| Objectives:To observe the effects of Liuwei Dihuang Fang on the DNA methylation and expression of estrogen receptor(ER)in postmenopausal atherosclerosis(AS),and find the key methylation sites of ER regulated by Liuwei Dihuang Fang.At the same time,to explore whether Liuwei Dihuang Fang can regulate ER expression through DNA methyltransferases(DNMTs)and demethylases(Ten-eleven translocations,TETs),thereby exerting anti-smooth muscle cell proliferation and postmenopausal AS effects.Methods:First,we collected aortic vessels from pre-and postmenopausal women,and observed the degree of vascular lesions by HE staining and transmission electron microscopy.Subsequently,we used LDLR-/-mice to replicate the postmenopausal AS mouse model by high-fat feeding combined with bilateral ovariectomy,and treated with Liuwei Dihuang Fang for 90 days.At the end of the experiment,the levels of blood lipids in mice were observed by biochemical kits.The lesion degree of mouse aorta was observed by oil red and Masson staining.The proliferation of mouse aortic root smooth muscle was observed by immunohistofluorescence.The levels of serum angiotensin Ⅱ(Ang II)and homocysteine(Hcy)were observed by ELISA kits.In vitro,we induced a proliferation model of human vascular smooth muscle cells(HVSMCs)with Hcy and intervened with Liuwei Dihuang Fang.The effects of different concentrations of Hcy on the viability of HVSMCs were observed by MTT.The effects of Liuwei Dihuang Fang on the proliferation,migration and cell cycle of HVSMCs were observed by MTT,cell scratch,Transwell and flow cytometry.The effect of Liuwei Dihuang Fang on the phenotype conversion of HVSMCs was observed by immunofluorescence staining.Through this part of the experiment,the effects of Liuwei Dihuang Fang on antipostmenopausal AS and inhibiting the proliferation of HVSMCs were revealed.The expression of ERs in the aorta of postmenopausal women and postmenopausal AS mice were observed by immunohistochemical staining.The effects of Liuwei Dihuang Fang on the expression of ERs in HVSMCs were observed by WB and PCR.We further observed the DNA methylation rate of each fragment of ESR1(the gene encoding the ERα protein)in the aorta of postmenopausal AS women and HVSMCs proliferation models by MethylTarget methylation sequencing.At the same time,the effect of Liuwei Dihuang Fang on the methylation rate of ESR1 was observed.The effects of Liuwei Dihuang Fang on ERs expression and DNA methylation in postmenopausal AS were revealed through this part of the experiment.The expression of DNMTs and TETs in the aorta of postmenopausal women and postmenopausal AS mice were observed by immunohistochemical staining.The levels of serum DNMTs and TETs in mice were observed by ELISA kits.The effects of Liuwei Dihuang Fang on the overall methylation of mice and HVSMCs were observed by methylation kits.The effects of Liuwei Dihuang Fang on the expression of DNMTs and TETs in HVSMCs were observed by WB and PCR.Through this part of the experiment,the effects of Liuwei Dihuang Fang on the expression of DNMTs and TETs in postmenopausal AS were revealed.According to the results of the previous step,we constructed HVSMCs cell models with high DNMT3a expression or low TET2 expression by transfecting DNMT3a overexpression plasmid or TET2 siRNA.On this basis,Hcy and Liuwei Dihuang Fang were used to intervene cells,and the expressions of DNMT3a and TET2 were verified by WB.The effects of Liuwei Dihuang Fang on the proliferation,migration and cell cycle of HVSMCs were observed by cell scratch,Transwell and flow cytometry.The effect of Liuwei Dihuang Fang on the phenotype conversion of HVSMCs was observed by immunofluorescence staining.The effect of Liuwei Dihuang Fang on the overall methylation of HVSMCs was observed by methylation kit.The effects of Liuwei Dihuang Fang on the expression of ERa were observed by WB and PCR.Through this part of the experiment,it was confirmed whether Liuwei Dihuang formula affected the expression of ERa by regulating DNMT3a and TET2.Finally,dCAS9-DNMT3A was used to target hypermethylated ESR1,the effects of Liuwei Dihuang Fang on ERα expression were observed by WB and PCR.The effects of Liuwei Dihuang Fang on the proliferation,migration and cell cycle of HVSMCs were observed by cell scratch,Transwell and flow cytometry.The effect of Liuwei Dihuang Fang on the phenotype conversion of HVSMCs was observed by cell immunofluorescence staining.The binding effect between ERα,TET2 and DNMT3a was observed by co-immunoprecipitation experiments.Through