Study On The Role Of ROS/NF-κB/NLRP3 Signaling Pathway In The Damage Of Vascular Endothelial Cells Induced By AngⅡ And The Protective Effect Of Qiqilian Capsule

Objective:In this study,the protective mechanism of Qiqilian Capsule on HUVECs was explored by establishing a model of HUVECs injury induced by AngⅡand using the ROS/NF-κB/NLRP3 signal pathway as a target to clarify the possible molecular mechanism of antihypertensive.And therefore,scientific basis for Qiqilian capsules’further clinical application was provided.Methods:1.Establish vascular endothelial cell inflammation injury model in hypertensive environment.Taking HUVECs as research objects,the expression of NLRP3,ASC and Caspase-1 in each group of cells was detected by Western blot with different concentrations of AngⅡsolutions of 0,10^-5,10^-7,10^-9and 10^-11mol/L being selected,and HUVECs being stimulated at different times for 0,3,6,12,24,and 48 hours.2.Explore the regulatory effect of AngⅡon the ROS/NF-κB/NLRP3signaling pathway by using HUVECs as the research object.After HUVECs hypertension model which was intervened by DPI,PDTC,MCC950,DPI+PDTC separately was established,the apoptosis of each group and ROS generation were detected by flow cytometry;the expression of NLRP3,ASC,Caspase-1,NF-κB protein in each group of cells was detected by Western blot;and the expression of IL-1βand IL-18 was detected by ELISA.3.Explore the protective mechanism of Qiqilian capsule on HUVECs with ROS/NF-κB/NLRP3 signal pathway as the target through HUVECs injury model induced by AngⅡ.After HUVECs hypertension models which was intervened by Qiqilian Capsules containing high-,medium-and low-dose groups separately was established,cell apoptosis and ROS production in each group was detected by flow cytometry;the expression of NLRP3,ASC,Caspase-1,NF-κB protein in each group of cells was detected by Western blot;the expression of IL-1βand IL-18 was detected by ELISA.Results:1.Imitate vascular endothelial cell inflammation injury model in simulated hypertension environment.1 The protein expression of NLRP3,ASC and Caspase-1 increased the most in HUVECs when the concentration of AngⅡsolution was 10^-7mol/L.2 The protein expression of NLRP3,ASC and Caspase-1 increased the most in HUVECs when the optimal intervention concentration was used to stimulate HUVECs,which lasted for 24 hours.2.Explore the regulatory effect of AngⅡon the ROS/NF-κB/NLRP3signaling pathway by using HUVECs as the research object.（1）Apoptosis can be caused by intervention of HUVECs with AngⅡ;however,compared with the model group,the apoptosis of HUVECs（P<0.01）can be reduced by intervention of DPI,PDTC,MCC950,DPI+PDTC.It is suggested that vascular endothelial cells can be protected by inhibiting ROS generation or（and）NF-κB protein expression and NLRP3 inflammatory corpuscle activity.（2）Activation of ROS/NF-κB/NLRP3 signaling pathway can be induced through intervention of HUVECs with AngⅡ.ROS generation or（and）NF-κB protein expression and NLRP3 inflammatory body activity can be inhibited respectively.Compared with the model group,the expression of NLRP3 inflammatory body-related proteins NLRP3,ASC,and Caspase-1（P<0.01）can be inhibited and the expression of inflammatory factors IL-1βand IL-18 downstream of ROS/NF-κB/NLRP3 signaling pathway（P<0.01）can be downregulated in each administration group.It is suggested that HUVECs injury can be induced by AngⅡ,which may be caused by promoting ROS generation and NF-κB protein upregulation in the body,and activating NLRP3 inflammatory bodies,thereby up-regulating the inflammatory factors IL-1βand IL-18 downstream of the ROS/NF-κB/NLRP3 signaling pathway Expression.3.Explore the protective mechanism of Qiqilian capsule on HUVECs with ROS/NF-κB/NLRP3 signal pathway as the target by using HUVECs injury model induced by AngⅡ.（1）Compared with the model group,HUVECs apoptosis（P<0.01）can be reduced in the high,medium and low Qiqilian capsules groups,which suggested that vascular endothelial cells can be protected by Qiqilian capsules.（2）Compared with the model group,the production of ROS（P<0.01）was reduced,the expression of NF-κB protein（P<0.01）was down-regulated,the expression of NLRP3 inflammatory bodies’related protein NLRP3,ASC,and Caspase-1 were inhibited,and the expression of inflammatory factors IL-1β,IL-18 downstream of the ROS/NF-κB/NLRP3signaling pathway（P<0.01）was down-regulated in the high,medium and low Qiqilian capsules groups.It is suggested that vascular endothelial cells may be protected by Qiqilian capsule through inhibiting ROS expression,down-regulating NF-κB protein expression,inhibiting NLRP3 inflammatory corpuscle activity,and down-regulating the expression of inflammatory factors IL-1βand IL-18 downstream of this pathway.Conclusion:1.AngⅡhas a damaging effect on HUVECs.The best intervention concentration is 10^-7mol/L,and the best intervention time is 24 hours.2.AngⅡmay up-regulate the expression of inflammatory factors IL-1βand IL-18 downstream of this pathway by activating the ROS/NF-κB/NLRP3 inflammatory corpuscle pathway,and therfore induce vascular endothelial cell injury.3.Vascular endothelial cells can be protected by Qiqilian capsule protects through inhibiting the activation of the ROS/NF-κB/NLRP3inflammatory corpuscle pathway and reducing the expression of inflammatory factors IL-1βand IL-18 downstream of this pathway.