Norcantharidin induces apoptosis through autophagosome accumulation in breast cancer MDA-MB-231 cells / 中国药科大学学报

@#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.

Antioxidation effect of dihydroquercetin pretreatment in isolatedrat hearts during myocardial ischemia reperfusion injury / 中国药理学通报

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.

Effects of acacetin on T47D cell proliferation / 中国药理学通报

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.

Improvement Effects of Baicalein against Myocardial Ischemia/Reperfusion Injury in Rats / 中国药房

OBJECTIVE:To study the improvement effects of baicalein against myocardial ischemia/reperfusion(I/R)injury in rats. METHODS:I/R injury model was induced by Langendorff method. Isolated heart of 40 rats were randomly divided into nor-mal group(continuous perfusion),model group(perfusion withdrawal 20 min)and baicalein high,medium and low concentration groups (K-H solution of baicalein 40,10 and 2.5 μmol/ml 10 min before perfusion withdrawal). The myocardial infarction rate, the activity of creatine kinase(CK)and lactate dehydrogenase(LDH)in coronary effluent liquid,SOD activity and MDA content, GSH/GSSG and apoptosis rate of cardiac muscle cell in myocardial tissue were detected. RESULTS:Compared with normal group, the myocardial infarction rate,apoptosis rate of cardiac muscle cell,the activities of CK and LDH and the content of MDA in myo-cardial tissue were increased in model group,while SOD activity and GSH/GSSG of myocardial tissue decreased(P0.05). CONCLUSIONS:Baicalein has cer-tain improvement effect on myocardial I/R injury in rats,and its mechanism may be associated with antioxidant and anti-apoptosis effect of baicalein.

Isoliquiritigenin induced apoptosis in human melanoma A375 cells / 中国药理学通报

Aim To evaluate the mechanism of apopto-sis induced by the isoliquiritigenin in A375 human ma-lignant melanoma cells. Methods Sulforhodamine B ( SRB) method was used to determine the A375 cell viability;acridine orange/ethidium bromide ( AO/EB) and Hoechst 33258 staining were used to observe the morphological changes of apoptotic cells; flow cytome-try was used to detect A375 cell apoptotic rate;DCFH-DA was applied to determine the changes of total intra-cellular ROS in A375 cells;JC-1 method was used to measure the changes of mitochondrial membrane poten-tial;the kits methods were used to determine the con-tent of ATP, lactic acid and glucose in A375 cell which was treated with different concentrations of isoliquiritigenin. Results Isoliquiritigenin could in-hibit A375 cell proliferation in a concentration-depend-ent manner; A375 cells showed obvious apoptosis charateristics after treatment by isoliquiritigenin, and the apoptosis rate increased with increasing concentra-tion of isoliquiritigenin. The level of total intracellular ROS in A375 cells increased obviously after dealing with different concentrations of isoliquiritigenin;in ad-dition, the mitochondrial membrane potential, the lev-els of intracellular ATP,lactic acid and the level of glu-cose uptake all declined. Conclusions These find-ings demonstrate that isoliquiritigenin can induce apop-tosis of A375 cells. The mechanism may be related to elevation of ROS level and reduction of aerobic glycoly-sis level.

Research on Mathematical Logic in Syndrome Theory of TCM and False Proposition Features of“Element of Syndrome” / 世界科学技术-中医药现代化

Through the study on mathematical logic relation in the syndrome theory of traditional Chinese medicine (TCM), it revealed that the mathematical logic relation among three basic elements in the judgment of syndromes, which were the disease cause (a), disease location (b), disease nature (c) and syndrome (z), was f(z)=a+b+c. The mathematical logic relation between syndrome (z) and symptom (zi) was f(z)= z1+z2+z3+…+zi. The obvious feature reflected by the complexity of syndromes was the construction number of symptoms. During the syndrome differentiation process, the mathematical logic relation between symptom and disease cause, location, nature and syndrome was z1+z2+z3+…+zi =a+b+c= f(z). However, syndromes generally exhibited a nonlinear relationship of point-set topology. After calculation, 79 major single TCM syndromes had a total of more than 6.5×105 different forms. The number within a certain range of syndrome group was approximately 6.6×10100. The super huge data “Googol” may be the root of complex TCM syndromes. The results showed that the researches on “element of syndrome” using the bayesian networks, the neural network algorithm and the algorithm of double levels of frequency power were irrelevant to the mathematical logic relation of the intrinsic relations of syndrome theory. Therefore, “element of syndrome” was a false proposition feature in the study of TCM basic theory. The established syndrome differentiation method with“element of syndrome” as its core was not conformed to the inherent law of TCM theory and clinical practice.

