| BackgroundCervical cancer(cervical cancer,CC)is a class of malignant tumors that occur in women's vagina and cervical canal.The morbidity rate is the second highest in all women with malignant tumors and is therefore a serious threat to the health and safety of women One of the tumors.In China,cervical cancer has become one of the most common and multiple gynecological malignancies,the death rate of women in China's cancer mortality rate of the second,so the incidence of cervical cancer in China is still at a high level.Although the current clinical use of surgery or combined radiotherapy and chemotherapy and other methods of treatment of early cervical cancer is significant,but for patients with advanced cervical cancer has little effect.In addition,the inhibition of cervical cancer cell growth,proliferation,invasion and metastasis of drugs are traditional anti-cancer drugs,which in the killing of tumor cells at the same time on the surrounding normal tissue cells caused by side effects,and can not be avoided.Chinese medicine in the treatment of cervical cancer has a significant advantage,a large number of basic and clinical research at home and abroad have confirmed that traditional Chinese medicine through multi-channel,multi-target,multi-link play a role,with comprehensive onset characteristics,so as to play a preventive and treatment of tumor efficacy.At present,the natural plant and its active ingredients of anti-tumor activity has been initially recognized by the international medical profession.Physcion 8-0-?-glucopyranoside(PG)is one of the chemical components contained in the hoof,and current studies have demonstrated that PG has anti-tumor effects but has not yet been studied on its effects on cervical cancer progression and development in vivo and in vitro.ObjectiveIn this study,we investigated the biological effects of physcion 8-0-0-?-glucopyranoside(PG)on cervical cancer in vitro and in vivo.We first observed the inhibitory effect of PG on the proliferation of cervical cancer HeLa cells by in vitro experiments,and studied whether it can induce apoptosis of HeLa cells and detect the expression of apoptosis-related proteins after PG treatment,so as to clarify the effect of PG on cervical cancer cell biological behavior Impact.Meanwhile,we established a model of human cervical cancer xenografts in nude mice to study the inhibitory effect of PG on the growth of HeLa cells in nude mice,and to further clarify the antitumor effect of PG in vivo.This study aims to clarify the role of PG anti-cervical cancer mechanism,so as to provide an experimental basis for its clinical application in the treatment of cervical cancer.Methods(1)HeLa cells were randomly divided into 7 groups of PG final concentration,the corresponding concentration of PG was added to each group of cells to the final concentration of 5,10,20,40,60,80,100 ?g/mL,the corresponding treatment 24 The cell proliferation was measured by CCK-8 method.The cells were treated with 40 ?g/m of HeLa cells at 0,12,24,36,48,72 h respectively.The cells were treated with CCK-8 at different time points Proliferation inhibition rate.The cells were treated with 20,40 and 60 ?g/mL PG for 24 hours,and the apoptosis of the cells was detected by DAPI staining.Transwell ? cells were used to detect the invasiveness of the cells.Cell scratch test The expression of caspase-3,caspase-9,Bax and Bcl-2 protein was detected by western blot.The expression of caspase-3,caspase-9,Bax and Bcl-2 were detected by flow cytometry.(2)HeLa cells were subcutaneously injected into nude mice to establish nude mice models.Twenty subcutaneous transplanted nude mice models were selected and randomly divided into control group,10,20,40 mg/kg PG group and treated accordingly.(L)and width(W)of the nude mice in each group at 0,5,8,12,and 15 days after the administration.The tumor volume was calculated and the growth of the transplanted tumor was drawn.The expression of caspase-3 and Bcl-2 protein in the xenografts of each group were detected by immunohistochemical method.Apoptosis rate of transplanted tumor cells was measured by TUNEL assay,and the apoptosis rate was calculated.Results(1)The inhibition rates of HeLa cells were(4.3± 0.4)%,(18.8 ± 1.7)%and(39.1±3.3),respectively,when the concentrations of PG were 5,10,20,40,60,80 and 100 ?/%,(42.2±4.2)%,(60.3±6.5)%?(63.5±6.8)%and(66.4±6.6)%respectively.The IC50 was 41.34 ?g/mL.The cell proliferation was inhibited by CCK-8 at the time point of 0,12,24,36,48 and 72 h after treatment with 40 ?g/m of HeLa cells.The results showed that the cell proliferation inhibition rates were 0,(36.4±7.8)%,(48.1 ? 