| Objective:To comfirm the antitumor effects of Ruyanning Decoction combined with exemestane by observing ovariectomized nude mice bearing MDA-MB-435 breast cancer in terms of the general state of mice,turmor weight,tumor inhibition rate and estrogen level;to address the underlying mechanism by examining Wnt/JNK signaling pathway effectors: JNK and AP-1,and downstream genes:STAT3,Bcl-2,p53 and caspase 8.Material and methods:1.Experimental materials Cell line: human breast cancer cell line MDA-MB-435Experimental animals: 4~6 weeks old,female BALB/C nude mice,20±2g,SPF class,42 in total.Drug preparation: Ruyanning Decoction consists of 10 g Bupleurum,15 g Radix Pseudostellariae,10 g Rhizoma Pinelliae,15 g Scutellariae,10 g Fried Glycyrrhiza,30 g keel,30 g oyster,20 g Hedyotis diffusa,20 g Scutellariae barbatae,15 g Prunella,15 g Zedoary,and 20 g Fritillaria thunbergii.These Chinese medicines were purchased from the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine.Preparation method: According to the traditional Chinese medicine decoction method,Ruyanning Decoction was decocted twice.Firstly,10 times water was added to decoct for 1 hour,then filtered and collected the liquid.Secondly,added 8 times water to the residue and decocted for half an hour,then filtered and collected the liquid again.All the liquid was combined and adjusted to 3.3 g/ml,stored at 4?.Exemestane was dissolved with water and adjusted to 3mg/ml.2.Establishment of MDA-MB-435 tumor bearing castrated nude mice modelFemale nude mice were anesthetized by intraperitoneal anesthesia.The skin was prepared at the lower edge of the rib arch of the back of the nude mice,0.5-1 cm at the side of the spine.The skin was disinfected by routine disinfection and iodine complex disinfection.This is an important anatomical landmark of the ovary.Lifting the muscular layer on the surface of the milky white fat mass gently with tweezers,cutting the muscular layer about 0.5 cm long along the lateral margin of psoas major muscle with scissors,we can find the pale red rice grain size "mulberry-like" ovaries in the fat mass of nude mice.After separation,clamp ligation,ovariectomy,compression hemostasis,the rest of the tissue put back into the abdominal cavity,layered suture muscle layer and skin,iodine complex incision disinfection.The contralateral ovaries were removed by the same method and then sutured and sterilized according to the above methods.After the operation,the anti-infection mice were fed for 7 days.The nude mice were free to move,eat and drink water,and were given the replacement of bedding every day.Iodophor disinfection was performed on the right chest wall of two ovariectomized nude mice.0.2 ml of cell fluid was extracted from each of the two 1 ml syringes and inoculated into the second breast pad of the right chest wall of the ovariectomized nude mice.3.Experimental grouping and Administration40 castrated nude mice bearing tumor were randomly divided into four groups: model control group,exemestane group,Ruyanning Decoction group and combined group.Each group had 10 castrated nude mice.(1)model control group: normal saline was given to the stomach;(2)exemestane group: given exemestane 4 mg/kg body weight was given,equal to 10 times the clinical dosage;(3)Ruyanning group: Ruyanning Decoction 33.3 g / kg body weigh was given,equal to 12 times the clinical dosage;(4)combined group: Ruyanning Decoction 33.3 g / kg body weight was given at the same timeand Intravagant administration of exemestane 4mg/kg.4 groups of tumor bearing castrated nude mice were administered 0.2ml/ D,once daily,and continuously administered with 21 d.4.Experimental materialsThe 4 groups of tumor bearing castrated nude mice were sampled after 21 days of administration.(1)Serum specimens: After eyeball extraction and congestion,the eyeballs of mice were quickly clamped with elbow ophthalmic forceps.The eyeballs were inverted,and the head was down.the heart of the mice was pressed lightly to speed up the blood flow from the orbit into the EP tube at the same time.The blood specimens were collected about 0.5ml-1ml.Blood samples were kept at room temperature for 2 hours,then centrifuged for 20 minutes at 1000 *g.The supernatant was stored in a 2 ml cryopreservation tube and stored in a refrigerator at-80 C for ELISA detection.(2)Tumor specimens: After taking blood,the tumor-bearing nude mice were killed by neck exfoliation.The tumor tissues were stripped completely,weighed and placed on the ice box.After observing the tumor tissues with naked eyes,the specimens were cut and taken,and the whole tumor tissue was cut.The tumor necrosis tissues were avoided by taking samples from different parts.The specimens were divided into two parts: 1)Western Bloting specimens: the tumor tissue was placed in EP tube and stored in-80 C refrigerator for reserve;2)RT-PCR specimens: 2-3 pieces of tumor tissue about 8 mm3 were taken and placed in 1 ml Trizol,marked with a marker pen,and immediately stored in-80 C refrigerator for reserve.5.Testing indicators and methods(1)To observe the changes of general state: The body weight,behavior,water intake and death of ovariectomized nude mice bearing tumor were observed after treatment.The weight of the nude mice was measured every other day and the weight change curve was drawn.The number of spontaneous activities of ovariectomized mice in each group within 5 minutes was recorded before administration,7,15 and 21 days after administration.The food and water intake of nude mice were recorded daily.In case of death,the time and number of death should be recorded.At the end of the experiment,the tumor-bearing nude mice were sacrificed and the tumor tissues were stripped completely.The tumor weight was weighed and the tumor inhibition rate was calculated.