Tangshen formula attenuates hepatic steatosis by inhibiting hepatic lipogenesis and augmenting fatty acid oxidation in db/db mice

Abstract

Tangshen formula (TSF), a well-prescribed traditional Chinese formula, has been used in the treatment of diabetic nephropathy. However, whether TSF ameliorates dyslipidemia and liver injury associated with diabetes remains unclear. In this study, we examined the effects of TSF on lipid profiles and hepatic steatosis in db/db mice. For this purpose, 8‑week-old db/db mice were treated with TSF or saline for 12 weeks via gavage and db/m mice were used as controls. Body weight and blood glucose levels were monitored weekly and bi-weekly, respectively. Blood samples were obtained for the analysis of lipids and enzymes related to hepatic function, and liver tissues were analyzed by histology, immunohistochemistry and molecular examination. The results revealed that TSF markedly reduced body weight, liver index [liver/body weight (LW/BW)] and improved lipid profiles, hepatic function and steatosis in db/db mice. TSF induced the phosphoralation of AMP-activated protein kinase and inhibited the activity of sterol regulatory element-binding protein 1 together with the inhibition of the expression of genes involved in de novo lipogenesis (DNL) and gluconeogenesis, such as fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), stearoyl CoA desaturase 1 (SCD1), glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1). Additionally, the silent mating type information regulation 2 homolog 1 (Sirt1)/peroxisome proliferator-activated receptor α (PPARα)/malonyl-CoA decarboxylase (MLYCD) cascade was potently activated by TSF in the liver and skeletal muscle of db/db mice, which led to enhanced fatty acid oxidation. These findings demonstrated that TSF attenuated hepatic fat accumulation and steatosis in db/db mice by inhibiting lipogenesis and augmenting fatty acid oxidation.

Figure 1

High performance liquid chromatography (HPLC) chromatogram of Tangshen formula (TSF). TSF was subjected to HPLC analysis, and its chemical fingerprint was revealed by a DAD detector. (A) Loganin, (B) calycosin-7-O-β-D-glucoside, (C) naringenine-7-rhamnosidoglucoside, (D) neohesperidin, (E) naringenin, (F) aloe-emodin were identified in the HPLC fingerprint at 254 nm. Chemical structures are depicted in Fig. 8.

Figure 2

Treatment with Tangshen formula (TSF) suppresses weight gain and reduces lipid accumulation in the livers of db/db mice. (A) The administration of TSF for 12 weeks effectively diminished body weight gain in db/db mice. An inhibitory trend was detected and reached statistical significance as early as week 6 of TSF treatment. (B and C) Treatment with TSF decreased liver weight and liver weight/body weight (LW/BW). (D) Fasting blood glucose (FBG) for each group. (E) Representative H&E and Oil Red O staining revealed markedly diminished vesicular steatosis and fat accumulation in the livers of TSF-treated mice. Scale bar, 100 μm. Data are presented as the means ± SEM. ^*P<0.05 and ^***P<0.001 vs. db/m group; ^#P<0.05 and ^##P<0.01 vs. db/db group; ns, no significance.

Figure 2

Treatment with Tangshen formula (TSF) suppresses weight gain and reduces lipid accumulation in the livers of db/db mice. (A) The administration of TSF for 12 weeks effectively diminished body weight gain in db/db mice. An inhibitory trend was detected and reached statistical significance as early as week 6 of TSF treatment. (B and C) Treatment with TSF decreased liver weight and liver weight/body weight (LW/BW). (D) Fasting blood glucose (FBG) for each group. (E) Representative H&E and Oil Red O staining revealed markedly diminished vesicular steatosis and fat accumulation in the livers of TSF-treated mice. Scale bar, 100 μm. Data are presented as the means ± SEM. ^*P<0.05 and ^***P<0.001 vs. db/m group; ^#P<0.05 and ^##P<0.01 vs. db/db group; ns, no significance.

Figure 3

Tangshen formula (TSF) inhibits the expression of hepatic lipogenic enzymes in the db/db mice. (A and C) Protein and mRNA (relative fold change) levels of lipogenic genes in the livers of db/db mice. (B) Representative images of hepatic sections stained for lipogenic enzymes fatty acid synthase (FAS), 3′-hydroxylmethyl glutaryl coenzyme A reductase (HMGCR) (left panel) and semi-quantitative analysis (right panel). Scale bar, 100 μm. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. db/m group; ^#P<0.05, and ^###P<0.001 vs. db/db group.

