Effect Of Yishenhuoxue Capsule On The Expression Of Inflammatory Factors In Rabbit Atherosclerosis Induced By Hyperhomocysteinemia

BackgroundAtherosclerosis(AS)is a cardiovascular disease which threatens the human health.It has been confirmed that hyperhomocysteinemia(Hhcy)is an independent risk factor for atherosclerosis in the coronary,cerebral,and peripheral vasculature. Lowering plasma Hcy levels and reverse the Hhcy-induced pathological changes are recognized as important mechanisms of drugs targeting atherogenesisAS is a chronic inflammatory process which refers to the damaged endothelial cells,mononuclear macrophages,smooth muscle cells,and amounts of cell factors and growth factors.Adhesion molecules,chemotatic factors and growth factors play important roles in the AS.Recently,the stimulatory effect of Hcy on the expression of inflammatory factors has been suggested to play an important role in Hcy-mediated AS.Hcy could activate nuclear factor-kappa B(NF-κB),upregulate the expression of inflammatory factors such as intercellular adhesion molecule-1(ICAM-1),monocyte chemoattractant protein-1(MCP-1),transformation growth factor-β₁(TGF-β₁)and matrix metalloproteinase-9(MMP-9)in the vascular endothelial cells,smooth muscle cells and monocytes,and increase the monocyte adhesion to aortic endothelium, promote the proliferation of smooth muscel cells and collogen synthesis.Yi-Shen-Huo-Xue-Capsule(YC),an extract of traditional Chinese herbal medicine,has been used as an anti-Hhcy agent for years in clinic.In this study,the Hhcy and AS model were induced by high methionine diet in rabbits,folic acid,VitB₆, VitB₁₂were indicated as the positive drug,the changes of the pathology and the ultrastructure of arterial media were observed,the variation of ICAM-1,MCP-1, TGF-β₁,MMP-9 mRNA and protein expression was detected by real-time PCR and Immunohistochemistry,the protein expression of the inhibitor of NF-κB(IκB-a)and the nuclear translocation of NF-κB were detected by Western blot and Immunohistochemistry.The possible mechanisms of YC in inhibiting vascular inflammatory in Hhcy was investigated,which could provide scientific evidence for the treatment of Hhcy and AS in clinic.AimTo investigate the possible mechanisms of YC in preventing and treating Hhcy and AS through observing the plasma Hey levels,changes of the morphology and the ultrastructure and detecting the variation of ICAM-1,MCP-1,TGF-β1 and MMP-9 mRNA and protein expression and NF-κB activation.Methods1.Experimental animals:2～3 months old male New Zealand White rabbits weighing 2.0-2.5 kg.2.Model:Hhcy and AS rabbit model was induced by feeding high methionine diet(which include 2%methionine)for 5 months after the balloon injury in abdominal aorta.3.Animals grouping:56 male New Zealand White rabbits were randomly divided into 7 groups(n=8):normal group,model group,preventive group,low dose YC group,middle dose YC group,high dose YC group and western medicine group (folic acid,VitB₁₂,VitB₆).4.Detection contents:(1)Measurement of the plasma Hey concentration by high performance liquid chromatography;(2)Observation of the pathological structure in aortic wall by HE staining;(3)Measurement of the morphological index of aortic wall: the intimal thickness(IT),medial thickness(MT)intimal area(IA),and ratio of intimal area to medial area(IA/MA);(4)Ultrastructural observation of the VSMCs in the media under transmission electron microscope;(5)Analysis of ICAM-1,MCP-1, TGF-β1 and MMP-9 mRNA expression by real-time PCR;(6)Analysis of ICAM-1, MCP-1,TGF-β₁ and MMP-9 protein expression by Immunohistochemistry.(7) Analysis of NF-κB activation:the protein experesion of IκB-a by Western blot and the nuclear expression of NF-κB by Immunohistochemistry.Results1.Comparison of plasma Hcy concentrationThe plasma Hcy concentration in the model group was significantly higher than that in the normal group(P＜0.01);After treatment with YC and western medicine,the Hcy concentration in each group was decreased in a different degree compared with the model group,and among those treatment groups,the preventive group had the lowest level of Hcy in plasma(P＜0.01).The Hcy concentration in the preventive group,the high dose YC group was lower than that in the western medicine group (P＜0.05);No significant difference was shown in the low dose YC group and middle dose YC group compared with the western medicine group(P＞0.05).2.Observation of sections of aortic wall stained with HEThe structure of the intima,the media and the adventitia of aortic wail was very clear.No thickening and no lipidoses were shown in the normal group rabbits.The VSMCs in media were shown a fusiform shape.The nucleus were stained in blue and arranged regularly as concentric circles.The thickened intima and foam cell aggregation were shown obviously and in the model group.The arrangement of its VSMCs was disordered,and a large amount of nucleus was shown in the unit area. The intima was shown