this part of the experiment,it was confirmed that Liuwei Dihuang Fang could inhibit the proliferation of HVSMCs by reducing the DNA methylation of ESR1 and up-regulating the expression of ERα.Results:In Chapter 1,we found that postmenopausal women with AS have increased apoptosis in the aortic endothelial layer and significant proliferation of smooth muscle layer cells compared with premenopausal women with AS.The levels of blood lipids TC,TG and LDL-c in postmenopausal AS mice were significantly increased,and the lipid deposition in the aorta was increased,confirming the successful replication of the postmenopausal AS model.The intervention of Liuwei Dihuang Fang significantly reduced the levels of TC,TG and LDL-c in the blood of postmenopausal AS mice,increased the level of HDL-c,reduced the lipid deposition in the aorta,and slowed down the lesions of AS.The smooth muscle in the blood vessels of the postmenopausal AS mice proliferated significantly,and the levels of AngⅡ and Hey in the blood were significantly increased.Liuwei Dihuang Fang significantly inhibited the proliferation of aortic root smooth muscle cells in postmenopausal AS mice,and decreased the levels of AngⅡ and Hey in the blood.250 μM Hey intervention for 24 hours could induce HVSMCs to reach a proliferation rate of 60%,while 12 μg/mL Liuwei Dihuang Fang could significantly inhibit the proliferation of HVSMCs.Hcy induced HVSMCs to proliferate and migrate significantly,the cells in G0/G1 phase were significantly reduced,and the cells in S phase and G2/M phase were increased.Liuwei Dihuang Fang significantly inhibited the proliferation and migration of HVSMCs induced by Hcy,and at the same time significantly increased the cells in G0/G1 phase and decreased the cells in S phase and G2/M phase.In Hcy-induced HVSMCs,the expression of contractile protein α-SMA was significantly decreased,while the expression of synthetic protein OPN was significantly increased.The intervention of Liuwei Dihuang Fang could significantly up-regulate α-SMA,down-regulate OPN,and inhibit the phenotypic transition of smooth muscle cells.In Chapter 2,we found that ERa was mainly expressed in the vascular endothelium and smooth muscle layer in premenopausal women with AS,and the expressions of ERα,ERβ and GPR30 were significantly decreased in postmenopausal women.The expression of ERs in the aortic root of postmenopausal AS mice was significantly decreased,and the treatment of Liuwei Dihuang Fang significantly upregulated the expression of ERs.In the Hcy-induced HVSMCs proliferation model,the expressions of ERα and ERβ were significantly decreased,but Liuwei Dihuang Fang could only significantly up-regulate the expression of ERα.The sequencing results showed that the methylation rate of ESR1 was significantly increased in postmenopausal AS women and HVSMCs.Liuwei Dihuang Fang could reduce the methylation rate of ESR1 in HVSMCs proliferation model.In Chapter 3,DNMT1 was significantly increased in endothelial layer of postmenopausal AS women,but DNMT3a and DNMT3b were not significantly changed;the expression of DNMT1 and DNMT3a in smooth muscle layer was significantly increased,but DNMT3b was not significantly changed.The content of 5mc in the blood of postmenopausal AS mice was significantly increased,the content of 5-hmc was significantly decreased,and the overall methylation level was increased.Liuwei Dihuang Fang decreased the content of 5-mc in the blood of postmenopausal AS mice,increased the content of 5-hmc,and decreased the overall methylation level.The expressions of DNMT1,DNMT3a and DNMT3b were significantly increased in the blood of postmenopausal AS mice,while only DNMT1 and DNMT3a were significantly increased in blood vessels.The intervention of Liuwei Dihuang Fang could significantly inhibit the increase of DNMTs in blood and blood vessels.Hcy caused a decrease in overall methylation in HVSMCs,and the intervention of Liuwei Dihuang Fang significantly up-regulated the content of 5-mc in cells.In the Hcyinduced HVSMCs proliferation model,the expression of DNMT1 was significantly decreased,and the expressions of DNMT3a and DNMT3b were significantly upregulated.The intervention of Liuwei Dihuang Fang significantly up-regulated the expression of DNMT1 in cells,but significantly down-regulated the expressions of DNMT3a and DNMT3b.Meanwhile,we found that compared with premenopausal women with AS,the expression of TET1,TET2 and TET3 in the vascular endothelium and smooth muscle layer were significantly increased in postmenopausal