| Objective:In recent years,with the improvement of people's living standard and the change of dietary habits,the incidence of atherosclerosis(AS)is increasing year by year,which is one of the main causes of death.Monocyte derived macrophage plays an important role in the development of AS,.the blood monocytes through the endothelium and differentiate into macrophages stimulatded by various external factors.The surface of A scavenger receptor of macrophage containing large amounts of oxidation low density lipoprotein(ox-LDL)when blood lipid concentration is too high,caused by lipid deposition within the cytoplasm of the final formation of foam cells and foam cells with characteristic pathological cells in AS plaques,there are a large number of plaques in AS.A large number of studies have confirmed that the occurrence of AS is the result of lipid metabolism disorder and chronic inflammation.Other studies have shown that LPS can increase the occurrence of AS by activating the LOX-1/NF-kappa B pathway.Paeoniflorin is a traditional Chinese medicine composition,clinical research shows that it has a good anti-inflammatory effects.In this study we observated the effect of albiflorin on foam cell formation and AS by establishing foam cell model and mouse model of atherosclerosis and explore its mechanism.Methods:1.The effect of paeoniflorin on the formation of THP-1 derived foam cells1)In order to study the effect of paeoniflorin on foam cell formation,a stable THP-1 derived foam cell model was established.THP-1 cells were divided into two groups respectively:THP-1 group and the group of macrophages.Macrophages were treated with 160nmol/L PMA and incubated for 24 hours.Then the induced cells were divided into two groups for the group of macrophages and foam cells group,foam cells treated with ox-LDL for 48 hours,and the cells were stained with oil red O,to observeo the intracellular lipid deposition.2)In order to study the effect of paeoniflorin on foam cell formation.The macrophages were divided into three groups: macrophage group(control group);foam cells group: macrophage cells treated with 50mg/L ox-LDL,and static culture for 48 hours to induce foam cells;albiflorin group: macrophages pretreatmented with albiflorin for 1 hours after adding 50mg/L ox-LDL and incubated 48 hours.The lipid deposition of cells in each group was observed by oil red O staining,and the content of cholesterol(TC)and triglyceride(TG)in the cell lysate were measured.3)In order to study the change of LOX-1/NF-kappa B pathway in foam cell formation.The THP-1 induced macrophages were treated with different concentrations of ox-LDL(25/50/100mg/L)for 48 hours,and untreated macrophages were used as control group.The total RNA and cytoplasmic protein were collected in each treatment group.The expression of LOX-1 and NF-kappa B in the transcription and protein levels were determined by quantitative PCR and Western blot,respectively.The changes of IL-6 and TNF-a expression were measured.4)In order to further determine the role of LOX-1/NF-kappa B in foam cell formation.The cells were divided into four groups: macrophage group(control group);ox-LDLgroup : cells treated with 50mg/L ox-LDL for 48 hr;LOX-1 antibody group: cells to LOX-1 neutralizing antibody pretreated for 1 hours,then added 50mg/L ox-LDL for 48 hours,the antibody concentration was 10ug/ml;NF-kappa B inhibitor(PDTC)group: cells pretreated with NF-kappa B inhibitorfor 24 hours,then added 50mg/L ox-LDL 48 hours,the final concentration of 40umol/L inhibitors.Oil red O staining was used to observe the cell lipid deposition in each group,and the expression levels of LOX-1 and NF-kappa B in the transcription and protein levels were determined by quantitative PCR and western,respectively.5)In order to observe the effect of paeoniflorin on the expression of LOX-1/NFkappa B pathway in foam cell formation.The THP-1 cells induced by PMA into macrophages were divided into three groups respectively: macrophage group:control group;foam cell group : macrophages treated with 50mg/L ox-LDLfor 48 hours induced into foam cells;albiflorin group: macrophages albiflorin pretreated with albiflorin for 1 hours after adding 50mg/L ox-LDL and coincubated for 48 hours.The total RNA and cell protein were extracted respectively.The expression levels of LOX-1 and NF-kappa B in the transcription and protein levels were detected by quantitative PCR and Western blot.The changes of IL-6 and TNF-a expression were measured.2 Effects of paeoniflorin on atherosclerosis in mice1)In order to study the effect of paeoniflorin on the formation of atherosclerosis in mice.The ApoE-/-mice were randomly divided into three groups: control group(NC),high fat group + saline group(HFD),high fat group + paeoniflorin group(HFD+A).Determing the content of triglycerides(TG),cholesterol(TC),low density lipoprotein(LDL)and high density lipoprotein(HDL)in serum by detection kit,and observing the pathological change of aorta and lipid deposition by HE and oil red O staining of the thoracic aorta.2)In order to study the effect of paeoniflorin on LOX-1/NF-kappa B pathway in atherosclerosis model in mice.The ApoE-/-mice were randomly divided into three groups: control group(NC),high fat group + saline group(HFD),high fat group + paeoniflorin group(HFD+A).The protein and total RNA were isolated from thoracic aorta.The changes of LOX-1 and NF-kappa B expression levels were measured by quantitative PCR and Western blot,and the changes of IL-6 and TNF-a expression were measured.Results:1.The effect of paeoniflorin on the formation of THP-1 derived foam cells1)After treatment of PMA,THP-1 cells were adherent growth and a large number of cells had pseudopodia into macrophages like changes and expressed CD14.After ox-LDL treatment,the oil red O staining were deposited in a large number of lipid deposits.THP-1 derived foam cell model was successfully established.2)The results showed that the lipid deposition in the cytoplasm of the cells decreased significantly compared with that of the cells treated with paeoniflorin,and the contents of cholesterol(TC)and triglyceride(TG)in the cell lysate were significantly decreased in the process of paeoniflorin pretreatment.3)After treatment of macrophages with different concentrations of ox-LDL.The results of quantitative PCR and Western blot showed that the expression level of LOX-1 and NF-kappa B increased with the increase of ox-LDL concentration,and the expression of IL-6 and TNF-a was also increased.4)The results of oil red O staining showed that the lipid deposition of cytoplasm was improved obviously,and the results of quantitative PCR and Western blot showed that the expression levels of LOX-1 and NF-kappa B were significantly decreased,and the expression of IL-6 and TNF-a were significantly reduced after cells treated with LOX-1 neutralizing antibody and NF-kappa B inhibitor.5)The results of quantitative PCR and Western blot showed that the expression levels of LOX-1 and NF-kappa B were significantly decreased,and the expression of IL-6 and TNF-a were significantly reduced after treated with paeoniflorin.2 Effects of paeoniflorin on atherosclerosis in mice1)The contents of serum triglyceride(TG),cholesterol(TC),low density lipoprotein(LDL)and high density lipoprotein(HDL)were decreased after treatment with paeoniflorin in mice model of atherosclerosis.And the results of HE and oil red O staining showed that the pathological changes and lipid deposition were significantly improved.2)The expression of LOX-1 and NF-kappa B in thoracic aorta tissue was significantly decreased and the expression of IL-6 and TNF-a were significantly decreased after the treatment with paeoniflorin.Conclusions:1)Paeoniflorin reduced the accumulation of lipid in the cytoplasm of macrophages by regulating the LOX-1/NF-kappa B signaling pathway and blocked the foaming process of ox-LDL.2)Ox-LDL promotes the formation of foam cells in plaque instability by up regulating the expression of LOX-1 and NF-kappa B.The degradation of atherosclerotic plaque formation by regulating expression of LOX-1/NF-kappa B pathway and inflammatory molecules inhibit lipid glycosides in Paeonia in this process and the inhibition of collagen fiber in plaque and plaque,plaque rupture prevention,to avoid the occurrence of adverse events. |
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