Geniposide and Chlorogenic Acid Combination Ameliorates Non-alcoholic Steatohepatitis Involving the Protection on the Gut Barrier Function in Mouse Induced by High-Fat Diet

Figures

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  Figure 1. Effect of GC combination on liver injury induced by HFD. (A) Hematoxylin-eosin staining for liver sections (200 times of magnification). (B) Oil red staining (for visualization of the lipid deposition) for liver sections (200 times of magnification). (C) NAS. Data represents the median and individual NAS in each group. (D) ALT level in plasma and hepatic TG. C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate, NAFLD, non-alcoholic fatty liver disease, NAS, NAFLD activity score. *P < 0.05, **P < 0.01.

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  Figure 2. Effects of GC combination on hepatic Kupffer cells infiltration, cytokines production and LBP in NAFLD induced by HFD. (A) Immunofluorescence Staining for F4/80 in liver tissue (600 times of magnification). (B) Hepatic IL-1β and TNF-α content. (C) Hepatic LBP content. C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate, LBP, lipopolysaccharide binding protein, NAFLD, non-alcoholic fatty liver disease. *P < 0.05, **P < 0.01.

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  Figure 3. Effects of GC combination on hepatic TLR4 expression in NAFLD induced by HFD. TLR4 protein was detected by western-blot. The relative expression levels of proteins were corrected by β-actin. The data represents the fold changes relative to that in control group. C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate, TLR4, Toll like receptor 4, NAFLD, non-alcoholic fatty liver disease. *P < 0.05, **P < 0.01.

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  Figure 4. Effects of GC combination on LPS and D-lactate in plasma and tight junction protein expression in colon tissue in NAFLD induced by HFD. (A) LPS and D-lactate in plasma. (B) Protein expression of tight junction (ZO-1, Occludin and Claudin-1) in colon tissue detected by western-blot. The relative expression levels of proteins were corrected by β-actin. The data represents the fold changes relative to that in control group. C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate, LPS, lipopolysaccharide, NAFLD, non-alcoholic fatty liver disease. *P < 0.05, **P < 0.01.

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  Figure 5. Immunofluorescence staining of tight junction (ZO-1, Occludin and Claudin-1) in colon. White arrows point out the tight junction disruption (600 times of magnification). C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate.

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  Figure 6. Effects of GC combination on phosphorylation of MLC2 in colon tissue in NAFLD induced by HFD. p-MLC2 was detected by western-blot. The relative expression levels of proteins were corrected by MLC2. The data represents the fold changes relative to that in control group. C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate, p-MLC2, phosphorylated myosin light chain 2, NAFLD, non-alcoholic fatty liver disease. **P < 0.01.

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  Figure 7. Effects of GC combination on phosphorylation of MYPT1, ROCK activity and ratio of active RhoA in colon tissue in NAFLD induced by HFD. (A) p-MYPT1 was detected by western-blot. The relative expression levels of proteins were corrected by MYPT1. The data represents the fold changes relative to that in control group. (B) ROCK activity and ratio of active RhoA in colon tissue. C, control, HFD, high-fat diet, GC, geniposide and chlorogenic acid combination, QHD, Qushi Huayu Decoction, NaB, sodium butyrate, p-MYPT, phosphorylated myosin phosphatase target subunit, ROCK, Rho-associated kinase, NAFLD, non-alcoholic fatty liver disease.*P < 0.05, **P < 0.01.

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  Figure 8. Protection on intestinal barrier function contributes to the therapeutic effects of GC combination on NASH. Overgrowth of the Gram-negative bacteria leads to increased content of LPS in the intestine and gut barrier dysfunction. LPS activates RhoA/ROCK signaling which induces activation of MLC2 via direct phosphorylation or phosphorylation of MYPT. The increased phosphorylation of MLC2 leads to the rearrangement of TJ proteins and increase of TJ permeability. The gut-derived LPS go into liver via portal circulation and activate KCs to produce inflammatory factors participating in the onset of NASH. GC combination was found to restore the TJs protein expression and down-regulate RhoA/ROCK signaling while ameliorated NASH. GC, Geniposide and chlorogenic acid combination, IL-1β, interleukin-1β, LBP, lipopolysaccharide binding protein, LPS, lipopolysaccharide, MLC, myosin light chain, MYPT, myosin phosphatase target subunit, NASH, non-alcoholic steatohepatitis, ROCK, Rho-associated kinase, TNF, tumor necrosis factor, TLR, Toll-like receptor, TJ, tight junction.