| Objective Breast cancer has the highest incidence of malignancy in women,so the level ? prevention of breast cancer is very important.If the breast cancer can be blocked in the precancerous lesions stage,it will help reducing the probability of breast cancer.Modern medicine generally adopts surgical resection or endocrine drugs in the treatment of breast precancerous lesions.However,due to the limitations of surgery and side effects of drugs,the treatment effect is relatively poor.Traditional Chinese Medicine,whereas,can take its advantages here.On the basis of the preliminary work,this study used Xiaotan Jieyu Prescription to intervene rat with breast precancerous lesions and precancerous lesions of breast cancer MCF-10 AT cells and hence verified the efficacy of Xiaotan Jieyu Prescription on precancerous lesions of breast cancer;clarified the relationship between PI3K-AKT signal transduction pathway and breast precancerous lesion.This study also discussed the possible mechanism and effective target of Xiaotan Jieyu Prescription from the perspective of PI3K-AKT signal transduction pathway and hence provided modern molecular biological basis for treatment of precancerous lesions of breast cancer with "Xiaotan Jieyu Prescription".Methods 1.In vivo experiments(1)Establishment of rat model of precancerous lesions of breast: 123 SD female rats were randomly divided into four groups: blank control group,15 rats;DMBA low dose group(5mg each rat),36 rats;DMBA middle dose group(10mg each rat),36 rats and DMBA high dose group(20mg each rat),36 rats.DMBA combined with estrogen and progesterone was used in sequential method to induce rat precancerous lesion model.Rats were sacrificed at week 8,week 10 and week 12,and breast tissues or tumors were taken to observe the pathological changes of mammary gland,then the DMBA optimal dose and optimal time node of the model of breast cancer were screened out.(2)Study on the mechanism of Xiaotan Jieyu Prescription on DMBA combined with estrogen and progesterone-induced breast cancer in SD rats: 120 rats were randomly divided into two groups: blank control group with 20 rats and model group with 100 rats.From the 11 th week,the model group was randomly divided into 5 groups: model control group,Xiaotan Jieyu Prescription low does group(LD group),Xiaotan Jieyu Prescription medium dose group(MD group),Xiaotan Jieyu Prescription high dose group(HD group),tamoxifen group(TAM group)and were given the corresponding intragastrical administration for 4 weeks.All rats were sacrificed at the end of the experiment and the second pair of breasts and the surrounding skin were taken under the sterile condition.Then the pathological sections were taken for pathological observation and analysis.The expression levels of PI3 K,AKT and PTEN were detected by immunohistochemistry and Western Blot.2.In vitro experimentsThe experiment sets 5 groups: the control group(CON),Xiaotan Jieyu Prescription group(XTJY)with a drug concentration of 11mg/ml(raw medicine),Tamoxifen group(TAM)with a drug concentration of 0.01mg/ml,inhibitor group(LY),given PI3 K inhibitor LY294002 at a concentration of 16.29 ?M and combined group of Xiaotan Jieyu Prescription and LY294002(XTJY + LY),with a concentration 11 mg / ml(raw medicine)for Xiaotan Jieyu Prescription and 16.29?M for LY294002.(1)Observe effects of Xiaotan Jieyu Prescription on growth inhibition rate of breast precancerous lesions MCF-10 AT cells by CCK-8 Method and observe the cells' growth state with optical microscope;(2)detect effect of Xiaotan Jieyu Prescription on apoptotic rate and cell cycle of breast precancerous lesions MCF-10 AT cells by flow cytometry;(3)detect the effect of Xiaotan Jieyu Prescription on PI3K-AKT signaling pathway and protein expression with Q-PCR and Western blot method.Results 1.In vivo experiments(1)Modeling results:Observation found that the first 8 weeks,10 weeks and 12 weeks,the blank control group did not appear precancerous lesions and invasive carcinoma,while there were different degrees of general hyperplasia,precancerous lesions and invasive carcinoma in the low,middle and high dose groups.This indicates that DMBA combined with estrogen and progesterone in sequential method can successfully replicate the pre-breast cancer model.In the 12 th week,the precancerous lesion and invasive carcinoma were thickened in different degrees compared with the 10 th week(P<0.05),and the higher the concentration of DMBA,the deeper the precancerous lesion and invasive cancer.With the advance of time,the model group of mammary gland tissue lesions is in line with breast cancer striped continuous process of Normal ? hyperplasia ? atypical hyperplasia ? carcinoma in situ ? invasive cancer.For DMBA mid-dose group,the success rate of modeling can reach 91.7% in 10 th week.Compared with the other groups,this was statistically significant(P<0.05).(2)Effect of Xiaotan Jieyu Prescription on breast precancerous lesions in ratsThere were 18 normal breast tissues in the blank control group,accounting for 90% of the rats,and only 2(10%)had normal hyperplasia and in the model control group,14(70%)rats were cancerous and 6(30%)had precancerous lesions.With the intervention by tamoxifen and Xiaotan Jieyu Prescription,the proportion of cancer in each treatment group was lower than that of model control group.Thereinto,4(20%)rats in HD group and TAM group turn to cancer,7(36.8%)rats in MD group turn to cancer,11(57.9%)rats in LD group turn to cancer,and most of them remained in the precancerous stage and the differences between each group and the model control group are statistically significant(P<0.05).The incidence of breast cancer in the HD group and TAM group were both 20%,indicating an identical anti-cancer effect.