Nano-curcumin simultaneously protects the blood-brain barrier and

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Biological and Medical Applications of Materials and Interfaces

Nano-curcumin simultaneously protects the blood-brain barrier and reduces M1-microglial activation during cerebral ischemia-reperfusion injury Ye Wang, Jun Luo, and Shi-Yong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20594 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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ACS Applied Materials & Interfaces

Nano-curcumin simultaneously protects the bloodbrain barrier and reduces M1-microglial activation during cerebral ischemia-reperfusion injury Ye Wanga#, Jun Luob*, Shi-Yong Lia,b,c* aDepartment

of Neurology, Second Affiliated Hospital of Nanchang University, Nanchang,.

China bDepartment

of Cardiology, Second Affiliated Hospital of Nanchang University, Nanchang,

China cDepartment

of Rehabilitation, Second Affiliated Hospital of Nanchang University, Nanchang,

China

ACS Paragon Plus Environment

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ABSTRACT

Oxidative stress and inflammation are two important pathophysiological mechanisms that arouse neuronal apoptosis and cerebral damage after ischemia/reperfusion (I/R) injury. Here, we hypothesized that curcumin-encapsulated nanoparticles (NPcurcumin) could reduce oxidative stress and inflammation in the ischemic penumbra via protecting the blood-brain barrier and inhibiting M1-microglial activation. Under oxidative stress conditions in vitro, we found that NPcurcumin protected microvascular endothelial cells against oxidative stress and reduced BBB permeability. In vivo, NPcurcumin could cross the blood brain barrier (BBB) and accumulate in the ischemic penumbra. At 3 d after I/R injury, NPcurcumin inhibited the increase in MMP9, attenuated the decrease in occludin and ZO-1, and maintained BBB integrity. NPcurcumin effectively reduce the number of activated M1-microglia, and weaken the increase of TNF-α and IL-1β. Furtherly, NPcurcumin also reduced infarcted size and improved function recovery.

Keywords: Curcumin, ischemia/reperfusion injury, blood brain barrier, antioxidation, antiinflammation


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1. INTRODUCTION Stroke is a leading cause of disability and death in worldwide.1 Thrombolysis with intravenous tissue plasminogen activator (tPA) can help restore blood flow to brain regions by quickly dissolving the clot, which is the first-line drug for ischemic stroke treatment.2,3 Although restoration of blood flow is critical for reducing neuronal apoptosis, it also results in local and systemic inflammatory responses and exacerbates oxidative stress and inflammatory damage.4 Therefore, our aim is to find effective methods to reduce oxidative stress and inflammatory damage after ischemia/reperfusion (I/R) injury. Curcumin from Curcuma longa possesses tremendous therapeutic potency, including antiinflammatory and antioxidative actions. For example, studies have demonstrated that curcumin can inhibit reactive oxygen species formation5 and reduce inflammatory injury.6 The poor water solubility and unstable chemical properties of curcumin largely limit its biomedical application.7,8 There has been various nanocarriers9,10 for encapsulating and delivering curcumin, including the use of microcapsules,11 micelles,12 hyalurosomes,13 emulsions14 and liposomes15 as delivery systems. These approaches can effectively increase the stability of curcumin and prolong its circulation time in vivo. In order to effectively deliver curcumin to the brain, the copolymer of poly(ethylene glycol)-bpoly(d,l-lactide) (referred to as mPEG-b-PLA) was used to prepare curcumin-encapsulated nanoparticles (referred to as NPcurcumin). This study aims to investigate whether NPcurcumin can reduce oxidative stress and maintain the integrity of blood-brain barrier (BBB), inhibit M1microglial activation and relieve inflammation, reduce the number of apoptotic neurons and function recovery after I/R injury. We will address a promising strategy to treat cerebral I/R injury