(2)The levels of E2 and P in serum of ovariectomized nude mice bearing tumor were detected by competitive inhibition enzyme-linked immunosorbent assay(ELISA).(3)Western blotting was used to detect the expression of JNK protein,STAT3 and Bcl-2 oncogenes.The expression levels of AP-1 gene,tumor suppressor gene P53 and Caspase 8 were detected by RT-PCR.Analyzing the counting data by ANOVA by SPSS20.0 software.The data was expressed by mean ± standard deviation(X ±S).The difference was statistically significant with P < 0.05.The difference was not statistically significant with P > 0.05.Results:1.General state,tumor weight and tumor inhibition rate: The general state of castrated nude mice with tumor in the exemestane group gradually showed a downward trend,showing weight loss,mental depression,loss of appetite,water consumption,skin color and brightness reduction,skin dryness and loose and easy to fall off,bow-back,joy pack,fewer activities and so on aftert one week.The general condition of the combined group decreased after 2 weeks of administration,which was lighter than that of the exemestane group.Ru Yan Ning Decoction Group and model control group had better state and similar performance.Until the end of the experiment,none of the four groups of castrated nude mice died.The weight of ovariectomized nude mice with tumor in each group decreased in varying degrees after 7,15 and 21 days of administration.The weight of exemestane group decreased significantly more than Ruyanning Decoction group and combined group(P < 0.05).On the 7th day of administration,the number of spontaneous activities of exemestane group and Ruyanning Decoction group decreased significantly,and the number of spontaneous activities of exemestane group decreased significantly compared with Ruyanning Decoction group(P < 0.05),while the number of spontaneous activities of combined group did not decrease significantly;on the 15 th and 21 st days of administration,the number of spontaneous activities of each group decreased significantly,and there was statistical significance among the three groups.Meaning(P<0.05).The tumor weight of Ruyanning Decoction group,exemestane group and combined group was significantly lighter than that of model control group(P < 0.05).The tumor weight of combined group and exemestane group was significantly lighter than that of Ruyanning Decoction group(P < 0.05),but there was no significant difference between combined group and exemestane group(P > 0.05).The inhibitory rates of Ru Yan Ning Decoction,exemestane group and combination group were 37.53%,60.83% and 66.09% respectively.2.Estrogen level: The levels of estradiol(E2)and progesterone(P)in serum of ovariectomized nude mice bearing tumor were measured.Compared with the model control group,the levels of estradiol(E2)and progesterone(P)in Ruyanning Decoction group,exemestane group and combined group were significantly decreased(P < 0.05).Yi(P < 0.05),but there was no significant difference between the Ru Yan Ning Decoction Group and the exemestane group(P > 0.05).3.Expression level of effector JNK and AP-1 protein: Compared with model control group,the expression of JNK protein and the relative expression of AP-1 gene in Ruyanning Decoction group,exemestane group and combined group were significantly decreased(P < 0.05),and the combined group was significantly lower than Ruyanning Decoction group and exemestane group(P < 0.05).Significance(P < 0.05).The relative expression of AP-1 gene in combination group was not significantly lower than that in exemestane group(P > 0.05).4.Expression level of oncogene STAT3 and Bcl-2 protein and expression level of tumor suppressor gene P53 and Caspase 8 gene: Ruyanning Decoction group,exemestane group and combined group could down-regulate the expression of STAT3 and Bcl-2 protein,up-regulate the expression level of P53 and Caspase 8 gene,which had statistical significance compared with model control group(P < 0.05).The level of STAT3 protein down-regulation in Ruyanning Decoction group was significantly higher than that in exemestane group and combined group(P < 0.05).There was no significant difference in the down-regulation level of Bcl-2 protein among Ruyanning Decoction group,exemestane group and combined group(P > 0.05).The expression levels of P53 and Caspase 8 were up-regulated in the combined group,which was significantly higher than those in Ruyanning Decoction group and exemestane group(P < 0.05).Conclusions:1.Ruyanning Decoction can maintain the body weight and physical status of ovariectomized breast-tumor-bearing nude mice and reduce the adverse side effects of exemestane,such as weight loss,decreased activity and poor mental status.2.Ruyanning Decoction can lower the serum level of estrogen in ovariectomized breast-tumor-bearing nude mice and show synergistic effect with exemestane.3.In ovariectomized breast-tumor-bearing nude mice,Ruyanning Decoction can down-regulate the expression of JNK and AP-1,which are effectors of Wnt/JNK signaling pathway,and this inhibitive effect are enhanced when combined with exemestane.4.Moreover,Ruyanning Decoction can regulate downstream genes of Wnt/JNK signaling pathway to control breast tumor growth.Ruyanning Decoction can down-regulate the expression of STAT3 and BCL-2 to inhibit the proliferation of tumor cells.Meanwhile,it can up-regulate the expression of p53 and Caspase 8 to promote the apoptosis of tumor cells.These regulations are more effective when Ruyanning Decoction is combined with exemestane. |
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