| Part Ⅰ:The Effect of Corosolic Acid study on Apoptosis of CaSki Cervical Carcinoma Epithelial CellsObjectiveCervical cancer is one of the most common malignancies in China.According to incomplete statistics,eervical cancer has become the second female malignant tumor,and the younger age of onset,morbidity and mortality increased year by year,thus a serious threat to the health and safety of women in our country.In the current clinical treatment of cervical cancer,chemotherapy drugs are usually the main,but as the main drug of Western medicine drug side effects and drug resistance,a serious threat to the patient’s clinical prognosis and rehabilitation.Therefore,from the small side effects and drug resistance of small Chinese medicine,screening of anti-cervical cancer drugs become the current clinical research hot and difficult.Corosive acid(C30H48O4)as a natural pentacyclic triterpenoid compounds,derived from calyx kiwi(cat ginseng),loquat,lagerstroemia and other plants.In a number of clinical and pharmacological studies have pointed out that Corosolic acid has anti-tumor,anti-oxidation,control of blood sugar and weight loss effect.In the development of weight loss and diabetes in the United States,tetraloxic acid has received FDA phase Ⅲ clinical trials.There are also a number of studies have shown that Corosolic acid can inhibit the tumor cell activity,promote tumor cell apoptosis and inhibition of angiogenesis within the tumor to play a very good anti-cancer effect.At the same time,tetraloxic acid is a natural product,rather than artificial synthesis,its biocompatibility,toxic side effects and low resistance characteristics,but also highlights its advantages in the development of anti-tumor drugs.Thus,tetraloxic acid has become one of the popular candidates for anti-tumor drugs.However,the anti-tumor research of colosol acid is concentrated in hepatocellular carcinoma,gastric cancer and lung cancer,but its research on cervical cancer has not been systematically reported.In order to clarify the role and clinical value of korosorbide in the treatment of cervical cancer,the effect of different concentrations of corosolic acid on the apoptosis activity of human cervical carcinoma epithelial cell CaSki was studied.The eflfects of different concentrations of corosolic acid The cell morphology,proliferative capacity,apoptosis rate,apoptosis rate,cell cycle,in vivotumorigenic ability of human cervical carcinoma epithelial cells and the proliferation of CaSki in vivowere observed by CaSki culture of human cervical carcinoma epithelial cells.So as to systematically and comprehensively reveal the effect of tetracycline on the apoptosis of human cervical carcinoma epithelial cell CaSki,and then provide new methods and strategies for the prevention and treatment of cervical cancer.MethodsIn this study,the effect of corosolic acid on the apoptosis of human cervical carcinoma epithelial cell CaSki was the breathrough point.The specific experimental method was as follows.1.The morphological changes of CaSki cells in cervical cancer epithelial cells were observed by different concentrations of Corosolic acid(0、10、50、100μg/mL)and cervical cancer epithelial cells.The morphological changes of CaSki cells were observed by different concentrations of Corosolic acid.2.The proliferation of CaSki cells was detected by MTT assay and CCK-8 method.The proliferation of CaSki cells was determined by different concentrations of corosolic acid(0,10,20,50,100μg/mL)in the impact of the rate.3.The cell structure and morphology of CaSki cells were detected by AO/PI staining and Hoechst 33258 staining.The changes of CaSki cell nucleus and the expression of apoptotic bodies were investigated in different concentrations of Corosolic acid.(P<0.05),and the apoptotic and necrotic cells were determined according to the structural changes of CaSki nucleus.The apoptotic and necrotic rates of different groups of CaSki cells were calculated,and then different concentrations of corosolic acid(0,10,50,100μg/mL)On the apoptosis of CaSki cells.4.The cell cycle of CaSki cells in different cervical cancer cells was analyzed by flow cytometry.The effects of different concentrations of corroxacylic acid(0,10,50,100μg/mL)on CaSki cell cycle were analyzed.And then analyzed the causes of apoptosis in CaSki cells.5.The early apoptosis rate,late apoptotic rate and total apoptosis rate of CaSki cells in different cervical cancer cells were analyzed by flow cytometry.Then,different concentrations of corosolic acid(0,10,50,100μg/ML)on the apoptosis of CaSki cells was verified.6.Construction of human cervical cancer epithelial cell CaSki to construct the mouse solid tumor model,respectively,the application of blank group,Corosolic acid group(intraperitoneal injection,drug concentration of 10,50,100μg/mL),by calculating the different groups of tumor relative Volume,weight and in vivotumor inhibition rate,to study the effects of different concentrations of tetralotine on CaSki in vivotumorigenie ability.7.The proliferation of CaSki in tumor tissue of tumor-bearing mice was detected by immunohistochemistry to determine the effect of different concentrations of tetraloxic acid on the growth of CaSki.So as to verify the effect of Corosolic acid on CaSki apoptosis of cervical cancer epithelial cells from animal and cell level.Results1.Corosolic acid can significantly inhibit the proliferation of cervical cancer cells CaSki activity,while microscopic observation of