Prognostic value of coronary CT angiography in heart failure patients with preserved ejection fraction.

OBJECTIVES: To investigate the prognostic value of coronary CT angiography (CCTA) in heart failure patients with preserved ejection fraction (HFpEF). METHODS: Between January 2009 and December 2013, 6497 participants (mean age 63 ± 9.4 [range 32-86] years; 4111 men) who underwent CCTA and echocardiography were prospectively included. Participants were divided into HFpEF group and without HFpEF group. The primary endpoint was major adverse cardiovascular events (MACEs), including cardiovascular mortality, nonfatal myocardial infarction (MI), or hospitalization for heart failure (HF). RESULTS: Among those participants, 3096 were identified with HFpEF and 3401 were without HFpEF. Higher prevalence of coronary atherosclerosis was observed in HFpEF group than those without (78.3% vs. 64.9%, p < 0.001). During a median of 11.0 [IQR: 9.0-12.0] years follow-up, participants with HFpEF exhibit a heightened risk of MACEs in CAD-RADS = 0, 1-2, and ≥ 3 respectively (p < 0.001 for all). In the risk-adjusted hazard analysis among participants with HFpEF, CAD-RADS = 1-2 increased a 2.5-time risk for non-fatal MI (adjusted HR: 2.5, 95% CI: 1.5 to 4.3, p < 0.001), while CAD-RADS ≥ 3 conferred 3.9-fold and 3.1-fold higher risk for cardiovascular mortality (adjusted HR: 3.9, 95% CI: 2.2 to 7.1, p < 0.001) and hospitalization due to HF (adjusted HR: 3.1, 95% CI: 1.9 to 5.3, p < 0.001) with reference to CAD-RADS = 0 respectively. CONCLUSIONS: Coronary artery disease is common in participants with HFpEF and associated with MACEs. Among those participants, the presence of CAD-RADS = 1-2 increased the risk of nonfatal MI, while CAD-RADS ≥ 3 were correlated with cardiovascular mortality and hospitalization due to HF. KEY POINTS: • Higher median of CACS and higher CAD-RADS categories were observed in the HFpEF group than those without (p < 0.001 for both). • Participants with HFpEF exhibit a heightened risk of MACEs in CAD-RADS = 0, 1-2, and ≥ 3 respectively (p < 0.001 for all). • In the risk-adjusted hazard analysis among participants with HFpEF, CAD-RADS =1-2 increased a 2.5-time risk for non-fatal MI (adjusted HR: 2.5, 95% CI: 1.5 to 4.3, p < 0.001) with reference to CAD-RADS = 0 respectively.

Plaque progression at coronary CT angiography links non-alcoholic fatty liver disease and cardiovascular events: a prospective single-center study.

OBJECTIVES: The presence of non-alcoholic fatty liver disease (NAFLD) has been associated with major adverse cardiovascular events (MACEs); however, the mechanisms that initiate the risk for MACEs in patients with NAFLD remain unknown. We sought to investigate whether plaque progression (PP), determined by coronary CT angiography (CCTA), moderate the relationship between NAFLD and MACEs. METHODS: A total of 1683 asymptomatic participants (mean age, 63.3 ± 9.4 [range, 38-85] years; 1117 men) who underwent baseline and follow-up CCTA examination were prospectively included in our study. All of the participants were divided into the NAFLD and non-NAFLD groups. PP was determined by follow-up CCTA. The primary endpoint was MACEs, defined as the composite of all-cause death, nonfatal myocardial infarction, and unplanned hospitalization for acute coronary syndrome leading to revascularization. RESULTS: At follow-up CCTA, participants with NAFLD showed higher incidence of PP than those without [33.0% (248/752) vs. 16.6% (155/931), p < 0.001]. Compared with non-NAFLD participants, participants with NAFLD had a lower 9.7-year event-free survival rate (80.9 vs. 66.4%, log-rank p < 0.001). Cox regression analysis revealed NAFLD was significantly associated with MACEs (HR = 1.63, 95% CI: 1.28 to 2.06, p < 0.001) after adjusting for covariables. However, this association was no longer significant after adjustment for PP (HR = 1.10, 95% CI: 0.84 to 1.45, p = 0.496). The mediation analysis revealed that PP had a significant indirect effect (ß = 0.0587, 95% CI: 0.0424 to 0.08, p < 0.001) and mediated 99.8% (p = 0.002) for the relationship between NAFLD and MACEs. CONCLUSIONS: Plaque progression, identified by follow-up CCTA, mediates the relationship between NAFLD and MACEs. KEY POINTS: The incidence of CCTA-identified PP was higher for participants with NAFLD than those without NAFLD (248/752 [33.0%] vs. 155/931 [16.6%], p < 0.001). Participants with NAFLD had a lower 9.7-year event-free survival rate than those without NAFLD (66.4% vs. 80.9%, log-rank p < 0.001). The mediation analysis revealed that PP had a significant indirect effect (ß = 0.0587, 95% CI: 0.0424 to 0.08, p < 0.001) and mediated 99.8% (p = 0.002) for the relationship between NAFLD and MACEs.

