EGCG promotes PRKCA expression to alleviate LPS-induced acute lung injury and inflammatory response

Abstract

Acute lung injury (ALI), which could be induced by multiple factors such as lipopolysaccharide (LPS), refer to clinical symptoms of acute respiratory failure, commonly with high morbidity and mortality. Reportedly, active ingredients from green tea have anti-inflammatory and anticancer properties, including epigallocatechin-3-gallate (EGCG). In the present study, protein kinase C alpha (PRKCA) is involved in EGCG protection against LPS-induced inflammation and ALI. EGCG treatment attenuated LPS-stimulated ALI in mice as manifested as improved lung injury scores, decreased total cell amounts, neutrophil amounts and macrophage amounts, inhibited the activity of MPO, decreased wet-to-dry weight ratio of lung tissues, and inhibited release of inflammatory cytokines TNF-α, IL-1β, and IL-6. PRKCA mRNA and protein expression showed to be dramatically decreased by LPS treatment while reversed by EGCG treatment. Within LPS-stimulated ALI mice, PRKCA silencing further aggravated, while PRKCA overexpression attenuated LPS-stimulated inflammation and ALI through MAPK signaling pathway. PRKCA silencing attenuated EGCG protection. Within LPS-induced RAW 264.7 macrophages, EGCG could induce PRKCA expression. Single EGCG treatment or Lv-PRKCA infection attenuated LPS-induced increases in inflammatory factors; PRKCA silencing could reverse the suppressive effects of EGCG upon LPS-stimulated inflammatory factor release. In conclusion, EGCG pretreatment inhibits LPS-induced ALI in mice. The protective mechanism might be associated with the inhibitory effects of PRKCA on proinflammatory cytokine release via macrophages and MAPK signaling pathway.

Figure 1

Epigallocatechin gallate (EGCG) alleviates LPS-induced acute lung injury (ALI) in mouse model. Mice were randomly divided into four groups: the control group, the EGCG group, the LPS group, and the LPS + EGCG group. Mice in each group were treated accordingly and examined for (A) the pathological changes in lung tissues by hematoxylin and eosin (H&E) staining (200 ×). scale bar = 50 μm; (B) lung injury scores following the methods reported before; (C) bronchoalveolar lavage fluid (BALF) cell numbers including total cells, neutrophils, macrophages and lymphocytes using a hemocytometer and Giemsa staining; (D) MPO activity using a spectrophotometer; (E) lung wet/dry weight ration. **P < 0.01 compared to Control group; #P < 0.05 compared to LPS group.

Figure 2

EGCG decreases the levels of the inflammatory cytokines in vivo. The BALF levels of inflammatory factors, including TNF-α, IL-6, and IL-1β in each group were examined by ELISA using corresponding mouse ELISA kits. ***P < 0.001 compared to Control group; ##P < 0.001 compared to LPS group.

Figure 3

EGCG modulates PRKCA expression in LPS-induced ALI mice. Mice were divided into four groups (the control group, the EGCG group, the LPS group, and the LPS + EGCG group) accordingly. (A) The expression levels of the top ten genes predicted to interact with EGCG were examined in the control group and the LPS group using real-time qPCR. (B) PRKCA mRNA expression in mouse lung tissues from each group was determined using real-time qPCR. (C) PRKCA protein levels in mouse lung tissues from each group were determined using Immunoblotting. *P < 0.05, **P < 0.01 compared to Control group; ##P < 0.01 compared to LPS group.

Figure 4

Effects of PRKCA on LPS-induced ALI. Mice were randomly divided into seven groups: the control group, the LPS group, the LPS + EGCG, the LPS + Lv-NC group, the LPS + Lv-shPRKCA group, the LPS + Lv-PRKCA group, and the LPS + EGCG + Lv-shPRKCA group. Mice in each group received treatment and/or injection accordingly. (A) PRKCA mRNA and protein expression levels in lung tissues of mice from each group was determined using real-time qPCR and Immunoblotting; (B) PRKCA expression levels in alveolar macrophages of mice lung from each group were determined using real-time qPCR; (C) pathological changes using H&E staining (200 ×). scale bar = 50 μm; (D) lung injury scores following the methods reported before; (E) cell numbers including total cells, neutrophils, macrophages and lymphocytes using a hemocytometer; (F) MPO activity using a spectrophotometer; (G) lung wet/dry weight ration. *P < 0.05, **P < 0.01, compared to the control group. #P < 0.05, ##P < 0.01, compared to the LPS + Lv-NC group.

Figure 5

Effects of PRKCA on the levels of the inflammatory cytokines in vivo through MAPK signaling pathway. Mice were grouped and treated accordingly. (A) The bronchoalveolar lavage fluid (BALF) levels of inflammatory factors, including TNF-α, IL-6, and IL-1β in each group were examined by ELISA using corresponding mouse ELISA kits. (B) The protein expression levels of MAPK signaling pathway-related proteins, including p-p38, p38, p-ERK1/2, ERK1/2, p-JNK and JNK in each group were examined by Immunoblotting. **P < 0.01, compared to the control group. #P < 0.05, compared to the LPS + Lv-NC group.

Figure 6

Effects of PRKCA on LPS-induced inflammatory responses through MAPK pathway in vitro. Mouse macrophage RAW 264.7 cells were divided into seven groups: the negative control group, the LPS group, the LPS + EGCG group, the LPS + Lv-NC group, the LPS + Lv-shPRKCA group, the LPS + Lv-PRKCA group, and the LPS + EGCG + Lv-shPRKCA group. Cells in each group were treated and/or infected accordingly. (A–E) The protein levels of PRKCA, IL-6, IL-1β, and TNF-α were determined using Immunoblotting. (F) The protein expression levels of MAPK signaling pathway-related proteins, including p-p38, p38, p-ERK1/2, ERK1/2, p-JNK and JNK in each group were detected using Immunoblotting. **P < 0.01 compared to the control group; #P < 0.05, ##P < 0.01 compared to the LPS + Lv-NC group.