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6TMR Modern Herbal Medicine  2019, Vol. 2 Issue (3): 131-139    DOI: 10.12032/TMRmhm2017A48
Orginal Article     
Effect of Tanshinone IIA on LPS-induced inflammatory response in a ROS-NLRP3 inflammasome dependent manner in RAW264.7 cells
Dan Li1,2, Shan Gao1,2, Sarhene Michael1,2, Yu-Ying Guo1,2, Hao Deng1,2, Shi-Xin Xu1,2, Guan-Wei Fan1,2,*()
1First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.
2Tianjin Key Laboratory of Translational Research of TCM Prescription and Syndrome, Tianjin, China.
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Abstract

Emerging evidence has demonstrated that Tanshinone IIA (Tan IIA) prevents cardiomyocytes injury, cardiac fibroblasts and atherosclerosis. However, the molecular mechanism underlying the effects of Tan IIA is still unclear. To investigate the role of Tan IIA in inflammatory response in a ROS-NLRP3 inflammasome dependent manner, RAW264.7 cells stimulated with LPS were recruited to produce a cell model of inflammatory response. Our results indicated that the production of NO was significantly increased after stimulated by LPS, and Tan IIA treated significantly decreased the level of NO. The mRNA expression of NLRP3, IL-1β and TNF-α was significantly inhibited by Tan IIA compared with LPS treated cells. The protein expression of NLRP3, IKBα, pp65/p65 and pp38/p38 was significantly decreased by Tan IIA, compared with LPS or LPS+ATP stimulated groups. Meanwhile, Tan IIA significantly inhibited the level of ROS induced by LPS+ATP. And NAC, a ROS inhibitor, could also inhibit the protein expression of NLRP3. Based on these findings, it could be speculated that the mechanism underlying the effect of Tan IIA may involve the regulation of ROS-NF-κB/ P38-NLRP3 pathway. This study further characterized the molecular mechanism of Tan IIA, and provided new thoughts to its clinical therapy.

Highlights

Tan IIA could inhibit the inflammatory response and NLRP3 expression stimulated by LPS or LPS+ATP. Acetylcysteine (N-acetyl-l-cysteine, NAC), a ROS inhibitor, could inhibit LPS+ATP-induced increase in NLRP3 level. The mechanism underlying the effects of Tan IIA may involve the regulation of ROS-NF-κB/ P38-NLRP3 pathway. This study further characterized the molecular mechanism of Tan IIA, and provided new thoughts to its clinical therapy.



Key wordsTanshinone IIA      Inflammation      NLRP3 inflammasome      ROS     
Published: 25 July 2019
Fund:  This study was funded by the Tianjin Outstanding Youth Science Foundation (No. 17JCJQJC46200), the National Natural Science Foundation of China (No.81774050), the Natural Science Foundation of Tianjin (17JCYBJC29000) and the Foundation of First Teaching Hospital of Tianjin University of Traditional Chinese Medicine (No. 201703).
Corresponding Authors: Fan Guan-Wei     E-mail: fgw1005@hotmail.com
Cite this article:

Dan Li, Shan Gao, Sarhene Michael, Yu-Ying Guo, Hao Deng, Shi-Xin Xu, Guan-Wei Fan. Effect of Tanshinone IIA on LPS-induced inflammatory response in a ROS-NLRP3 inflammasome dependent manner in RAW264.7 cells. 6TMR Modern Herbal Medicine, 2019, 2(3): 131-139. doi: 10.12032/TMRmhm2017A48

URL:

https://www.tmrjournals.com/mhm/EN/10.12032/TMRmhm2017A48     OR     https://www.tmrjournals.com/mhm/EN/Y2019/V2/I3/131

Figure 1. Tan IIA possessed regulatory effect on NO production, NLRP3, IL-1β, TNF-α mRNA expression
A. RAW264.7 Cells NO production of different groups.The cells were pretreated with Tan IIA 10μM, 1μM, and 0.1μM for 30 min and stimulated with LPS (1 μg/mL) for 24 h. * P < 0.05 vs ctrl, # P < 0.05 vs LPS. B-D. Tan IIA possessed regulatory effect on mRNA expression of TNF-α, NLRP3 and IL-1β. The cells were pretreated with Tan IIA 10μM, 1μM, and 0.1μM for 30 min and stimulated with LPS (1 μg/mL) for 6 h. * P < 0.05 vs ctrl, # P < 0.05 vs LPS.
Figure 2. Tan IIA possessed regulatory effect on protein expression of NLRP3, IKBα, pp65/p65 and pp38/p38.
The cells were pretreated with Tan IIA 10μM for 30 min and stimulated with LPS (1 μg/mL) for 5.5 h. ATP was added 30 min before cells were harvested. A. Representative Western blot of NLRP3, IKBα, pp65/p65 and pp38/p38. B-F. Quantitative data of average across three separate experiments. *P < 0.05 vs ctrl, #P < 0.05 vs LPS/LPS+ATP.
Figure 3. Tan IIA possessed regulatory effect on ROS expression (scale bar 50μm).
The cells were pretreated with Tan IIA 10μM for 30 min and stimulated with LPS (1 μg/mL) for 5.5 h. ATP was added 30 min before cells were harvested. A. Representative fluorescence images of cell ROS expression. B. Quantitative data of average across three separate experiments. * P < 0.05 vs ctrl, # P < 0.05 vs LPS/LPS+ATP.
Figure 4. NAC possessed regulatory effect on protein expression of NLRP3.
The cells were pretreated with Tan IIA 10μM for 30 min and stimulated with LPS (1 μg/mL) /LPS (1 μg/mL) +NAC (1mM) for 5.5 h. ATP was added 30 min before cells were harvested. A. Representative Western blot of NLRP3. B. Quantitative data of average across three separate experiments. * P < 0.05 vs ctrl, # P < 0.05 vs LPS+ATP.
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