Hypaconite Pretects Endothelial Cells Against Damage Through Regulating High-mobility Group Box1

ObjectiveHigh-mobility group box1(HMGB1) is a nuclear non-histone structural protein, expression, translocation and release of which have a close relationship with cell damage. The protein could get translocation and release in the later period of cells injury with its acetylation. The accumulation of HMGBl in cytoplasm could overactivate cellular autophagy to induce cells damage. And the HMGB1proteins leak out to extracellular matrix may promote expression of NF-κB-mediated inflammatory factors such as TNF-a, VCAM-1, ICAM-1and ILs through RAGE or TLR4receptors. Meanwhile the protein in extracellular matrix could induce more HMGBl proteins to secrete and release, thus to development a regenerative loop in cells to trigger damage amplification. So, the translocation and release of HMGB1play critical roles in cells damage, and the protein is considered as damage-associated molecular pattern in many pathologic processes. In addition, due to the late occurrence and long-time maintain in cells injury, HMGBl is thought to be a promising target of many acute and severe diseases, so that get a good value to be researched.Traditional Chinese herb Fuzi, is thought to have effect on reviving yang for resuscitation and supplementing fire and Yang. It is always used to treat shocks in clinic. In clinical observation and animal experiment, evidence has been gathered that Shenfu injection and Sini Decoction which contain Aconite could protect animals’endothelial cells from damage, but the detail antioxidant function of Fuzi is still unclear. In this study, we used hydrogen peroxide and oxidized LDL (oxLDL) respectively to establish models of damaged endothelial cell, to explore the material basis of Fuzi in protecting endothelial cells from damage, and discuss the mechanism of its protective effect. Methods1. Used MTT assay to explore the effect of hydrogen peroxide and oxLDL on inducing damages of endothelial cells. And then Hypaconitine, Aconitine, Mesaconitine and Benzoylaconitine, all of which were main monomer component of traditional Chinese herb Fuzi, were used to treated endothelial cells stimulated with hydrogen peroxide or oxLDL, to explore which one(s) of those above were the active substance(s) of Fuzi in protecting endothelial cells from damage.2. Western blot analysis was employed to detect the effect of hydrogen peroxide and oxLDL on expression of activate caspase3. Thereafter, active substance of Fuzi was used to treat endothelial cells, and Western blot analysis was recruited to explore the effect of active substance in Fuzi on activate caspase3in damaged endothelial cells.3. ELISA and Western blot analyses were used to explore the effect of oxLDL and active substance in Fuzi on translocation and release of HMGB1in endothelial cells.4. Microarray analysis was used to screen the transcription factors could be regulated by active substance of Fuzi. And then we predict the target gene of Fuzi on the basis of its regulation function of HMGB1.5. QRT-PCR, Western blot analyses were recruited to explore the effect of active substance in Fuzi on transcription and expression of predicted target gene.6. We suppressed the effect of active substance in Fuzi on predicted target gene, and then used ELISA and Western blot analyses to ovserve whether it regulated translocation and release in this condition.7. The effect of active substance in Fuzi on regulating HMGB1was depressed, and then we used Western blot analysis to investigate the effect of active substance of Fuzi on expression of activate caspase3, which is a apoptosis-associated protein.Results1. Hypaconitine, one of substance in Fuzi, could improve cells viability of endothelial cells stimulated by hydrogen peroxide and oxLDL.2. Hypaconitine could down-regulate expression of activate caspase3in endothelial cells stimulated by hydrogen peroxide and oxLDL.3. Hypaconitine could prevent the cytoplasmic translocation and extracelluar release of HMGB1in oxLDL-stimulated endothelial cells.4. Hypactine could promote transcription and expression of HDAC3in oxLDL-stimulated endothelial cells.5. When HDAC3gene was silent, Hypaconitine would not suppress translocation and release of HMGB1any more.6. When translocation and release of HMGB1stayed in high level, Hypaconitine would not show its anti-apoptotic effect.Conclusion1. Hypaconitine is the active ingredient of Fuzi to protect endothelial cells from damage.2. Hypaconine prevents the cytoplasmic translocation and extracelluar release of HMGB1through promoting the expression of HDAC3.3. Hypaconitine protects endothelial cells from damage, through preventing the translocation and release of HMGB1.