Nicotinamidemononucleotide Improves Hypercholesterolemia Throughthe HNF-1α/PCSK9/LDLR Pathway

Objectives: To explore the effect and mechanism of nicotinamide mononucleotide(NMN)on hypercholesterolemia.Methods:(1)The cholesterol kit was used for detecting serum low density lipoproteincholesterol(LDL-C),total cholesterol(TC),triglyceride(TG)and high density lipoproteincholesterol(HDL-C)of SD rats fed normal diet,high fat diet and high fat diet treated with NMN.(2)Huh7 cells were treated with different concentrations of NMN(0,12.5,25,50,100,200 μmol/L)for 24 hours,and CCK-8 kit was used to detect the survival rate of the cells.(3)Pretreatment with 50 mg/L LDL for 2hours made Huh7 cells lipid-filled,and then added different concentrations of NMN to treat the cells for 24 hours,and oil red O staining was used to observe the intracellular lipid accumulation.(4)Pretreated with different concentrations of NMN Huh7 cells for 24 hours,and then cultured with 20μg/m L of Dil-LDL for another 4 hours and the ability of cells to take up Dil-LDL was detected by fluorescence microscopy.(5)Huh7 cells were treated with NMN at different concentration gradients for 24 hours.The expressions of LDLR,PCSK9,HNF1α,SREBP-2 protein and mRNA were detected by Western blot and q RT-PCR.(6)Huh7 cells were treated with 100 mol/L NMN at different time gradients(0,4,8,16,24 hours),and the protein and mRNA expressions of LDLR,PCSK9,HNF1α and SREBP-2 were detected by Western blot and q RT-PCR.Results:(1)The results showed that the serum levels of TG,TC and LDL-C in SD rats fed with high-fat diet were higher than those of SD rats fed with ordinary diet,and HDL-C levels were not significantly affected;SD rats fed with high-fat diet after NMN drug treatment,the serum levels of TG,TC,and LDL-C were lower than that of SD rats fed with a high-fat diet,and HDL-C levels had no significant effect.(2)CCK-8 test results showed that different concentration gradients of NMN had no obvious differenceon the survival rate of Huh7 cells.(3)Oil red O staining showed that NMN increased the number of orange-red lipid droplets in a concentrationdependent mannerin Huh7 cells.(4)Dil-LDL assay results showed that NMN increased the uptake of Dir-LDL by Huh7 cells in a concentration dependent manner.(5)Western blot and q RT-PCR results showed that NMN significantly up-regulated the expression of LDLR and down-regulated the expression of PCSK9 and HNF1α in Huh7 cells at the concentration of 100 and 200 mol/L,but had no significant effect on the expression of SREBP-2.(6)Western blot and q RT-PCR results showed that NMN significantly up-regulated the expression of LDLR in Huh7 cells at 24 hours,while down-regulated the expression of PCSK9 and HNF1α,but had no significant effect on the expression of SREBP-2.Conclusion: NMN can regulate the HNF1α/PCSK9/LDLR pathway to increase the uptake of cholesterol by hepatocytes,thereby reducing thehighcholesterol level in plasma and improving the blood lipid level.