Inhibitory effects of Licochalcone A on proliferation of melanoma B16 F10 cells / 中国药理学通报

Aim To investigate the mechanism of the melanoma B16 F10 cells proliferation induced by Lico-chalcone A in vitro. Methods The proliferation of B16 F10 cells induced by Licochalcone A was deter-mined by SRB method. The morphological changes were observed using Giemsa staining under the phase contrast microscope equipped with a digital camera. The melanin level was assessed by colorimetric meth-od. The apoptotic rate was determined by Annexin V-FITC/PI assay. Cell cycle distribution was determined by flow cytometry. The mRNA expression levels of B cell lymphoma/lewkmia-2 ( Bcl-2 ) , Bcl-2 associated X protein ( Bax) , the cell cycle protein CyclinE2 and cyclin-dependent kinase-2 ( CDK2 ) CDK2 were detec-ted using Q-PCR analysis. Results The proliferation of B16 F10 cells treated with Licochalcone A was effec-tively inhibited in a concentration and time-dependent manner. A clear morphological change was observed with the increasing concentration of Licochalcone A in B16F10 cells, the dendrite-like projections changed to the narrowing ball shape, which was associated with the increasing melanin level. The low concentration of Licochalcone A could induce B16F10 differentiation, and the high concentration of Licochalcone A could in-duce B16F10 apoptosis, which was accompanied with the increasing G1 phase in cell cycle. The mRNA ex-pression levels of Bcl-2 /Bax, CyclinE2 and CDK2 were markedly reduced. Conclusion Licochalcone A can effectively inhibit the proliferation of B16 F10 cells, induced cell cycle arrest at G1 phase, and fur-ther induced differentiation and apoptosis.

Study on Key Scientific Problem of Dynamic Evolving Rule of Syndrome of Traditional Chinese Medicine / 世界科学技术-中医药现代化

Based on the theory of topological structure within the syndrome of tradition Chinese medicine (TCM), the high order multiple dimensions and nonlinear structure of syndrome can be cracked. And the panorama of complex and changeable syndrome can be showed. The dynamic evolving law of sub-sets of syndromes and the changing rule of the main signs and symptoms with the secondary signs and symptoms can be interpreted. Therefore, the evolving law of sub-sets of syndromes and the accurate corresponding law of prescriptions and medicines can be realized. And the scientific connotation of dynamic evolving rule of syndrome can be revealed. Studying on the occurrence regularity of syndrome, the structure characterization of TCM syndrome can be carried out. And the mathematical structure-physical structure-biological structure can be revealed. Along with the mass new specific forms of syn-dromes to be cracked, the simulation data platform of therapeutic methods with prescriptions and medicines of syn-dromes can be built through the simulation technology of computer. Using the clinical data, through the literature of carding and contrast, the scientific connotation of judgment and diagnostic standard of syndromes can be interpreted. The distracting law of syndromes of diseases can also be revealed through the clinical data of syndrome-groups. And the theoretical basis for the formulation of judgments and diagnostic standard of syndromes of diseases which con-forming to the dynamic evolving law of syndromes can be provided. Evaluation on TCM efficacy can be carried, in order to make clear the curative effect evaluation index system of TCM syndrome in multiple dimensions and multi-ple levels for the promotion of the substantially improvement of syndrome differentiation level.

The mechanism of alteronol inhibiting the proliferation of human promyelocytic leukemia HL-60 cells / 药学学报

This study is to investigate the mechanism of human promyelocytic leukemia HL-60 cells proliferation induced by alteronol in vitro. Human promyelocytic leukemia HL-60 cells cultured in vitro were treated with different concentrations of alteronol. Inhibition rate was detected by SRB assay. Cellular morphological changes were observed by Hoechst and AO/EB (acridine orange/ethidium bromide dye) staining. The apoptosis rate was determined by Annexin V-FITC/PI assay. Cell cycle distribution was determined by flow cytometry. Western blotting analysis was carried out to determine the cell cycle related proteins. The proliferation of HL-60 cells treated with alteronol was inhibited in a concentration-dependent manner. Based on cell viability assay, observation on cell morphology and apoptosis rate, it confirmed that alteronol played an obvious role in proliferation inhibition of human promyelocytic leukemia HL-60 cells, but it did not induce apoptosis in human promyelocytic leukemia HL-60 cells in different concentrations groups. Alteronol could effectively inhibit the proliferation of human promyelocytic leukemia HL-60 cells inducing cell cycle arrest at G1 phase, as well as, alteration expression of cell cycle proteins level of CyclinD1 and pRb.

Protective effect of total flavonoids from Sorbus TianschanicaL leaf against myocardial ischemia/reperfusion injury in rats / 中国药理学通报

Aim To investigate the protective effect and the underline mechanisms of total flavonoids from Sorbus Tianschanica L leaf (TFST) against myocardial ischemia/reperfusion (I/R) injury.Methods The I/R injury model of rat isolated heart was prepared by improved Langendorff retrograde perfusion method.Following the treatment,the coronary blood flow levels (CF),left ventricular developed pressure (LVDP) and maximal rise/fall rate of left ventricular pressure (±dp/dt_(max)) were monitored as the myocardial function.The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in myocardial tissue were measured by the commercial kits. The in vitro anti-oxidative capacity of TFST was detected by the DPPH and lipid peroxidation reaction system.Results Compared with I/R injury group, pre-incubation with TFST (6.0 mg·L~(-1)) significantly improved the LVDP,±dp/dt_(max) and CF during reperfusion. TFST (6.0 mg·L~(-1)) treatment significantly increased the SOD activities and reduced the MDA levels in myocardium tissue.Moreover,TFST (from 6.25 to 100.0 mg·L~(-1)) scavenged the DPPH, hydroxyl radical and superoxide anion free radicals, and inhibited the lipid peroxidation reaction in a concentration-dependent way.Conclusions All the results demonstrate that TFST possesses the cardioprotective effect on ischemia/reperfusion injury.This efficacy may be due to its antioxidative activity.