Chlorogenic Acid Downregulates The Expression Of NPC1L1 And HMGCR Through PXR And SREBP2 Signaling Pathways And Their Interactions With HSP90 To Maintain Cholesterol Homeostasis

Background:Hypercholesterolemia plays a crucial role in the development of several diseases,such as cardiovascular disease,non-alcoholic fatty liver disease,diabetes,neurodegenerative diseases,and cancer,particularly atherosclerosis.Therefore,maintaining the balance of cholesterol metabolism is extremely important.Sterol regulatory elements binding protein 2(SREBP2)is a major transcription factor that regulates cholesterol homeostasis.It can regulate 3-hydroxy-3-methylglutaryl-Co A reductase(HMGCR)and Niemann-pick C1-like 1(NPC1L1)to maintain cholesterol homeostasis.Nuclear receptor pregnane X receptor(PXR)is a key molecule in regulating cholesterol homeostasis.Activation of PXR increases cholesterol in the serum and several potential mechanisms have been reported,including increased hepatic cholesterol synthesis,induction of PCSK9,increased intestinal absorption,and decrease of bile acid synthesis.Chlorogenic acid(CA)is a phenolic acid composed of caffeic acid and quinic acid,which has the effect of regulating cholesterol metabolism.However,it is not clear how CA regulates cholesterol metabolism.Therefore,the purpose of this study was to investigate the molecular mechanism of CA lowering cholesterol based on SREBP2/PXR regulatory pathways in vitro and in vivo.Objectives:In this study,a model of hypercholesterolemia in vitro and in vivo was established.CA can down-regulate the expression of NPC1L1 and HMGCR based on the SREBP2/PXR dual signaling pathway and its interaction with HSP90 thus maintaining cholesterol homeostasis.It provides theoretical basis and scientific basis for the prevention and treatment of hypercholesterolemia and drug development.Methods:1.This study investigates the regulatory effect of CA on cholesterol by constructing a mouse model of hypercholesterolemia.Lipid-related biochemical indexes such as TC,TG and LDL-C in mouse serum,liver and feces were determined;Bile acid changes were analyzed by targeted metabonomics,and histopathological changes of liver were observed by H&E staining;The effects of NPC1L1,ABCG5 and HMGCR expression in liver and intestinal tissues and SREBP2 and PXR expression and distribution(nucleus and plasma)were determined by RT-q PCR,Western blot and immunohistochemistry.2.To explore the molecular regulation mechanism of cholesterol metabolism by CA,a hypercholesterolemic cell model was used as the research object.Using ezetimibe as control drugs,the TC levels of cells were detected;By constructing a Caco2 cell monolayer model,the effect of CA on cholesterol uptake was studied;The effects of CA on the expression of NPC1L1,ABCG5,and HMGCR,as well as the expression and distribution of SREBP2 and PXR in cells were studied using RT-q PCR and Western blot.3.Rifampin and ketoconazole were used as control drugs to explore the regulation of cholesterol metabolism by CA through the interaction of SREBP2 and PXR.Exploring the effect of CA on the binding of HSP90 to PXR and SREBP2 in a high cholesterol cell model by using immunoprecipitation and immunofluorescence techniques.4.Small interfering RNA technology was used to construct si SREBP2,si PXR,and si HSP90.By measuring the level of TC content in cells and examining the protein expression of cholesterol metabolism related genes SREBP2,PXR,NPC1L1,and HMGCR,the relationship between SREBP2 and PXR was further studied to explore the regulatory effect of CA on cholesterol in hypercholesterolemic cell models.5.This study used AMPK inhibitor Compound C as the control drug.The effect of CA on AMPK-SREBP2 signaling pathway and its downstream cholesterol homeostasis related target genes was explored by Western blot,immunoprecipitation,and immunofluorescence techniques,and the possible interaction between AMPK and SREBP2 and its mechanism was investigated.The possible interaction between AMPK and SREBP2 and its mechanism were investigated to further clarify the molecular targets of CA for blood lipid reduction.Result:1.CA could dose-dependently reverse the elevation of serum TC,TG,LDL-C,ALT,AST,and TBA,attenuate the hepatic lipid accumulation and promote the excretion of TC,TG and TBA in hypercholesterolemia mice.The PCA plot and heatmap results showed that CA can inhibit the absorption of cholesterol by altering the composition of BA.The treatment with CA significantly decreased the TC content of cells in a dose-dependent manner.Caco2 cell intestinal barrier model evaluation demonstrated that CA treatment significantly decreased the rate of cholesterol uptake.2.In the state of high cholesterol,CA could decrease the nuclear accumulation of PXR and SREBP2 protein,which consequently affected the expression of downstream target genes,including NPC1L1 and HMGCR.3.CA regulates cholesterol homeostasis in a hypercholesterolemic cell model by regulating the interaction between SREBP2 and PXR.Co-immunoprecipitation and immunofluorescence showed that HSP90 can bind to SREBP2 and PXR.After treatment with CA,the binding of HSP90 and SREBP2 is significantly reduced,while the binding of HSP90 and PXR is significantly increased in Caco2 and Hep G2 cells.Rifampicin could markedly reversed the effect of CA on the binding of HSP90 to SREBP2 and HSP90 to PXR.Ketoconazole could further enhance the effect of CA on SREBP2 and PXR through HSP90.In summary,SREBP2 can interact with the PXR signaling pathway through HSP90 to jointly regulate the expression of downstream target genes.Rifampicin,ketoconazole,and CA can all regulate the expression of downstream target genes NPC1L1 and HMGCR through the interaction of SREBP2/HSP90/PXR.4.The effect of PXR,HSP90,SREBP2 gene silencing on the regulation of cholesterol metabolism by chlorogenic acid.PXR silencing decreased PXR nuclear protein,but increased SREBP2 in the nucleus,and finally showed no significant changes in the expression of downstream target proteins NPC1L1 and HMGCR regulated by both.HSP90 gene silencing remarkably reduced the nuclear aggregation of SREBP2 but increased PXR nuclear aggregation,which finally attenuated the protein expression of NPC1L1 and HMGCR.SREBP2 gene silencing significantly reduced the nuclear aggregation of SREBP2 and PXR,and down-regulated the expression of NPC1L1 and HMGCR.These results also demonstrate that the regulation of CA on genes related to cholesterol metabolism is principally related to the SREBP2 pathway.5.Compound C,an AMPK specific inhibitor,can significantly increased the intracellular cholesterol content and almost completely abolished the cholesterol lowering effect of CA in Hep G2 and Caco2 cells.The levels of SREBP2 and PXR nuclear proteins,HMGCR and NPC1L1 total proteins were significantly increased when AMPK was prevented by compound C.CA exhibited concentration-dependent effects on the activation of AMPK.Co-immunoprecipitation experiments further demonstrated that CA enhanced the binding of AMPK to SREBP2,while compound C decreased the binding of AMPK and SREBP2,which can markedly reverse the effect of CA on the binding of AMPK and SREBP2 in Hep G2 cells.However,no obvious protein-protein binding between AMPK and PXR was observed in this study.Conclusion:1.CA can regulate genes involved in cholesterol metabolism by affecting the dual signaling pathways of PXR and SREBP2,in which SREBP2 plays a dominant role.2.There is an interaction mediated by HSP90 in the regulation of SREBP2 and PXR signaling pathways by CA.3.CA downregulates NPC1L1 and HMGCR expression by acting on the AMPK/SREBP2 direct pathway and AMPK/SREBP2/HSP90/PXR indirect pathway,thus retaining cholesterol homeostasis.