| Objective:To study the effects of Huatan Huoxue Jiedu Recipe on the secretion of inflammatory factors,proliferation,migration,apoptosis,and phenotypic changes of RASMC induced by LPS and whether the mechanism is affected by MAPKs signaling pathway.Methods:1.Preparation of drug-containing serum:Sixteen SD Japanese whit e rabbits weighing 2.5±0.5 kg were randomly divided into four groups:rosuvastatin group(RS,2.5 mg/kg/d)and Huatan Huoxue Jiedu Recip e.Low-dose group(ZD,17.2mg/kg/d),Huatan Huoxue Jiedu Recipe hi gh-dose group(ZG,34.4mg/kg/d)and blank group(Control,equal volu me of normal saline)were continuously fed for 7 days,last time The blood was taken from the abdominal aorta for 3 hours,and the supe rnatant was filtered,inactivated,and stored in a-80? freezer.2.Experimental grouping:The experiment was divided into 5 groups:blank group,LPS group,LPS + rosuvastatin group,LPS + Huatan Huoxue Jiedu Recipe low-dose group,LPS + Huatan Huoxue Jiedu Recipe high dose group.3.MTT assay to detect the effect of LPS on proliferation of RASMC.4.MTT assay for the effect of Huatan Huoxue Jiedu Recipe on L PS-induced proliferation of RASMC.5.ELISA was used to detect the effect of Huatan Huoxue Jiedu Recipe on LPS-induced inflammatory cytokines and TIMP-1 expression in RASMC.6.Flow Cytometry Assays the Apoptosis of RASMC with Drug-con taining Serum.7.Transwell chamber was used to detect the effect of drug-containing serum on LPS-induced migration of RASMC.8.RT-PCR and Weston blotting were used to detect MAPKs signal ing pathway related genes and proteins(p38MAPK,ERK,JNK),major phenotypic marker genes and proteins(?-SM-actin,OPN)and related apoptotic genes and proteins(caspase-3,Bax,Bcl-2)expression level s in RASMC.Results:1.The effects of LPS on the growth and proliferation of RASMC:The cell viability of RASMC treated with LPS at concentrations of 0.01,0.1,0.5,1,5,and 10 ?g/ml was significantly increased at 6,12,24,and 48 h(P<0.05);100 ?g/ml The cell viability increased 6 h,12 h,24 h after LPS treatment(P<0.05),but cell viability decreased significantly after 48 h(P<0.05).The effect of LPS at a concentration of 0.5 ?g/ml for 24 h was the most significant(P<0.05).2.Effect of Huatan Huoxue Jiedu Recipe on the proliferative activity of RASMC under LPS:The effects of rosuvastatin and high-dose Huatan Huoxue Jiedu Recipeat 10%,20%,and 40%concentration can significantly inhibit the promotion of LPS at 24 and 48 hours(P<0.05),there was no significant difference between the two(P>0.05);10%concentration of Huatan Huoxue Jiedu Recipe low dose group for 24h and 20%,40%concentration of Huatan Huoxue Jiedu Recipe low dose The effect of LPS on proliferation was inhibited at 24 and 48 h(P<0.05),but it was lower in rosuvastatin group and high dose group of Huatan Huoxue Jiedu Recipe(P<0.05).3.Intervention Effects of Huatan Huoxue Jiedu Recipe on LPS-Indu ced Secretion of inflammatory Factors in RASMC:Compared with Con trol Group,LPS Group,TNF-?,IL-6,MCP-1,MMP-9 Levels and MMP-9/TIMP-1 was significantly increased and the TIMP-1 level was signi ficantly decreased(P<0.05).Compared with LPS group,rosuvastatin g roup,Huatan Huoxue Jiedu Recipe low dose group,Huatan Huoxue Ji eduRecipe high dose group TNF-?,IL-6,MCP-1,MMP-9 Levels and t he proportion of MMP-9/TIMP-1 were significantly decreased,and the TIMP-1 level was significantly increased(P<0.05).There was no signif icant difference