Elsevier

Neuroscience

Volume 441, 10 August 2020, Pages 46-57
Neuroscience

Research Article
Sodium Tanshinone IIA Sulfonate Protects Against Cerebral Ischemia–reperfusion Injury by Inhibiting Autophagy and Inflammation

https://doi.org/10.1016/j.neuroscience.2020.05.054Get rights and content

Highlights

  • Sodium tanshinone IIA sulfonate (STS) protected against brain damage induced by stroke.

  • STS reduced neuroinflammation by inhibiting the infiltration of immune cells after stroke.

  • STS inhibited the expression of autophagy associated proteins after stroke.

Abstract

Sodium tanshinone IIA sulfonate (STS) can protect against brain damage induced by stroke. However, the neural protection mechanism of STS remains unclear. We investigated whether STS performs its protective function by suppressing autophagy and inflammatory activity during brain injury. We established a transient middle cerebral artery occlusion and reperfusion (MCAO/R) model by blocking the left middle cerebral artery with a thread inserted through the internal carotid artery for 1 h, followed by reperfusion for 48 h either with or without STS and the autophagy inhibitor 3-methyladenine (3-MA). Neuroprotective effects were determined by evaluating infarction, brain edema, and neurological deficits. The numbers of microglia-derived macrophages, monocyte-derived microglia, T cells, and B cells in the brains were measured, based on the surface marker analyses of CD45, CD11b, B220, CD3, and CD4 using fluorescence-assisted cell sorting. STS (10, 20, 40 mg/kg) was able to significantly reduce infarct volumes, improve neurological deficits, and reduce brain water contents. STS treatment reduced neuroinflammation, as assessed by the infiltration of macrophages and neutrophils, corresponding with reduced numbers of macrophages, T cells, and B cells in ischemia/reperfusion (I/R) brains. In addition, STS treatment also attenuated the upregulation of autophagy associated proteins, such as LC3-II, Beclin-1 and Sirt 6, which was induced by MCAO. These results demonstrated that STS can provide remarkable protection against ischemic stroke, possibly via the inhibition of autophagy and inflammatory activity.

Introduction

Stroke, especially its most common form, acute ischemic stroke (AIS), remains the leading cause of mortality and long-term disability, worldwide. Stroke can be treated with appropriate reperfusion therapy within a strict time window; however, many other factors contribute to the prognosis of ischemic stroke (Macrez et al., 2011, French et al., 2016, Zerna et al., 2016). Numerous studies have revealed that programmed cell death processes, including apoptosis, autophagy, and necrosis, play significant roles in the progression of neural injury and the prognosis of AIS (Fuchs and Steller, 2011). Among these processes, the autophagy pathway is the primary mechanism that maintains the balance between the degradation and formation of cellular proteins and damaged organelles, which has a great influence on cell survival (Shintani and Klionsky, 2004, Yoshimori, 2007, Shibutani and Yoshimori, 2014).

Autophagy is the type II programmed cell death pathway, during which autophagosomes engulf and degrade damaged or aging organelles and cytosolic proteins for further processing and recycling (Yoshimori, 2007, Shibutani and Yoshimori, 2014). This process serves diverse protective roles, such as emergency energy supply and detrimental material clearance (Rabinowitz and White, 2010, Russell et al., 2014). Studies have shown the involvement of autophagy during neural injury and death following cerebral ischemia (Zheng et al., 2012, Yang et al., 2015, Zhao et al., 2016). Despite several reports that autophagy is ubiquitous during ischemic stroke (Zheng et al., 2012, Yang et al., 2015), whether autophagy plays a detrimental or beneficial role in general or during different phases of ischemic stroke remains unclear. Many different pathways are involved in autophagy during cerebral ischemic stroke, such as the phosphoinositide 3-kinase (PI3K)-protein kinase b (Akt)-mammalian target of rapamycin complex 1 (mTORC1) pathway and the 5′ AMP-activated protein kinase (AMPK)-mTORC1 pathway (Poels et al., 2009, Li and McCullough, 2010, Chong et al., 2012). The beclin-1-Bcl-2 complex and the tumor suppressor p53 also play key roles during autophagy (Wang et al., 2009). The microtubule-associated protein 1 light chain 3 (LC3) and beclin-1 are considered to be significant biomarkers for the detection of autophagosomes (Maiuri et al., 2010). LC3-I can be hydrolyzed into LC3-II during the activation of autophagy; therefore, the LC3-II/LC3-I ratio is widely used as a marker of the autophagic activation level (Oberstein et al., 2007). The transformation from LC3-I to LC3-II has been detected in the cerebral hemisphere 24–72 h following hypoxia–ischemia (Zhu et al., 2005). Beclin-1 expression levels were also elevated, peaking 24–72 h after cerebral ischemic injury (Carloni et al., 2008). Therefore, autophagy plays an essential role during ischemic stroke and autophagy interference may effectively contribute to ischemic stroke treatment.

