Nucleosides isolated from Ophiocordyceps sinensis inhibit cigarette smoke extract-induced inflammation via the SIRT1-nuclear factor-κB/p65 pathway in RAW264.7 macrophages and in COPD mice

Abstract

Background: Ophiocordyceps sinensis (C. sinensis) extracts have been found to have a therapeutic effect on patients with chronic obstructive pulmonary disease (COPD). Silent information regulator 1 (SIRT1) plays an important role in the regulation of inflammatory mediators and correlates with lung function and COPD exacerbations. The objective of this work was to explore the anti-inflammatory effect and preliminary pathways of nucleosides from cultured C. sinensis on RAW264.7 macrophages and COPD mice.

Materials and methods: The nucleosides were extracted from cultured C. sinensis powder and further purified by macroporous resin D101 and glucan G10 columns. Inflammation and oxidative stress models in RAW264.7 macrophages and in mice were established by injection of cigarette smoke extract (CSE). We then examined how the isolated nucleosides regulated the production of the associated inflammatory mediators in vitro and in vivo by enzyme-linked immunosorbent assay, reverse transcription polymerase chain reaction, and Western blot.

Results: The nucleosides inhibited inflammatory mediator expression of tumor necrosis factor-α, interleukin-6, interleukin-1β, and nitric oxide in both the CSE-stimulated RAW264.7 macrophages and mice. Moreover, the nucleosides elevated SIRT1 activation and suppressed nuclear factor-κB (NF-κB)/p65 activation in vitro and in vivo. Nucleoside treatment significantly decreased the levels of the inflammatory mediators in the bronchoalveolar lavage fluid (BALF) and serum of the CSE-induced mice. The nucleosides also altered the recruitment of inflammatory cells in BALF and improved characteristic features of the lungs in the CSE-induced mice.

Conclusion: These results show that the nucleosides suppressed COPD inflammation through the SIRT1-NF-κB/p65 pathway, suggesting that the nucleosides may be partly responsible for the therapeutic effects of cultured C. sinensis on COPD patients.

Keywords: COPD; NF-κB/p65; Ophiocordyceps sinensis; SIRT1; anti-inflammatory; nucleosides.

Figure 1

Typical chromatograms of (A) nucleoside standards and (B) unknown peaks in C. sinensis determined by high-performance liquid chromatography. Notes: Unknown nucleoside peaks in C. sinensis were tentatively identified as: 1, cytidine; 2, adenine; 3, guanine; 4, uracil; 5, hypoxanthine; 6, uridine; 7, adenosine; 8, 2′-deoxyadenosine; 9, guanosine; 10, thymidine. Abbreviation: C. sinensis, Ophiocordyceps sinensis.

Figure 2

Effects of the isolated nucleosides on NO and iNOS expression in RAW264.7 cells. Notes: The isolated nucleosides decreased (A) CSE-induced NO production, (B) iNOS mRNA expression, (C) iNOS protein, and (D) its quantitative level in RAW264.7 cells. **P<0.01, CSE-induced group vs control group (without any treatment); ^#P<0.05, ^##P<0.01, nucleoside treatment groups (25 and 50 µg/mL) vs CSE-induced group. All experiments were repeated three times. Data shown as mean ± SEM (n=3). Abbreviations: CSE, cigarette smoke extract; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; NO, nitric oxide.

Figure 3

Effects of the nucleosides on CSE-induced TNF-α, IL-6, and IL-1β expression in RAW264.7 cells. Notes: The nucleosides suppressed CSE-induced expression of (A) TNF-α, (B) IL-6, and (C) IL-1β, and (D) their mRNA expression in RAW264.7 cells. *P<0.05, **P<0.01, ***P<0.001, CSE-induced group vs control group (without any treatment); ^#P<0.05, ^##P<0.01, nucleoside treatment groups (25 and 50 µg/mL) vs CSE-induced group. All the experiments were repeated three times. Data shown as mean ± SEM (n=3). Abbreviations: CSE, cigarette smoke extract; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.

