Flavonoids of Tetrastigma hemsleyanum Diels et Gilg Against Acute Hepatic Injury by Blocking PI3K/AKT Signaling Pathway

Abstract

Objective: This study aims to investigate the mechanism of Tetrastigma hemsleyanum Diels et Gilg flavonoids (THF) on acute hepatic injury (AHI). Methods: First, high-performance liquid chromatography (HPLC) fingerprints were established to obtain the main chemical components of THF. According to the network pharmacology databases, collect active targets of AHI and potential targets. Using interaction targets to construct a protein-protein interaction (PPI) network, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Finally, the affinity between the core targets and the main active ingredients was verified by molecular docking. Next, verified network pharmacology predictions with animal experiments. Mice were treated with THF (20, 40, and 80 mg/kg) for 7 days, and then built an acute liver injury model (lipopolysaccharide [LPS], 10 mg/kg). Detecting the liver biochemical indices, observe the liver pathological changes, and verify the key signaling pathway targets. Results: HPLC showed that the main components of THF were quercetin and kaempferol. Seven active ingredients and 193 potential targets were screened, and 259 disease targets related to acute liver injury, quercetin, and kaempferol may be the main active ingredients in THF. PPI network analysis showed that tumor necrosis factor (TNF), interleukin-6 (IL-6), and tumor protein 53 (TP53) were potential targets of THF for the treatment of AHI. KEGG analysis showed that the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway might be one of the main pathways in the treatment of AHI. The molecular docking results showed that active compounds both have strong binding activity with potential targets in PPI. In vivo experiments showed that THF could reduce the fibrosis and inflammation of liver tissue etc. Meanwhile, it could downregulate the alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-6, tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP) levels, and the protein expressions of phosphorylated phosphoinositide 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), and the ratio of BCL2-associated X (BAX)/B-cell lymphoma-2 (BCL-2) in the liver tissue of the mice with acute liver injury and upregulate the level of interleukin-10 (IL-10). Conclusion: The treatment of acute liver injury with THF is characterized by multicomponents and multitargets, and its mechanism may be related to the alleviation of the inflammatory response, reduction of apoptosis, and regulation of the PI3K/AKT signaling pathway.

Keywords: Tetrastigma hemsleyanum Diels et Gilg; acute hepatic injury; flavonoids; molecular dock; network pharmacology.

Figure 1

HPLC of the standard and THF sample at 372 nm: (A) standard diagram and (B) sample diagram. 1, kaempferol; 2, quercetin; 3, nicotifiorin; 4, vitexin.

Figure 2

Network pharmacology figures. (A) Venn diagram of common targets of THF and AHI. (B) THF–compound–AHI–target network. (Active THF names are marked by green squares, and the yellow circles indicate the THF treatment of AHI-related common targets.) (C) PPI network of related THF targets in the treatment of AHI. (D) GO enrichment analysis. (E) Enrichment analysis of KEGG pathways. AHI, acute hepatic injury; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction; THF, Tetrastigma hemsleyanum Diels et Gilg flavonoids.

Figure 3

The protein–ligand of the docking simulation (3D and 2D figures). (A) Kaempferol with TNF. (B) Quercetin with TNF. (C) Kaempferol with IL-6. (D) Quercetin with IL-6. (E) Kaempferol with TP53. (F) Quercetin with TP53. IL-6, interleukin-6; TNF, tumor necrosis factor.

Figure 4

Histopathological examination of liver tissue (HE staining, 200×; Masson staining 400×). CON, control; H&E, hematoxylin and eosin; MOD, model; THF-H, Tetrastigma hemsleyanum Diels et Gilg flavonoids of high dose; THF-L, Tetrastigma hemsleyanum Diels et Gilg flavonoids of low dose; THF-M, Tetrastigma hemsleyanum Diels et Gilg flavonoids of medium dose.

Figure 5

Effect of THF on ALT and AST levels in the liver of AHI. Data are presented as the mean ± standard deviation (SD), n = 10. ^#p < 0.05 compared with the CON group; ⁣^∗p < 0.05, and ⁣^∗∗p < 0.01 compared with the MOD group. AHI, acute hepatic injury; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CON, control; MOD, model; THF-H, Tetrastigma hemsleyanum Diels et Gilg flavonoids of high dose; THF-L, Tetrastigma hemsleyanum Diels et Gilg flavonoids of low dose; THF-M, Tetrastigma hemsleyanum Diels et Gilg flavonoids of medium dose.

Figure 6

Effect of THF on TNF-α, IL-6, IL-10, and CRP in serum of mice with LPS-induced AHI. And the production of IL-6, IL-10, TNF-α, and CRP was detected by ELISA kits. Data are presented as the mean ± standard deviation (SD), n = 10. ^#p < 0.05, ^##p < 0.01 compared with the CON group; ⁣^∗p < 0.05, ⁣^∗∗p < 0.01 compared with the MOD group. AHI, acute hepatic injury; CON, control; CRP, C-reactive protein; IL-6, interleukin-6; IL-10, interleukin-10; LPS, lipopolysaccharide; MOD, model; THF-H, Tetrastigma hemsleyanum Diels et Gilg flavonoids of high dose; THF-L, Tetrastigma hemsleyanum Diels et Gilg flavonoids of low dose; THF-M, Tetrastigma hemsleyanum Diels et Gilg flavonoids of medium dose.

Figure 7

Effect of THF on hepatic PI3K/AKT pathway in AHI mice (400×). Data are presented as the mean ± standard deviation (SD), n = 3. ^#p < 0.05, ^##p < 0.01 compared with the CON group; ⁣^∗p < 0.05, ⁣^∗∗p < 0.01 compared with the MOD group. AHI, acute hepatic injury; AKT, protein kinase B; CON, control; MOD, model; p-AKT, phosphorylated protein kinase B; PI3K, phosphoinositide 3-kinase; p-PI3K, phosphorylated phosphoinositide 3-kinase; THF-H, Tetrastigma hemsleyanum Diels et Gilg flavonoids of high dose; THF-L, Tetrastigma hemsleyanum Diels et Gilg flavonoids of low dose; THF-M, Tetrastigma hemsleyanum Diels et Gilg flavonoids of medium dose.

Figure 8

Effect of THF on liver apoptotic proteins BAX and BCL-2 in AHI mice. Data are presented as the mean ± standard deviation (SD), n = 3. ^##p < 0.01 compared with the CON group; ⁣^∗p < 0.05, ⁣^∗∗p < 0.01 compared with the MOD group. AHI, acute hepatic injury; BAX, BCL2-associated X; BCL-2, B-cell lymphoma-2; CON, control; MOD, model; THF-H, Tetrastigma hemsleyanum Diels et Gilg flavonoids of high dose; THF-L, Tetrastigma hemsleyanum Diels et Gilg flavonoids of low dose; THF-M, Tetrastigma hemsleyanum Diels et Gilg flavonoids of medium dose.