Comparison Of ACTA And Smads Signal Pathway Between Bushen Treatment And Shugan Treatment In Vitro Culture Ovarian Granulose Cells Of IVF-ET Patients

Objective: Preliminary studies have confirmed that Bushen treatment and Shugan treatment can improved the clinical pregnancy rate, pregnancy outcomes and oocyte quality in patients with IVF-ET(in vitro fertilization-embryo transfer, IVF-ET). This study continued to compare the Bushen with Shugan methods in vitro culture IVF-ET patients ACTA in ovarian granulosa cells and its role in regulating signaling pathways, to explore the similarities and differences in mechanism and detail pathways between the two methods.Methods:1 Preparation of serum containing drugs The serum contained Bushentiaojing recipe and Xiaoyaosan recipe: Female volunteers 10 were chosen at the age of 20-30 years old, in good health, with menstrual regularity, mature follicles showed by B-ultrosound, without organic diseases of, ovaries and uterus respectively, taking the Bushentiaojing recipe and Xiaoyasan recipe orally. Females took 300 ml of Bushentiaojing and Xiaoyaosan recipe each respectively day, in the morning and evening for 3 days. In the fourth day early in the morning on an empty stomach-a day,s dose of the drug(medication before fasting 12h), 1h after the last medication venous blood 5ml, for 2h at room temperature, 10 min for 3000 r per min, supernatant, 56 ℃ water bath for 30 min to inactivate complement, 0.22μm sterile filter filter-sterilized, aliquot preparation Bushentiaojing party Xiaoyaosan serum containing two kinds of people. Normal serum: Five female volunteers were selected, at the age of 20-30 years old, in good health, with menstrual regularity, with mature follicles by B-ultrosound, and without ovarian and uterine organic disease, fasting venous blood 5ml, above normal preparation serum, normal serum control set. Serum were saved at- 20℃ for later using. 2 The culture of granule cells in vitro Put the granulosa cells after centrifugation in 35 mm dish set 2ml nutrient solution, 37 ℃, 5% CO2(V/V), 100% humidity. Change the solution 24 h after the cells attached to the bottom of the dish and put Bushentiaojing Recipe and Xiaoyao san recipe serum. The follicles were divided into 8 groups according to the serum and doses level, named Bushen low,medium and high dose groups,Shugan low, medium and high dose groups, normal group and blank control group. Cultured after 24 h, granulosa cells were collected. The first batch of granulosa cells were collected by mechanical methods, stored at-80℃(for real-time fluorescent quantitative PCR). Using mechanical and enzymatic methods to collect the second batch, and fixed quickly by 2% paraformaldehyde(each for immunohistochemistry and immunofluorescence). Bushen high dose group and Shugan high dose group after 24 h incubation, adding SB431542(concentration of 10μmol/L), incubated for 2h, respectively, Bushen and Shugan Inhibitor group, collection and experimental methods were got as above. The ACTA, ACTRIIB, ALK4, Smad2, Smad3 and Smad4 m RNA expressions of them were analyzed by real-time fluorescent quantitative PCR.The protein content of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 and P-Smad2, P-Smad3 were detected by immunofluorescence and immunohistochemistry.Results:1 Comparison of the expression of ACTA, ACTRⅡB, ALK4, Smad2, Smad3, Smad4 m RNA among groups in vitro culture of granulosa cells. 1.1 Comparison of the expression of ACTA, ACTRⅡB, ALK4, Smad2, Smad3, Smad4 m RNA in vitro culture of granulosa cells among Normal group, Blank control group and each treatment group. The expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 m RNA in normal group was higher than that in Blank control group,(P <0.05). Compare with control group and normal group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 m RNA in Shugan groups was higher, the difference was statistically significant(P <0.05). The expression in Shugan high dose was the highest(P<0.05). Compare with control group and normal group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 m RNA in Bushen groups was higher, the difference was statistically significant(P<0.05). The expression in Bushen high dose were increased significantly(P<0.05). Compared the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 m RNA between Shugan groups and Bushen groups, the expression in Bushen high dose was increased statistically(P<0.05). The following were Bushen middle dose, Shugan high dose, Shugan middle dose, Bushen low dose group, Shugan low dose group. The Bushen high group was increased more than that in Shugan group,the difference was not statistically significant(P>0.05). 1.2 Comparison of the expression of ACTA, ACTRⅡB, ALK4, Smad2, Smad3, Smad4 m RNA between Shugan, Bushen inhibitor groups and other non inhibitor groups. Compared with Shugan Inhibitor group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 m RNA in Shugan high dose group was increased statistically(P<0.05); the expression of Shugan middle dose was no difference(P>0.05); the expression of Shugan low dose group, Blank Control and Normal group were decreased(P<0.05). Compare with Bushen Inhibitor group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 m RNA in Shugan high dose group was increased statistically(P<0.05); the expression of Shugan middle dose was no difference(P>0.05); the expression of Shugan low dose group, blank Control and normal group were decreased(P<0.05). 2. Comparison of the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, Smad4 protein in vitro culture of granulosa cells among groups. 