| Objective:Based on the clinical effect of Pishenshuangbu Formula treating early threatened abortion,our study aims at further exploring its action mechanism for maintaining the pregnancy.Through Pishenshuangbu Formula medicated serum legislated by tonifying Pishenshuangbu Formula in vitro human pregnancy villus HTR-8 cells,our study observe the total number of HTR-8 cells,the number of apoptosis and the related apoptosis proteins:the Bcl-2,Bax and TNF-a Caspase-3 expression to explore the influence of Pishenshuangbu Formula in increasing human pregnancy villinourish cells,reducing the apoptosis,protecting cells survive,by which it can effectively prevent threatened abortion,explore the cellular and molecular mechanisms for maintaining the pregnancy,provide experimental support for fetus,and enrich TCM theory of reproductive.Methods:80 SPF healthy adult female SD rats are randomly divided into 8 groups: blank control group,the flexion progesterone group,Zishenyutai pill for high,medium and low dose group,Pishenshuangbu Formula for high,medium and low dose group,each group of ten only.In blank control group given saline lavage,flexor progesterone group to fill the stomach to bend progesterone tablet distilled water preparation,Zishenyutai pill for high,medium and low dose group were given Zishenyutai pill for high,medium and low dose of saline lavage,Pishenshuangbu Formula fill double side high,medium and low dose group were given the spleen kidney fill double side high,medium and low dose water decoction lavage,in the morning and once,for seven days.In the last 7 days for 1 h after femoral artery blood,blood before 12 h fast water.Blood specimen was performed after the let stand at room temperature for 4h 3000 r/min 15 min,the centrifugal separation of serum,0.22 um aseptic filtration membrane filtration,56 degrees Centigrade water bath inactivated 30 min,buy 4 degrees Centigrade refrigerator used for in vitro culture nourish cells.The HTR-8 cells inoculated in a sterile culture bottle,after waiting for cells to stick wall evenly extend.Randomly selected eight bottles as the eight groups of sample cells,each group of samples after six holes,each group were added with 15% volume fraction of the corresponding medicated serum culture,giving each HTR-8 cells to complete medium containing medicine serum of rats 48 h after training HE dyeing observation of cell number and the total number of apoptosis,immune histochemical method to detect groups of apoptosis protein TNF-a,Bcl-2,Bax,Caspase-3 expression.Results: 1.The influence of Pishenshuangbu Formula medicated serum of rats on the total number of human pregnancy villus nourish cells HTR-8 in vitro culturedCompared with the blank control group,Pishenshuangbu Formula high,middle dose group,Zishenyutai pill of high,middle dose group of HTR-8 cells were increased,the total difference was statistically significant(P<0.05),Pishenshuangbu Formula,double side low dose group and Zishenyutai pill low dose group of HTR-without increasing the total 8 cells,there was no statistically significant difference(P>0.05),and flexor progesterone group of HTR-8 cells than increased significantly compared with blank control group,the total difference was statistically significant(P< 0.01);Compared with group to bend progesterone,Pishenshuangbu Formula the high,middle dose group of HTR-8 cell has the tendency of increase,the total but there was no statistically significant difference(P>0.05),Pishenshuangbu Formula in low dose group of the total number of cells is parent flexor progesterone could reduce cell group,the difference was statistically significant(P<0.05),Pishenshuangbu Formula,double side of high,middle dose group of HTR-8 cell differences had no statistical significance(P>0.05);And Zishenyutai pill compared high,medium and low dose group,Pishenshuangbu Formula fill double side high,medium and low dose group of HTR-8 cell total difference has no statistical significance(P>0.05).2.The influence of Pishenshuangbu Formula medicated serum of rats on the number of apoptosis cells of human pregnancy villus nourish cells HTR-8 in vitro culturedPrescription of Pishenshuangbu Formula of high,middle dose group,Zishenyutai pill of high,middle dose group of HTR-8 cell apoptosis number is lower than the blank control group,the difference was statistically significant(P<0.05),Pishenshuangbu Formula,double side low dose group and Zishenyutai pill low dose group of HTR-8 apoptosis number is no lower than the blank control group,there was no statistically significant difference(P>0.05);To bend progesterone group number of HTR-8 cell apoptosis was significantly lower than blank control group,the difference was statistically significant(P<0.01);Compared with the group to bend progesterone,Pishenshuangbu Formula the high,middle dose group of HTR-8 no increase in the number 8 cell apoptosis(P>0.05),Pishenshuangbu Formula,apoptosis of low dose group,the difference was statistically significant(P<0.05),Pishenshuangbu Formula,double side between high,middle dose group of HTR-8 apoptosis number difference had no statistical significance(P>0.05);And Zishenyutai pill compared high,medium and low dose group,Pishenshuangbu Formula fill double side high,medium and low dose group of HTR-8 apoptosis number difference is no statistical significance(P>0.05).3.The