7.4)%(63.3±6.4)%,(72.7%)at 0,12,24,36,48,72 hours after PG±8.6)%.With the increase of PG concentration,the proliferation inhibition rate of HeLa cells was also increased,and with the prolongation of PG time,the proliferation inhibition of HeLa cells also increased with the increase of PG concentration.DAPI staining showed that there were almost no apoptotic cells in the control group.When the PG cells were treated with 20,40 and 60?g/mL PG cells,the number of apoptotic cells gradually increased,showing typical apoptotic morphological features,Chromatin condensation can be seen under the microscope,cell membrane blistering,but also observed nuclear pyknosis and so on.With the increase of PG concentration,the number of apoptotic cells increased gradually.In control group,the apoptotic rate of PG group was 0,(13.6±3.5)%,(38.4± 6.7)%and(59.5±9.6)%,respectively,,The apoptosis rate of HeLa cells was gradually increased.The results of Transwell chamber assay showed that the number of invasive cells in control group was(147± 22),(115 ± 17),(78 ± 15),(20 ±5)52±12).The number of invasive cells in each PG group was significantly lower than that in the control group(P<0.05).The results showed that the number of invasive cells in HeLa cells decreased gradually with the increase of PG concentration.The results of cell scratch test showed that the migration distance of the control group was(365.7±49.5)?m,(288.3±37.3)?m,(231.4±34.6)?m,(20±5)(P<0.05),and the migration distance of HeLa cells decreased with the increase of PG concentration.The migration distance of HeLa cells was significantly decreased with the increase of PG concentration(P<0.05).he apoptotic rates of the control group were(0.4± 0.1)%,(12.3±2.9)%,(36.7±7.1)%,(61.3±8.3)%.The apoptotic rate of HeLa cells was significantly increased with the increase of PG concentration.The apoptotic rate of each PG group was significantly higher than that of the control group(P<0.05).Western blot analysis showed that the relative expression of Bax protein in the control group,20 ?g/mL PG group,40 ?g/mL PG group and 80 ?g/mL PG group were(0.08± 0.02),(0.11± 0.06)(P<0.05),and the expression of Bax protein in the PG group was significantly higher than that in the control group(0.38 ±0.15)and(0.74±0.22),respectively.The expression of Bcl-2 protein in the control group,20 ?g/mL PG group,40?g/mL PG group and 80?g/mL PG group were detected respectively.The relative expression of Bax protein in each group was(0.67±0.22),(0.41±0.19),(0.28 ± 0.11),(0.05±0.02),the expression of Bcl-2 protein in each PG group was significantly lower than that in the control group(P<0.05)The expression of Caspase-3 protein was detected in the control group,20 ?g/mL PG group and 40 jig/mL PG group.The expression of Caspase-3 protem in the control group was significantly lower than that in the untreated group(0.13±0.05),(0.28 ± 0.11),and(0.64±0.27),respectively.The expression of Caspase-3 protein in PG group was(0.04± 0.01),(0.13± 0.05)(P<0.05),and the expression of Caspase-9 protein increased with the increase of the concentration of PG The expression of Caspase-9 protein in each group was significantly higher than that in the control group(P<0.05)(0.08 ± 0.01),(0.12±0.05),(0.18(0.12±0.05))in PG group,PG group,PG group and PG group at 80 ?g/mL,PG group and control group,respectively.The relative expression of Caspase-(P<0.05),and the expression of Caspase-9 protein was significantly higher in the PG group than in the control group(P<0.05),and the expression of Caspase-9 protein also increased with the increase of PG concentration.(2)In this experiment,20 nude mice were all tumor.(P<0.05).With the increase of PG concentration,the volume of tumor in nude mice was decreased(P<0.05),and the volume of tumor in nude mice was significantly lower than that in control group at each time point(P<.The results of TUNEL showed that the number of positive cells in each group was significantly decreased(P<0.05)as compared with the control group.The number of positive cells decreased with the increase of PG concentration(P<0.05).Immunohistochemical results showed that the positive sites of Caspase-3 protein staining were mainly distributed in the transplanted tumor cells in nude mice,brown granules.Compared with 0 mg/kg group,the expression of Caspase-3 protein was significantly increased in all groups(P<0.05),and the expression of Caspase-3 protein in nude mice was significantly higher than that in control group(P<0.05)The expression of Caspase-3 protein in the transplanted tumor cells also increased.Between the 10 mg/kg group and the 20 mg/kg group(x2= 4.319,P = 0.036),between the 10 mg/kg and 40 mg/kg groups(x2 = 5.211,P(P<0.05).There was significant difference in the expression of Caspase-3 between 20 mg/kg group and 40 mg/kg group(x2 = 8.419,P = 0.003).Bcl-2 protein staining of the positive sites were mainly distributed in nude mice transplanted tumor cells were pulp,brown granules.Compared with 0 mg/kg group,the expression of Bcl-2 protein was significantly decreased in all groups(P<0.05).The expression of Bcl-2 protein in nude mice xenograft tumor cells was significantly lower-2 protein expression levels also decreased.Between the 10 mg/kg and 20 mg/kg groups(x2 = 5.233,P = 0.031),between the 10 mg/kg and 40 mg/kg groups(x2 = 6.719,P(P<0.05).There was significant difference in the expression of Bcl-2 protein between 20 mg/kg group and 40 mg/kg group(x2 = 9.822,P = 0.002).ConclusionPG can inhibit the growth of cervical cancer Hela cells and transplanted tumors in vivo and in vitro experiments,and promote its apoptosis.The mechanism of the above process is related to inhibiting the expression of Bcl-2 and promoting the expression of caspase-3,caspase-9 and Bax. |
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