| ObjectivesHyperlipidemia is usually caused by excess nutrition and is characterized by a disturbance in lipid metabolism.Chronic hyperlipidemia,abnormal lipid accumulation in surrounding tissues,can lead to hepatic fatty lesions,atherosclerosis,insulin resistance and other pathological changes.Therefore,early detection and treatment of hyperlipidemia is of great significance for reducing cardiovascular and cerebrovascular events and preventing non-alcoholic fatty liver disease and type 2 diabetes.The disorder of cholesterol metabolism is the core problem of hyperlipidemia,so it is very important to regulate the expression of genes related to cholesterol metabolism in the treatment of hyperlipidemia.SCAP is a key regulator for the maturation of SREBP-2,which induces the transfer of SREBP-2 from endoplasmic reticulum to Golgi apparatus,thereby promoting the synthesis of intracellular cholesterol and playing an important role in regulating cholesterol homeostasis.In addition,SCAP/SREBP2 acts as a signal transduction center to integrate the inflammation and cholesterol metabolism.SCAP/SREBP2 can activate NLRP3 which induce inflammation by activating subsequent maturation of inflammatory cytokines such as interleukin-1 beta(IL-1β)and interleukin-18(IL-18),and initiating a type of inflammation.In our study,we use the hyperlipidemia(HLP)rats as model,and investigated the effect of electroacupuncture(EA)on SCAP and SREBP-2 and the expression of downstream cholesterol metabolism target genes HMGCR,PCSK9 and inflammatory reactions NLRP3,IL-1β,IL-18.Trying to provide experimental evidence for application of EA in treatment of HLP and its related diseases.MethodsSeventy two SPF adult male SD rats with a body mass of(250±50)g were obtained from certificated institution.After 1 week of adaptive feeding,10 rats were selected randomly to the normal group(NG)and fed with normal diet,the remaining 62 rats were fed with high-fat diet for 28 days to establish HLP model,finally,57 rats were successfully modeled.After modeling,50 rats were randomly selected and divided into HLP model(MG),and electroacupuncture group(EAG),sham electroacupuncture group(SEG),electroacupuncture and overexpression group(EOG)and interference group(IG),10 rats in each group.After allocation,Rats in EAG and EOG received electroacupuncture treatment(2/100 Hz,1m A)at acupoint ST40,SP9 for 30 minutes each time daily for 28 days.Rats in SOG received acupuncture at the local skin of ST40,SP9 without electricity.In the meantime,rats in EOG and IG received lentivirus which overexpression or interfered the expression of SCAP.Body mass were measured once a week.Serum were collected form the abdominal aorta for lipid,AST,ALT and hemorheology detecting before executed.The level of TC in liver was measured by high performance liquid chromatography and the morphological changes of liver and aortic arch were observed by hematoxylin-eosin staining.Fresh liver tissue were deprived to detect the protein expression of SCAP,SREBP-2,HMGCR and PCSK9 by Western Blotting,detect mRNA level of SCAP,SREBP-2,HMGCR and PCSK9 by Realtime-PCR.Immunofluorescence were apply to investigate the expression of SCAP.The immunohistochemical(IHC)stain was used to observe the NLRP3 in liver and aortic arch.The protein expression of NLRP3,IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay(ELISA).Results:1.Effects of EA on body mass,lipid and hemorheology of HLP rats.(1)Body mass:Body mass in all groups increased persistently during intervention.Compared with NG,rats in MG have higher body mass before and after intervention(P<0.01).Body mass of rats in EAG and IG were emerging lower than that in MG after 2 weeks(P<0.01).After 3 weeks,body mass of rats in EOG were lower than that in MG(P<0.01).After 4 weeks,body mass of rats in SEG were lower than that in MG(P<0.05).Body mass in EAG were lower than that in EOG(P<0.05).There was no significant difference in body mass between the rats in IG and the EAG during the whole intervention.