| Objective: To establish an atherosclerosis(AS)model of rabbit common carotid artery intimal injury,to simulate the AS in restenosis after PTCA in clinical practice,and to observe the intervention effect of Huotan Jiedu Tongluo Decoction on this model.To investigate whether Huotan Jiedu Tongluo Decoction can inhibit the progression of atherosclerosis through endoplasmic reticulum stress(ERS)-autophagy signaling pathway.Methods: Thirty male Japanese rabbits were randomly divided into 5 groups: 6 in each group,which were sham operation group,model group,Chinese medicine group,western medicine group and high dose Chinese medicine group.Except for the sham operation group fed with normal feed,the other groups were fed with 2% L-methionine feed for 2 weeks,then the left common carotid artery intima of the balloon injury was performed to establish the rabbit common carotid artery AS model.The sham operation group and the model group were given normal drinking water for gavage,the Chinese medicine group was given Huotan Jiedu Tongluo Decoction for intragastric administration,the western medicine group was given atorvastatin for intragastric administration,and the traditional Chinese medicine high dose group was given high dose of Huotan Jiedu Tongluo Decoction for intragastric administration.Each group was administered by intragastric administration for 4 weeks.1.Vascular ultrasound examination: Ultrasound detection was performed on each of the five common carotid arteries on the left side of the sham operation group,the right side of the model group,and the left side of the model group(injury side).After the photograph,the intima-media thickness of the common carotid artery was measured,and three measurement points were selected for each vessel,which were measured at the distal end of the common carotid artery,the proximal end of the common carotid artery,and the midpoint between them.2.Enzyme-linked immunosorbent assay(ELISA):(1)detection of homocysteine(Hcy)content in rabbit serum at 0,2,and 6 weeks in the model group;(2)detection of the content of homocysteine(Hcy)in serum of each group at sixth week.3.Pathological examination:(1)HE staining was used to detect the intimal hyperplasia of the common carotid artery in the injured side;(2)Masson staining was used to observe the expression of collagen fibers in the common carotid artery;(3)Immunohistochemical staining was used to detect the expression of eIF2 a in the common carotid artery of each group.(4)The ultrastructure of intima of rabbit common carotid artery was detected by electron microscopy.4.Real-time quantitative polymerase chain reaction(Real-time PCR)detection:(1)The expression of mRNA levels of GRP78,PERK,eIF2 a,which are endoplasmic reticulum stress markers,in the rabbit common carotid arteries of each groups.(2)The expression of mRNA levels of beclin1,Atg12,and ULK1,which are autophagy marker molecules in each groups of arterial tissue.5.Western blot analysis: protein expression levels of ERS marker molecules GRP78,PERK,p-eIF2 a,and autophagy marker molecules beclin1,Atg12,ULK1 in common carotid artery tissues.Results:1.The results of vascular ultrasound showed that compared with the left side of the sham operation group and the right side of the model group,the intima-media thickness of the left side of the model group was significantly widened,and the intravascular-medial hyperplasia was extremely obvious,and the statistical difference was extremely significant.(P<0.01).There was no significant difference between the right side of the model group and the left side of the sham operation group(P>0.05).The results of the three measuring points in the telecentric,midpoint and proximal end of each group were consistent,and there was no statistical difference between the three groups(P>0.05).2.The results of ELISA showed that:(1)The serum Hcy content in the model group was significantly higher than that in the 0th week(P<0.01).Compared with week 0,the content of Hcy in serum was significantly increased at the 6th week and there was a significant difference(P<0.01).Compared with the second week,the serum Hcy content also increased(P<0.05).(2)At the 6th week,the expression of Hcy in the serum of each group showed that the model group compared with the sham operation group,Hcy the expression level was significantly increased in serum(P<0.01).The levels of Hcy were significantly lower in the Chinese medicine group,western medicine group and high-dose group than the model group(P<0.01).3.Pathological examination:(1)HE staining results: Compared with the sham operation group,the model group had obvious intimal hyperplasia and adventitial inflammatory cell infiltration;the three groups of drugs compared with the model group,the degree of intimal hyperplasia and injury were significantly reduced.The inflammatory infiltration of the outer membrane was reduced between the sham operation group and the model group.(2)Masson staining results showed that the sham-operated group had predominantly red muscle fibers after Masson staining,and no obvious collagen fibers were observed.The rabbit common carotid artery in the model group had obvious intimal hyperplasia,intima,media,The outer membrane showed different degrees of blue staining,and the collagen fibers increased significantly.Compared with the model group,the collagen fibers were significantly reduced and the intimal hyperplasia was alleviated in the three groups.(3)Immunohistochemical results: the expression of eIF2 a in the sham operation group was negative,and the color of the nucleus was basically blue.Compared with the sham operation group,the intima of the model group was significantly thickened,and the expression of eIF2 a in the neointimal was positive,showing a large number of brown-yellow particles.A small amount of brown-yellow particles were observed in the three groups of drugs,and the expression of eIF2 a was significantly reduced compared with the model group.(4)Electron microscopy showed that there were no autophagic vacuoles in the intima of the common carotid artery in rabbits,and a small amount of lipid droplets were observed.Compared with the sham operation group,the autophagic vacuoles in the intima of the common carotid artery in the model group increased significantly.A large number of autophagosome lysosomes were observed;the autophagosomes of the three groups were significantly reduced compared with the model group.4.The results of RT-PCR showed that compared with the sham operation group,the mRNA levels of ERS markers GRP78,PERK,eIF2 a,autophagy markers beclin1,Atg12 and ULK1 were significantly increased in the model group of rabbit common carotid arteries(P< 0.01),the levels of GRP78,PERK,eIF2 a,beclin1,Atg12,and ULK1 mRNA were significantly lower in the Chinese medicine group,the western medicine group,and the high-dose group of traditional Chinese medicine compared with the model group(P<0.01).5.Western blot results showed that compared with the sham operation group,the expression levels of ERS markers GRP78,PERK,p-eIF2 a,and autophagy markers beclin1,Atg12,and ULK1 were significantly increased in the model group(P< 0.01).Compared with the model group,the protein expression levels of GRP78,PERK,p-eIF2 a,beclin1,Atg12,and ULK1 in the carotid tissues of the three groups were significantly lower(P<0.01).Conclusion: 1.Successfully established AS model of rabbit common carotid artery balloon injury.It has a high rate of formation and constant lesions and can be used to mimic the mechanism of restenosis after PTCA in humans.2.Huotan Jiedu Tongluo Decoction can significantly improve rabbit carotid atherosclerosis and significantly inhibit the intimal hyperplasia after balloon injury.Huotan Jiedu Tongluo Decoction can reduce the content of Hcy in rabbit serum.3.Endoplasmic reticulum stress(ERS)-autophagy signaling pathway molecule over-expression in AS model after balloon injury,Huotan Jiedu Tongluo Decoction can inhibit the excessive activation of ERS-autophagy pathway,providing new therapeutic targets and theoretical basis for the treatment of AS after PTCA. |
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