Effect Of Neferine On LPS-induced Human Umbilical Vein Endothelial Cells Under Co-culture System

Objective:As a chronic vascular inflammatory disease,atherosclerosis(AS)has become a major disease that endangers human's health.Some study found that t he main cause of the formation and development of AS is that human body in fected by some microorganism,the inflammatory cytokines and acute phase prot ein expression was also increased,which caused by abnormal function of huma n vascular endothelial cells(HUVECs).Lipopolysaccheride(LPS)is a strong infla mmatory promoter of AS,which can be attached to HUVECs,and the interaction between HUVECs and smooth muscle cells(SMCs)leads to inflammatory syn drome.Neferine has many pharmacological effects,such as anti-arrhythmia,reduci ng blood fat,anti-inflammatory,scavenging oxygen free radical.In this experim ent,through the establishment of HUVECs-SMCs co-culture system to study the SMCs co-culture system HUVECs on LPS-induced damage secretion,proliferat ion and adhesion functions,while observing whether the NF-?B signaling path way involve in the AS,clarify whether Neferine involves in the regulation of i nflammatory damage by affecting the expression of VECs NF-?B signaling pat hway,providing new targets for the treatment of AS.Methods:Human vascular endothelial cells(HUVECs)were cultured,and observe the injuring condition of HUVECs after stimulating HUVECs using LPS,the c oncentration of LPS was selected according the degree of injury.In order to si mulate the environment in human body,we construct an indirect co-cultivating s ystem between HUVECs and SMCs by planting HUVECs in Transwell cell's u pper chamber and SMCs in lower chamber.CCK8 was used to detect the influe nce of Neferine in different concentration on LPS-induced VECs injury.Nitric o xide(NO)content was measured by Griess Reagent.The nitric-oxide synthase(N OS)activity was assessed by commercially available kits.A flow cytometry wit h double staining of Annexin and PI was used to detected the apoptosis rate o f HUVECs.Meanwhile,the protein levels of I?B-?,NF-?B p65 were detected by Western blotting,we use ELISA method to detect the expression level of TNF-a and ICAM-1,and their mRNA expression was assessed by Real-time quantitive PCR.Results:The inhibitory rate of HUVECs was suitable after 24h,which induced b y LPS in the concentration of 100ug·mL-1(P<0.05).The results of CCK8 assay and flow cytometry showed that Neferine could inhibit the apoptosis rate of H UVECs stimulated by LPS,and showed a certain concentration(P<0.05).Neferi ne also reversed the increase in NO content and total NOS(tNOS)and iNOS activity in HUVECS induced by LPS under co-culture conditions(P<0.05).The result of western blot show that the expression of NF-xB and p65 was decreas ed,while I?B-? was increased(P<0.05).ELISA and RT-PCR results showed that Neferine reduced the inflammatory infection of TNF-a and ICAM-1 secretion,a nd in a concentration dependent manner(P<0.05).Conclusion:The results show that Neferine has protective effect on HUVECs in duced by LPS.In the condition of co-cultivating system between HUVECs and SMCs,Neferine can regulate NO release and NOS activity,while inhibiting NF-?B signaling pathway and decreasing expression of TNF-a/ICAM-1 inflammatory cytokines.Eventually,inflammation was reduced,and Neferine play a positive rol e in anti-atherosclerosis.