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ObjectiveTo explore the mechanism of plumbagin as a novel ferroptosis inducer in bladder cancer inhibition. MethodBladder cancer T24 cells were used in this study. The effect of different concentrations of plumbagin （0.1， 1， 2， 3， 6， 12， 24， 48 μmol·L-1） on the viability of T24 cells was detected by cell counting kit-8 （CCK-8）. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the apoptosis of T24 cells was detected by annexin V-fluorescein isothiocyanate （Annexin V FITC）/PI apoptosis kit. Different inhibitors （ferroptosis inhibitor Fer-1， apoptosis inhibitor VAD， and necroptosis inhibitor Nec-1） were used in combination with plumbagin （6 μmol·L-1）. Reactive oxygen species （ROS） fluorescent probe （DCFH-DA）， malonaldehyde （MDA）， and glutathione （GSH） kits were used to detect the effects of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the level of ROS and the content of MDA and GSH in T24 cells， respectively. The effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on peroxide levels in T24 cells was detected by C11-BODIPY fluorescent probe. Western blot was used to detect the effect of different concentrations of plumbagin （1.5， 3， 6 μmol·L-1） on the protein expression of solute carrier family 7 member 11 （SLC7A11）， glutathione peroxidase 4 （GPX4）， nuclear factor E2-related factor-2 （Nrf-2）， and Kelch-like ECH-associated protein 1 （Keap1）. ResultCompared with the blank group， plumbagin could inhibit the activity of T24 cells （P<0.05） with IC50 of 3.52 μmol·L-1. At the concentrations of 1.5， 3， 6 μmol·L-1， plumbagin significantly promoted the apoptosis of T24 cells （P<0.05） as compared with the blank group. Compared with the plumbagin group at 6 μmol·L-1， the ferroptosis inhibitor and apoptosis inhibitor groups could reverse the inhibitory effect of 6 μmol·L-1 plumbagin on the proliferation of T24 cells （P<0.05）. Compared with the blank group， the plumbagin groups at 1.5， 3， 6 μmol·L-1 showed increased content of ROS， MDA， and lipid peroxides in T24 cells， decreased GSH level， and reduced SLC7A11， GPX4， and Nrf-2/Keap1 （P<0.05）. Conclusionplumbagin can induce ferroptosis， and its mechanism is related to the Nrf-2/Keap1 signaling pathway.
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Objective To investigate the inhibitory effect and mechanism of temozolomide on migration and invasion of U251 human glioma cells enhanced by plumbagin. Methods CCK-8 method was used to study the effects of plumbagin, temozolomide and plumbagin+temozolomide on the proliferation of glioma U251 cells. Cell scratch test was used to detect the migration of U251 cells in the control (DMSO), plumbagin, temozolomide and plumbagin+temozolomide groups for 48h. Transwell assay was used to detect the effect of the combination therapy on the invasion of U251 cells. Western blot was used to detect the relative expression levels of E-cadherin in three groups. Results CCK-8 showed that the proliferation inhibition rate of U251 cells treated with plumbagin (1.25 μmol/L) combined with temozolomide (200 μmol/L) for 48h was 75.69%, significantly higher than that treated with plumbagin alone (P=0.012) or temozolomide alone (P=0.034). Cell scratch assay showed that the combination of plumbagin and temozolomide could significantly enhance the inhibition effect of temozolomide on the migration of U251 cells (P=0.023). Transwell assay showed that the invasion ability of U251 cells was significantly decreased after the combination therapy (P < 0.05). The protein expression of E-cadherin in the combination group was significantly higher than those in plumbagin or temozolomide groups (P < 0.05). Conclusion Plumbagin combined with temozolomide can inhibit the migration and invasion of glioma cells and enhance the sensitivity of glioma cells to temozolomide. And the effect is achieved by the protein expression of E-cadherin.
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In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.
