The Research Of The Effective Components Of Guizhi In Inhibiting The Invasion And Migration Of Adenomyosis Derived Cell

Objective: In this study,selecting Ramulus Cinnamomi volatile oil and Trans-Cinnamaldehyde as the effective components of Guizhi,main to explore the mechanism of action inhibiting the invasion and migration of adenomyosis derived cells,elucidating its inhibitory effect on adenomyosis derived cell may through Rho A/ROCK signaling pathways,so as to provide experimental support for drugs on the metastasis of adenomyosis,and provide new ideas and methods for controlling the metastasis of adenomyosis.Methods:(1)Choosing the patients who consent to surgical treatment with adenomyosis in hospitalization,extracting the primary cell.(2)Culturing adenomyosis derived cells in vitro,Immunofluorescence of characteristics for fluorescence expression capability with Vimentin,CK7,Von Willebrand Factor,E-cadherin,alpha smooth muscle.(3)Medication for cell activity RCVO(0,0.4,0.8,1.6,3.2,6.4 mg/m L)、TC(0,8,16,32,64,128 μg/m L)and toxicity RCVO(0,0.5,1,1.5 mg/m L)、TC(0,6,12,18μg/m L)of adenomyosis derived cells was detected by CCK-8 assay.(4)Transwell assay examined the effects of medication on cell invasion,grouping model control group、RCVO 0.5 mg/m L、RCVO 1 mg/m L,model control group、TC 6 μg/m L、TC 12 μg/m L.(5)Cell wound scratch assay examined the effects of medication on cell migration,grouping model control group、RCVO 0.5 mg/m L、RCVO 1 mg/m L,model control group、TC 6 μg/m L、TC 12 μg/m L.(6)Western blotting detected the expression after medication intervention through the protein with Rho A、ROCK1、ROCK2,grouping model control group、RCVO 0.5 mg/m L、RCVO 1 mg/m L,model control group、TC 6 μg/m L、TC 12 μg/m L.Results:(1)Adenomyosis derived cells fostering successfully.(2)Immunofluorescence characterized with strongly positive staining for Vimentin and almost negative for CK7,Von Willebrand Factor,E-cadherin,alpha smooth muscle with cells.(3)CCK-8 assay results show that Ramulus Cinnamomi volatile oil has an inhibitory effect on cells grow and is concentration-dependent,selecting the least cytotoxic concentration for subsequent experiments.(4)The results of transwell assay show that compared with model control group,cell invasion ability was diminished with Ramulus Cinnamomi volatile oil 0.5 mg/m L group,and these were significantly downregulated with Ramulus Cinnamomi volatile oil 1 mg/m L group(P<0.05).(5)The results of cell wound scratch assay show that compared with model control group,cell migration ability was diminished with Ramulus Cinnamomi volatile oil 0.5 mg/m L group,and these were significantly downregulated with Ramulus Cinnamomi volatile oil 1 mg/m L group(P<0.05).(6)The results of Western blotting show that compared with model control group,the protein expression Rho A、ROCK1、ROCK2 were diminished with Ramulus Cinnamomi volatile oil 0.5mg/m L group,and these were significantly downregulated with Ramulus Cinnamomi volatile oil 1 mg/m L group(P<0.05).(7)CCK-8 assay results show that Trans-Cinnamaldehyde has an inhibitory effect on cells grow and is concentration-dependent,selecting the least cytotoxic concentration for subsequent experiments.(8)The results of transwell assay show that compared with model control group,cell invasion ability was diminished with Trans-Cinnamaldehyde 6 μg/m L group,and these were significantly downregulated with Trans-Cinnamaldehyde 12 μg/m L group(P<0.05).(9)The results of Cell wound scratch assay show that compared with model control group,cell migration ability was diminished with Trans-Cinnamaldehyde 6 μg/m L group,and these were significantly downregulated with Trans-Cinnamaldehyde12 μg/m L group(P<0.05).(10)The results of Western blotting show that compared with model control group,the protein expression Rho A、 ROCK1、ROCK2 were diminished with Trans-Cinnamaldehyde 6 μg/m L group,and these protein index were significantly downregulated with Trans-Cinnamaldehyde 12μg/m L group(P<0.05).Conclusions: The two patients cells both with the characteristics phenotype of adenomyosis derived cells,the mechanism may be related to Rho A、ROCK1、ROCK2 inhibiting the activity of cells to limit the invasion and migration ability with Ramulus Cinnamomi volatile oil and Trans-Cinnamaldehyde,providing experimental support for effective components of Guizhi to treat adenomyosis.