Computer simulation of molecular docking between methylene blue and some proteins of Porphyromonas gingivalis / 北京大学学报(医学版)

OBJECTIVE@#To study the binding target of photosensitizer and bacteria in antimicrobial photodynamic therapy with computer-simulated target prediction and molecular docking research methods and to calculate the binding energy.@*METHODS@#The protein names of Porphyromonas gingivalis (Pg) were obtained and summarized in Uniprot database and RCSB PDB database; the structure diagrams of methy-lene blue were screened in SciFinder database, PubChem database, ChemSpider database, and Chemical Book, and ChemBioDraw software was used to draw and confirm the three-dimensional structure for target prediction and Cytoscape software was used to build a visual network diagram; a protein interaction network was searched and built between the methylene blue target and the common target of Pg in the String database; then we selected FimA, Mfa4, RgpB, and Kgp K1 proteins, used AutoDock software to calculate the docking energy of methylene blue and the above-mentioned proteins and performed molecular docking.@*RESULTS@#The target prediction results showed that there were 19 common targets between the 268 potential targets of methylene blue and 1 865 Pg proteins. The 19 targets were: groS, radA, rplA, dps, fabH, pyrG, thyA, panC, RHO, frdA, ileS, bioA, def, ddl, TPR, murA, lepB, cobT, and gyrB. The results of the molecular docking showed that methylene blue could bind to 9 sites of FimA protein, with a binding energy of -6.26 kcal/mol; with 4 sites of Mfa4 protein and hydrogen bond formation site GLU47, and the binding energy of -5.91 kcal/mol, the binding energy of LYS80, the hydrogen bond forming site of RgpB protein, was -5.14 kcal/mol, and the binding energy of 6 sites of Kgp K1 protein and the hydrogen bond forming site GLY1114 of -5.07 kcal/mol.@*CONCLUSION@#Computer simulation of target prediction and molecular docking technology can initially reveal the binding, degree of binding and binding sites of methylene blue and Pg proteins. This method provides a reference for future research on the screening of binding sites of photosensitizers to cells and bacteria.

Effects of ophiopogonin D on fatty acid metabolic enzymes in cardiomyocytes / 中国中药杂志

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.

Ophiopogonin D interferes with ferroptosis to reduce the damage of cardiomyocytes induced by ophiopogonin D' / 药学学报

This study investigated the intervention effect and possible mechanism of ophiopogonin D (OPD) in protecting cardiomyocytes against ophiopogonin D' (OPD')-induced injury, and provided relevant experimental data for the clinical use of Ophiopogon japonicas. Cell counting kit-8 (CCK-8) assay was used to evaluate the effect of OPD and OPD' on H9c2 cell viability. The content of reaction oxygen species (ROS) in cells were detected by flow cytometry. The contents of Fe2+ in cells were detected by FerroOrange's fluorescence imaging. The content of glutathione (GSH) and glutathione peroxidase (GSH-Px) were detected by kits. The expression of transferrin receptor 1 (TFR1), cyclooxygenase 2 (COX2), NADPH oxidase 1 (NOX1), long-chain acyl-CoA synthetase 4 (ACSL4), cationic amino acid transporter 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and ferritin heavy chain 1 (FTH1) was detected by Western blot. Results showed that OPD' (1 μmol·L-1) significantly induced the expression of ferroptosis-related proteins, the contents of Fe2+, ROS, and GSH-Px were increased, and the content of GSH were decreased. In addition, different concentrations of OPD (0.5, 1, and 2 μmol·L-1) could partially reverse the myocardial cell injury caused by OPD', and the best effect was obtained when the dose range was 1-2 μmol·L-1. The experimental results show that OPD can interfere with the ferroptosis caused by OPD', and then have a protective effect on H9c2 cells.

Research progress in pharmacology and toxicology of evodiamine / 中国中药杂志

Evodiamine, a bioactive indole alkaloid from Evodia rutaecarpa, E. rutaecarpa var. officinalis, or E. rutaecarpa var. bodinieri, has been extensively investigated due to its pharmacological activities in recent years. At present, evodiamine is proved to significantly suppress the proliferation of a variety of cancer cells and mediate cell processes such as cell cycle arrest and cell migration. In addition, evodiamine displays significant pharmacological activities against cardiovascular diseases(hyperlipidemia, etc.), and tinea manus and pedis. Recently, evodiamine has been found to have potential toxic effects, such as hepatotoxicity, nephrotoxicity, and cardiotoxicity. However, the pharmacological and toxicological mechanism of evodiamine is not clear, and its toxicity in vitro and in vivo has been rarely reported. Therefore, this study reviewed the pharmacological and toxicological articles of evodiamine in recent years, aiming at providing new ideas and references for future research.

