Cryptotanshinone from Salvia miltiorrhiza Bunge (Danshen) inhibited inflammatory responses via TLR4/MyD88 signaling pathway

Xin-Xing Li; Xiaoting Zheng; Zhenjie Liu; Qiongming Xu; Hongzhen Tang; Jianfang Feng; Shilin Yang; Chi Teng Vong; Hongwei Gao; Yitao Wang

doi:10.1186/s13020-020-00303-3

Cryptotanshinone from Salvia miltiorrhiza Bunge (Danshen) inhibited inflammatory responses via TLR4/MyD88 signaling pathway

Chin Med. 2020 Mar 2:15:20. doi: 10.1186/s13020-020-00303-3. eCollection 2020.

Authors

Xin-Xing Li^#^{1

2}, Xiaoting Zheng^#³, Zhenjie Liu^{1

2}, Qiongming Xu^{1

4}, Hongzhen Tang^{1

2}, Jianfang Feng^{1

2}, Shilin Yang^{1

2}, Chi Teng Vong³, Hongwei Gao^{1

2}, Yitao Wang³

Affiliations

¹ 1College of Pharmacy, Guangxi University of Chinese Medicine, Nanning, 530000 China.
² Guangxi Engineering Technology Research Center of Advantage Chinese Patent Drug and Ethnic Drug Development, Nanning, 530020 China.
³ 3State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Room 1050, N22 Research Building, Macao, China.
⁴ 4College of Pharmaceutical Science, Soochow University, Suzhou, 215123 China.

^# Contributed equally.

Abstract

Background: Cryptotanshinone (CPT), as a major component of Salvia miltiorrhiza Bunge (Danshen), displays many pharmacological activities including anti-inflammatory effects. However, the exact cellular and molecular mechanisms of the anti-inflammatory activities of CPT remain to be elucidated. The present study was aimed to clarify its mechanisms on lipopolysaccharide (LPS)-induced inflammatory responses in mouse macrophages, RAW264.7 cells.

Methods: In the current study, the anti-inflammatory properties of CPT were evaluated using LPS-stimulated RAW264.7 cell model. MTT assay was used to determine the viability of RAW264.7 cells. The anti-inflammatory effects of CPT were measured based on the detection of nitric oxide (NO) production (Griess and flow cytometry assay), and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release (ELISA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzyme expressions were also determined by western blotting. Besides, by using flow cytometry, we also evaluated the effect of CPT on LPS-induced calcium influx. Finally, the underlying anti-inflammatory mechanisms of CPT were investigated using western blotting to assess the protein levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K)/AKT, nuclear factor erythroid 2 related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB) pathways.

Results: Our data showed that CPT inhibited LPS-induced pro-inflammatory cytokine release like IL-6, and TNF-α, as well as NO production. It displayed a significant inhibitory effect on the protein expressions such as iNOS, COX-2, NF-κB pathway like inhibitor of kappa B kinase (IKK)α/β, inhibitor of kappa B (IκB)-α and NF-κB/p65, PI3K/AKT pathway like PI3K and AKT, and MAPK pathway like c-Jun N-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and p38, in LPS-stimulated RAW264.7 macrophages. Moreover, the immunofluorescence results indicated that CPT suppressed NF-κB/p65 translocation from the cytoplasm into the nucleus. Further investigations showed that CPT treatment increased NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) expressions together with its upstream mediator, Nrf2. In addition, CPT inhibited LPS-induced toll-like receptor 4 (TLR4) and MyD88 expressions in RAW264.7 macrophages.

Conclusions: Collectively, we suggested that CPT exerted significant anti-inflammatory effects via modulating TLR4-MyD88/PI3K/Nrf2 and TLR4-MyD88/NF-κB/MAPK pathways.

Keywords: Anti-inflammation; Cryptotanshinone; Nrf2; PI3K/AKT; TLR4-MyD88.