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Humans , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , High-Throughput Screening Assays , Lung Neoplasms , Transcription Factor 4/genetics , beta Catenin/geneticsABSTRACT
Aim To investigate the effects of plumbagin on reactive oxygen species (ROS) level and expressions of a-smooth muscle actin (ct-SMA), Smad2/ 3, p-Smad2/3, and Smad7 proteins. Methods We isolated primary hepatic stellate cell (HSC) of rat and inoculated the activated HSC. Then these cells would be divided into TGF-ßl treatment group,NADPH oxidase inhibitor (DPI) group,plumbagin treatment group, and control group. MIT was used to test cell proliferation and fluorescent probe was used to detect ROS level. Moreover, the protein expressions of Smad2/3, pSmad2/3, and Smad7 were determined using Western blot. Results Results of fluorescent probe showed that the positive rate of a-SMA was up to 91%. MTT test indicated the optimal concentrations of plumbagin were 2,4, and 8 u.mol • L " 1 . A decreased level of ROS could be found in DPI and plumbagin treatment groups. In addition, results of Western blot showed that a decreased expression of Smad2/3, p-Smad2/3 and an increased expression of Smad7 could be found in DPI and plumbagin treatment groups compared to control group. Conclusions Plumbagin may decrease the expression of smad2/3 and p-smad2/3 by down-regulating the level of ROS,and thus play an anti-HSC activation role.
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Objective: To explore the effects of plumbagin (PLB) on the proliferation and apoptosis of hepatocellular carcinoma hepG2R cells resistant to sorafenib, and to clarify its mechanism. Methods: The hepatocellular carcinoma cells hepG2 were cultured in vitro and the models of sorafenib-resistant HepG2R cells were set up. MTT assay was used to identify the resistance factor and screen the concentration and time of drug. According to the results, the concentrations of sorafenib and PLB were confirmed as 5 μmol · L-1 and 2 μmol · L-1. The action time in various groups was 48 h. The HepG2R cells were randomly divided into control group, sorafenib (5 μmol · L-1) group, PLB (2 μmol · L-1) group, sorafenib (5 μmol · L-1) combined PLB (2 μmol · L-1) group (combination group). MTT assay was used to detect the cell vitality in various groups. The clone formation rates of cells in various groups were detected by clone formation assay. The apoptotic rates of cells in various groups were determined with Hoechst33342 assay and flow cytometry (FCM). The expression levels of cleaved Caspase-3, Bax and Bel-2 proteins in the cells in various groups were examined by Western blotting method, and the Bax/Bcl-2 ratio was calculated. The reactive oxygen (ROS) levels in the cells in various groups were detected by ROS detector kit. Results: As the increasing of concentrations of sorafenib, the cell vitalities of HepG2 and HepG2R cells were gradually decreased; the IC5o of sorafenib-resistant HepG2R cells was 4. 5 times as much as HepG2 cells (P<0. 05). The sensitivities of sorafenib resistant HepG2R cells were increased with the increasing of PLB concentrations and prolongation of time. Compared with control group, sorafenib group and PLB group, the clone formation rate of the cells in combination group was decreased (P<0. 05 or P<0. 01). The Hoechst 33342 assay results showed the nuclei were lightly stained; a few of the nuclei in sorafenib group and PLB group were strongly stained and bright; while in combination group, the nuclei were mostly stained and chromatin was condensed and bright. The FCM results showed that compared with control group, sorafenib group and PLB group, the apoptotic rate of cells in combination group was significantly increased (P< 0. 05 or P< 0. 01). The Western blotting results showed that the expression level of cleaved Caspase-3 in the cells and the Bax/Bcl -2 ratio in combination group were increased significantly (P<0. 05 or P<0. 01). The ROS level in the cells in combination group was higher than those in the other groups (P< 0.05 or P<0. 01). Conclusion: PLB can improve the resistance of hepartocellular carcinoma to sorafenib and the mechanism may be related to increasing the ROS level.
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Objective: To prepare plumbagin transfersomal (PBG-T) gel and investigate its transdermal penetration characteristics in vitro. Methods: Plumbagin transfersomes were prepared by film-ultrasonic dispersion method. The optimal prescription condition of PBG-T was selected by central composite design and response surface method. The formula of PBG-T gel was optimized by orthogonal test. The Franz diffusion cell was used to investigate transdermal penetration characteristics of PBG-T gel in vitro. Results: The optimal prescription condition of transfersomes was determined as drug 10.0 mg, phospholipids 700.0 mg, Tween-80 91.5 mg, ultrasonication time 13 min. The optimal prescription condition of transfersomal gel was 1% carbomer 940 as gel matrix, and 5% glycerol as the humectant. According to the optimized prescription, the entrapment efficiency, the mean particle size, and Zeta potential of PBG-T were (79.88 ± 2.26)%, (125.64 ± 4.54) nm, and (-30.97 ± 1.13) mV. The cumulative penetration rate of PBG-T gel was 70.0% at 12 h. Conclusion: The optimal preparation technique is stable and feasible. Transfersomal gel features a sustained release in vitro, the transfersomal gel can increase penetration rate of plumbagin through the skin of rats.
