Ginkgo biloba Extract 50 (GBE50) Ameliorates Insulin Resistance, Hepatic Steatosis and Liver Injury in High Fat Diet-Fed Mice

Abstract

Background: Ginkgo biloba extract 50 (GBE50) has a variety of pharmacological functions such as anti-inflammatory, antioxidant and maintenance of glucose and lipid metabolism homeostasis. However, the therapeutic effects and mechanisms of GBE50 on non-alcoholic fatty liver disease (NAFLD) remain unknown. Therefore, in this study, we evaluated the therapeutic effects of GBE50 in NAFLD by using a high-fat diet (HFD) mice model.

Methods: C57BL/6J mice were fed a HFD diet for 15 weeks and were given respectively 25, 50, and 100 mg/kg GBE50 daily by gavage from 3 to 15 weeks. After the administration, blood samples and liver tissues were collected for biochemical detection, histological measurement, immunohistochemistry and Western blot, respectively.

Results: We found that GBE50 treatment could ameliorate insulin resistance (IR), glucose intolerance, lipid accumulation, hepatic steatosis and liver injury in HFD-fed mice. Further mechanism exploration discovered that the hepatoprotective effects of GBE50 on NAFLD may be related to the strengthening of IRS-1 signal activation and the weakening of NF-κB, Akt and endoplasmic reticulum stress signals activation.

Conclusion: GBE50 is a potentially powerful therapeutic agent for the treatment of NAFLD.

Keywords: Ginkgo biloba extract 50; hepatic steatosis; inflammatory response; insulin resistance; liver injury; non-alcoholic fatty liver disease.

Figure 1

GBE50 attenuated IR in HFD-fed mice. (A–C) The levels of fasting blood glucose (FBG) (A), fasting insulin (FINS) (B) and the homeostasis model assessment of IR (HOMA-IR) values (C); n=11 mice/group. (D and E) The results of glucose tolerance test (GTT) (D) and insulin tolerance test (ITT) (E); n=5 mice/group. All data are presented as mean±SD, *P<0.05, **P<0.01 vs HFD alone group; ^##P<0.01 vs NC group.

Figure 2

GBE50 ameliorated HFD-induced hepatic lipid metabolism disorder. (A) The results of the ratio of liver weight to body weight. (B and C) Representative Oil red O staining images (B) and the quantification of lipid droplet (C). (D–K) The levels of serum total cholesterol (TC) (D), serum triglyceride (TG) (E), liver TC (F), liver TG (G), serum high-density lipoprotein cholesterol (HDL-C) (H), serum low-density lipoprotein cholesterol (LDL-C) (I), serum free fatty acids (FFA) (J), and liver FFA (K). All data are presented as mean±SD, n=11 mice/group, *P<0.05, **P<0.01 vs HFD alone group; ^##P<0.01 vs NC group.

Figure 3

GBE50 ameliorated HFD-induced liver injury. (A–D) Representative liver sections images of hematoxylin-eosin (HE) staining, Masson trichrome staining and Sirius red staining (A), the quantitative data of hepatic steatosis from the HE staining (B), Masson trichrome positive staining area (C), and Sirius red staining positive staining area (D). (E and F) The levels of serum alanine aminotransferase (ALT) (E) and aspartate aminotransferase (AST) (F). All data are presented as mean±SD, n=11 mice/group, *P<0.05, **P<0.01 vs HFD alone group; ^##P<0.01 vs NC group.

Figure 4

GBE50 attenuated HFD-induced inflammatory cell infiltration and the expression levels of pro-inflammatory cytokines in liver. (A–D) Representative immunohistochemical staining images of the expressions of F4/80, CD45, and Ly6G (A), and the quantification of F4/80 (B), CD45 (C), and Ly6G (D) positive staining areas in liver tissues. (E–G) The levels of IL-6 (E), IL-1β (F) and TNF-α (G) in liver tissues. All data are presented as mean±SD, n=11 mice/group, *P<0.05, **P<0.01 vs HFD alone group; ^##P<0.01 vs NC group.

Figure 5

GBE50 promoted IRS-1 signal activation and inhibited Akt signal activation in HFD-fed mice. (A–D) The phosphorylation levels of IRS-1 (Tyr608) (A and B) and IRS-1 (Ser307) (C and D) under baseline (control) and in response to an intraperitoneal injection of insulin (1.0 IU/kg for 15 min) in HFD alone group and HFD+100 mg/kg GBE50 group; n=5 mice/group. (E–G) The expression levels of p-Akt (Thr308) (E and F) and p-Akt (Ser473) (E and G) proteins in liver tissues in various of groups were detected by Western blot; n=6 mice/group. All data are presented as mean±SD, *P<0.05, **P<0.01 vs HFD alone group; ^††P<0.01 vs HFD+insulin group; ^##P<0.01 vs NC group.

Figure 6

GBE50 attenuated endoplasmic reticulum stress in HFD-fed mice. (A–C) Representative Western blot bands of the expressions of p-eIF2α, eIF2α, ATF-4, CHOP and GRP78 in liver tissues in various of groups. (D–G) The quantification of the expression levels of p-eIF2 (D), ATF-4 (E), CHOP (F) and GRP78 (G). All data are presented as mean±SD, n=6 mice/group, **P<0.01 vs HFD alone group; ^##P<0.01 vs NC group.