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2. MATERIALS AND METHODS 2.1. Materials. The block copolymer mPEG5K-b-PLA8K was synthesized based on our previous study.16 Curcumin was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Western blotting and immunofluorescence staining were carried out with the following primary antibodies purchased from Abcam (Cambridge, UK): anti-MMP9, anti-Iba-1, anti-CD86, antioccludin, anti- zona occluden-1( ZO-1), and anti-cleaved-caspase-3. The secondary antibodies used in this study were Alexa Fluor 488- and 594-conjugated donkey anti-mouse, rabbit, or goat IgG from Invitrogen (Carlsbad, CA, USA). HPR-conjugated anti-IgG was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Milli-Q Synthesis System (Millipore, Bedford, MA, USA) was used to produce ultra-purified water. 2.2. Preparation of nanoparticles loaded with curcumin. The single-emulsion method was used for preparing nanoparticles. In a typical experimental protocol, 10 mg curcumin and 100 mg mPEG-b-PLA were dissolved in 2 mL of mixed solvent containing chloroform and dimethyl sulfoxide (DMSO) (3:1, v/v), and then 8 mL of ultra-purified water was added to mixed solvent. In an ice bath, the mixture was emulsified by sonication (450 W for 2 min) to form an oil-inwater emulsion. A rotary vacuum evaporator was used to evaporate the organic solvent chloroform, and the nanoparticles were further purified by dialysis for 12 h using membrane dialysis tubing (Spectrum Laboratories, Rancho Dominguez, CA, USA). To determine the size and zeta potential, NPcurcumin (containing 0.2 mg/mL curcumin in aqueous solution) was detected using the Malvern Zetasizer Nano ZS90 apparatus (Worcester, UK). These measurements were conducted at 25 °C. The stability of NPcurcumin was tested by measuring the change of its size at different time.


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2.3. Establishment of the BBB model and measurement of trans-endothelial electric resistance. According to a previous study,17 we isolated and cultured primary murine brain microvascular endothelial cells (MBMECs). MBMECs (5×104 cells/cm2) were resuspended in endothelial cell medium, and seeded into 24-well transwell filters (Corning, USA).18 The quantitative analysis of barrier integrity was performed by detecting the electrical resistance of a cellular monolayer.19 The transendothelial electrical resistance (TEER) of the MBMEC layer was measured by using the Epithelial Voltohmmeter together with the Endohm-12 chamber (World Precision Instruments).20 The ohms per square centimeter of the transwell insert was used to analyze the TEER of the MBMEC layer. 2.4. Drug treatment in vitro. Cells experiment in vitro was divided the following four groups: (1) Control group, cultured in endothelial cell medium for 48 h, (2) H2O2 group, MBMECs cultured in endothelial cell medium with H2O2 (200 µM) for 48 h, (3) H2O2 (200 µM)+Curcumin (20 μM) group, MBMECs cultured in endothelial cell medium with H2O2(200 µM)+Curcumin (20 μM) for 48 h, and (4) H2O2+NPcurcumin group, MBMECs cultured in endothelial cell medium with H2O2 (200 µM) NPcurcumin (20 μM curcumin) for 48 h. 2.5. Detection of the mitochondrial membrane potential. The normal cells have high mitochondrial membrane potential. When the cationic dye tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was internalized by mitochondria in normal cells, the orange-colored emission light (590±17.5 nm) was produced by JC-1 aggregation. The low mitochondrial membrane potential in apoptotic cells increases the cellular efflux of JC-1 and decreases its intracellular concentration, which yields green fluorescence with emission at 530±15 nm. At 10 minutes after JC-1 was added to the cells, the cells were washed 3 times and then observed and analyzed with image analysis software (Image-Pro Plus).


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2.6. Cell apoptosis. According to the manufacturer's instructions, the Annexin-V Fluorescein (FITC) apoptosis detection kit (Oncogene, San Diego, CA, USA) was used to test cell apoptosis. After 1×105 cells were suspended in 100 μL of binding buffer, FITC-Annexin (5 μL) and PI (5 μL) were added to the cell suspension, and then mixed gently and incubated for 20 min in the dark. This cell suspension was washed with ice-cold PBS for 3 times, then be analyzed by BD FACSCalibur flow cytometer. 2.7. Real-time polymerase chain reaction (PCR). We extracted total RNA using the RNeasy RNA isolation kit (Qiagen). The Prime Script RT Reagent Kit (Takara Japan) was used for reverse transcription total RNA into cDNA. The FastStart Universal Probe Master (Roche Applied Science, Indianapolis, IN, USA) was used for the real-time PCR. These reactions were started at 95 °C for 10 min and then amplified for 30 cycles. Each cycle was comprised of 94 °C for 50 s, 55 °C for 30 s, and 4 °C to end the reaction. Primer Sequences: Occludin Forward: 5′CTCCCATCCGAGTTTCAGGT-3 ′ , Reverse: 5’-GCTGTCGCCTAAGGAAAGAG-3 ′ ; MMP-9

Forward:

5’-AAGGACGGCCTTCTGGCACACGCCTTT-3’,

GTGGTATAGTGGGACACATAGTGG-3′; ZO-1 CCACCTCTGTCCAGCTCTTC-3’, 3’GAPDH

Reverse:

Forward:

5’5’-

Reverse: 5 ′ -CACCGGAGTGATGGTTTTCT-

Forward: 5’- AGAGGGAAATCGTGCGTGAC-3’,

Reverse:

5’-

CAATAGTGATGACCTGGCCGT-3’. The average expression of different genes was normalized to GADH as an endogenous housekeeping gene. 2.8. Western blotting analysis. PRO-PREPTM (Boca Scientific) was used to extract proteins from the cells and tissues. The protein concentration was qualified by bovine serum albumin (BSA) and the BCA protein kit. After samples were electrophoresed and separated in a sodium dodecyl sulfate polyacrylamide gel, these proteins were transferred to polyvinylidene difluoride


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(PVDF) membranes. The membranes were incubated with different primary antibodies overnight at 4°C, and the ECL system (Pierce, Rockford, IL, USA) was used to detect protein expression. The Image-Pro Plus software was used to analyze these blots. 2.9. Accumulation of NPcurcumin in the brain. The brain was removed from mice at 3 h after tail vein injection with NPcurcumin (loading curcumin 25 mg/kg) or free curcumin (25 mg/kg). Then, the ipsilateral cortex was lysed, homogenized, and centrifuged at 10,000 x g for 5 min, and then the supernatant was collected. Quantitative analysis of curcumin was performed by reverse phase high-performance liquid chromatography (RP-HPLC, Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase was a mixture of acetonitrile and 2% acetic acid (50:50, v/v), and its flow rate was 1.2 ml/min. 21,22 The detection wavelength was 425 nm, and the standard curve of curcumin was prepared for the HPLC analysis. 2.10. Murine model. C57BL/6J male mice (weighing 26 to 28 g) were purchased from Beijing HFK Bioscience Co., Ltd. All mice were cared for and used according to the Guide of the Care and Use of Laboratory Animals, and the procedure was approved by the Animal Care and Use Committee of the University of Nanchang. The mouse middle cerebral artery model was consistent with our previous research.23 After the mice were anesthetized by inhaling isoflurane, the left carotid artery was exposed by a midline neck incision, and then a 5-0 nylon suture with blunted tip was inserted into the MCA origin. After 30 min, the nylon suture was withdrawn to establish reperfusion. 2.11. Immunohistochemistry. The brain tissues were fixed with 4% paraformaldehyde,and then cut into 12 µm thick slices. After being blocked with blocking buffer for 1 h, these sections incubated with an anti-Iba-1 or anti-cleaved-caspase-3 antibody at 4 °C overnight. These sections


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were then incubated with a secondary antibody for 2 h in the dark at room temperature. After a final wash with PBS, the sections were observed with the Zeiss Axiovert 200M microscope. 2.12. Evaluation of neurological deficits and the infarct size. C57BL/6J mice were randomly divided into 4 groups as follows: (1) sham group, (2) PBS group, I/R mice treated with PBS, (3) curcumin group, I/R mice treated with curcumin, (4) NPcurcumin group, I/R mice treated with NPcurcumin. At 30 mins after I/R injury, PBS, curcumin (25 mg/kg) and NPcurcumin (loading 25 mg/kg curcumin) were injected by tail vein. The neurological function of all mice was estimated double-blindly by the modified Neurological Severity Score (mNSS) with a total score of 18. A score of 0 represents normal, and the higher score represents more significant injury. To assess infarct size, 2,3,5-triphenyltetrazolium chloride (TTC) was used for staining 2 mm-thick brain slices. After the slices were scanned, the image analysis software was used to determine the ischemic area. 2.13. Statistical analysis. Values are expressed as the means ± standard deviations. One-way analysis of variance (ANOVA) was used to determine significant differences among groups. Student’s t-test was performed to determine the difference from each other. 3. RESULTS AND DISCUSSION 3.1. Characterization of NPcurcumin. Curcumin, which is a hydrophobic polyphenol compound, is insoluble in aqueous solution. The mPEG-b-PLA copolymer is an amphiphilic polymer with good stability that has been used for the delivery of therapeutic agents.24 Curcumin was encapsulated in mPEG-b-PLA block copolymer nanoparticles through the single emulsion method. We demonstrated that curcumin was effectively encapsulated in the nanoparticle core. The drug loading content (DLC %) and drug loading efficiency (DLE %) of NPcurcumin were 4.92±0.23% and 51.7±3.1%, respectively. As shown in Figure 1A and B, NP had a narrow size