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Objective:To investigate the cytotoxic, apoptotic and inhibitory activities on cell migration and invasion of plumbagin in the human cholangiocarcinoma (CCA) cell line (CL-6) in comparison with human embryonic fibroblast cell line (OUMS).Methods:Cytotoxicity activity was evaluated using MTT assay. Inhibitory effect on cell migration and invasion were investigated using label-free real-time cell analysis and QCM ECMatrix cell invasion chamber, respectively. Apoptotic activity was evaluated using flow cytometry and CellEvent™ Caspase 3/7 assay.Results:Based on results of the cytotoxicity test in CL-6 cells, 50% inhibitory concentration (ICConclusions:The cytotoxic activity and inhibition of migration and invasion including apoptosis induction in the human CCA cell line (CL-6) suggest that plumbagin could be a promising candidate for CCA chemotherapeutics. However, its relatively low selective cytotoxic effect on CCA cells is a major concern.
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Objective: To investigate the cytotoxic, apoptotic and inhibitory activities on cell migration and invasion of plumbagin in the human cholangiocarcinoma (CCA) cell line (CL-6) in comparison with human embryonic fibroblast cell line (OUMS). Methods: Cytotoxicity activity was evaluated using MTT assay. Inhibitory effect on cell migration and invasion were investigated using label-free real-time cell analysis and QCM ECMatrix cell invasion chamber, respectively. Apoptotic activity was evaluated using flow cytometry and CellEvent™ Caspase 3/7 assay. Results: Based on results of the cytotoxicity test in CL-6 cells, 50% inhibitory concentration (IC
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OBJECITVE: To study the inhibitive effect of plumbagin on Lewis lung cancer. METHODS: Cell proliferation was determined by CCK8 assay. Apoptosis was determined by flow cytometry. The expression of Bcl-2 and VEGF protein was studied by Western blot assay. The model of C57BL/6 mice bearing Lewis lung cancer was established by subcutaneous seeding of Lewis lung cancer cells, and randomly divided into 5 groups (n = 6). Tumor-bearing mice were injected with normal saline, plumbagin(low, medium, high dose) or cyclophosphamide (CTX) in each group. The tumor volume was measured. All mice were sacrificed on Day 22nd under aseptic condition for the tumor collection. The transplanted tumors were weighed for calculation of the tumor inhibition rate; Immunohistochemical method was applied to assessing the VEGF expression in tumor tissue. RESULTS CCK-8 assay showed that plumbagin had an obvious inhibition on Lewis lung carcinoma cells line in a dose-dependent manner(r = 0.953, P < 0.05). Plumbagin significantly increased cell apoptosis rate(P<0.05). The protein levels of Bcl-2 and VEGF were significantly reduced by plumbagin (0, 2.5, 5, 10 μmol·L-1) treatment(P < 0.05). In plumbagin(low, medium, high dose) groups and CTX group, the tumour volume, tumour weight and the expression of VEGF were significantly less than those in the control group (P < 0. 05). CONCLUSION: The plumbagin effectively inhibits Lewis lung carcinoma cells proliferation and tumor growth of Lewis lung carcinoma cells in mice. The mechanism involved is down-regulating the expression of Bcl-2, VEGF and inducing cell apoptosis.