(3)Effect of Xiaotan Jieyu Prescription on expression of PI3K-AKT signal transduction pathway in breast precancerous lesion rats:Western Blot test results show: Compared with the model control group,the expression of PI3 K and P-Akt protein in LD,MD and HD group,TAM group and PI3 K and P-Akt protein decreased in different degrees,and the decrease of Xiaotan Jieyu Prescription group and tamoxifen group was significant(P<0.05).PTEN protein expression also increased in different degrees,amongst which Xiaotan Jieyu high dose group and tamoxifen group increased significantly(P<0.05).The expression of PI3 K and p-Akt protein in breast tissue of breast cancer precancerous model rats was down-regulated by Xiaotan Jieyu recipe and PTEN expression was up-regulateed,where the HD group has the best effect,which indicates that Xiaotan Jieyu prescription has a dose-effect relationship in the treatment of breast precancerous lesions in rats.Compared with TAM group,the effect of Xiaotan Jieyu prescription on PI3 K and p-Akt was higher than that of tamoxifen(P <0.05).The immunohistochemical results showes: compared with the model control group,the expression of PTEN protein was the highest in LD,MD and HD groups and TAM group,and all four groups have enhanced expressions at different extents;the expression of PI3 K and P-Akt in LD,MD and HD groups and TAM group were decreased in different degrees,and with the increase of the dose of Xiaotan Jieyu Prescription groups,PI3 K protein and AKT protein weakened to higher levels,showing a dose-effect relationship.The expression of PI3 K and P-Akt protein in HD group was more obvious than that in other groups.2.In vitro experiments(1)Effect of Xiaotan Jieyu Prescription on proliferation of MCF-10 AT cells in breast precancerous lesionsThe CCK-8 test results indicate the 24 h and 48 h OD values of breast precancerous lesions MCF-10 AT cells in the group of Xiaotan Jieyu Prescription group,TAM group,LY group and XTJY + LY group were lower than those in control group and the difference was with statistical significance(P<0.05).The inhibitory rates of 48 h drug intervention are 69.20 ± 2.53% in XTJY group,43.20 ± 3.87% in TAM group,75.40± 3.52% in LY group and 77.82 ± 2.64% in XTJY + LY group and the inhibitor rate of each group is obviously higher than the rates of 24 h drug intervention.The inhibitory effect is positively related with intervention time.Under the microscope,the cells in the control group were densely grown and the refraction was good;the number of breast precancerous lesions MCF-10 AT cells of each intervened group decreased significantly after 24 h intervention and the cells in each group showed different degrees of necrosis,meanwhile,suspended cells appeared and the refractive properties deteriorated.(2)Effect of Xiaotan Jieyu Prescription on cell cycle and apoptosis of MCF-10 AT cell in breast precancerous lesionsDetect with the employment of ANNEXIN V-FITC / PI double staining flow cytometry: the apoptotic rate of MCF-10 AT cells in the control group was 7.73±1.41%,while the apoptotic rates of each drug group are 43.40±2.71% for Chinese medicine group,33.03±1.60% for tamoxifen group,40.97±5.70% for inhibitor group,44.83±8.40% for traditional Chinese medicine + inhibitor group,which are obviously higher than the control group and the difference is statistically significant.With the prolonged intervention time the apoptotic rate increases.The cells in G1/G0 phase of XTJY group,TAM group,LY group and XTJY + LY group were 68.93 ± 0.72%,60.50 ± 1.04%,65.10 ± 3.98%,77.37 ± 1.20%.Compared with the control group 39.73 ± 2.87%,the proportion was significantly higher.This indicates the above groups may lead to breast precancerous lesions MCF-10 AT cells being blocked in the G1/G0 phase,the difference was statistically significant(P<0.05).Cell cycle results showed each group of drugs can block cell differentiation and the block mainly occurred in the G1/G0 period.(3)Inhibitory effect of Xiaotan Jieyu Prescription on PI3 K / Akt signaling pathway in breast precancerous lesions MCF-10 AT cellsWestern blot and Q-PCR results showed,compared with the control group,the expressions of PI3 K,P-Akt,AKT protein and gene in XTJY group,TAM group,LY group and XTJY + LY group were decreased in different degrees,PTEN protein and gene expression also increased to varying degrees and the difference is statistically significant(P<0.05).Conclusions 1.DMBA with 10 mg each rat is the ideal model dose,10 weeks is also more suitable modeling time node,and can be used to successfully prepare breast cancer model rats.2.PI3K-AKT signaling pathway has been activated in the stage of precancerous lesions of breast cancer and is closely related to the development and reversal of breast precancerous lesions.3.Xiaotan Jieyu Prescription can induce the apoptosis of breast precancerous lesions MCF-10 AT cell and it has a definite therapeutic effect on DMBA combined with estrogen and progesterone-induced SD female rat precancerous lesion model.Its working mechanism is related with affecting the PI3K-AKT signal transductionpathway and inducing apoptosis of cells. |
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