Ameliorative effects of Compound K and ginsenoside Rh1 on non-alcoholic fatty liver disease in rats

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease, which has no standard treatment available. Panax notoginseng saponines (PNS) have recently been reported to protect liver against hepatocyte injury induced by ethanol or high fat diet (HFD) in rats. Compound K and ginsenoside Rh1 are the main metabolites of PNS. In this study, we evaluated the effects of CK and Rh1 on NAFLD. Rats fed HFD showed significant elevations in liver function markers, lipids, glucose tolerance, and insulin resistance. Treatment with CK or Rh1 either alone or in combination dramatically ameliorated the liver function impairment induced by HFD. Histologically, CK and Rh1 significantly reversed HFD-induced hepatocyte injury and liver fibrosis. In vitro experiments demonstrated that treatment with CK or Rh1 alone or in combination markedly induced cell apoptosis, and inhibited cell proliferation and activation in HSC-T6 cells. Additionally, CK and Rh1, either alone or in combination, also repressed the expression of fibrotic factors TIMP-1, PC-I, and PC-III. Taken together, our results demonstrate that CK and Rh1 have positive effects on NAFLD via the anti-fibrotic and hepatoprotective activity.

Figure 1. Chemical structures of CK and Rh1.

(a) CK; (b) Rh1.

Figure 2. Effects of CK or Rh1 alone or in combination on insulin resistance in HFD-fed rats.

(a) Blood glucose levels during GTT. (b) Insulin resistance index values between 0 and 120 min of GTT. Insulin resistance index = [fasting glucose (mmol/L) × fasting insulin (μU/ml)]/22.5. Control, normal diet alone; HFD, high fat diet alone; RSG, Rosiglitazone plus high fat diet; P, phospholipid plus high fat diet; PNS + P, the combination of panax notoginseng saponins and phospholipid plus high fat diet; CK + P, the combination of compand K and phospholipid plus high fat diet; Rh1 + P, the combination of Rh1 and phospholipid plus high fat diet; CK + Rh1 + P, the combination of CK, Rh1 and phospholipid plus high fat diet. n = 20. ^aP < 0.05 versus control group, ^bP < 0.05 versus HFD group, ^cP < 0.05 versus phospholipid group, ^dP < 0.05 versus PNS group.

Figure 3. Effects of CK or Rh1 alone or in combination on liver structures in HFD-fed rats.

(a–c) HE staining (a) and Masson’s trichrome staining (b) of liver sections are shown. Quantitative analysis of NAFLD activity, inflammation and fibrosis are shown (c). The thick yellow arrows represent inflammation. The asterisks represent steatosis, and the fine yellow arrows represent fibrosis. NAFLD activity score, inflammation score and the extent of liver fibrosis were graded by 4-grade scores. Three sections per liver were used for each animal and twenty mice were used for each group for staining. (d and e) Electron micrographs of liver sections (d) are shown. Quantitative analysis of mitochondia is presented (e). Data are the mean ± SD of thirty-eight independent graphs. Vvm: volume density of the mitochondrial; Svm: surface density of the mitochondrial; Num: Numerical density of the mitochondrial; Qm: Specific surface area of the mitochondrial. Control, normal diet alone; HFD, high fat diet alone; RSG, Rosiglitazone plus high fat diet; P, phospholipid plus high fat diet; PNS + P, the combination of panax notoginseng saponins and phospholipid plus high fat diet; CK + P, the combination of Compound K and phospholipid plus high fat diet; Rh1 + P, the combination of Rh1 and phospholipid plus high fat diet; CK + Rh1 + P, the combination of CK, Rh1 and phospholipid plus high fat diet. n = 20. ^aP < 0.05 versus control group, ^bP < 0.05 versus HFD group, ^cP < 0.05 versus phospholipid group.

Figure 4. Effects of CK or Rh1 alone or in combination on cell proliferation and apoptosis in HSCs.

(a) Cells were cultured in the presence of Rosiglitazone (RSG), CK, Rh1 or the combination of CK and Rh1 for 6 hours, before they were subjected to MTT assay. All results are the means ± SD of three independent experiments. *P < 0.05 versus control group. (b) Flow cytometric plots show cells in live, early apoptosis and late apoptosis when cells were treated with RSG, PNS, CK, Rh1 or the combination of CK and Rh1 for 6 hours. (c) Bar graphs show the percentages of early apoptotic cells. Data are the mean ± SD of three independent experiments. ^aP < 0.05 versus control group, ^bP < 0.05 versus PNS group.

Figure 5. Effects of CK or Rh1 alone or in combination on cell activation in HSCs.

(a) HSC-T6 cells were cultured in the presence of Rosiglitazone (RSG), CK, Rh1 or the combination of CK and Rh1 for 6 hours. Cells were then subjected to immunostaining for α-SMA. Original magnification ×400. (b) Quantitative analysis of the relative expression is shown. Data are the mean ± SD of three independent experiments. ^*P < 0.05 versus control group. (c) Total RNA was extracted and subjected to real-time PCR. All results are normalized to the levels of β-actin and are the means ± SD of three experiments. ^*P < 0.05 versus control group.

Figure 6. Effects of CK or Rh1 alone or in combination on the expression levels of fibrosis-associated proteins in HSCs.

(a) Expression levels of TIMP-1, PC-I and PC-III in HSC-T6 cells were determined by Western blotting. β-actin was used as the loading control. Cells were cultured in the presence of Rosiglitazone (RSG), CK, Rh1 or the combination of CK and Rh1 for 6 hours. Representative blots show the expression of TIMP-1, PC-I and PC-III in different groups. (b) Bar graphs show the relative expression of TIMP-1, PC-I and PC-III in different groups. Data are the mean ± SD of three independent experiments. ^*P < 0.05 versus control group.