Expert consensus on Antiviral Oral Liquid in treatment of influenza in clinical practice / 中国中药杂志

Antiviral Oral Liquid is modified on the basis of Baihu Decoction in Treatise on Febrility Diseases by ZHANG Zhongjing and Qingwen Baidu Yin in Qing Dynasty, with effects in clearing toxic heat, repelling dampness and cooling blood. It is widely used in clinical treatment of common colds, influenza and upper respiratory tract infection, mumps, viral conjunctivitis and hand-foot-mouth disease, with a good clinical efficacy and safety. Based on a questionnaire survey of clinicians and a systematic review of study literatures on Antiviral Oral Liquid, the international clinical practice guidelines development method was adopted to analyze the optimal available evidences and expert experiences in the "evidence-based, consensus-based and experience-based" principles. The consensus was jointly reached by more than 30 multidisciplinary experts nationwide, including clinical experts of traditional Chinese and Western medicine in the field of respiratory diseases and infectious diseases, and methodological experts. In the study, literatures were retrieved based on clinical problems in the clinical survey as well as PICO clinical problems. The GRADE system was used for the classification and evaluation of evidence, and fully combined with clinical expert experience, so as to reach expert consensus by the nominal grouping method. This expert consensus recommended or suggested indications, usage and dosage, course of treatment, intervention time for treatment, and the safety and precautions of Antiviral Oral Liquid for treatment of influenza, and can provide reference for the rational use of this drug in clinical practice.

Ophiopogonin D protects cardiomyocytes against ophiopogonin D'-induced injury through suppressing endoplasmic reticulum stress / 中国中药杂志

This study is aimed to investigate the intervention effect and possible mechanism of ophiopogonin D( OPD) in protecting cardiomyocytes against ophiopogonin D'( OPD')-induced injury,and provide reference for further research on toxicity difference of saponins from ophiopogonins. CCK-8 assay was used to evaluate the effect of OPD and OPD' on cell viability. The effect of OPD on OPD'-induced cell apoptosis was measured by flow cytometry. Morphologies of endoplasmic reticulum were observed by endoplasmic reticulum fluorescent probe. PERK,ATF-4,Bip and CHOP mRNA levels were detected by Real-time quantitative polymerase chain reaction( PCR) analysis. ATF-4,phosphorylated PERK and e IF2α protein levels were detected by Western blot assay. RESULTS:: showed that treatment with OPD'( 6 μmol·L-1) significantly increased the rate of apoptosis; expressions of endoplasmic reticulum stress related genes were increased. The morphology of the endoplasmic reticulum was changed. In addition,different concentrations of OPD could partially reverse the myocardial cell injury caused by OPD'. The experimental results showed that OPD'-induced myocardial toxicity may be associated with the endoplasmic reticulum stress,and OPD may modulate the expression of CYP2 J3 to relieve the endoplasmic reticulum stress caused by OPD'.

Aconitine ameliorates cardiomyocyte hypertrophy induced by angiotensin Ⅱ / 中国中药杂志

This paper was aimed to investigate the inhibitory effect of aconitine(AC) on angiotensin Ⅱ(Ang Ⅱ)-induced H9 c2 cell hypertrophy and explore its mechanism of action. The model of hypertrophy was induced by Ang Ⅱ(1×10-6 mol·L-1),and cardiomyocytes were incubated with different concentrations of AC. Western blot was used to quantify the protein expression levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain(β-MHC),and α-smooth muscle actin(α-SMA). Real-time quantitative PCR(qRT-PCR) was used to quantify the mRNA expression levels of cardiac hypertrophic markers ANP,BNP and β-MHC. In addition,the fluorescence intensity of the F-actin marker,an important component of myofibrils,was detected by using laser confocal microscope. AC could significantly reverse the increase of total protein content in H9 c2 cells induced by Ang Ⅱ; qRT-PCR results showed that AC could significantly inhibit the ANP,BNP and β-MHC mRNA up-regulation induced by AngⅡ. Western blot results showed that AC could significantly inhibit the ANP,BNP and β-MHC protein up-regulation induced by AngⅡ. In addition,F-actin expression induced by Ang Ⅱ could be inhibited by AC,and multiple indicators of cardiomyocyte hypertrophy induced by Ang Ⅱ could be down-regulated,indicating that AC may inhibit cardiac hypertrophy by inhibiting the expression of hypertrophic factors,providing new clues for exploring the cardiovascular protection of AC.

