Isochlorogenic Acid C Alleviates High-Fat Diet-Induced Hyperlipemia by Promoting Cholesterol Reverse Transport

Abstract

Background: Promoting cholesterol reverse transport (RCT) has been proven to be a promising hyperlipidemia therapy since it is more effective for the treatment of atherosclerosis (AS) caused by hyperlipidemia. Liver X receptor (LXR) agonists can accelerate RCT, but most of them trigger undesirable liver steatosis due to the activation of liver LXRα. Aim: We aim to figure out whether isochlorogenic acid C (ICAC) facilitates RCT without causing hepatic steatosis. Methods: In vitro study, we established foam macrophages and macrophages with loaded NBD-cholesterol models to investigate the competence of RCT promoting ICAC. RT-qPCR and Western blot were used to verify ICAC's regulation of RCT and NF-κB inflammatory pathways. In this in vivo study, male 6-week-old C57BL/6 mice were fed a high-fat diet (HFD) to investigate ICAC's anti-hyperlipidemic effect and its functions in regulating RCT. The anti-hyperlipidemic effect of ICAC was evaluated by blood and liver lipid levels, liver hematoxylin, oil red o staining, and liver coefficient. Finally, mRNA levels of genes involved in RCT and inflammation pathways in the liver and intestine were detected by RT-qPCR. Results: ICAC prevented macrophages from foaming by up-regulating the LXRα mediated RCT pathway and down-regulating expression of the cholesterol absorption genes LDLR and CD36, as well as suppressing iNOS, COX2, and IL-1β inflammatory factors. In HFD-fed mice, ICAC significantly lowered the lipid level both in the serum and the liver. Mechanistic studies showed that ICAC strengthened the RCT pathway in the liver and intestine but didn't affect liver LXRα. Furthermore, ICAC impeded both adipogenesis and the inflammatory response in the liver. Conclusion: ICAC accelerated RCT without affecting liver LXRα, thus resulting in a lipid-lowering effect without increasing liver adipogenesis. Our results indicated that ICAC could be a new RCT promoter for hyperlipidemia treatment without causing liver steatosis.

Keywords: cholesterol efflux; foam cells; hyperlipemia; inflammatory; isochlorogenic acid C; reverse cholesterol transport.

FIGURE 1

ICAC triggers the reverse cholesterol transport pathway and promotes cholesterol efflux. (A) Chemical structure of isochlorogenic acid C (ICAC). (B) Cell survival rate assay of ICAC in BMMs. (C) Fluorescence in BMMs under light microscopy. (D) Extracellular fluorescent cholesterol intensity was measured with a microplate reader. (E–G) Gene expression level of LXRα, ABCA1 and ABCG1 in BMMs. (H–K) Protein expression level of LXRα, ABCA1, and ABCG1 in BMMs. (L–N) Gene expression level of CD36, LDLR, and SR-BI in BMMs. (O–R) Protein expression level of CD36, LDLR, and SR-BI in macrophages. All data in (B,D–G,I–N,P–R) were represented as mean ± SEM. Statistical difference was determined by one -way ANOVA test. Compare with a vehicle, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. n = 3 for each group.

FIGURE 2

ICAC inhibits ox-LDL-induced foam macrophage formation and inflammatory response. (A) Oil red O staining of lipid accumulation in BMMs stimulated by ox-LDL. (B) Quantification of ORO staining in BMMs (n = 3). (C–F) Gene expression level of NF-κB, iNOS, COX, and IL-1β in BMMs. (G–J) Protein expression level of iNOS, COX2, and IL-1β in BMMs. (K–M) Protein expression level of NF-κB pathway. All data in (B–F, H–J, N, M, O, P) were represented as mean ± SEM. Statistical difference was determined by one -way ANOVA test. Compare with a vehicle, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. n = 3 for each group.

FIGURE 3

ICAC decreases serum cholesterol and increases bile acid levels in hyperlipidemic mice. (A) Weekly weight of mice in each group. (B)Weight of each group in the 12-week. (C) Coefficients of brown adipose tissue. (D–G) Serum TC, LDL-C, HDL-C, ox-LDL of C57BL/6 mice fed a chow diet or a high fat diet with or without T0901317 or icac for 12 weeks. (H,I) Coefficients of the heart and kidney. (J) Serum TG levels of all groups at the end of treatment. All data were represented mean ± SEM. Statistical difference was determined by one -way ANOVA test. Compare with HFD, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. n = 10 for each group. Compared with AT, ^# p < 0.05.

FIGURE 4

ICAC decreases hepatic tissue lipid and inflammation levels in HFD-induced hyperlipidemic mice. (A) Liver morphological investigation, HE and ORO staining of liver biopsy. (B–F) ORO staining area, TC, TG level and the gene expression levels of iNOS and COX2 in liver of C57BL/6 mice fed a chow diet or a high fat diet with or without AT, T0901317 or ICAC for 12 weeks. All data in (B–F) were represented mean ± SEM. Statistical difference was determined by one -way ANOVA test. Compared with HFD, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. Compared with AT, ^# p < 0.05, ^## p < 0.01. Compared with T090, ^ο p < 0.05, ^οοοο p < 0.0001. n = 10 for each group.

FIGURE 5

ICAC upregulates liver and intestine RCT pathways without inducing lipogenesis gene expression. (A–C) Gene expression levels of ABCA1, ABCG1, and CYP7A1, in the liver of mice. (D–F) Gene and protein expression levels of LDLR in the liver of mice. (G–I) Gene expressions levels of LXRα, ABCG5, and ABCG8 in the intestine of mice. (J–N) Gene expression levels of LXRα, SREBP-1c, ACC, FAS, and SCD-1 in the liver of mice. All data were represented mean ± SEM. Statistical difference was determined by one -way ANOVA test. Compared with HFD, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. Compared with AT, ^# p < 0.05, ^## p < 0.01. Compared with T090, ^ο p < 0.05, ^οοοο p < 0.0001. n = 10 for each group.