| Objective: Prostate cancer is one of the common tumors in men with high morbidity and mortality,as well as poor prognosis,easy to relapse after surgery,especially in elderly patients,and serious threat to the health of elderly patients.Search for drugs that can effectively relieve prostate cancer is of great significance for the treatment of prostate cancer actively.As an important source of drug development,traditional Chinese medicine(TCM)has less adverse reactions and more accurate curative effect,so it has a good development prospect,and has become a research hotspot in the field of medicine.Saikosaponin is extracted from Bupleurum chinense DC of Umbelliferae family and has many pharmacological effects such as anti-inflammation,liver protection,angiogenesis inhibition and anti-tumor.The most active subtype of saikosaponin-d has been proved to have strong anti-tumor,anti-inflammatory,anti-fibrosis and other pharmacological activities.However,at present,the anti-tumor mechanism of saikosaponin-d has not been fully elucidated.In this study,the effects of saikosaponin-d on apoptosis and autophagy of LNCap cells of prostate cancer were studied by cell experiments in vitro so as to provide reference for clinical treatment.Methods:The effect of SSd on LNCap cell viability was measuredusing MTS method.Apoptosis rates of the cells were detected by Hoechst 33258 fluorescence staining,Annexin V-FITC/PI staining flow cytometry and theexpressions of Bcl-2 and Bax were detected by Western Blot.In addition,the expression of autophagy marker LC3?/? was observed by immunofluorescence staining,and the expressions of autophagy related proteins LC3?/? and p62 were detected by Western Blot,and the effects of autophagy inhibitors and autophagy activators were studied in this process.Results:1.MTS method to detect cell viability: the results showed that SSd could inhibit the proliferation of LNCap cells.compared with the control group,with the increase of administration concentration,cell viability decreased.At the same administration concentration,the cell viability decreased with the increase of administration time.The results showed that SSd inhibited the proliferation of LNCap cells in a time-dependent and dose-dependent manner.2.Hoechst 33258 fluorescence staining was used to observe the changes of cell morphology: The nucleus morphology of control group was regular and uniform blue fluorescence.With the increase of administration concentration,the number of irregular nuclear cells gradually increased and dense granular blue fluorescence appeared in cytoplasm.Most of the nuclei are cleaved and have lost their normal nuclear morphology when the administration concentration reached 20?mol/L.3.Annexin V-FITC/PI double-staining flow cytometry was used to detect the apoptosis rate.The results showed that the apoptosis rates and total apoptosis rates increased with the increase of concentrations,and the difference was statistically significant(P<0.01).The results suggested that SSd could induce apoptosis of LNCap cells in a dose-dependent manner.4.The expressions of Bcl-2 and Bax was detected by Western Blot: the expression of Bcl-2 decreased gradually and the expression of Bax increased gradually with the increase of administration concentration,the difference was statistically significant(P<0.05).5.The expression of LC3 was observed by immunofluorescence staining: the fluorescence expression of LC3 increased gradually under confocal microscope with the increase of concentrations,and the results showed that LC3 aggregation increased and autophagy increased.6.The expressions of autophagy related proteins LC3?/? and p62 were detected by Western Blot: the results showed that the expression of p62 protein decreased gradually and the expression of LC3?/LC3? increased gradually with the increase of concentrations,the difference was statistically significant(P<0.05).7.The effects of autophagy inhibitor and autophagy activator on the autophagy process of SSd induced LNCap cells were detected by Western Blot.The results showed that the relative protein expression levels of LC3?/LC3?andp62 were changed correspondingly after using autophagy inhibitor(3-MA)and autophagy activator rapamycin respectively.Conclusion: 1.SSd could inhibit the proliferation of LNCap cells and decrease the cell viability in a time-dependent and dose-dependent manner.2.SSd induced apoptosis in LNCap cells of human prostate cancer,including the expression of Bcl-2 decreased Bax increased.3.SSd can increase autophagy level in LNCap cells which is influenced by autophagy activator and autophagy inhibitor. |
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