Ginsenoside Rg3 attenuates sepsis-induced injury and mitochondrial dysfunction in liver via AMPK-mediated autophagy flux

Abstract

Sepsis-led mitochondrial dysfunction has become a critical pathophysiological procedure in sepsis. Since ginsenosides have been applied in the treatment of mitochondrial dysfunction, ginsenoside Rg3 was employed to study its effects on the mitochondrial dysfunction induced by sepsis. The apoptosis rate, oxygen consumption rate (OCR), reactive oxygen species (ROS), antioxidant glutathione (GSH) pools, and mitochondrial transmembrane potential (MTP) were determined in LPS-induced sepsis hepatocytes treated with different concentrations of Rg3. Then, the protein expression levels of mitochondrial biogenesis related transcription factors, autophagy-related proteins, and AMP-activated protein kinase (AMPK) signal pathway related proteins were determined by Western blotting in both in vitro and in vivo sepsis models. Rg3 shows functions of promotion of OCR, attenuation of ROS, and maintenance of GSH pools, and its conjugating activity in the in vitro sepsis models. Rg3-treated cells were observed to have a higher MTP value compared with the LPS only induced cells. Moreover, Rg3 treatment can inhibit mitochondrial dysfunction via increasing the protein expression levels of mitochondrial biogenesis related transcription factors. Rg3 treatment has the function of inhibitor of apoptosis of human primary hepatocytes, and Rg3 can up-regulate the autophagy-related proteins and activate AMPK signal pathway in sepsis models. Meanwhile, the mitochondrial protective function exerted by Rg3 decreased after the autophagy inhibitors or AMPK inhibitor treatment in LPS-induced human primary hepatocytes. Rg3 can improve mitochondrial dysfunction by regulating autophagy in mitochondria via activating the AMPK signal pathway, thus protecting cell and organ injuries caused by sepsis.

Keywords: Autophagy; Liver injury; Mitochondrial dysfunction; Rg3; Sepsis.

Figure 1. Rg3 inhibits LPS-induced apoptosis in sepsis model

(A) Representative dot plots of apoptosis rate measured by flow cytometry. (B) Quantitative analysis of % apoptotic death. ^###, P<0.001, compared with the control group. ###, P <0.001; **, P<0.01, compared with LPS group.

Figure 2. Effects of Rg3 on OCR, ROS production, and antioxidant GSH pools in sepsis model

Rg3 promotes OCR (A), attenuates the LPS-induced ROS production (B), and maintain antioxidant GSH pools (C,D) and its conjugating activity (E,F) in a dose-dependent manner. ^##, P<0.01, compared with the control group; *, P<0.05; **, P<0.01, compared with the LPS group.

Figure 3. Rg3 inhibits LPS-induced mitochondrial dysfunction in sepsis model

(A) Representative fluorescence microscopy images of JC-1. LPS-treated human primary hepatocytes were observed to have JC-1 monomer form (cells with green fluorescence), indicating lower MTP. However, both the negative control cells and Rg3-treated cells were observed to have JC-1 aggregate form (cells with red fluorescence), indicating high MTP values. (B) Quantitative analysis of JC-1 fluorescence. (C,D) Expression levels of respiratory chain complexes assembly proteins in mitochondria determined by Western blotting. the expression levels of OPA1, complex I, and complex II were down-regulated with LPS treatment, which were reversed by the Rg3 treatment. (E,F) Expression levels of mitochondrial biogenesis related transcription factors PGC1-α, NRF-1, and Tfam-1 as determined by Western blotting. *, P<0.05; **, P<0.01, ***, P<0.001, compared with the LPS group.

Figure 4. Rg3 recovers LPS- and CLP-induced mitochondrial dysfunction via autophagy in vitro

(A,B) Expression levels of autophagy-related proteins determined by Western blotting. LC3B II/LC3B I and Beclin-1 levels were higher in Rg3-treated group than in the control group and the LPS-treated group, while Rg3 treatment showed no effect on the p62 levels. (C) Representative fluorescence microscopy images of JC-1. The green fluorescence in LPS-treated human primary hepatocytes was increased following exposure to autophagy inhibitors. When combined with the autophagy inhibitors, Rg3 treatment cannot inhibit the LPS-induced JC-1 monomer production. (D) Quantitative analysis of JC-1 fluorescence. (E,F) Expression levels of mitochondrial biogenesis related transcription factors PGC1-α, NRF-1, and Tfam-1, as determined by Western blotting. When combined with the autophagy inhibitors, expression levels of mitochondrial biogenesis related transcription factors PGC1-α, NRF-1, and Tfam-1 cannot be up-regulated even when treated with 25 μM Rg3. *, P<0.05; **, P<0.01, the groups of comparison were indicated in data.

Figure 5. Rg3 promotes mitochondrial autophagy via AMPK signal pathway activation in vitro

(A,B) Expression levels of AMPK signal pathway related proteins determined by Western blotting. LPS treatment decreased the levels of p-AMPK and ACC (p-ACC) in hepatocytes, while the Rg3 treatment reversed the inhibition effect brought by LPS, further promoting the phosphorylation of the AMPK signal pathway related proteins. (C) Representative fluorescence microscopy images of JC-1, the green fluorescence in Rg3-treated, LPS-induced hepatocytes was increased, while it decreased when exposed to Compound C. (D) Quantitative analysis of JC-1 fluorescence. (E,F) Expression levels of mitochondrial biogenesis related transcription factors determined by Western blotting. The expression levels of PGC1-α, NRF-1, and TFAM-1 were down-regulated when treated with Compound C compared with the LPS + Rg3 group. (G,H) Expression levels of autophagy-related proteins determined by Western blotting. Compound C treatment reversed the up-regulation effects of autophagy-related proteins brought by Rg3 treatment. *, P<0.05; **, P<0.01, the groups of comparison were indicated in data.

Figure 6. Rg3 promotes mitochondrial autophagy via AMPK signal pathway activation in vivo

(A) Kaplan–Meier curves of rat survival rate. Rats treated with 10 and 20 mg/kg Rg3 exhibited a significantly higher survival rate 72 h post-CLP induction compared with the CLP only group. (B,C) Expression levels of mitochondrial biogenesis related transcription factors PGC1-α, NRF-1, and Tfam-1, as determined by Western blotting. (D,E) Expression levels of autophagy-related proteins determined by Western blotting. LC3B II/LC3B I and Beclin-1 levels were significantly up-regulated in Rg3-treated group than in the sham group and the CLP-treated group, while Rg3 treatment showed no effect on the p62 levels. (F,G) Expression levels of AMPK signal pathway related proteins determined by Western blotting. CLP treatment decreased the levels of p-AMPK and ACC (p-ACC) in rat models, while the Rg3 treatment reversed the inhibition effect brought by CLP treatment, further promoting the phosphorylation of the AMPK signal pathway related proteins. *, P <0.05, compared with the CLP only group.