Hollow Prussian Blue Nanozymes Drive Neuroprotection against

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Hollow Prussian Blue Nanozymes Drive Neuroprotection against Ischemic Stroke via Attenuating Oxidative Stress, Counteracting Inflammation, and Suppressing Cell Apoptosis Kai Zhang, Mengjiao Tu, Wei Gao, Xiaojun / Cai, Fahuan Song, Zheng Chen, Qian Zhang, Jing Wang, Chentao Jin, Jingjing Shi, Xiang Yang, Yuankai Zhu, Weizhong Gu, Bing Hu, Yuanyi Zheng, Hong Zhang, and Mei Tian Nano Lett., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

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Hospital, ultrasound Zhang, Hong; Department of Nuclear Medicine and PET-CT Center, The Second Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, P.R. China; Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China; Tian, Mei; Department of Nuclear Medicine and PET-CT Center, The Second Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, P.R. China

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Hollow Prussian Blue Nanozymes Drive Neuroprotection against Ischemic Stroke via Attenuating Oxidative Stress, Counteracting Inflammation, and Suppressing Cell Apoptosis Kai Zhang1, Mengjiao Tu1, Wei Gao2, Xiaojun Cai2*, Fahuan Song1, Zheng Chen4, Qian Zhang5, Jing Wang1, Chentao Jin1, Jingjing Shi1, Xiang Yang1, Yuankai Zhu1, Weizhong Gu6, Bing Hu2, Yuanyi Zheng2*, Hong Zhang1,3*, and Mei Tian1* 1Department

of Nuclear Medicine and PET-CT Center, The Second Hospital, Zhejiang

University School of Medicine, Hangzhou, Zhejiang 310009, P.R. China. 2Shanghai

Institute of Ultrasound in Medicine, Sixth People's Hospital, Shanghai Jiao Tong

University Affiliated, Shanghai 200233, P.R. China. 3Shanxi

Medical University, Taiyuan, Shanxi 030001, P.R. China.

4Department

of Neurosurgery, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai

200082, P.R. China. 5Department

of Oncology, Tenth People’s Hospital, Tongji University, Shanghai 200072, P.

R. China. 6Department

of Pathology, Children’s Hospital, Zhejiang University School of Medicine,

Hangzhou, Zhejiang 310051, P.R. China.

Corresponding Authors * Email: [email protected]; [email protected]; [email protected]; [email protected];

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ABSTRACT: Ischemic stroke is a devastating disease and one of the leading causes of mortality worldwide. Overproduction of reactive oxygen and nitrogen species (RONS) following ischemic insult is known as a key factor in exacerbating brain damage. Thus, RONS scavengers that can block excessive production of RONS have great therapeutic potential. Herein, we propose an efficient treatment strategy in which an artificial nanozyme with multienzyme activity drives neuroprotection against ischemic stroke primarily by scavenging RONS. Specifically, through a facile, Bi3+-assisted, template-free synthetic strategy, we developed hollow Prussian blue nanozymes (HPBZs) with multienzyme activity to scavenge RONS in a rat model of ischemic stroke. The comprehensive characteristics of HPBZs against RONS were explored. Apart from attenuating oxidative stress, HPBZs also suppressed apoptosis and counteracted inflammation both in vitro and in vivo, thereby contributing to increased brain tolerance of ischemic injury with minimal side effects. This study provides a proof of concept for a novel class of neuroprotective nanoagents that might be beneficial for treatment of ischemic stroke and other RONS-related disorders.