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Objective@#To study the effect of plumbagin on epithelial-mesenchymal transition (EMT) and underlying mechanisms in human tongue squamous cell carcinoma (TSCC) cells.@*Methods@#Methyl thiazolyl tetrazolium assay was apllied to examine the proliferation inhibition effect and half maximal inhibitory concentration (IC50) of plumbagin (0.1, 1.0, 5.0, 10.0, 20.0 μmol/L) in 12, 24, 48 h in TSCC cells. Transwell assay was used to count the number of transmembrane cells and scratch test was performed to examine cells mobility. The flow cytometry was applied to measure intracellular reactive oxygen species (ROS) level in control group, plumbagin group (1.0 μmol/L, 24 h) and glutathione (GSH)+plumbagin group. The expression of E-cadherin, vimentin, Slug, p38 mitogen activated protein kinases (p38MAPK) and phospho-p38MAPK (p-p38MAPK) proteins were determined by Western blotting. The expression of E-cadherin, vimentin and Slug were detected by Western blotting in control group, plumbagin group, activator combined group (p38MAPK activator+plumbagin) and inhibitor combined group (p38MAPK inhibitor+plumbagin).@*Results@#After the treatment of plumbagin for 12, 24, and 48 h, the IC50 of TSCC cells were 10.3, 3.1, 1.5 μmol/L. After treated by 1.0 μmol/L plumbagin for 24 h, the number of transmembrane cells were significantly reduced ([50±13], P<0.05) in comparison to control group (204±6), as well as the cells mobility ([18.2±2.3]%, P<0.05) in comparison to control group ([49.3±1.2]%). Compared to control group (2.32±0.52), the ROS level was increased in plumbagin group (902.20±10.69), while compared to plumbagin group, the ROS level was reduced in GSH combined group (2.18±0.15). In comparison to control group, the expression of E-cadherin was up-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were down-regulated in plumbagin group (P<0.05). In comparison to plumbagin group, the expression of E-cadherin was down-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were up-regulated in GSH combined group (P<0.05). Treatment of cells with p38MAPK activator could decrease the expression of E-cadherin significantly (P<0.05) and increase the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group. Treatment of cells with p38MAPK inhibitor could increase the expression of E-cadherin significantly (P<0.05) and decrease the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group.@*Conclusions@#Plumbagin inhibits EMT via ROS/p38MAPK-mediated pathway in human TSCC cells.
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Plumbagin (Plumbago zeylanica L.) has a wide spectrum of anticancer activity with a relatively lower toxicity. The molecular mechanisms of proliferation inhibition and apoptosis induction by plumbagin on esophageal squamous carcinoma cell lines may be important for the structure modification and clinical application of plumbagin. After treatment of KYSE-30, KYSE-70 and KYSE-140 cells with 0-20 μmol·L-1 of plumbagin for 24, 48, 72 h, CCK8 was used to examine the proliferation, Annexin V and PI immunofluorescence staining for apoptosis, real-time PCR and Western blot for FoxM1 mRNA and protein expression, dual-luciferase reporter gene assay for the transcriptional activity of FoxM1, respectively. In addition, the relationship between anti- tumor effect of plumbagin and FoxM1 was investigated in vivo. Plumbagin significantly inhibited proliferation and induced apoptosis of esophageal squamous carcinoma cell in vitro and in vivo. Moreover, plumbagin down-regulated the expression of FoxM1 through suppression of its gene transcription. Our findings suggest that plumbagin may inhibit the proliferation of esophageal squamous carcinoma cell in vivo and in vitro through down-regulating the expression of FoxM1.
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Plumbagin, a hydroxy 1,4-naphthoquinone compound from plant metabolites, exhibits anticancer, antibacterial, and antifungal activities via modulating various signaling molecules. However, its effects on vascular functions are rarely studied except in pulmonary and coronary arteries where NADPH oxidase (NOX) inhibition was suggested as a mechanism. Here we investigate the effects of plumbagin on the contractility of skeletal artery (deep femoral artery, DFA), mesenteric artery (MA) and renal artery (RA) in rats. Although plumbagin alone had no effect on the isometric tone of DFA, 1 µM phenylephrine (PhE)-induced partial contraction was largely augmented by plumbagin (ΔT(Plum), 125% of 80 mM KCl-induced contraction at 1 µM). With relatively higher concentrations (>5 µM), plumbagin induced a transient contraction followed by tonic relaxation of DFA. Similar biphasic augmentation of the PhE-induced contraction was observed in MA and RA. VAS2870 and GKT137831, specific NOX4 inhibitors, neither mimicked nor inhibited ΔT(Plum) in DFA. Also, pretreatment with tiron or catalase did not affect ΔT(Plum) of DFA. Under the inhibition of PhE-contraction with L-type Ca²⁺ channel blocker (nifedipine, 1 µM), plumbagin still induced tonic contraction, suggesting Ca²⁺-sensitization mechanism of smooth muscle. Although ΔT(Plum) was consistently observed under pretreatment with Rho A-kinase inhibitor (Y27632, 1 µM), a PKC inhibitor (GF 109203X, 10 µM) largely suppressed ΔT(Plum). Taken together, it is suggested that plumbagin facilitates the PKC activation in the presence of vasoactive agonists in skeletal arteries. The biphasic contractile effects on the systemic arteries should be considered in the pharmacological studies of plumbagin and 1,4-naphthoquinones.
