Ginsenoside Rg1 ameliorates aging‑induced liver fibrosis by inhibiting the NOX4/NLRP3 inflammasome in SAMP8 mice

Abstract

Aging is often accompanied by liver injury and fibrosis, eventually leading to the decline in liver function. However, the mechanism of aging‑induced liver injury and fibrosis is still not fully understood, to the best of our knowledge, and there are currently no effective treatment options available for liver aging. Ginsenoside Rg1 (Rg1) has been reported to exert potent anti‑aging effects due to its potential antioxidant and anti‑inflammatory activity. The present study aimed to investigate the protective effect and underlying mechanism of action of Rg1 in aging‑induced liver injury and fibrosis in senescence‑accelerated mouse prone 8 (SAMP8) mice treated for 9 weeks. The histopathological results showed that the arrangement of hepatocytes was disordered, vacuole‑like degeneration occurred in the majority of cells, and collagen IV and TGF‑β1 expression levels, that were detected via immunohistochemistry, were also significantly upregulated in the SAMP8 group. Rg1 treatment markedly improved aging‑induced liver injury and fibrosis, and significantly downregulated the expression levels of collagen IV and TGF‑β1. In addition, the dihydroethylene staining and western blotting results showed that Rg1 treatment significantly reduced the levels of reactive oxygen species (ROS) and IL‑1β, and downregulated the expression levels of NADPH oxidase 4 (NOX4), p47phox, p22phox, phosphorylated‑NF‑κB, caspase‑1, apoptosis‑associated speck‑like protein containing a C‑terminal caspase recruitment domain and the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which were significantly upregulated in the liver tissues of elderly SAMP8 mice. In conclusion, the findings of the present study suggested that Rg1 may attenuate aging‑induced liver injury and fibrosis by reducing NOX4‑mediated ROS oxidative stress and inhibiting NLRP3 inflammasome activation.

Keywords: NADPH oxidase 4; NLR family pyrin domain containing 3 inflammasome; ginsenoside Rg1; liver aging; oxidative stress.

Figure 1.

Effects of Rg1 treatment on histopathological changes in the liver in SAMP8 mice. (A) H&E staining of the liver (Scale bar, 100 µm; Magnification, ×200). The black arrows indicate that the cells of the SAMP8 group were not clearly defined, the nuclei were absent or deeply stained, and were squeezed to one side. (B) PAS staining of the liver (Scale bar, 50 µm; Magnification, ×400). (C) Positive PAS staining area (normalized to SAMR1 group). Data are presented as the mean ± SD; n=4. **P<0.01 vs. SAMR1; ^##P<0.01 vs. SAMP8. SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1; PAS, periodic acid-Schiff; Rg1, ginsenoside Rg1.

Figure 2.

Effects of Rg1 treatment on liver fibrosis in SAMP8 mice. (A) Masson's trichrome staining in the liver (Scale bar, 50 µm; magnification, ×400). The arrows indicate collagen deposition. (B) Mean density of the positive area of staining in the liver (Masson's trichrome staining). Data are presented as the mean ± SD; n=4. **P<0.01 vs. SAMR1; ^#P<0.05, ^##P<0.01 vs. SAMP8. Rg1, ginsenoside Rg1; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1.

Figure 3.

Effects of Rg1 treatment on the expression levels of collagen IV and TGF-β1 in the liver of SAMP8 mice. (A) Expression of collagen IV in the liver (Scale bar, 50 µm; Magnification, ×400). (B) Expression of TGF-β1 in the liver (Scale bar, 50 µm; Magnification, ×400). (C) Mean density of positive (C) collagen IV and (D) TGF-β1 staining area in the liver. Data are presented as the mean ± SD; n=4. **P<0.01 vs. SAMR1; ^##P<0.01 vs. SAMP8. Rg1, ginsenoside Rg1; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1.

Figure 4.

Effect of Rg1 treatment on ROS production in the liver of SAMP8 mice. (A) Accumulation of ROS production in the liver was measured via DHE fluorescence and Hoechst 33258 staining (Scale bar, 20 µm; Magnification, ×400). (B) Mean density of ROS production (red fluorescence) in the liver. Data are presented as the mean ± SD; n=3. **P<0.01 vs. SAMR1; ^##P<0.01 vs. SAMP8. Rg1, ginsenoside Rg1; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1; DHE, dihydroethylene.

Figure 5.

Effect of Rg1 treatment on NOX4 expression in the liver of SAMP8 mice. (A) Western blotting was used to determine the expression levels of NOX4, p22phox and p47phox in the liver of SAMP8 mice. Semi-quantitative analysis of the relative expression of (B) NOX4, (C) p22phox and (D) p47phox. Protein expression levels were normalized to GAPDH. Data are presented as the mean ± SD; n=3. **P<0.01 vs. SAMR1; ^#P<0.05, ^##P<0.01 vs. SAMP8. Rg1, ginsenoside Rg1; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1; NOX4, NADPH oxidase 4.

Figure 6.

Effects of Rg1 treatment on the expression levels of NLRP3, ASC, caspase-1 and IL-1β in the liver of SAMP8 mice. (A) Western blotting was used to analyze the expression levels of NLRP3, ASC, caspase-1 and IL-1β in the liver of SAMP8 mice. Semi-quantitative analysis of the relative expression of (B) NLRP3, (C) ASC, (D) caspase-1 and (E) IL-1β. Protein expression levels were normalized to GAPDH. Data are presented as the mean ± SD; n=3. *P<0.05, **P<0.01 vs. SAMR1; ^#P<0.05, ^##P<0.01 vs. SAMP8. Rg1, ginsenoside Rg1; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain.

Figure 7.

Effects of Rg1 treatment on the expression levels of NLRP3 in the liver of SAMP8 mice. (A) Expression of NLRP3 in the liver (Scale bar, 50 µm; Magnification, ×400). (B) Mean density of the NLRP3-positive staining area in the liver. Data are presented as the mean ± SD; n=4. **P<0.01 vs. SAMR1; ^#P<0.05, ^##P<0.01 vs. SAMP8. Rg1, ginsenoside Rg1; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1; NLRP3, NLR family pyrin domain containing 3.

Figure 8.

Effects of Rg1 treatment on the expression levels of p-NF-κB in the liver of SAMP8 mice. (A) Western blotting was used to analyze the expression levels of p-NF-κB and NF-κB in the liver of SAMP8 mice. Semi-quantitative analysis of the relative expression of (B) NF-κB and (C) p-NF-κB/NF-κB. Data are presented as the mean ± SD; n=3. **P<0.01 vs. SAMR1; ^#P<0.05 vs. SAMP8. Rg1, ginsenoside Rg1; SAMP8, senescence-accelerated mouse prone 8; SAMR1, senescence-accelerated resistant mouse 1; p-, phosphorylated.