Plumbagin ameliorates LPS-induced acute lung injury by regulating PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling pathways

Abstract

Acute lung injury (ALI) is a major pathophysiological problem characterized by severe inflammation, resulting in high morbidity and mortality. Plumbagin (PL), a major bioactive constituent extracted from the traditional Chinese herb Plumbago zeylanica, has been shown to possess anti-inflammatory and antioxidant pharmacological activities. However, its protective effect on ALI has not been extensively studied. The objective of this study was to investigate the protective effect of PL against ALI induced by LPS and to elucidate its possible mechanisms both in vivo and in vitro. PL treatment significantly inhibited pathological injury, MPO activity, and the wet/dry ratio in lung tissues, and decreased the levels of inflammatory cells and inflammatory cytokines TNF-α, IL-1β, IL-6 in BALF induced by LPS. In addition, PL inhibited the activation of the PI3K/AKT/mTOR signalling pathway, increased the activity of antioxidant enzymes CAT, SOD, GSH and activated the Keap1/Nrf2/HO-1 signalling pathway during ALI induced by LPS. To further assess the association between the inhibitory effects of PL on ALI and the PI3K/AKT/mTOR and Keap1/Nrf2/HO-1 signalling, we pretreated RAW264.7 cells with 740Y-P and ML385. The results showed that the activation of PI3K/AKT/mTOR signalling reversed the protective effect of PL on inflammatory response induced by LPS. Moreover, the inhibitory effects of PL on the production of inflammatory cytokines induced by LPS also inhibited by downregulating Keap1/Nrf2/HO-1 signalling. In conclusion, the results indicate that the PL ameliorate LPS-induced ALI by regulating the PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling, which may provide a novel therapeutic perspective for PL in inhibiting ALI.

Keywords: ALI; Keap1‐Nrf2/HO‐1 signalling; PI3K/AKT/mTOR signalling; plumbagin.

FIGURE 1

Effect of PL on lung inflammatory response induced by LPS. (A) Pathological changes of lung tissue (×100). (B) Pathological score of lung tissue. (C) Lung wet‐to‐dry weight ratio. (D) MPO activity in lung tissues. ^## p < 0.01 versus Control group; ^* p < 0.05, ^** p < 0.01 versus LPS group.

FIGURE 2

Effect of PL on inflammatory medium production in BALF induced by LPS. (A) The concentration of TNF‐α in BALF. (B) The concentration of IL‐1β in BALF. (C) The concentration of IL‐6 in BALF. (D) The level of total cells in BALF. (E) The level of neutrophils in BALF. (F) The level of macrophages in BALF. (G) The concentration of total protein in BALF. ^## p < 0.01 versus Control group; ^* p < 0.05, ^** p < 0.01 versus LPS group.

FIGURE 3

Effects of PL on PI3K/AKT/mTOR signalling during LPS‐induced ALI. The protein content of P13K, p‐P13K, AKT, p‐AKT, mTOR and p‐mTOR were detected by western blot, and GAPDH was used as internal reference protein. ^## p < 0.01 versus Control group; ^* p < 0.05, ^** p < 0.01 versus LPS group.

FIGURE 4

Effect of PL on Keap1‐Nrf2/HO‐1 signalling during LPS‐induced ALI. (A) SOD activity in lung tissues. (B) GSH activity in lung tissues. (C) CAT activity in lung tissues. (D) MDA activity in lung tissues. (E) The protein content of Keap1, Nrf2, and HO‐1 were detected by western blot, and GAPDH was used as internal reference protein. ^## p < 0.01 versus Control group; ^* p < 0.05, ^** p < 0.01 versus LPS group.

FIGURE 5

Effect of PL on inflammatory response in the RAW264.7 cells induced by LPS. (A) Viability of RAW264.7 cells after exposure to PL was detected by MTT. (B) The concentration of TNF‐α in cell‐cultured supernatant. (C) The concentration of IL‐1β in cell‐cultured supernatant. (D) The concentration of IL‐6 in cell‐cultured supernatant. ^## p < 0.01 versus Control group; ^* p < 0.05, ^** p < 0.01 versus LPS group.

FIGURE 6

Effect of PL on PI3K/AKT/mTOR and Keap1‐Nrf2/HO‐1 signalling in the RAW264.7 cells induced by LPS. The protein content of P13K, p‐P13K, AKT, p‐AKT, mTOR, p‐mTOR, Keap1, Nrf2 and HO‐1 were detected by western blot, and GAPDH was used as internal reference protein. ^## p < 0.01 versus Control group; ^* p < 0.05, ^** p < 0.01 versus LPS group.

FIGURE 7

Activation of PI3K/AKT/mTOR signalling reversed the protective effect of PL on LPS‐induced inflammatory response in the RAW264.7 cells. (A) The protein content of P13K, p‐P13K, AKT, p‐AKT, mTOR and p‐mTOR were detected by western blot, and GAPDH was used as internal reference protein. (B) The concentration of TNF‐α in cell‐cultured supernatant. (C) The concentration of IL‐1β in cell‐cultured supernatant. (D) The concentration of IL‐6 in cell‐cultured supernatant. ^** p < 0.01.

FIGURE 8

Inhibition of Keap1‐Nrf2/HO‐1 signalling reversed the protective effect of PL on LPS‐induced inflammatory response in the RAW264.7 cells. (A) The protein content of Keap1, Nrf2 and HO‐1 were detected by western blot, and GAPDH was used as internal reference protein. (B) The concentration of TNF‐α in cell‐cultured supernatant. (C) The concentration of IL‐1β in cell‐cultured supernatant. (D) The concentration of IL‐6 in cell‐cultured supernatant. ^** p < 0.01.