(2)Lipid: Compared with NG,the serum TC and LDL-C in MG were significant increased(P<0.01).After 4 week’s treatment,serum TC and LDL-C in EAG were lower than that in MG(P<0.01).Compared to MG,the serum TC in SEG was decreased(P<0.05),but there was no significant difference in serum TG between the two groups(P>0.05).Serum TC and LDL-C in EOG were lower than that in MG,and higher than that in EAG(P<0.01,P<0.05).Serum TC,TG and LDL-C in IG were lower than that in MG and EAG,but higher than that in NG(P<0.01,P<0.05).There was no significant difference in serum TG and HDL-C levels among groups,Except IG compared with MG.(3)Hemorheology: Compared with NG,the whole blood viscosity,plasma viscosity and hematocrit and deformation index in MG were significantly increased(P<0.01);Compared to MG,the whole blood viscosity,plasma viscosity and erythrocyte aggregation index were significantly decreased in EAG,EOG and IG(P<0.01).It was also decreased in SEG,but with no significance(P>0.05).Compared with EAG,the EOG rats’ viscosity 200/S,viscosity 30/S,viscosity 5/S were higher(P<0.01,P<0.05),the IG rats’ viscosity30 /S were lower(P<0.01).2.Effects of EA on TC level in liver,AST,ALT and morphology of liver and aortic arch of HLP rats.(1)HPLC results: Compared with NG,TC in liver of rats in MG were significantly increased(P<0.01);Compared with MG,TC in liver of rats in EAG ? EOG and IG were significantly decreased(P<0.01).It was also decreased in SEG,but with no significance(P>0.05).Compared with EAG,TC in liver of rats in EOG were higher(P<0.05),while IG were lower(P<0.01).(2)ALT,AST: Compared to NG,the levels of AST and ALT in MG were significantly increased(P<0.01);Compared with MG,the levels of AST and ALT in EAG,EOG and IG were significantly decreased(P<0.05,P<0.01).The level of AST and ALT was also decreased in SEG,but only the ALT level is significantly different compared with MG(P<0.05).Compared with EAG,the level of ALT of rats in EOG were higher(P<0.01,P<0.05),while the level of AST in IG were lower(P<0.05).(3)HE staining results in liver:Histopathology showed that the structure of liver lobules in NG was clear,the cells were closely connected,and the cytoplasm was uniform and rich,without vacuoles,steatosis and inflammatory cells.Diffuse steatosis was found in MG,the cytoplasm was loose,lipid droplets and inflammatory cells was obvious.The vacuolation of lipid droplets and inflammatory cells in SEG was slightly less than that in MG.Compared with MG,it showed less fat vacuoles and inflammatory cells in EOG.Cellular lipid drops and inflammatory cells in EAG were further reduced.Cellular lipid drops,and inflammatory cells were also reduced in IG.(4)HE staining results in aortic arch:The thickness of aortic cavity in NG was uniform,and no lipid or inflammation was observed in the subcutaneous area.The cell arrangement was slightly irregular,the wall of aortic arch was thicker and inflammatory cells were observed in MG.The inflammatory cells was slightly reduced in SEG.The aortic wall in EAG and IG was similar compared with MG,but only a small amount of inflammatory cells was observed under the intima.The inflammatory cells in aortic architis in the EOG was lower than that in MG,while higher than that in EAG.3.Effects of EA on expression of SCAP/SREBP-2 signaling pathway proteins levels in liver.(1)SCAP/SREBP-2 proteins levels in liver :Compared with NG,protein expression of SCAP and SREBP-2 in HLP liver increased significantly(P<0.01).After 4 week’s intervention,compared with MG,protein expression of SCAP and SREBP-2 in EAG,EOG and IG were significantly decreased(P<0.01,P<0.05)It was also decreased in SEG,but with no significance(P>0.05).Compared with EAG,protein expression of SCAP and SREBP-2 in EOG were higher(P<0.05),while it was lower in IG(P<0.01).(2)HMGCR/PCSK9 proteins levels in liver: Compared with NG,protein expression of HMGCR and PCSK9 in liver of HLP rats increased significantly(P<0.01).Compared with MG,protein expression of HMGCR and PCSK9 in EAG were significantly decreased(P<0.01,P<0.05),while it did not show significant alternation in SEG(P>0.05).Protein expression of HMGCR and PCSK9 in EOG were lower than that in MG(P<0.05),while higher than that in EAG.Protein expression of HMGCR and PCSK9 in IG were lower than that in MG and EAG(P<0.01,P<0.05).