2.1. Animals and Ethics Statement

Male C57BL/6 background mouse (Shanghai SIPPR-Bk Lab Animal Co., Ltd., Shanghai, China) had free access to water and food under the following conditions: 22 ± 3°C temperature, 60 ± 5% humidity, and 12 h light/dark cycle. Our team treated animals following Guide for Care and Use of Laboratory Animals, and investigation protocols were made in accordance with guidelines of Ethics Committee in Pudong New Area Gongli Hospital (Shanghai, China). Surgery processes were made to minimize suffering after anesthesia.

2.2. Middle Cerebral Artery Occlusion (MCAO) Mouse Model

We anesthetized mouse by intraperitoneal injection with sodium pentobarbital at 30 mg/kg. We monitored body temperature to maintain it to 36.5~37.5°C. Modified MCAO-treated mouse model was utilized to induce permanent focal ischemia following [12]. In brief, we occluded right middle cerebral artery (MCA) via inserting monofilament nylon suture with 0.24~0.26 mm diameter through heat-rounded tip into internal carotid artery. It was further advanced until it closed MCA origin. Each group included a total of six mice.

2.3. Animal Treatments

We randomly assigned cerebral ischemia and sham-operated mice to the vehicle or CL group. Mice in the CL group were intraperitoneally injected with 1 mg/kg of CL immediately dissolved in 0.9% NaCl and 1% dimethyl sulfoxide (InvivoGen, San Diego, CA, USA) and on postoperative day 1. All mice were reanesthetized and sacrificed 3 days post MCAO.

2.4. Infarct Volume Measurement

Infarct volumes were analyzed following [13]. We decided infarct volume applying 2,3,5-triphenyltetrazolium chloride (TTC) at 3 d post MCAO. Our group sliced brain tissue to thick sections, which we stained with 2% solution TTC for twenty minutes at 37°C and then fixed with 4% paraformaldehyde. Our team imaged and analyzed TTC-stained sections utilizing Image-Pro Plus 5.1 (Media Cybernetics, Bethesda, MA, USA). Lesion volume is calculated as (total infarct volume + intact contralateral hemisphere volume − intact ipsilateral hemisphere volume)/contralateral hemisphere volume.

2.5. Histopathological Analyses

Technician fixed brain tissues in 10% buffered formalin, routinely processed, embedded in paraffin, and sliced into 4 μm thick sections, which we placed on slides and stained for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis (Shanghai Yeasen Biotech Co., Ltd., Shanghai, China). The slides were then evaluated under a light microscope (Motic Hong Kong Limited, Hong Kong, China).

2.6. BV2 Cell Culture and Transfection

BV2 cells were purchased from Wuhan Biofavor Biotechnology Service Co., Ltd. (Wuhan, China), which we maintained in DMEM (Invitrogen Corporation, Carlsbad, CA, USA) and supplied with 10% FBS (Invitrogen Corporation, Carlsbad, CA, USA) under atmosphere of 95% air/5% CO₂. Our team seeded BV2 cells into 24-well plates with density 1 × 10⁵ cells/mL, which we incubated with serum-free DMEM over the night. On the following day, we pretreated cells with CL (1 μM), MCC950 (10 μg/mL), or Ac-YVAD-CMK (10 ng/mL) for 6 h in prior stimulation under OGD conditions for 3 h. Afterward, we cultured cells with normal conditions for 1 d and then collected for analysis.

Our team transfected BV2 cells with small interfering RNA against Nrf2 (GeneCopoeia, Shanghai, China) employing Lipofectamine 2000 Transfection Reagent (Invitrogen Corporation). The cells were utilized for further experiments post 48 h. The following primer sequences were used: (sense) 5′-GCA GCC AUG ACU GAU UUA A-3′ and (antisense) 5′-UUA AAU CAG UCA UGG CUG CUU-3′.

2.7. Apoptosis Flow Cytometric Analysis

Our team collected BV2 cells from various groups, which we resuspended in 1x binding buffer. Adding 5 μL Annexin V-fluorescein isothiocyanate (FITC) solution and 5 μL propidium iodide solution following protocols of Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA), we incubated cells under room temperature for fifteen minutes in dark. Immediately afterward, we subjected cells to flow cytometry to calculate viable and apoptosis cell percentages.

2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

IL-18, IL-1β, and TNF-α expression levels were assessed utilizing commercial ELISA kits (Abcam, Cambridge, UK) following manufacturer protocols. In brief, we collected 100 μL cell culture supernatants or mouse serum, which we put to 96-well plates coated with appropriate antibody. We incubated them with enzyme conjugation solution for 60 min at 37°C. Post washing the plates five times, we added substrates I and II to the wells and incubated plates with room temperature for an additional 15 min. Finally, we terminated reactions by stopping solution addition. Our team determined absorbance through measuring optical density by microplate reader (Thermo, Waltham, MA, USA) at 450 nm wavelength.

2.9. Western Blot Analysis

Our team lysed cells or brain tissues in Triton X-100 lysis buffer (25 mM Tris-HCl and pH 7.5). Proteins that purified were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis utilizing 12% separating and 5% stacking gel followed by transferred protein from the gels onto nitrocellulose membranes, which were blocked with 5% nonfat milk and TBS-T buffer (0.2% Tween 20, 150 mM NaCl, 50 mM Tris-HCl, and pH 7.5) for 1 h at room temperature. The membranes were probed with primary antibodies against Nrf2, HO-1, NLRP3, caspase-1, IL-1β, NF-κB, and GAPDH. Following incubation with respective secondary antibodies (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA), we incubated membranes utilizing Lumi-Phos™ WB chemiluminescent substrate (Pierce Biotechnology, Waltham, MA, USA) for 5 min. Our team measured protein expression levels via ImageJ software (NIH, Bethesda, MD, US).

