| Thyroid carcinoma is one of the most common malignant tumor of the human endocrine system and one of the fastest rising velocity of malignant tumor in recent years.At present the main treatment is surgery,but still there is the risk of recurrence and metastasis after surgery.Our team found that the incidence of thyroid tumors in rats decreased significantly by given Prunella vulgaris L.powder through the mouth for two years.As a traditional commonly used Chinese herbal of Ruanjian Sanjie,Prunella vulgaris L.used in preventing and controlling thyroid disease for a history of two thousand years.It is found that Prunella vulgaris L.had obvious therapeutic effect with treating goiter in the clinical treatment,but the mechanism is not clear yet.Therefore,this study chose the types of thyroid cancer with the highest incidence as the test object,intends to explore the effect of Prunella vulgaris L.to thyroid papillary carcinoma cells K1? apoptosis and the function of mitochondrial apoptotic pathways in the process of Prunella vulgaris L.inducing thyroid papillary carcinoma cells apoptosis,and so as to provide a scientific basis for prevention and treatment of thyroid papillary carcinoma by Prunella vulgaris L..Objective To explore the effect of Prunella vulgaris L.to thyroid papillary carcinoma cells? apoptosis and the function of mitochondrial apoptotic pathways in the process of Prunella vulgaris L.inducing cancer cells apoptosis,and to provide a scientific basis for the clinical application of Prunella vulgaris L..Methods 1.Experimental substances and preservation method Prunella vulgaris L.extract(1 g extract is equivalent to 6.95 g medicinal materials)was brown viscous semisolid.It extracted by traditional enrichment method ?Water extract-alcohol precipitation? and stored at 4 ? in a plastic bottle.According to proper Prunella vulgaris L.extract with sterilized water dissolve configured to 400 mg/ml.The solution was stored at 4? after micropore filter of 22?m.2.Cell lines Thyroid follicular epithelial cells(HUM-CELL-0097),Papillary thyroid cancer cells(K1).3.Experimental method 3.1 Cell culture HUM-CELL-0097 cells were cultured in Epi CM 4101 media.K1 cells were cultured in RPMI-1640 media.All cells were cultured at 37 ? in a 5% incubator.3.2 Experimental protocol 3.2.1 The toxic effect of Prunella vulgaris L.on thyroid follicular epithelial cells HUM-CELL-0097 Cells were treated with PV in 0,2,5,10,20,40,80,160,200 mg/ml doses.The exposure time was 24 h.The cell proliferate ability was detected at the end of experiments,and the IC50 was calculated.3.2.2 The toxic effect of Prunella vulgaris L.on thyroid papillary carcinoma cells K1 Cells were treated with PV in 0,2,5,10,20,40,80,160,200 mg/ml doses.The exposure time was 24 h.The cell proliferate ability was detected at the end of experiments,and the IC50 was calculated.Observation of cell morphology was conducted in control and five treatment groups with 1/8,1/4,1/2,1,2 IC50.3.2.3 The effect of inducing thyroid papillary carcinoma cells apoptosis under Prunella vulgaris L.treatment K1 Cells were treated with Prunella vulgaris L.in 1/8,1/4,1/2,1,2 IC50 doses for 24 h.The cell apoptosis of K1 cells was detected by flow cytometry and Hoechst method.3.2.4 The role and mechanism of mitochondrial apoptotic pathways on thyroid papillary carcinoma cell apoptosis under Prunella vulgaris L.treatment K1 Cells were treated with PV in 1/8,1/4,1/2,1,2 IC50 doses for 24 h.The MMP of K1 cells was detected by flow cytometry.K1 Cells were treated with PV in 1/2,1,2 IC50 doses for 24 h.The protein expression of Bcl-2,Bax,Cyto C,Caspase-9,Caspase-3,Cleaved Caspase-3 were determined by western blotting method.3.3 Indexes and detection General toxicity observation including cell viability(MTT methods)and cell morphology(inverted microscope).Apoptosis damage observation including cell apoptosis(flow cytometry and Hoechst).Mitochondria toxicity observation including MMP(flow cytometry)and protein expression(western blotting method).Results 1.The toxic effect of Prunella vulgaris L.on thyroid follicular epithelial cells With the increase of Prunella vulgaris L.?s concentration,the cell viability in the treatment group above 20 mg/ml were significantly decreased(P<0.05).The IC50 of PV to HUM-CELL-0097 cells for 24 h is 12.908 mg/ml.2.The toxic effect of Prunella vulgaris L.on thyroid papillary carcinoma cells 2.1 Cell proliferate ability With the increase of Prunella vulgaris L.?s concentration,the cell viability in all treatment group were significantly decreased(P<0.05).The IC50 of Prunella vulgaris L.to K1 cells for 24 h is 2.427 mg/ml.2.2 Cell state Normal K1 cells are ovoid,strong-adherent ability.Compared with the control group,the number of K1 cells decreased significantly,cells became shrinking and round in the Prunella vulgaris L.dose of 2.4 mg/ml and 4.8 mg/ml.3.The effect of inducing thyroid papillary carcinoma cells apoptosis under Prunella vulgaris L.treatment 3.1 Hoechst staining to observe apoptosis Compared with the control group,the numbers of apoptosis cells were significantly increased in treatment group with Prunella vulgaris L.in 2.4 mg/ml and 3.4 mg/ml dose.3.2 Flow cytometry to observe apoptosis Compared with the control group,the rate of apoptosis cells increased significantly in treatment group with Prunella vulgaris L.in 2.4 mg/ml and 3.4 mg/ml dose(P<0.05).4.The role and mechanism of mitochondrial pathways on thyroid cancer cell apoptosis under Prunella vulgaris L.treatment 4.1 Mitochondrial membrane potential Compared with the control group,the MMP was reduced in treatment group with Prunella vulgaris L.in 0.6~4.8 mg/ml dose(P<0.05).3.2 Protein expression level of mitochondrial apoptotic pathways Compared with the control group,the expressions of apoptosis protein Bcl-2,Caspase-9,Caspase-3 were decreased and the expressions of Bax,Cyto C,Cleaved Caspase 3 were increased(P<0.05).Conclusions 1.The IC50 within 24 hours of Prunella vulgaris L.to thyroid follicular epithelial cells(HUM-CELL-0097),thyroid papillary carcinoma cells(K1)were 12.908,2.427 mg/ml respectively,which indicates that the Prunella vulgaris L.has good security..2.Prunella vulgaris L.in 2.4~4.8 mg/ml can induce apoptosis of thyroid papillary carcinoma cells.3.Prunella vulgaris L.could activate mitochondrial apoptotic pathways to induce the apoptosis of thyroid papillary carcinoma cells. |
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