(3)Immunofluorescence results:SCAP was highly expressed in hepatocytes and localized in the cytoplasm.The expression trend between groups was consistent with the results of WB.Compared with NG,protein expression of SCAP in HLP liver increased significantly(P<0.01).After 4 week’s intervention,compared with MG,It was decreased in SEG,but with no significance(P>0.05).Protein expression of SCAP in EAG,EOG and IG were significantly decreased(P<0.01,P<0.05).Compared with EAG,protein expression of SCAP in EOG were higher(P<0.05).4.Effects of EA on expression of SCAP/SREBP-2 signaling pathway mRNA levels in liver.(1)SCAP/SREBP-2 mRNA levels in liver :Compared with NG,mRNA expression of SCAP and SREBP-2 in liver of HLP rats increased significantly(P<0.01).Compared with MG,mRNA expression of SCAP and SREBP-2 in EAG ? EOG and IG were significantly decreased(P<0.01,P<0.05).While,there was no significant changes in SCAP and SREBP-2 levels between SEG and MG(P>0.05).The mRNA expression of SCAP in EOG were higher than that in EAG(P<0.01).The mRNA expression of SCAP and SREBP-2 in IG were lower than that in MG and EAG(P<0.01).(2)HMGCR/PCSK9 mRNA levels in liver:Compared with NG,mRNA expression of HMGCR and PCSK9 in iver of HLP rats increased significantly(P<0.01).Compared with MG,mRNA expression of HMGCR and PCSK9 in EAG were significantly decreased(P<0.01,P<0.05),while it did not show significant alternation in SEG(P>0.05).The mRNA expression of HMGCR and PCSK9 in EOG were lower than that in MG(P<0.05),and higher than that in EAG(P<0.05,P<0.01).The mRNA expression of HMGCR and PCSK9 in IG were lower than that in MG(P<0.01).Besides,mRNA expression of HMGCR in IG were also lower than that in EAG(P<0.01).5.Effects of EA on expression of NLRP3,,IL-1β,IL-18 in liver and aortic arch.(1)NLRP3 levels in liver and aortic arch by IHC: Compared with NG,NLRP3 levels in liver and aortic arch was significantly increased(P<0.01).Compared with MG,both EAG,EOG and IG showed decreased NLRP3 levels(P<0.01).Compared with EAG,NLRP3 level in EOG was higher(P<0.01),NLRP3 level in IG was lower(P<0.01).(2)IL-1β and IL-18 levels in liver and aortic arch by ELISA: Compared with NG,the relative expression levels of IL-1β,IL-18 in liver and aortic arch were significantly increased(P<0.01).Compared with MG,expression levels of IL-1β,IL-18 in liver and aortic arch were significantly decreased in EAG,EOG and IG(P<0.01).Expression levels of IL-1β in liver were significantly decreased in SEG(P<0.01).No significant changes regarding to IL-18 levels in liver and IL-18 and IL-1β levels in aortic arch were observed between SEG and MG(P>0.05).The relative expression levels of IL-1β and IL-18 in liver and aortic arch in EOG were higher than that in EAG(P<0.01,P<0.05).The the relative expression levels of IL-1β and IL-18 in liver and aortic arch in IG were lower than that in MG and EAG(P<0.01).Conclusion:1.Electroacupuncture therapy can suppress the the body mass increasing of HLP rats,regulate lipid metabolism,especially the serum TC and LDL-C levels,reduce the synthesis of TC in liver,improve the hyperviscosity and hyperhysteresis of blood caused by hyperlipidemia,and inhibit the accumulation of lipid in liver and the formation of atherosclerotic plaque in the arteries.2.Electroacupuncture can down-regulate the protein and mRNA expression of SCAP,SREBP-2 and its downstream target genes HMGCR and PCSK9 in the liver of HLP rats,and inhibit cholesterol synthesis and regulate cholesterol metabolism finally.3.Electroacupuncture has the effect of ameliorate inflammation of liver and aortic arch,via regulation of SCAP/SREBP-2 and reduction level of NLRP3 and its downstream inflammatory cytokines IL-1β and IL-18.4.Electroacupuncture can down-regulate the expression of cholesterol metabolism-related target genes HMGCR,PCSK9 and inflammation-related target genes NLRP3,IL-1β,IL-18 through SCAP/SREBP-2 pathway,so as to improve hyperlipidemia related hepatic steatosis and atherosclerotic plaque formation. |
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