OBJECTIVE To reveal the under mechanism of how Liuwei Dihuang(LWDH)inhibits the phenotypic con⁃version of VSMCs.METHODS 24 ApoE-/-mice were divided into 4 groups:sham group,model group,E2 group,and LWDH group.Six C57B...OBJECTIVE To reveal the under mechanism of how Liuwei Dihuang(LWDH)inhibits the phenotypic con⁃version of VSMCs.METHODS 24 ApoE-/-mice were divided into 4 groups:sham group,model group,E2 group,and LWDH group.Six C57BN/L6 mice were used as control group.The primary VSMCs were divided into control group,model group,E2 group,LWDH group,LWDH+MPP group,and LWDH+PHTPP group with or without control siRNA,ERαsiRNA,ERβsiRNA,and myocardin siRNA.Oil red staining was used to evaluate the lipid deposition in the cardiac aorta.Serum chemistry analysis to test serum TG,TC,LDL,and HDL.Immunofluorescence staining was used to testα-SMA,osteopontin and F-actin.Immunohistochemical staining was performed to check out the myocardin in the cardiac aorta.The mRNA levels ofα-SMA,osteopontin,ERα,ERβ,SRC3 and myocardin were detected by real time-PCR,and the protein expression levels of them were detected by Western blotting.Co-immunoprecipitation was proceeded to test the interaction between ERαand SRC3 and SRC3 and myocardin.Flow cytometry was used to check out the cell cycle.Wound healing assay and Transwell were managed to evaluate the migration capacity of VSMCs.RESULTS In vivo administration of LWDH suppressed atherosclerotic symptoms,decreases phenotypic marker of vascular endothelial cell,and increases phenotypic marker of VSMC in ovariectomized ApoE-/-female mice.Moreover,LWDH significantly increased the mRNA and protein expression levels of ERα,ERβ,SRC3 and myocardin in the cardiac aorta of ovariecto⁃mized ApoE-/-female mice.In vitro,LWDH altered cell cycle and reduced the elevated cyclinD protein expression migra⁃tion capacity and in the model VSMCs.In addition,LWDH inhibited phenotypic conversion and promoted the expression of ER,SRC3,and myocardin of the primary VSMC phenotypic conversion model.Inhibition of ERαalmost completely eliminated the impacts of LWDH onα-SMA and osteopontin.Furthermore,LWDH promoted the interaction between ERαand SRC3 and up-regulated the co-activation of SRC3 and myocardin.CONCLUSION LWDH could inhibit the pheno⁃typic conversion of VSMCs in vitro and in vivo by increasing the activity of myocardin through up-regulating the expres⁃sion of ERαand promoting the interaction between ERαand SRC3.Our research reveals the under mechanism of how LWDH inhibits the phenotypic conversion of VSMCs.展开更多
AIM: The application of strychnine(S) is limited due to its toxicity; strychnine N-oxide(SNO) is a derivative of strychnine. The aim was to employ zebrafish embryos to investigate and compare the developmental toxicit...AIM: The application of strychnine(S) is limited due to its toxicity; strychnine N-oxide(SNO) is a derivative of strychnine. The aim was to employ zebrafish embryos to investigate and compare the developmental toxicity induced by S and SNO. METHODS: The toxicity of S and SNO was examined through the hatching rate and survival rate. Morphological changes of the zebrafish were observed with a dissecting microscope. Apoptosis was detected through acridine orange(AO) staining and flow cytometry. Apoptotic genes were measured by RT-PCR. RESULTS: Embryo malformation was observed in the embryos exposed to S at 200 μmol·L–1. When SNO concentration was increased to 1 mmol·L–1, scoliolosis, and pericardial edema could be seen in some embryos. Results from fluorescence microscopy and flow cytometry analysis showed that S at 200 μmol·L–1 induced apoptosis, whereas the apoptotic rate in the SNO-treated group(200 μmol·L–1) was much lower than that in the S group. RT-PCR analysis showed that p53 mRNA expression and the ratio of Bax/Bcl-2 in the S group were significantly altered compared with the control group(*P < 0.05). Moreover, Bax mRNA expression in both S and SNO group were significantly different from that in the control group(**P <0.01). CONCLUSION: These results lead to the conclusion that SNO has significantly lower toxicity than S in zebrafish embryos.展开更多
