Sodium Tanshinone IIA Sulfonate Decreases Cigarette Smoke-Induced Inflammation and Oxidative Stress via Blocking the Activation of MAPK/HIF-1α Signaling Pathway

Abstract

Aberrant activation of hypoxia-inducible factor (HIF)-1α is frequently encountered and promotes oxidative stress and inflammation in chronic obstructive pulmonary disease (COPD). The present study investigated whether sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA, can mediate its effect through inhibiting HIF-1α-induced oxidative stress and inflammation in cigarette smoke (CS)-induced COPD in mice. Here, we found that STS improved pulmonary function, ameliorated emphysema and decreased the infiltration of inflammatory cells in the lungs of CS-exposed mice. STS reduced CS- and cigarette smoke extract (CSE)-induced upregulation of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the lungs and macrophages. STS also inhibited CSE-induced reactive oxygen species (ROS) production, as well as the upregulation of heme oxygenase (HO)-1, NOX1 and matrix metalloproteinase (MMP)-9 in macrophages. In addition, STS suppressed HIF-1α expression in vivo and in vitro, and pretreatment with HIF-1α siRNA reduced CSE-induced elevation of TNF-α, IL-1β, and HO-1 content in the macrophages. Moreover, we found that STS inhibited CSE-induced the phosphorylation of ERK, p38 MAPK and JNK in macrophages, and inhibition of these signaling molecules significantly repressed CSE-induced HIF-1α expression. It indicated that STS inhibits CSE-induced HIF-1α expression likely by blocking MAPK signaling. Furthermore, STS also promoted HIF-1α protein degradation in CSE-stimulated macrophages. Taken together, these results suggest that STS prevents COPD development possibly through the inhibition of HIF-1α signaling, and may be a novel strategy for the treatment of COPD.

Keywords: COPD; cigarette smoke; hypoxia-inducible factor-1α; inflammation; oxidative stress; sodium tanshinone IIA sulfonate.

FIGURE 1

STS ameliorated cigarette smoke (CS)-induced COPD in mice. (A) Chemical structure of STS. (B,C) 3 months after CS inhalation, lung function parameters including total lung capacity and FEV100/FVC among different groups were calculated. (D) Lung pathology was determined by H&E staining. Scale bars = 100 μm. (E) Average alveolar intercept, representing the degree of emphysema was quantified by Image Pro Plus 6.0 software. Data are expressed as mean ± SEM, n = 6, ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 2

STS reduced the numbers of inflammatory cells and inflammatory factors in the CS-induced COPD in mice. Three months after CS inhalation, lung tissues and BALF was collected. (A) The total cells in BALF were calculated. (B) The mRNA levels of TNF-α and IL-1β in the lung tissues were analyzed by Quantitative real-time PCR (RT-PCR). (C,D) The relative protein levels of TNF-α and IL-1β in the lung tissues were analyzed by ELISA. (E) Immunohistochemical staining of IL-1β-positive cells in the lungs. Data are presented as mean ± SEM, n = 6, ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 3

STS inhibited CSE-induced production of inflammation cytokines in RAW 264.7 macrophages. RAW 264.7 macrophages were cultured with and without CSE and/or 1, 2.5, or 5 μM STS for 24 h. (A) The viabilities of macrophages treated as indicated were measured by CCK-8 assay. (B,C) The supernatant was collected to detect the content of TNF-α and IL-1β by ELISA. (D,E) The protein level of IL-1β in the macrophages was analyzed by Western blot. Data are presented as mean ± SEM, n = 3, ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 4

STS inhibited CSE-induced oxidative stress and MMP-9 upregulation in macrophages. RAW 264.7 macrophages were treated with CSE and different concentrations of STS (1, 2.5, and 5 μM) for 24 h. (A) The intracellular ROS was determined using a fluorescence microplate according to the ROS assay kit. The relative protein levels of NOX1 (B), HO-1 (C), and MMP-9 (D) were determined by Western blot. The mRNA levels of HO-1 (E) and MMP-9 (F) in the lung tissues were analyzed by RT-PCR. Data are presented as mean ± SEM, n = 3, ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 5

STS suppressed CS-induced HIF-1α expression in the lungs and in macrophages. (A–C) 3 months after CS inhalation, lung tissues were collected and prepared for RT-PCR and Western blot analysis using antibodies against HIF-1α and β-actin. Data are presented as mean ± SEM, n = 6, ^∗∗P < 0.01. (D,E) Macrophages were treated with CSE and different concentrations of STS (1, 2.5, and 5 μM) for 24 h. Cells were then collected and prepared for Western blot analysis using antibodies against HIF-1α and β-actin. Data are presented as mean ± SEM, n = 3, ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 6

HIF-1α siRNA repressed CSE-induced elevation of inflammation cytokines and HO-1 expression in macrophages. Macrophages were transfected with HIF-1α siRNA by using Lipofectamine 2000 according to the manufacturer’s instructions. (A,B) 6 h after transfection, Western blot analysis was performed to evaluate the changes of HIF-1α expression. (C,D) Transfected macrophages were deprived of FCS for 6 h and incubated with CSE for 24 h. The supernatant was collected to examine the content of TNF-α and IL-1β by ELISA. (E–G) Transfected macrophages were deprived of FCS for 6 h and incubated with CSE for 24 h. The cell samples were collected to measure the content of IL-1β and HO-1 by Western blot. Data are presented as mean ± SEM, n = 3, ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 7

STS inhibited CSE-induced MAPK activation in macrophages. RAW 264.7 macrophages were treated as indicated for 24 h. Cell lysates were prepared and immunoblotted with antibodies for p-ERK, p-P38 and p-JNK. (A,B) Western blot was used to analyze the phosphorylation levels of p-ERK, p38 MAPK and JNK. Results are representative of different experiments. (C–E) Scanning densitometry of western blot on different samples was analyzed quantitatively. Expression of p-ERK, p-P38 and p-JNK was normalized to ERK, P38 MAPK and JNK level, respectively. (F,G) The relative protein level of HIF-1α was determined by Western blot. Data are presented as mean ± SEM, n = 3, ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 8

STS promoted CSE-induced HIF-1α protein degradation in macrophages. RAW 264.7 macrophages were treated with CSE for 24 h with or without 5 μM STS, followed by incubation with cycloheximide (CHX) from 0 to 30 min. (A) Cell lysates were then collected and prepared for Western blot analysis using antibodies against HIF-1α or β-actin. (B) The relative intensity of HIF-1α protein content was quantified. Data are presented as mean ± SEM, n = 3.

FIGURE 9

A schematic pathway illustrating the protective effects of STS against CS-induced COPD in mice. STS decreases the phosphorylation of ERK, p38 MAPK and JNK, which is responsible for HIF-1α down-regulation, resulting in protection against inflammatory responses, oxidative stress, emphysema and lung function decline in CS-exposed lungs.