Figure 3

Tangshen formula (TSF) inhibits the expression of hepatic lipogenic enzymes in the db/db mice. (A and C) Protein and mRNA (relative fold change) levels of lipogenic genes in the livers of db/db mice. (B) Representative images of hepatic sections stained for lipogenic enzymes fatty acid synthase (FAS), 3′-hydroxylmethyl glutaryl coenzyme A reductase (HMGCR) (left panel) and semi-quantitative analysis (right panel). Scale bar, 100 μm. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. db/m group; ^#P<0.05, and ^###P<0.001 vs. db/db group.

Figure 3

Tangshen formula (TSF) inhibits the expression of hepatic lipogenic enzymes in the db/db mice. (A and C) Protein and mRNA (relative fold change) levels of lipogenic genes in the livers of db/db mice. (B) Representative images of hepatic sections stained for lipogenic enzymes fatty acid synthase (FAS), 3′-hydroxylmethyl glutaryl coenzyme A reductase (HMGCR) (left panel) and semi-quantitative analysis (right panel). Scale bar, 100 μm. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. db/m group; ^#P<0.05, and ^###P<0.001 vs. db/db group.

Figure 4

Tangshen formula (TSF) activates the phosphorylation of AMPK and attenuates the nuclear translocation of sterol regulatory element-binding protein 1 (SREBP1) in the livers of db/db mice. (A) Representative photomicrographs of p-AMPK staining and semi-quantitative analysis. Scale bar, 100 μm. (B) Western blot analysis assessed the protein levels of total and thephosphorylation of AMPK, precursor forms of hepatic SREBP1 (preSREBP1), and nuclear form (nSREBP1). The SREBP1 mRNA level was quantified by RT-qPCR. (C) Representative images of immunofluorescence staining for SREBP1 (green) and nucleus (blue) was photographed by confocal microscopy. Scale bar, 25 μm. ns, not significant. Data are presented as the means ± SEM. ^*P<0.05 vs. db/m group; ^#P<0.05 vs. db/db group.

Figure 4

Tangshen formula (TSF) activates the phosphorylation of AMPK and attenuates the nuclear translocation of sterol regulatory element-binding protein 1 (SREBP1) in the livers of db/db mice. (A) Representative photomicrographs of p-AMPK staining and semi-quantitative analysis. Scale bar, 100 μm. (B) Western blot analysis assessed the protein levels of total and thephosphorylation of AMPK, precursor forms of hepatic SREBP1 (preSREBP1), and nuclear form (nSREBP1). The SREBP1 mRNA level was quantified by RT-qPCR. (C) Representative images of immunofluorescence staining for SREBP1 (green) and nucleus (blue) was photographed by confocal microscopy. Scale bar, 25 μm. ns, not significant. Data are presented as the means ± SEM. ^*P<0.05 vs. db/m group; ^#P<0.05 vs. db/db group.

Figure 4

Tangshen formula (TSF) activates the phosphorylation of AMPK and attenuates the nuclear translocation of sterol regulatory element-binding protein 1 (SREBP1) in the livers of db/db mice. (A) Representative photomicrographs of p-AMPK staining and semi-quantitative analysis. Scale bar, 100 μm. (B) Western blot analysis assessed the protein levels of total and thephosphorylation of AMPK, precursor forms of hepatic SREBP1 (preSREBP1), and nuclear form (nSREBP1). The SREBP1 mRNA level was quantified by RT-qPCR. (C) Representative images of immunofluorescence staining for SREBP1 (green) and nucleus (blue) was photographed by confocal microscopy. Scale bar, 25 μm. ns, not significant. Data are presented as the means ± SEM. ^*P<0.05 vs. db/m group; ^#P<0.05 vs. db/db group.

Figure 5

Tangshen formula (TSF) promotes silent mating type information regulation 2 homolog 1 (Sirt1) upregulation and activates the Sirt1/peroxisome proliferator-activated receptor (PPARα)/malonyl-CoA decarboxylase (MLYCD) signaling pathway. (A and B) Western blot analyses assessed the abundance of Sirt1, PGC1α, PPARα, LXRα, carnitine palmitoyltransferase 1A (CPT1A) and MLYCD in the liver. (C) Hepatic mRNA levels (expressed as fold change) of genes involved in fatty acid oxidation were quantified by RT-qPCR. (D) Hepatic mRNA expression of lipoprotein lipase (LPL) and fatty acid translocase CD36 (FAT/CD36). Data are presented as the means ± SEM. ^*P<0.05 and ^**P<0.01 vs. db/m group; ^#P<0.05 and ^##P<0. 01 vs. db/db group.

