Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however, the dose-dependent cardiotoxicity associated with DOX significantly limits its clinical application. In the present study, we investigated whether Rb1 could prevent DOX-induced apoptosis in H9C2 cells via aryl hydrocarbon receptor (AhR). H9C2 cells were treated with various concentrations (−μM) of Rb1. AhR, CYP1A protein and mRNA expression were quantified with Western blot and real-time PCR analyses. We also evaluated the expression levels of caspase-3 to assess the anti-apoptotic effects of Rb1. Our results showed that Rb1 attenuated DOX-induced cardiomyocytes injury and apoptosis and reduced caspase-3 and caspase-8, but not caspase-9 activity in DOX-treated H9C2 cells. Meanwhile, pre-treatment with Rb1 decreased the expression of caspase-3 and PARP in the protein levels, with no effects on cytochrome c, Bax, and Bcl-2 in DOX-stimulated cells. Rb1 markedly decreased the CYP1A1 and CYP1A2 expression induced by DOX. Furthermore, transfection with AhR siRNA or pre-treatment with AhR antagonist CH-223191 significantly inhibited the ability of Rb1 to decrease the induction of CYP1A, as well as caspase-3 protein levels following stimulation with DOX. In conclusion, these findings indicate that AhR plays an important role in the protection of Ginsenoside Rb1 against DOX-triggered apoptosis of H9C2 cells.
Apoptosis* ; Blotting, Western ; Cardiotoxicity ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cytochrome P-450 CYP1A1 ; Cytochrome P-450 CYP1A2 ; Cytochromes c ; Doxorubicin ; Myocytes, Cardiac ; Real-Time Polymerase Chain Reaction ; Receptors, Aryl Hydrocarbon* ; RNA, Messenger ; RNA, Small Interfering ; Transfection

OBJECTIVE To study the cardiotoxicity of ophiopogonin D′(OPD′) for rat H9c2 cardio? myocytes. METHODS H9c2 cells were exposed to OPD′ 0.1, 1, 5, 10, 20, 25 and 50 μmol·L-1 for 24 h. Cell viability was examined by MTS assay, and the morphological changes in H9c2 cells were quanti? fied. The cell nucleus injury was examined by high content immune fluorescence screening and the morphological changes were observed under a fluorescence microscope. After treatment with OPD′ 0.1, 1, 5 and 10 μmol·L- 1 for 24 h, the effect on reactive oxygen species (ROS), mitochondrial mem? brane potential(MMP) and apoptosis was detected by flow cytometry. RESULTS The viability was sig? nificantly reduced following exposure to OPD′ 0.1, 1, 5, 10, 20, 25 and 50 μmol·L- 1 (P<0.05,P<0.01). The IC50 value was 9.9 μmol ·L- 1 and cell shrinkage and apoptosis occurred. The levels of ROS and apoptosis rate of H9c2 cells were significantly increased after exposure to OPD′ 0.1, 1, 5 and 10 μmol·L-1 for 24 h (P<0.05,P<0.01) and MMP markedly declined (P<0.05,P<0.01). CONCLUSION OPD′ has significent cytotoxicity on H9c2 cells. It may be related to inducing apopotsis pathways.

OBJECTIVE To investigate the hepatotoxicity mechanisms of ethanol extract of Radix Polygoni Multiflori (RPM) and Radix Polygoni Multiflori Praeparata (RPMP) by high-content screen assay.METHODS HepG2 cells were treated with RPM (10,25,50,100,200 and 300 mg·L-1) and RPMP (10,50,100,300,600 and 1200 mg· L-1) for 3-24 h,respectively.The cell viability was detected by a CellTiter-GloTM luminescent cell viability assay kit.Cell count,reactive oxygen species (ROS),mitochondrial membrane potential (MMP),glutathione (GSH),superoxide dismutase 2 (SOD2),activating transcription factor 4 (ATF4),apoptosis,and cell cycles were investigated by high-content screen assay.Besides,SOD2 and ATF4 levels were confirmed by Western blotting.RESULTS RPM 300 mg· L-1 showed nearly 48 ％ reduction in cell viability compared with cell control (P＜0.01),while RPMP had no significant effect at the same concentration.Both RPM and RPMP decreased the level of MMP (P＜0.05) but incresed levels of GSH,ROS,SOD2 and ATF4 significantly (P＜0.05).Besides,RPM 200 mg· L-1 significantly increased the expression of SOD2 (P＜0.05) at 3 h by high-content screen assay,and the enhanced expression of ATF4 was shown at 6 h (P＜0.05).RPMP 300 mg· L-1 markedly increased the expression of ATF4 at 6 h (P＜0.05),while the expression of SOD2 significantly increased at 24 h (P＜0.05).CONCLUSION Both RPM and RPMP have some cytotoxicity,and the cytotoxicity of RPM is stronger than that of RPMP.The hepatotoxicity mechanisms of RPM and RPMP may be related to cell apoptosis caused by long-term oxidative stress and endoplasmic reticulum stress.

