| Objective:A mouse model of atherosclerosis vulnerable plaque（VP）after carotid artery cannulation was established to simulate atherosclerosis-prone plaque in the clinical setting,to investigate the intervention effect of Huotan Jiedu Tongluo Formula in this model,and to investigate whether Huotan Jiedu Tongluo Formula might inhibit the progression of atherosclerosis vulnerable plaque through the endoplasmic reticulum autophagy-NLRP3 inflammatory vesicle signaling pathway.Methods:In vivo experiments:12 male C57BL/6J mice and 36 male ApoE-/-mice were divided into four groups of 12 mice each:sham-operated group（C56BL/J6）,model group(ApoE-/-),herbal group(ApoE-/-),and western medicine group(ApoE-/-).Except for sham operation,all groups were given carotid cannulation+high-fat diet containing 0.25%cholesterol and 15%cocoa cream for 8 weeks to establish mouse carotid artery atherosclerosis vulnerable plaque model.The mice in the Chinese medicine group were given Huotan Jiedu Tongluo Formula by gavage,the mice in the Western medicine group were given a subcutaneous injection of Evolocumab.The mice in the sham-operated group and model group were given an equal volume of distilled water by gavage.The material was taken from each group 8 weeks after administration.1.Body weight of mice:Body weight of mice in each group was measured and recorded weekly.2.Histopathological staining:HE staining to detect the intima of carotid arteries in each group of mice;Masson staining to observe the expression of collagen fibers in each layer of carotid arteries;immunohistochemical staining to detect the expression of MMP in carotid arteries in each group of mice.Real-time PCR:mRNA expression levels of NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-α in carotid artery tissues of mice;mRNA expression levels of endoplasmic reticulum autophagy in carotid artery tissues of mice.The mRNA expression levels of GRP78,ULK1,mTOR,Beclin1,Atg7,LC3B,and P62 in carotid artery tissues of all groups of mice.5.Protein immunoblotting（Western blot）assay:inflammatory indexes:NLRP3,ASC,Caspase-1 and autophagy marker molecules ULK1,mTOR,Beclinl,Atg7,LC3B,and P62 protein expression levels in mouse carotid artery tissues.In vitro experiments:After lipopolysaccharide（LPS）induced mouse peritoneal macrophages RAW264.7 to establish a cellular inflammatory injury model,the cells were divided into control group,LPS group,Chinese medicine group and western medicine group,and the activity of RAW264.7 was detected by CCK-8 method;protein immunoblotting method was used to detect NLRP3,Caspase-1,Beclin1,LC3B and P62 protein expression levels were detected by protein immunoblotting.Results:1.The results of mouse body weight measurement showed that the mean weekly body weight of mice in the model group increased significantly compared with that in the sham-operated group,which was statistically different（P<0.05）,and the increasing trend of weekly body weight was apparent;compared with the model group,the bodyweight of mice in the Chinese medicine and western medicine groups decreased significantly（P<0.05）,and the increasing trend of weekly body weight was lower than that in the model group.2.The ELISA results showed that the serum triglyceride（TG）and low-density lipoprotein（LDL）in mice in the model group were significantly higher compared with the sham-operated group（P<0.01）,and the high-density lipoprotein（HDL）was not statistically different compared with the sham-operated group（P>0.05）;the serum TG and LDL in mice in the Chinese medicine and western medicine groups were significantly lower compared with the model group（P<0.01）There was no statistically significant difference between the TG and LDL in the serum of mice in the Chinese herbal medicine group and the Western medicine group compared with the model group（P>0.05）.Compared with the sham-operated group,the serum levels of inflammatory factors IL-1β and TNF-α in mice in the model group were significantly higher（P<0.01）,and the serum levels of IL-1β and TNF-α in mice in each group were significantly lower（P<0.01）after the administration of traditional Chinese medicine and western medicine compared with the model group,and the results were statistically significant.3.Pathological examination:HE staining results showed that compared with the sham operation,the intima-media thickening in the carotid artery of mice in the model group was significantly thickened and the percentage of intima-media area increased significantly（P<0.01）,while the intima-media thickening in the carotid