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OBJECTIVES@#To explore the effects of hypoxic and hypobaric conditions on blood gas and erythrocyte-related indicators in rats.@*METHODS@#SD male rats were exposed to low-pressure hypoxic conditions simulating an altitude of 6500 m in a small or a large experimental cabin. Abdominal aortic blood samples were collected and blood gas indicators, red blood cells (RBCs) count, and hemoglobin (Hb) content were measured. The effects of exposure to different hypoxia times, different hypoxia modes, normal oxygen recovery after hypoxia, and re-hypoxia after hypoxia preconditioning on blood gas indicators, RBCs count and Hb content were investigated.@*RESULTS@#The effect of blood gas indicators was correlated with the length of exposure time of hypoxia and the reoxygenation after leaving the cabin. Hypoxia caused acid-base imbalance and its severity was associated with the duration of hypoxia; hypoxia also led to an increase in RBCs count and Hb content, and the increase was also related to the time exposed to hypoxia. The effects of reoxygenation on acid-base imbalance in rats caged in a small animal cabin were more severe that those in a large experimental cabin. Acetazolamide alleviated the effects of reoxygenation after leaving the cabin. Different hypoxia modes and administration of acetazolamide had little effect on RBCs count and Hb content. Normal oxygen recovery can alleviate the reoxygenation and acid-base imbalance of hypoxic rats after leaving the cabin and improve the increase in red blood cell and hemoglobin content caused by hypoxia. The improvement of hypoxia preconditioning on post hypoxia reoxygenation is not significant, but it can alleviate the acid-base imbalance caused by hypoxia in rats and to some extent improve the increase in red blood cell and hemoglobin content caused by hypoxia.@*CONCLUSIONS@#Due to excessive ventilation and elevated RBCs count and Hb content after hypoxia reoxygenation, oxygen partial pressure and other oxygenation indicators in hypoxic rats are prone to become abnormal, while blood gas acid-base balance indicators are relatively stable, which are more suitable for evaluating the degree of hypoxia injury and related pharmacological effects in rats.
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Rats , Animals , Male , Acetazolamide , Hypoxia , Oxygen , Erythrocytes , Hemoglobins , Acid-Base ImbalanceABSTRACT
ObjectiveTo explore the activating effects of ten important effective components in seven medicinal and edible substances on human pregnane X receptor (PXR), including Glycyrrhizae Radix et Rhizoma (liquiritin and glycyrrhizic acid), Houttuyniae Herba (quercetin and houttuyfonate), Prunellae Spica (rosmarinic acid), Cassiae Semen (aurantio-obtusin), Poria (pachymic acid), Lilii Bulbus (Lilium brownii saponin and colchicine), and Lycii Fructus (Lycium barbarum polysaccharide) and screen potentially toxic components. MethodCell counting kit-8 (CCK-8) assay was used to investigate the cytotoxic effect of liquiritin, glycyrrhizic acid, quercetin, houttuyfonate, rosmarinic acid, pachymic acid, aurantio-obtusin, and colchicine (10, 20, and 50 μmol·L-1), and L. brownii saponin and L. barbarum polysaccharide (10, 20, and 50 mg·L-1) on normal human hepatocyte cell line (L02). The release of lactate dehydrogenase (LDH) in L02 cells after drug treatments was detected by the biochemical analyzer. The apoptosis induced by ten effective components was explored by Hoechst 33342 staining. The secreted luciferase reporter system was used to co-transfect the PXR expression vector and reporter gene vector containing cytochrome P450 3A4 (CYP3A4) transcriptional regulatory region into L02 cells, with 10 μmol·L-1 rifampicin (RIF) as a positive control. After treated with liquiritin, glycyrrhizic acid, quercetin, houttuyfonate, rosmarinic acid, aurantio-obtusin, pachymic acid, and colchicine (5, 10, and 20 μmol·L-1) and L. brownii saponin and L. barbarum polysaccharide (5, 10, and 20 mg·L-1) for 24 h, the cells were tested for secreted luciferase activity. ResultCompared with the control group, colchicine, L. brownii saponin, and quercetin decreased the cell viability (P<0.05, P<0.01). Compared with the control group, quercetin, rosmarinic acid, glycyrrhizic acid, colchicine, aurantio-obtusin, and pachymic acid increased the release rate of LDH in L02 cells (P<0.05, P<0.01). The proportion of hyperchromatic nuclei increased gradually after rosmarinic acid, liquiritin, and L. barbarum polysaccharide treatments as compared with the control group (P<0.05, P<0.01). In terms of co-transfection of pcDNA3.1-PXR and pGLuc-CYP3A4 into L02 cells, compared with the control group, aurantio-obtusin and pachymic acid showed activating effects on PXR (P<0.05), whereas liquiritin and glycyrrhizic acid showed inhibitory effects (P<0.05). ConclusionThe findings suggest that when medicinal and edible substances are taken for a long time, attention should be paid to their influence on drug-metabolizing enzymes and possible interactions, so as to improve their safety.