Effect of six components in Polygoni Multiflori Radix on regulation of CYP3A4 mediated by human pregnane X receptor / 中国中药杂志

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.

Retrospective analysis and discussion on 74 cases of adverse reactions of traditional Chinese medicine injection / 中国中药杂志

As a modern dosage form drug with rapid effect, traditional Chinese medicine (TCM) injection has been more and more used in clinical practice. Meanwhile the safety of TCM injection has attracted more and more attention. The retrospective analysis on 74 cases of adverse reaction of TCM injections collected from 2007 to 2016 in the Third Affiliated Hospital of Beijing University of Chinese Medicine showed that the proportion of men and women with adverse reactions was 0.54:1; the average age was 62.5 years old; 21 kinds of TCM injections were involved. Among them, the most reported were blood-regulating agents. The top four kinds of TCM injections with highest adverse drug reactions (ADRs) were Tanreqing injection, Danhong Injection, Shuxuening Injection and Xuesaitong for injection. The top three clinical manifestations of adverse reactions were lesions of skin and its appendages, damage of circulatory system and damage of nervous system. The potential causes of the adverse reactions of TCM injections were analyzed, and it was believed that individual difference, medicine, pharmaceutical excipients, solvent and TCM syndrome differentiation may be the main five causes for the adverse reactions of TCM injections. In order to reduce the adverse reactions of TCM injections, it is suggested that the clinical pharmacists should participate in the application management of TCM injections in the hospital; the production enterprises shall strengthen the whole life cycle management of the drugs; and at the same time, the drug control and administration authorities should improve the drug management methods constantly and encourage the development of TCM injections to the high quality level.

Transcriptional regulation effect of constituents in Polygonum multiflorum Thunb.based on human progesterone X receptor(hPXR)mediated CYP3A4 rapid screening system / 中国药理学与毒理学杂志

OBJECTIVE This study investigated transcriptional regulation of the main chemical con-stituents of Polygonum multiflorum Thunb. including Stilbene Glucoside (THSG) and anthraquinone constituents (Emodin, Rhein, Aloeemodin, Chrysophanol and Physcion) and six potential liver injury constituents(gallic acid,quercetin,luteolin,kaempferol,resveratrol)on mediated by PXR CYP3A4.Early establishment of pregnane X receptor mediated CYP3A4 drug induced rapid screening technique was used to determine the effects of these constituents. METHODS First,effect of constituents on the cell activity was detected by MTS cell viability assay. IC50was calculated. Second, the expression vector and reporter vector were co-transfected into Hep G2 cells,10 μmol·L-1Rifampicin as a positive control, 10 μmol·L-1Ketoconazole as a negative control. After treated with different concentrations of (the an-thraquinone constituents concentrations were 2.5,5 and 10 μmol·L-1;the concentrations of Gallic Acid, Quercetin,Luteolin,Kaempferol,Apigenin,Resveratrol concentrations were 5,10 and 20 μmol·L-1)for 24 h,the cells were tested for dual luciferase activity.RESULTS The results show that the inhibitory ef-fect of THSG,Chrysophanol,Emodin,Rhein and Aloeemodin on CYP3A4 was inhibited by co-transfec-tion of pcDNA3.1 and pGL4.17-CYP3A4. The expression of pcDNA3.14-PXR and pGL4.17-CYP3A4 was induced by the four constituents. Besides, Emodin has a directly inducing effect. Four anthraqui-none constituentscan induce the effect of CYP3A4 by PXR, but Emodin can directly induce CYP3A4. THSG can inhibit CYP3A4,but in the presence of PXR plasmid can induce CYP3A4.For the six poten-tial liver injury constituents, results show that the plasmid pcDNA3.1 was cotransfected with pGL4.17-CYP3A4 regulation of Gallic Acid and Resveratrol on CYP3A4 inhibitory effects of Quercetin,Luteolin, Kaempferol have an induce effect; after pcDNA3.14-PXR and pGL4.17-CYP3A4 cotransfected, Quercetin, Luteolin, Kaempferol, Apigenin, Resveratrol have induced effect, three constituents'induc-tion effect had significant difference.CONCLUSION 12 kinds of Polygonum multiflorum Thunb.constit-uents have inhibitory or activating effects on CYP3A4, after the participation of PXR, 9 components have induced effects on CYP3A4, and the induction effect of 6 components has significant difference. The results suggested that we should pay attention to potential drug interactions when combined with Polygonum multiflorum Thunb.,and improve safety and efficacy.

