Ingredients from Litsea garrettii as Potential Preventive Agents against Oxidative Insult and Inflammatory Response

Abstract

Oxidative stress and inflammation undoubtedly contribute to the pathogenesis of many human diseases. The nuclear transcription factor erythroid 2-related factor (Nrf2) and the nuclear factor κB (NF-κB) play central roles in regulation of oxidative stress and inflammation and thus are targets for developing agents against oxidative stress- and inflammation-related diseases. Our previous study indicated that the EtOH extract of Litsea garrettii protected human bronchial epithelial cells against oxidative insult via the activation of Nrf2. In the present study, a systemic phytochemical investigation of L. garrettii led to the isolation of twenty-one chemical ingredients, which were further evaluated for their inhibitions on oxidative stress and inflammation using NAD(P)H:quinone reductase (QR) assay and nitric oxide (NO) production assay. Of these ingredients, 3-methoxy-5-pentyl-phenol (MPP, 5) was identified as an Nrf2 activator and an NF-κB inhibitor. Further studies demonstrated the following: (i) MPP upregulated the protein levels of Nrf2, NAD(P)H:quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase regulatory subunit (GCLM); enhanced the nuclear translocation and stabilization of Nrf2; and inhibited arsenic [As(III)]-induced oxidative insult in normal human lung epithelial Beas-2B cells. And (ii) MPP suppressed the nuclear translocation of NF-κB p65 subunit; inhibited the lipopolysaccharide- (LPS-) stimulated increases of NF-κB p65 subunit, COX-2, iNOS, TNF-α, and IL-1β; and blocked the LPS-induced biodegrade of IκB-α in RAW 264.7 murine macrophages. Taken together, MPP displayed potential preventive effects against inflammation- and oxidative stress-related diseases.

Figure 1

Chemical structures of the purified ingredients.

Figure 2

QR including activities of purified ingredients in hepa 1c1c7 cells. Cells were incubated with indicated doses of ingredients for 24 h, and then QR-inducing activity was measured. SF (2.0 μM) was used as a positive control. Values were presented as mean ± SD (n = 3). ^∗∗∗p < 0.0001, treated versus control. C: control.

Figure 3

Inhibitory effect of purified ingredients on NO production in RAW 264.7 cells. Cells were treated with indicated concentrations of ingredients along with LPS (1 μg/mL) for 24 h, and then the accumulation of nitrite from the supernatants was evaluated by Griess reagent. Didox was used as a positive control and possessed an inhibitory rate of about 70% at 100 μM. Values were presented as mean ± SD (n = 3). ^∗p < 0.05, relative NO level; ^#p < 0.05, cell viability, treated versus LPS group. Column, relative NO level; dot, cell viability. C: control.

Figure 4

MPP activates Nrf2 signaling pathway in human lung epithelial Beas-2B cells. (a) MPP had no cytotoxicity up to 50 μM. Cells were treated with indicated doses of MPP for 48 h, and then cell viability was determined using MTT assay. (b) MPP induced the ARE-dependent luciferase activity in a dose-dependent manner. Cells were transfected with ARE-firefly luciferase and TK-Renilla luciferase plasmids and then treated with the indicated doses of MPP or SF (5.0 μM) for 16 h. (c) MPP dose dependently induced the protein levels of Nrf2 and its downstream genes. Cells were treated with or without indicated doses of MPP or SF (5.0 μM) for 16 h, and then the total cell lysates were subjected to immunoblot analysis. (d and e) MPP induced the nuclear translocation of Nrf2. For (d), cells were treated with or without MPP (25 μM) or SF (5 μM) for 8 h and then subjected to indirect fluorescence staining. For (e), cells were treated with SF (5 μM) and SF (5 μM) and indicated doses of MPP for 8 h, and then the nuclear extracts were collected and subjected to immunoblot analysis. (f) MPP increased the half-life of Nrf2. Cells were left untreated or treated with MPP (25 μM) for 4 h. Cycloheximide (50 μM) was added to block protein synthesis. Cells were harvested at the indicated time points, and then total cell lysates were subjected to immunoblot analysis. (g) MPP induced Nrf2, NQO1, and GCLM but had no effect on Keap1 in the time course study. Cells were treated with MPP (25 μM) for the indicated times, and then the protein levels were measured by immunoblot analysis. (h) MPP induced Nrf2, NQO1, and GCLM in a dose-dependent manner but had no effect on Keap1. Cells were treated with indicated doses of MPP for 16 h, and then the protein levels were measured by immunoblot analysis. Values were presented as mean ± SD (n = 3). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.0001, treated versus control; C: control.

Figure 5

MPP protects human lung epithelial Beas-2B cells against As(III)-induced oxidative insults. (a) MPP enhanced intracellular GSH levels. Cells were exposed to the indicated doses of MPP or SF (5 μM) for 24 h, and the induced GSH level was assessed using GSH detection kit. (b) MPP prevented Beas-2B cells against As(III)-induced cell death. Cells were pretreated with indicated doses of MPP for 6 h and cotreated with 5 μM As(III) for 24 h. Then the cell viability was measured by MTT assay. (c) MPP inhibited the As(III)-stimulated ROS production. Cells were pretreated with or without MPP (25 μM) or SF (5 μM) for 6 h and then cotreated with 5 μM As(III) for another 12 h. The level of ROS was evaluated using ROS detection kit. (d) MPP inhibited AS(III)-induced cell apoptosis. Cells were pretreated with or without MPP (25 μM) or SF (5 μM) for 6 h and cotreated with 5 μM As(III) for 24 h, and then the cell viability was measured by AO/EB assay. Values were presented as mean ± SD (n = 3). ^∗∗∗p < 0.0001, treated versus control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.0001, treated versus As. C: control.

Figure 6

MPP inhibits LPS-induced inflammatory response in RAW 264.7 macrophages. (a) MPP had no cytotoxicity at doses ≤ 100 μM. Cells were treated with indicated doses of MPP for 48 h, and the cell viability was determined by MTT assay. (b and c) MPP inhibited the LPS-induced NF-κB nuclear translocation. After being pretreated with 25 μM MPP for B or indicated doses of MPP for C or didox (100 μM) for 1 h, cells were cotreated with LPS (1 μg/mL) for 1 h and then subjected to indirect fluorescence staining and immunoblot analysis. (c) MPP inhibited the NF-κB-dependent luciferase activity. After being cotransfected with NF-κB firefly luciferase and TK-Renilla luciferase, cells were pretreated with MPP (25 μM) or didox (100 μM) for 1 h and cotreated with LPS (1 μg/mL) for 16 h. (d) MPP inhibited the LPS-stimulated activation of NF-κB p65 subunit, IκB-α, iNOS, and COX-2. Cells were cotreated with indicated doses of MPP or didox (100 μM) and LPS for 16 h, and then the protein levels were measured by immunoblot analysis. (e) MPP inhibited the LPS-stimulated activation of inflammatory cytokines TNF-α and IL-1β. The concentrations of cytokines were detected using the ELISA kits. Values were presented as mean ± SD (n = 3). ^###p < 0.0001, treated versus control; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.0001, treated versus LPS. C: control.