The Role Of Glucocorticoids And Berberine In The Metabolism Of Low Density Lipoprotein And PCSK9

BackgroundSystemic lupus erythematosus(SLE)is a kind of immune disease which involves complicated inflammation.It is demonstrated that SLE can lead to premature atherosclerosis.This may be caused by the disease itself and the side effect of treatment.Glucocorticoid is the main therapeutic drug.Researches showed that glucocorticoid can cause hypertension,dyslipidemia,glucose intolerance and obesity.All these side effects can contribute to the atherosclerosis.In terms of lipid metastasis,it is shown that glucocorticoid can increase the level of low density lipoprotein(LDL)by reducing hepatic low density lipoprotein receptor(LDLR).Our research group previously found that the level of Proprotein convertase subtilisin kexin type 9(PCSK9)of the patient in acute phase increased significantly comparing to that of patient in stable phase.PCSK9 associates with LDL metastasis closely.It can reduce the clearance of LDL via decomposing the hepatic LDLR,and thus increase the level of LDL in the circulation.As a result,PCSK9 lead to atherosclerosis.Some researches showed that berberine have the effect of reducing hepatic expression of PCSK9 as well as increasing LDLR.Our research selected the SLE patients in Peking Union Medical College Hospital who registered in the Chinese Rheumatology Information Platform.We collected the data and detected the PCSK9 level of these patients,and then analyzed statistically.We also use the HepG2 cell to explore the relationship between glucocorticoid and LDL metastasis and the relationship between the berberine and LDL metastasis.Section one:The influence of glucocorticoid to the lipid level of SLE patientObjectiveAnalyze the levels of TC?TG?HDL and LDL in the SLE patients who use different glucocorticoid doses.Study the atherosclerosis risk factors which associate with PCSK9 possibly.MethodsSelected 67 SLE patients aged between 20-45 years old from Peking Union Medical College Hospital who have registered in the Chinese rheumatology platform.33 patients use no or low dose glucocorticoid(less than 0.5mg/kg/d prednisone)and 34 patients use moderate or high dose glucocorticoid(more than 0.5mg/kg/d prednisone).Measured serum PCSK9 levels of these patients by ELISA.Then acquire the patients'information from the Chinese rheumatology platform.Analyze the data by SPSS software.Finally analyze the relationship between PCSK9 and atherosclerosis related indicator by stepwise multiple linear regression.Results1.The patients who used moderate or high dose of glucocorticoid in recent one month had a significantly higher level of TG,TC and LDL than those who used no or low dose.The level of HDL had no statistical significance between the two groups.2.The patients who used moderate or high dose of glucocorticoid in recent one month had a significantly higher level of PCSK9 than those who used no or low dose.3.The analysis of stepwise multiple linear regression indicated that the factors influences the level of PCSK9 are SLEDAI score,daily glucocorticoid dose and diastolic blood pressure.Conclusions1.Moderate or high dose glucocorticoid cause SLE patients increased TC,TG and LDL level.2.The level of PCSK9 may be affected by the disease activity,blood pressure and daily glucocorticoid dose.Section two:The effect of glucocorticoid on the LDL metastasis in HepG2 cellObjectiveExplore the effect of glucocorticoid on LDLR and PCSK9 in cell level.Study the effect of glucocorticoid on the LDL uptake in HepG2 cell.Methods:Resuscitated HepG2 cell and then passage until the amount of the cell meet the required.Digested the cells and seeded them on 6 or 12 wells plates.After 24 hours,change with LPDS medium to cultured 8 hours.Add different concentrations hydrocortisone to the wells.Some cells were lysed to extract the RNA after 24 hours.Measured mRNA relative expression by RT-Q-PCR.Some cells were added with Dil-labeled LDL.Cultured the cells in dark place for 5 hours.Examine the uptake by fluorescence microscope.Digested the cell off and measured the mean fluorescence intensity by flow cytometry.Results1.Within the concentration of 0-200ng/ml,glucocorticoid decreases the expression of LDLR mRNA in HepG2,and is in time and concentration dependent.2.Within the concentration of 0-200ng/ml,glucocorticoid decreases the ability of uptake LDL in HepG2.3.Within the concentration of 0-200ng/ml,glucocorticoid decreases expression of PCSK9 mRNA in HepG2.Conclusions1.Glucocorticoid decrease HepG2 LDLR in transcriptional level and weaken the function of LDLR uptake.2.Glucocorticoid decrease HepG2 PCSK9 in transcriptional level.Section three:The effect of Berberine(BBR)on LDL metastasis in HepG2 and its antagonism against the LDL disorder caused by glucocorticoid.Objective:Study the mechanism of the BBR effect on LDL in cell level.Explore the BBR antagonism against the LDL disorder caused by glucocorticoid.Methods:Resuscitated HepG2 cell and then passage until the amount of the cell meet the required.Digested the cells and seeded them on 6 or 12 wells plates.After 24 hours,change with LPDS medium to cultured 8 hours.Add different concentrations hydrocortisone to the wells.Some cells were lysed to extract the RNA after 24 hours.Measured mRNA relative expression by RT-Q-PCR.Some cells were added with Dil-labeled LDL.Cultured the cells in dark place for 5 hours.Examine the uptake by fluorescence microscope.Digested the cell off and measured the mean fluorescence intensity by flow cytometry.Seeded cells to new plates,added different concentration of glucocorticoid and BBR to the wells together.Measured mRNA expression an Dil-LDL mean fluorescence intensity.Results1.BBR increase LDLR mRNA expression in HepG2 in time and concentration dependent manner.2.BBR increase the ability of LDL uptake in HepG2.3.BBR decrease PCSK9 mRNA expression in HepG2 in time and concentration dependent manner.4.BBR decrease the expression of SREBP-2 and HNF-1 mRNA,which are two upstream regulators of PCSK9.5.Glucocorticoid decrease LDLR mRNA expression in HepG2 and BBR act against this effect.6.Glucocorticoid decrease PCSK9 mRNA expression in HepG2 and BBR strengthen this effect.7.Glucocorticoid decrease LDL uptake in HepG2 and BBR act against this effect.Conclusions1.SREBP-2 and HNF-1,which are two upstream regulators of PCSK9,are decreased by BBR in transcriptional level in HepG2.And PCSK9 mRNA is also decreased by BBR.2.BBR can enhance LDLR mRNA expression as well as the function of LDL uptake in HepG2.