Effects Of Notoginsenoside R1 On Atherosclerosis By Ang ? And Its Possible Mechanism

Objective:The aim of this study was to investigate the preventive and therapeutic effects of Notoginsenoside R1(NR1)on apolipoprotein E gene knockout(Apo E-/-)mice induced by angiotensin ?(Ang?)in vivo and in vitro.The mechanism of NR1 in the prevention and treatment of atherosclerosis at the cellular and genetic levels was explored by using the mitogen-activated protein kinase(MAPK)/ nuclear factor kappa-B(NF-?B)signaling pathway as a breakthrough point,providing a new experimental basis for targeted therapy of AS.Methods:1.Theoretical part:The paper reviewed the research progress between Ang? and AS;the correlation between Ang?-mediated MAPK/NF-kappa B signaling pathway and AS was analyzed in depth;combined with the theory of traditional Chinese medicine and the pharmacolo-gical mechanisms of NR1 on cardiovascular system,which provided a theoretical basis for the intervention of NR1 on AS by using MAPK/ NF-kappa B signaling pathway as therapeutic target.2.In vivo experiment:The replication method of Apo E-/-mouse AS model was induced by Ang?.The ALZET osmotic pump containing Ang? 1000ng/kg/min was implanted into the subcutaneous combined normal diet of mice for 4 weeks.Forty Apo E-/-male mice were randomly divided into two groups:Ang? group(control group)and NR1 intervention Ang? group(administration group)with 20 mice in each group.On the second day of modeling,the administration group was intraperitoneally injected with NR1 25mg/kg daily,and the control group was given daily intraperitoneal injection of saline of the same volume as NR1.(1)The body weight of mice was measured 1 week before operation and 1,2 and 4 weeks after operation,and the dose of NR1 was adjusted according to the change of body weight.(2)Kent non-invasive blood pressure monitoring system was used to monitor the changes of blood pressure in mice.(3)Serum levels of total cholesterol(TC),triglycerides(TG),high-density lipopro-tein-cholesterol(HDL-C)and low-density lipoprotein-cholesterol(LDL-C)in mice were measured by biochemical method.(4)Hematoxylin eosin staining(HE)was used to detect the aortic root plaque area and the aortic root and head arm necrosis center area.(5)Lipid deposition in mouse aortic plaque was measured by oil red O staining.(6)Picric Sirius red Staining was used to detect collagen fiber content in plaque.(7)The expression of a-SMA and CD68 in mice plaques were detected by immunohistochemistry.(8)Serum levels of tumor necrosis factor-a(TNF-a),interleukin-6(IL-6),interleu-kin-1 beta(IL-1b)and monocyte chemoattratant protein-1(MCP-1)were measured by enzymelinked immunosorbent assay.(9)Real-Time PCR was used to detect the expression of vascular inflammation-related genes(TNF-a?IL-6?IL-1b?MCP-1),intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)messenger ribonucleic acid(m RNA).(10)Western blot was used to detect the expression of I?B kinase(IKK),p-IKK,P65,p-P65,?B inhibitory protein(Inhibitor of ?B,I?B)and p-I?B protein in blood vessels.3.In vitro experiments:To investigate the effect of NR1 on the Ang? pathway itself,we selected MOVAS cells(mouse aortic smooth muscle cell)as a carrier for the action of NR1.Ang?-induced MOVAS cells were used to establish a vascular smooth muscle cell(VSMC)proliferation model to simulate the role of VSMC in AS.Movas cells were divided into four groups(control group,NR1 group,Ang? group,Ang?+NR1 group).Ang? stimulation dose was 5?M and NR1 treatment concentration was 50?M.(1)The proliferation activity of each group of cells was measured by cell counting kit-8(CCK-8).(2)DNA synthesis in cell proliferation was detected by 5-bromodeoxyuridine(Brd U)assay.(3)Transwell chambers were used to detect the longitudenal migration ability of each group of cells.(4)The lateral migration ability of each group was tested by scratch healing test.(5)Western blot was used to detect the levels of angiotensin type 1 receptor(AT1R),angiotensin type 2 receptor(AT2R)and activetion of MAPKs(ERK,JNK,P38MARK)pathways in Ang?group and Ang?+NR1 group.Results:1.In vivo experiment: Ang? subcutaneously induced Apo E-/-mice for 4 weeks and successfully established an AS model.(1)Comparison of body weight changes:There was no significant difference in body weight between the two groups(P>0.05).(2)Comparison of blood pressure:The systolic blood pressure(SBP)level of the two groups before treatment was comparable(P>0.05),and the SBP was significantly increased at 2 and 4 weeks after the pumping of Ang?,but there was no significant difference between the groups(P>0.05).