Aim To study whether Ophiopogonin D has an effect inhibitory on myocardial hypertrophy induced by AngiotensinⅡand its possible mechanism. Methods Rat myocardial cell line H9 c2 were cultured in vitro. The effect of Ophiopogonin D on cell vitality was tested by;H9 c2 cells were treated with AngⅡ 1μmol ·L-1 after 24h to induce the cardiac hypertrophy,then it was co-treated with different concentrations of Ophio-pogonin D were added for 24h. After above,the total protein content was detected by BCA method;Quantita-tive real-time PCR ( qRT-PCR ) technique was used to examine the expression of marker genes BNP and β-MHC mRNA ,which representing the function of hear-ing; Western blot was used method to detect the ex-pression of autophagy protein LC3 B and high-through-put screening technology was emptoyed to verify it. In addi-tion, the changes of mitochondrial membrane po-tential in H9c2 myocardial cell were also examined. Results The cell viability results showed that H9 c-2 cells exposed to different concentrations of AngⅡ had no significant effect on vitality compared with the con-trol group after 24 h,but high concentrations of Ophio-pogonin D ( 50 ~100μmol · L-1 ) could obviously in-hibit the cell activity. Ot-her experimental results showed that myocardial cells treated with AngⅡ for 24h could cause myocardial hypertrophy,which appar-ently displayed the growth level of specific hypertrophic gene mRNA expression and the marked increase of the total protein expression. As hypertrophy was activated by AngⅡ, cells autophagy would be significantly en-hanced at the same time, more-over, the mitochondrial membrane potential would be reduced. But the effects of Ophiopogonin D could significantly reverse those pathological changes. Conclusion All above experi-mental results indicate that Ophiopogonin D can in-hibitmyocardial hypertrophy induced by AngⅡand pos-sibly plays a critical role in cardiovascular protection.

Aim To observe the cytochrome 450 effect of ginsenoside Re on H9c2 cells, in order to clarify the molecular mechanism of ginsenoside Re. Methods H 9 c 2 cells were separately treated with ginsenoside Re for 1, 5, 10, 50, 100 μmol·L-1 or 6, 24, 36, 48, 60 h. CYP2C11, 2J3, 4A1, 4A3, 4F4 and ANP mR-NA expressions were analyzed by Real time PCR, and CYP4 A1 , 2 J3 protein expressions were detected by Western blot. Results Compared with control group, ginsenoside Re could effectively upregulate CYP2 C11 , CYP2 J3 , ANP mRNA expression to 1. 6 , 1. 8 , 3. 2 fold, and downregulate CYP4A1, CYP4A3, CYP4F4 mRNA expression to 0. 4, 0. 15, 0. 3 fold. Ginsen-oside Re could decrease CYP4 A1 protein expression in a concentration-dependent manner, while ginsenoside Re could increase CYP2 J3 protein expression in a con-centration-dependent manner. Conclusion Ginsen-oside could regulate CYP450 enzyme and change ANP gene expression, which might be the molecular mecha-nism of ginsenoside Re.