women with AS.At the same time,the expressions of TET1,TET2 and TET3 in the blood and blood vessels of postmenopausal AS mice were significantly increased,and the intervention of Liuwei Dihuang Fang could significantly reduce the expression of TETs.Hcy significantly increased the expression of TET1 and inhibited the expression of TET2 in HVSMCs,but had no significant effect on the expression of TET3,while the intervention of Liuwei Dihuang Fang could significantly down-regulate the expression of TET1 and up-regulate the expression of TET2.In Chapter 4 and 5,we transfected DNMT3a overexpression plasmid and TET2 siRNA,the expression of DNMT3a was significantly increased,and the expression of TET2 was significantly decreased,confirming the success of transfection.HVSMCs proliferated and migrated significantly after high expression of DNMT3a and low expression of TET2.The number of cells in G0/G1 phase decreased,and the number of cells in S phase and G2/M phase increased;the expression of contractile phenotype protein α-SMA was significantly decreased,and the expression of synthetic phenotype protein OPN was significantly increased;the overall methylation level was increased,and the expression of ERα was significantly decreased.Compared with before transfection,the effect of Liuwei Dihuang Fang on inhibiting the proliferation,migration and phenotype conversion of HVSMCs was weakened after high expression of DNMT3a and low expression of TET2,and the effect of up-regulating ERαexpression was also weakened.The results confirmed that Liuwei Dihuang Fang regulated the expression of ERa by reducing DNMT3a and up-regulating TET2.ESR1 has a total of 23 CpG islands,of which fragment 5,fragment 10 and fragment 21 are located before the promoter and on exons.After directed hypermethylation of these three fragments,it was found that methylated fragment 5 had the most significant effect on inhibiting the expression of ERα,and Liuwei Dihuang Fang could significantly reduce the methylation rate of this fragment.After targeting hypermethylated fragment 5,HVSMCs proliferated and migrated significantly.The number of cells in G0/G1 phase decreased,the number of cells in S phase and G2/M phase increased,the expression of contractile phenotype protein α-SMA was significantly decreased,and the expression of synthetic phenotype protein OPN was significantly increased.Compared with the undirected hypermethylation group,the effect of Liuwei Dihuang Fang on inhibiting the proliferation,migration and phenotype switch of HVSMCs was also attenuated after the hypermethylated fragment 5.The results confirmed that Liuwei Dihuang Fang could inhibit the proliferation of HVSMCs by inhibiting the methylation of ESR1 and up-regulating the expression of ERα.In addition,the co-immunoprecipitation results revealed that ERα,TET2 and DNMT3a combined with each other to form a protein complex.Conclusion:1.Liuwei Dihuang Fang significantly improved postmenopausal AS aortic lesions,reduced lipid deposition,and inhibited the proliferation of smooth muscle cells.2.The expression of ERa in postmenopausal AS was significantly decreased,and the DNA methylation rate of ESR1 was significantly increased.Among them,the methylation of CpG island fragment 5 has the most significant effect on inhibiting the expression of ERα.Liuwei Dihuang Fang could inhibit the proliferation of HVSMCs by reducing the methylation rate of ESR1 and increasing the expression of ERα.3.The expressions of DNMT1 and DNMT3a were significantly increased in postmenopausal AS.In Hcy-induced HVSMCs,the expression of DNMT1 was significantly decreased,and the expressions of DNMT3a and DNMT3b were significantly increased.Liuwei Dihuang Fang could significantly reduce the expression of DNMT1 and DNMT3a in postmenopausal AS mice,and Liuwei Dihuang Fang could inhibit the proliferation of HVSMCs by reducing DNMT3a and up-regulating the expression of ERα.4.The expressions of TET1,TET2 and TET3 were significantly increased in postmenopausal AS.However,in Hcy-induced HVSMCs,the expression of TET1 was significantly increased,the expression of TET2 was significantly decreased,and the expression of TET3 had no significant change.Liuwei Dihuang Fang could significantly reduce the expression of TETs in postmenopausal AS mice,and Liuwei Dihuang Fang could up-regulate the expression of ERa by increasing TET2,thereby inhibiting the proliferation of HVSMCs.5.ERα,TET2,and DNMT3a bound to each other to form protein complexes. |
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