influence of Pishenshuangbu Formula medicated serum of rats on cells apoptosis induced proteins TNF-a expression of human pregnancy villus nourish cells HTR-8 in vitro culturedCompared with the blank control group,Pishenshuangbu Formula high,middle dose group,Zishenyutai pill of high,middle dose group of HTR-8 expression of apoptosis protein TNF-a area of the total sum,integral optical density and the mean average blackness is reduced,the difference was statistically significant(P<0.05),Pishenshuangbu Formula,double side low dose group and Zishenyutai pill low dose group of TNF-a expression of the area of the total sum,integral optical density and average black degrees higher average(P>0.05),the flexor progesterone group expression of HTR-8 cells TNF-a area of the total sum,integral optical density and the mean average blackness was significantly lower than the blank control group(P< 0.01);Compared with group to bend progesterone,Pishenshuangbu Formula the high dose group of TNF-a expression of the area of the total sum,integral optical density higher and the mean average blackness,there was no statistically significant difference(P>0.05),Pishenshuangbu Formula fill double side dose group of TNF-a combined expression of integral optical density and the difference between the mean average blackness has no statistical significance(P>0.05),Pishenshuangbu Formula fill double side between high,middle dose group of the expression of TNF-a difference had no statistical significance(P>0.05);And Zishenyutai pill compared high,medium and low dose group,Pishenshuangbu Formula the high,medium and low dose group of TNF-a expression of the area of the total sum,integral optical density and the difference between the mean average blackness has no statistical significance(P > 0.05).4.The influence of Pishenshuangbu Formula medicated serum of rats on cells apoptosis regulatory proteins the Bcl-2,Bax and the influence of the ratio of expression of human pregnancy villus nourish cells HTR-8in vitro culturedCompared with the blank control group,Pishenshuangbu Formula high,middle dose group,Zishenyutai pill of high,middle dose group of HTR-8 cells apoptosis protein expression of Bcl-2 the suppression of the total sum,integral optical density and the mean average blackness were higher(P<0.05),Pishenshuangbu Formula,double side low dose group and Zishenyutai pill low dose group the Bcl-2 expression of the area of the total sum,integral optical density and the mean average blackness is reduced,there was no statistically significant difference(P>0.05),and flexor progesterone group of HTR-8 Bcl – 2 cells express the area of the total sum,integral optical density and the mean average blackness is significantly more than the blank control group(P<0.01);Compared with group to bend progesterone,Pishenshuangbu Formula the high,middle dose group of HTR-8 Bcl-2cells express the area of the total sum,integral optical density and the difference between the mean average blackness has no statistical significance(P>0.05),Pishenshuangbu Formula,the high,middle dose group of the Bcl-2 expression between the area of the total sum,integral optical density and the mean average blackness differences had no statistical significance(P>0.05);Pishenshuangbu Formula high,medium and low dose group of HTR-8 Bcl-2 cells express the area of the total sum,integral optical density and the mean average blackness is Zishenyutai for high,medium and low dose group than the difference has no statistical significance(P>0.05).Compared with the blank control group,Pishenshuangbu Formula high,middle dose group,Zishenyutai pill of high,middle dose group of HTR-8 cell and promote apoptosis protein expression of Bax area of the total sum,integral optical density and the mean average blackness being reduced(P<0.05),Pishenshuangbu Formula,double side low dose group and Zishenyutai pill low dose group Bax expression in the area of the total sum,integral optical density higher and the mean average blackness,there was no statistically significant difference(P>0.05);To bend progesterone group of HTR-8 cell Bax expression of area of the total sum,integral optical density and the average mean blackness was significantly lower than in blank control group(P<0.01);Compared with group to bend progesterone,Pishenshuangbu Formula high dose group and the dose of HTR-8 Bax apoptosis protein expression differences no statistical significance(P>0.05),Pishenshuangbu Formula Bax expression differences between high,middle dose group had no statistical significance(P>0.05);Compared with Zishenyutai pills high-dose group,Pishenshuangbu Formula the high dose group of HTR-8 Bax apoptosis protein expression of combined lower(P<0.05),and zi kidney in the womb pill compared,low dose group,Pishenshuangbu Formula,low dose group of HTR-8 cells in the area of the expression of Bax total sum,integral optical density and average black for the mean difference is no statistical significance(P>0.05).Compared with the blank control group,Pishenshuangbu Formula high,middle dose group,Zishenyutai pill of high,middle dose group of HTR-8 Bcl-2cells/Bax ratio of area of the total sum,integral optical density and the mean average blackness were higher(P<0.05),Pishenshuangbu Formula,double side low dose group and Zishenyutai pill low dose group of the