artery of mice in the Chinese medicine and western medicine groups was significantly reduced compared with the model group and the percentage of intima-media area decreased significantly（P<0.01）;compared with the model group,the intima-media thickening in the Chinese medicine and western medicine groups was reduced and the percentage of intima-media area increased significantly（P<0.01）.Masson staining results showed that the Masson staining of the common carotid artery in the sham-operated group showed mainly red myofibers with no significant collagen fiber deposition,while the model group showed significantly enhanced blue staining（P<0.01）and significant collagen fiber deposition.In contrast,both the Chinese medicine group and the western medicine group showed a small amount of blue staining,but collagen fiber deposition was reduced compared with the model group（P<0.01）.The blue staining was significantly enhanced in the model group（P<0.01）and the collagen fiber deposition was significant in both the Chinese medicine and western medicine groups.Immunohistochemistry results showed that the expression of MMP-9 in the sham-operated group was negative,and the nucleus staining was basically blue,while the endothelium of the model group was significantly thickened compared with that of the sham-operated group,and the expression of MMP-9 in the new endothelium was positive,and a large number of brown-yellow particles were seen,while a small number of brownyellow particles were seen in the Chinese medicine group and the western medicine group,and the expression of MMP-9 was significantly reduced compared with that of the model group.4.RT-PCR results showed that compared with the sham-operated group,the expression of NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-α,inflammatory vesiclerelated molecules in the tissue of common carotid artery of mice in the model group was significantly higher（P<0.01）,and the expression of autophagy-related indexes GRP78,ULK1,Beclin1,Atg7 and LC3B,was significantly decreased（P<0.01）,and mTOR and P62 expression increased（P<0.01）;compared with the model group,NLRP3,ASC,Caspase-1,IL-1β,IL-18,TNF-α mRNA expression significantly decreased（P<0.01）,and GRP78,ULK1,Beclinl,Atg7,LC3B expression significantly The expression of GRP78,ULK1,Beclin1,Atg7 and LC3B was significantly increased（P<0.01）,and the expression of mTOR and P62 was decreased（P<0.01）.5.Western blot results showed that compared with sham operation,the expression of NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-α,inflammatory vesicle-related molecules,and autophagy-related indexes GRP78,ULK1,Beclinl,Atg7 and LC3B,were significantly increased in the tissues of the common carotid artery of mice in the model group（P<0.01）.expression was significantly decreased（P<0.01）,and mTOR and P62 expression was increased（P<0.01）;compared with the model group,NLRP3,ASC,Caspase-1 protein expression was significantly decreased（P<0.01）,GRP78,ULK1,Beclin1,Atg7,LC3B expression was significantly increased（P<0.01）,and mTOR and P62 expression was decreased（P<0.01）.6.In vitro experiments:Expulsion of phlegm and detoxification of Tongluo formula can increase the activity of LPS-induced mouse peritoneal macrophages RAW264.7 cells（P<0.01）and improve the level of cellular autophagy to inhibit the overexpression of NLRP3 inflammatory vesicles.Conclusion:1.The experiment successfully established a mouse atherosclerosis vulnerable plaque model with high modeling rate and constant lesions,which can be used to mimic the mechanism study of human atherosclerosis-prone plaque.2.The formula can significantly inhibit carotid atherosclerosis-prone plaque in mice,and has a significant inhibitory effect on intimal hyperplasia and plaque fragility after carotid artery cannulation.3.Huotan Jiedu Tongluo Formula can reduce body weight and blood lipids in ApoE-/mice.4.Endoplasmic reticulum autophagy-NLRP3 inflammatory vesicle signaling pathway molecules have significant effects in atherosclerosis vulnerable plaque model after carotid artery cannulation.5.Huotan Jiedu Tongluo Formula can regulate autophagy and NLRP3 inflammatory vesicles in mouse peritoneal macrophages RAW264.7 to inhibit LPS-induced excessive inflammatory expression.6.The formula can regulate the endoplasmic reticulum autophagy-NLRP3 inflammatory vesicles signaling pathway to inhibit atherosclerosis vulnerable plaque,which provides a new therapeutic target and theoretical basis for the treatment of atherosclerosisprone plaques in Chinese medicine. |
PDF Full Text Request