cervical cancer cells in the form of CaSki cells can be seen,with the addition of cervical epithelial cells to enhance the concentration of corosolic acid in cervical cancer epithelial cells CaSki cells in apoptotic state gradually increased,apoptotic CaSki cells mostly rounded,and from the cell clumps out,and suspended in the culture medium,and more to one or several crisp sheet-like existence,and the volume Compared with other tumor cells,the cell structure was unclear and the apoptotic trend was observed.2.Corosolic acid can promote the apoptosis of CaSki cells in cervical cancer cells.AO/PI staining and Hoechst 33258 staining showed that corosolic acid could induce crescent-shaped and broken-down apoptotic bodies in CaSki cells.Also appeared irregular nuclear shift to promote CaSki apoptosis.3.10,50μg/mL Corosolic acid can make the CaSki of cervical cancer epithelium more stay in the GO/G1 phase,thereby reducing the mitotic ability of cervical cancer epithelial cell CaSki,and then reduce its proliferation;10,50,100 μg/mL Corosolic acid carn significantly reduce the proportion of CaSki in cervical squamous cell epithelium in S period,thus shortening the preparation time of mitosis in the S phase of CaSki,leading to the loss of genetic material after G2/M phase,50,100μg/mL Corosolic acid can make CaSki epithelial cells in G2/M phase,thereby inducing CasKi apoptosis.4.Corosolic acid can increase the early apoptosis rate,late apoptosis rate and total apoptosis rate of CaSki in cervical cancer epithelial cells.5.Corosolic acid can reduce the tumor volume and weight of CaSki in vivoand enhance its inhibition rate in vivoto reduce the tumorigenic ability of CaSki in vivo.6.Corosolic acid can inhibit the proliferation of cervical cancer cell line CaSki in tumor tissue by reducing the number of proliferating tumor cells and reducing the tumor proliferation index of the tumor tissue.Conclusions1.Corosolic acid can promote the cell cycle of CasKi cells in GO/G1 phase,G2/M phase and shorten S period by inhibiting the proliferation of CasKi in cervical cancer epithelial cells and increasing the apoptosis rate of CaSki cells.The apoptosis of Cervical Carcinoma Epithelial Cell.2.Corosolic acid can reduce the ability of cervical cancer epithelial cell CaSki in mice subcutaneous tumorigenesis;can inhibit cervical cancer epithelial cell CaSki in tumor tissue proliferation activity.Part Ⅱ:Effects of Corosolic Acid on PI3K/Akt Signaling Pathway in CaSki Cervical Cancer Epithelial CellsObjectivePI3K-Akt signaling pathway is one of the most important signal regulation pathways in the process of cervical cancer.It is mainly involved in the proliferation,apoptosis,invasion,migration and neovascularization of cervical cancer cells.In modem life Medical research is to be used as cervical cancer markers and therapeutic targets for research.It has been agreed that the activation of threonine protein kinase AKT must be carried out under the catalysis of PI3K protein to form AKT phosphorylated protein(p-AKT),and the activation of thrombolytic protein kinase(PI3K-Akt),And then activate PI3K-AKT-mTOR downstream signaling protein,to promote the proliferation of cervical cancer cells and inhibit cervical cancer cell apoptosis.It can be deduced that the effect of tetracycline on apoptosis of cervical cancer epithelial cell CaSki is closely related to PI3K-Akt signaling pathway.In order to validate this inference,we also explored the biological mechanism of tetracycline acid in promoting the apoptosis of human cervical carcinoma epithelial cell CaSki.The expression of PI3K,p-PI3K,Akt and p-Akt in human cervical carcinoma epithelial cells were detected by western blot.The expression of PI3K,p-PI3K5 Akt and p-Akt in human cervical carcinoma epithelial cells were measured by different concentrations of corosolic acid and human cervical carcinoma epithelial cell CaSki.Effects of Corosolic Acid on PI3K/Akt Signaling Pathway in Human Cervical Carcinoma Epithelial Cells,revealing the mechanism of Corosolic Acid to regulate CaSki apoptosis in cervical cancer epithelial cells by acting on PI3K/Akt signaling pathway,and then for further development Cervical cancer treatment of natural medicine to provide theoretical basis and technical support.Methods1.Different concentrations of corosolic acid(0,10,50,100μg/mL)and cervical cancer epithelial cells CasKi mixed culture.2.The expression of PI3K,p-PI3K,Akt and p-Akt of CaSki was determined byWestern Blot in order to determine the effect of Corosolic acid on PI3K/AKT signaling pathway in cervical cancer epithelial cells.CaSki apoptosis process.Results1.Corosolic acid can inhibit the expression of PI3K protein in CaSki cervical epithelial cells,while low concentration of Corosolic acid can promote the phosphorylation of PI3K protein and induce high expression of p-PI3K,while high concentration of Corosolic acid Acid can inhibit PI3K protein phosphorylation,thereby reducing the p-PI3K expression.2.Corosolic acid can inhibit the expression of Akt protein in CaSki cervical epithelial cells,while low concentration of Corosolic acid can promote the phosphorylation of Akt protein,and induce the high expression of p-Akt.Acid can inhibit Akt protein phosphorylation,thereby reducing the p-Akt expression.ConclusionsEffect of Corosolic acid on Proliferation and Proliferation of CaSki cervical cancer epithelial cells by regulation of PI3K/Akt signaling pathway. |
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