Role of vitamin K-dependent protein Gas6 in the expression of endothelial cell adhesion molecule-1 and chemokines induced by Porphyromonas gingivalis lipopolysaccharide / 北京大学学报(医学版)

OBJECTIVE@#Growth-arrest-specific protein 6 (Gas6) is a vitamin K-dependent protein and involved in cell proliferation, survival, adhesion and migration . Also it has been shown to play an important role in the inflammatory response .The aim of present study was to investigate the role of Gas6 in the process of the expression of adhesion molecules and chemokines of human umbilical vein endothelial cells (HUVECs) induced by Porphyromonas gingivalis lipopolysaccharide(P.g-LPS).@*METHODS@#After up-regulation and down-regulation of the expression of Gas6, the vascular endothelial cells were stimulated with 1 mg/L P.g-LPS for 3 h and 24 h. Real-time quantitative polymerase chain reaction(real-time PCR) was taken to detect the expression of the cell adhesion molecules:intercellular adhesion molecule-1 (ICAM-1) and E-selectin, as well as chemokines:interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1). Wound healing assay was taken to observe the migration ability of endothelium cells in different groups.@*RESULTS@#After 3 h of P.g-LPS stimulation, the expression of adhesion molecules and chemokine in the down-regulation group was not significantly different from that in the control group,while in the up-regulation group the decrease of E-selectin, ICAM-1, IL-8 and MCP-1 was 81%±0%, 47%±3%, 76% ± 3%, 26% ± 6% respectively. After 24 h of P.g-LPS stimulation, the expression of adhesion molecules and chemokine in down-regulation group was significantly higher than that in control group (2.06±0.07, 1.99±0.11, 3.14±0.15, 1.84±0.03 flod), while these molecules in the down-regulation group was significantly lower than in the control group (29%±1%, 62%±3%, 69%±1%, 41%±2%). Differences were statistically significant (P<0.01). Wounding healing assay showed that down-regulation of Gas6 enhanced migration ability of endothelial cells while up-regulation of Gas6 weakened this ability,which was consistent with the trend of real-time PCR result.@*CONCLUSION@#Down-regulation of the Gas6 gene enhanced the expression of ICAM-1, E-selectin, IL-8 and MCP-1 in HUVECs after P.g- LPS stimulating, while up-regulaiton of the Gas6 gene weakened the expression of ICAM-1, E-selectin, IL-8 and MCP-1 in HUVECs after P.g-LPS stimulating,suggesting that Gas6 may play a role in the process of endothelial cell adhesion.

Expert consensus on prescription comment of Chinese traditional patent medicine for promoting the rational use of drugs in Beijing / 中国中药杂志

With the growth of number of Chinese patent medicines and clinical use, the rational use of Chinese medicine is becoming more and more serious. Due to the complexity of Chinese medicine theory and the uncertainty of clinical application, the prescription review of Chinese patent medicine always relied on experience in their respective, leading to the uncontrolled of clinical rational use. According to the traditional Chinese medicine (TCM) theory and characteristics of the unique clinical therapeutics, based on the practice experience and expertise comments, our paper formed the expert consensus on the prescription review of Chinese traditional patent medicine for promoting the rational use of drugs in Beijing. The objective, methods and key points of prescription review of Chinese patent medicine, were included in this expert consensus, in order to regulate the behavior of prescription and promote rational drug use.

