Compound Shouwu Jiangzhi Granule regulates triacylglyceride synthesis to alleviate hepatic lipid accumulation.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease with few therapeutic options currently available. Traditional Chinese medicine has been used for thousands of years and exhibited remarkable advantages against such complicated disease for its "multi-component, multi-target and multi-pathway" characteristics. Compound Shouwu Jiangzhi Granule (CSJG) is a clinical empirical prescription for the treatment of NAFLD, but its pharmacological mechanism remains unknown. METHODS: The clinical efficacy of CSJG was retrospectively analyzed in NAFLD patients by comparing blood biomarkers levels and liver MR images before and after CSJG treatment. Then, high-fat/high-fructose (HFHF) diet-induced NAFLD mice were used to further confirm CSJG's effect against hepatic lipid accumulation through hepatic lipid determination and histopathological staining of liver samples. Next, the ingredients of CSJG were determined, and network pharmacology analysis was performed to predict potential targets of CSJG, followed by quantitative PCR (qPCR) and western blotting for verification. Then, lipidomics study was carried out to further explore the anti-NAFLD mechanism of CSJG from the perspective of triacylglyceride (TAG) synthesis but not free fatty acid (FFA) synthesis. The enzymes involved in this process were assayed by qPCR and western blotting. The potential interactions between the key enzymes of TAG synthesis and the active ingredients of CSJG were analyzed by molecular docking. RESULTS: CSJG attenuated blood lipid levels and hepatic fat accumulation in both NAFLD patients and mice. Although network pharmacology analysis revealed the FFA synthesis pathway, CSJG only slightly affected it. Through lipidomics analysis, GSJG was found to significantly block the synthesis of diglycerides (DAGs) and TAGs in the liver, with decreased DGAT2 and increased PLD1 protein expression, which diverted DAGs from the synthesis of TAGs to the production of PEs, PCs and PAs and thus lowed TAGs level. Molecular docking suggested that rhein, luteolin and liquiritigenin from CSJG might be involved in this regulation. CONCLUSION: Clinical and experimental evidence demonstrated that CSJG is a promising agent for the treatment of NAFLD. CSJG regulated TAGs synthesis to alleviate hepatic lipid accumulation. Rhein, luteolin and liquiritigenin from CSJG might play a role in it.

An UHPLC-QTOF-MS-based strategy for systematic profiling of chemical constituents and associated in vivo metabolites of a famous traditional Chinese medicine formula, Yinchenhao decoction.

Yinchenhao decoction (YCHD), a famous traditional Chinese medicine formula, has been applied for relieving jaundice in China for more than 1800 years. However, the material basis for YCHD is still unclear, and the chemical composition and metabolism characteristic in vivo are undefined, making the potential effective constituents and mechanism of action unclear. Herein, an ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS)-based strategy was applied for the chemical profiling of YCHD, as well as their in vivo prototypes and global metabolites that defined the metabolome. Our results showed that a total of 139 chemicals were identified in YCHD, including 28 organic acids, 12 monoterpenoids, five diterpenes, three triterpenoids, 17 iridoids, 23 anthraquinones, 26 flavonoids, four coumarins and 21 other types. Moreover, 58 prototypes and 175 metabolites were found in rat biological samples after oral administration of YCHD; those distributed in plasma, liver, intestine and feces were suggested to be potentially effective substances. Oxidation, hydrogenation, decarboxylation and conjugations with methyl, sulfate and glucuronate were considered as the predominant metabolic pathways in vivo. In conclusion, this is a systemic study of chemical constituents and in vivo metabolome profiles of YCHD, contributing to the material basis understanding and further mechanism research.

Improved pharmacokinetics of tenofovir ester prodrugs strengthened the inhibition of HBV replication and the rebalance of hepatocellular metabolism in preclinical models.