Coronary CT Angiography in Asymptomatic Adults with Hepatic Steatosis.

Background The long-term prognostic value of coronary CT angiography (CCTA) in asymptomatic adults with hepatic steatosis (HS) remains unknown. Purpose To evaluate the long-term prognostic value of CCTA in asymptomatic adults with HS. Materials and Methods Between January 2009 and December 2013, consecutive asymptomatic adults who underwent CCTA evaluation and unenhanced abdominal CT were prospectively enrolled. All participants were divided into two groups-with HS and without HS according to abdominal CT results. The primary end point was major adverse cardiovascular events (MACEs), defined as cardiac death, stroke, myocardial infarction, and angina requiring hospitalization. Multivariable Cox regression analysis and Kaplan-Meier analysis were used to compare survival rates. Results One thousand thirteen participants with HS and 1940 participants without HS who completed the follow-up were included (mean age, 66 years ± 10 [standard deviation] [range, 29-90 years]; 1940 men). During a median of 7.2 years of follow-up (interquartile range, 6.3-8.1), MACEs were observed in 96 of 1013 participants with HS (10%), whereas 80 of 1940 participants without HS (4%) had MACEs. In participants with a Coronary Artery Disease Reporting and Data System (CAD-RADS) category of 0, both participants with and without HS had a similar 8.8-year event-free survival rate (99.2% event-free survival rate in participants with HS vs 99.0% event-free survival rate in participants without HS, P = .77). As for participants with CAD-RADS categories 1 or 2 or 3-5, the 8.8-year event-free survival rate was lower in participants with HS than in those without HS (70.6% vs 85.2%, P < .001; 51.4% vs 71.7%, P = .03, respectively). The risk of MACEs was higher for participants with HS than for those without HS in CAD-RADS categories 1 and 2 (adjusted hazard ratio = 2.3; 95% CI: 1.4, 3.9; P < .001) and CAD-RADS categories 3-5 (adjusted HR = 2.1; 95% CI: 1.2, 3.6; P = .006) but not in the setting of CAD-RADS category 0 (adjusted HR = 5.1; 95% CI: 0.1, 398; P = .47). Conclusion Asymptomatic participants with hepatic steatosis (HS) had a worse prognosis than those without HS in the presence of coronary artery disease (CAD) at coronary CT angiography, whereas participants with HS and without CAD might have excellent clinical outcomes during a median follow-up of 7.2 years. © RSNA, 2021 Online supplemental material is available for this article.

[Effects of ophiopogonin D on fatty acid metabolic enzymes in cardiomyocytes].

To explore the effect of ophiopogonin D on main fatty acid metabolic enzymes in human cardiomyocyte AC-16,so as to provide reference for cardiovascular protection mechanism and safe clinical application of Ophiopogon japonicus.CCK-8 (cell counting kit-8) was used to detect the effect of different concentrations of ophiopogonin D on the viability of cardiomyocytes.Meanwhile,the effect of different concentrations of ophiopogonin D on the morphology and quantity of cardiomyocytes was observed under microscope.The effect of ophiopogonin D on the mRNA expression of CYP2J2,CYP4F3,CYP4A11,CYP4A22 and CYP4F2 in cardiomyocytes was detected by RT-PCR.Western blot was used to detect the protein expression of CYP4F3 in different concentrations of ophiopogonin D.Compared with the control group,low-concentration ophiopogonin D had no effect on the viability of cardiomyocytes.However,ophiopogonin D with a concentration of higher than 20µmol·L~(-1)could promote the viability.Under the microscope,ophiopogonin D with a concentration of below 100µmol·L~(-1)had no significant effect on the morphology and number of cardiomyocytes.RT-PCR results showed that compared with the control group,5µmol·L~(-1)ophiopogonin D could slightly up-regulate mRNA expressions of CYP2J2 and CYP4F3,while high-concentration ophiopogonin D (10 and 20µmol·L~(-1)) could significantly induce mRNA expressions of CYP2J2and CYP4F3 in a dose-dependent manner (P<0.05).The same concentration of ophiopogonin D had a little effect on the mRNA expressions of CYP4A11,CYP4A22 and CYP4F2.Western blot results showed that 20µmol·L~(-1)ophiopogonin D could significantly induce the protein expression of CYP4F3 in a dose-dependent manner (P<0.05).Based on the above results,ophiopogonin D (less than100µmol·L~(-1)) has no effect on the viability of AC-16 cardiomyocytes.Ophiopogonin D (less than 100µmol·L~(-1)) can selectively induce the expressions of CYP2J2 and CYP4F3,regulate the metabolic pathway of fatty acid signaling molecules,and thus protecting the cardiovascular system.