Figure 4

Effects of the nucleosides on (A) mRNA, (B) Western blot, and (C) quantity of SIRT1, as well as (D) Western blot and (E) quantity of NF-κB/p65 in CSE-induced RAW264.7 cells. Notes: **P<0.01, CSE-induced group vs control group (without any treatment); ^#P<0.05, ^##P<0.01, nucleoside treatment groups (25 and 50 µg/mL) vs CSE-induced group. All the experiments were repeated three times. Data shown as mean ± SEM (n=3). Abbreviations: CSE, cigarette smoke extract; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-κB; NO, nitric oxide; SIRT1, silent information regulator 1.

Figure 5

Impact of the SIRT1 antagonist EX527 on treatment effects of the nucleosides on the CSE-induced RAW264.7 cells. Notes: (A) mRNA levels of SIRT1, TNF-α, IL-6, IL-1β, and iNOS; (B) Western blots of SIRT1, NF-κB/p65, and iNOS; (C) quantification histograms of SIRT1, NF-κB/p65, and iNOS. Study groups: 1, C: control, without any treatment; 2, CSE: CSE treatment; 3, CSE + N: CSE + nucleoside (50 µg/mL) treatment; 4, EX + CSE + N: EX527 (10 µM) + CSE + nucleosides (50 µg/mL) treatment; 5, EX + CSE: EX527 (10 µM) + CSE treatment; 6, EX: EX527 (10 µM) treatment. ^#P<0.05, CSE + N group vs CSE group; *P<0.05, C: CSE + EX + N group vs CSE + EX group. Abbreviations: CSE, cigarette smoke extract; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1β, interleukin-1β; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-κB; NO, nitric oxide; SIRT1, silent information regulator 1.

Figure 6

Effects of the nucleosides on (A) NO, (B) TNF-α, (C) IL-1β, and (D) inflammatory cells in CSE-induced mice. Notes: Study groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after CSE challenge; 20: nucleosides dosed at 20 mg/kg; 40: nucleosides dosed at 40 mg/kg. **P<0.01, ***P<0.001, model group vs control group; ^#P<0.05, ^##P<0.01, ###P<0.001, treatment group vs model group. Data shown as mean ± SEM (n=4, each group). Abbreviations: BALF, bronchoalveolar lavage fluid; CSE, cigarette smoke extract; IL-1β, interleukin-1β; NO, nitric oxide; TNF-α, tumor necrosis factor-α.

Figure 7

Effects of the nucleosides on lung histological changes measured by H&E staining and the associated protein expression by immunohistochemistry and Western blot analysis. Notes: (A) H&E staining analysis of the effect of nucleosides (100×) and immunohistochemistry analysis of SIRT1 and NF-κB/p65 expression (400×). (B) Western blot analysis of iNOS, SIRT1, and NF-κB/p65 expression in the CSE-stimulated mice. (C) Quantitative levels of (B). Study groups: C: control, orally treated with the vehicle only; M: model, intraperitoneal injection of CSE; P: positive control, orally treated with budesonide (2 mg/kg) after CSE challenge; 20: nucleosides dosed at 20 mg/kg; 40: nucleosides dosed at 40 mg/kg. *P<0.05, **P<0.01, model group vs control group; ^#P<0.05, treatment group vs model group. Data shown as mean ± SEM (n=4, each group). Abbreviations: CSE, cigarette smoke extract; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-κB; SIRT1, silent information regulator 1.

Figure 8

Proposed therapeutic mechanism of the nucleosides on cigarette smoke extract-induced inflammation via the SIRT1–NF-κB/p65 pathway. Abbreviations: COPD, chronic obstructive pulmonary disease; IL-1β, interleukin-1β; IL-6, interleukin-6; NF-κB, nuclear factor-κB; NO, nitric oxide; SIRT1, silent information regulator 1; TNF-α, tumor necrosis factor-α.