KEYWORDS: ischemic stroke, hollow Prussian blue, neuroprotection, nanozyme, reactive oxygen and nitrogen species

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Ischemic stroke is one of the leading causes of mortality worldwide, accounting for 5.2% of deaths globally.1 It generally refers to a transient or permanent reduction in cerebral blood flow caused by local blockage of the cerebral artery via a thrombus or embolus.2 The transient blood flow reduction can trigger ischemia/reperfusion injury, which initiates overwhelming production of reactive oxygen and/or nitrogen species (RONS, i.e., ROS and/or RNS), including superoxide anion (O2·-), hydrogen peroxide (H2O2), hydroxyl radical (·OH), nitric oxide (·NO), and peroxynitrite (ONOO-).3 These eruptible RONS have been thought to mediate the majority of the damage that follows ischemia/reperfusion injury.4 They can induce mitochondrial damage and caspase-mediated apoptosis and lead to severe disruption of cerebral tissue architecture and loss of brain function by strongly oxidizing lipids, proteins, and DNA.3,

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In addition, overproduced RONS can serve as crucial signaling molecules to

trigger activation of microglia, induce infiltration of peripheral leukocytes, and stimulate secretion of cytokines from inflammatory cells.2, 6 Moreover, the excessive accumulation of RONS may induce inactivation and overconsumption of endogenous antioxidases, resulting in oxidative damage to brain regions, particularly the ischemic penumbra, the ischemic brain region that can potentially be salvaged.7-10 Endogenous antioxidases, such as superoxide dismutase and catalase, participate in regulation of the production of O2·-, which is the main constituent of RONS.3 However, these natural enzymes are unstable, with a short circulation half-life, and are difficult to adequately replenish during disease progression.7, 8 Of note, O2·- can further react with H2O2 and ·NO to generate ·OH and ONOO-, respectively. Both ·OH and ONOO- are considered extremely chemically reactive and can dramatically deteriorate oxidative injury,9,

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and there are no

specific enzymes that target them. Thus, RONS-targeting nanozymes should be an ideal approach to protecting neurons against RONS-induced impairments and further promoting therapeutic outcomes for ischemic stroke.

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Currently, to protect ischemia-injured neurons, special attention has been devoted to developing feasible nanomaterials with enzyme-like activity (nanozymes), including ceria, iron oxide-based, metal-based, and carbon nanozymes, which present stable enzyme-like activity, robust antioxidative activity, great physiological stability and have a low synthetic cost.3, 11 Among these nanoparticles, ceria nanoparticles have been highlighted as promising artificial enzymes for the treatment of ischemic stroke.12 Nevertheless, their potential toxicity should still be deliberated.13 In addition, the enzyme-like activity of ceria nanoparticles is limited by the particle size, atomic ratio of surface Ce3+ to Ce4+, and specific surface area.14, 15 Other artificial nanozymes, including melanin, carbon nanotubes, and manganese oxide nanozymes, also have several disadvantages, such as a relatively low RONS catalytic efficiency, potential nanotoxicity, or complex preparation procedures, which may hinder their wide application in counteracting oxidative damage in vivo.11, 16, 17 Prussian blue (PB) which presents excellent biosafety,18 is an FDA-approved antidote for caesium and thallium intoxication.19 PB-based nanoparticles have been previously developed as sophisticated contrast agents for ultrasound, photoacoustic, and magnetic resonance imaging; as photothermal conversion agents; and as drug delivery systems for theranostic applications in tumors.18, 20-23 Recently, PB nanoparticles have been found to be efficient ROS scavengers, which was attributed to their catalase-, superoxide dismutase-, and peroxidase-like activities.24-26 Unlike many iron-based nanoparticles, which are harmful in biological systems and may generate toxic effects by producing ·OH via the Fenton reaction,27, 28 PB nanozymes are reported to inhibit the production of ·OH, likely due to their reducing ability and low redox potential.25, 29 In addition to ROS, RNS (including ·NO and ONOO-) are also implicated in the development of ischemic stroke. ·NO is overproduced during ischemic stroke and can interact with O2·- to generate ONOO- and ·OH. However, to date, the RNS-scavenging efficiency of PB nanozymes has not been investigated.