Differences in the Hemolytic Behavior of Two Isomers in Ophiopogon japonicus In Vitro and In Vivo and Their Risk Warnings.

Ophiopogonin D (OPD) and Ophiopogonin D' (OPD') are two bioactive ingredients in Ophiopogon japonicus. Previously published studies have often focused on the therapeutic effects related to OPD's antioxidant capacity but underestimated the cytotoxicity-related side effects of OPD', which may result in unpredictable risks. In this study, we reported another side effect of OPD', hemolysis, and what was unexpected was that this side effect also appeared with OPD. Although hemolysis effects for saponins are familiar to researchers, the hemolytic behavior of OPD or OPD' and the interactions between these two isomers are unique. Therefore, we investigated the effects of OPD and OPD' alone or in combination on the hemolytic behavior in vitro and in vivo and adopted chemical compatibility and proteomics methods to explain the potential mechanism. Meanwhile, to explain the drug-drug interactions (DDIs), molecular modeling was applied to explore the possible common targets. In this study, we reported that OPD' caused hemolysis both in vitro and in vivo, while OPD only caused hemolysis in vivo. We clarified the differences and DDIs in the hemolytic behavior of the two isomers. An analysis of the underlying mechanism governing this phenomenon showed that hemolysis caused by OPD or OPD' was related to the destruction of the redox balance of erythrocytes. In vivo, in addition to the redox imbalance, the proteomics data demonstrated that lipid metabolic disorders and mitochondrial energy metabolism are extensively involved by hemolysis. We provided a comprehensive description of the hemolysis of two isomers in Ophiopogon japonicus, and risk warnings related to hemolysis were presented. Our research also provided a positive reference for the development and further research of such bioactive components.

Author Correction: Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/ EETs and suppressing ER stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mitochondrial Iron Overload-Mediated Inhibition of Nrf2-HO-1/GPX4 Assisted ALI-Induced Nephrotoxicity.

Aristolactam I (ALI) is an active component derived from some Traditional Chinese medicines (TCMs), and also the important metabolite of aristolochic acid. Long-term administration of medicine-containing ALI was reported to be related to aristolochic acid nephropathy (AAN), which was attributed to ALI-induced nephrotoxicity. However, the toxic mechanism of action involved is still unclear. Recently, pathogenic ferroptosis mediated lipid peroxidation was demonstrated to cause kidney injury. Therefore, this study explored the role of ferroptosis induced by mitochondrial iron overload in ALI-induced nephrotoxicity, aiming to identify the possible toxic mechanism of ALI-induced chronic nephropathy. Our results showed that ALI inhibited HK-2 cell activity in a dose-dependent manner and significantly suppressed glutathione (GSH) levels, accompanying by significant increases in intracellular 4-hydroxynonenal (4-HNE) and intracellular iron ions. Moreover, the ALI-mediated cytotoxicity could be reversed by deferoxamine mesylate (DFO). Compared with other inhibitors, Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, obviously alleviated ALI-induced cytotoxicity. Furthermore, we have shown that ALI could remarkably increase the levels of superoxide anion and ferrous ions in mitochondria, and induce mitochondrial damage and condensed mitochondrial membrane density, the morphological characteristics of ferroptosis, all of which could be reversed by DFO. Interestingly, ALI dose-dependently inhibited these protein contents of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4), which could be partly rescued by Tin-protoporphyrin IX (SnPP) and mitoTEMPO co-treatment. In conclusion, our results demonstrated that mitochondrial iron overload-mediated antioxidant system inhibition would assist ALI-induced ferroptosis in renal tubular epithelial cells, and Nrf2-HO-1/GPX4 antioxidative system could be an important intervention target to prevent medicine containing ALI-induced nephropathy.

