| Hypertension has close relationship with ECs dysfunction.Endothelial dysfunction is a phenotypic change of the vascular endothelium that occurs before adverse cardiovascular events,and indicates future cardiovascular risks,In the classic Chinese medicine prescription for the treatment of cardiovascular disease,the study found a common Chinese medicine,Rhubarb.In ancient books,the treatment of hypertension is mainly based on the principle of invigorating deficiency and relieving substance.On the basis of dialectics,using rhubarb to relieve fire and defecate is beneficial to the liver and improve the symptoms.Modern research also shows that rhubarb has pharmacological effects such as lowering blood pressure,lowering blood lipids,and anti-inflammatory.AE is an effective anthraquinone component of rhubarb.AE is reported having anti-inflammatory effect.This study explored the cardiovascular protective effect of Aloe-Emodin on angiotensin Ⅱ(AngⅡ)-induced hypertension mouse model and its mechanism through in vivo and in vitro experiments.ObjectiveThrough the use of angiotensin Ⅱ to induce mouse hypertension models and mouse microvascular endothelial cell dysfunction,this research explored the therapeutic effects and mechanisms of different concentrations of Aloe-Emodin on endothelial injury models,in order to guide the prevention and treatment of hypertension-related cardiovascular diseases Medication provides scientific basis.Methods(1)Pharmacodynamic study of Aloe-Emodin in the treatment of AngⅡ-induced vascular endothelial injury:AngⅡ was injected subcutaneously,and AngⅡ was injected at three fixed time points a day to establish a mouse hypertension model.Fifty-six mice were randomly divided into 6 groups,including control group,AngⅡ group,low-concentration AE group(10mg/kg),and medium-concentration AE group(20mg/kg),high concentration AE group(30mg/kg),Losartan group(20mg/kg).The mouse cuff-tail method was used to detect the blood pressure of the mice.After the blood pressure of the mice increased,the mice were given normal saline,Aloe-Emodin suspension,and Losartan suspension for the corresponding groups.After administration,the blood pressure was measured.The mice were sacrificed and samples were collected.The heart of mice were frozen and sliced into 8μm thick slices on the attached glass slide,and frozen and stored.The immunofluorescence twocolor co-localization method was used to detect the integrity of mouse heart vascular endothelial cell tight junction protein ZO-1/2,and platelet endothelial cell adhesion molecule(PECAM)was used as a marker of vascular endothelium.AngⅡ(20nM)was used to stimulate mouse vascular endothelial cells to establish an endothelial inflammation model,and different concentrations of AE(0.05 μM,0.1μ,0.3μM,0.5μM)were given to endothelial cells.The cells were grouped into normal group,Angll model group,and Aloe-Emodin administration group with different concentrations.After the endothelial cells adhered to the wall,Angll and Aloe-Emodin were administered at the same time.After 24h,the supernatant or cell protein was collected for the next experiments.MTT and cell proliferation experiments were used to detect cell viability,and we selected the appropriate concentration from the source.Immunofluorescence method was used to detect the expression and connection of endothelial connexin ZO-1,and endothelial cell membrane permeability test and trans-endothelial cell resistance test were used to detect the damage of endothelial cell membrane.(2)Study on the mechanism of Aloe-Emodin in the treatment of AngⅡ-induced hypertensive endothelial injury in mice:The mouse heart slices cryopreserved in the prepharmacodynamic experiment were used for the mechanism experiment.The immunofluorescence method was used to detect the expression of NLRP3 in mouse cardiac vascular endothelial cells,and the enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of IL-1β and HMGB1 in mouse serum.In the study of cell mechanism,AngⅡ(20nM)was used to stimulate mouse microvascular endothelial cells to establish an endothelial inflammation model.and different concentrations of Aloe-Emodin were used to interfere with endothelial cells.The cell experiment was divided into control group,AngⅡgroup,and AE group(0.05μM,0.1 μM,0.3μM,0.5μM).The test was conducted 24 hours after the simultaneous administration of AngⅡ and Aloe-Emodin.WB detected NLRP3,ProCasp1,Cle-Casp1 expression;IF detected the cocolization ofNLRP3 and Pro-Casp1,NLRP3 and ASC;ELISA method detected the content