(3)Comparison of blood lipid indexes:There was no signifycant difference in TC,TG,HDL-C and LDL-C between the two groups before treatment(P>0.05).After treatment:Compared with the same group,the serum TG and LDL-C indexes of the drug-administered group were significantly improved(P<0.01 or P<0.05);compared with the control group,the drug-administered group was superior to the control group in reducing serum TG and LDL-C(P<0.01).(4)HE staining: Compared with the control group,the administration group signifycantly reduced the AS plaque area in the aortic root of the mouse(P<0.01),and significantly reduced the unit necrosis center area of the aortic root and the brachiocephalic trunk(P<0.01).(5)Oil red O staining:Compared with the control group,the drug-administered group significantly reduced lipid accumulation in mouse aortic plaques(P<0.05).(6)PSR staining:Compared with the control group,the drug-administered group significantly inhibited the production of collagen fibers in the plaque.(7)Immunofluorescence assay: Compared with the control group,the administration group signifycantly reduced the infiltration of macrophages in the aortic wall of the mouse;increased the content of SMC in the plaque.(8)Serological indicators:Compared with the control group,serum levels of TNF-a,IL-6,IL-1?and MCP-1 were improved in the drug-administered group(P<0.05 or P<0.01).(9)Real-Time PCR:Compared with the control group,the administration group significantly reduced the expression of TNF-a,IL-6,IL-1?,MCP-1,ICAM-1 and VCAM-1 m RNA in the blood vessels(P<0.01 or P<0.05).(10)Western blot:Compared with the control group,the administration group could significantly increase the level of I-kappa B protein(P<0.01),signifycantly inhibit the phosphorylation of IKK and I-kappa B(P<0.01).2.In vitro experiments:Ang? induced Movas cells to success-fully establish VSMC proliferation model.(1)CCK-8 experiment:Compared with the control group,there was no significant difference in the absorbance values measured at 450 nm wavelength in NR1 group at different time periods(P>0.05),and the proliferative effect of Ang? group persisted with the prolongation of action time(12-60 h)(P<0.01).When Ang?+NR1 acted for 12 hours,the inhibition of proliferation of NR1 was obvious,and the absorbance at 12,24,48 and 60 hours had significant difference(P<0.05 or P<0.01).(2)Brd U experiment:Compared with the control group,the absorbance of NR1 group at 450 nm wavelength had no significant difference(P>0.05);the absorbance of Ang?group increased significantly(P<0.01).Compared with Ang?group,the absorbance of Ang?+NR1 group decreased significantly(P<0.01).(3)Transwell experiment:Compared with the control group,there was no significant differrence in the number of cells passing through the ventricular membrane in NR1 group(P>0.05),while the number of cells passing through the ventricular membrane in Ang? group increased signifycantly(P<0.01).Compared with Ang?group,the number of cells passing through the ventricular membranes in Ang?+ NR1 group decreased significantly(P<0.01).(4)Cell scratch test:Compared with Control group,the healing rate of NR1 group had no significant difference(P>0.05),while that of Ang?group decreased signifycantly(P<0.01).Compared with Ang?group,the repair rate of cell damage in Ang?+NR1 group was significantly higher(P<0.01).(5)Western blot method: Compared with Ang?group,AT1 R level in Ang?+NR1 group decreased significantly(P<0.05),but AT2 R level had no difference(P>0.05).Compared with Ang?group,the phosphorylation levels of ERK,P38 and JNK in Ang?+NR1 group were signifycantly inhibited(P<0.01).Conclusion:1.The model of atherosclerosis was successfully established in Apo E-/-mice induced by Ang? for 4 weeks.2.Panax notoginseng saponin R1 has a definite effect on anti-AS plaque induced by Ang? in Apoe-/-mice.3.The anti-AS effect of Panax notoginseng saponin R1 was related to increasing the content of smooth muscle cells in plaque and reducing macrophage aggregation.4.Panax notoginseng saponin R1 has the effect of reducing blood lipid in atherosclerotic mice,and could prevent and treat diseases by reducing the risk factors of AS.5.The anti-AS plaque effect of notoginsenoside R1 was not only related to the reduction of inflammatory factors in the circulation,but also significantly inhibited the proliferation and migration of Movas cells induced by Ang?,and inhibited the activation of MAPKs(p-ERK,p-P38 MARK,p-JNK)by down-regulating AT1 R.Furthermore,it inhibited the activation of NF-?B signaling pathway in aortic wall tissue,and down-regulated the expression of downstream cytokines(TNF-?,IL-6,IL-1?)chemokines(MCP-1)and adhesion molecules(ICAM-1 and VCAM-1),thereby delaying the pathological process of AS.