Research On Regulatory Effect Of Pyranoanthocyanin Vitisin A On Cholesterol Metabolism

Hypercholesterolemia is the pathological basis for inducing cardiovascular disease(Cardiovascular disease,CVD).The regulation of cholesterol is an effective means to treat hypercholesterolemia and prevent CVD.The commonly used statins in clinical practice can bring certain side effects to the body.Therefore,seeking natural functional active substances to improve the body's cholesterol metabolism has become a research hotspot.Anthocyanin is a kind of flavonoid compound,which is widely found in dark fruits and vegetables,it has strong physiological activity.As an anthocyanin derivative,pyrananthocyanin is produced during the fermentation and aging process of berry rich in anthocyanin.Its structure is that the hydroxyl group of proanthocyanidin between C₄ and C₅ undergoes cyclization addition condensation to form the fourth pyran ring.Compared with the prototype anthocyanin,the pyrananthocyanin has better stability,and also has good anti-inflammatory,anti-oxidant and other biological activities,but it is difficult to prepare high-purity pyrananthocyanin,which limits further research on it.And there is a lack of research reports on the metabolism of pyrananthocyanins.Based on this,our subject has carried out the following work:(1)First,we use Cyanidin-3-O-glucoside(Cyanidin-3-O-glucoside,C3G)and pyruvate to synthesize Vitisin A,and use a medium-pressure preparation device to separate high-content Vitisin A,and use the liquid phase Chromatography and mass spectrometry(Liquid chromatography-mass spectrometry,LC-MS)to carry out qualitatively and quantitatively analysis,and the prepared Vitisin A content had reached 94.8%,which can be used for subsequent cell experiments.(2)To explore the regulation of Vitisin A on the intracellular cholesterol metabolism of HepG2 cells under high cholesterol environment,and further explore the mechanism of Vitisin A on cholesterol metabolism.40?M cholesterol and 4?M 25-hydroxycholesterol were used to establish a HepG2 cell high cholesterol model,and 0-200?M C3G and Vitisin A were used for intervention.After 24 hours of intervention,the CCK8 method was used to detect cell viability,and the cell morphology was observed with an inverted microscope.In order to determine the optimal dose of the intervention substance,the total cholesterol content of HepG2 cells was further measured with a kit to evaluate the cholesterol-lowering effect of the intervention substance.The results show that high concentrations of C3G and Vitisin A have the effect of lowering cholesterol,but the effect of C3G is not significant,and Vitisin A can significantly reduce the amount of intracellular total cholesterol.The cholesterol-lowering effect of different concentrations of Vitisin A is dose-dependent,and reaches the maximum when the maximum dose used is 200?M.(3)Using qPCR and Western Blot technology to analyze the molecular mechanism of Vitisin A regulating cholesterol metabolism.Through the determination and analysis of cholesterol metabolism-related genes and proteins,it can be seen that Vitisin A up-regulates the expression of SREBP2,LDLR,ABCA1 and PPAR?at the m RNA level,and reduces the transcription level of HMGCR and ABCG5,while there is no significant difference in the gene expression of LXR?,PCSK9 and ABCG8.At the protein level,it increases the protein level of LDLR and inhibits the expression of HMGCR,ABCG5,PCSK9 and LXR?,but has no significant effect on the expression of CYP7A1.It shows that Vitisin A can reduce the cholesterol content in HepG2 cells by inhibiting the synthesis of endogenous cholesterol,promote the reverse transport of cholesterol,and maintain the balance of the steady-state level of cholesterol in the liver.This study can be used as a theoretical basis for Vitisin A's natural functional factors to prevent hypercholesterolemia.