Bcl-2/Bax ratio of area of the total sum,integral optical density and average blackness average decreased,there was no statistically significant difference(P>0.05),to bend progesterone group of HTR-8Bcl-2 cells/Bax ratio of area of the total sum,integral optical density and the mean average blackness was significantly higher than blank control group(P< 0.01);Compared with group to bend progesterone,Pishenshuangbu Formula high,middle dose group of the Bcl-2/Bax average ratio of total area,the average black level without the rising trend,there was no statistically significant difference(P > 0.05),Pishenshuangbu Formula,the high,middle dose group of the ratio of the Bcl-2/Bax differences had no statistical significance(P>0.05);Compared with Zishenyutai pills high-dose group,Pishenshuangbu Formula the high dose group of HTR-8 Bcl-2 cells/Bax ratio of the sum of the integral optical density increases,the difference was statistically significant(P<0.05),Pishenshuangbu Formula,double side,low dose group of the Bcl-2/Bax ratio of area of the total sum,integral optical density and the mean average blackness is Zishenyutai pill,there was no statistically significant difference than the low dose group(P > 0.05).5.The influence of Pishenshuangbu Formula medicated serum of rats on apoptosis protein expression of Caspase-3 execution of human pregnancy villus nourish cells HTR-8 in vitro culturedCompared with the blank control group,Pishenshuangbu Formula high,middle dose group,Zishenyutai pill of high,middle dose group of HTR-8 apoptosis protein expression of Caspase-3 area of the total sum,integral optical density and average blackness mean lower(P<0.05),Pishenshuangbu Formula,double side low dose group and Zishenyutai pill low dose group of Caspase-3 area of the total sum,integral optical density and average blackness average rise,there was no statistically significant difference(P>0.05),and flexor progesterone group of HTR-8 Caspase-3cells express the area of the total sum,integral optical density and the mean average blackness was significantly lower than the blank control group(P< 0.01);Compared with group to bend progesterone,Pishenshuangbu Formula high,middle dose group of Caspase-3 area of the total sum,integral optical density and average blackness mean no statistically significant difference(P>0.05),Pishenshuangbu Formula,the high,middle dose group of Caspase-3 expression between area of the total sum,integral optical density and average blackness mean differences had no statistical significance(P>0.05);Compared with Zishenyutai pills high-dose group,Pishenshuangbu Formula high dose group of Caspase-3 combined integral optical density,the average black degree average is reduced,the difference was statistically significant(P<0.05);Compared with Zishenyutai pill dose group,Pishenshuangbu Formula,the average dose group of Caspase-3 in black for the mean is reduced,the difference was statistically significant(P<0.05);Compared with Zishenyutai pill low dose group,Pishenshuangbu Formula low dose group of Caspase-3 area of the total sum,integral optical density and average black for the mean difference is no statistical significance(P>0.05).6.The comprehensive control of Pishenshuangbu Formula medicated serum of rats to nourishcells apoptosis related proteins expression of human pregnancy villus nourish cells HTR-8 in vitro culturedCaspase-3 groups of HTR-8 cells combined with integral optical density of each group was positively related to the level of the expression of TNF-a corresponding and negatively correlated with the expression of Bcl-2 levels,and the expression of Bax levels were positively correlated,and the ratio of the Bcl-2/Bax negatively correlated;Caspase-3 integral optical density summation expression level significantly correlated with corresponding the Bcl-2/Bax ratio,and the expression of TNF-a level no obvious correlation.Conclusion: 1.Pishenshuangbu Formula medicated serum can promote human pregnancy villus sertoli cell number increase,its mechanism might be: reduce pregnancy villi of sertoli cell apoptosis,protect cells survive,and conducive to the maintenance of early pregnancy.The role of approaches may be:(1)the Pishenshuangbu Formula medicated serum induced by reducing cell apoptosis protein expression of TNF-a,inhibition of death receptor pathway,reduce apoptosis protein expression of Caspase-3,thus inhibiting human pregnancy villus sertoli cell apoptosis,protect cells survive,maintain pregnancy;(2)the Pishenshuangbu Formula medicated serum by increasing cell apoptosis inhibiting protein expression of Bcl-2,reduce cells and promote the expression of apoptosis proteins Bax,raise the ratio of the Bcl-2/Bax,reduce apoptosis protein expression of Caspase-3,inhibiting apoptosis mitochondria,reduce pregnancy villi of sertoli cell apoptosis,protect cells survive,maintain growth,promote the normal development of embryos in the first trimester,and maintain the normal conduct of early pregnancy.2.Pishenshuangbu Formula medicated serum by influencing the apoptosis pathway can not only protect a pregnancy villus sertoli cell survival,growth,but also through regulating the ratio of the Bcl-2/Bax,won't make the apoptosis imbalance,will not lead to trophocyte diseases caused by insufficient apoptosis. |
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