Figure 5

Tangshen formula (TSF) promotes silent mating type information regulation 2 homolog 1 (Sirt1) upregulation and activates the Sirt1/peroxisome proliferator-activated receptor (PPARα)/malonyl-CoA decarboxylase (MLYCD) signaling pathway. (A and B) Western blot analyses assessed the abundance of Sirt1, PGC1α, PPARα, LXRα, carnitine palmitoyltransferase 1A (CPT1A) and MLYCD in the liver. (C) Hepatic mRNA levels (expressed as fold change) of genes involved in fatty acid oxidation were quantified by RT-qPCR. (D) Hepatic mRNA expression of lipoprotein lipase (LPL) and fatty acid translocase CD36 (FAT/CD36). Data are presented as the means ± SEM. ^*P<0.05 and ^**P<0.01 vs. db/m group; ^#P<0.05 and ^##P<0. 01 vs. db/db group.

Figure 6

Tangshen formula (TSF) stimulates the phosphorylation of AMP-activated protein kinase (AMPK), silent mating type information regulation 2 homolog 1 (Sirt1) and fatty acid oxidation in the skeletal muscle of db/db mice. (A) Skeletal muscle Sirt1, p-AMPK, PPARγ co-activator (PGC1α) and malonyl-CoA decarboxylase (MLYCD) were analyzed by western blot analysis and (B) semi-quantitative analysis. (C) Expression of genes involved in fatty acid oxidation and (D) mitochondrial biogenesis. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. db/m group; ^#P<0.05 and ^##P<0.01 vs. db/db group.

Figure 6

Tangshen formula (TSF) stimulates the phosphorylation of AMP-activated protein kinase (AMPK), silent mating type information regulation 2 homolog 1 (Sirt1) and fatty acid oxidation in the skeletal muscle of db/db mice. (A) Skeletal muscle Sirt1, p-AMPK, PPARγ co-activator (PGC1α) and malonyl-CoA decarboxylase (MLYCD) were analyzed by western blot analysis and (B) semi-quantitative analysis. (C) Expression of genes involved in fatty acid oxidation and (D) mitochondrial biogenesis. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. db/m group; ^#P<0.05 and ^##P<0.01 vs. db/db group.

Figure 7

Tangshen formula (TSF) treatment induces the phosphorylation of Akt and mammalian target of rapamycin (mTOR) and restores glycogen deposition. (A) TSF induced the upregulation of PI3K, p-Akt and blunted the phosphorylation of mTOR and (B) semi-quantitative analysis. (C) TSF regulated the expression of the glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1) genes in the livers of db/db mice. (D) Representative photomicrographs of PAS staining shows glycogen deposition in the livers of db/db mice treated with TSF. Scale bar, 100 μm. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. db/m group; ^#P<0.05 and ^###P<0.001 vs. db/db group.

Figure 7

Tangshen formula (TSF) treatment induces the phosphorylation of Akt and mammalian target of rapamycin (mTOR) and restores glycogen deposition. (A) TSF induced the upregulation of PI3K, p-Akt and blunted the phosphorylation of mTOR and (B) semi-quantitative analysis. (C) TSF regulated the expression of the glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1) genes in the livers of db/db mice. (D) Representative photomicrographs of PAS staining shows glycogen deposition in the livers of db/db mice treated with TSF. Scale bar, 100 μm. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. db/m group; ^#P<0.05 and ^###P<0.001 vs. db/db group.

Figure 8

Representative compounds identified in Tangshen formula (TSF) by high performance liquid chromatography (HPLC). HPLC chemical fingerprint chromatogram of TSF including (A) loganin (PubChem CID: 87691), (B) calycosin-7-O-β-D-glucoside (PubChem CID: 442813), (C) naringenine-7-rhamnosidoglucoside (PubChem CID: 25075), (D) neohesperidin (PubChem CID: 442439), (E) naringenin (PubChem CID: 932), (F) Aloe-emodin (PubChem CID: 10207).