The Effect Of Bushenhuoxue Decoction On Decidual Stromal Cells Of Unexplained Recurrent Spontaneous Abortion Based On PI3K/AKT Pathway

Objective:To explore the influence of Bu Shen Huo Xue decoction to decidual stromal cells apoptosis and proliferation related signal pathways and factors,discuss its etiology and pathogenesis mechanism for unexplained recurrent spontanous abortion,as a treatment method of unexplained recurrent spontanous abortion.BSHXD proposed an effective prescription of tonify the kidney and promote blood circulation for treating decidual stromal cells in basis for the treatment of this disease.Materials and Methods:1.Through clinical observation of BSHXD's influence on unexplaine recurrent spontanous abortion patiernts' expression of IL-17A on peripheral blood,using Elisa to determine the the level of IL-17A,comparing in 30 cases of patients with unexplained recurrent spontanous abortion separately before and after treatment with BSHXD+dydrogesterone or dydrogesterone for 4 weeks;2.To determine the harmacodynamic material basis of BSHXD,serum pharmacology method is applied to analyze material composition of BSHXD and blood transitional components.Mass spectrometry and HPLC analysis each herb and the chemical composition of the whole decoction,and then compared with the BSHXD drug-derived serum,ultimately determine the drug ingredients of BSHXD drug-derived serum;3.10 cases of gestational failure unexplained recurrent spontanous abortion patients with deficiency of kidney and blood stasis and 10 cases of normal early pregnancy women decidual tissue,applied layered centrifugation to establish a system of decidual stromal cells in vitro.Flow of the fluorescence technique to detect the apoptosis of unexplained recurrent spontanous abortion and normal early pregnancy women decidual stromal cells;CCK-8 assay is used to test the influence of BSHXD and IL-17A on DSCs'proliferation.Using the methord of Real-timePCR and WesternBlot to observe BSHXD and IL-17A intervening to decidual stromal cells PI3K/AKT signal pathway and the change of mRNA and protein level,suggestive of decidual stromal cells on the regulatory mechanism of PI3K/AKT signal pathway.Result:1.The level of IL-17A on the unexplained recurrent spontanous abortion with deficiency of kidney and blood stasis peripheral blood is22.45�10.85 before the treatment of BSHXD+dydrogesterone,after four weeks'treatment,the level of IL-1 7A is 12.89�4.95,shows significant statistical differences(p<0.001);the efficay of BSHXD+dydrogesterone is higher than the dydrogesterone only group(p<0.05).2.BSHXD drug-derived serum is identified to 12 prime constituents,contains 15,16-Dihydrotanshinone ??tanshinol B?Neotanshinone A?Hydroxytanshinone ? A?Isocryptotanshinone?Tanshinone ? B?Tanshinone ? A?D?sobutyl phthalate?logaric acid 6,-O-?-D-glucoside?kaempferide?Quercetin-7-O-?-L-rhamnoside and Linolic acid.3.The proportion of unexplained recurrent spontanous abortion decidual stromal cells of early apoptosis is higher than the normal early pregnancies(p<0.05).4.IL-17A can promote DSCs proliferation activity at a concentration of 30ng/ml,compared to the control group with statistical differences(p<0.05);PI3K inhibitors could inhibite proliferation activity of DSCs at a concentration of 10uM as the apprptiate statistical differences(p<0.001);IL-1 7A has no effect on DSCs after PI3K inhibitors intervening,compared to PI3K inhibitors group with no statistical difference(p>0.05);5.5%BSHXD+5%blank serum has the most obvious effect on the proliferation activity of DSCs from unexplained recurrent spontanous abortion,compared to the 10%blank serum group with significant statistical difference(p<0.01);BSHXD has no obvious influence on decidual stromal cells with IL-1 7A stimulate,compared to the IL-1 7A group with no significant statistical difference(p>0.05),BSHXD could eliminate the inhibition of PI3K inhibitors on DSCs proliferation,then IL-17A could promote the proliferation activity of DSCs,compared to no intervention group with statistical difference(p<0.05);6.BSHXD can increase the IL-17A?IL-17RA?PI3K?AKT?p-AKT?PTEN mRNA expression level on decidual stromal cells from unexplained recurrent spontanous abortion;7.BSHXD could up-regulate Bcl-xL and down-regulate BAD mRNA expression level on decidual stromal cells from unexplained recurrent spontanous abortion.Conclusion:1.BSHXD can down-regulate the level of IL-1 7A in the peripheral blood from unexplained spontanous abortion to adjust the immune and inhibition a inflammatory response coused by IL-17A;2.12 prime constituents of BSHXD drug-derived serum shows that BSHXD drug-derived serum has the consistent with the decoction.3.BSHXD can up-regulate IL-17A and IL-17RA mRNA level to activate the PI3K/AKT pathway to promote decidual stromal cells proliferation,finally achieve the purpose of invigorating the kidney and promoting blood circulation.4.BSHXD can regulate the Bcl-xL and BAD mRNA expression in decidual stromal cells from unexplained recurrent spontanous abortion,and ameliorate its abnormal apoptosis statement to prevent miscarriages.