestly showed negative regulatory effect on basal SULT1A3 expression. Furthermore, a combination of luciferase reporter gene and CHIP assays showed that FXR repressed SULT1A3 transcription through direct binding to the region at base pair positions -664 to -654. In conclusion, this study for the first time confirmed FXR was a negative transcriptional regulator of human SULT1A3 enzyme.

O-Sulfation disposition of curcumin and quercetin in SULT1A3 overexpressing HEK293 cells: the role of arylsulfatase B in cellular O-sulfation regulated by transporters.

Extensive phase II metabolic reactions (i.e., glucuronidation and sulfation) have resulted in low bioavailability and decreased biological effects of curcumin and quercetin. Compared to glucuronidation, information on the sulfation disposition of curcumin and quercetin is limited. In this study, we identified that BCRP and MRP4 played a critical role in the cellular excretion of curcumin-O-sulfate (C-O-S) and quercetin-O-sulfate (Q-O-S) by integrating chemical inhibition with transporter knock-down experiments. Inhibited excretion of sulfate (C-O-S and Q-O-S) caused significant reductions in cellular O-sulfation of curcumin (a maximal 74.4% reduction) and quercetin (a maximal 76.9% reduction), revealing a strong interplay of sulfation with efflux transport. It was further identified that arylsulfatase B (ARSB) played a crucial role in the regulation of cellular O-sulfation by transporters. ARSB overexpression significantly enhanced the reduction effect of MK-571 on the cellular O-sulfation (fmet) of the model compound (38.8% reduction for curcumin and 44.2% reduction for quercetin). On the contrary, ARSB knockdown could reverse the effect of MK-571 on the O-sulfation disposition of the model compound (29.7% increase for curcumin and 47.3% increase for quercetin). Taken together, ARSB has been proven to be involved in cellular O-sulfation, accounting for transporter-dependent O-sulfation of curcumin and quercetin. A better understanding of the interplay beneath metabolism and transport will contribute to the exact prediction of in vivo drug disposition and drug-drug interactions.

Cinobufotalin inhibits the epithelial-mesenchymal transition of hepatocellular carcinoma cells through down-regulate ß-catenin in vitro and in vivo.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and mortality. The prognosis of HCC is poor due to the high postoperative recurrence rate and metastasis rate. Epithelial-mesenchymal transition (EMT) plays a key role in the metastasis of HCC, which is closely related to the invasion, intrahepatic metastasis and low survival rate. Here we demonstrated that cinobufotalin can upregulate epithelial markers (E-cadherin) and downregulate mesenchymal markers (N-cadherin, snail, slug and ZEB1) in HepG2, SMMC-7721 and SNU-368 cells. We further found that the mRNA and protein expression of ß-catenin and its target genes (i.e. MMP7 and DKK1), which are related to tumor invasion and metastasis, were decreased after cinobufotalin treatment. Overexpression of ß-catenin promoted EMT of HepG2 and SMMC-7721 cells, and cinobufotalin could antagonize this induction. While Knockdown of ß-catenin could inhibit EMT and cinobufotalin enhanced this inhibition. In addition, cinobufotalin significantly suppressed the tumor EMT, as demonstrated by increased E-cadherin expression and decreased N-cadherin and vimentin expression, and inhibited formation and metastasis of lung metastases in vivo. In conclusion, our study has revealed a novel anticancer mechanism of cinobufotalin, which inhibits EMT progress by downregulating ß-catenin, and then prevents the migration and invasion of HCC. These results provide convincing evidence for the development of cinobufotalin as a potential HCC metastasis inhibitor.

Corrigendum: PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC.

[This corrects the article DOI: 10.3389/fphar.2020.577108.].

P300-dependent acetylation of histone H3 is required for epidermal growth factor receptor-mediated high-mobility group protein A2 transcription in hepatocellular carcinoma.

High-mobility group protein A2 (HMGA2) is highly expressed in hepatocellular carcinoma (HCC) cells and contributes to tumor metastasis and poor patient survival. However, the molecular mechanism through which HMGA2 is transcriptionally regulated in HCC cells remains largely unclear. Here, we showed that the expression HMGA2 was upregulated in HCC, and that elevated HMGA2 could promote tumor metastasis. Incubation of HCC cells with epidermal growth factor (EGF) could promote the expression of HMGA2 mRNA and protein. Mechanistic studies suggested that EGF can phosphorylate p300 at Ser1834 residue through the PI3K/Akt signaling pathway in HCC cells. Knockdown of p300 can reverse EGF-induced HMGA2 expression and histone H3-K9 acetylation, whereas a phosphorylation-mimic p300 S1834D mutant can stimulate HMGA2 expression as well as H3-K9 acetylation in HCC cells. Furthermore, we identified that p300-mediated H3-K9 acetylation participates in EGF-induced HMGA2 expression in HCC. In addition, the levels of H3-K9 acetylation positively correlated with the expression levels of HMGA2 in a chemically induced HCC model in rats and human HCC specimens.

PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC.

High expression of programmed death-ligand-1 (PD-L1) in hepatocellular carcinoma (HCC) cells usually inhibits the proliferation and functions of T cells, leading to immune suppression in tumor microenvironment. However, very little has been described regarding the mechanism of PD-L1 overexpression in HCC cells. In the present study, we found epidermal growth factor (EGF) stimulation promoted the expression of PD-L1 mRNA and protein in HCC cells. Inhibition of epidermal growth factor receptor (EGFR) could reverse EGF-induced the expression of PD-L1 mRNA and protein. Subsequently, we also observed that the phosphorylation level of Pyruvate kinase isoform M2 (PKM2) at Ser37 site was also increased in response to EGF stimulation. Expression of a phosphorylation-mimic PKM2 S37D mutant stimulated PD-L1 expression as well as H3-Thr11 phosphorylation in HCC cells, while inhibition of PKM2 significantly blocked EGF-induced PD-L1 expression and H3-Thr11 phosphorylation. Furthermore, mutation of Thr11 of histone H3 into alanine abrogated EGF-induced mRNA and protein expression of PD-L1, Chromatin immunoprecipitation (ChIP) assay also suggested that EGF treatment resulted in enhanced H3-Thr11 phosphorylation at the PD-L1 promoter. In a diethylnitrosamine (DEN)-induced rat model of HCC, we found that the expression of phosphorylated EGFR, PKM2 nuclear expression, H3-Thr11 phosphorylation as well as PD-L1 mRNA and protein was higher in the livers than that in normal rat livers. Taken together, our study suggested that PKM2-dependent histone H3-Thr11 phosphorylation was crucial for EGF-induced PD-L1 expression at transcriptional level in HCC. These findings may provide an alternative target for the treatment of hepatocellular carcinoma.

EGF promotes DKK1 transcription in hepatocellular carcinoma by enhancing the phosphorylation and acetylation of histone H3.

The protein Dickkopf-1 (DKK1) is frequently overexpressed at the transcript level in hepatocellular carcinoma (HCC) and promotes metastatic progression through the induction of ß-catenin, a Wnt signaling effector. We investigated how DKK1 expression is induced in HCC and found that activation of the epidermal growth factor receptor (EGFR) promoted parallel MEK-ERK and PI3K-Akt pathway signaling that converged to epigenetically stimulate DKK1 transcription. In HCC cell lines stimulated with EGF, EGFR-activated ERK phosphorylated the kinase PKM2 at Ser37, which promoted its nuclear translocation. Also in these cells, EGFR-activated Akt phosphorylated the acetyltransferase p300 at Ser1834 Subsequently, PKM2 and p300 mediated the phosphorylation and acetylation, respectively, of histone H3 at the DKK1 promoter, which synergistically enhanced DKK1 transcription. The mechanism was supported with mutational analyses in cells and in a chemically induced HCC model in rats. The findings suggest that dual inhibition of the MEK and PI3K pathways might suppress the expression of DKK1 and, consequently, tumor metastasis in patients with HCC.

Characterization of Formononetin Sulfonation in SULT1A3 Overexpressing HKE293 Cells: Involvement of Multidrug Resistance-Associated Protein 4 in Excretion of Sulfate.

Formononetin is one of the main active compounds of traditional Chinese herbal medicine Astragalus membranaceus. However, disposition of formononetin via sulfonation pathway remains undefined. Here, expression-activity correlation was performed to identify the contributing of SULT1A3 to formononetin metabolism. Then the sulfonation of formononetin and excretion of its sulfate were investigated in SULT1A3 overexpressing human embryonic kidney 293 cells (or HKE-SULT1A3 cells) with significant expression of breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4). As a result, formononetin sulfonation was significantly correlated with SULT1A3 protein levels (r = 0.728; p < 0.05) in a bank of individual human intestine S9 fractions (n = 9). HEK-SULT1A3 cells catalyzed formononetin formation of a monosulfate metabolite. Sulfate formation of formononetin in HEK-SULT1A3 cell lysate followed the Michaelis-Menten kinetics (Vmax = 13.94 pmol/min/mg and Km = 6.17 µM). Reduced activity of MRP4 by MK-571 caused significant decrease in the excretion rate (79.1%-94.6%) and efflux clearance (85.3%-98.0%) of formononetin sulfate, whereas the BCRP specific inhibitor Ko143 had no effect. Furthermore, silencing of MRP4 led to obvious decrease in sulfate excretion rates (>32.8%) and efflux clearance (>50.6%). It was worth noting that the fraction of dose metabolized (fmet), an indicator of the extent of drug sulfonation, was also decreased (maximal 26.7%) with the knockdown of MRP4. In conclusion, SULT1A3 was of great significance in determining sulfonation of formononetin. HEK-SULT1A3 cells catalyzed formononetin formation of a monosulfate. MRP4 mainly contributed to cellular excretion of formononetin sulfate and further mediated the intracellular sulfonation of formononetin.