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@#The aim of this study was to investigate the effect of norcantharidin (NCTD) on the proliferation and apoptosis of triple-negative breast cancer cell line MDA-MB-231.Western blot was used to detect the effect of NCTD on the expression levels of apoptosis-related proteins Bax/Bcl-2, cleaved-PARP/PARP/PARP, cleved-caspase-9, cleaved-caspase-3 and MCL-1 in MDA-MB-231 cells.Also, the expression levels of autophagy-related proteins LC3-II/LC3-I, Parkin and PINK1 in MDA-MB-231 cells were measured by Western blot.Flow cytometry was used to measure the effect of NCTD on the changes of mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS).The effect of NCTD on autophagy flow in cells expressing mCherry-EGFP-LC3 was detected by a confocal microscope.Moreover, the effects of NCTD combined with chloroquine (CQ) or 3-methyladenine (3-MA) on the apoptosis of MDA-MB-231 cells were detected by flow cytometry.The results showed that NCTD significantly increased the expression levels of Bax/Bcl-2, cleaved-PARP/PARP, cleaved-caspase-9, cleasved-caspase-3 and LC3-II/LC3-I proteins, and promoted the mitochondrial translocation of Parkin, and blocked the autophagic flow in MDA-MB-231 cells. Moreover, NCTD combined with CQ accelerated apoptosis, while NCTD combined with 3-MA decreased apoptosis.These results suggest that NCTD can induce autophagy accumulation and lead to apoptosis of MDA-MB-231 cells.
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Aim To investigate the effect of acacetin on cell proliferation and the influence of acacetin on estrogen receptor expression in vitro.Methods The proliferation rates and the cell cycle changes of acace-tin-treated T47D cells were measured by sulforhodam-ine B(SRB)assay and flow cytometry,respectively. Moreover,the mRNA expressions of estrogen receptor-alpha(ERα),estrogen receptor-beta(ERβ)and pro-liferating antigen(Ki67)were determined by quantita-tive real time PCR (qPCR).Western blot was em-ployed to detect the ERαand ERβprotein expression. Results Acacetin significantly promoted the prolifera-tion and increased the amount of cells arrested in S and G2 /M phase under the concentration of 0.001 ~1 0μmol·L -1 .Ki67 mRNA level and the ERαprotein level in T47D cells were remarkably upregulated after acacetin treatment.To clarify which estrogen receptors played a role in acacetin induced the proliferation of T47D cells,the combination treatment of acacetin and ERαinhibitor (MPP)/ERβ inhibitor (PHTPP) was employed.We found that MPP could reverse the cell proliferation,the cell arrested in S and G2 /M phase and the increased Ki67 mRNA level induced by acace-tin.PHTPP also alleviated the T47D cell proliferation induced by acacetin,whereas no significant changes were found in cell cycle and Ki67 mRNA level.Con-clusion Acacetin stimulates the cell proliferation of T47D cells in the concentration from 0.001 μmol · L -1 to 1 0 μmol·L -1 ,which is mainly mediated by ERα.
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Aim To investigate the protective effects of dihydroquercetin(DDQ) against myocardial ischemis reperfusion injury(MIRI) in rats.Methods Male Sprague-Dawley rats were randomly divided into 4 groups(n=10):normal,control,I/R model, and I/R model+DDQ(5,10 mg·L-1).This study used an isolated Langendorff rat heart model.The left ventricu-lar developed pressure(LVDP),heart rate(HR) and the maximum rise and fall rate of the left ventricular pressure(±dp/dtmax) were monitored and documented using a physiological recorder.The levels of lactate dehydrogenase(LDH) and creatine kinase(CK) were analyzed using enzyme-linked immunosorbent assay(ELISA).Infarct size was measured using 2,3,5-triphenyltetrazolium chloride staining.The levels of superoxide dismutase(SOD) and malondialdehyde(MDA), as well as the ratio of glutathione/glutathione disulfide(GSH/GSSG) were measured via ELISA.HE staining was used to observe the pathological changes of myocardial tissue.Results Compared with the I/R model group, the I/R model+DDQ groups raised hemodynamic parameters, SOD level, and GSH/GSSG ratio;and reduced the amount of CK, LDH, MDA levels.Moreover, the I/R model+DDQ groups had lower infarct size and pathological changes in myocardial tissue than I/R model group.Conclusion DDQ exertes cardioprotective effects against I/R via improving the oxygen free radical scavenging ability, the inhibition of oxygen free radical and reducing lipid peroxidation.
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Objective To develop an apoptosis model of nucleus pulposus cells in cell culture.Methods To mimic the nutrient-deficient microenvironment of degenerative intervertebral disc,nucleus pulposus cells derived from infant SD rat disc were cultured under serum limiting conditions.Nucleus pulposus cells were cultured in culture medium contai-ning 1%, 3%, 5%, 8%and 10%fetal bovine serum( FBS) respectively to select the optimum FBA concentration.Apoptosis was assessed by flow cytometry, Western blot,cell counting kit, and immunofluorescence technique.Results The flow cy-tometry revealed that apoptosis rate of the nucleus pulposus cells increased with decreasing concentration of FBS, and 3%FBS used in the experimental group was the most effective concentration to induce apoptosis(P<0.05).Western blot dem-onstrated significantly higher expression of Bax and caspase-3 enzyme in the 3%FBS group than in the 10%FBS group, while bcl-2 activity decreased.The results of CCK-8 test indicated that the nucleus pulposus cells got slower proliferation in the medium containing 3%FBS.Immunofluoresence analysis showed that FAS expression was significantly higher in the 3%FBS group than in the 10%FBS group.Conclusions 3%FBS condition may induce apoptosis in the nucleus pulposus cells and compromise the cell function to induce intervertebral disc degeneration.The caspase family should be involved in the process.
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Objective To explore the effect of caspase-9 inhibitor on low fetal bovine serum ( FBS)-induced apop-tosis in cartilage endplate chondrocytes in SD rat vertebrae.Methods Disc cartilage endplates were obtained from 3-month old SD rats and subjected to sequential digestion to harvest chondrocytes for primary culture, and apoptosis was in-duced by 1%FBS for 48 hours.Three groups of chondrocytes were treated by 1% FBS, caspase-9 inhibitor ( Z-LEHD-FMK) and DMSO, respectively.After 48 hours, apoptosis was detected by DAPI staining and flow cytometry.The expres-sion of procaspase-9, active caspase-9 and active caspase-3 was monitored by Western blot.Results Compared with the 1%FBS group (40.8 ±0.84)%and DMSO group (40.2 ±1.56)%, the apoptosis rate of the caspase-9 inhibitor group (26.3 ±2.56)% was significantly lower (P<0.05).The expressions of active caspase-9 and active caspase-3 in the caspase-9 inhibitor group were significantly lower than those in the other two groups (P<0.05).Conclusions Caspase-9 inhibitor can inhibit low FBS-induced apoptosis in cartilage endplate chondrocytes of rat vertebrae, and might become a new drug for the treatment of disc degeneration.