| Objective: This study established a rat model of atherosclerosis(AS)after balloon injury of the common carotid artery,simulated clinical restenosis(RS)after PTCA,and observed the effect of Huotan Jiedu Tongluo Yin on the AS model after balloon injury.To explore whether Tan Jiedu Tongluo Decoction inhibits the formation of atherosclerosis through the endoplasmic reticulum stress(ERS)-autophagy signaling pathway.Methods: Forty 5-week-old SPF male Sprague-Dawley rats were randomly divided into 4 groups,each with 10 rats,namely sham operation group,balloon injury group,Huotan Jiedu Tongluo Decoction group,and atorvastatin group.The 2F Fogarty balloon was used to establish a balloon injury model of the left common carotid artery in rats.After the operation,the treatment group was given Hutan Jiedu Tongluo Decoction and atorvastatin by gavage,while the sham operation group and the balloon injury group were given an equal volume of purified water.On the 28 th day after surgery,the injured carotid artery tissue of each group was extracted.Hematoxylin-Eosin(HE)staining method was used to observe the pathological remodeling of the carotid artery in each group;Masson staining method was used to observe the fibrosis of the injured carotid artery wall in each group;immunohistochemical method(IHC)To detect the positive cell expression percentages of proliferating cell nuclear antigen(PCNA),α-smooth muscle actin(α-SMA),and endoplasmic reticulum stress marker signal molecules CRP78 and PERK in the common carotid artery of the injured side of each group of rats;Transmission electron microscope observation of the changes in the ultrastructure of the injured common carotid artery wall;Real-time PCR detection: 1.Determination of the endoplasm in the injured common carotid artery tissue of each group of rats The m RNA expression levels of network stress marker signal factors GRP78,PERK,el IF2 a,CHOP;2.Determine the m RNA expression levels of key autophagy signaling factors m TOR,LC3 B,ATG7,Beclin-1 in the injured common carotid artery tissue of each group of rats.Western blot test: Endoplasmic reticulum stress marker signal factors GRP78,p-PERK,PERK,el IF2 a,CHOP and key autophagy signal factors m TOR,LC3 B,The protein expression level of ATG7 and Beclin-1.Result:1.The results of HE showed that the endothelium of the common carotid artery of rats in the sham-operated group had a complete and smooth structure,and the tissues were arranged uniformly and regularly,consisting of only a layer of flat vascular endothelium;in the injured common carotid artery of the rats in the balloon injury group The membrane is dense and disorderly arranged.Its neointima makes the blood vessel wall thicker and the lumen area is obviously narrowed.Statistics show that compared with the sham operation group,the neointimal area(NIA)and the ratio of NIA/media area(MA)in the balloon injury group were significantly higher(P<0.01),while the phlegm Jiedu Tongluo drink group The NIA and NIA/MA ratios of the atorvastatin group and the atorvastatin group were significantly lower than the model group(P<0.01),and the lumen stenosis was significantly improved.2.The results of Masson staining showed that the common carotid artery in the sham operation group showed a red color rich in muscle fibers after staining;while the intima of the common carotid artery on the injured side of the model group was significantly thickened,and the blood vessel wall appeared to be Compared with the sham operation group,the original volume fraction(AVF)was significantly higher for collagen fiber-based blue staining(P<0.01);while the Huotan Jiedu Tongluo Decoction group and the atorvastatin group were compared with the balloon injury group.The blue staining of collagen fibers was significantly reduced,while the AVF value was significantly reduced(P<0.01),indicating that Huotan Jiedu Tongluo Decoction can effectively delay the process of arterial fibrosis after PTCA;3.The results of immunohistochemistry showed:(1)No obvious brown-yellow PCNA positive expression particles were observed in the sham operation group,while the positive expression of α-SMA was more significant;compared with the sham operation group,the average optical density value of PCNA positive expression particles in the vessel wall of the balloon injury group(IOD)was significantly higher than that in the sham operation group(P<0.01),while the IOD value ofα-SMA was significantly lower(P<0.01),indicating that balloon injury promoted the proliferation and cell phenotype of the smooth muscle cells of the common carotid artery intima Conversion: Compared with the balloon injury group,the