[Effect of six components in Polygoni Multiflori Radix on regulation of CYP3A4 mediated by human pregnane X receptor].

This paper aimed to study the six chemical components of Polygoni Multiflori Radix (gallic acid, quercetin, luteolin, kaempferol, resveratrol, apigenin). By the established pregnane X receptor (human pregnant X receptor, PXR) CYP3A4 mediated drug induced rapid screening technique, the effect of chemical components on the cell activity was detected by MTS cell method, and the value of IC50 was calculated. The dual luciferase reporter system was used to co-transfect PXR reporter gene expression vector containing transcriptional regulation and CYP3A4 with HepG2 cells, with 10 µmol·L⁻¹ rifampicin (RIF) as a positive control, and 10 µmol·L⁻¹ of ketoconazole (TKZ) as negative control. Gallic acid, quercetin, luteolin, kaempferol, apigenin, resveratrol(5, 10, 20 µmol·L⁻¹) were used to incubate for 24 h, and the luciferase activity was detected. The results showed that when plasmid pcDNA3.1 was co-transfected with pGL4.17-CYP3A4, gallic acid and resveratrol had an inhibitory effect on the regulation of CYP3A4, and quercetin, luteolin, kaempferol had an inductive effect on CYP3A4; when pcDNA3.14-PXR was co-transfected with pGL4.17-CYP3A4, quercetin, luteolin, kaempferol, apigenin, resveratrol had an inductive effect. To sum up, the 6 reported liver injury components had inhibitory or activating effects on CYP3A4. After PXR plasmid was involved, 5 components had an inductive effect on CYP3A4, and the inductive effects of 2 components were significantly different. In this experiment, we found that 2 kinds of potential liver injury components in Polygoni Multiflori Radix had been induced by CYP3A4, which was achieved through PXR regulation. It suggested that attention shall be paid to potential drug interactions when combined with Polygoni Multiflori Radix, so as to improve the safety and efficacy.

[Protective effect of ginsenoside Rb1 on doxorubicin-induced myocardial autophagy].

Ginsenoside Rb1 (Rb1), which is one of the main ingredients derived from Panax ginseng, has been found to have extensive pharmacological activities including antioxidant, anti-inflammatory, anticancer properties. In this study, the effect of Rb1 on doxorubicin-induced myocardial autophagy was studied with H9c2 as the study object. CCK-8 method, transmission electron microscope observation, fluorescence staining observation and Western blot were used to detect changes in H9c2 cell proliferation and autophagy after treatment. According to the results, doxorubicin could cause cell viability decrease, significant increase in the LC3-Ⅱ/LC3-I ratio and down-regulation of the expression of p62. Pretreatment with ginsenoside Rb1 inhibited cell viability decrease and increase in doxorubicin-induced autophagic structure and LC3-Ⅱ/LC3-I ratio, and down-regulation of the expression of p62. In conclusion, doxorubicin could induce H9c2 cell death and induce autophagy, and ginsenoside Rb1 showed a protective effect on DOX-induced cardiotoxicity, which may be correlated with suppression of DOX-induced autophagy.

[Effect of clinical doses of Realgar-Indigo Naturalis formula and large-dose of realgar on CYP450s of rat liver].

To investigate the effect of clinical dose of Realgar-Indigo Naturais formula (RIF) and large-dose of Realgar on main drug-metabolizing enzymes CYP450s of rat liver, as well as its regulatory effect on mRNA expression. Wistar rats were administrated orally with tested drugs for 14 days. A Cocktail method combined with HPLC-MS/MS was used in the determination of 4 cytochrome P450 isozymes (CYP1A2, CYP2B, CYP3A and CYP2C) in liver of the rats, and the mRNA expression levels of the above subtypes were detected by real-time fluorescent quantitative PCR. The results showed that RIF can significantly induce CYP1A2 and CYP2B enzyme activity, and inhibit CYP3A enzyme activity. This result was consistent with the mRNA expression. However, its single compound showed weaker or even contrary phenomenon. Different doses of Realgar also showed significant inconsistencies on CYP450 enzymes activity and mRNA expression. These phenomena may be relevant with RIF compatibility synergies or toxicity reduction. The results can also prompt drug interactions when RIF is combined with other medicines in application.

