Protective Effects of Red Ginseng Against Tacrine-Induced Hepatotoxicity: An Integrated Approach with Network Pharmacology and Experimental Validation

Abstract

Introduction: Tacrine, an FDA-approved acetylcholinesterase inhibitor, has shown efficacy in treating Alzheimer's disease, but its clinical use is limited by hepatotoxicity. This study investigates the protective effects of red ginseng against tacrine-induced hepatotoxicity, focusing on oxidative stress.

Methods: A network depicting the interaction between compounds and targets was constructed for RG. Effect of RG was determined by MTT and FACS analysis with cells stained by rhodamine 123. Proteins were extracted and subjected to immunoblotting for apoptosis-related proteins.

Results: The outcomes of the network analysis revealed a significant association, with 20 out of 82 identified primary RG targets aligning with those involved in oxidative liver damage including notable interactions within the AMPK pathway. in vitro experiments showed that RG, particularly at 1000μg/mL, mitigated tacrine-induced apoptosis and mitochondrial damage, while activating the LKB1-mediated AMPK pathway and Hippo-Yap signaling. In mice, RG also protected the liver injury induced by tacrine, as similar protective effects to silymarin, a well-known drug for liver toxicity protection.

Discussion: Our study reveals the potential of RG in mitigating tacrine-induced hepatotoxicity, suggesting the administration of natural products like RG to reduce toxicity in Alzheimer's disease treatment.

Keywords: AMPK; liver toxicity; oxidative stress; red ginseng; tacrine.

Figure 1

A compound-target network of RG. Circles and diamonds denote protein targets and compounds, respectively. The circle edge and body color indicate targets and signaling path-ways related to oxidative stress and NAFLD, respectively. Edges denote interactions between compound and target.

Figure 2

Effect of RG on Tacrine cytotoxicity. (A) Effect of tacrine on hepatotoxicity. MTT assay was performed in HepG2 cells. Toxicity of Tacrine (30, 100, 300, 1000 mg/mL) (left), RG (30, 100, 300, 1000 mg/mL) concentration and 300 μM Tacrine were simultaneously administered (right). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. (B) Western blotting of apoptosis-related proteins, PARP, procaspase 3 and Bcl-xL. HepG2 cells were simultaneously treated with 300 μg/mL of Tacrine and 1000 μg/mL of RG for 6 hours. (C) The cytoprotective effect of RG was also evaluated under a fluorescence microscope as an experiment to determine the toxicity of Tacrine in hepatocytes. HepG2 cells were treated with RG (1000 μg/mL) and Tacrine (300 μg/mL) for 6 hours. Cells were then stained with Calcein and Pi (0.5 μM each). Data are presented as the average of repeated samples, with error bars representing standard deviations (vs control ** p < 0.01; vs Tacrine; ^#p < 0.05, ^##p < 0.01).

Figure 3

Effect of RG on Mitochondrial Damage in Tacrine Hepatotoxicity. (A) Fluorescence-activated cell sorting analysis. (B) Mitochondrial membrane potential measurement. HepG2 cells were treated with RG (1000μg/mL) and Tacrine (300μg/mL) for 6 hours. Subsequently, fluorescence intensity was measured by FACS for mitochondrial membrane permeability (MMP) after Rhodamine 123 staining. Data are presented as the average of repeated samples, with error bars representing standard deviations (vs control **p < 0.01; vs Tacrine; ^##p < 0.01).

Figure 4

Effect of RG on the AMPK pathway. (A) Immunoblot analysis of p-AMPK, p-ACC and β-actin was performed on lysates of HepG2 cells treated for RG times (0, 10’, 30’, 1 h, 3 h, 6 h). The results were confirmed by repeat experiments below. (B) Huh7 cells were treated with RG (0, 10’, 30’, 1 h, 3 h, 6 h) and immunoblot analysis of p-AMPK, p-ACC and β-actin was performed. Data are presented as the average of repeated samples, with error bars representing standard deviations (vs control *p < 0.05, **p < 0.01).

Figure 5

Effect of RG on LKB1 activation. (A) Western blotting of key signaling molecules in-volved in the p-LKB1 pathway in HepG2 cells. HepG2 cells were treated with RG (1000μg/mL) for the indicated times. β-actin used as loading control. Results are representative of three independent experiments. Relative protein Levels represent mean ± SD. (**p < 0.01). (B) Effect of RG on Tacrine apoptosis on HepG2 and LKB1-deficient HeLa cells. Cells were co-treated with Tacrine 300 μg/mL and 1000 μg/mL RG for 6 hours and MTT test was performed to measure cell viability. Graphs represent mean ± SD. From three independent experiments presented as percentage of control (**p < 0.01 vs vehicle-treated control group; ^##P<0.01 vs tacrine-treated group). Western blotting of LKB1 on HepG2 cells and LKB1-deficient HeLa cells RG treatment for 3 h (below).

Figure 6

Effect of red RG on YAP signal. (A) Phosphorylation of YAP and LATS1. Immunoblot analysis was performed on HepG2 cells treated with RG (1000 μg/mL) over time. (B) Effects of RG on YAP and LATS1in LKB1-deficient HeLa cells determined by Western blotting. Data are pre-sented as mean of replicate samples with error bars representing standard deviation (vs control *p < 0.05).

Figure 7

Effects of RG on Tacrine-induced liver injury in rats. 100 and 300 mg/kg RG or 200 mg Silymarin were orally administrated to rats for three consecutive days. Subsequently, 30mg/kg tacrine was injected orally. (A) Serum ALT and AST level in rats confirmed the effect of RG on liver injury. The data represent the mean ± S.E.M. (**p < 0.01 between the vehicle control; ^##p < 0.01, ^#p < 0.05 between tacrine treatments). (B) Histo-chemical analysis of liver tissue in rats was performed by H&E staining (bar = 200μm). Arrows indicate the degeneration of hepatocytes.