A Study Of Lipoprotein Metabolic Regulation In Coronary Heart Disease With Phlegm Turbidity Syndrome Based On PCSK9/LDLR Pathway

1 Objective:To investigate the regulatory mechanism of lipoprotein in coronary heart disease(CHD)with phlegm turbidity syndrome(PTS),we examined the related index of PCSK9/LDLR and oxLDL/LOX-1 pathway.We observed and compare the differences between CHD patients and healthy control,and difference between CHD with and without PTS.We supplement biological evidence for clinical diagnosis of patients with CHD and PTS.We also provide reference for diagnosis of CHD with PTS,by comparing the level and ratio of HDL and LDL subtypes.2.Method:2.1 Study on the levels of serum PCSK9,LRP1,oxLDL,LOX-1,HSP27 and the expression of LRP1 in peripheral blood mononuclear cells in patients with CHD and PTSWe collected 131 patients with CHD from cardiovascular department of Guanganmen Hospital of China Academy of Chinese Medical Sciences from September 2016 to January 2017,and 30 healthy persons as control over the same period from Guanganmen hospital.In accordance with the TCM differential diagnosis,CHD patients were divided into PTS group and non-PTS group.Fasting blood samples were collected from all subjects in the morning.The levels of serum PCSK9,LRP1,oxLDL,LOX-1,HSP27 were measured by ELISA method,and the expression of LRP1 mRNA in PBMC was measured by RT-PCR.The above indexes were compared between patients with CHD and healthy people,and between PTS and non-PTS.2.2 Study on plasma HDL,LDL subtypes and serum sd-LDL levels in patients with CHD and PTSWe collected 69 patients with CHD from cardiovascular department of Guanganmen Hospital of China Academy of Chinese Medical Sciences from September 2016 to January 2017,and 22 healthy persons as control over the same period from the Guanganmen hospital.In accordance with the TCM differential diagnosis,CHD patients were divided into PTS group and non-PTS group.Lipoprint system was used to detect the distribution and level of HDL and LDL in plasma,and the serum sdLDL was detected using ELISA.The above indexes were compared between CHD patients and healthy people,and between PTS and non-PTS.2.3 Statistical analysisSpss20.0 statistical software was used for statistical processing.The measurement were input as mean + standard deviation(x±s).The t test was used for groups close to normal distribution,and the variance test was used when the sample is consistent to normal distribution and the homogeneity of variance.Enumeration data were described in terms of frequency and composition ratio,Chi square test was used for comparison between groups.3.Result:3.1 The level of serum PCSK9,LRP1,oxLDL,LOX-1,HSP27 and the expression of LRP1 mRNA in PBMC in CHD patients and healthy people.3.1.1 Results of serum lipid level in CHD patients and healthy people.CHD group:TC increased,showed a significant difference(p<0.05);TG,LDL-C,VLDL increased,showed a very significant difference(p<0.01);HDL-C decreased,showing a very significant difference(p<0.01).3.1.2 The levels of serum PCSK9,LRP1,oxLDL,LOX-1 and HSP27 in CHD patients.CHD group:LOX-1,oxLDL increased,HSP27 decreased,with significant difference(p<0.05);PCSK9,LOX-1 increased,showed a very significant difference(p<0.01).3.1.3 Results of LRP1 mRNA expression in PBMC in CHD patients.CHD group(compared with healthy people):LRP1 mRNA decreased,showing a very significant difference(P<0.01).3.2 The level of PCSK9,LRP1,oxLDL,LOX-1,HSP27 and the expression of LRP1 mRNA in PBMC in CHD patients with PTS and without PTS.3.2.1 The results of blood lipid level in CHD patients with PTS.PTS group:LDL-C,ApoB increased,showed a significant difference(p<0.05).3.2.2 Results of serum PCSK9,LRP1,oxLDL,LOX-1 and HSP27 level in CHD patients with PTS.PTS group:LRP1 and LOX-1 increased,HSP27 decreased,there was significant difference(p<0.05);PCSK9,oxLDL increased,showed a very significant difference(p<0.01).3.2.3 Results of LRP1 mRNA expression in PBMC in CHD patients with PTS.PTS group:LRP1 mRNA decreased,showing a very significant difference(P<0.01).3.3 The results of level and constituent ratio of plasma HDL,LDL subtypes,and serum sdLDL level in CHD patients and healthy people.3.3.1 Results of serum