Influences of Realgar-Indigo naturalis, A Traditional Chinese Medicine Formula, on the Main CYP450 Activities in Rats Using a Cocktail Method.

The purpose of this work was to study the influences of Realgar-Indigo naturalis (RIF) and its principal element realgar on 4 main cytochrome P450 enzymes activities in rats. A simple and efficient cocktail method was developed to detect the four probe drugs simultaneously. In this study, Wistar rats were administered intragastric RIF and realgar for 14 days; mixed probe drugs were injected into rats by caudal vein. Through analyzing the pharmacokinetic parameter of mixed probe drugs in rats, we can calculate the CYPs activities. The results showed that RIF could inhibit CYP1A2 enzyme activity and induce CYP2C11 enzyme activity significantly. Interestingly, in realgar high dosage group, CYP3A1/2 enzyme activity was inhibited significantly, and different dosage of realgar manifested a good dose-dependent manner. The RIF results indicated that drug coadministrated with RIF may need to be paid attention in relation to drug-drug interactions (DDIs). Realgar, a toxic traditional Chinese medicine (TCM), does have curative effect on acute promyelocytic leukemia (APL). Its toxicity studies should be focused on. We found that, in realgar high dosage group, CYP3A1/2 enzymes activity was inhibited. This phenomenon may explain its potential toxicity mechanism.

[Transcriptional regulation effect of THSG and anthraquinones in tubers of Polygonum multiflorum based on human progesterone X receptor (PXR) mediated CYP3A4 rapid screening system].

The rapid screening technology was used to investigate the transcriptional regulation effect of main chemical constituents in tubers of Polygonum multiflorum, including 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside(THSG) and anthraquinones (such as rhein, chrysophanol, aloe-emodin, emodin) on CYP3A4 drug inducers induced by human pregnancy X receptor (PXR).The effect of chemical composition on the cell activity was detected by MTS cell viability assay. IC50 was calculated. The expression vector and the reporter vector were co-transfected into HepG2 cells, with 10 µmol•L⁻¹ rifampicin (RIF) as a positive control, and 10 µmol•L⁻¹ ketoconazole (TKZ) as a negative control. After treated with different concentrations of anthraquinones (2.5, 5, 10 µmol•L⁻¹) for 24 h, the cells were tested for dual luciferase activity. The results show that the inhibitory effect of THSG, chrysophanol, emodin, rhein and aloe-emodin on CYP3A4 was inhibited by co-transfection of pcDNA3.1 and pGL4.17-CYP3A4. The expressions of pcDNA3.14-PXR and pGL4.17-CYP3A4 were induced by the four compounds. Besides, emodin had a direct inducing effect. In conclusion, the four anthraquinone compounds have an inducing effect on CYP3A4 by PXR, but emodin can directly induce CYP3A4. THSG can inhibit CYP3A4, but plasmid can induce CYP3A4 after intervened with PXR.These results suggest that we should pay attention to the liver function and avoid liver damage in the combined administration of drugs.

Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/EETs and suppressing ER stress.

AIM: CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis. METHODS: Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca(2+) concentrations ([Ca(2+)]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA. RESULTS: Angiotensin II (10(-6) mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca(2+)]i in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules (GRP78, CHOP, p-JNK and cleaved caspase-12) and ER stress-mediated apoptosis. OPD (100, 250 and 500 nmol/L) dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca(2+) homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca(2+) homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca(2+) homeostasis. Furthermore, the intracellular Ca(2+) chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells. CONCLUSION: OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca(2+) homeostasis and ER stress.

Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation.

