Anti-Inflammatory Activity of Sanghuangporus sanghuang Mycelium

Abstract

Acute lung injury (ALI) is characterized by inflammation of the lung tissue and oxidative injury caused by excessive accumulation of reactive oxygen species. Studies have suggested that anti-inflammatory or antioxidant agents could be used for the treatment of ALI with a good outcome. Therefore, our study aimed to test whether the mycelium extract of Sanghuangporus sanghuang (SS-1), believed to exhibit antioxidant and anti-inflammatory properties, could be used against the excessive inflammatory response associated with lipopolysaccharides (LPS)-induced ALI in mice and to investigate its possible mechanism of action. The experimental results showed that the administration of SS-1 could inhibit LPS-induced inflammation. SS-1 could reduce the number of inflammatory cells, inhibit myeloperoxidase (MPO) activity, regulate the TLR4/PI3K/Akt/mTOR pathway and the signal transduction of NF-κB and MAPK pathways in the lung tissue, and inhibit high mobility group box-1 protein 1 (HNGB1) activity in BALF. In addition, SS-1 could affect the synthesis of antioxidant enzymes Heme oxygenase 1 (HO-1) and Thioredoxin-1 (Trx-1) in the lung tissue and regulate signal transduction in the KRAB-associated protein-1 (KAP1)/nuclear factor erythroid-2-related factor Nrf2/Kelch Like ECH associated Protein 1 (Keap1) pathway. Histological results showed that administration of SS-1 prior to induction could inhibit the large-scale LPS-induced neutrophil infiltration of the lung tissue. Therefore, based on all experimental results, we propose that SS-1 exhibits a protective effect against LPS-induced ALI in mice. The mycelium of S. sanghuang can potentially be used for the treatment or prevention of inflammation-related diseases.

Keywords: HNGB; HO-1; KAP1/Nrf2 pathway; PI3K/Akt/mTOR pathways; acute lung injury; lipopolysaccharide; mycelium of Sanghuangporus sanghuang.

Figure 1

SS-1 inhibited lipopolysaccharide (LPS)-induced cell inflammation in RAW264.7 cells. Cytotoxicity (A) of SS-1 in LPS-stimulated RAW264.7 cells. Cells were treated with SS-1 at 125, 250 and 500 μg/mL for 24 h, and cell viability was assayed by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. NO (B); TNF-α (C); IL-1β (D); and IL-6 (E) production in LPS-stimulated RAW264.7 cells. Cells were incubated with or without LPS (100 ng/mL) in the presence of various doses (125, 250 and 500 μg/mL) of SS-1 for 24 h. Data are expressed as the means ± SD of three independent experiments; ^### compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 1

SS-1 inhibited lipopolysaccharide (LPS)-induced cell inflammation in RAW264.7 cells. Cytotoxicity (A) of SS-1 in LPS-stimulated RAW264.7 cells. Cells were treated with SS-1 at 125, 250 and 500 μg/mL for 24 h, and cell viability was assayed by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. NO (B); TNF-α (C); IL-1β (D); and IL-6 (E) production in LPS-stimulated RAW264.7 cells. Cells were incubated with or without LPS (100 ng/mL) in the presence of various doses (125, 250 and 500 μg/mL) of SS-1 for 24 h. Data are expressed as the means ± SD of three independent experiments; ^### compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 2

Effects of SS-1 on iNOS, COX-2, IκBα, p-IκBα, and NF-κB protein expression (A); and MAPK phosphorylation (B) in LPS-induced RAW264.7 cells. Cells were incubated with or without LPS (100 ng/mL) in the presence of various concentrations (125, 250 and 500 μg/mL) of SS-1 for 24 h. The data were presented as mean ± SD for the three different experiments performed in triplicate; ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); * p < 0.05, ** p < 0.01 and *** p < 0.001 were compared with LPS-alone group.

Figure 2

Effects of SS-1 on iNOS, COX-2, IκBα, p-IκBα, and NF-κB protein expression (A); and MAPK phosphorylation (B) in LPS-induced RAW264.7 cells. Cells were incubated with or without LPS (100 ng/mL) in the presence of various concentrations (125, 250 and 500 μg/mL) of SS-1 for 24 h. The data were presented as mean ± SD for the three different experiments performed in triplicate; ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); * p < 0.05, ** p < 0.01 and *** p < 0.001 were compared with LPS-alone group.

