Effects And Mechanisms Of Cultivating Granules On Invasion And Proliferation Of Human Villus Trophoblasts

Objectives:To observe the effect of Peiyu particles on the proliferation, apoptosis and invading function of human trophoblasts as well as its correlation between ERK pathway.Methods and results:Normal human placental villus with 5-10 weeks of gestation was selected for trophoblasts primary culture. Gavage administration was performed on normal female SD rats to get pharmacological serum. Following even passage, the trophoblasts was divided into Peiyu particles large-dosage serum group, Peiyu particles medium- dosage serum group, Peiyu particles small-dosage serum group, Duphaston serum group, normal serum group and serum-free group. For each group of culture solution,20% serum was added. Then, another 6 groups of trophoblasts were extracted and the ERK pathway was blocked with UO126 prior to the addition of serum, followed by adding the aforesaid groups of serum. The indexes were tested 48 hours after the cultivation.TW transwell methods were used to test the effect of Peiyu particles on the invading functions of trophoblasts. According to the testing results, there was statistically significant difference in invading trophoblasts count between all the Peiyu particles serum groups of varying dosages and the normal group when the ERK pathway was unblocked. Of all the Peiyu particles serum groups, the medium-dosage serum group experienced the most invading trophoblasts count. When the ERK pathway was blocked, there was statistically significant difference in invading trophoblasts count between all the Peiyu particles serum groups of varying dosages and the normal group. Of all the Peiyu particles serum groups, the medium-dosage serum group experienced the most invading trophoblasts count. As to groups sharing the same pharmacological serum, there was statistically significant difference in invading trophoblasts count before and after the blocking of ERK pathway. After the blocking of ERK pathway, all the groups sharing the same pharmacological serum experienced reduction in invading trophoblasts count.MTT methods were used to test the effect of Peiyu particles on the proliferative ability of trophoblasts. According to the testing results, there was statistically significant difference in absorbancy between all the Peiyu particles serum groups of varying dosages and the normal group when the ERK pathway was unblocked. Of all the Peiyu part icles serum groups, the medium-dosage group had the highest absorbancy. After the ERK pathway was blocked, there was statistically significant difference in absorbancy between all the Peiyu particles serum groups of varying dosages and the normal group. Of all the Peiyu particles serum groups, the medium-dosage serum group ranked first in absorbancy. As to groups sharing the same pharmacological serum, there was statistically significant difference in absorbancy before and after the blocking of ERK pathway. After the blocking of ERK pathway, all the groups sharing the same pharmacological serum experienced decline in absorbancy.Flow cytometry was adopted to test the effect of Peiyu particles on the PCNA expression of trophoblasts. According to the testing results, there was statistically significant difference in PCNA positive rate between all the Peiyu particles serum groups of varying dosages and the normal group when the ERK pathway was unblocked. Of all the Peiyu particles serum groups, the medium-dosage s.erum group topped the list of PCNA positive rate. When the ERK pathway was blocked, there was statistically significant difference in PCNA positive rate between all the Peiyu particles serum groups of varying dosages and the normal group. Of all the Peiyu particles serum groups, the medium-dosage serum group ranked first in PCNA positive rate. As to groups sharing the same pharmacological serum, there was statistically significant difference in PCNA positive rate before and after the blocking of ERK pathway. After the blocking of ERK pathway, all the groups sharing the same pharmacological serum experienced decline in PCNA positive rate.Annexin V-FITC/PI double staining was adopted to test the effect of Peiyu particles on inhibiting trophoblasts apoptosis. According to the testing results, there was statistically significant difference in trophoblasts non-apoptosis rate between all the Peiyu particles serum groups of varying dosages and the normal group when the ERK pathway was unblocked. Of all the Peiyu particles serum groups, the medium-dosage serum group saw the highest trophoblasts non-apoptosis rate. When the ERK pathway was blocked, there was statistically significant difference in trophoblasts non-apoptosis rate between all the Peiyu particles serum groups of varying dosages and the normal group. As to groups sharing the same pharmacological serum, there was statistically significant difference in trophoblasts non-apoptosis rate before and after the blocking of ERK pathway. After the blocking of ERK pathway, all the groups sharing the same pharmacological serum experienced decline in trophoblasts non-apoptosis rate.Western Blotting was used to test the effect of Peiyu particles on the expression of protein Bax and protein Bcl-2 relevant to the apoptosis of human trophoblasts. According to the testing results, there was no statistically significant difference in protein Bcl-2 relative expression level between all the Peiyu particles groups and the normal group when the ERK pathway was unblocked. From the Peiyu particles small- dosage serum group to medium- dosage serum group and to large-dosage serum group, protein Bcl-2 relative