AIM: Pregnane X receptor (PXR) is a nuclear receptor that regulates a number of genes encoding drug metabolism enzymes and transporters and plays a key role in xeno- and endobiotic detoxification. Ginkgolide B has shown to increase the activity of PXR. Here we examined whether ginkgolide B activated PXR and attenuated xenobiotic-induced injuries in endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with ginkgolide B. The expression of PXR, CYP3A4, MDR1, VCAM-1, E-selectin and caspase-3 were quantified with qRT-PCR and Western blot analysis. Cell apoptosis was analyzed with flow cytometry. Fluorescently labeled human acute monocytic leukemia cells (THP-1 cells) were used to examine cell adhesion. RESULTS: Ginkgolide B (30-300 µmol/L) did not change the mRNA and protein levels of PXR in the cells, but dose-dependently increased nuclear translocation of PXR protein. Ginkgolide B increased the expression of CYP3A4 and MDR1 in the cells, which was partially reversed by pretreatment with the selective PXR signaling antagonist sulforaphane, or transfection with PXR siRNA. Functionally, ginkgolide B dose-dependently attenuated doxorubicin- or staurosporine-induced apoptosis, which was reversed by transfection with PXR siRNA. Moreover, ginkgolide B suppressed TNF-α-induced THP-1 cell adhesion and TNF-α-induced expression of vascular adhesion molecule 1 (VCAM-1) and E-selectin in the cells, which was also reversed by transfection with PXR siRNA. CONCLUSION: Ginkgolide B exerts anti-apoptotic and anti-inflammatory effects on endothelial cells via PXR activation, suggesting that a PXR-mediated endothelial detoxification program may be important for protecting endothelial cells from xeno- and endobiotic-induced injuries.

[3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation].

3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for HepG2 cells, their glycogen storage capacity under 2D and 3D culture conditions were detected by immunohistochemistry technology, and the mRNA expression levels of phase Ⅰ and Ⅱ drug metabolism enzymes, drug transporters, nuclear receptors and liver-specific marker albumin(ALB) were compared between 2D and 3D culture conditions by using RT-PCR method. Immunohistochemistry results showed that HepG2 cells had abundant glycogen storage capacity under 3D culture conditions, which was similar to human liver tissues. The mRNA expression levels of major drug metabolism enzymes, drug transporters, nuclear receptors and ALB in HepG2 cells under 3D culture conditions were up-regulated as compared with 2D culture conditions. For drug hepatotoxicity evaluation, the typical hepatotoxic drug acetaminophen(APAP), and most reported drugs Polygonum multiflorum Thunb.(Chinese name He-shou-wu) and Psoraleae corylifolia L.(Chinese name Bu-gu-zhi) were selected for single dose and repeated dose(7 d) exposure. In the repeated dose exposure test, 3D HepG2 cells showed higher sensitivity. This established 3D HepG2 cells model with magnetic levitation 3D culture techniques was more close to the human liver tissues both in morphology and functions, so it was a better 3D hepatotoxicity evaluation model.

[Effects of aqueous extract of Cassiae Semen on activity hepatic microsomal CYP450 isozymes in rats].

To study the effect of aqueous extract of Cassiae Semen on the activity, mRNA and protein expressions of cytochrome P450(CYP450) system in rat liver microsomes, microsomes of rat liver were prepared after the oral administration with aqueous extract of Cassiae Semen for 14 days. The enzyme activity was quantified by Cocktail method. Meanwhile, the mRNA and protein expressions of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1 in the livers were detected by RT-PCR and Western blot. The result of this experiment was that aqueous extract of Cassiae Semen obviously induced the enzyme activities of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1. Low dose of aqueous extract of Cassiae Semen significantly reduced the activity of CYP2D2, but the activity of CYP2D2 was significantly induced by middle dose and high dose of aqueous extract of Cassiae Semen. These subtypes were increased in a dose-dependent manner except for CYP3A1. The mRNA levels of CYP1A2, CYP2C11, CYP2D2 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant effect in CYP2B1 and CYP3A1 mRNA expressions. The protein levels of CYP2C11 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant difference. Since the enzyme activity, mRNA and protein expressions of CYP450, particularly CYP2C11and2E1subtypes, were induced or inhibited by aqueous extract of Cassiae Semen to varing degrees, suggesting the potential drug-drug interactions should be concerned.

[Effects of Reduning injection on activity of hepatic microsomal CYP450 isozymes in rats].

