Tanshinone Ⅱ A On Vascular Endothelial Damage Class Estrogen-like Protective Effect And Mechanism Research

Menopausal women are more susceptible to coronary heart disease than young women. Estrogen replacement therapy (ERT) became the focus of the medical profession. However, due to a series of side effects of estrogen replacement therapy, such as venous thrombosis, stroke, breast cancer, estrogen replacement therapy can not be used extensively. Salvia (Salvia miltiorrhiza) is one of the Chinese traditional medicines which can promote the circulation of blood and eliminate stasis. Tanshinone IIA, as one of primary fat-soluble ingredients of Salvia, can significantly inhibit the contraction of the coronary artery, improve coronary blood supply, prevent atherosclerosis formation and platelet aggregation, and repair endothelial damage.Preliminary study found that tanshinone IIA could strongly bind with estrogen receptor. We speculated that tanshinone IIA may play an estrogen-like effect in terms of biological activity. In our study, we first observed the relationship between the serum estrogen levels, lipid levels, sICAM-1and blood stasis syndrome (BSS) in postmenopausal women with coronary heart disease. With ox-LDL-induced endothelial cell damage model and ovariectomized apoE-/-mice atherosclerosis (AS) model, we investigated the impact of the Tanshinone IIA to the endothelial, vasomotor state, plaque stability, levels of serum estrogen and inflammatory factors, and observed the activation of the signal transduction pathway protein(ERK,MAPK, AKT), and explored whether the Vascular protective effect of Tanshinone IIA is similar as it of estrogen and the mechanism.This paper consists of three parts:literature review, clinical study and experimental research.1. Literature Review:①Estrogen and Atherosclerosis②Anti-atherosclerotic effect of Tanshinone IIA2. Clinical study:Relationship between serum estrogen levels and blood stasis syndrome in postmenopausal women with coronary heart diseaseObjective:To observe the difference of serum estrogen levels in postmenopausal women with coronary heart blood stasis syndrome and non-blood stasis syndrome.Methods:CHD patient were diagnosed according to the coronary heart disease diagnostic criteria developed by the American College of Cardiology and the American Heart Association in1999. Patient with BSS were diagnosed by the diagnostic criteria of BSS in1986. Select40healthy postmenopausal women,43postmenopausal female with CHD first visiting a doctor for the CHD,23patients with BSS,20patients with non-blood stasis. The serum lipids were determined in blood samples taken after patients’ admition by Beijing Anzhen Hospital. The levels of estrogen and sICAM-1were measured by HLISA.Results: Compared with the healthy control group, the levels of plasma TG. TC, sICAM-1in coronary heart disease group were all significantly incresed (P<0.05), E2were significantly decreased (P<0.05), the levels of E2of patients with BSS were decreased further(P>0.05), and there is an increasing trend of serum sICAM-1levels (P>0.05). Pearson correlation analysis showed that: there were negative significant correlations between serum E2levels and TC, sICAM-1levels in patient with coronary heart disease.Conclusion: The estrogen level of menopausal women with coronary heart disease is slightly lower than healthy menopausal women.3. Experimental study: Including the Cell experiments and Animal experiments two parts.Cell experiments: Effect of Tanshinones ⅡA on the injury of human umbilical vein endothelial cell induced by ox-LDLObjective: To explore the protective mechanism of tanshinone ⅡA on impaired endothelial cells and compare the effect with estrogen.Methods: HUVECs were cultured in vitro and ox-LDL l00μg/ml was added in all the groups except the normal group to manufacture oxidative damaged endothelial cell model. There are eight groups: normal control group (NOR), model group (MOD), estrogen group (E2,0.01), estrogen+ICT182780group (E2+ICO, E2:0.01μM,ICT:0.1μM). tanshinone ⅡA low-dose group (TLD,0.1), tanshinone ⅡA middle-dose group (TMD,1μM). tanshinone ⅡA high-dose group (THD,10μM), Tanshinone ⅡA+ICI182780group(TMD+ICI, TMD:1μM, ICI: O.luM). All the groups were cultured in the media with different drugs above for24hours, then we determined the cell activity of each group and took cell culture supernatant and the cells to detect NO, ET-1, NF-kappaB, AP-1, sICAM-1, E-selectin content by ELISA and cell p-p38, p-Akt, p-ERK1/2protein levels by Western-blot.Results:①Cell activity detection determined by MTT method: Compared with NOR group, the cell activity of MOD group’s cell was decreased significantly (P’<0.01); compared with MOD group, the cell activity of E2and Tanshinone IIA groups’ cell were increased significantly (P<0.01).