| Objective.1.To observe the lipid-modulating effects of herbal cake-separated moxibustion on atherosclerosis(AS)vulnerable plaque rabbits.2.To investigate the effects of herbal cake-separated on the expression of Ras homologue genome member A(Rho A),Ras homologue-associated convoluted helix protein kinase(ROCK)and related proteins in rabbits with AS vulnerable plaques,and to explore the mechanism of herbal cake-separated moxibustion in stabilizing vulnerable plaques in rabbits with AS by inhibiting Rho A/ROCK to protect vascular endothelial function.Methods.Sixty-five New Zealand rabbits were randomly divided into 10 in the blank group and 11 each in the model group,direct moxibustion group,herbal cake-separated moxibustion group,atorvastatin group and inhibitor group.The AS vulnerable plaque model was established in all groups except for the blank group by high-fat feeding,balloon injury of abdominal aorta,gene transfection,and trigger plaque rupture.The atorvastatin group was fed with atorvastatin calcium tablets 1.96mg/(kg·d),and the inhibitor group was injected with fasudil hydrochloride 10 mg/(kg·d)along the ear edge veins.Direct moxibustion group and herbal cake-separated moxibustion group used moxibustion either directly on the acupoints or with a herbal cake;the two acupoints groups,Juque(CV14),bilateral Tianshu(ST25)and Fenglong(ST40)as group 1,or bilateral Xinshu(BL15),Ganshu(BL18)and Pishu(BL20)as group 2,should be used alternatively once daily,three moxa cones for each acupoint;the treatement course of all the groups lasted for 8 weeks.After 13 weeks,the serum total cholesterol(TC),triglycerides(TG),low-density lipoprotein(LDL),high-density lipoprotein(HDL),apolipoprotein A(APO-A)and apolipoprotein B(APO-B)were measured by ELISA method;the lipid area of abdominal aortic plaque tissue was observed by oil red O staining method,and the percentage of positive lipid area was calculated.HE staining was performed to observe the structure of abdominal aortic plaque tissue and to measure intima thickness and intima-media thickness(IMT);immunohistochemistry method was performed to observe abdominal aortic Rho A,ROCK,Tumour necrosis factor-α(TNF-α),p38 mitogen-activated proteinkinase(p38MAPK),Toll-like receptor 4(TLR4),nuclear factor kappa B(NF-κB),endothelial nitric oxide synthase(e NOS)for antigenic activity.Results.1.Compared with the blank group,serum TC,TG,LDL and APO-B levels were significantly increased and HDL and APO-A levels were significantly decreased in the model group(P < 0.01);compared with the model group,serum TC,TG,LDL and APO-B levels were significantly decreased and HDL and APO-A levels were significantly increased in the direct moxibustion,herbal cake-separated moxibustion and atorvastatin groups(P < 0.05 or P < 0.01);compared with the atorvastatin group,serum APO-B levels were significantly decreased in the direct moxibustion,herbal cake-separated moxibustion and atorvastatin groups(P < 0.01).Compared with the direct moxibustion group,the rabbit serum APO-B level in the herbal cake-separated moxibustion group was significantly reduced(P < 0.01);compared with the atorvastatin group,the rabbit serum APO-B level in theherbal cake-separated moxibustion group was significantly reduced(P < 0.05).2.Compared with the blank group,the model group showed extensive lipid deposition in the vessel wall,resulting in severe intimal thickening and luminal stenosis,with a significant increase in the percentage of lipid-positive area(Ρ < 0.01);compared with the model group,the extent of lipid droplet deposition in the direct moxibustion group was comparable to that in the model group,and the extent of lipid droplet deposition in the herbal cake-separated moxibustion group and atorvastatin group was smaller than that in the direct moxibustion group,with less stenosis.The percentage of lipid-positive area was significantly lower in the three intervention groups(P < 0.05 or P < 0.01).3.HE staining: compared with the blank group,the intima and intima-media of the model group were thickened and protruded into the lumen,lipid deposition,intraplaque hemorrhage,thrombus formation,destruction of intima-media structure,irregular arrangement of elastic fibres,poorly defined structure,loose cytoplasm of smooth muscle cells,a large number of foam cells were seen,intima thickness and IMT were significantly thickened in the model group(P < 0.01).Compared with the model group,the degree of vascular damage in the direct moxibustion group was comparable to that in the model group,and the degree of mesothelial damage in the herbal cake-separated moxibustion and atorvastatin groups was less than that in the direct moxibustion group.No thrombus formation was seen in the three intervention groups,and the intima thickness and IMT were significantly reduced(P < 0.05 or P <0.01).5.Immunohistochemical assays: compared with the blank group,the model group showed significantly higher(Ρ < 0.01)and lower(Ρ < 0.01)values of Rho A,ROCK,TNF-α,p38 MAPK,TLR4,NF-κB optical density and e NOS optical density in the model group;compared with the model group,the direct moxibustion,herbal cake-separated moxibustion,atorvastatin and inhibitor groups showed significantly higher(Ρ < 0.05 or Ρ < 0.01)and lower(Ρ < 0.01)values of Rho A,ROCK,TLR4,NF-κB optical density and e NOS optical density in the model group.TNF-α,p38 MAPK,TLR4,and NF-κB optical density values were significantly decreased(Ρ< 0.05 or Ρ < 0.01)and e NOS optical density values were significantly increased(Ρ <0.05 or Ρ < 0.01)in the direct moxibustion,herbal cake-separated moxibustion,atorvastatin,and inhibitor groups;compared with the direct moxibustion group,TNF-α and NF-κB optical density values were significantly decreased(Ρ < 0.05)and e NOS optical density values were significantly increased(Ρ < 0.05)in the herbal cake-separated moxibustion group,and e NOS optical density values were significantly increased(Ρ < 0.01 or Ρ<0.05),and NF-κB optical density values decreased(Ρ < 0.05)and e NOS optical density values increased(Ρ < 0.01)in the atorvastatin group.Conclusion.1.The positive effect of herbal cake-separated moxibustion on lipid and apolipoprotein levels in AS-prone plaque rabbits.2.Herbal cake-separated moxibustion can reduce lipid deposition in AS vulnerable plaque,improve endothelial structure and protect vascular endothelium to stabilize AS vulnerable plaque.3.Rho A/ROCK-mediated TNF-α/e NOS/NO signalling pathway can be inhibited by herbal cake-separated moxibustion to protect vascular endothelial cell function and stabilize AS vulnerable plaque.4.Rho A/ROCK-mediated P38MAPK/e NOS/NO signalling pathway was inhibited by herbal cake-separated moxibustion to protect vascular smooth muscle cell function and stabilize AS vulnerable plaque.5.Rho A/ROCK-mediated TLR4/NF-κB/e NOS/NO signaling pathway is inhibited by herbal cake-separated moxibustion to protect vascular macrophage function and stabilize AS vulnerable plaque.6.The effect of Rho A/ROCK in mediating TNF-α,38 MAPK,and TLR4/NF-κB signaling pathways inhibition by herbal cake-separated moxibustion to protect vascular endothelial function and stabilize AS vulnerable plaque may be achieved by regulating the mutual conduction of e NOS/NO signaling. |
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