| Objective: In this study,we aim to explore the potential therapeutic effects of Zhilong Huoxue Tongyu capsule on the improvement of early carotid atherosclerosis by studying the downstream molecules of the regulatory T cells,and their inhibitory effects on cell proliferation and inflammation in the atherosclerotic lesions based on the TGF-??Smads pathway.To observe the beneficial effects of Zhilong Huoxue Tongyu capsule,we designed the rabbit model of carotid atherosclerosis by feeding high-fat diet and carotid intimal air drying.Parameters such as level of lipids in blood and related pathological changes were observed after treatment with Zhilong Huoxue Tongyu capsule,atorvastatin calcium tablet and the mixed medication of Zhilong Huoxue Tongyu capsule and atorvastatin calcium tablet.Methods:1.Fifty New Zealand white rabbits were adaptively fed for 1 week and randomly divided into 5 groups according to the random number table method,the normal control group(n=10),the model group(n=10),the Zhilong Huoxue Tongyu capsule group(n=10),the atorvastatin calcium tablet group(n=10),Zhilong Huoxue Tongyu capsule + atorvastatin calcium tablets(n=10).One week later,the right carotid intimal air drying operation was performed,and the high-fat diet was continued to establish a carotid atherosclerosis model of rabbits.Formally administered 1 week after surgery,respectively: 3.12 g crude drug / kg / d of the Zhilong Huoxue Tongyu capsule group,0.51 mg / kg / d of atorvastatin calcium tablet group,3.12 g crude drug / kg / d + 0.51 mg / kg / d of mixed medication group,each group of rabbits was fed with an equal amount(4 mL?kg)of saline.Single cage feeding,unlimited drinking water,each experimental rabbit was fed 150 g of feed daily.2.After 4 weeks of drug intervention of experimental rabbits in each group,blood samples were collected from the heart after anesthesia to collect serum samples.Seven serum samples were randomly selected from each group,the concentrations of triglyceride,total cholesterol,high-density lipoprotein cholesterol,and low-density lipoprotein cholesterol were measured by using a fully automated biochemical analyzer.Three samples were randomly selected from each group,the expression levels of TGF-?1,IL-10,IL-6 and IL-17 were measured by ELISA method.3.All animals were sacrificed by air embolization after blood collection,take the right common carotid artery for conventional paraffin embedding and prepare HE stained sections,observe the pathological changes of the intima of each specimen.4.Three carotid tissue samples were randomly selected from each group,the protein expressions of Foxp3,MMP-2,and MMP-9 in each group of specimens were determined by western blot;the contents of TGF-?1,Foxp3,IL-10,MMP-2,and MMP-9 mRNA in each group were measured by RT-PCR.5.All experimental data were statistically analyzed by using Graphpad prism 8.0.1 software,all are expressed as mean ± standard deviation(),the comparison between multiple groups was performed by one-way ANOVA Tukey's multiple test,P<0.05 was considered statistically significant.Results: 1.The experimental process was relatively smooth,there were no death records throughout the experiment.2.The results of the blood lipid: Compared with the normal control group,all blood lipid indices in the model group were significantly increased(P<0.001).The blood lipid levels of experimental rabbits in each treatment group were reduced to varying degrees after drug intervention,P <0.05 was statistically significant.3.The results of histopathological changes seen under the microscope: In the normal group,the layers of carotid artery wall of rabbits were clear,and no lipid deposition and carotid atherosclerotic plaques were seen;The carotid intimal hyperplasia of rabbits in the model group experiment was severe,visible plaque formation with a lipid core and a thin fibrous cap in the lumen can be seen.Foam cell aggregation,inflammatory cell infiltration and irregular thickening of the middle and outer membranes were also observed.It showed that the rabbits carotid artery wall were damaged to some extent after modeled,which conformed with the pathological changes of CAS.The carotid arterial intimal thickening of the experimental rabbits in the Zhilong Huoxue Tongyu capsule group and the atorvastatin calcium tablet group were thinner than the model group,the endothelial part were bulged with less plaques,a small amount of foam cells and inflammatory cells infiltration,and the media thickened to varying degrees.In the mixed-medicine group,the morphology of rabbit endothelial cells was still intact,but there was slight intimal hyperplasia and some inflammatory cells were visible.4.The results of ELISA: Compared with the normal group,the levels of TGF-?1 and IL-10 in the model group were significantly reduced,and the levels of IL-6 and IL-17 were significantly increased with statistical significance(P<0.01).After the intervention of the Zhilong Huoxue Tongyu Capsule or the Atorvastatin Calcium Tablets,the levels of IL-6 and IL-17 in each experimental rabbit decreased,and the level of IL-10 increased as compared with the model group with statistical significance of P<0.01.TGF-?1 increased in the Zhilong Huoxue Tongyu Capsule group but did not change significantly in the atorvastatin calcium tablet group.After the combined intervention,the improvement of the above indicators was more obvious than the single medicine group.5.The results of Western blot: Compared with the normal group,Foxp3 protein expression in the model group was significantly reduced and MMP-2 and MMP-9 protein expressions were significantly increased(P<0.01).After the intervention of Zhilong Huoxue Tongyu capsule or atorvastatin calcium tablets,the expression of Foxp3 protein increased,while the level of MMP-2 protein decreased significantly(P<0.05).as compared with the model group.MMP-9 protein level was significantly(P <0.05)reduced in the Zhilong Huoxue Tongyu capsule group but there was no significant change in the atorvastatin calcium tablet group.However,after the treatment with combined Chinese and the western medicine group,the changes of the above three proteins were more prominent than the single medicine group.6.The results of RT-PCR: The levels of Foxp3,TGF-?1,and IL-10 mRNA in the model group were significantly reduced compared with the normal group,while the levels of MMP-2 and MMP-9 mRNA were significantly increased(P<0.01).After the combined intervention,the mRNA levels of the above five genes changed more significantly(P<0.01).Conclusion: 1.It is suggested that Zhilong Huoxue Tongyu Capsule can improve early atherosclerosis by reducing lipids.2.Zhilong Huoxue Tongyu Capsule may stimulates Foxp3 to promote the differentiation and maturation of Tregs through the TGF-??Smads pathway,immunosuppressive effector T lymphocytes and upregulate the expression levels of IL-10 and TGF-?1-the downstream signalling molecules.Moreover,it also results in the downexpression of pro-inflammatory factors IL-17,IL-6,thereby reducing the local inflammation and immune response of the body,stabilize vulnerable plaque and improve early carotid atherosclerosis.3.Zhilong Huoxue Tongyu Capsule can inhibit the expression of MMP-2 and MMP-9 through the TGF-??Smads pathway,reducing extracellular matrix degradation and stabilizing vulnerable plaque. |
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