Inflammation featuring the infiltration of peripheral blood leukocytes and microglia/macrophages has been shown to play an important role in brain injury after ischemic stroke. In addition to microglia/macrophages and neutrophils, lymphocytes, including T cells and B cells with diverse surface markers, are noteworthy. The roles of T cells and B cells during the pathogenesis and progression of brain inflammation after ischemic stroke have been widely studied. Researchers have detected infiltrating lymphocytes in post-mortem ischemic brain specimens (Song et al., 2012). In mice subjected to middle cerebral artery occlusion (MCAO) and photothrombotic stroke, lymphocyte infiltration was found to last for 14 days in the ischemic brain, accompanied by the elevated production and release of inflammatory cytokines and reactive oxygen species (ROS). Therefore, inhibiting the infiltration of inflammatory cells and reducing the production of inflammatory cytokines may be pivotal for the prognosis and treatment of ischemic stroke.

Tanshinone IIA (TSA) is the major bioactive and effective ingredient extracted from a type of Chinese traditional medicine, known as Salvia miltiorrhiza, which has been widely reported to be beneficial for patients with cardiovascular disease (Li et al., 2019, Zhang et al., 2019). The water-soluble derivative of TSA, sodium tanshinone IIA sulfonate (STS), has also long been reported to be a cardioprotective agent when administered following myocardial infarction and injury (Fu et al., 2007, Tian and Wu, 2013, Zhang et al., 2014). Recently, several studies revealed that both TSA and STS exert protective effects against focal cerebral ischemia/reperfusion (I/R) injury in rodent models.

Brain infarction volumes and edema, as well as neurological dysfunction, were markedly attenuated by TSA administration, and this protective effect was linked to the inhibition of neutrophil infiltration and the expression of macrophage migration inhibitory factor (MIF) and cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 (Chen et al., 2012). Another study used both in vitro and in vivo experiments to demonstrate the beneficial effects of TSA on the inhibition of neurocyte apoptosis. They further revealed that the PI3K/Akt signaling pathway may be involved in the neuroprotective mechanism of TSA (Tang et al., 2012). Studies have also demonstrated the involvement of autophagy during neural injury and death following cerebral ischemia (Zheng et al., 2012, Yang et al., 2015, Zhao et al., 2016). Furthermore, a recent study showed that TSA could decrease the production of inflammatory cytokines such as IL-1, TNF-α, C-X-C motif chemokine 10, and chemokine (C–C motif) ligand 12 (Fang et al., 2018). Together, this evidence indicated that STS may play a protective role by modulating programmed cell death, cytokine expression and release, and the infiltration of inflammatory leukocytes. Whether autophagy and inflammation are associated with STS treatment remains worthy of further investigation to better understand this traditional medicine and to develop more effective therapeutic strategies for ischemic stroke.

Given the above, we designed the present experiment to investigate the effects of STS treatment on I/R-induced autophagy and neuroinflammation. Our findings provide further evidence to support the application of STS during treatment for cerebral diseases, such as stroke.

Section snippets

Chemicals and reagents

STS was obtained from Shanghai No. 1 Biochemical & Pharmaceutical Co. (568-72-9, Shanghai, China). The chemicals 3-methyladenine (3-MA; M9281, Sigma-Aldrich, St. Louis, MO, USA) and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich, (17779, St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime Biotech (P0012, Jiangsu, China). Anti-LC3 (Ab48394), anti-beclin-1 (Ab55878), and anti-Sirt 6 (Ab62739) antibodies were obtained from

Effects of STS on infarct volume and neurological deficit score following MCAO

Fig. 1 shows the experimental design and animal treatments. Fig. 2 shows photographs of typical coronal sections from each group. The infarct volume of the STS treatment group decreased significantly compared with the MCAO + saline group (F4,35 = 42.41, P < 0.0001; Supplementary Table 1, Fig. 3A). The STS at 40 mg/kg showed the strongest protective effect among all treatments examined. In addition, 3-MA, an autophagy inhibitor that was used as a positive control, also reduced the infarct volume

Discussion

In the present study, we examined the hypothesis that STS protects against I/R brain injury and further explored whether anti-autophagy and anti-neuroinflammatory effects contribute to the beneficial effects of STS. Our results confirmed the neuroprotective effects of STS, at doses of 10, 20, and 40 mg/kg, on MCAO model mice. STS treatment remarkably reduced the infarct volume, improved neurological scores, and decreased the brain water content after 1 h of ischemia, followed by 2 days of

Funding

This study was supported by the National Natural Science Foundation of China [grant numbers 81870939, 81771283, 81901994, and 81571147]; and the Natural Science Foundation of Hubei Province [grant number 2019CFC847].

Conflicts of interest

None declared.

Acknowledgment

We thank Lisa Giles, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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