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. 2012 Apr;55(4):1271-81.
doi: 10.1002/hep.24792. Epub 2012 Mar 1.

Rosmarinic acid and baicalin epigenetically derepress peroxisomal proliferator-activated receptor γ in hepatic stellate cells for their antifibrotic effect

Melissa D Yang  1 , Yi-Ming Chiang, Reiichi Higashiyama, Kinji Asahina, Derek A Mann, Jelena Mann, Clay C C Wang, Hidekazu Tsukamoto
Affiliations

Rosmarinic acid and baicalin epigenetically derepress peroxisomal proliferator-activated receptor γ in hepatic stellate cells for their antifibrotic effect

Melissa D Yang et al. Hepatology. 2012 Apr.

Abstract

Hepatic stellate cells (HSCs) undergo myofibroblastic transdifferentiation (activation) to participate in liver fibrosis and identification of molecular targets for this cell fate regulation is essential for development of efficacious therapeutic modalities for the disease. Peroxisomal proliferator-activated receptor γ (PPARγ) is required for differentiation of HSCs and its epigenetic repression underlies HSC activation. The herbal prescription Yang-Gan-Wan (YGW) prevents liver fibrosis, but its active ingredients and molecular mechanisms are unknown. Here we demonstrate YGW prevents and reverses HSC activation by way of epigenetic derepression of Pparγ involving reductions in MeCP2 expression and its recruitment to Pparγ promoter, suppressed expression of PRC2 methyltransferase EZH2, and consequent reduction of H2K27di-methylation at the 3' exon. High-performance liquid chromatography / mass spectrometry (HPLC/MS) and nuclear magnetic resonance (NMR) analyses identify polyphenolic rosmarinic acid (RA) and baicalin (BC) as active phytocompounds. RA and BC suppress the expression and signaling by canonical Wnts, which are implicated in the aforementioned Pparγ epigenetic repression. RA treatment in mice with existing cholestatic liver fibrosis inhibits HSC activation and progression of liver fibrosis.

Conclusion: These results demonstrate a therapeutic potential of YGW and its active component RA and BC for liver fibrosis by way of Pparγ derepression mediated by suppression of canonical Wnt signaling in HSCs.

