Study On The Anti-Atherosclerosis Mechanism Of Buyang Huanwu Decotion Based On The Cross-Dialogue Of PI3K And TLR4 Signal Transduction Pathway

Objective:In this experimental study,the effects of Buyang Huanwu Decoction(补阳还五汤)containing drug serum on the cross-talk between TLR4/NF-κB and PI3K/AKT/NF-κB signaling pathways were investigated,and the mechanism and target of the anti-atherosclerotic effect of Buyang Huanwu Decoction were explored.Methods:1.Fifteen SD male rats were divided into a blank group and a Buyang Huanwu Decoction group.The blank group was gavaged with saline for 7 days,and the Buyang Huanwu Decoction group was gavaged with Buyang Huanwu Decoction for 7 days.Blood collection was performed in the abdominal aorta on day 8 to produce a blank serum and a Buyang Huanwu Decoction containing drug serum.2.The proliferative activity of different concentrations of lipopolysaccharide and different concentrations of Buyang Huanwu Decoction containing drug serum on human umbilical vein endothelial cells was determined by CCK-8 method,and the optimal concentrations of lipopolysaccharide and drug were screened.3.Well-grown human umbilical vein endothelial cells were selected and divided into7 groups,namely,blank group,model group,Chinese medicine group,LY group,TAK group,LY+Chinese medicine group and TAK+Chinese medicine group.The blank group was pretreated with 10% blank serum for 24h;the model group was pretreated with 10%blank serum for 2h and then treated with 1μg/ml lipopolysaccharide for 24h;the Chinese medicine group was pretreated with 10% Buyang Huanwu Decoction containing drug serum for 2h and then treated with 1μg/ml lipopolysaccharide for 24h;the LY group was pretreated with 10μM LY294002 for 2h,then pretreated with 10% blank serum for 2h,then treated with 1μg/ml LPS for 24h;the TAK group was pretreated with5μM TAK242 for 2h,then pretreated with 10% blank serum for 2h and then treated with1μg/ml LPS for 24h;the LY+Chinese medicine group was pretreated with 10 u M LY294002 for 2h,then pretreated with 10% Buyang Huanwu Decoction containing drug serum for 2h,and then treated with 1μg/ml lipopolysaccharide for 24h;the TAK+Chinese medicine group was pretreated with 5u M TAK242 for 2h,then pretreated with 10% Buyang Huanwu Decoction containing drug serum for 2h,and then treated with 1μg/ml lipopolysaccharide for 24 h.At the end of treatment in the thermostat,the cells were collected and proteins were extracted,and the protein expression of TLR4,P-PI3 K,P-AKT,NF-κB p65,MMP-9,ICAM-1,and TNF-α was determined by Western blot.Results:1.The results of CCK-8 method showed that the cell proliferation rate increased with the increase of lipopolysaccharide concentration,and human umbilical vein endothelial cells proliferated when the lipopolysaccharide concentration reached 1 μg/ml or 10 μg/ml(P < 0.05).2.The results of CCK-8 method showed that when 10% concentration of Buyang Huanwu Decoction containing drug serum significantly promoted the proliferation of human umbilical vein endothelial cells(P < 0.01).3.The results of the Western blot method showed that:1)Compared with the blank group,the protein expression of TLR4,p-PI3 K,p-AKT,NF-κB p65,MMP-9,ICAM-1,and TNF-α was significantly increased in the model group(P<0.01).2)Compared with the model group,the protein expression of TLR4,p-PI3 K,p-AKT,NF-κB p65,MMP-9,ICAM-1,and TNF-α was significantly lower in the Chinese medicine,LY+Chinese medicine,and TAK+Chinese medicine groups(P<0.01).3)After administration of LY294002,the expression of p-PI3 K,p-AKT,NF-κB p65,TNF-α,ICAM-1 and MMP-9 proteins were significantly lower in the LY group compared with the model group(P<0.01),but there was no significant difference in the expression of the above proteins in the TAK242 group(P>0.05).4)After administration of TAK242,the expression of TLR4,NF-κB p65,TNF-α,ICAM-1 and MMP-9 proteins were significantly lower in the TAK group compared with the model group(P<0.01),but there was no significant difference in the expression of the above proteins in the LY group(P>0.05).5)The expression of NF-κB p65,TNF-α,ICAM-1 and MMP-9 protein were significantly lower in the LY+Chinese medicine group and TAK+Chinese medicine group compared with the Chinese medicine group(P<0.01).6)There were no significant differences in NF-κB p65,TNF-α,ICAM-1 and MMP-9 protein expression in the TAK+Chinese medicine group compared with the LY+Chinese medicine group(P>0.05).Conclusion:1.Buyang Huanwu Decoction containing drug serum is used to inhibit the expression of NF-k B and downstream TNF-α,ICAM-1 and MMP-9 through TLR4/NF-κB and PI3K/AKT/NF-κB signaling pathways,thus achieving anti-atherosclerotic effects.2.The effects of Buyang Huanwu Decoction containing drug serum on TLR4/NF-κB and PI3K/AKT/NF-κB signaling pathways were essentially the same.