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distribution (147.8 ± 5.7 nm) and its zeta potential was -4.14±0.18 mV in deionized water. The TEM images showed that nanoparticles loaded with curcumin had spherical structures and smooth surfaces. To examine its stability, NPcurcumin was incubated in PBS with 10% fetal bovine serum at 37 °C. Our data showed that the size of the nanoparticles was consistent within 48 h (Figure 1C), suggesting that NPcurcumin was stable in serum. In addition, the release of the curcumin was detected at pH 7.4 and 37 °C. Our data demonstrated that NPcurcumin released approximately 85% of the curcumin within 72 h (Figure 1D).

Figure 1. Nanoparticle characterization. (A) Particle size and distribution (B) Zeta potential of NPcurcumin. (C) The size change of NPcurcumin. (D) Cumulative release of curcumin from NPcurcumin. 3.2. The protective roles of curcumin and NPcurcumin for MBMECs and the BBB in vitro. Mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III


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and IV), which is generally used to evaluate the mitochondrial function and cell viability. In this study, the ΔΨm was significantly decreased by H2O2 treatment (Figure 2A and B).The ΔΨm of MBMECs in Curcumin and NPcurcumin were significantly higher than cells in H2O2 group. These data suggested that curcumin or NPcurcumin could inhibit the mitochondrial decrease induced by H2O2. This is because curcumin, as an excellent scavenger of most ROS, effectively reduced mitochondrial damage.25 Consistent with the change in ΔΨm, the flow cytometry results demonstrated that curcumin or NPcurcumin effectively reduced the number of apoptotic MBMECs induced by H2O2. These results illustrated that curcumin or NPcurcumin could protect MBMECs against oxidative stress in vitro.


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Figure 2. NPcurcumin protects endothelial cells and trans-endothelial electric resistance (TEER) of the BBB in vitro. (A) NPcurcumin significantly inhibits the decrease in ΔΨm induced by H2O2 in endothelial cells. (B) Quantitative analysis of the red/green fluorescence ratio. (C) The apoptosis rate was measured by flow cytometry analysis after staining with Annexin V-FITC and propidium iodide. (D) The effects of NPcurcumin on the TEER of the BBB in vitro. (mean ± standard deviation, ** P < 0.01，* P < 0.01). The BBB is an important physical barrier of the brain tissue, which is critical to maintaining the brain homeostasis.26 Trans-endothelial electrical resistance (TEER), as an effective method, has been used to analyze the integrity of BBB models in vitro.20 According to previous studies, the BBB in vitro model was prepared using a MBMEC monolayer and then exposed to H2O2 with curcumin or NPcurcumin. We found that the TEER value of MBMEC was significantly decreased by H2O2 treatment, suggesting that H2O2 could induce the breakage of the BBB in vitro model (Figure 2D). However, the decreased TEER values induced by H2O2 stimulation could be weakened by curcumin or NPcurcumin treatment. These data showed that curcumin or NPcurcumin treatment could protect MBMECs against oxidative stress, which was beneficial for maintenance of BBB integrity. 3.3. NPcurcumin may cross the BBB and enter the penumbra. I/R injury can arouse oxidative stress and inflammation cascade damage, which result in the destruction of the structure and function of tight junctions. These factors will rapidly disrupt the BBB and exacerbate its leakage. It is well known that the leaky BBB can increase the rate of nanoparticles crossing the BBB.27,28 For example, studies have demonstrated that nanoparticles, as drug delivery vehicles, can cross the BBB and deliver therapeutic agents to injured brain tissue.29 In this study, we investigated whether NPcurcumin could deliver curcumin into the ischemic


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penumbra (Figure 3A). To confirm whether the nanoparticles could deliver curcumin into the ischemic penumbra, the curcumin concentration in the cortex was detected by RP-HPLC at 3 h after injection of curcumin (25 mg/kg) or NPcurcumin (loading 25 mg/kg curcumin). As shown in Figure 3B, the curcumin concentration in the cortex in NPcurcumin group was over 20 times higher than that in the curcumin group. These data suggested that the nanoparticles effectively delivered curcumin to the ischemic brain tissue. Furtherly, a strong fluorescent signal of curcumin was found in the ipsilateral cortex in NPcurcumin group (Figure 3C), which suggested that NPcurcumin could cross the BBB and enter the penumbra.