基金National Natural Science Foundation of China(8177319081774029)Jiangsu Provincial Science and Technology Department Social Development Fund(BE2011846)
文摘OBJECTIVE To reveal the under mechanism of how Liuwei Dihuang(LWDH)inhibits the phenotypic con⁃version of VSMCs.METHODS 24 ApoE-/-mice were divided into 4 groups:sham group,model group,E2 group,and LWDH group.Six C57BN/L6 mice were used as control group.The primary VSMCs were divided into control group,model group,E2 group,LWDH group,LWDH+MPP group,and LWDH+PHTPP group with or without control siRNA,ERαsiRNA,ERβsiRNA,and myocardin siRNA.Oil red staining was used to evaluate the lipid deposition in the cardiac aorta.Serum chemistry analysis to test serum TG,TC,LDL,and HDL.Immunofluorescence staining was used to testα-SMA,osteopontin and F-actin.Immunohistochemical staining was performed to check out the myocardin in the cardiac aorta.The mRNA levels ofα-SMA,osteopontin,ERα,ERβ,SRC3 and myocardin were detected by real time-PCR,and the protein expression levels of them were detected by Western blotting.Co-immunoprecipitation was proceeded to test the interaction between ERαand SRC3 and SRC3 and myocardin.Flow cytometry was used to check out the cell cycle.Wound healing assay and Transwell were managed to evaluate the migration capacity of VSMCs.RESULTS In vivo administration of LWDH suppressed atherosclerotic symptoms,decreases phenotypic marker of vascular endothelial cell,and increases phenotypic marker of VSMC in ovariectomized ApoE-/-female mice.Moreover,LWDH significantly increased the mRNA and protein expression levels of ERα,ERβ,SRC3 and myocardin in the cardiac aorta of ovariecto⁃mized ApoE-/-female mice.In vitro,LWDH altered cell cycle and reduced the elevated cyclinD protein expression migra⁃tion capacity and in the model VSMCs.In addition,LWDH inhibited phenotypic conversion and promoted the expression of ER,SRC3,and myocardin of the primary VSMC phenotypic conversion model.Inhibition of ERαalmost completely eliminated the impacts of LWDH onα-SMA and osteopontin.Furthermore,LWDH promoted the interaction between ERαand SRC3 and up-regulated the co-activation of SRC3 and myocardin.CONCLUSION LWDH could inhibit the pheno⁃typic conversion of VSMCs in vitro and in vivo by increasing the activity of myocardin through up-regulating the expres⁃sion of ERαand promoting the interaction between ERαand SRC3.Our research reveals the under mechanism of how LWDH inhibits the phenotypic conversion of VSMCs.
基金supported by The Opening Project of Jiangsu Key Laboratory for Chinese Medicine Processing(ZYPZ010)the National Natural Science Foundation of China(81373295)
文摘AIM: The application of strychnine(S) is limited due to its toxicity; strychnine N-oxide(SNO) is a derivative of strychnine. The aim was to employ zebrafish embryos to investigate and compare the developmental toxicity induced by S and SNO. METHODS: The toxicity of S and SNO was examined through the hatching rate and survival rate. Morphological changes of the zebrafish were observed with a dissecting microscope. Apoptosis was detected through acridine orange(AO) staining and flow cytometry. Apoptotic genes were measured by RT-PCR. RESULTS: Embryo malformation was observed in the embryos exposed to S at 200 μmol·L–1. When SNO concentration was increased to 1 mmol·L–1, scoliolosis, and pericardial edema could be seen in some embryos. Results from fluorescence microscopy and flow cytometry analysis showed that S at 200 μmol·L–1 induced apoptosis, whereas the apoptotic rate in the SNO-treated group(200 μmol·L–1) was much lower than that in the S group. RT-PCR analysis showed that p53 mRNA expression and the ratio of Bax/Bcl-2 in the S group were significantly altered compared with the control group(*P < 0.05). Moreover, Bax mRNA expression in both S and SNO group were significantly different from that in the control group(**P <0.01). CONCLUSION: These results lead to the conclusion that SNO has significantly lower toxicity than S in zebrafish embryos.