in TNF-? levels between the rosuvastatin and the low-dose group of Huaqi Huoxue Jiedu Recipe group(P>0.05).The levels of TNF-? in the rosuvastatin and the low-dose group of Huatan Huo xue Jiedu Recipe group were higher that the low-dose Huoxue Huoxue Jiedu Recipe group(P<0.05);There was no significant difference in IL-6 levels between the rosuvastatin group,the low-dose group of Hu atan Huoxue Jiedu Recipe,and the high dose of Huatan Huoxue Jiedu Recipe(P>0.05).The level of MCP-1 between Huatan Huoxue Jiedu R ecipe high dose group was significantly lower than that of Huatan Hu oxue Jiedu Recipe low dose group(P<0.05).The MCP-1 level in rosu vastatin group was significantly lower than that of Huatan Huoxue Jie du Recipe low dose group and high dose group(P<0.05).There were n o significant differences in MMP-9 levels between the rosuvastatin gr oup and the low-dose group of Huatan Huoxue Jiedu Recipe(P>0.05).Huatan Huoxue Jiedu Recipe had high doses.The level of MMP-9 in the group was significantly lower than that in the rosuvastatin group(p<0.05).There was no significant difference in the level of MMP-9 between the low-dose group of Huatan Huoxue Jiedu Recipe and the high-dose group of Huatan Huoxue Jiedu Recipe(P>0.05).The level of TIMP-1 was significantly higher in rosuvastatin group than in low d ose group of Huatan Huoxue Jiedu Recipe and high dose group of Hu atan Huoxue Jiedu Recipe(P<0.05).The expression of TIMP-1 was si gnificantly lower in the high-dose group of Huatan Huoxue Jiedu Reci pe.Huatan Huoxue Jiedu Recipe low-dose group(P<0.05);MMP-9/TI MP-1 ratio between rosuvastatin group,Huatan Huoxue Jiedu Recipe 1 ow-dose group and Huatan Huoxue Jiedu Recipe high-dose group had Significant difference(P>0.05).4.Effect of Huatan Huoxue Jiedu Recipe on the apoptosis of RA SMC induced by LPS:Compared with blank group,the apoptosis rate of RASMC in LPS group was significantly decreased(P<0.05);comp ared with LPS group,rosuvastatin group The apoptotic rate of the pat ients in the low-dose group of Huatan Huoxue Jiedu Recipe,the high-dose group of Huatan Huoxue Jiedu Recipe group was significantly hi gher(P<0.05);There was no significant difference in apoptotic rate b etween high dose group of rosuvastatin group and Huatan Huoxue Jied u Recipe group(P>0.05),apoptotic rate between high dose group of r osuvastatin group and Huatan Huoxue Jiedu Recipe Significantly highe r than the low-dose group of Huatan Huoxue Jiedu Recipe(P<0.05).5.Effects of Huatan Huoxue Jiedu Recipe on the migration of RASMC induced by LPS:compared with the blank group,the migration ability of RASMC in LPS group was significantly increased(P<0.05);compared with LPS group,rosuvastatin group The migratory capacity of Huatan Jiexue Jiedu Recipe low-dose group,Huatan Huoxue Jiedu Recipe high-dose group RASMC significantly decreased(P<0.05);Rosuvastatin group and Huatan Huoxue Jiedu Recipe high-dose group There was no significant difference in the migration ability between the two groups(P>0.05).The migration ability of rosuvastatin and Huatan Huoxue Jiedu Recipe high dose group was significantly higher than that of Huatan Huoxue Jiedu Recipe low dose group(P<0.05).6.Effect of Huatan Huoxue Jiedu Recipe on phenotypic conversion of RASMC:Compared with blank group,the expression of ?-SM-acti n mRNA and protein in LPS group was decreased(P<0.05),and the e xpression of OPNmRNA and protein was increased.