Figure 3. NPcurcumin crosses the BBB. (A) Time schedule for treatment of mice with I/R injury with curcumin or NPcurcumin. (B) The concentration of curcumin in cortex was determined by RP-HPLC (mean ± SD, ** P < 0.01). (C) Sections of the injured hemisphere were observed by


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confocal microscopy. The endothelial cells of the BBB were stained with CD31 (red), the green fluorescent signal was curcumin, and the cell nuclei were stained with DAPI (blue). 3.4. NPcurcumin ameliorates alterations of MMP-9, Occludin and ZO-1. MMP-9 belongs to the matrix metalloproteinase gene family and plays important roles in degrading extracellular matrix components. MMP-9 is also closely related to blood-brain barrier disruption, the infarct volume, stroke severity, and the functional outcome and is significantly upregulated after ischemic stroke.30 MMP-9 inhibition can protect neurons from brain injury.31 Therefore, we examined whether NPcurcumin could inhibit MMP-9 expression (Figure 4A). Compared with that of the sham group, a significant increase in MMP-9 mRNA and protein expression was observed in the mice with I/R injury. However, the enhancement was inhibited by NPcurcumin but not curcumin treatment (Figure 4B and C). These data demonstrated that NPcurcumin elicited better inhibitory effects on MMP9 expression in the ischemic penumbra than curcumin, which might be related to accumulation of NPcurcumin in the injured brain areas.


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Figure 4. Effects of NPcurcumin on MMP9, occludin, and ZO-1. (A) Time schedule for analysis of MMP9 expression in the ischemic cortex of mice with I/R injury by Western blotting (B) and PCR (C). Occludin and ZO-1 expression in the ischemic penumbra was detected by PCR (D) and real-time PCR (E) (mean ± SD, n=3, ** P < 0.01). The tight junctions in BBB were formed by ZO-1 and occludin, which are keys to maintenance of BBB integrity. The loss or decrease of ZO-1 or occludin results in the increased permeability of BBB, which aggravates inflammatory cell infiltration into the penumbra and oxidative stress.32,33 Our data showed that the occludin and ZO-1 expression levels in PBS group were significantly lower than that in sham group (Figure 4D and E). NPcurcumin treatment effectively prevent occludin and ZO-1 downregulation at day 3 after I/R cerebral injury. These data demonstrated that NPcurcumin protected the BBB by inhibiting the decrease in tight junction proteins, which was closely associated with nanoparticle-based drug-delivery systems.


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3.5. NPcurcumin inhibits M1 microglia polarization and inflammatory damage. Microglia is the most important defense against exogenous threats in brain. There are two polarized microglial subtypes: pro-inflammatory microglia (M1-microglia) and antiinflammatory microglia (M2-microglia).34 After brain I/R injury, microglia polarized into M1microglia are rapidly recruited into the ischemic penumbra. At the same time, M1-microglia can also produce and release pro-inflammatory factors, such as ROS, TNF-α and IL-1β, which amplify the inflammatory response and oxidative stress. Ionizing calcium-binding adaptor molecule 1 (Iba1) is a microglial marker, and CD68 is a marker of activated M1-microglia. Therefore, Iba1+ and CD68+ double-positive cells were considered activated M1-microglia in this study. Here, higher numbers of Iba1+ and CD68+ microglia were present in the penumbra in mice treated with PBS or curcumin than in mice in the sham group (Figure 5A and B). However, significantly fewer Iba1+ and CD68+ double-positive microglia were detected in the mice treated with NPcurcumin. These data demonstrated that NPcurcumin effectively inhibited M1-microglia activation induced by I/R injury.