Tenofovir (TFV) ester prodrugs, a class of nucleotide analogs (NAs), are the first-line clinical anti-hepatitis B virus (HBV) drugs with potent antiviral efficacy, low resistance rate and high safety. In this work, three marketed TFV ester drugs, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF) and tenofovir amibufenamide fumarate (TMF), were used as probes to investigate the relationships among prodrug structures, pharmacokinetic characteristics, metabolic activations, pharmacological responses and to reveal the key factors of TFV ester prodrug design. The results indicated that TMF and TAF exhibited significantly stronger inhibition of HBV DNA replication than did TDF in HBV-positive HepG2.2.15 cells. The anti-HBV activity of TMF was slightly stronger than TAF after 9 days of treatment (EC50 7.29 ± 0.71 nM vs. 12.17 ± 0.56 nM). Similar results were observed in the HBV decline period post drug administration to the HBV transgenic mouse model, although these three TFV prodrugs finally achieved the same anti-HBV effect after 42 days treatments. Furthermore, TFV ester prodrugs showed a correcting effect on disordered host hepatic biochemical metabolism, including TCA cycle, glycolysis, pentose phosphate pathway, purine/pyrimidine metabolism, amino acid metabolism, ketone body metabolism and phospholipid metabolism. The callback effects of the three TFV ester prodrugs were ranked as TMF > TAF > TDF. These advantages of TMF were believed to be attributed to its greater bioavailability in preclinical animals (SD rats, C57BL/6 mice and beagle dogs) and better target loading, especially in terms of the higher hepatic level of the pharmacologically active metabolite TFV-DP, which was tightly related to anti-HBV efficacy. Further analysis indicated that stability in intestinal fluid determined the actual amount of TFV prodrug at the absorption site, and hepatic/intestinal stability determined the maintenance amount of prodrug in circulation, both of which influenced the oral bioavailability of TFV prodrugs. In conclusion, our research revealed that improved pharmacokinetics of TFV ester prodrugs (especially intestinal stability) strengthened the inhibition of HBV replication and the rebalance of hepatocellular metabolism, which provides new insights and a basis for the design, modification and evaluation of new TFV prodrugs in the future.

[Comparison of in vivo pharmacokinetics of five constituents in Zhishi Zhizi Chi Decoction in normal rats and CUMS-induced depressive rats].

A LC-MS/MS method was developed for the rapid and simultaneous determination of genipin-1-ß-D-gentiobioside,geniposide,naringin,hesperidin and neohesperidin in SD rat plasma.The linear relationships of these five constituents in rats were validated,and the specificity,accuracy,precision and stability met the requirements.Their pharmacokinetic parameters were calculated by DAS 3.2.2,and the results showed that the metabolic process in vivo of the five constituents accorded with the characteristics of noncompartmental model.Their main pharmacokinetic parameters were listed as follows:(1) genipin-1-ß-D-gentiobioside:t_(1/2)(3.20±0.51)h,C_(max)(403.15±96.93)µg·L~(-1)and AUC_(0-t)(612.56±148.50)µg·L~(-1)·h for the model group,while t_(1/2)(3.07±0.75) h,C_(max)(229.50±60.63)µg·L~(-1)and AUC_(0-t)(413.14±76.37)µg·L~(-1)·h for the normal group;(2) geniposide:t_(1/2)(3.24±0.68) h,C_(max)(2 961.40±688.02)µg·L~(-1),and AUC_(0-t)(10 972.87±1 992.96)µg·L~(-1)·h for the model group,while t_(1/2)(4.56±0.96) h,C_(max)(1 833.27±558.13)µg·L~(-1),and AUC_(0-t)(8 996.27±3 053.48)µg·L~(-1)·h for the normal group;(3) naringin:t_(1/2)(1.64±0.59) h,C_(max)(415.13±259.54)µg·L~(-1),and AUC_(0-t)(608.62±289.05)µg·L~(-1)·h for the model group,while t_(1/2)(1.02±0.25) h,C_(max)(355.08±180.00)µg·L~(-1),and AUC_(0-t)(501.07±242.68)µg·L~(-1)·h for the normal group;(4) hesperidin:t_(1/2)(0.86±0.29) h,C_(max)(95.17±22.80)µg·L~(-1)and AUC_(0-t)(141.19±54.63)µg·L~(-1)·h for the model group,while t_(1/2)(0.95±0.31) h,C_(max)(46.48±18.33)µg·L~(-1)and AUC_(0-t)(69.51±14.73)µg·L~(-1)·h for the normal group;(5) neohesperidin:t_(1/2)(0.89±0.29) h,C_(max)(828.78±361.56)µg·L~(-1)and AUC_(0-t)(1 292.29±553.73)µg·L~(-1)·h for the model group,while t_(1/2)(0.90±0.31) h,C_(max)(314.68±172.45)µg·L~(-1)and AUC_(0-t)(385.99±138.55)µg·L~(-1)·h for the normal group.