(P<0.05);Compare d with LPS group,rosuvastatin group,Huatan Huoxue Jiedu Recipe lo w dose group,Huatan Huoxue Jiedu Recipe high dose group a-SM-act inmRNA and protein Expression increased(P<0.05)and OPN mRNA a nd protein expression decreased(P<0.05);rosuvastatin group,Huatan Huoxue Jiedu Recipe high-dose group had no significant difference in inhibition of RASMC phenotypic changes(P>0.05).The phenotypic i nhibition of RASMC in low doses of Huatan Huoxue jiedu Recipe wa s weaker than that of rosuvastatin and high dose of Huatan Huoxue Ji edu Recipe(P<0.05).7.Effect of Huatani Huoxue Jiedu Recipe on the expression of ER k,p-38MAPK,JNKmRNA and p-ERK1/2,p-p38MAPK and p-JNK prote in in LPS-induced RASMC:Compared with blank group,LPS group E RK1/2 The expression levels of JNK,p-38MAPKmRNA and p-ERK1/2,p-p38MAPK and p-JNK protein were significantly increased(P<0.05).Compared with LPS group,ERK1/2,JNK,p-38MAPKmRNA and p-ER K1/2,p-p38MAPK,p-JNK in rosuvastatin group,low dose group of H uatan Huoxue Jiedu Recipe,and high dose group of Huatan Huoxue Ji edu Recipe The expression level of JNK protein was significantly dec reased(P<0.05);the expression levels of ERK,JNK,p38MAPKmRNA and p-ERK1/2,p-JNK protein in rosuvastatin group and high dose gro up of Huatan Huoxue Jiedu Recipe were not significant difference(P>0.05),ERK,JNK,p38MAPKmRNA and p-ERK1/2,p-JNK protein expr ession levels in the low dose group of Huatan Huoxue Jiedu Recipe were significantly higher than those in rosuvastatin group and Huatan Huoxue Jiedu Recipe(P<0.05),The expression of p-p38MAPK protein in the high-dose group of Huatan Huoxue Jiedu Recipe was significa ntly lower than that in the rosuvastatin group and the low-dose group of Huatan Huoxue Jiedu Decoction(P<0.05).Huayu Huoxue Jiedu Re cipe The p-p38MAPK protein level in the low dose group was signifi cantly higher than that in the rosuvastatin group(P<0.05).8.Effect of Huatan Huoxue Jiedu Recipe on the expression of Bax,caspase3,Bcl-2 mRNA and protein in RASM-induced RASMC:Comp ared with blank group,the expression of pro-apoptotic proteins Bax,c aspase3mRNA and protein decreased in LPS group(P<0.05),anti-apo ptotic protein Bcl-2mRNA and protein expression increased;compared with LPS group,rosuvastatin group,Huatan Huoxue Jiedu Recipe low-dose group,Huatan Huoxue Jiedu Recipe prescription high-dose group promote apoptosis The expression of Bax,caspase3 mRNA and protei n were increased(P<0.05),and the expression of anti-apoptotic protei n Bc1-2mRNA and protein was decreased(P<0.05).Conclusion:1.LPS can significantly promote the proliferation,migration,and phenotypic conversion of RASMC and induce RASMC to secrete TNF-?,IL-6,MCP-1,MMP-9 and reduce the expression of TIMP-1.2.Huatan huoxue Jiedu Recipe can significantly reduce the level of inflammatory cytokines secreted by LPS-induced RASMC and up-regulate the expression of TIMP-1,induce apoptosis of RASMC,inhibit the migration of RASMC and phenotype transformation.3.Huatan Huoxue Jiedu Recipe interferes with inflammatory response,inhibits the proliferation,migration,and phenotypic conversion of RASMC by inhibiting MAPKs signaling pathway.4.Huatan Huoxue Jiedu Recipe promote LPS-indused RASMC apop tosis may be through inhibition of ERK signaling pathway,down regula tion of Bcl-2,upregulation of Bax,caspase-3 achieved. |
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