of IL-1β and HMGB1 in the cell supernatant.Transfection of gNLRP3 into endothelial cells to prepare cells that knock down NLRP3 gene.At the same time,AngⅡ and Aloe-Emodin were administered.Western blot was used to detect transfection efficiency,endothelial tight connective protein ZO1/2,NLRP3 inflammasome pathway related proteins Caspase-1,IL-1β,HMGB1.Finally,western blot was used to detect the ubiquitination of NLRP3 by Aloe-Emodin,and the proteasome inhibitor MG-132 was given to detect the expression of NLRP3 inflammasome constituent protein and activated protein.Results(1)Pharmacodynamic study of Aloe-Emodin in the treatment of AngⅡ-induced vascular endothelial injury:In vivo experiments,this study found that Aloe-Emodin and Losartan can significantly reduce blood pressure in mice,compared with the AngⅡ model group.At the same time,this research found that the expression of tight junction protein ZO-1 and ZO-2 in the heart and blood vessel endothelium of hypertensive mice were significantly reduced,indicating that the vascular endothelium of hypertensive mice was damaged.However,the tight junction protein of the heart and blood vessels of the mice in the Aloe-Emodin drug group was significantly increased,while the Losartan group did not change,indicating that Aloe-Emodin could reduce the blood pressure of hypertensive mice and restore the vascular endothelial connection function.In cell experiments,Mtt showed that AE of 0.05μM,0.01μM,0.03μM,and 0.05μM is not toxic to cells and could restore cell proliferation.So this study chose these 4 concentrations for the next experiment.This study found that after Angll stimulates endothelial cells,endothelial connexin is destroyed,cell membrane permeability increases,and cell resistance decreases,indicating that AngⅡ causes endothelial cell dysfunction.When Aloe-Emodin was administered,endothelial connexin was restored,cell membrane permeability decreased,and cell resistance increased,indicating that AloeEmodin has a protective effect on endothelial cell damage induced by AngⅡ,and as the drug concentration increased,the efficacy increased.(2)Study on the mechanism of Eloe-Emodin in the treatment of AngⅡ-induced hypertensive endothelial injury in mice:in vivo experimental results showed that the expression of NLRP3 protein was significantly higher in the heart and blood vessel endothelium of mice in the AngⅡ group,while the Aloe-Emodin group was significantly higher.The decreasing change indicates that the mechanism of Aloe-Emodin to protect endothelial cells may be closely related to the NLRP3 inflammasome.This study further found that Aloe-Emodin could significantly reduce the inflammatory factor IL-1β and HMGB1 levels in the serum of AngⅡ mice.In in vitro experiments,western blot and ELISA results are consistent with in vivo experiments.Aloe-Emodin significantly reduced the protein content of cle-caspase-1 and the release of IL-1β and HMGB1,indicating that AloeEmodin can inhibit the activation of NLRP3 inflammasome.Immunofluorescence colocalization proved that AngⅡ could significantly increase the co-localization of NLR3/Caspsel and NLRP3/ASC,while Aloe-Emodin could significantly reverse this change.After successfully knocking down the NLRP3 gene in endothelial cells,it was found that there was no significant change in endothelial tight junction protein ZO-1 and ZO-2,Cle-IL1β and HMGB1 in either the AngⅡ group or the Aloe-Emodin group.It shows that AloeEmodin does protect endothelial cells through the consistent NLRP3 inflammasome pathway.Interestingly,this study found that Aloe-Emodin could significantly reduce the content of NLRP3 protein.This study tested NLRP3 ubiquitination and found that Aloe-Emodin could promote NLRP3 ubiquitination.The experimental results of protease inhibitor MG-132 also further supported this point.Because Aloe-Emodin significantly increased the ubiquitination level of NLRP3,and after administration of MG-132,the NLRP3 protein content and Clecaspase-1 protein content were significantly increased compared with the AngⅡ and AloeEmodin group.Conclusion(1)Aloe-Emodin could reduce AngⅡ-induced hypertension in mice while protecting the connection function of endothelial cells.(2)Aloe-Emodin protects endothelial cells by inhibiting the formation and activation of NLRP3 inflammasomes induced by AngⅡ.(3)Aloe-emodin inhibits AngⅡ-induced endothelial NLRP3 inflammasome activation may be related to the promotion of NLRP3 protein ubiquitination... |
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