Bufei Qingyu Granules Inhibit the Development of Systemic Sclerosis via Notch-1/Jagged-2 Signaling Pathway.

Systemic sclerosis (SSc) is a rare chronic autoimmune disorder, mainly characterized by skin sclerosis. In this study, Bufei Qingyu Granules (BQG), a Chinese herbal formula, was used to treat SSc. To better understand the effects and molecular mechanisms of BQG, we successfully established a Bleomycin- (BLM-) induced SSc mouse model, and the mice were treated by BQG. Meanwhile, transcriptomic and bioinformatics analyses were conducted on those samples. As a result, we visually showed that BQG ameliorated the overall health of mice, including body weight, spleen, and thymus index. Thus, it also significantly alleviated inflammation presented by Chemokine (C-X-C motif) ligand 2 (Cxcl2), vasculopathy characterized by α-smooth muscle actin (α-SMA), and fibrotic changes elaborated by not only pathological images, but also the hydroxyproline (HYP) content. After testing by transcriptomic analysis, Cxcl2, Synaptosomal-associated protein 25 (Snap25), and Eukaryotic translation initiation factor 3, and subunit J2 (Eif3j2) which were differentially expressed genes, were verified, so that the data were credible. We further found that BQG could regulate Notch signaling pathway by significantly decreasing both mRNA and protein expression levels of Notch-1 and Jagged-2. Hence, this study demonstrated that BQG could ameliorate the sclerotic skin in mice model involved in inflammation, vascular changes, and fibrosis effects, which was partly mediated by Notch signaling pathway.

Pharmacokinetics of the prototype and hydrolyzed carboxylic forms of ginkgolides A, B, and K administered as a ginkgo diterpene lactones meglumine injection in beagle dogs.

Ginkgo diterpene lactones meglumine injection (GDLI) is a commercially available product used for neuroprotection. However, the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI, i.e., ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), have never been fully evaluated in beagle dogs. In this work, a simple, sensitive, and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was developed, and the prototypes and total amounts of GA, GB, and GK were determined in beagle dog plasma. The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations. For the first time, the pharmacokinetics of GA, GB, and GK were fully assessed in three forms, i.e., the prototypes, the hydrolyzed carboxylic forms, and the total amounts, after intravenous administration of GDLI in beagle dogs. It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma, and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio. All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages. GA, GB, and GK showed a constant half-life approximately 2.7, 3.4, and 1.2 h, respectively, which were consistent for the forms at three dose levels (0.3, 1.0, and 3.0 mg·kg-1) and after a consecutive injection of GDLI for 7 days (1.0 mg·kg-1).

Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC-MS/MS method.