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Figures

Fig. 1
Fig. 1
YGW prevents and reverses hepatic stellate cell (HSC) activation in culture. A. Phase contrast microscopy of activating day 3 or fully-activated day 7 rat HSCs cultured for the last 48 hr with YGW extract, vehicle control, or no addition. Note a morphologic reversal of activated HSCs to quiescent cells. B. Immunostaining for SMA. Note a marked reduction in SMA with YGW extract. C. Oil red O staining after retinol and palmitate addition is increased in YGW-treated 7 day HSCs. D. The mRNA levels for activation marker genes, α1(I)collagen, αSMA, TGF-β1 are conspicuously suppressed in day 7 HSC by the 48-hr treatment with YGW extract while PPARγ mRNA is induced. *p<0.05, **p<0.01 compared to the vehicle control treatment.
Fig. 2
Fig. 2
PPARγ epigenetic repression is lifted with YGW extract. A. Recruitment of Ser2-p RNA polymerase II to the Pparγ gene is significantly reduced in day 7 culture-activated HSCs with no addition or with the vehicle control treatment, and this reduction is attenuated by the YGW extract treatment. B. Increased MeCP2 recruitment to Pparγ promoter in day 7 culture-activated HSCs is normalized with the YGW extract. C. MeCP2 protein detected by immunoblotting in day 5 HSCs cultured for 24 and 48 hr with the vehicle control becomes undetectable by the YGW treatment. D. Increased H3K27me2 at the Pparγ exon 2 locus in day 7 HSCs is reduced with the YGW extract. E. Increased mRNA levels of the PRC2 component EZH2, Suz12, and EED in day 7 HSCs are reduced by the YGW treatment. F. H3K4me2 at the Pparγ promoter locus is increased by the YGW extract treatment in day 7 HSCs compared to HSCs treated with the vehicle. G. Reduced H3 acetylation (H3Ac) in 7 day HSCs is attenuated with the YGW extract. *p<0.05 compared to day 1 HSCs, †p<0.05 compared to the vehicle control.
Fig. 3
Fig. 3
Suppression of IKK and NF-κB with YGW. A. Day 5 HSCs cultured with the YGW extract vs. the vehicle control for 6 or 24 hr in serum-free medium, show reduced IKK activity as assessed by phosphorylation of IκBα-GST fusion protein. A positive control for IKK activation is shown with LPS-stimulated RAW macrophage cell line (last lane). B. Day 5 HSCs cultured with the YGW extract for 24 or 48 hr, show marked reductions in the levels of IκBα and IκBβ proteins, as well as in type I collagen protein. C. The activity of κB promoter is significantly reduced by the YGW extract in the rat HSC line (BSC) as assessed by a transient transfection-reporter analysis. *p<0.05.
Fig. 4
Fig. 4
Identification of active components. A. Treatment of day 7 HSCs with a gel filtration fraction with a molecular mass range of 200~750Da, causes a morphologic reversal of HSCs as compared to the cells treated with the elution buffer control (phase contract microscopy). B and C. Addition of the fraction to 7 day HSC culture reduces increased α1(I)collagen mRNA and MeCP2 enrichment to the Pparγ promoter as shown with the YGW extract. D. A summary of chromatographic methods for separation of YGW’s active ingredients. E. Butanol (BuOH) fraction A and B eluted with 10% acetonitrile-90% water and 40% acetonitrile-60% water, respectively, produce reproducible effects of HSC morphologic reversal as shown by phase contrast microscopy and oil red O staining. F. LC/MS tracing of butanol fractions identifies 5 peaks of which two are identified to be RA and BC. G. Molecular structures of rosmarinic acid and baicalin.
Fig. 4
Fig. 4
Identification of active components. A. Treatment of day 7 HSCs with a gel filtration fraction with a molecular mass range of 200~750Da, causes a morphologic reversal of HSCs as compared to the cells treated with the elution buffer control (phase contract microscopy). B and C. Addition of the fraction to 7 day HSC culture reduces increased α1(I)collagen mRNA and MeCP2 enrichment to the Pparγ promoter as shown with the YGW extract. D. A summary of chromatographic methods for separation of YGW’s active ingredients. E. Butanol (BuOH) fraction A and B eluted with 10% acetonitrile-90% water and 40% acetonitrile-60% water, respectively, produce reproducible effects of HSC morphologic reversal as shown by phase contrast microscopy and oil red O staining. F. LC/MS tracing of butanol fractions identifies 5 peaks of which two are identified to be RA and BC. G. Molecular structures of rosmarinic acid and baicalin.
Fig. 5
Fig. 5
Rosmarinic acid (RA) and baicalin (BC) are the YGW’s active components to render epigenetic de-repression of Pparγ. A. Note both rosmarinic acid (RA) and baicalin (BC) reverse activated HSC to quiescent cells as shown by phase contrast and UV-excited autofluorescence microscopy. B. RA and BC (270 µM) reduce mRNA expression for α1(I)collagen and increase that for PPARγ. C. RA and BC reduce MeCP2 protein level in day 7 HSCs. D. MeCP2 enrichment to the Pparγ promoter is reduced with RA and BC. E. EZH2 mRNA level is reduced equally by RA and BC. F. H3K27me2 at the Pparγ exon 2 is reduced by RA and BC. * p<0.05 compared to the solvent control. G. RA and BC (270 µM) reduce the expression of Wnt10b, Wnt3a, and Necdin in day 7 HSCs compared to the vehicle control as determined by qPCR analysis. H. RA (shaded bar) and BC (black bar) reduces the TOPFLASH promoter activity in day 7 primary HSCs ad determined by a transient transfection using an electroporation method. I. RA treatment (ip injection daily at 0.1mg/25g body weight) given during the last one week of the 2-week cholestasis caused by the bile duct ligation, attenuates liver fibrosis in mice as assessed by digital morphometric analysis of Sirius red staining. *p<0.05 compared to the vehicle control. J. Hepatic expression of α1(I)procollagen and SMA are also significantly reduced by the RA treatment. *p<0.05 compared to Sham. +p<0.05 compared to vehicle-treated (Cont) mice.
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