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The specific surface area (i.e., surface-to-volume ratio) plays a critical role in the RONS-scavenging efficiency of PB nanozymes.30 Therefore, PB nanozymes with a hollow structure, which can provide a large specific surface area, are considered to have excellent RONS scavenging efficiency.31, 32 Concerning the construction of hollow PB nanoparticles, several efforts have been made previously.33-36 Through the “miniemulsion periphery polymerization” method, PB nanoboxes with a hollow cubic nanostructure were prepared.33, 35 However, the PB nanoboxes demonstrated limited specific surface area and amorphous inner shells with poor crystallinity.33, 35 By developing the “surface protected and interior etched” method, uniformly dispersed hollow PB nanoparticles with high crystallinity and a large surface area were synthesized.34 Nonetheless, this synthetic method requires multiple steps under high temperature and high pressure.34 Moreover, it is difficult to achieve large-scale production of hollow PB nanoparticles via the abovementioned synthetic strategies. Thus, developing a facile synthetic strategy is essential for large-scale production of hollow PB nanoparticles. In addition, the great RONS scavenging capability of hollow PB nanoparticles indicates their potential for treatment of RONS-related disorders, such as ischemic stroke; however, this potential has not yet been explored. Herein, we propose an efficient disease-modifying treatment strategy for ischemic stroke through developing artificial nanozymes, namely, hollow PB nanozymes (HPBZs), which can drive neuroprotection against ischemic stroke primarily via scavenging RONS. In the present study, we adopt a facile Bi3+-assisted, template-free synthetic strategy to achieve HPBZs with multienzyme-like activities. Compared with previously reported research,33-36 our template-free synthetic strategy possesses several advantages, including easy bulk preparation and no need for any post-treatment or complex preparation processes. The prepared HPBZs with a size of approximately 65 nm possess an inner cavity and demonstrate good physiological stability with stable hydrodynamic diameters in a physiological environment. Furthermore, owing to the variable valence state and extraordinary redox capability, HPBZs 5 ACS Paragon Plus Environment

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with multienzyme-like activity not only converted harmful ROS into harmless molecules (i.e., H2O and O2) but also scavenged detrimental RNS. The unique hollow structure endows HPBZs with a large specific surface area to counteract RONS and greatly improve the RONS scavenging capacity of HPBZs. As a proof-of-concept, HPBZs were shown to effectively protect neurons against hypoxic/ischemic injury in vitro and in vivo, primarily through antioxidative, anti-inflammatory, and anti-apoptotic effects with negligible side effects (Scheme 1). HPBZs were constructed by mixing bismuth nitrate, potassium ferricyanide, polyvinylpyrrolidone (PVP), and hydrochloric acid (1 M) under magnetic stirring and then were maintained at a temperature of 80 °C (Figure 1a). During the synthetic process, a metastable Bi3+/[Fe(CN)6]3- intermediate was generated and acted as an unstable template. Meanwhile, [Fe(CN)6]3- was reduced by PVP to generate [Fe(CN)6]4-, and Fe3+ ions were ionized from [Fe(CN)6]3- ions. [Fe(CN)6]4- ions were then combined with the Fe3+ ions to form PB nanozymes on the surface of the metastable Bi3+/[Fe(CN)6]3--PVP, ultimately forming the HPBZs with a cavity inside (Figure 1a). As demonstrated in the schematic diagram of the preparation of HPBZs, Bi3+ ions played a vital role (Figure 1a, Figure S1-S3), and the detailed mechanisms of HPBZ formation is illustrated in the Supporting Information. This synthetic strategy for HPBZs is facile and low-cost, providing an alternative to hard- or soft-templating fabrication of hollow nanomaterials. Unlike the previously reported strategies for preparing PB nanoparticles with a hollow structure,33-36 our Bi3+ ion-assisted, template-free strategy possesses several advantages: first, the current strategy is easy but efficient without the need for a template; second, the synthetic process is simple; and third, it is easy to achieve large-scale production of HPBZs. Transmission electron microscopy demonstrated that HPBZs with a size of approximately 65 nm were uniformly dispersed and possessed a hollow cavity (Figure 1b). The selected area electron diffraction pattern (Figure 1c) and X-ray diffraction pattern 6 ACS Paragon Plus Environment