②Biochemical parameters: Compared with NOR group, MOD group’s NO、ET-1、NF-κ B、AP-1、sICAM-1and E-selectin were increased significantly(P<0.01);compared with MOD group, the levels of NO、ET-1、NF-κ B、AP-1、sICAM-1and E-selectin of E2and Tanchinone ⅡA group were decreased significantly(P<0.01or P<0.05). and the effect was dose-dependent, E2+ICI and TMD+ICI group’s NO、ET-1、NF-κB、AP-1、sICAM-1and E-selectin were not decreased (P>0.05).③Related protein content:Compared with NOR group, the p-p38、p-ERK1/2、p-Akt protein content of MOD group were increased significantly(P<0.01or P<0.05); compared with MOD group, E2and Tanshinone ⅡA groups’ protein content were decreased (P<0.01or P<0.05), but E2+ICI and TMD+ICI groups’ protein content were no difference (P>0.05).Conclusion:Tanshinone IIA could play a estrogen-like protective role on the ox-LDL injured HUVECs by anti-oxidation, anti-inflammatory, regulation of endothelial function. Estrogen could bind with its nuclear receptor to achieve the non-transcribed effect to play a vascular protective role. The protective effect of Tanshinone IIA could be partly inhibited by estrogen receptor inhibitor.Animal experiments:Effect of Tanshinones ⅡA on vascular endothelial injury of ovariectomized atherosclerotic ApoE-/-mice.Objective:To investigate the Effect of Tanshinones IIA on vascular endothelial injury of ovariectomized atherosclerotic ApoE-/-mice and explore whether Tanshinone IIA play a estrogen-like anti-atherosclerotic role on blood vessels.Methods:A high-fat diet was given to the female ovariectomized apoE-/-mice to build the atherosclerotic model, and all the mice were divided into7groups:normal control group (NOR), model group (MOD), the estrogen group (E2,0.00013mg/g/d), the estrogen+estrogen receptor antagonist group(E2+ICI, fulvestrant injection,0.065mg/g/m), Tanshinone IIA low dose group (TLD,30mg/kg/d). Tanshinone IIA high dose group (THD,60mg/kg/d), Tanshinone IIA low-dose+estrogen receptor antagonist group(TLD+ICI, fulvestrant injection,0.065mg/g/m). The mice were given normal diet and high-fat diet respectively for12weeks, fasted for12hours, weighed, and removed the eye for blood serum, which was separated for ELISA test of E2, NO. ET-1, NF-κ B, sICAM-1indicators and biochemical testing of SOD, MDA.The0.5crn section of aortic arch from the openning was carried for HE and Oil Red O staining. The aortas section from opening to the bifurcation of the abdominal aorta were carried for Western-blot detection of protein p-p38,p-ERK1/2, p-Akt.Results:①Histopathology showed:Oil Red O:the part riched in lipid was red. In addtion to the NOR group, the other groups" aortas have AS plaques formation.②E2:Compared with the NOR group, MOD group’s serum E2levels was significantly lower (P<0.01).Compared with MOD group,E2and E2+ICI group’s E2was significantly higher (P <0.01),but TLD. THD and TLD+ICI group’s E2levels were no difference.(P<0.01).③NO and ET-1:Compared with NOR group, the level of NO in serum of MOD group was decreased significantly (P<0.05), while, ET-1in serum was increased (P<0.01),compared with MOD, E2and Tanshinone ⅡA groups’ NO were increased (P<0.05), ET-1were decreased (P<0.01), but there were no difference between MOD and E2+ICI、TLD+ICI groups (P>0.05).④Inflammatory and immune related cytokines: Compared with NOR group, The levels of NF-κ B、AP-1、sICAM-1、E-selectin in serum of MOD group were increased significantly (P<0.01); compared with MOD group,E2、TLD、THD group’s NF-κB、AP-1、sICAM-1、 E-selectin were decreased (P<0.01),but E2+ICI and TLD+ICI group’s NF-κB、AP-1、 sICAM-1、E-selectin were no difference (P<0.05).⑤SOD and MDA: Compared with NOR group, MOD group’s SOD content was decreased significantly(P<0.05), but MDA content was increased significantly(P<0.05); compared with MOD group,E2、TLD、THD group’s SOD content were increased significantly(P<0.01or P<0.05), MDA content were decreased significantly(P<0.05), E2+ICI and TLD+ICI group’s SOD and MDA were no difference(P<0.05).⑥Related protein expression:Compared with NOR group, the p-p38> p-ERK1/2、p-Akt protein content in mice aorta of MOD group were increased significantly(P<0.01or P<0.05); compared with MOD group, E2and Tanshinone ⅡA groups’ protein content were decreased (P<0.01or P<0.05), but E2+ICI group’s protein content were no dilTerence(P>0.05).Conclusion: Tanshinone IIA could play a estrogen-like endothelial protective role on the atherosclerosis of ovariectomized apoE-/-mice by anti-oxidation, anti-inflammatory. regulation of endothelial function. However, there is different between the mechanisms of Tanshinone ⅡA and estrogen.