Experimental Study And Clinical Verification Of Hesperidin On Atherosclerosis

Objective:(1)Experimental study: To investigate the effect of Hesperetin on the proliferation,migration and phenotypic transformation of primary rat thoracic aortic vascular smooth muscle cells(RVSMCs)induced by platelet derived growth factor-BB(PDGF-BB).To explore the effect of Hesperetin on carotid atherosclerosis(As)in hamsters at animal level.(2)Clinical study: Hesperetin-rich orange juice was used to conduct a small sample nursing diet intervention pre-trial on high-risk groups of As,and the role of Hesperetin in As was clinically verified.Methods:(1)Experimental research : In the experimental study in vitro,we first improved the tissue attachment method to isolate and culture the primary RVSMCs;The morphology,quantity and growth state of primary RVSMCs were observed and recorded,and the expression of smooth muscle actin ?(?-SMA),smooth muscle SM22? protein(SM22?)and Calponin were detected by cellular immunofluorescence chemistry,and the phenotype and purity of primary RVSMCs were identified.The proliferative activity of the third generation RVSMCs was detected by CCK-8 kit,and RVSMCs was treated with different concentrations of Hesperetin(10?mol/L,50?mol/L,100?mol/L,150?mol/L,200?mol/L,250?mol/L,300?mol/L)for 24 h,and the effect of Hesperetin on RVSMCs activity was detected with CCK-8 kit to observe the cytotoxicity of Hesperetin on RVSMCs.RVSMCs was treated with different concentrations of PDGF-BB(2ng/m L,5ng/m L,10ng/m L,25ng/m L,50ng/m L)for 24 h,0ng/m L was used as a blank control group.RVSMCs proliferation was detected by CCK-8 kit.RVSMCs was treated with different concentrations of PDGF-BB(25ng/m L,50ng/m L,100ng/m L)for 24 h,and the protein expression of ?-SMA and SM22? were detected by Western Blot to select the most suitable drug concentration for PDGF-BB to establish RVSMCs proliferation model.The effects of different concentrations of Hesperetin(0?mol/L,50?mol/L,100?mol/L,150?mol/L,200?mol/L,250?mol/L,300?mol/L)on the proliferation activity of RVSMCs stimulated by PDGFBB were detected by CCK-8 kit,and the most suitable intervention concentration of Hesperetin for follow-up experiment was selected.RVSMCs was divided into three groups: control group,model group(PDGF-BB group)and treatment group(PDGFBB+Hesperetin).The migration ability of RVSMCs was detected by cell scratch test,and the protein expression levels of contractile phenotypic markers ?-SMA,SM22? and synthetic phenotypic markers ELN and cell proliferation factor PCNA were detected by Western Blot.In the in vivo experimental study,the model of anthropomorphic hamster carotid artery As was established by high fat diet combined with carotid artery ligation,and different concentrations of Hesperetin were given by intragastric administration.30 experimental animals were randomly divided into blank control group(NC group),model group(Model group),low dose group(L-Hesperetin group),middle dose group(M-Hesperetin group)and high dose group(H-Hesperetin group).After 7 weeks of experiment,the carotid arteries of hamsters in each group were taken for frozen section and oil red O staining,and the As pathological changes of carotid arteries in each group were observed.(2)Clinical study: As risk screening was carried out with arteriosclerosis detector in the physical examination center of the affiliated Hospital of Guizhou Medical University from February 2018 to September 2018.16 samples were randomly selected from the high risk population of As and randomly divided into the control group and the treatment group.The control group received routine health education,and the treatment group drank 500 m L-rich orange juice every day for diet nursing intervention in addition to routine health education.After 8 weeks,two groups of people were screened for As risk,to observe the effect of orange juice rich in Hesperetin on As risk index arm-ankle pulse wave velocity(brachialal ankle pulse wave velocity,Ba PWV),and to verify the role of Hesperetin in As.Results:(1)Experimental study: In the experimental study in vitro,the primary RVSMCs,cells of SD rats were successfully isolated and cultured in a long fusiform or triangular adherent growth,and the RVSMCs after passage was closely arranged,showing a "peak-valley" feature.The cell immunofluorescence results showed that the purity of the cultured cells was more than 95%.The results of CCK-8 detection showed that the proliferation ability of RVSMCs was the strongest at 3-5 days after passage,which was the best time for follow-up experiments.All concentrations of Hesperetin did not produce cytotoxicity to RVSMCs,PDGF-BB at the concentration of 25ng/m L was the most suitable intervention concentration for the establishment of RVSMCs proliferation model in vitro,and Hesperetin at the concentration of 100?mol/L was the best intervention concentration for the treatment group.the results of scratch test showed that 25ng/m L PDGF-BB could significantly increase the migration ability of RVSMCs,and the migration ability of RVSMCs induced by PDGF-BB decreased after the addition of 100?mol/L Hesperetin.Western Blot results showed that 25ng/m L PDGF-BB could significantly decrease the protein expression of RVSMCs contractile phenotypic markers ?-SMA and SM22?,and increase the protein expression of synthetic phenotypic markers ELN and PCNA.With the addition of 100?mol/L Hesperetin,the protein expression of RVSMCs contractile phenotypic markers ?-SMA and SM22? increased,while the protein expression levels of synthetic phenotypic markers ELN and PCNA decreased.In the in vivo experimental study,the anthropomorphic hamster carotid artery As model was successfully established and intervened by Hesperetin.Compared with the NC group,the weight gain of the Model group increased,and the carotid artery oil red O staining showed that the carotid intimamedia thickened,the lumen area decreased and a large number of As lipid plaques were formed in the Model group,indicating that the carotid artery As model was successful.Compared with Model group,different concentrations of Hesperetin could reduce the weight gain of hamsters.The results of oil red O staining showed that the intima-media thickness of carotid artery,the degree of lumen stenosis,lipid deposition and the lesion of As plaque were reduced in different concentrations of Hesperetin group.(2)Clinical study: The orange juice rich in Hesperetin was given diet nursing intervention to the people at high risk of As,and it was found that orange juice could effectively reduce the value of Ba PWV,the risk index of As.Conclusion:(1)Experimental study: In vitro,it was found that Hesperetin could inhibit the proliferation and migration of RVSMCs induced by PDGF-BB,and then affect the phenotypic transformation of RVSMCs.In vivo,it was found that Hesperetin could inhibit the formation of As plaque in the carotid artery of anthropomorphic hamster.(2)Clinical study: Orange juice rich in Hesperetin can effectively reduce the risk index Ba PWV,of As and thus reduce the risk of As.