Effect of ophiopogonin D in resisting vascular endothelial cell apoptosis induced by AngⅡthrough up-regulating CYP2J2/EETs / 中国中药杂志

This study aimed to investigate the effect and mechanism of ophiopogonin D (OP-D) on Ang Ⅱ-induced HUVECs apoptosis, in order to provide a reliable basis for the safety and efficacy of traditional Chinese medicines. The effect of Ang Ⅱ on survival and total proteins content of HUVECs were measured by MTT and Western blotting. The effect of OP-D on Ang Ⅱ-induced lactate dehydrogenase (LDH) release rate in HUVECs was measured by enzyme standard instrument. The effects of OP-D and 11,12-EET on phosphorylation of JNK/c-Jun induced by Ang Ⅱ were measured by Western blot and RT-PCR with the help of JNK specific inhibitor SP600125 and CYP450 isozymes selective inhibitor 6-(2-propargyloxyphenyl) hexanoic acid (PPOH). The cell apoptosis was assayed by flow cytometry. According to the results, different doses of Ang Ⅱ had no significant effect on cell survival; treatment with Ang Ⅱ at 1×10⁻⁶ mol·L⁻¹ could increase the release of LDH (<0.001). The phosphorylation of JNK and c-Jun could be inhibited by the pre-treatment with SP600125, 11,12-EET and OP-D. Pre-treatment with OP-D could significantly reduce the release of LDH induced by Ang Ⅱ stimulation, decrease the expression of caspase-3, and diminish the apoptosis of cells. The protective effect of OP-D was suppressed, when being pretreated with PPOH. The experimental results showed that the apoptosis of HUVECs induced by Ang Ⅱ may be associated with JNK/c-Jun signaling pathway. OP-D-mediated CYP2J2 expression increased 11,12-EET levels, and could remarkably resist Ang Ⅱ-induced injury and apoptosis of cells, which is associated with the maintenance of endothelium homeostasis.

Establishment and verification of risk assessment scale for clinical safety medication of aconitine / 中国中药杂志

Chinese patent medicine containing aconitine is the key in clinical rational drug use. These drugs contain Chuanwu, Caowu or Fuzi, and Aconitum brachypodum with functions of expelling wind-dampness or tonifying Yang, all of which shall be used by strictly following the indications and dosage. However, there are many kinds of such drugs. Not only the unfamiliar knowledge of some Chinese and Western physicians about the characteristics of them, but also the combination of multiple drugs from different clinical departments, would increase the risk of aconitine poisoning. Based on the previous research, this paper proposed three core elements "syndrome differentiation-dosage differentiation-toxicity differentiation" from the prescription review and pharmacy consulting work, and objective and standardized evaluation was used to build a risk assessment scale containing 3 categories, 9 items and 36 indicators with Hulisan Jiaonang and Qufeng Zhitong Jiaonang as the example. This scale was used to evaluate the risk of a therapeutic regimen before and after the implementation. According to the verification of the existing adverse reaction cases, the risk assessment scale can be used to indicate the risk of drug treatment program and identify the risk level of drug treatment status. This paper tried to provide a methodological paradigm for scientific and objective evaluation on the safety of Chinese patent medicines, and help to identify the key links and risk prevention in the rational use by Chinese medicine physicians and pharmacists.

Construction of semantic relations of the top-level ontology of military medicine / 军事医学

Objective To explore a construction method of semantic relations of the top-level ontology of military medicine in order to construct the top-level ontology semantic network.Methods The military medical corpus was selected,and the relationships between the concepts were extracted using text analysis.On the basis of inheriting the semantic relations of UMLS,the special semantic relations of military medicine were added.Results A method of establishing the semantic relations of the top-level ontology of military medicine was proposed,the applicability of which was verified by example of the branch of military medical equipment.Conclusion The proposed method is effective and feasible,which can provide important support to the establishment of top-level semantic networks of military medicine.

Transcriptional regulation effect of THSG and anthraquinones in tubers of Polygonum multiflorum based on human progesterone X receptor (PXR) mediated CYP3A4 rapid screening system / 中国中药杂志

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-β-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.

Protective effect of ginsenoside Rb₁ on doxorubicin-induced myocardial autophagy / 中国中药杂志

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 / 中国中药杂志

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.

3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation / 中国中药杂志

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 / 中国中药杂志

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.