Aqueous Extract of Descuraniae Semen Attenuates Lipopolysaccharide-Induced Inflammation and Apoptosis by Regulating the Proteasomal Degradation and IRE1α-Dependent Unfolded Protein Response in A549 Cells

Abstract

Background: Lipopolysaccharide (LPS)-induced acute lung injury (ALI) induces endoplasmic reticulum stress, unfolded protein response (UPR), apoptosis, and inflammation. Inositol-requiring enzyme 1 (IRE1)-α is important for adaptive and apoptotic UPR determination during ER stress. The aqueous extract of Descuraniae Semen (AEDS) is reported to be a safe and effective herb for the treatment of pulmonary edema as it shows anti-inflammatory activities.

Methods: We investigated the effects of AEDS on LPS-induced ALI in A549 cells with respect to the regulation of IRE1α-dependent UPR, proteasomal degradation, mitochondrial membrane potential (MtMP), inflammation, and apoptosis.

Results: AEDS attenuated ER stress by regulating the proteasomal degradation. LPS induced ER stress [binding immunoglobulin protein (BiP), phosphorylated IRE1α, sliced X-box binding protein 1 [XBP1s], phosphorylated cJUN NH2-terminal kinase (pJNK), B-cell lymphoma (Bcl)-2-associated X (Bax), Bcl-2], inflammation (nucleus factor-kappa B (NF-κB) p65 nuclear translocation, nucleus NF-κB, pro-inflammatory cytokines] and apoptosis [C/EBP homologous protein (CHOP), cytochrome c, caspase-8, and caspase-6, and TUNEL] were significantly attenuated by AEDS treatment in A549 cells. AEDS prevents LPS-induced decreased expression of MtMP in A549 cells.

Conclusions: AEDS attenuated LPS-induced inflammation and apoptosis by regulating proteasomal degradation, promoting IRE1α-dependent adaptive UPR, and inhibiting IRE1α-dependent apoptotic UPR. Moreover, IRE1α-dependent UPR plays a pivotal role in the mechanisms of LPS-induced ALI. Based on these findings, AEDS is suggested as a potential therapeutic option for treating patients with ALI.

Keywords: Descuraniae semen; IRE1α; acute lung injury; apoptosis; inflammation; lipopolysaccharide; unfolded protein response.

Figure 1

Effects of lipopolysaccharide (LPS) and the aqueous extract of Descuraniae Semen (AEDS) on cell viability and endoplasmic reticulum (ER) stress in A549 cells. (A) LPS (50 µg/mL) decreased the cell viability in a time-dependent manner after 8 h of induction. (B) LPS (50 µg/mL) increased the binding immunoglobulin protein (BiP) expression levels in a time-dependent manner after 16 h of induction. (C) AEDS exhibited no effects on the cell viability in a dose-dependent manner in 1 h (D) AEDS decreased the BiP expression levels in a dose-dependent manner in 1 h (E) Experimental protocols with five experiment groups: Control, LPS, AEDS, AEDS pre-treatment followed by LPS induction (preAEDS), and LPS induction followed by AEDS treatment (postAEDS) groups. Cell counting-kit (CCK)-8 assay was used to detect the cell viability. All data are expressed as the mean ± standard deviation (SD). All p-values were calculated using one-way analysis of variance followed by Tukey’s post-hoc test. The experiment was performed five times in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 2

AEDS decreases ER stress by regulating the proteasomal degradation and attenuates LPS-induced ER stress and inositol-requiring enzyme 1 (IRE1)-α-dependent apoptotic unfolded protein response (UPR) cascades in A549 cells. (A) Effects of MG132 (10 µM, 6 h), AEDS (200 ug/mL, 1 h), and MG132 + AEDS on binding immunoglobulin protein (BiP) expression in A549 cells. (B) Western blotting results of ER stress and IRE1α-dependent apoptotic UPR cascades. (C–H) Quantitative Western blotting results of ER stress and IRE1α-dependent apoptotic UPR cascades. (I) Mitochondrial membrane potential (MtMP) assay results. MtMP was measured using the JC-1 mitochondrial membrane potential assay kit. All data are expressed as the mean ± SD. All p-values were calculated using one-way analysis of variance followed by Tukey’s post-hoc test. The experiment was performed five times in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 3

AEDS attenuates LPS-induced nuclear factor-kappa B (NF-κB) nuclear translocation and inflammation in A549 cells. (A) Microphotographs (200X) of immunofluorescence analysis of NF-κB p65 and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI). (B) Western blotting results of nucleus NF-κB. (C) Quantitative Western blotting results of nucleus NF-κB. All p-values were calculated using one-way analysis of variance followed by Tukey’s post-hoc test. The experiment was performed five times in duplicate. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 4

AEDS attenuates LPS-induced elevation in pro-inflammatory cytokine levels. ELISA results of (A) tumor necrosis factor (TNF)-α (B) interleukin (IL)-1β, (C) IL-6, and (D) IL-8 levels in the culture medium. All p-values were calculated using one-way analysis of variance followed by Tukey’s post-hoc test. The experiment was performed five times in duplicate. **p < 0.01, ****p < 0.0001. ns, not significant.

Figure 5

AEDS attenuates LPS-induced apoptotic UPR and apoptosis in A549 cells (A) Microphotographs (200X) of immunofluorescence analysis of terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) and DAPI. (B) Western blotting results of CCAAT-enhancer-binding protein homologous protein (CHOP), cytochrome c (Cyt C), caspase-8, caspase-6, and cleaved caspase-3 (cCaspase-3). (C–G) Quantitative Western blotting results of CHOP, Cyt C, caspase-8, caspase-6, and cCaspase-3. All p-values were calculated using one-way analysis of variance followed by Tukey’s post-hoc test. The experiment was performed five times in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 6

Mechanisms of LPS-induced acute lung injury (ALI) and protective role of AEDS. AEDS decreases LPS-induced inflammation and apoptosis by regulating the proteasomal degradation, promoting IRE1α-dependent adaptive UPR, and inhibiting IRE1α-dependent apoptotic UPR. IRE1α-dependent UPR plays a pivotal role in LPS-induced ALI. AEDS tends to induce adaptive UPR, whereas LPS induces apoptotic UPR.