| ObjectiveThis study analyzes the chemical compounds of JSP,then uses human uterine fibroid cells as the carrier to investigate the role of Wnt/?-catenin and NF-?B signaling pathways in human uterine fibroid cells.On this basis,to study the pharmacodynamic effect of Juli Sanjie Pill(JSP)on human uterine fibroid cells and to discuss the molecular mechanism and action mechanism of JSP intervening human uterine fibroid cells through Wnt/?-catenin and NF-?B double signaling pathways.MethodsFirst,the chemical constituents of JSP were analyzed by UHPLC-QE-MS technique.Human primary uterine fibroid cells were cultured and identified by immunohistochemistry.KYA1797K,Wnt/?-catenin pathway inhibitor and PDTC,NF-?B pathway inhibitor,were used to intervene human uterine fibroid cells,and the expression levels of signal molecules and downstream target genes in Wnt/NF-?B double signaling pathways were detected by Western blotting and RT-qPCR.CCK8 and flow cytometry were used to detect the proliferation inhibition rate,apoptosis rate and cell cycle of human uterine fibroid cells treated by JSP.The expression levels of signal molecules and downstream target genes in the Wnt/NF-?B double signaling pathways after JSP intervention in human uterine fibroid cells were detected by Western blotting and RT-qPCR.Results1.Based on UHPLC-QE-MS,the total ion flow diagram and fingerprint of chemical components of JSP were established.A total of 663 compounds were isolated,including 195 terpenoids,105 flavonoids,79 alkaloids,69 phenylpropanoids,43 phenols and other compounds.2.KYA1797K acting on uterine fibroid cells,compared with blank group,the protein expressions of Wnt 5b,IKK?,p-IKB?,P65,TNFa and IL-6 in uterine fibroid cells increased(P<0.05),and the protein expressions of p-GSK-3?,?-catenin,EGFR and MMP2 decreased(P<0.05),while there were no significant differences in GSK-3? and IKB?(P>0.05).The expressions of ?-catenin,EGFR and MMP2 mRNA in uterine fibroid cells decreased(P<0.05),and the expressions of IKK?,IKB?,P65,TNF? and IL-6 mRNA increased(P<0.05),while there were no significant differences in Wnt 5b and GSK-3? mRNA(P>0.05).3.PDTC acting on uterine fibroid cells,compared with blank group,the protein expressions of Wnt 5b,p-GSK-3?,?-catenin,EGFR and MMP2 increased(P<0.05),and the protein expressions of IKK?,p-IKB?,P65,TNF? and IL-6 decreased(P<0.05),while there were no significant differences in GSK-3? and IKB?(P>0.05).The mRNA expressions of ?-catenin,EGFR and MMP2 in uterine fibroid cells increased(P<0.05),and the mRNA expressions of IKK?,IKB?,P65,TNF? and IL-6 decreased(P<0.05),while there were no significant differences in Wnt 5b and GSK-3? mRNA(P>0.05).4.Compared with Omg/mL group,the inhibitory rates of proliferation of uterine fibroid cells in JSP groups were significantly different(P<0.05)after 12H,24H and 48H culture.Compared with the blank group,the early and late apoptotic rates of JSP high and low concentration groups and mifepristone group were significantly different(P<0.05).The early and late apoptotic rates in JSP high concentration group and mifepristone group were higher than that in JSP low concentration group(P<0.05).Compared with the blank group,the cell numbers of G0/G1 phase and G2/M phase in JSP high and low concentration groups and mifepristone group were significantly different(P<0.05),and JSP high concentration group and mifepristone group were higher than the low concentration group(P<0.05).There was no significant difference between the JSP high concentration group and mifepristone group(P>0.05).5.Compared with the blank group,the protein expressions of Wnt 5b,p-GSK-3?,?-catenin,EGFR and MMP2 in JSP high and low concentration groups and mifepristone group decreased(P<0.05),while the protein expression of GSK-3? had no significant difference(P>0.05).Compared with JSP low concentration group,the protein expressions of Wnt 5b,p-GSK-3?,?-catenin,EGFR and MMP2 in JSP high concentration group and mifepristone group decreased(P<0.05),while the protein expression of GSK-3? had no significant difference(P>0.05).Compared with mifepristone group,the protein expression of Wnt 5b in JSP high concentration group were lower(P<0.05),and the protein expressions of p-GSK-3?,GSK-3?,?-catenin,EGFR and MMP2 were not significantly different(P>0.05).6.Compared with blank group and low concentration group,the protein expressions of IKK?,p-IKB?,P65 and TNF? in JSP high concentration group and mifepristone group increased(P<0.05),but there was no significant difference in the protein expression of IKB?(P>0.05).Compared with the blank group,the protein expressions of IKKa and P65 in JSP low concentration group increased(P<0.05),but the protein expressions of p-IKB? and IKB? had no significant difference(P>0.05).Compared with JSP high concentration group,the protein expressions of p-IKBa and P65 in mifepristone group increased(P<0.05),but the protein expressions of IKK?,IKB? and TNF? had no significant difference(P>0.05).The expression of IL-6 protein in mifepristone group was higher than that in JSP low concentration group(P<0.05),which there was no significant difference between the JSP high and low concentration groups(P>0.05).7.Compared with the blank group,the mRNA expressions of Wnt 5b,GSK-3?,?-catenin,EGFR and MMP2 in JSP high and low concentration groups and mifepristone group decreased(P<0.05);the mRNA expressions of IKK?,IKB?,P65 and IL-6 increased(P<0.05);the expression of TNF? mRNA in JSP high concentration group and mifepristone group increased(P<0.05),while there was no significant difference in JSP low concentration group(P>0.05).Compared with the low concentration group,the mRNA expressions of Wnt 5b,GSK-3?,?-catenin,EGFR and MMP2 in JSP high concentration group and mifepristone group decreased(P<0.05);the mRNA expressions of IKB?,P65 and IL-6 increased(P<0.05);there was no significant difference in IKKa mRNA expression(P<0.05).The expression of TNFa mRNA in mifepristone group was higher than that in JSP low concentration group(P<0.05),while there was no significant difference of which between JSP high and low concentration groups(P>0.05).There was no significant difference in protein mRNA expression between JSP high concentration group and mifepristone group(P>0.05).Conclusion1.UHPLC-QE-MS technology can quickly and efficiently identify the chemical components of JSP,which provides the basis for the research on the medicinal substances of JSP.JSP has many chemical components,including terpenoids,flavonoids,alkaloids,phenylpropanoids,phenols,etc.2.KYA1797K,Wnt/?-catenin pathway inhibitor and PDTC,NF-?B pathway inhibitor,can both affect the expression levels of Wnt/?-catenin signaling pathway and NF-?B signaling pathway molecules and downstream target genes in uterine fibroid cells,suggesting that there may be crosslink between two signaling pathways in uterine fibroid cells.3.JSP can significantly inhibit the proliferation of uterine fibroid cells,increase the early and late apoptotic rates,block the cell cycle,which confirmed the pharmacodynamic effect of JSP on uterine fibroid cells.4.JSP affected the expression levels of Wnt/?-catenin pathway and NF-?B signaling pathway molecules and downstream target genes in uterine fibroid cells,indicating that JSP interferes the proliferation and apoptosis of uterine fibroid cells through the Wnt/NF-?B double signaling pathway. |
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