Figure 3

Effects of SS-1 on TLR4/PI3K/Akt/mTOR/IKK protein expression (A); and anti-oxidative enzymes, HO-1, Trx-1, and Nrf2/KAP1 protein expression (B) in LPS-induced RAW264.7 cells. Cells were incubated with or without LPS (100 ng/mL) in the presence of various concentrations (125, 250 and 500 μg/mL) of SS-1 for 24 h. The data were presented as mean ± SD for the three different experiments performed in triplicate; ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01 and *** p < 0.001 were compared with LPS-alone group.

Figure 3

Effects of SS-1 on TLR4/PI3K/Akt/mTOR/IKK protein expression (A); and anti-oxidative enzymes, HO-1, Trx-1, and Nrf2/KAP1 protein expression (B) in LPS-induced RAW264.7 cells. Cells were incubated with or without LPS (100 ng/mL) in the presence of various concentrations (125, 250 and 500 μg/mL) of SS-1 for 24 h. The data were presented as mean ± SD for the three different experiments performed in triplicate; ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01 and *** p < 0.001 were compared with LPS-alone group.

Figure 4

SS-1 attenuated pulmonary inflammation in vivo. Six hours after LPS injection with or without SS-1 pretreatments, mice were exsanguinated and their left lower lungs were fixed. Then, tissue sections were stained with hematoxylin and eosin (H&E): (A) Control; (B) LPS; (C) LPS + Dex; (D) LPS + SS-1-L; (E) LPS + SS-1-M; (F) LPS + SS-1-H. The figure demonstrates a representative view (×400) from each group; each bar represents the mean ± SD of six mice. (G) Severity of lung injury was analyzed by the lung injury scoring system. Each value represents as mean ± SD of six mice; ^### compared with sample of control group; ** p < 0.01, and *** p < 0.001 were compared with LPS-alone group. The infiltrating neutrophils were more abundant in (B) LPS group as shown by arrows.

Figure 4

SS-1 attenuated pulmonary inflammation in vivo. Six hours after LPS injection with or without SS-1 pretreatments, mice were exsanguinated and their left lower lungs were fixed. Then, tissue sections were stained with hematoxylin and eosin (H&E): (A) Control; (B) LPS; (C) LPS + Dex; (D) LPS + SS-1-L; (E) LPS + SS-1-M; (F) LPS + SS-1-H. The figure demonstrates a representative view (×400) from each group; each bar represents the mean ± SD of six mice. (G) Severity of lung injury was analyzed by the lung injury scoring system. Each value represents as mean ± SD of six mice; ^### compared with sample of control group; ** p < 0.01, and *** p < 0.001 were compared with LPS-alone group. The infiltrating neutrophils were more abundant in (B) LPS group as shown by arrows.

Figure 5

SS-1 improved pulmonary edema (A); Myeloperoxidase (MPO) activity (B); MPO and HMGB1 protein level (C) in vivo; and reduced cellular counts (D); and total protein (E) in BALF. Six hours after LPS injection with or without SS-1 pretreatments, mice were sacrificed and their lungs were lavaged. The right lower lungs were used to assess wet to dry (W/D) ratio of lung. Cells in the BALF were collected and cytospin preparations were made. Total cells and total proteins in BALF were analyzed. Data represent mean ± SD of six mice; ^### compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); * p < 0.05, ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 5

SS-1 improved pulmonary edema (A); Myeloperoxidase (MPO) activity (B); MPO and HMGB1 protein level (C) in vivo; and reduced cellular counts (D); and total protein (E) in BALF. Six hours after LPS injection with or without SS-1 pretreatments, mice were sacrificed and their lungs were lavaged. The right lower lungs were used to assess wet to dry (W/D) ratio of lung. Cells in the BALF were collected and cytospin preparations were made. Total cells and total proteins in BALF were analyzed. Data represent mean ± SD of six mice; ^### compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); * p < 0.05, ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 6

SS-1 down regulated: TNF-α (A); IL-6 (B); IL-1β (C); and NO (D); and increased IL-10 (E) in BALF. Six hours after LPS injection with or without SS pre-treatments, mice were sacrificed, their lungs were lavaged and the BALF were collected. TNF-α, IL-6, IL-1β, NO and IL-10 were detected by ELISA. Data represent mean ± SD of six mice; ^### compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); * p < 0.05 ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 7