Aim With metabolomics method, to study Shen-Mai decoction’ s function on protecting the myo-cardial injured rats caused by doxorubicin for probing into the functioning mechanism of Shen-Mai decoction’ s medical effect. Methods By means of UPLC-TOF-MS, the metabolites of urine of the rats treated by Shen-Mai decoction were analyzed. Then, the differ-ences between each group of the metabolites were sought with PLS-DA ( the partial least square discrimi-nant analysis ) and OPLS-DA ( the orthogonal partial least squares discriminant analysis ) . VIP ( variable importance in projection ) and t test were used to screen out potential biomarkers. Results Fourteen endogenous metabolites such as succinyladenosine, a-denosine 2′, 3′-cyclic phosphate, S-( 3-methylbu-tanoyl )-dihydrolipoamide-E, cis-4-hydroxycyclohexy-lacetic acid, phenylbutyrylglutamine, 3-butyn-1-al, 3-hydroxytetradecanedioic acid, dihydrolipoamide and pyruvic acid, etc. were characterized. Conclusions The results indicate that Shen-Mai decoction can pro-tect the body from myocardial injury by regulating pu-rine metabolism, some acid metabolism, fat metabo-lism and energy metabolism, etc. The study expounds the functioning mechanism for Shen-Mai decoction ’ s medical effect in the body and provides theoretical grounds for the rationality of the two medical herbs ’ compatibility and their combination in clinical treat-ment of diseases.

Aim To study the influence of Si-Wu De-coction (SWD ) and its active components on cyto-chrome P450 activity and mRNA expression in rats in order to provide an experimental basis for compatibility of SWD.Methods SWD and its active components were intragastrically administrated for seven days,the doses of SWD was 10 g · kg -1 · d -1 ,the doses of fructose,ferulic acid,ligustrazine,peoniflorin were 0.334,0.002,0.011 and 0.022 g·kg -1 ·d -1 ,re-spectively.After administration for seven days,rats were executed,and liver microsomes were prepared. The effects of SWD and its active components on cyto-chrome P450 in rats were investigated by hybrid probe and liver microsomes incubation method.The level of mRNA expression in liver was detected by real-time quantitative polymerase chain reaction using specific target primers for CYP450 genes.The level of protein expression of CYP2B1 was detected by Western blot. Results Compared with the control group,fructose significantly decreased the activity of CYP1A2, CYP2B6,CYP2C9,CYP2D6;ferulic acid significantly decreased the activity of CYP2C9,CYP2B6;ligus-trazine significantly decreased the activity of CYP1A2, CYP2C9,CYP2B6;peoniflorin significantly decreased the activity of CYP2D6,CYP2B6;fructose,ferulic acid,peoniflorin inhibited the mRNA expression of CYP2B1;fructose,ferulic acid,ligustrazine and peon-iflorin also inhibit the protein expression of CYP2B1. Conclusion Fructose,ferulic acid,peoniflorin inhib-it the activity of CYP2B1,decrease the expression lev-els of mRNA and protein of CYP2B1.

OBJECTIVE To study the effect of the ethanol extract of Fructus Psoraleae(EEFP)on endogenous metabolites in rat urine based on metabolomics. METHODS Male SD rats were orally administered with EEFP at the doses of 0.54,1.08 and 1.62 g · kg-1,respectively,once a day for two consecutive weeks. Urine samples were collected for 12 h after the last administration. Data were acquired with the MassLynx software based on ultra-performance liquid chromatography quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS). The principal component analysis(PCA)and partial least squares-discriminant analysis(PLS-DA)were used to analyze the difference of endogenous metabolites in different groups,then putative biomarkers were found through the orthogonal partial least-squares-discriminant analysis(OPLS-DA),variable importance in the projection(VIP)and t test and their relative intensity were determined. RESULTS The results of PCA showed that samples of each group were clustered,all the groups were separated,and that the distance between the EEFP groups and the blank control group was increased in a dose-dependent manner. The relative contents of p-cresol glucuronide and galactose-beta-1,4-xylose were 40.0 ± 11.2,2.7 ± 2.6,16.8 ± 6.3 and 45.9 ± 16.4,32.6±22.1,8.0±8.3 in the EEFP 0.54,1.08 and 1.62 g·kg-1 groups,respectively,significantly lower than those of the control group,which were 107.0 ± 26.9 and 82.3 ± 13.6(P<0.01),respectively. The relative contents of 5-L-glutamyl-taurine,and gluconolactone were 22.4 ± 10.0,47.6 ± 19.1 and 138.2 ± 18.8,337.3±64.0 in EEFP 1.08 and 1.62 g·kg-1groups,respectively,significantly higher than those of the blank control group,which were 2.6±1.6 and 20.5±6.8,respectively(P<0.01). The relative content of D-pantothenoyl-L-cysteine was 74.2 ± 31.5 in the EEFP 1.62 g · kg-1 group,significantly higher than that in the blank control group(0.6±0.5)(P<0.01). As the dose of EEFP increased,D-pantothenoyl-L-cysteine,5-L-glutamyl-taurine,and gluconolactone had an upward trend(P<0.01),while galactose-beta-1,4-xylose and p-cresol glucuronide had a downward trend(P<0.01). CONCLUSION The two-week administration of EEFP has effect on the endogenous metabolites in urine. The substances identified are mainly related to energy metabolism,taurine,tyrosine and glucose metabolism.