2.10. RNA Extraction and qRT-PCR

Our team extracted total RNA utilizing TRIzol reagent (Sigma-Aldrich Corporation). We determined RNA concentrations through Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). Our group performed quantification of Nrf2 and endogenous control GAPDH applying TaqMan assays via supplemented assay-particular primers (Applied Biosystems, Carlsbad, CA, USA). The statistician analyzed data applying 2^−ΔΔCt method.

2.11. Statistics Analyses

We presented data by means ± SD. Statistics analyses were made via GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA) to find significant differences between groups. p values ≤ 0.05 were regarded as statistically significant. Two-tailed Student’s t-tests were applied to calculate significant differences among groups, and two-way ANOVAs with post hoc Bonferroni tests were employed to determine significant differences among at least three groups.

3.1. CL Treatment Suppressed Middle Cerebral Artery Occlusion- (MCAO-) Induced BI

Previous studies have found that CL has therapeutic and protective effects in a variety of diseases, including traumatic BI [14], cancer [15, 16], diabetes [17, 18], and myocardial ischemia [19]. Hence, the present study is aimed at determining if CL conveys a protective effect against AIS-induced BI. In a mouse model, CL was found to reverse MCAO-induced BI by decreasing the infarct volume, as determined by TTC staining (Figures 1(a) and 1(b)). Immunohistochemical staining showcased that apoptotic cell portions in brain tissue of infarct region had increased significantly after MCAO-induced AIS. Also, CL treatment decreased the rate of apoptosis in the infarct region (Figures 1(c) and 1(d)), suggesting that CL conveys a protective effect against MCAO-induced BI.

Increasing evidence has shown that CL has anti-inflammatory effect [20]. To confirm anti-inflammatory activity of CL, TNF-α, IL-1β, and IL-18 serum expression levels, they were detected with commercial ELISA kits. Data showcased that TNF-α, IL-18, and IL-1β expression levels in the MCAO group had increased, which were suppressed by CL treatment (Figures 2(a)–2(c)). Data imply that CL could suppress the inflammatory factor releases. Data of WB analyses validated that CL inhibited AIS-induced activation regarding the NLRP3 inflammasome and suppressed AIS-induced pyroptosis by reducing caspase-1 and IL-1β expression levels (Figures 2(d)–2(f)), suggesting that NLRP3 inflammasome activation functions importantly in pyroptosis inductions.

Former investigations found that Nrf2/HO-1 pathway functions anti-inflammatory role importantly [10, 21]. Likewise, data of the present study showcased that CL treatment promoted Nrf2/HO-1 activation and suppressed AIS-induced NF-κB expression (Figures 2(g)–2(k)). These data are concordant to those of former study that Nrf2/HO-1 pathway activations suppress NLRP3 inflammasome-dependent pyroptosis [22].

3.2. Oxygen-Glucose Deprivation- (OGD-) Induced Microglial Injury Relative to the NLRP3/Caspase-1 Pathway Activations

To determine if NLRP3 inflammasome and caspase-1 are involved in AIS-induced BI, BV2 cells were cultured under OGD conditions to simulate AIS in an in vitro cell model as previously reported [23]. The result showed that the portion of apoptotic BV2 cells had increased under OGD conditions for 3 h. But pretreatment with NLRP3 inhibitor MCC950 or caspase-1 inhibitor Ac-YVAD-CMK decreased apoptotic BV2 cell portions (Figures 3(a) and 3(b)). ELISA results showed that blocking activation of NLRP3/caspase-1 pathway suppressed OGD-induced IL-18, IL-1β, and TNF-α expressions (Figures 3(c)–3(e)). WB data showcased that NLRP3/caspase-1 pathway activations blocked the decrease in IL-1β expression (Figures 4(a)–4(d)).

3.3. CL Treatment Reversed OGD-Induced Microglial Injury by HO-1/Nrf2 Pathway Activations

In order to decide if CL treatment reversed OGD-induced microglial injury through Nrf2/HO-1 pathway activations, Nrf2 silencing in BV2 cells was conducted, which showed that silencing of Nrf2 significantly decreased Nrf2 expression at protein and mRNA levels (Figures 5(a) and 5(b)). Furthermore, flow cytometry data showcased that OGD induction promoted BV2 cell apoptosis, which was reversed by CL treatment. However, the protective effect of CL decreased after downregulation of Nrf2 (Figures 5(c) and 5(d)). Our data advise that Nrf2 functions importantly in CL-mediated neuroprotection in AIS. Meanwhile, ELISA data showcased that Nrf2 downregulation reversed CL anti-inflammatory effect (Figures 5(e)–5(g)). WB data showcased that CL treatment suppressed OGD-induced NLRP3/caspase-1/IL-1β-mediated cell pyroptosis, but downregulation of Nrf2 reversed the protective effect of CL (Figures 6(a)–6(d)) and suppressed HO-1 expression, which caused NF-κB expression upregulations under OGD conditions even with CL treatment (Figures 6(e)–6(h)).