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(Figure S4) revealed good crystallinity and PB composition (JCPDS card no. 73-0687). Element mapping confirmed the elemental components of the hollow structure and the C, Fe, N, Bi, and K elemental distribution (Figure 1d-e). The HPBZs were observed to exhibit the characteristic absorption peak of PB at a wavelength of 721 nm, which can be ascribed to the intermetallic charge transfer band from Fe Ⅱ to Fe Ⅲ in PB (Figure 1f). In addition, Fourier transform infrared spectroscopy of HPBZs revealed a characteristic peak at the wavenumber 2090 nm-1, demonstrating the Fe Ⅱ -CN-Fe Ⅲ chemical group in the PB structure (Figure 1g). These results demonstrated successful preparation of HPBZs with good crystallinity. The average hydrodynamic diameter of HPBZs was 78 nm as measured by dynamic light scattering (Figure 1h), and the zeta potential of HPBZs was -26.0 mV (Figure S5). To evaluate the stability of HPBZs in a physiological environment, changes in the hydrodynamic diameter of HPBZs at various time points were detected. The results demonstrated that the hydrodynamic diameter of HPBZs in saline solution remained stable for at least 3 d (Figure S6), indicating the potential of HPBZs for further in vitro and in vivo applications. Oxidative damage, along with the subsequent inflammatory response, which is triggered by the overproduction of RONS, is of critical significance in contributing to the damage in ischemic brain tissues.37 Thus, scavenging RONS may be an efficient strategy for treatment of ischemic stroke. Therefore, we investigated the effects and mechanisms of HPBZs against RONS. To study the peroxidase-like activity of HPBZs, the natural peroxidase substrates 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic

acid)

(ABTS)

and

3,5,3',5'-tetramethylbenzidine (TMB) were selected. HPBZs could catalyze H2O2 to oxidize the toxic ABTS and TMB into nontoxic substances, indicating peroxidase-like activity of the prepared HPBZs (Figure 2a). A TiO2/UV system generating ·OH was chosen to test the effect of HPBZs on scavenging ·OH. The characteristic signal intensity of BMPO/·OH was reduced by HPBZs, indicating excellent ·OH scavenging capacity of HPBZs (Figure 2b-c). 7 ACS Paragon Plus Environment

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The ·OH scavenging property of HPBZs might be ascribed to the low oxidation-reduction potential, which can transform ·OH into H2O (standard redox potential, ·OH/H2O: 2.9 V, Berlin Green/PB: 0.9 V, Prussian Yellow/Berlin Green: 1.4 V).25 We further investigated whether HPBZs could scavenge H2O2. Addition of HPBZs into a 100 μM H2O2 solution led to a greatly reduced H2O2 level compared with the negative control, which might be attributed to the catalase-like activity of HPBZs by decomposing H2O2 into H2O and O2 (2H2O2  2H2O + O2).38 Moreover, HPBZs induced H2O2 consumption in a concentration- and time-dependent manner (Figure 2d). O2·− is the initial RONS and can be eliminated by superoxide dismutase. Since the xanthine/xanthine oxidase system can induce the generation of O2·−, it was selected to investigate the superoxide dismutase-like activity (2O2·− + 2H+  H2O2 + O2) of HPBZs. The signal intensity of BMPO/O2·− was distinctly reduced after the addition of HPBZs (Figure 2e), indicating a high O2·−-scavenging capacity of HPBZs. The standard redox potential of O2·−/H2O2, Berlin Green/PB, and Prussian Yellow/Berlin Green is 1.5 V, 0.9 V, and 1.4 V, respectively. Considering the multienzyme-like activities of HPBZs, previous studies have reported that PB nanoparticles can mimic catalase-, superoxide dismutase-, and peroxidase-like activities, likely due to their abundant redox potential in various forms, including Prussian blue, Prussian White, Berlin Green, and Prussian Yellow, making them robust electron transporters.24-26 In addition to ROS, overproduction of ·NO can also induce oxidative damage.3 ·NO can react with O2·− to generate ONOO- and ·OH. ONOO- owns high penetration capacity across lipid bilayers and plays an important role in disruption of the blood-brain barrier.3 The scavenging effect of HPBZs on ONOO- was explored via an ONOO--induced pyrogallol red bleaching assay (Figure 2f). The inhibition rate of ONOOreached up to 72% (Figure 2g). In addition, the inhibition rates of H2O2, O2·−, and ·OH were 88%, 79%, and 62%, respectively (Figure 2g). Overall, these results demonstrate that HPBZs can robustly scavenge RONS through converting harmful RONS into harmless molecules (Figure 2h). 8 ACS Paragon Plus Environment