OBJECTIVE To study the mitochondrial toxicity effect of Radix Aconiti Lateralis Praepa?rata(Fuzi)on H9c2 cardiomyocytes. METHODS H9c2 cells were exposed to Fuzi decoction 6.25, 12.5,25,50 and 100 g·L-1 for 24 h. Fluorescence staining and CCK-8 assay were used to detect cell viability. H9c2 cells were exposed to Fuzi decoction 6.25,12.5 and 25 g · L-1 for 24 h,while the effect on mitochondrial membrane potential and reactive oxygen species(ROS)was detected by flow cytometry. The fluorescence molecular probe and laser scanning confocal microscope were used to observe the effect on Ca2+ in cells,Ca2+ and superoxide in mitochondria. The effect on ATP concentration in cells was detected via firefly luciferin and the expression of Pgc-1α,Bcl-2 and Bax mRNA evaluated by real-time PCR,while the expression of Pgc-1α protein was measured by Western blotting. RESULTS H9c2 cell viability was significantly inhibited by Fuzi decoction 12.5-100 g · L-1(P<0.05,P<0.01). The IC50 value was 47.4669 g · L-1,while the 95%confidence limit was 32.5997-69.1145 g · L-1. After treatment with Fuzi decoction 25 g · L-1 ,the fluorescence intensity of ROS in the normal control group increased from 204±67 to 454±78(P<0.05),that of mitochondrial superoxide increased from 5.4±1.8 to 26.8±8.5 (P<0.01),mitochondrial membrane potential decreased from 1.7±0.5 to 0.8±0.4(P<0.05),the fluores?cence intensity of intracellular Ca2+increased from 7.8±0.8 to 22.1±0.5(P<0.05)while that of mitochon?drial Ca2+decreased from 38.0±4.3 to 9.2±1.6(P<0.01),and intracellular ATP concentration decreased from (10.6 ± 0.4)μmol · g-1 to (5.3 ± 1.1)μmol · g-1 protein (P<0.05). qPCR and Western blotting test results showed that compared with the normal control group ,Pgc-1αand Bcl-2 mRNA relative expression level in Fuzi decoction 25 g·L-1 group was decreased from 1.00±0.10 and 1.00±0.10 to 0.09±0.06(P<0.01)and 0.43±0.06(P<0.01),respectively, while the relative expression of Bax mRNA was increased from 1.00 ± 0.03 to 1.17 ± 0.06 (P<0.05),and the expression of Pgc-1α protein was decreased from 0.906±0.034 to 0.541±0.003(P<0.01). CONCLUSION Fuzi has some mitochondrial toxicity to cardiomy?opathy. This effect arises from the combined action of different mechanisms. Mitochondrial toxicity of myocytes may account for the cardiac toxicity of Fuzi.

Aim To study the effect of ginsenoside F1 on the enzyme activity and expression of gene of CYP3 A4 through activation of pregnane X receptor ( PXR ) . Methods With different concentrations of ginsenoside F1 treated on LS174T cells, the expression of CYP3A4 mRNA was determined by Q-PCR, and the enzyme activity was measured by P450-GloTM CYP3A4 assay according to the manufacturer′s instructions, fur-ther PXR-CYP3 A4 stable translation HepG2 cell lines were used to test ginsenoside F1 activates PXR by re-porter gene screening assay. Results The results re-vealed that the levels of CYP3 A4 gene and protein ex-pression were significantly increased by ginsenoside F1 in a concentration-dependent manner. At the same time, reporter gene screening showed that ginsenoside F1 could also enhance the transcriptional activity of PXR. Conclusion Ginsenoside F1 can significantly up-regulate the gene expression and enzyme activity of CYP3A4 via the PXR-CYP3A4 pathway.