Based On Estrogen Receptor Alpha To Investigate The Effect Of Catalpol On Endothelial Cell Pyroptosis In Peri-menopausal AS

Aim:Atherosclerosis(AS),a chronic inflammatory disease of the arterial intima,has became the major cause of death in humans.Its morbidity is increasing in recent years.Epidemiological studies have shown that the incidence of cardiovascular diseases in perimenopausal women is significantly higher than that in young women due to the lack of endogenous estrogen.Endothelial injury is the initiating factor of AS.Endothelial cell dysfunction,and further release of a variety of active substances and adhesion molecules would lead to chronic inflammation,which could promote the development of AS.Pyroptosis is a programmed inflammatory cell death that is dependent on the activation of caspase-1.Studies have shown that autophagy can inhibit the formation of inflammatory signaling protein complexes,meanwhile estrogen receptors can regulate autophagy-related genes and proteins.Previous studies have shown that the catalpol has high binding capacity with the estrogen receptor and has anti-cardiovascular effect,but its mechanism is still unknown.Therefore,this study is to observe whether the catalpol has protective effects on endothelial cell pyroptosis and whether this effect is achieved by autophagy.Therefore,this study is to observe the effects of catalpol on endothelial estrogen receptors,endothelial cell pyroptosis and autophagy,and to explore whether the catalpol can regulate autophagy by upregulating estrogen receptor,consequently,inhibit endothelial cell pyroptosis and finally resist the anti-permenopausal atherosclerosis.Method1.Replication of peri-menopausal AS model and the effect of catalpol on it:Female ApoE-/-and C57BL/6 mice were used.C57BL/6 control group mice were cut from both sides of the back,the a small amount of fat was removed near the ovary and all these SPF-level experimental mice were fed with pellets.Female ApoE-/-sham operation group operation was the same as C57BL/6 blank control group and all these SPF-level experimental mice were fed with pellets one week and then fed with high fat diet(HFD).The peri-menopausal AS model mice were resected from the bilateral ovaries and fed to the normal diet for one week.The peri-menopausal AS model mice were randomly divided into the following five groups and then fed with high fat:the model group,the E2 group(0.13mg/kg),the simvastatin group(4.3mg/kg)and the catalpol group(7.22mg/kg,1.80mg/kg).The administration group was intragastrically administrated once a day,and the control group,the ApoE-/-control group and the model group were given the same amount of saline daily,total of 90 days.Weighing the mice weekly,and the dose volume was adjusted according to the weight.After 90 days of administration,the kits were used to detect the expression of plasma lipids,inflammatory factors and Hcy;Oil red staining was used to observe the aortic lipid deposition;2.The effect of catalpol on Hcy-induced pyroptosis of endothelial cells:Cell viability was measured by MTT assay,and the effect of different concentrations of Hcy on cell viability was observed.Flow cytometry and Western Blot were used to observe the time-effect relationship of Hcy-HUVECS pyroptosis and determine the time required for modeling.The cells were divided into blank group,Hcy group,Hcy+40μl catalpol group,Hcy+20μl catalpol group,Hcy+10μl catalpol group and Hcy+10nM E2 group.Hoechst-PI staining was used to detect membrane integrity of endothelial cell,WB and PCR were used to detect the expression of pyroptosis-related factors NKRP3,ASC,caspase-1,GSDMD-N,IL-1β and IL-18 in aorta and human umbilical vein endothelial cells.3.The effect of catalpol on endothelial cell pyroptosis after autophagy inhibition:The expressions of autophagy-related factors P62,LC3 and beclin1 in aorta and human umbilical vein endothelial cells were detected by WB and PCR.The expression of autophagy protein LC3 was detected by immunofluorescence assay.Interference with autophagy inhibitor 3-MA,Hoechst-PI staining was used to detect the integrity of endothelial cell membranes,and western blot and PCR were used to detect the expression of pyroptosis-related factors,and to observe the role of autophagy in the regulation of endothelial cell pyroptosis.4.The effect of catalpol on endothelial cell pyroptosis after estrogen receptor interference:The expression of estrogen receptor in mouse aorta and human umbilical vein endothelial cells was detected by WB