IOD value of PCNA in the Huotan Jiedu Tongluo Decoction group and the atorvastatin group were significantly reduced(P<0.01),while the IOD value of α-SMA was significantly increased(P<0.01),which indicates that Huotan Jiedu Tongluo Decoction can inhibit the proliferation of vascular smooth muscle after balloon injury;(2)In the sham operation group,there were no obvious brown-yellow CRP78 and PERK positive expression particles;compared with the sham-operation group,the intima of the balloon injury group was significantly thickened,the lumen was severely narrowed,and a large number of brown-yellow particles were seen in the neointima.The IOD values of CRP78 and PERK were significantly increased(P<0.01);compared with the model group,the positive expression particles of CRP78 and PERK in the Huotan Jiedu Tongluo Decoction group and the atorvastatin group were significantly reduced,and the IOD of the two groups The values were also significantly reduced(P<0.01),indicating that the Huotan Jiedu Tongluo Decoction group can inhibit the activation of vascular endoplasmic reticulum stress;4.The results of transmission electron microscopy showed that no endoplasmic reticulum swelling and autophagosomes were found in the neointima of the common carotid artery in the sham operation group;compared with the sham operation group,the neointima of the common carotid artery was visible in the balloon injury group.Plasma reticulum edema is obvious,and the number of autophagosomes and autophagolysosomes are also significantly increased;and the autophagosomes and autophagolysosomes of the two drug treatment groups are significantly reduced compared with the model group,indicating that Huotan Jiedu Tongluo Decoction effectively reduce the stress load of the endoplasmic reticulum and promote the restoration of homeostasis;5.Real-time PCR detection results show that: compared with the sham operation group,the ERS marker signal molecules GRP78,PERK,e IF2α,CHOP and autophagy marker signal molecules LC3 B,ATG7,Beclin-1 in the common carotid artery tissue of the model group The m RNA expression level was significantly increased(P<0.01),while the m RNA expression level of m TOR was significantly decreased(P<0.01).Compared with the balloon injury group,the ERS in the carotid artery tissue of the rats in the two drug treatment groups The m RNA expression levels of signaling factors GRP78,PERK,e IF2α,CHOP,and autophagy signaling factors LC3 B,ATG7,and Beclin-1 were significantly reduced(P<0.01),while the expression level of m TOR was significantly increased(P<0.01),indicating Huotan Jiedu Tongluo Decoction can effectively inhibit the excessive activation of ERS-autophagy in balloon-injured blood vessels at the gene level.6.Western blot detection results showed that compared with the sham operation group,the ERS marker signal factors GRP78,p-PERK,PERK,el IF2 a,CHOP,and the key autophagy signal factors LC3 B,ATG7,Beclin-1 in the common carotid artery tissue of the model group The protein expression level of the drug group was significantly increased(P<0.01),while the expression level of the autophagy protein molecule m TOR was significantly reduced(P<0.01);compared with the model group,GRP78,p-PERK,PERK,and PERK in the carotid artery tissue of the drug group The expression levels of el IF2 a,CHOP,and key autophagy signaling factors LC3 B,ATG7,and Beclin-1 molecules were significantly reduced(P<0.01),while the expression level of m TOR protein was significantly increased(P<0.01),indicating that Huotan Jiedu Tongluo Decoction can effectively inhibit the excessive activation of ERS-autophagy in the carotid artery after balloon injury at the protein level.Conclusion:1.This study successfully established an AS model of rat common carotid artery balloon injury,which can be used in the study of vascular restenosis caused by new atherosclerosis after clinical PTCA;2.Huotanjiedu Tongluo Decoction can be used Significantly reduce the degree of fibrosis of the vessel wall tissue,and significantly inhibit the proliferation of balloon-injured vascular smooth muscle,and have a significant inhibitory effect on the formation of postoperative neointima,thereby effectively improving the balloon-injured carotid artery AS in rats after PTCA;3.In the AS model after balloon injury,the ERS-autophagy signal pathway is over-activated;Huotan Jiedu Tongluo Decoction can effectively inhibit the ERS-autophagy signal pathway in the balloon-injured arterial tube in this model Overexpression,which provides new therapeutic targets and coping strategies for clinical treatment of AS after PTCA. |
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