To research the influence of Reduning injection on the activity and mRNA expression of cytochrome P450 (CYP450) system in rat liver microsomes. Rat liver microsomes were prepared after a seven-days continuous administration of Reduning injection. An HPLC-MS method was applied to determine the specific metabolites of CYP450 probe substrates in rat liver microsomal incubations. The activity of CYP450 isozymes were represented by the formation of metabolites. The Real-time quantitative polymerase chain reaction (Q-PCR) was applied to determine the mRNA expression levels of CYP450. Reduning injection significantly reduced the activity of CYP2B1, 2C12, 2C13 (P < 0.01), but did not affect CYPlA2; low dose and high dose of Reduning injection had an inhibition trend on the activity of CYP2D2, but did not statistically differ from control group; low dose of Reduning injection significantly induced the activity of CYP3A1 (P < 0.01), high dose of Reduning injection had an induce trend on the activity of CYP3A1, but did not statistically differ from control. At the mRNA level, low and high dose of Reduning injection had an induce trend on the expression of CYP1A2, 2C11, 2D1, 2E1, 3A1, but did not statistically differ from control. Reduning injection significantly induced the activity of CYP2B1. Reduning injection significantly induced the activity of CYP3A1 in mRNA expression and enzyme activity levels, which may result adverse drug reaction after being combined with macrolides antibiotics. Reduning injection significantly reduced the activity of CYP2B1, 2C12, 2C13, 2D2 in enzyme activity levels, when combined with other drugs, it should be fully taken into account of the possible drug-drug interaction in order to avoid adverse side effects.

[Preliminary study on hepatotoxicity induced by dioscin and its possible mechanism].

Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor (AhR) mRNA and CYP1A mRNA and protein in HepG2 cells in vitro. Dioscin 0.5-32 µmol · L(-1) exposed to HepG2 cells for 12 h, cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase (LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope, and the effect on production of reactive oxygen species (ROS) was detected by flow cytometry. The mRNA expression of CYP1A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot. The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 0.5-32 µmol · L(-1). Compared with the control, the LDH release rate and ROS were significantly increased. The expression of CYPlA and AhR mRNA was increased. The expression of CYP1Al protein was increased after dioscin treatment, and resveratrol, an AhR antagonist, could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor (AhR) and induce the expression of CYP1A.

Serum Pharmacochemistry Analysis Using UPLC-Q-TOF/MS after Oral Administration to Rats of Shenfu Decoction.

The purpose of this study was to study the serum pharmacochemistry of SFD as well as the material basis through analyzing the constituents absorbed in blood. The SFD was orally administrated to Wistar rats at 20 g·kg(-1), and Ultra Performance Liquid Chromatography (UPLC) fingerprints of SFD were created. Serum samples were collected for analysis, and further data processing used MarkerLynx XS software. 19 ginsenosides and 16 alkaloids were detected in SFD. The absorption of alkaloids (mainly monoester diterpenoid alkaloids) increased when Aconitum carmichaeli Debx. was combined with Panax ginseng, while the ginsenosides remained stable. Diester diterpenoid alkaloids were not present in the serum samples. A suitable serum pharmacochemistry method was successfully established to study pharmacological effects and potential improvements in formulation. This may also be useful for toxicity reduction. We suspect that the increased absorption of the monoester diterpenoid alkaloids from the mixture of Panax and Radix, compared to the Panax only extract, may be the reason for the combination of the two herbs in popular medicine formulas in China.

Panax ginseng inhibits intestinal absorption of toxic Aconitum carmichaeli alkaloids in Vitro.

OBJECTIVE: To evaluate the rationality and compatibility of Shenfu Formula (, SFF), a typical Chinese medicine (CM) comprised of Panax ginseng and Aconitum carmichaeli. METHODS: Caco-2 cells were used to study the permeability of Aconitum carmichaeli marker compounds when the CM preparation was combined with Panax ginseng. P-glycoprotein (P-gp) activity and protein as well as multidrug resistance 1 (MDR1) mRNA were analyzed with rhodamine123 efflflux, western blot and real time quantitative polymerase chain reaction. RESULTS: Aconitine (AC), mesaconitine (MA), hypaconitine (HA) and fifive other active alkaloids in Aconitum carmichaeli were selected as marker compounds. Panax ginseng inhibited intestinal absorption of highly toxic AC, MA and HA from Aconitum carmichaeli in Caco-2 cells. P-gp and breast cancer resistance protein (BCRP) were observed to be involved in AC, MA and HA efflflux. Panax ginseng induced P-gp activity in Caco-2 cells via increased MDR1/P-gp expression. Thus, Panax ginseng facilitated P-gp-mediated efflflux of toxic Aconitum carmichaeli alkaloids and restricted their intestinal absorption without inflfluencing other active components. CONCLUSION: Future studies to elucidate mechanism of reduced toxicity of Aconitum carmichaeli when combined with Panax ginseng will guide future formula optimization.