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Considering the outstanding antioxidative property of HPBZs, we hypothesized that HPBZs were likely to be of great benefit in protecting cells against hypoxic/ischemic injury. SH-SY5Y (human neuroblastoma) and Raw 264.7 (mouse peritoneal macrophages) cells were cultured to assess the cytotoxicity of HPBZs and more importantly to study the potential protective effects of HPBZs against ischemia-induced oxidative damage. To evaluate the toxicity of HPBZs in SH-SY5Y cells, the viability of cells after treatment with HPBZs at various concentrations (ranging from 0 to 640 μg/mL) was tested via cell counting kit-8 assays. The results demonstrated that HPBZs did not induce any obvious cytotoxicity at HPBZs concentrations up to 160 μg/mL (Figure 3a). Since neurons are susceptible to oxidative damage, SH-SY5Y cells were selected to examine the neuroprotective effect of HPBZs against oxidative damage induced by H2O2. SH-SY5Y is a human-derived neuroblastoma cell line that retains various properties of human neurons can be readily maintained under culture conditions.39 SH-SY5Y cells have been used as a common in vitro neuronal model to explore the neuroprotective effects of novel pharmaceuticals.40,

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When

exposed to H2O2 (400 μM), a great loss in the number of viable cells was observed (Figure S7); However, the cell viability was enhanced from 17.15% to 50.13% as the concentration of HPBZs was increased from 0 to 40 μg/mL, which indicated a significant neuroprotective effect of HPBZs under oxidative conditions (Figure S7). To further evaluate the cytoprotective effects of HPBZs under ischemic conditions, we constructed an in vitro ischemic model by culturing the SH-SY5Y cell line with cobalt chloride (CoCl2). CoCl2 has been extensively used as an available reagent to induce a hypoxic microenvironment and subsequent overproduced ROS in different cell lines, which has been deemed a common in vitro model for exploring the mechanisms of antioxidants against ischemia-associated disorders.3,

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In line with this, upon stimulation with CoCl2 (300 M), the cells exhibited

decreased cell viability (Figure 3b) and excessive production of ROS (Figure 3c), whereas HPBZs treatment dramatically promoted cell survival (Figure 3b) and suppressed the ROS 9 ACS Paragon Plus Environment

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production (Figure 3c). These results suggest that HPBZs can drive cell protection via antioxidative activity. Upregulation of pro-apoptotic p53 and downregulation of anti-apoptotic Bcl-2 are associated with ischemia-induced cell death/apoptosis,43, 44 while upregulation of Bcl-2 can suppress the generation of ROS.45, 46 We conducted western blot studies with SH-SY5Y cells to assess whether regulation of p53 and Bcl-2 proteins is implicated in the neuroprotective effect of HPBZs in vitro. Compared with SH-SY5Y cells without CoCl2 stimulation, those stimulated with CoCl2 exhibited increased expression of pro-apoptotic p53 and decreased expression of anti-apoptotic Bcl-2 (Figure 3d). Conversely, HPBZs pretreatment explicitly downregulated the expression of pro-apoptotic p53 protein and upregulated the expression of anti-apoptotic Bcl-2 protein (Figure 3d). These findings indicate that HPBZs can exert anti-apoptotic effects to protect cells from ischemia-induced injury by regulating the expression of anti- and pro-apoptotic proteins. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is documented to be of great importance in mediating cell survival and apoptosis.47, 48

