| Purpose:To observe the effect of Ruyanning combined with Exemestane on the tumor weight and tumor inhibition rate in MDA-MB-435 breast cancer post-menopause nude mice and to explore the mechanism of their inhibiting tumor growth;To investigate the mechanism of Ruyanning combined with Exemestane on MDA-MB-435 breast cancer post-menopause nude mice through PI3K-AKT pathway.;To observe the expression of m-TOR,HIF-1,LDH-A and other proteins in MDA-MB-435 breast cancer post-menopause nude mice by combining Chinese herbal compound Ruyanning with exemestane to explore its regulation mechanism on the metabolism of breast cancer post-menopause nude mice.Material and method:1.Cell line:human breast cancer MDA-MB-435 cell lineAnimals:Female BALB/C Athymic nude mice 50(SPF grade),4 to 6 weeks old,body weight 20±2gPreparation of traditional Chinese medicine:Ruyanning Fang(including Bupleurum 10g,Pseudostellariae 15g,Scutellaria 15g,France Pinellia 10g,Baked Licorice 10g,raw keel,raw oyster each 30g,etc.)are purchased from Liaoning University of Traditional Chinese Medicine Hospital.Drugs are decocted twice,respectively,plus 10 times,8 times the water decoction 60 min,40 min,combined decoction,discarded dregs,adjusted to a concentration of 3.3g/ml,4℃refrigerator spare.Exemestane,25mg/tablet,dissolved in distilled water,adjust the concentration to 3mg/ml spare.2.Preparation of the model bilateral ovariectomy:Nude mice are injected with 10%chloral hydrate at a concentration of 100 mg/ml and 4 ml/kg into nude mice by intraperitoneal injection.The nude mouse is put on a clean operating table.Its limbs are fixed in the center of the operating table.Skin is prepared in the back of the middle(lower edge of the costal arch)with a skin care device,and disinfected with complex iodine,cuted the skin with a scalpel and gently.The incision is located 0.5cm next to the spine,0.5cm away from the lower edge of the rib arch.Cut the muscles and the peritoneum.And milky tissue is seen In the abdominal cavity.Then raising the peritoneal cavity,rice size pink"mulberry-like"ovaries coated by peritoneal can be seen in the abdominal cavity.Then ligation pliers,isolated ovaries,separated.Then check bleeding or not,stitch with the skin,with iodine for the incision disinfection.Using the above method to remove the contralateral ovary,the mice is incubated at about 20℃,waiting for waking.Afer surgery the mice are injected penicillin(60,000 U/ml)by musle,0.25ml/day.The incition is disinfected by iodophor for 3 days.Mice can eat and drink.It is feed with standard SPF for 30 days.The model is successful.3.The creation of model of nude mouse with MDA-MB-435 breast cancer cell:The MDA-MB-435 cells in the logarithmic growth phase are digested with trypsin/EDTA and the PBS solution is washed twice with an appropriate amount of cell culture medium and then uniformly mixed.Cells is stained with 0.4%trypan blue,viable cells(>95%)are counted,cell count plates are counted,and the viable cell concentration are adjusted to 1 x 10~7/ml.The nude mice after ovariectomized are disinfected with iodophor on the right chest wall and the cells are inoculated with the second breast pad of the right chest wall of two nude mice by using a syringe(1 ml).The concentration of the cytosol is 0.2 ml per animal.Afer about 10 days,showing the site of breast milk inoculated tumor nodules,its texture is hard,and growing rapidly.When the primary tumor grow up to 0.8cm~3 size,the tumor is removed by surgery.Bone marrow aspirate the primary tumor is transplanted to the remaining 48 nude mice under the breast pad,10%chloral hydrate(0.02ml/10g body weight)by intraperitoneal injection is injected to nude mice.Under sterile conditions,Taking the primary tumor tissue,it is placed in DMEM culture medium,while cuted into small pieces about 1mm~3,and small pieces is put into the bone marrow puncture needle standby.Before inoculation,the right chest wall in nude mice is disinfected with iodophor,and the needle is inserted into the needle at 1 cm next to the second breast pad on the right chest wall.When the needle is inserted under the breast pad,the needle core is inserted.Immediately the tumor tissue is off the right chest wall under the second breast pad,and then the skin is disinfected with iodine,not to suture the wound.The injection of penicillin 15000 U one-time,nude mice is proceed to feed in the SPF boxes.After the 4 to 5 days from transplantation,solid tumors are seen growth on the pad.There are tumor formation rate of 100%and the shape and size of the neat.After about 15 days from transplantation,the tumor can grow to 9±2mm.3.Experimental groups and administration:Choosing 48 mice from 50 successful modeling nude mice were randomly divided into four groups:(1) model control group;(2)Chinese medicine group(Ruyanning group);(3)western medicine group(exemestane group);(4)combined group(exemestane and Ruyanning group).Each group administered according to the human and mouse dose conversing formula.