slightly thickened in the YC and western medicine treated groups.The arrangement of their VSMCs was slightly disordered,and the amount of nucleus shown in the unit area in these four groups was smaller than that in the model group.3.Comparison of morphological index of aortic wall(1)IT:IT was increased significantly in the model group compared with the normal group(P＜0.01).After treatment with YC and western medicine,IT in each group was decreased in a different degree compared with the model group,IT was shown a more significant decrease in the preventive group and the high dose YC group compared with that in the model group(P＜0.01),however,no significant difference was shown in the low dose YC group and the western medicine group compared with the model group(P＞0.05).IT in the preventive group and the high dose YC group was lower than that in the western medicine group(P＜0.05).(2)MT:MT was increased in different degree in the model group and all treatment groups compared with the normal group,but no significant difference was shown in comparison among these seven experimental groups(all P＞0.05).(3)IA:IA was increased significantly in the model group compared with the normal group(P＜0.01).After treatment with YC and western medicine,IA in each group was decreased in a different degree compared with the model group,IA was shown a more significant decrease in the preventive group and the high dose YC group compared with that in the model group(P＜0.01),however,no significant difference was shown in the western medicine group compared with the model group (P＞0.05).IA in the preventive group was lower than that in the western medicine group(P＜0.05).(4)IA/MA:The ratio of IA/MA was significantly higher in the model group than than in the normal group(P＜0.01).The ratio of IA/MA was decreased in a different degree in each treatment group compared with the model group(all P＜0.05).The ratio of IA/MA in the preventive group and the high dose YC group was lower than that in the western medicine group(P＜0.05).4.Ultrastructural observation of the VSMCs in the aortic mediaIn the normal group,the VSMCs exhibited typical characteristics of contractile phenotype in the aortic media.The VSMCs were shown the fusiform shape with the presence of abundant myofilaments,normally arranged dense areas and dense bodies and a small quantity of mitochondria and endoplasmic reticulum in the normal rabbits. In the model group,the migration of VSMCs from media to endothelium was observed.The VSMCs appeared typical characteristics of synthetic phenotype,with depletion or decrease of myofilaments,swollen mitochondria and increase of cellular organelles such as rough endoplasmic reticulum,ribosome,as well as apparato reticulare.However,these changes were ameliorated when treated with YC.The mitochondria with slight swelling,dense areas,dense bodies and myofilaments were observed.No significant amelioration was shown in the VSMCs of western medicine group rabbits.5.Comparison of expression of ICAM-1,MCP-1,TGF-β₁ and MMP-9 mRNA in aortic wall(1)Comparison of expression of ICAM-1 mRNA:The expression of ICAM-1 mRNA in the model group was higher than that in the normal group(P＜0.01).After treatment with YC and western medicine,ICAM-1 mRNA expression in each group was decreased in a different degree compared with the model group,and among those treatment groups,the preventive group had the lowest level of ICAM-1 mRNA expression(P＜0.01).No significant difference was shown in comparison between the low dose YC group and the model group(P＞0.05).The expression of ICAM-1 mRNA was decreased significantly in the preventive group,the middle dose YC group,the high dose YC group and the western medicine group compared with that in the model group(all P＜0.05).The expression of ICAM-1 mRNA in the preventive group and the high dose YC group were lower than that in the western medicine group(P＜0.05).(2)Comparison of expression of MCP-1 mRNA:The expression of MCP-1 mRNA in the model group was higher than that in the normal group(P＜0.01).The expression of MCP-1 mRNA was decreased significantly in the YC treatment groups and the western medicine group compared with that in the model group(all P＜0.05). The expression of MCP-1 mRNA in the preventive group,the middle dose YC group and the high dose YC group were lower than that in the western medicine group(all P＜0.05).