lipid level in CHD patients and healthy people.CHD group:VLDL increased,showed a significant difference(p<0.05);TG,LDL-C,increased,showed a very significant difference(p<0.01);HDL-C decreased,showing a very significant difference(p<0.01).3.3.2 The constituent ratio of HDL subtypes in CHD patients.CHD group:HDL1%,HDL4%decreased and showed a significant difference(p<0.05);HDL2%,HDL3%,Large(1-3)%deceased,showed a very significant differences(p<0.01),HDL6%,HDL7%,HDL8%,HDL9%,HDL 10%,Intermediate(4-7)%,Small%(8-10)increased,showed a very significant difference(p<0.01).3.3.3 The level of HDL subtypes in CHD patients.CHD group:HDL1,HDL3,HDL4 decreased and showed a significant difference(p<0.05);HDL2,Large(1-3)decreased and showed a very significant differences(p<0.01),HDL6,HDL7,HDL8,HDL9,HDL10,Small(8-10)increased,showed a very significant difference(p<0.01).3.3.4 The constituent ratio of LDL subtypes in CHD patients.CHD group:LDL1%decreased,showing significant difference(p<0.05);LDL3%,LDL4%,LDL5%increased,showed a very significant difference(p<0.01).3.3.5 Results of level of LDL subtypes and mean LDL particle size in CHD patients and healthy subjects.CHD group:LDL2,LDL3,LDL4,LDL5 increased,showed a very significant difference(p<0.01);the average size of LDL particles decreased,showed a very significant difference(p<0.01).3.3.6 Results of serum sdLDL level in CHD patients.CHD group:serum sdLDL level increased showed a significant difference(p<0.05).3.4 The results of level and constituent ratio of plasma HDL,LDL subtypes,and serum sdLDL levels in CHD patients with PTS.3.4.1 Results of serum lipid level in CHD patients with PTS.PTS group:LDL-C,ApoB increased,showed a very significant difference(p<0.01).3.4.2 The constituent ratio of HDL subtypes in CHD patients with PTS.PTS group:HDL2%,HDL3%,HDL4%,Large(1-3)%deceased,showed a very significant differences(p<0.01),HDL6%,HDL7%,HDL8%,HDL9%,HDL10%,Intermediate(4-7)%,Small%(8-10)increased,showed a very significant difference(p<0.01).3.4.3 The level of HDL subtypes in CHD patients with PTS.PTS group:HDL2,HDL3,HDL4,Large(1-3)decreased and showed a very significant differences(p<0.01),HDL8,Small(8-10)increased,showed a significant difference(p<0.05).3.4.4 The constituent ratio of LDL subtypes in CHD patients with PTS.PTS group:LDL 1%decreased,showed a very significant difference(p<0.01);LDL3%,LDL4%,LDL5%increased,showed a very significant difference(p<0.01).3.4.5 Results of level of LDL subtypes and mean LDL particle size in CHD patients with PTS.CHD group:LDL3,LDL4,LDL5 increased,showed a very significant difference(p<0.01);the average size of LDL particles decreased,showed a very significant difference(p<0.01).3.4.6 Results of serum sdLDL level in CHD patients with PTS.PTS group:serum sdLDL level increased and showed a significant difference(p<0.05).4.Conclusion:4.1 CHD patients shows increase serum level of PCSK9,LRP1,oxLDL,LOX-1,decrease level of HSP27,and down-regulation of LRP1 mRNA expression in PBMC.The level of LDL-C and VLDL is presumably regulated through PCSK9/LDLR and oxLDL/LOX-1 pathway,so the accumulation of cholesterol is mediated by inflammation and oxidative stress,and finally leads to formation of CHD.4.2 CHD patient with PTS shows increase serum level of PCSK9,LRP1,oxLDL,LOX-1,decrease level of HSP27,and down-regulation of LRP1 mRNA expression in PBMC,suggesting that LDL-C、apoB are mediated through PCSK9/LDLR and oxLDL/LOX-1 pathway,and participate in regulating the formation of PTS.4.3 The CHD patient shows decreased large particles and increased small particles in HDL composition,and increased small particles of LDL(LDL3-7%),increased serum sdLDL and decreased mean LDL particle size,indicating that small particles of HDL and small particles LDL participate in formation of CHD,large particles of HDL may have preventive function on CHD.4.4 The CHD patient with PTS shows decreased large particles and increased small particles in HDL composition,and increased small particles of LDL(LDL3-7%),increased serum sdLDL and decreased mean LDL particle size,indicating that small particles of HDL and small particles LDL participate in formation of CHD and PTS,large particles of HDL may have preventive function on CHD and PTS.