(1)model control group:0.2ml saline is gavaged for every mouse everyday.(2)Chinese medicine group(Ruyanning group):Ruyanning is according to 33g.kg(equivalent to 12 times the amount of clinical).0.2ml is gavaged for every mouse everyday.(3)western medicine group(exemestane group):Exemestane is administration according to4mg.kg for every mouse(equivalent to 10 times the amount of clinical).0.2ml is gavaged for every mouse everyday.(4)combined group(exemestane and Ruyanning group):Ruyanning is according to 33g.kg.0.2ml is gavaged for every mouse everyday and Exemestane is according to 4mg.kg.All are administered continuously for 21 days.4.Experimental drawing:At the end of the experiment,nude mice in each group are drawn.The nude mice are sacrificed by cervical dislocation and placed on the ice box.The tumor is completely peeled off,weighed and placed on top of the ice box.Macroscopic observation of tumor tissue morphology,size,and then cut it into a number of tumor full-thickness tissue,while drawing more to avoid tumor necrosis.A total of 2 copies of the material:(1)RT-PCR specimens:2-3tissue block about 8mm~3are placed in 1mlTrizol,and marked the marker,immediately placed in-80℃freezer reserve;(2)Western Blot specimens:tissue block are placed in EP tube,frozen in-80℃in the refrigerator.5.Detection indicators:(1)After 21 days with medicine,the nude mice is executed by cervical.The tumor is stripped out completely and weighted.The tumor inhibition rate is calculated.The tumor inhibition rate=(the tumor weigh of model controlling group-its of group of experiment)/its of model controlling group×100%.(2)Anti-tumor effect:The expression of P53 and caspase3 in tumor is detected by Western blot.The expression of NF-kB in tumor is detected by RT-PCR.And the results are statistical analyze.(3)PI3K-AKT pathway:The expression of PI3K and AKT in tumor is detected by Western blot.The expression of PI3K、AKT in tumor is detected by RT-PCR.And the results are statistical analyze.(4)Metabolic regulation mechanism:The expression of mTOR and HIF-1 in tumor is detected by Western blot.The expression of mTOR、LDH-A、HIF-1 and GLUT1 in tumor is detected by RT-PCR.And the results are statistical analyze.Results:1.Tumor weight:Compared with control group,Ruyanning group,Exemestane group and the combined group of tumor weight are less than the control group(P<0.05).The combined group is the least among all(P<0.05).2.Anti-tumor effect:P53and caspase3:Western blot:Compared with control group,Ruyanning group,Exemestane group and the combined group of the expression of P53 is more than the control group(P<0.05).The combined group is more significant especially(P<0.05).RT-PCR:NF-kB:Compared with control group,Ruyanning group,Exemestane group and the combined group of the expression on NF-kB is less than the control group(P<0.05).The combined group is more significant especially(P<0.05).3.PI3K-AKT pathway:Western blot:PI3K、AKT:Compared with control group,Ruyanning group,Exemestane group and the combined group of the expression of PI3K and AKT are less than the control group(P<0.05).The combined group is the least among all(P<0.05).RT-PCR:PI3K、AKT:Compared with control group,Ruyanning group,Exemestane group and the combined group of the expression on PI3K、AKT are less than the control group(P<0.05).The combined group is more significant especially(P<0.05).4.Metabolic regulation mechanism:Western blot:mTOR、HIF-1 and GLUT1:Compared with control group,Ruyanning group,Exemestane group and combined group of the expression on mTOR、LDH-A、HIF-1and GLUT1 are less than control group(P<0.05).The combined group is the least among all(P<0.05).RT-PCR:mTOR、LDH-A、HIF-1 and GLUT1:Compared with control group,Ruyanning group,Exemestane group and combined group of the expression on mTOR、LDH-A、HIF-1 and GLUT1 are less than control group(P<0.05).The combined group is the least among all(P<0.05).Conclusion:1.Inhibitory effect of Ruyanning combined Exemestane on postmenopausal breast cancer in nude mice:Ruyanning combined Exemestane may significantly inhibited tumor in nude mice with postmenopausal breast cancer by up-regulating the expression of P53,caspase 3 and down-regulating NF-kB,and the effect of combination therapy is more obvious.2.Effect of Ruyanning combined Exemestane on PI3K-AKT Pathway in nude mice with postmenopausal breast cancer:Ruyanning combined Exemestane inhibits the activity of the PI3K-AKT pathway by down-regulating the expression of PI3K,AKT.3.Metabolic regulation of exemestane on the metabolism of nude mice in postmenopausal breast cancer:Ruyanning combined Exemestane can down-regulate the expression of downstream genes in the PI3K-AKT pathway to affect the metabolic activity of tumor cells and inhibit the growth of tumor cells. |
