Evaluation of the Liver Toxicity of Pterocephalus hookeri Extract via Triggering Necrosis

Abstract

Pterocephalus hookeri (C. B. Clarke) Höeck, recorded in the Chinese Pharmacopoeia (2015 version) as a Tibetan medicine for the treatment of various diseases, especially rheumatoid arthritis, was believed to possess a slight toxicity. However, hardly any research has been carried out about it. The present study aimed to evaluate the toxicity in vivo and in vitro. Toxicity was observed by the evaluation of mice weight loss and histopathological changes in the liver. Then, the comparison research between ethyl acetate extract (EAE) and n-butanol extract (BUE) suggested that liver toxicity was mainly induced by BUE. The mechanical study suggested that BUE-induced liver toxicity was closely associated with necrosis detected by MTT and propidium iodide (PI) staining, via releasing lactate dehydrogenase (LDH), reducing the fluidity, and increasing the permeability of the cell membrane. Western blot analysis confirmed that the necrosis occurred molecularly by the up-regulation of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3), as well as the activation of the nuclear factor-kappa-gene binding (NF-κB) signaling pathway in vivo and in vitro. This finding indicated that the liver toxicity induced by BUE from P. hookeri was mainly caused by necrosis, which provides an important theoretical support for further evaluation of the safety of this folk medicine.

Keywords: Pterocephalus hookeri; inflammation; liver toxicity; n-butanol extract; necrosis.

Figure 1

Effects on body weight in mice treated with PH (5 g/kg) during 16 days. Values are expressed as mean ± SD (n = 8), # p < 0.05, ## p < 0.01 compared with the control group.

Figure 2

Histopathological analysis of heart, liver, spleen, lung, and kidney treated with PH (hematoxylin and eosin (H&E); ×200).

Figure 3

Effects of EAE and BUE on the serum levels of ALT, AST, ALP, TBIL and DBIL (A), and histopathological changes (B) in the liver. Data are represented as mean ± SD (n = 8), * p < 0.05 and ** p < 0.01 compared with the control group (H&E; ×200).

Figure 4

Effects of EAE and BUE on (A) NF-κB signaling pathway and (B) RIP1 and RIP3 for liver tissues in carrageenan-induced mice. Results are expressed as mean ± SD (n = 3), * p < 0.05, ** p < 0.01 compared with the control group.

Figure 5

Effects of (A–C) EAE and (D–F) BUE on viabilities of L-02, RAW264.7, and HUVEC cells. Results are expressed as a percentage of the control group, which was set at 100%; values are expressed as means ± SD (n = 5), * p < 0.05, ** p < 0.01 compared with the control group.

Figure 6

Effects of (A–C) EAE and (D–F) BUE on viabilities in L-02, RAW264.7, and HUVEC cells treated with Z-VAD-FMK, 3-MA, and Nec-1. Results are expressed as a percentage of the control group, which was set at 100%; values are expressed as means ± SD (n = 5), ## p < 0.01 compared with the control group, * p < 0.05, ** p < 0.01 compared with the BUE group.

Figure 7

Necrotic effects induced by EAE and BUE in L-02, RAW264.7, and HUVEC cells by PI staining (×200).

Figure 8

Effects of EAE and BUE on (A) NF-κB signaling pathway and (B) RIP1 and RIP3 in L-02 cells. Results are expressed as mean ± SD (n = 3), * p < 0.05, ** p < 0.01 compared with the control group.

Figure 9

Effects of EAE and BUE (A) on LDH release content and (B) cell membrane fluidity in L-02 cells. Results are expressed as mean ± SD (n = 5), * p < 0.05, ** p < 0.01 compared with the control group.

Figure 10

Effects of EAE and BUE on membrane permeability changes in L-02 cells. The green presented FDA⁺; the orange presented FDA⁺/PI⁺; the red presented PI⁺ (×200).