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Radiation-induced skin injury refers to the acute and chronic skin damage caused when skin is exposed to radiation. Radiation-induced skin damage may be created by nuclear disaster, radiation accident, radiation therapy, occupational exposure and so on. Approximately 95% of patients receiving radiotherapy will eventually develop into radiation-induced dermatitis during or after the treatment. As a consequence, how to appropriately prevent and remedy radiation-induced skin injury is of great practical significance. According to traditional Chinese medicine, radiation-induced skin injury belongs to fire, heat and toxin, blocking Qi and blood, injuring the muscle surface, affecting the distribution of Qi, blood and body fluid in the body, and damaging the function of viscera. This paper summarizes the cognition and development of traditional Chinese medicine theory of radiation-induced skin injury, as well as the research progress of internal and external treatment of traditional Chinese medicine, so as to provide a basis for the research and treatment of radiation-induced skin injury with traditional Chinese medicine.
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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.
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Tanshinone IIA (Tan IIA) is a pharmacologically active substance extracted from the rhizome of Salvia miltiorrhiza Bunge (also known as the Chinese herb Danshen), and is widely used to treat atherosclerosis. The pregnane X receptor (PXR) is a nuclear receptor that is a key regulator of xenobiotic and endobiotic detoxification. Tan IIA is an efficacious PXR agonist that has a potential protective effect on endothelial injuries induced by xenobiotics and endobiotics via PXR activation. Previously numerous studies have demonstrated the possible effects of Tan IIA on human umbilical vein endothelial cells, but the further mechanism for its exerts the protective effect is not well established. To study the protective effects of Tan IIA against hydrogen peroxide (H₂O₂) in human umbilical vein endothelial cells (HUVECs), we pretreated cells with or without different concentrations of Tan IIA for 24 h, then exposed the cells to 400 μM H₂O₂ for another 3 h. Therefore, our data strongly suggests that Tan IIA may lead to increased regeneration of glutathione (GSH) from the glutathione disulfide (GSSG) produced during the GSH peroxidase-catalyzed decomposition of H₂O₂ in HUVECs, and the PXR plays a significant role in this process. Tan IIA may also exert protective effects against H₂O₂-induced apoptosis through the mitochondrial apoptosis pathway associated with the participation of PXR. Tan IIA protected HUVECs from inflammatory mediators triggered by H₂O₂ via PXR activation. In conclusion, Tan IIA protected HUVECs against H₂O₂-induced cell injury through PXR-dependent mechanisms.
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Humans , Apoptosis , Asian People , Atherosclerosis , Endothelial Cells , Glutathione , Glutathione Disulfide , Human Umbilical Vein Endothelial Cells , Hydrogen Peroxide , Inflammation , Oxidative Stress , Regeneration , Rhizome , Salvia miltiorrhiza , Triacetoneamine-N-Oxyl , XenobioticsABSTRACT
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.
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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.
Subject(s)
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 , TransfectionABSTRACT
Aim To investigate the induction effect of ginsenoside Rc, Re, Rf and Rg1 on CYP1A1, and further validate the role of aryl hydrocarbon receptor in CYP1A1 expression. Methods Dual luciferase re-porter gene system was performed. Four kinds of gin-senoside were screened for aryl hydrocarbon receptor activation by reporter assays, and TCDD as the positive control. Further with different concentrations of ginsen-oside Rc, Re, Rf and Rg1 treated on LS174T cells, RNA and total protein were extracted to detect the reg-ulating effect of ginsenosides on CYP1 A1 mRNA and protein expression with Real-time PCR and Western blot technology respectively. Results Reporter gene screening showed that the ginsenoside Rc, Re, Rf and Rg1 could activate AhR and had potential effects on the induction of CYP1A1 enzyme. Meanwhile, dose-de-pendent induction of the gene expression were observed in response to ginsenoside Rc, Re, Rf and Rg1 and the levels of CYP1 A1 protein expression were increased by ginsenoside Rc, Re, Rf and Rg1 in varying de-grees. Conclusion Ginsenoside Rc, Re, Rf and Rg1 can up-regulate the gene and protein expression of CYP1 A1 possibly via the AhR-mediated CYP1 A1 path-way.