Tenofovir (TFV), a first-line anti-viral agent, has been prepared as various forms of prodrugs for better bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV-DP) in target cell to inhibit viral DNA replication. Hence, TFV-DP is the key active metabolite that exhibits anti-virus activity, its intracellular exposure and half-life determine the final activity. Therefore, simultaneous monitoring prodrug, TFV and TFV-DP in target cells will comprehensively evaluate TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV-DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide (TAF) as a representative of TFV prodrugs. A sensitive LC-MS/MS method was developed, and TAF, TFV and TFV-DP were separated on a XSelect HSS T3 column (4.6mm×150mm, 3.5µm, Waters) with gradient elution after protein precipitation. The method provided good linearity for all the compounds (2-500nM for TFV and TAF; 20-5000nM for TFV-DP) with the correlation coefficients (r) greater than 0.999. Intra- and inter-day accuracies (in terms of relative error, RE<10.4%) and precisions (in terms of coefficient of variation, CV<14.1%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.

Exploring the neuroprotective effects of ginkgolides injection in a rodent model of cerebral ischemia-reperfusion injury by GC-MS based metabolomic profiling.

Cerebral ischemia-reperfusion (I/R) injury usually contributes to mortality and disability after ischemic stroke. Ginkgolides injection (GIn), a standard preparation composed of ginkgo diterpene lactones extract, is clinically used for neuroprotective treatment on reconvalescents of cerebral infarction. However, the understanding about its therapeutic mechanism is still lacking. In this study, a gas chromatography-mass spectrometry (GC-MS) based metabolomic approach coupled with multivariate data analysis (MVDA) was applied to explore the neuroprotective effects of GIn in a rodent model of focal ischemic stroke induced by transient middle cerebral artery occlusion (tMCAO). Metabolomic profiling revealed a series of metabolic perturbations that underlie the cerebral I/R pathological events. GIn can reverse the I/R induced brain metabolic deviations by modulating multiple metabolic pathways, such as glycolysis, Krebs cycle, pentose phosphate pathway (PPP), γ-aminobutyrate (GABA) shunt and lipid metabolism. Moreover, the main bioactive components of GIn were distributed to brain tissue much more easily in tMCAO rats than in normal rats after an intravenous administration, suggesting that the increased cerebral exposure to ginkgolides in I/R pathological condition potentially facilitated the neuroprotective effects of GIn by directly targeting at brain. The present study provided valuable information for our understanding about metabolic changes of cerebral I/R injury and clinical application of GIn.

Pharmacokinetics and tissue distribution of ginkgolide A, ginkgolide B, and ginkgolide K after intravenous infusion of ginkgo diterpene lactones in a rat model.

Ginkgo diterpene lactones are compounds that are extracted from the Ginkgo biloba leaf and possess pharmacologic activities with neuroprotective effects. To address the poor bioavailability of ginkgo diterpene lactones, ginkgo diterpene lactone meglumine injection (GDLI) was formulated and is commercially available. In this study, a simple, sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for assessing the total amount and the amount of the prototype forms of ginkgolides A (GA), B (GB) and K (GK) in rat plasma and tissues. This method was used to calculate the concentrations of the hydrolysed carboxylic forms and assess the pharmacokinetics of the ginkgolides after intravenous (i.v.) GDLI administration in rats. Generally, all three ginkgolide forms showed dose-dependent plasma concentrations, and no obvious differences in pharmacokinetic parameters, i.e., area under the curve (AUC) of plasma concentration versus time and half-life, were observed after GDLI administration on 7 consecutive days. These ginkgolides primarily existed in the carboxylic form in the plasma, and the systemic concentrations of the carboxylic forms of GA and GB were 11- to 17- and 3- to 4-fold higher than those of their prototype forms, respectively. In contrast, dramatically increased levels of the GA and GB prototype lactones were detected in the liver and heart. GA, GB, and GK were extensively distributed in various organs/tissues; the highest levels were found in the kidneys, liver, and intestine, and the lowest levels were found in the brain. These data suggest that ginkgolides have difficulty crossing the blood-brain barrier and that their targets for protecting against cerebral ischaemia are located outside the central system.