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Figure 5. Roles of NPcurcumin on M1-microglia activation. (A) M1-microglia activation was observed by detecting IBa-1 (green), CD68 (red) and cell nuclei (blue) in the ischemic penumbra. IBa-1+ and CD68+ dual-positive cells were counted as activated M1-microglia. (B) Quantitative analysis of M1-microglia activation in the ipsilateral hemispheres. The change of TNF-α (C) and IL-1β (D) in the ischemic penumbra (mean ± SD, n = 3, ** P < 0.01, * P < 0.05). Pro-inflammatory cytokines35 are considered important causes of neuronal cell damage and apoptosis. Therefore, we evaluated changes in TNF-α and IL-1β expression in the penumbra. The significant increases in TNF-α and IL-1β were found in the ischemic penumbra in the PBS and curcumin groups (Figure 5 C and D). The lower expression of TNF-α and IL-1β was found


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in the NPcurcumin group than in the other groups (P < 0.05). These data showed that curcumin did not play a significant inhibitory role in the inflammatory response, which might be related to its poor stability in vivo. Similarly, the poor stability of curcumin had also been reported in previous studies.36 Our data demonstrated that NPcurcumin could deliver curcumin to the ischemic penumbra and reduce the inflammatory response. 3.6. NPcurcumin reduced neuronal apoptosis in the penumbra. Caspase-3 belongs to the endoprotease family. As a key mediator of neuronal apoptosis, caspase-3 can be activated by cerebral I/R injury.37 Nissl staining is a classic methods for labelling neurons in tissue sections. Therefore, cleaved caspase-3/Nissl double-positive neurons in the cerebral penumbra were considered dying neurons and analyzed by confocal microscopy. As shown in Figure 6A, significantly more activated caspase-3/Nissl double-positive neurons were present in the PBS and curcumin groups than in the sham group. However, fewest activated caspase-3/Nissl doublepositive neurons were detected in the NPcurcumin group than in the other groups. Importantly, lower activated caspase-3 gene expression was detected on day 3 after injury in the NPcurcumin group than in the PBS and curcumin groups (Figure 6B and C). These data demonstrated that NPcurcumin treatment could reduce the number of apoptotic neurons, suggesting that NPcurcumin could protect neurons against I/R injury.


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Figure 6. The roles of NPcurcumin on neurons. (A) Activated caspase-3 (red) and Nissl staining (green) was detected by confocal microscopy (scale bar, 20 µm). Activated caspase-3 gene expression was analyzed by PCR (B) and real-time PCR (C) (mean ± SD, n=3, ** P < 0.01). 3.7. Effects of NPcurcumin on the infarct volume and nerve function. 2,3,5Triphenyltetrazolium chloride (TTC) staining, an effective method to evaluate the infarct size and volume, has been used to differentiate between metabolically active and inactive tissues.38 In this study, the infarct volumes in the PBS, curcumin, and NPcurcumin groups were 40.1±5.6%, 32.4±7.6 and 18.3±4.1, respectively (Figure 7A and B). Compared with those of the PBS and curcumin groups, NPcurcumin treatment significantly reduced the infarct volume on day 3 after I/R injury, which showed the protective of NPcurcumin on neurons against I/R injury.


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Figure 7. Effects of NPcurcumin on the infarct volume and functional recovery. Representative TTC staining of at day 3 after cerebral I/R injury (A). The infarct sizes (B) and neurological scores (C) in the different groups (mean ± SD, n = 5, ** P < 0.01). The mNSS was used to estimate neurological functions on days 1, 2, and 3 after I/R injury. As shown in Figure 7C, severe behavioral deficits were found in the PBS group. Compared with PBS group, mice in the NPcurcumin group had lower mNSS scores on days 1, 2, and 3 after I/R injury. These data showed that NPcurcumin could effectively promote functional recovery in mice with I/R injury. 4. CONCLUSIONS In this study, we proved that NPcurcumin could deliver curcumin to the ischemic penumbra and enhance its antioxidative and anti-inflammatory actions in vivo. Furthermore, we confirmed


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that NPcurcumin treatment contributed to maintenance of BBB integrity and inhibited activation of M1-microglia and the release of inflammatory factors into the ischemic penumbra, which could prevent neuronal apoptosis and substantially improve brain function recovery in mice after I/R injury. ASSOCIATED CONTENT Supporting Information Supplementary Figures S1: The protective roles of curcumin and NPcurcumin in MBMECs against oxidative stress. Supplementary Figures S2: The effect of NPcurcumin on systemic toxicity and neurological function. AUTHOR INFORMATION Corresponding Author Jun Luo, Email: [email protected]. Shi-Yong Li, Email: [email protected]. Funding Sources National Natural Science Foundation of China (No: 81760417; 81660230), Science and Technology Program of Jiangxi, China (2016ACB21019; 2018ACB20020). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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Authors are grateful for the insightful input from Pro Jun Wang.

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Nano-curcumin simultaneously protects the blood-brain barrier and

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