The Mechanism Of Danshensu Against Atheroscleroticendothelial Cell Injury In Mice Through The MAPKs Pathway

Background:Atherosclerosis(AS)is the main cause of cardiovascular and cerebrovascular diseases,vascular endothelial dysfunction is the initial step of AS.oxidative low-density lipoprotein(ox-LDL)can cause changes in the structure and function of vascular endothelial cells.Based on the previous experimental study,Dansen sodium(DSS),an effective active ingredient of Salvia miltiorrhiza,was used to study the mechanism of relieving vascular endothelial injury and further expand its pharmacological mechanism for the treatment of anti-AS in traditional Chinese medicine Prevent AS from finding new targets.Objectives:In this study,To investigate the protective effect and possible mechanism of DSS on vascular endothelial cell injury induced by ox-LDL in atherosclerotic mice from animal and cell level.Methods:(1)Animal experimentsSPF grade 8 weeks old male ApoE gene knockout(Apo E-/-)mice 50,were used to establish the model of AS vascular injury induced by human serum low density lipoprotein(LDL).The mice were randomly divided into 5 groups(10 rats in each group):① control group,②model group,③low dose DSS group(DSS-L),④middle dose DSS group(DSS-M),⑤high dose DSS group(DSS-H),②,③,④,⑤ group were given ox-LDL 50 mg/L intraperitoneal injection once a day;The mice in group ① were given equal doses of normal saline,weekly body weight once,the experimental period of 16 weeks.(2)Cell experimentsDSS concentration gradient group:HUVEC was treated with DSS at concentration of 0,50,100,200 μg · L-1 for 24 h.DSS time gradient group:HUVEC was treated with 100μg · L-1 DSS for 0,6,12,24 and 48 h,respectively.Drug intervention group:HUVEC was inoculated into six-well plate according to certain concentration.①Control group:HUVEC was incubated with DMEM for 24 hours;②Model group:HUVEC was incubated with ox-LDL containing the appropriate concentration for 24 hours;③ox-LDL was incubated with DSS group;④ox-LDL group;⑤ox-LDL+SB203580;⑥ox-LDL+SP600125。Results:(1)Blood lipid detect in mice indicated that the levels of serum TG,TC and LDL-C in model group were significantly higher than those in control group(P<0.05).HDL-C was higher in drugs group than that in model group.Compared with model group,serum TC,TG and LDL-C of DSS-M group were significantly lower than those of model group(P<0.05).(2)Morphological observation of the mouse aorta AS plaque:The model group had obvious AS plaque formation compared with the control group,and DSS could inhibit the formation of AS plaque in a dose-dependent manner(P<0.05).(3)The expression of LDL in the aorta of the mouse aorta was significantly lower than that in the DSS-L group(P<0.05).The expression of LDL in the DSS-H group was significantly lower than that in the DSS-L group(P<0.05).(4)Mouse aorta LDL protein expression:Compared with model group,DSS-M and DSS-H groups significantly inhibited LDL protein expression(P<0.05).(5)The serum ET-1 test results showed:Compared with the control group,the plasma ET-1 level in the model group was significantly higher than that in the control group(P<0.05),and the DSS-M group was significantly lower than the model group(P<0.05).(6)Effects of ox-LDL on HUVEC Proliferation:Concentration gradient group:Compared with model group,25,50 and 100 mg/L ox-LDL groups could significantly inhibit the proliferation of HUVEC(P<0.05),and there was no significant difference between 25,50 and 100 mg/L ox-LDL group(P>0.05).Time gradient group:Compared with the control group,50 mg/L ox-LDL could inhibit the proliferation of HUVEC at 24 h(P<0.05).(7)Effects of ox-LDL on apoptosis of HUVEC:Concentration gradient group:Compared with model group,50 and 100 mg/L ox-LDL could significantly increase the early and late apoptotic rates(P = 0.00),and there was no significant difference between the two groups(P>0.05).Time gradient group:50 mg/L ox-LDL increased the apoptotic rate of early after 12 and 24 h(P = 0.001).(8)Effects of ox-LDL on the expression of p-p38 MAPK protein in HUVEC:Concentration gradient group:100 mg/L ox-LDL reached a peak at 24 h(P ＝0.00).Time gradient group:50 mg/L ox-LDL could increase the expression of p-p38 MAPK protein in HUVEC at 3h and 6h(P<0.05).(9)Effects of ox-LDL on p-JNK protein expression in HUVEC:Concentration gradient group:25 mg/L ox-LDL could significantly increase the expression of p-JNK protein in HUVEC after 12 h(P = 0.00).Time gradient group:50 mg/L ox-LDL could increase the expression of p-JNK protein in HUVEC at 3 and 6 hours(P<0.05).There was no significant difference between the two groups(P>0.05).(10)Effects of DSS on the expression of p-p38 MAPK and p-JNK protein in HUVEC cells induced by ox-LDL:Compared with model group,DSS-M and DSS-H groups could significantly inhibit the expression of p-p38 MAPK and p-JNK protein in HUVEC(P = 0.00).There was no significant difference between the two groups(P>0.05).(11)Effects of DSS on ox-LDL on the secretion of TNF-α and IL-6 by HUVEC:Compared with the model group,the level of IL-6 secreted by HUVEC was significantly lower in HUVEC treated with 25,200 mg/L DSS for 24 h(P<0.05).and there was no significant difference between the two groups(P>0.05).50,200mg/L DSS pretreatment HUVEC decreased the secretion of TNF-a in HUVEC(P<0.05)and there was no significant difference between the two groups(P>0.05).(12)Effects of DSS and MAPKs pathway inhibitors on the proliferation of HUVEC induced by ox-LDL:Compared with model group,100 mg/L DSS group could reduce the inhibitory effect of ox-LDL on HUVEC proliferation(P =0.00),SP600125 and SB203580 could reduce the inhibition of ox-LDL on HUVEC proliferation(P<0.05).(13)Effect of DSS and MAPKs pathway inhibitor on the apoptosis of HUVEC induced by ox-LDL:Compared with the model group,50,100 mg/L DSS could significantly reduce the early and late apoptotic rate(P = 0.00),There was no significant difference between the two groups(P>0.05),SP600125 and SB203580 can reduce the early and late apoptotic rate(P = 0.001).Conclusions:Animal experiments confirmed that DSS can delay the development of AS by lowering blood fat,ET-1,reducing IL-6,TNF-α,inhibiting AS plaque formation and down-regulating LDL expression.Cell experiments confirmed that ox-LDL can inhibit HUVEC proliferation and promote its apoptosis,Can induce HUVEC secretion of inflammatory factors,The results showed that ox-LDL could inhibit the proliferation of HUVEC and promote the apoptosis of HUVEC,and could induce the secretion of inflammatory factors in HUVEC.DSS could exert anti-AS effect by increasing the proliferation of HUVEC,decreasing the apoptosis of HUVEC and the secretion of inflammatory factors.DSS can play an anti-AS role by increasing HUVEC proliferation,decreasing HUVEC apoptosis and secretion of inflammatory factors,and its mechanism may be related to inhibition of MAPKs signaling pathway.