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OBJCTIVE To investigate the protective effect of ferulic acid (FA) on lipopolysaccharide (LPS)-induced damage to PC12 cells and hippocampal neurons in Sprague-Dawley (SD) rats and its potential mechanisms. METHODS ① in vitro study:PC12 cells were pretreated with FA 2.5-40 μmol · L-1 for 12 h and treated with LPS for another 8 h. CCK-8 kit was used to test PC12 cell viability. Inflammatory cytokines tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were de?tected by ELISA kits. Laser scanning confocal microscopy was performed to measure F-actin expres?sion in the cells. ②in vivo study:FA 25,50 and 100 mg · kg-1 was ip given to Sprague-Dawley(SD) rats once a day for 35 d,and from the 29th day,ip co-administered with LPS(0.2 mg · kg-1)for 7 d. Immunohistochemistry method was used to determine protein expression of phophodiestera 4B (PDE4B)in the hippocampus of rats. The protein expression of cAMP response element-binding protein (CREB) and phospho CREB (p-CREB) was determined by Western blotting. RESULTS In the in vitro study,compared with LPS group,cell viability was significantly increased in FA 10,20 and 40μmol·L-1 groups(P<0.05),while the production of inflammatory cytokines TNF-α and IL-1β decreased(P<0.05). The structure and distribution of cytoskeletal protein F-actin were ameliorated markedly in PC12 cells. In the in vivo study,hematoxylin-eosin(HE)staining showed that pretreatment with FA(50 and 100 mg·kg-1) alleviated the damage to the hippocampus induced by LPS in SD rats. Immunohistochemistry showed that FA(50 and 100 mg·kg-1)pretreatment effectively prevented LPS-induced up-regulation of PDE4B expression in the hippocampus of rats(P<0.05). Western blotting analysis showed that the inhibitory effects on the protein expressions of CREB and p-CREB induced by LPS were altered by FA(50 and 100 mg · kg-1)pretreatment(P<0.05). CONCLUSION FA can protect against LPS induced damage to PC12 cells and hippocampal neurons of rats. The resistant effect on neuron-inflammation of FA may be conferred by inhibiting LPS-induced up-regulation of PDE4B and stimulating signaling pathways of cAMP/CREB.
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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.
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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.
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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.
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Aim To study the effect of emodin in Po-lygonum multiflorum on the expression of CYP450 isoenzymes in L02 hepatocytes and explore its mecha-nism of cytotoxicity. Methods L02 cells were treated with different concentrations of emodin. Cell viability was examined by MTS assay kit, and cell membrane injury was examined by detecting the release rate of lactate dehydrogenase( LDH) . The expression of cyto-chrome P450 mRNA was detected by real time PCR. Results The result of MTS assay showed that L02 cells viability was significantly reduced following expo-sure to emodin in a concentration and time dependent manner. The LDH release rate of L02 cells significant-ly increased after exposure to emodin for 48 h com-pared with the control group. On the mRNA level, compared with the control group,emodin had inductive effects on mRNA of each CYP450 enzyme, while had significant inductive effects on mRNA of CYP1 A1 and CYP1 B1 in a concentration and time dependent man-ner. Conclusion Emodin in Polygonum multiflorum may generate significant liver injury in L02 cells and has inductive effects on CYP450 enzyme activity.