expression level declined with the increase in dosage. After the ERK pathway was blocked, there was difference between the serum-free group and the normal group; however, there was no statistically significant difference in protein Bcl-2 relative expression level between the rest of the serum groups and the normal group. From the Peiyu particles small-dosage serum group to medium-dosage serum group and to large-dosage serum group, protein Bcl-2 relative expression level rose with the increase in dosage. Except the normal serum group, all the serum groups saw statistically significant difference in protein Bcl-2 relative expression level before and after the blocking of ERK pathway. When the ERK pathway was unblocked, there was no statistically significant difference in protein Bax relative expression level between all the serum groups and the normal group. From the Peiyu particles small-dosage serum group to medium-dosage serum group and to large-dosage serum group, protein Bax relative expression level declined with the increase in dosage. After the ERK pathway was blocked, there was no statistically significant difference in protein Bax relative expression level between all the serum groups and the normal group. From the Peiyu particles small- dosage serum group to medium- dosage serum group and to large- dosage serum group, protein Bax relative expression level rose with the increase in dosage. Except the Peiyu particles small-dosage serum group, all the serum groups saw statistically significant difference in protein Bax relative expression level before and after the blocking of ERK pathway. When the ERK pathway was unblocked, there was no statistically significant difference in Bax/Bcl-2 ratio between all the Peiyu particles serum groups and the normal group. Unexceptionally each pharmacological serum group had a smaller Bax/Bcl-2 ratio than the normal group. And for the Peiyu particles serum groups, the Bax/Bcl-2 ratio fell with the rise in dosage. After the ERK pathway was blocked by U0126, there was no statistically significant difference in Bax/Bcl-2 ratio between all the Peiyu particles serum groups and the normal group. Unexceptionally each pharmacological serum group had a greater Bax/Bcl-2 ratio than the normal group. And for the Peiyu particles serum groups, the Bax/Bcl-2 ratio fell with the rise in dosage. After the ERK pathway was blocked, unexceptionally all groups sharing the same pharmacological serum experienced a smaller Bax/Bcl-2 ratio compared with that before the blocking. There was no statistically significant difference in Bax/Bcl-2 ratio between groups sharing the same pharmacological serum.Western Blotting was adopted to test the effect of Peiyu particles on the expression of ERK1/2 and pERK1/2 relevant to ERK pathway phosphorylation of human trophoblasts before and after the blocking of ERK pathway respectively. When ERK pathway was unblocked, there was no statistically significant difference in ERK1/2 relative expression levels between all the Peiyu particles serum groups and the normal group. In Peiyu particles serum groups of varying dosages, the ERK1/2 relative expression levels declined with the increase in dosage. After the ERK pathway was blocked, there was no statistically significant difference in ERK1/2 relative expression levels between all the Peiyu particles serum groups and the normal group. And in Peiyu particles serum groups of varying dosages, the ERK1/2 relative expression levels rose with the increase in dosage. The Peiyu particles small-dosage serum group saw statistically significant difference in ERK1/2 relative expression levels before and after the blocking of ERK pathway; however, no saw statistically significant difference in ERK1/2 relative expression levels was found on the rest of the Peiyu particles serum groups. Prior to the blocking of ERK pathway, there was no statistically significant difference in ERK1/2 relative expression levels between all the Peiyu particles serum groups and the normal group. And in Peiyu particles serum groups of varying dosages, the ERK1/2 relative expression levels declined with the increase in dosage. After the blocking of ERK pathway,ERK1/2 expressions were found only among the Peiyu particles dosage- serum groups, and the statistically significant difference in ERK1/2 relative expression levels compared with the normal group was found only among the Peiyu particles dosage-serum groups. Except the Peiyu particles dosage-serum groups, the rest of the groups sharing the same pharmacological serum unexceptionally saw statistically significant difference in ERK1/2 relative expression levels before and after the blocking of ERK pathway. When the ERK pathway was unblocked, there was no statistical ly significant difference in pERK1/2/ERK1/2 ratios between various serum groups and the normal group. In the Peiyu part icles medium -dosage the pERK1/2/ERK1/2 ratios serum groups is the highest. After the blocking of ERK pathway, relatively shallow strip was found only in the Peiyu particles medium-dosage group, and there was statistically significant difference in pERK1/2/ERK1/2 ratios compared with that of the normal group. After the ERK pathway was blocked, each Peiyu particles serum group saw decreased pERK1/2/ERK1/2 ratios, and groups sharing the same pharmacological serum saw significant difference in pERK1/2/ERK1/2 ratios before and after the blocking of ERK pathway.Conclusions:Peiyu particles is able to facilitate the invading and proliferative abilities of human trophoblasts and associated with ERK pathway. Besides, it may boost the invasion and proliferation of human trophoblasts in other ways.