Effects of SS-1 on LPS-induced iNOs, COX-2, IκB-α, and NF-κB protein expression in lung (A); and MAPK phosphorylation (B) expression in ALI mice. Mice were pretreated with different concentrations of SS for 1 h and stimulated with LPS. Western blotting using specific antibodies was used for the detection of iNOs, COX-2, IκB-α phosphorylated, NF-κB nuclear and cytosol, and total forms of three MAPK molecules, ERK, p38, and JNK. Data represent mean ± SD of six mice; ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 7

Effects of SS-1 on LPS-induced iNOs, COX-2, IκB-α, and NF-κB protein expression in lung (A); and MAPK phosphorylation (B) expression in ALI mice. Mice were pretreated with different concentrations of SS for 1 h and stimulated with LPS. Western blotting using specific antibodies was used for the detection of iNOs, COX-2, IκB-α phosphorylated, NF-κB nuclear and cytosol, and total forms of three MAPK molecules, ERK, p38, and JNK. Data represent mean ± SD of six mice; ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 8

Effects of SS-1 on LPS-induced TLR4, PI3K, AKT, mTOR, and IKK protein expression (A); and antioxidative enzymes and HO-1, Trx-1, Nrf2/KAP1 protein expression (B) in lung in ALI mice. Mice were pretreated with different concentrations of SS for 1 h and stimulated with LPS. The Western blotting using specific antibodies was used for the detection of TLR4, PI3K, AKT, mTOR, and IKK protein expression. Data represent mean ± SD of six mice; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 8

Effects of SS-1 on LPS-induced TLR4, PI3K, AKT, mTOR, and IKK protein expression (A); and antioxidative enzymes and HO-1, Trx-1, Nrf2/KAP1 protein expression (B) in lung in ALI mice. Mice were pretreated with different concentrations of SS for 1 h and stimulated with LPS. The Western blotting using specific antibodies was used for the detection of TLR4, PI3K, AKT, mTOR, and IKK protein expression. Data represent mean ± SD of six mice; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 were compared with sample of control group (one-way ANOVA followed by Scheffe’s multiple range tests); ** p < 0.01, and *** p < 0.001, were compared with LPS-alone group.

Figure 9

HPLC profile of SS-1 (A); and scheme of its mechanism for the protective effect on LPS-induced inflammation (B). HPLC chromatogram of the polyphenol standards (500 μg/mL) at 280 nm. Peaks: 1. Protocatechuic acid (4.53 min); 2. Protocatechvaldehyde (5.73 min); 3. Caffeic acid (7.53 min); 4. Syringic acid (8.18 min); 5. DTA (9.89 min); and 6. DBL (12.3 min). Representative chromatograms of SS-1 are shown: 1. Protocatechuic acid (96.8 μg/mg extract); 2. Protocatechvaldehyde (57.2 μg/mg extract); 3. Caffeic acid (59.3 μg/mg extract); 4. Syringic acid (42.6 μg/mg extract); 5. DTA (2,5-dihydroxyterephtalic acid, 80.7 μg/mg extract); and 6. DBL (3,4-dihydroxybenzalacetone, 90.2 μg/mg extract).

Figure 9

HPLC profile of SS-1 (A); and scheme of its mechanism for the protective effect on LPS-induced inflammation (B). HPLC chromatogram of the polyphenol standards (500 μg/mL) at 280 nm. Peaks: 1. Protocatechuic acid (4.53 min); 2. Protocatechvaldehyde (5.73 min); 3. Caffeic acid (7.53 min); 4. Syringic acid (8.18 min); 5. DTA (9.89 min); and 6. DBL (12.3 min). Representative chromatograms of SS-1 are shown: 1. Protocatechuic acid (96.8 μg/mg extract); 2. Protocatechvaldehyde (57.2 μg/mg extract); 3. Caffeic acid (59.3 μg/mg extract); 4. Syringic acid (42.6 μg/mg extract); 5. DTA (2,5-dihydroxyterephtalic acid, 80.7 μg/mg extract); and 6. DBL (3,4-dihydroxybenzalacetone, 90.2 μg/mg extract).