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Objective To investigate the relationship between serum prealbumin (PA) and inflammation in acute myocardial infarction (AMI) patients. Methods A prospective observational study was conducted. AMI patients hospitalized in the cardiovascular department of the General Hospital of Chinese People's Armed Police Forces from June 2014 to June 2016 were enrolled in the study. At the same time, healthy cases were enrolled as control. Venous blood was taken from patients at admission. Serum PA was detected by immune projection turbidimetry method and high-sensitivity C-reactive protein (hs-CRP) was measured by latex enhanced immune turbidimetry. High-sensitivity cardiac troponin T (hs-cTnT), and interleukin (IL-6, IL-8) was measured by electrochemical luminescence method. Creatine kinase-MB isoenzyme (CK-MB) was detected by rate method. PA, inflammatory factor and myocardial enzyme were compared between two groups. The correlation between PA and inflammatory factors was analyzed by Pearson linear correlation; The diagnostic value of PA was analyzed by receiver operating characteristic (ROC) curve. Results 173 AMI patients and 86 healthy controls were enrolled in the study. There were no significant differences in gender, age, history of smoking, hypertension and diabetes. Compared with the control, the levels of serum PA in AMI patients was lower [PA (g/L): 0.215±0.056 vs. 0.280±0.057], hs-CRP, IL-6, IL-8, hs-cTnT and CK-MB were higher [hs-CRP (mg/L): 6.63±3.52 vs. 2.25±1.45, IL-6 (ng/L): 38.03±22.43 vs. 6.13±3.38, IL-8 (ng/L): 295.61±98.70 vs. 17.24±7.31, hs-cTnT (μg/L): 4.789±2.874 vs. 0.009±0.008, CK-MB (U/L): 244.48±165.54 vs. 12.20±5.24], the difference was statistical significant (all P 0.05). It was shown by ROC curve analysis that area under ROC curve (AUC) of PA for diagnosis of AMI was 0.783±0.039, and the 95% confidence interval (95%CI) was 0.706-0.860 (P < 0.05). When the cut-off value was 0.190 g/L, the sensitivity was 29.63%, and the specificity was 62.22%. Conclusion PA may be involved in the inflammatory process of AMI and had a diagnostic value for AMI.
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Aim To evaluate the effects of ferulic acid ( FA ) on lipopolysaccharide ( LPS )-induced neuroin-flammation in microglia cells and its potential mecha-nisms. Methods Microglial activation was induced by stimulation with LPS, and the effects of FA pretreat-ment on microglial activation and production of proin-flammatory mediators, nitric oxide/iNOS were investi-gated. The role of the mitogen-activated protein kinases in the antiinflammatory actions of FA in LPS-stimulated microglia was further elucidated. Results Cell viabil-ity experiments revealed that FA did not produce cyto-toxicity in microglia. FA significantly inhibited LPS-in-duced production of tumour necrosis factor-alpha ( TNF-α) , interleukin-6 ( IL-6 ) , interleukin-1 beta ( IL-1β) , and nitric oxide ( NO ) . Protein and mRNA levels of COX-2 and inducible nitric oxide synthase ( iNOS) were also attenuated by FA. Further experi-ments on intracellular signalling mechanisms showed that inhibition of extracellular regulated kinase ( ERK) contributed to the anti-neuroinflammatory actions of FA. Conclusion The results suggest that FA inhibits LPS-induced microglial inflammation by partial targe-ting of ERK signalling and attenuation of ERK.
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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.
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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.
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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.
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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.
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OBJECTIVE Tostudythehepatotoxicityofveratrinehydrochloride(VH)anditsmecha-nismoninductionofapoptosisinvitro.METHODS HepG2cellswereexposedtoVH0.1-0.6g·L-1 for 24 h,cell viability was examined by CCK-8 assay,and the morphologic changes in HepG2 cells were quantified.After the treatment with VH 0.1 -0.5 g·L-1 for 24 h,cell membrane injury was examined by detecting the release rate of lactate dehydrogenase (LDH).The effect on reactive oxygen species (ROS),mitochondrial membrane potential and apoptosis was detected by flow cytometry.The mRNA expression of p53,Bax,cytochrome c,caspase 9,caspase 3 was evaluated by real-time PCR. RESULTS HepG2cellviabilitywassignificantlyreducedfollowingexposuretoVH0.1-0.5g·L-1. The IC50 value was 0.4 g·L-1 .The 95%confidence limit was 0.2558-0.6965 g·L-1 .The LDH release rate,ROS and apoptosis rate of HepG2 cells were significantly increased after exposure to VH 0.1 -0.5 g·L-1 for 24 h (P<0.05,P<0.01 ),and the mitochondrial membrane potential markedly declined (P<0.05,P<0.01 ).The expression of p53,Bax,cytochrome c,caspase 9 and caspase 3 was increased(P<0.05,P<0.01).CONCLUSION VHhascytotoxicpotential.Damagetocell me mbrane and mitochondria and initiation of apoptosis-related genes of caspase 9 and caspase 3 mRNA expression may be the mechanis m of apoptosis.