Effect Of Yunnanterpene G On The Regulation Of EMMPRIN And MMPs Expression In Macrophages

Objective:1.To extract and screen a new triterpenoid compound from the rhizome of C.foetida in Yunnan Province,which has activity on macrophages and their expression of extracellular matrix metalloproteinase inducer(EMMPRIN)and matrix metalloproteinases(MMPs).2.To study the effect of this compound on the biological behavior of macrophages and the expression of EMMPRIN and MMPs(MMP-2,MMP-9,MMP-14)in macrophages,and to explore its mechanism.This study will lay a foundation for the development of new drugs for anti-atherosclerosis and stabilizing atherosclerotic plaque in the future.Methods:1.High performance liquid chromatography is used to extract and separate effective active ingredients from rhizome of C.foetida in Yunnan.2.The structure of the new compound Yunnanterpene G was determined by NMR spectroscopy and X-ray crystal structure analysis.3.PMA induced THP-1 cells to differentiate into human macrophages,and macrophages were identified by flow cytometry.4.MTT was used to determine the effect of Yunnanterpene G on macrophage viability.5.Wound-healing migration assay was used to determine the effect of Yunnanterpene G on macrophage migration.6.The total RNA of macrophages was extracted by Trizol.RT-qPCR was used to determine the effect of Yunnanterpene G on mRNA expression level of EMMPRIN and MMPs in macrophages.7.Western blot assay was used to determine the effect of Yunnanterpene G on the expression level of EMMPRIN and MMPs proteins in macrophages.8.The shRNA adenoviral vector was constructed to interfere with EMMPRIN expression.To study the migration of macrophages and the mRNA expression of EMMPRIN,MMPs and TIMP-1 in macrophages after inhibiting the expression of EMMPRIN.9.Western Blot assay was used to explore the possible signal pathways of Yunnanterpene G affecting macrophages.10.The experiment of each group was repeated at least 3 times.SPSS 22.0 was used for statistical analysis.Counting data were represented by integers.Measurement data were presented as mean ± SD.Statistical analysis of data was performed with Student’s t-test for unpaired data in two trails.(*)P<0.05,(**)P<0.01,and(***)P<0.001 were considered significant.Results:1.For the first time,we isolated a new active triterpenoid from the rhizome of C.foetida in Yunnan and named it"yunnanterpene G".2.Yunnanterpene G was a highly oxygenated 9,19-Cycloartane triterpenes containing a six-ring skeleton.The molecular formula of Yunnanterpene G is C3pH4406.3.Yunnanterpene G inhibits the migration of macrophages without affecting cell viability.4.Yunnanterpene G inhibited the mRNA expression levels of EMMPRIN and MMPs(MMP-2,MMP-9 and MMP-14)in macrophages in a concentration-dependent manner(P<0.05).5.Yunnanterpene G inhibited the protein expression levels of EMMPRIN and MMPs(MMP-2,MMP-9 and MMP-14)in macrophages in a concentration-dependent manner(P<0.05).6.Interference with EMMPRIN expression in macrophages inhibited macrophage migration,up-regulated TIMP-1 mRNA expression and down-regulated MMPs(MMP-2,MMP-9 and MMP-14)mRNA expression(P<0.05).7.NF-κB and p3 8 MAPK signaling pathways may be involved in the regulation of Yunnanterpene G on macrophages.Conclusion(s):1.For the first time,"yunnanterpene G",a new active triterpenoid compound was extracted from the rhizome of C.foetida in Yunnan.2.Yunnanterpene G inhibits macrophage migration and down-regulates the expression of MMPs(MMP-2,MMP-9 and MMP-14)by inhibiting EMMPRIN expression.It may play an important role in stabilizing atherosclerotic plaques.