(3)Comparison of expression of TGF-β₁ mRNA:The expression of TGF-β₁ mRNA was higher in the model group than that in the normal group(P＜0.01).The expression of TGF-β₁ mRNA was decreased significantly in the YC treatment groups and the western medicine group compared with that in the model group(all P＜0.05). The expression of TGF-β₁ mRNA in the preventive group was lower than that in the western medicine group(P＜0.05). (4)Comparison of expression of MMP-9 mRNA:The expression of MMP-9 mRNA was higher in the model group than that in the normal group(P＜0.01).The expression of MMP-9 mRNA was decreased significantly in the YC treatment groups and the western medicine group compared with that in the model group(all P＜0.01). The expression of MMP-9 mRNA in the preventive group and high dose YC group were lower than that in the western medicine group(P＜0.05).6.Comparison of expression of ICAM-1,MCP-1,TGF-β1 and MMP-9 protein in aortic wall(1)Comparison of expression of ICAM-1 protein:The expression of ICAM-1 protein in the model group was higher than that in the normal group(P＜0.01).After treatment with YC and western medicine,the expression of ICAM-1 protein in each group was decreased in a different degree compared with the model group,however, no significant difference was shown in the low dose YC group and the western medicine group compared with the model group(P＞0.05).The expression of ICAM-1 protein was decreased significantly in the preventive group,the middle dose YC group and the high dose YC group compared with that in the model group(all P＜0.05).The expression of ICAM-1 protein in the preventive group and the high dose YC group was lower than that in the western medicine group,and also lower than that in the low dose YC group(all P＜0.01).(2)Comparison of expression of MCP-1 protein:The expression of MCP-1 protein in the model group was higher than that in the normal group(P＜0.01).The expression of MCP-1 protein was decreased significantly in the YC treatment groups and the western medicine group compared with that in the model group(all P＜0.01). The expression of MCP-1 protein in the preventive group and the high dose YC group was lower than that in the western medicine group(P＜0.01).(3)Comparison of expression of TGF-β₁ protein:The expression of TGF-β₁ protein was higher in the model group than that in the normal group(P＜0.01).After treatment with YC and western medicine,the expression of TGF-β₁ protein in each group was decreased in a different degree compared with the model group,however, no significant difference was shown in the low dose YC group,the middle dose YC group and the western medicine group compared with the model group(all P＞0.05). The expression of TGF-β₁ protein in the preventive group and the high dose YC group was lower than that in the western medicine group,in the low dose YC group and in the middle dose YC group,respectively(all P＜0.05).(4)Comparison of expression of MMP-9 protein:The expression of MMP-9 protein was higher in the model group than that in the normal group(P＜0.01).The expression of MMP-9 protein was decreased in a different degree in the YC treatment groups and the western medicine group compared with that in the model group(all P＜0.05),and among those treatment groups,the high dose YC group had the lowest level of MMP-9 protein expression(P＜0.01).A significant difference was shown between the high dose YC group and the western medicine group(P＜0.01).7.Comparison of the activation of NF-κB in aortic wallWestern blot showed that the protein expression of IκB-a in the model group was lower that in the normal group(P＜0.01),which indicated that HHcy promoted the degradation of IκB-a protein.The protein expression of IκB-a was increased in a different degree in the YC treatment groups and the western medicine group compared with that in the model group,and among those treatment groups,the high dose YC group had the highest level of IκB-a protein expression(P＜0.01),however, no significant difference was shown in comparison between the low dose YC group and the model group(P＞0.05).The protein expression of IκB-a in the high dose YC group was higher than that in the western medicine group(P＜0.01).Immunohistochemistry showed that the nuclear translocation of NF-κB was higher in the model group than that in the normal group(P＜0.01),which indicated that HHcy promoted the nuclear translocation of NF-κB.Compared with the model group,the nuclear translocation of NF-κB was decreased in a different degree in the YC treatment groups and the western medicine group,and among those treatment groups,the preventive group had the lowest level of NF-κB protein expression in the nuclear(P＜0.01).The nuclear translocation of NF-κB in the preventive group was lower than that in the western medicine group(P＜0.05).Conclusion 1.YC could decrease the plasma Hcy concentration.2.An inhibitory effect of YC on the inflammation in HHcy rabbits was observed in our present study.YC could decrease the mRNA and protein expression of ICAM-1,MCP-1,TGF-β1 and MMP-9.The mechanisms were possibly associated with the suppressive effect of YC on plasma Hcy concentration and aorta NF-κB activation.3.An anti-atherosclerosis effect of YC was observed in our present study.YC could decrease intimal thickness and intimal area,and inhibit the transformation of VSMCs from systolic phenotype to synthetic one.The mechanisms were possibly associated with the inhibitory effect of YC on the expression of inflammatory factors such as ICAM-1,MCP-1,TGF-β₁,MMP-9 and NF-κB activation.