1,Study On Molecular Mechanism Of Lipid Metabolism In Inflammatory Mice Caused By Rnai-mediated Gene Inhibition Of Insig-1 2,Analysis Of The Correlation Between Non-High Density Lipoprotein Cholesterol And Coronary Heart Disease In Elderly

Objective: To construct the Insig-1 siRNA plasmid in mice and identify the interference efficiency.Methods: Using molecular biological methods,three siRNA plasmid of Insig-1 and one non-homologous negative control vector were designed and synthesized and inserted into pGenesil-1 to generate the recombinant vectors. The recombinant vectors were named pGen-Insig-1-1(si-1), pGen-Insig-1-2(si-2), pGen-Insig-1-3(si-3) and pGen-Insig-1-nc(si-nc). After identification by restriction enzyme digestion and sequencing, the vectors were transfected into RAW264.7 cells by Lipofectamine 2000 liposome. The interference efficiency of pGen-Insig-1 was identified by reverse transcription PCR (RT-PCR) and western blot.Result: The recombinant vectors were verified correctly by result of DNA sequencing .The mRNA and protein expression of Insig-1 significantly decreased in RAW264.7 cells transfected with pGen-Insig-1 compared with untransfected cells by RT-PCR and western blot(P<0.05). Of three interference plasmids, interference efficiency of pGen-Insig-1-2 was the best.Conclusion: The siRNA plasmid of Insig-1 in mice was successfully constructed. Objective: To detect the effect of RNAi-mediated Insig-1 inhibition on lipid metabolism of RAW264.7on inflammation.Methods: RAW264.7 cells were cultured in 24-well plates. The interference vector pGen-Insig-1-2 and pGen-Insig-1-nc was transfected by Lipofectamine 2000 liposome after 24h and the medium containing 300μg/ml G418 was added after 24h. RAW264.7 cells transfected pGen-Insig-1-2 and the control plasmid pGen-Insig-1-nc were cultured in selection medium for 14 days, and a single growth was picked. The mRNA and protein expression of Insig-1 in the stable interference were identified by RT-PCR, western blot and immunocytochemistry. The optimal concentration of LDL and LPS were decided according to the protein expression of LDLr .Total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) levels were detected in pGen-Insig-1-2/RAW264.7, pGen-Insig-1-nc/RAW264.7 and RAW264.7 cells loading LDL and LPS by enzyme. Intracellular lipid accumulation situation was detected by Oil red O staining. Total cellular RNA and protein were extracted and expression levels of SCAP, LDLr and SREBP-2 were checked by RT-PCR and western blot.Results: Stable cell line transfected pGen-Insig-1 were identified by G418 and named pGen-Insig-1-2/RAW264.7 cells and pGen- Insig-1-nc/RAW264.7 cells. The mRNA and protein expression of Insig-1 significantly decreased in pGen-Insig-1-2/RAW264.7 cells compared with pGen-Insig-1-nc/RAW264.7 cells and untransfected cells by RT-PCR,western blot and immunocytochemistry(P<0.05).The concentration of LDL and LPS was 50μg/ml and 200ng/ml respectively according to the protein expression of LDLr. By Oil red O staining ,intracellular lipid droplets of red dye increased in RAW264.7cells loading 50μg/ml LDL and 200ng/ml LPS compared with untreated cells(blank control ); The intracellular lipid droplets of red dye were no significant difference between pGen-Insig-1-2-nc/RAW264.7 cells with the RAW264.7cells untransfected with pGen-Insig-1 (negative control) when stimulated by the same conditions; The red stained lipid droplets significantly increased in pGen-Insig-1-2/RAW264.7 cells compared with the cells untransfected pGen-Insig-1 (negative control) when stimulated by the same conditions. The change of TC , FC and CE by enzyme was similar with by Oil red O staining . By RT-PCR and western blot,the mRNA and protein levels of SCAP, LDLr and SREBP-2 decreased in RAW264.7cells loading 50μg/ml LDL and 200ng/ml LPS compared with untreated cells(blank control )(P<0.05) ;The mRNA and protein levels of SCAP, LDLr and SREBP-2 decreased in pGen-Insig-1-2/RAW264.7 cells compared with the RAW264.7cells untransfected pGen-Insig-1 when stimulated by the same conditions (P <0.05); The mRNA and protein levels of SCAP, LDLr and SREBP-2 were no significant difference between pGen-Insig-1-nc /RAW264.7 cells with the RAW264.7 cells untransfected pGen-Insig-1 (negative control) when stimulated by the same conditions (P>0.05).Conclusion: Under inflammatory stress, Insig-1 inhibition increased the mRNA and protein levels of SCAP, LDLr and SREBP-2 and promoted the intake of LDL in RAW264.7 cells, leading to cellular cholesterol ester accumulation and causing foam cell formation. Objective: To detect the effect of RNAi-mediated Insig-1 inhibition on lipid metabolism of mice on inflammation.Methods: The inflammatory model of mice was prepared by intraperitoneal injection