Celastrol Treatment Ameliorated Acute Ischemic Stroke-Induced Brain Injury by Microglial Injury Inhibition and Nrf2/HO-1 Pathway Activations

Background Stroke is the third main reason of mortality, which is the leading reason for adult disability in the globe. Poststroke inflammation is well known to cause acute ischemic stroke- (AIS-) induced brain injury (BI) exacerbation. Celastrol (CL) has exhibited anti-inflammatory activities in various inflammatory traits though underlying mechanisms remain unknown. So, the present investigation is aimed at studying CL protective mechanism against AIS-induced BI. Methods A mouse model regarding middle cerebral artery occlusion and an oxygen-glucose deprivation (OGD) cell model with or not CL treatment were constructed to study CL protective effects. NF-E2-related factor 2 (Nrf2) was then silenced in BV2 microglia cells (BV2) to study Nrf2 role regarding CL-mediated neuroprotection. Results The results showed that CL treatment suppressed AIS-induced BI by inhibiting NLRP3/caspase-1 pathway activations and induction of apoptosis and pyroptosis in vivo and in vitro. NLRP3/caspase-1 pathway blocking activation suppressed OGD-induced cell pyroptosis and apoptosis. Also, CL treatment reversed OGD-induced microglial injury by promoting Nrf2/heme oxygenase-1 (HO-1) pathway activations. Nrf2 downregulation reversed CL protective effects against OGD-induced microglial injury, pyroptosis, and apoptosis. Conclusion The findings advise that CL treatment ameliorated AIS-induced BI by inhibiting microglial injury and activating the Nrf2/HO-1 pathway.


Background
Acute ischemic stroke-(AIS-) induced brain injury (BI) and subsequent functional recovery are mediated by neuroinflammation [1]. NLR pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein complex composed of ASC, NLRP3, and caspase-1 [2], which serves as a central innate immune sensor triggered by endogenous "danger" signaling in response to pathogenic infection, metabolic dysregulation, and tissue damage [3]. NLRP3 inflammasome activations enhance caspase-1 activations besides maturation and release of proinflammatory cytokines such like interleukin-(IL-) 1β and IL-18, causing pyroptosis promotions [4]. Pyroptosis is a proinflammatory programmed cell apoptosis process, which has distinct signaling mechanisms from those of apoptosis and necrosis [4]. So, we surmised that the suppression of pyroptosis may reverse ASIinduced BI.
Therefore, the current research was to investigate if CL possesses protective effects against AIS-induced BI. Briefly, NLRP3 inflammasome-induced pyroptosis and apoptosis were assessed applying in vitro cell model and an AIS mouse model. Results indicate that CL treatment ameliorated AISinduced BI by inhibiting microglial injury and Nrf2/HO-1 pathway activations.

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.

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.

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 ðtotal infarct volume + intact contralateral hemisphere volume − intact ipsilateral hemisphere volumeÞ/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% CO 2 . Our team seeded BV2 cells into 24-well plates with density 1 × 10 5 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.

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 96well 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.

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 (Graph-Pad 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.

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 antiinflammatory 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 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 AISinduced NF-κB expression (Figures 2(g)-2(k)). These data
The data also showed that CL treatment reversed AISand OGD-induced nerve injury by suppressing NLRP3/caspase-1 pathway activations. CL was widely applied for various autoimmune disease treatments [29][30][31]. Previous investigations showcased that CL suppressed NLRP3 inflammasome-mediated IL-18 and IL-1β release, along with pyroptosis activation [9]. Meanwhile, CL treatment reversed AIS-induced BI by microglial injury inhibition and Nrf2/HO-1 pathway activations, whereas downregulation of Nrf2 reversed CL protective effect. HO-1/Nrf2 activation suppressed oxidative stress and inflammatory cytokine productions by decreasing NF-κB expressions. Nrf2 is a main factor in cytoprotection, which activated under stress conditions resulted from electrophilic and reactive oxygen species productions. Inflammasomes are central inflammation regulators [32]. Dihydromyricetin suppresses NLRP3 inflammasome-dependent pyroptosis by Nrf2 signaling      Figure 7: The regulatory mechanism of celastrol. CL treatment inhibits acute ischemic stroke-induced and NLRP3/caspase-1 pathway-mediated pyroptosis. At the same time, CL treatment promoted Nrf2/HO-1 pathway, which inhibit inflammatory cytokine-mediated apoptosis. 8 BioMed Research International pathway activations in vascular endothelial cells [22]. Outputs of current investigations showcased that CL treatment ameliorated AIS-induced BI by inhibiting microglial injury and Nrf2/HO-1 pathway activations. Previous investigations advised that celastrol can be used as novel drug to cause cerebral vascular dilation in cases where endothelial and/or BK channel function is impaired [33]. But if CL treatment can improve the vascular system after acute ischemic stroke is still unclear.

Conclusions
Our results highlight the pyrogenic properties of NLRP3 inflammatories and the antipyrogenic properties of CL, as well as their role in the pathogenesis of AIS-induced BI.
Our study found that CL treatment inhibited acute ischemic stroke-induced and NLRP3/caspase-1 pathway-mediated pyroptosis. Meanwhile, CL treatment promoted Nrf2/HO-1 pathway and inhibited apoptosis mediated by inflammatory cytokines (Figure 7). The data may have indispensable implications for new therapeutic strategy developments to treat this severe AIS complication.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Ethical Approval
All animals were treated following Guide for Care and Use of Laboratory Animals, and all investigation protocols were conducted in accordance with guidelines of Ethics Committee in Pudong New Area Gongli Hospital, Shanghai, China.

Conflicts of Interest
The authors declare that they have no conflicts of interest.