Subject(s)
Animals , Rats , 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt , Arteries , Catalase , Coronary Vessels , Femoral Artery , Mesenteric Arteries , Muscle, Smooth , NADPH Oxidases , Phenylephrine , Plants , Protein Kinase C , Relaxation , Renal Artery , Vasoconstrictor AgentsABSTRACT
Aim To investigate the effect of plumbagin on invasion and apoptosis of human hepatocellular car-cinoma ( HepG2 ) . Methods HepG2 cells were cul-tured in vitro with different concentrations of plumba-gin, then cell proliferation was observed by MTT as-say; cell invasion was observed by transwell invasion assay; cell apoptosis was detected by flow cytometry, and the protein expression of Bax, Bcl-2 was detected by immunocytochemistry. Results MTT results showed that plumbagin could significantly inhibit cell proliferation compared with the control group, and in a dose-dependent manner ( P ( P < 0. 01 ) . Flow cytometry showed that apoptosis rate was significantly higher in 4 , 8 μmol · L-1 group of plumbagin compared with that of the control group ( P<0. 01 ) . Immunocytochemistry showed that, with the increasing concentration of plumbagin, Bax protein expression increased, Bcl-2 protein expression was de-creased, both in a dose-dependent manner ( P <0. 01 ) . Conclusion Plumbagin can inhibit HepG2 cell proliferation and accelerate apoptosis of HepG2 cells, but also has the ability to inhibit HepG2 cell in-vasion.
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According to WHO about 80% of the world’s population relies on traditional medicine for their primary health care. Plumbago zeylanica L. is a medicinal plant, belong to the family of Plumbaginaceae and the root of P. zeylanica contains several bioactive constituent like L-dopa, Plumbagin (naphthoquinone), droseron, chitranone, triterpenoid, anthraquinone. This study is designed to evaluate the effect of P. zeylanica and naphthoquinone on the level of various amine neurotransmitters namely epinephrine, norepinephrine, serotonin, 5-hydroxyindole 3- acetic acid (5-HIAA), and dopamine on the discrete regions of the brain tissues. Wistar male albino rats were treated separately with ethanoloic extract of P. zeylannica (root) and the commercially purchased phytochemical naphthoquinone at the dose of 2mg/kg b. wt with different experimental groups. The brain tissue homogenates (cerebral cortex, cerebellum, hypothalamus, pons-medulla, midbrain, and corpus striatum) were analyzed to quantify the aforementioned neurotransmitters by high performance liquid chromatography (HPLC). The results showed that, administration of P. zeylanica and NQ does not alter the many of the studied neurotransmitter at significant level; however, there is a change in the neurotransmitter profile in few specific regions of Wistar rat brain and striatum was found to be affected more.
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A protocol was developed for the micropropagation of Plumbago scandens L. from the shoot tip and node explants.The best response of shoot elongation (10.18±2.01 mm) was observed on MS basal medium supplemented with 0.02 mg/L IAA – 0.02 mg/L GA3. The maximum number of root induction (10.0±2.21) and shoot elongation (8.24±3.24 mm) was observed on medium containing 0.01 mg/L IBA and 0.01 mg/L GA3. The in vitro propagated plants were transferred to soil with 80% survival rate. Profuse compact callus was induced and proliferated from several explants (cotyledons, internodes, hypocotyls and roots) cultured on MS medium supplemented with all the combinations of 2,4-D – GA3 or 2,4-D alone and combinations of IAA – BAP or IAA alone, and the highest percentage of friable callus (90%) were induced in the sections of compact callus using 2.0 mg/L IAA – 0.02 mg/L BAP – 0.5 mg/L GA3.The qualitative determination of chemical constituents in the extracts was evaluated by a gas chromatography coupled to a mass spectrometry, and it was verified the presence of plumbagin only in root extracts but not in in vitro plantlets.The antibacterial activity of root extracts against various pathogenic bacteria, and the minimum inhibitory concentrations (MICs) was determined. Chloroform extracts showed good antibacterial activity against Neisseria gonorrhoeae between 0.4 to 1.0 mg/L with 20.4 to 30.0 mm (diameter zone of inhibition); inhibition against Staphylococcus aureus was moderate, and lower against Escherichia coli. Chloroform extracts had the lowest MICs for N. gonorrhoeae (<0.1 mg/mL per disc), and the activities against S. aureus (MIC 0.2 mg/mL) and E. coli (MIC 0.4 mg/mL) were less pronounced.