of casein, and the concentrations of IL-6 and SAA in serum were measured to determine inflammatory model by ELLISA. PGen-Insig-1-2 and pGen-Insig-1-nc were injected into C57BL/6 inbred mice by tail vein. The total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) in mice serum were detected by enzyme; Lipid accumulation in liver and kidney were detected by Oil red O staining; Total cellular RNA and protein of liver were extracted, and SCAP, LDLr and SREBP-2 in the mRNA and protein expression levels were checked by RT-PCR and western blot.Result: IL-6 increased slightly but SAA significantly in serum of mice injected casein in vivo and fed high fat diet compared with mice injected with PBS in vivo and fed normal diet ( blank control ). The levels of TC, LDL-C, HDL-C and TG in serum increased in mice fed high fat diet and injected casein in vivo compared with mice fed normal diet and injected PBS in vivo(blank control) (P<0.05); The levels of TC, LDL-C, HDL-C and TG in serum decreased in mice fed high fat diet, injected casein in vivo and PGen-Insig-1-2 transfected compared with mice fed high fat diet , injected casein in vivo but untransfected (negative control) (P<0.05);But no significant difference between mice fed high fat diet, injected casein in vivo and PGen-Insig-1-nc trasfected mice fed high fat diet and injected casein in vivo (negative control) (P>0.05). Lipid droplets of red dye of liver and kidney increased in mice fed high fat diet and injected casein in vivo compared with mice fed normal diet and injected PBS in vivo (blank control) (P<0.05); Significantly increased in mice fed high fat diet , injected casein in vivo and PGen-Insig-1-2 transfected compared with mice fed high fat diet and injected casein in vivo but untransfected (negative control) (P<0.05); But no significant difference between mice fed high fat diet, injected casein in vivo and PGen-Insig-1-nc trasfected mice fed high fat diet and injected casein in vivo but untransfected (negative control) (P>0.05). By RT-PCR and western blot ,the mRNA and protein levels of SCAP, SREBP-2 and LDLr increased in mice fed high fat diet and casein in vivo compared with mice fed normal diet and injected PBS in vivo (blank control) (P<0.05); Significantly increased in mice fed high fat diet , injected casein in vivo and PGen-Insig-1-2 transfected compared with mice fed high fat diet and injected casein in vivo but untran sfected( negative control) (P<0.05) ;But no significant difference between mice fed high fat diet, injected casein in vivo and PGen-Insig-1-nc trasfected mice fed high fat diet and injected casein in vivo but untransfected (negative control) (P>0.05).Conclution: Under inflammatory stress, Insig-1 inhibition increased the mRNA and protein levels of SCAP, LDLr and SREBP-2 in liver and promoted the intake of LDL in liver and kidney ,leading to cellular cholesterol ester accumulation and causing foam cell formation. Objective: To investigate the correlation between non-high density lipoprotein cholesterol (non-HDL-C) level and CHD and coronary lesions in an aged population.Methods: 1272 cases of old patients who were more than 65 years old and accepted for coronary angiography were analyzed retrospectively. Based on the result of coronary angiography, the patients were divided into control group and CHD group. Further, 767 CHD patients were divided into subgroups according to the number of branches with pathological changes and Gensini score. Serum TC, HDL-C, LDL-C, and TG were assayed to measure the level of non-HDL-C. The differences in non-HDL-C among groups were compared, and the correlation between non-HDL-C and coronary artery disease degree was also analyzed.Results: The non-HDL-C level in the CHD group was significantly higher than that in the control group (P<0.01). Further, the serum non-HDL-C showed an increasing tendency accompanied by the increase in branches with lesions. Compared with patients with single or double coronary artery branches lesions, the non-HDL-C in those with multiple lesions in branches was significantly increased (P<0.01, P<0.05). With the aggravation of coronary artery stenosis, the serum non-HDL-C level was gradually increased. The most significant increase was observed in the group with Gensini score of more than 40. There was a significant difference between Gensini score >40 group and Gensini score of 20-40 group and Gensini score <20 group, respectively (P<0.05, P<0.01). Meanwhile, there were significant positive correlations between the serum non-HDL-C level and coronary lesions area and severity (r=0.147, P<0.01; r=0.152, P<0.01).Conclusion: In conclusion, serum non-HDL-C was closely associated with development of CHD and coronary artery lesions severity. It is also a key index to assess CHD risk.