To determine the potential involvement of the MAPK/ERK signaling pathway, the

expression levels of key proteins in this pathway were preliminarily evaluated through western blot studies. CoCl2 dramatically induced the activation of ERKs, evidenced by an increase in p-ERK levels normalized to total ERK levels, whereas HPBZs pretreatment clearly suppressed this response (Figure 3d). Since ROS can induce phosphorylation of epidermal growth factor receptor and subsequently trigger the activation of ERK,49 ERK activation might be associated with the elevated ROS level induced by CoCl2. In contrast, the CoCl2-induced increase in the ROS level was attenuated by HPBZs pretreatment, which might account for the deactivation of ERKs in HPBZs-pretreated cells. The inflammatory response is recognized as another crucial contributor to cell death after ischemic stroke.50 Primary immune cells, including microglia and astrocytes, can be 10 ACS Paragon Plus Environment

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dramatically activated following ischemia, which leads to secretion of inflammatory mediators and further contributes to the invasion of peripheral inflammatory cells.2, 3 These, in turn, can further exacerbate neuronal injury in the cerebral ischemic penumbra and exaggerate damage to the surrounding regions.51 To identify whether HPBZs could protect cells from damage via their anti-inflammatory effect in vitro, we analyzed the expression levels of pro-inflammatory mediators and the cytokine interleukin-1β (IL-1β) in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells (mouse peritoneal macrophages), which is a common in vitro inflammatory model.3 Before that, the toxicity of HPBZs in Raw 264.7 macrophages was evaluated, and similarly, no obvious cytotoxicity was found (Figure S8). Cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), both of which are inducible enzymes, are regarded as crucial mediators of the inflammatory response to ischemic injury.52 COX-2, acting as a rate-limiting enzyme for the synthesis of prostanoids, has been revealed to be involved in the cytotoxicity resulting from ischemia-induced inflammation and in the production of reactive free radicals.53 The expression of COX-2 can be remarkably upregulated by inflammatory stimuli.54 We therefore examined the expression of COX-2 in LPS-stimulated macrophages in the absence vs. presence of HPBZs through immunofluorescence and western blot studies. HPBZs pretreatment effectively attenuated the upregulation of COX-2 in macrophages stimulated by LPS (500 ng/mL) (Figure 3e-f), which indicated an anti-inflammatory effect of HPBZs. Additionally, the expression of iNOS, which serves as the key enzyme generating toxic ·NO,55 was also decreased in LPS-stimulated macrophages after pretreatment with HPBZs compared to those without pretreatment (Figure 3f). The reduced level of iNOS contributed to a dramatic reduction of the generation of ·NO upon LPS (500 ng/mL) stimulation (Figure 3g). Meanwhile, HPBZs markedly inhibited the expression of the cytokine IL-1β (Figure 3f). In addition, the expression of nuclear factor-κB (NF-κB) p-P65 normalized to total NF-κB P65 was increased in cells stimulated with LPS compared to those without LPS stimulation, whereas pretreatment with HPBZs suppressed the 11 ACS Paragon Plus Environment

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NF-κB activation (Figure 3f). Emerging evidence has revealed that NF-κB, a transcription factor, plays a key role in activating glial cells and regulating the expression of COX-2, iNOS, and IL-1β.56-58. NF-κB might be activated by the increased generation of ischemia-induced free radicals.58 Thus, these findings indicate that HPBZs pretreatment might exert anti-inflammatory effects through suppression of NF-κB activation, which further inhibits the secretion of pro-inflammatory mediators and cytokines following ischemic injury. Considering the antioxidative, anti-apoptotic, and anti-inflammatory activities of HPBZs in vitro, we further investigated their disease-modifying effects in a rat model of ischemic stroke induced via a 90-min middle cerebral artery occlusion (MCAO) surgery (Figure S9). Positron emission tomography (PET), serving as the representative molecular imaging modality, enables noninvasive visualization of molecular changes during biological processes in vivo by utilizing radiolabeled molecules59. PET imaging with [(18)F]fluoro-2-deoxyglucose (18F-FDG), a glucose analog, has been used to detect subtle glucose metabolic alterations in vivo in a wide range of neurological diseases.60, 61 In this study, significantly higher 18F-FDG uptake was observed in the ipsilateral ischemic brain area in the HPBZs-pretreated group than in the saline-pretreated MCAO group (Figure 4a, c, P