and PCR.Using estrogen receptor alpha,beta(MPP,PHTPP)inhibitors and ERa siRNA interference technology,WB and PCR were used to detect the expression of autophagyrelated factors,immunofluore-scence was used to detect the expression of autophagy protein LC3;Hoechst-PI staining was used to detect the membrane integrity of endothelial cell,WB and PCR were used to detect the expression of NLRP3,ASC,caspase-1,GSDMD-N,IL-1β and IL-18,and to observe the role of estrogen receptor in the regulation of endothelial cell pyroptosis by catalpol.Result1.The effect of catalpol on peri-menopausal AS model mice:Oil red staining showed no plaque formation in the aortic root of blank group mice,and partial lipid deposition in the aortic root of ApoE-/-control group mice.The levels of serum lipids,inflammatory factors IL-1β,IL-18 and Hcy increased.There were more plaque deposition in the aortic root of perimenopausal AS mice,and the levels of serum lipids,inflammatory factors IL-1 beta,IL-18 and Hcy increased further.After treatment with E2,simvastatin and catalpol,the formation of AS plaque in the aortic root of mice was significantly reduced,the level of blood lipid was lowered,the expression of IL-1 beta and IL18 in plasma was inhibited,the level of Hcy was decreased,and the pathological process of AS was slowed down.2.The effect of catalpol on Hcy-induced endothelial cell pyroptosis:MTT,flow cytometry and WB results showed that 60 mmol/L Hcy could induce human umbilical vein endothelial cells to replicate the model of endothelial cell pyroptosis for 12 hours.MTT results further showed that catalpol could inhibit endothelial cell injury at 10-40 micromol/L.WB,PCR and Hoechst-PI staining results showed that the expression of NLRP3/ASC/caspase-1/GSDMD-N/IL-1β/IL-18 was significantly increased in the model group.Catalpol group and E2 group could significantly inhibit the expression of coke-related factors in endothelial cells and maintain the integrity of endothelial cell membrane in a dose-dependent manner.3.The effect of catalpol on endothelial cell pyroptosis after autophagy inhibition:The results showed that compared with the blank group,the expressions of LC3 and Beclinl were inhibited and P62 were increased in AS and Hcy,while catalpol could significantly up-regulate the expressions of LC3 and Beclinl and promote autophagy.After the intervention of autophagy inhibitor 3-MA,compared with catalpol group,the effect of catalpol on NLRP3/ASC/caspase1/GSDMD-N/IL-1beta/IL-18 was reversed by autophagy inhibitor 3-MA.Hoechst-PI staining also showed that 3-MA could reverse the effect of catalpol on endothelial cell membrane integrity.4.The effect of catalpol on endothelial cell pyroptosis after estrogen receptor interference:Compared with the blank group,the expression of estrogen receptor was significantly inhibited in AS group and Hcy grou,while catalpol and E2 could up-regulate the expression of ERalpha and ERbeta.Estrogen receptor alpha inhibitor MPP could reverse the inhibition of catalpol on pyroptosis-related proteins and the protective effect of catalpol on endothelial cells.However,the effect of Estrogen receptor beta inhibitor PHTPP on catalpol was weak.MPP could significantly reduce the increase of LC3 fluorescence intensity induced by catalpol,decrease the expression of autophagy protein LC3 and Beclinl,meanwhile significantly increase the expression of P62 protein.The effect of PHTPP on autophagy was weak.Lentivirus transfection interferes the expression of ER alpha in endothelial cells.The expression of LC3 and Beclinl induced by catalpol was significantly decreased and the expression of P62 protein was significantly increased after ER alpha interference.Furthermore,ER alpha interference could significantly reverse the expression of pyroptosis-related proteins and cell membrane integrity in endothelial cells.These results suggest that catalpol could inhibit endothelial cell pyroptosis by up-regulating autophagy by ER alpha,thus achieving anti-AS effect.Conclusion1.Catalpol significantly reduces the occurrence of AS in perimenopausal mice.Catalpol plays a resistant role on the perimenopausal AS by regulating inflammatory factors,homocysteine and pyroptosis-related proteins in aortic endothelial cells.2.Catalpol inhibits Hcy-induced endothelial cell pyroptosis.3.Catalpol inhibits endothelial cell pyroptosis by upregulating autophagy levels.4.Catalpol inhibits endothelial cell pyroptosis by up-regulating autophagy through ER alpha,thus resisting perimenopausal AS.