2.1 Immunohistochemical results 2.1.1 Comparison of the expression of ACTA, ACTRⅡB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein among normal group, blank control group and each treatment group in vitro culture of granulosa cells. The expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in normal group was higher than that in blank control group,the difference was statistically significant(P<0.05). Compare with control group and normal group,the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Shugan groups was higher, the difference was statistically significant(P<0.05). The expression in Shugan high dose was the highest(P<0.05). Compare with Control group and Normal group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Bushen groups was higher, the difference was statistically significant(P<0.05).The expression in Bushen high dose were increased significantly(P<0.05). Compared the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein between Shugan groups and Bushen groups, the expression in Bushen high dose was increased than Shugan high dose, the difference was not statistically significant(P>0.05). The Bushen middle dose was increased than Shugan middle dose, the difference was statistically significant(P<0.05). The Bushen low dose was increased than Shugan low dose, the difference was not statistically significant(P>0.05). 2.1.2 Comparison of the expression of ACTA, ACTRⅡB, ALK4, Smad2, Smad3, Smad4 protein between Shugan, Bushen inhibitor groups and other non inhibitor groups. Compared with Shugan inhibitor group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Shugan high dose was increased significantly(P<0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Shugan middle dose has no significant difference(P>0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Blank control group, Normal group and Shugan low dose group was decreased significantly(P<0.05). Compared with Bushen inhibitor group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3,P-Smad2, P-Smad3, Smad4 protein in Bushen high dose was increased significantly(P<0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3,P-Smad2, P-Smad3, Smad4 protein in Bushen middle dose has no significant difference(P>0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in blank control group, normal group and Bushen low dose group was decreased significantly(P<0.05). 2.2 Cell immunofluorescence test results 2.2.1 Comparison of the expression of ACTA, ACTRⅡB, ALK4, Smad2, P-Smad2, Smad3, P-Smad3, Smad4 protein in vitro culture of granulosa cells among normal group, blank control group and each treatment group. The expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 proteins in normal group were higher than that in blank control group, the difference was statistically significant(P<0.05). Compared with control group and normal group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Shugan groups was higher, the difference was statistically significant(P<0.05). The expression in Shugan high dose was the highest(P<0.05). Compared with control group and normal group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Bushen groups was higher, the difference was statistically significant(P<0.05). The expression in Bushen high dose were increased significantly(P<0.05). Compare the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein between Shugan groups and Bushen groups, the expression in Bushen high dose was increased than Shugan high dose, the difference was not statistically significant(P>0.05). The Bushen middle dose was increased than Shugan middle dose, the difference was statistically significant(P<0.05). The Bushen low dose was increased than Shugan low dose, the difference was not statistically significant(P>0.05). 2.2.2 Comparison of the expression of ACTA, ACTRⅡB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in Shugan, Bushen inhibitor groups and other non inhibitor groups. Compared with Shugan inhibitor group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3 and Smad4 protein in Shugan high dose was increased significantly(P<0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3 and Smad4 protein in Shugan middle dose had no significant difference(P>0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3, Smad4 protein in blank control group,normal group and Shugan low dose group was decreased significantly(P<0.05). Compared with Bushen inhibitor group, the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3 and Smad4 protein in Bushen high dose were increased significantly(P<0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3 and Smad4 protein in Bushen middle dose had no significant difference(P>0.05); the expression of ACTA, ACTRIIB, ALK4, Smad2, Smad3, P-Smad2, P-Smad3 and Smad4 protein in blank control group,normal group and Bushen low dose group was decreased significantly(P<0.05).Conclusions:1 Xiaoyao San and Bushen Tiaojing Recipe can increase the expression of ACTA, ACTRIIB and ALK4 m RNA and protein of granulosa cells in vitro and improve the growth and development of the follicular. 2 Xiaoyao San and Bushen Tiaojing Recipe impacting ACTA signaling pathway is in a dose-dependent manner, and the effect of high doses is the best. 3 Bushen Tiaojing Recipe improving the expression of ACTA, ACTRII B, ALK4 m RNA and protein of granulosa cells in vitro is more significant than Xiaoyao San, suggesting that the effect of Bushen Tiaojing Recipe on promoting the growth and development of follicles is more significant than Xiaoyao San. 4 Xiaoyao San and Bushen Tiaojing Recipe can raise ACTA Smads signal pathway to develop its promoting follicle growth and improving the quality of oocyte, the mechanism may be induced ACTA with its receptor ⅡACTRⅡB to form complex, thus to raise Ⅰtype receptors ALK4, making its phosphorylation and activation, further phosphorylation intracellular signal factor Smad2 and Smad3 and Smad4, signal transduction and regulation.