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Objective: To investigate the propensity of plumbagin to inhibit the three isoforms of human cytochrome P450 (CYP), i.e., CYP1A2, CYP2C19, and CYP3A4 using human liver microsomes in vitro. Methods: Inhibitory effects of plumbagin on the three human CYP isoforms were investigated using pooled human liver microsomes. Phenacetin O-deethylation, omeprazole hydroxylation and nifedipine oxidation were used as selective substrates for CYP1A2, CYP2C19 and CYP3A4 activities, respectively. Concentrations of paracetamol, 5-hydroxyomeprazole, and oxidized nifedipine were determined in microsomal incubation mixture using high-performance liquid chromatography. Results: Plumbagin showed significant inhibitory effects on all CYP isoforms, but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation. The IC
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This article was aimed to study the effects of plumbagin to human hepatic stellate cells.Observations were made on the influence of proliferation inhibition rate,apoptosis,secretion of extracellular matrix function and matrix metalloproteinase (MMP) by plumbagin.HSC-LX2 and drug were co-incubated for 48 hours.Then,MTT assay was used in the detection of inhibition of cell proliferation.The flow cytometry was used in the detection of apoptosis.Immunohistochemical method was used to observe typeⅠ and Ⅲ collagen,MMP-1 and MMP-13 expression location and area.The results showed that the low,medium and high concentrations of plumbagin inhibited cell proliferation rate of HSC-LX2,induced apoptosis of cells,reduced the secretion of typeⅠ and Ⅲ collagen,and increased the secretion ability of MMP-1 and MMP-13.Effects mentioned above were dose-dependent with statistical difference (P < 0.05).Effects in the medium and high concentrations groups were stronger than colchicine group.It was concluded that plumbagin had the ability to inhibit cell proliferation rate of HSC-LX2,induce apoptosis,reduce the secretion of extracellular matrix,and increase the secretion ability of fibrin degradation enzyme.Therefore,it had intervention effect on the process of liver fibrosis.All effects mentioned above were dose-dependent.And effects in the medium and high concentrations groups were stronger than colchicine group.
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AIM:To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide ade -nine dinucleotidephosphate oxidase 4 ( Nox4 ) , reactive oxygen species ( ROS ) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro.METH-ODS:HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium-and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups .After incubated with each drug for 72 h, the mRNA expression of Nox4 was detec-ted by RT-PCR.ROS levels were tested by in situ loading probe method.The protein contents of Nox4 and α-SMA were measured by Western blot .RESULTS:Compared with model group , after treated with plumbagin for 72 h, the mRNA ex-pression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01).CONCLUSION:Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels .
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OBJECTIVE@#To investigate the propensity of plumbagin to inhibit the three isoforms of human cytochrome P450 (CYP), i.e., CYP1A2, CYP2C19, and CYP3A4 using human liver microsomes in vitro.@*METHODS@#Inhibitory effects of plumbagin on the three human CYP isoforms were investigated using pooled human liver microsomes. Phenacetin O-deethylation, omeprazole hydroxylation and nifedipine oxidation were used as selective substrates for CYP1A2, CYP2C19 and CYP3A4 activities, respectively. Concentrations of paracetamol, 5-hydroxyomeprazole, and oxidized nifedipine were determined in microsomal incubation mixture using high-performance liquid chromatography.@*RESULTS@#Plumbagin showed significant inhibitory effects on all CYP isoforms, but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation. The IC50 (concentration that inhibits enzyme activity by 50%) values of plumbagin and nootkatone (selective inhibitor) for CYP2C19 were (0.78 ± 0.01) and (27.31 ± 0.66) μM, respectively. The inhibitory activities on CYP1A2-mediated phenacetin O-deethylation and CYP3A4-mediated nifedipine oxidation were moderate. The IC50 values of plumbagin and α-naphthoflavone (selective inhibitor) for CYP1A2 were (1.39 ± 0.01) and (0.02 ± 0.36) μM, respectively. The corresponding IC50 values of plumbagin and ketoconazole (selective inhibitor) for CYP3A4 were (2.37 ± 0.10) and (0.18 ± 0.06) μM, respectively.@*CONCLUSIONS@#Clinical relevance of the interference of human drug metabolizing enzymes should be aware of for further development scheme of plumbagin as antimalarial drug when used in combination with other antimalarial drugs which are metabolized by these CYP isoforms.