*Contributed equally
The NLR family pyrin domain-containing 3 (NLRP3) inflammasome, which is composed of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and pro-caspase-1 protein complexes, is activated by the reactive oxygen species (ROS) that are associated with ischemia-reperfusion (I/R) and are involved in brain damage. Pomelo peel oil (PPO) exhibits antioxidant activity. However, it is unclear whether PPO is able to attenuate NLRP3 inflammasome-induced inflammation and pyroptosis. Healthy male Sprague-Dawley rats were subjected to 7 min of cardiac arrest via trans-esophageal electrical stimulation, followed by cardiopulmonary resuscitation (CPR). The rats were then treated with PPO prior to reperfusion for 24 h. Hematoxylin and eosin staining was used to evaluate brain tissue and cell damage. In the brain tissues, reactive oxygen species (ROS) were assayed, immunofluorescence was used to analyze the expression of NLRP3 and western blotting was performed to determine the expression levels of neuroenolase (NSE), NF-κB, interleukin-1β (IL-1β), gasdermin D (GSDMD) and the NLRP3 inflammasome. Treatment of the rats with PPO significantly decreased the pathological damage of the brain tissue and reduced the expression of NSE, production of ROS and secretion of NF-κB, NLRP3, IL-1β and GSDMD. In conclusion, these results demonstrate the ability of PPO to protect the brain against I/R injury in rats after CPR by a mechanism involving inhibition of the inflammation and pyroptosis mediated by NLRP3 inflammasome activation.
Cardiac arrest (CA) and subsequent cardiopulmonary resuscitation (CPR) induces systemic organ tissue ischemia/reperfusion (I/R) injury, particularly cerebral I/R injury (CIRI), which directly affects the prognosis and quality of life of patients. Numerous pathophysiological mechanisms of I/R injury, including oxidative stress, amino acid toxicity, energy metabolism, calcium overload, apoptosis and autophagy, have been targeted by interventions for brain protection (
A previous study has demonstrated that, in most organs, including the brain, heart, liver, lungs and intestines, activation of the NLRP3 inflammasome aggravates CIRI (
Pomelo (
Fresh and mature pomelo peels [
The compounds in the PPO were qualitatively analyzed using GC-MS (TRACE™ 1300; Thermo Fisher Scientific, Inc.). Helium was injected as a carrier gas at a flow rate of 1 ml/min. The temperature was increased from 45 to 250˚C according to the procedure. The heating program is maintained at 45˚C for 1 min, then at 10˚C/min to 165˚C for 2 min, then at 1.5˚C/min to 180˚C for 2 min and finally at 10˚C/min to 250˚C for 2 min. The temperatures of the injector and detector were set at 250˚C. Mass spectra were scanned from m/z 41-400 amu. The electron impact ionization energy was 70 eV. Identification of the detected compounds was performed by comparing the mass spectra with published data. Samples were identified using the Retention Time Locked database (NIST MS Search 2.3;
In total, 60 male Sprague-Dawley (SD) rats (body weight, 220-250 g; age 7-8 weeks) were provided by the Experimental Animal Center of Guangxi Medical University (License number SYXK Gui 2014-0003). All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (
The CA model was established according to the method reported by Chen
All rats were anesthetized using pentobarbital (30 mg/kg) by intraperitoneal injection 24 h post reperfusion. Three rats in each group were perfused with 4% paraformaldehyde for hematoxylin and eosin (H&E) staining and immunofluorescence experiments. Cerebral cortices were immediately harvested from the remaining 5 rats in each group and stored at -80˚C for subsequent western blot analysis.
Fresh cerebral cortex was homogenized with phosphate buffer in a weight (g):volume (ml) ratio of 1:20. Following centrifugation at 1,000 x g for 10 min, the supernatant was used for the measurement of ROS and protein concentrations. Thereafter, 190 µl supernatant was mixed with 10 µl DCFH-DA from an ROS assay kit (WLA070; Wanleibio Co., Ltd.) in a 96-well plate and incubated at 37˚C in the dark for 30 min. ROS were detected via fluorescence with an excitation wavelength of 500 nm and emission wavelength of 525 nm. The protein concentration was determined using the BCA method (P0010; Beyotime Institute of Biotechnology) and the results were expressed as fluorescence intensity/mg protein.
The rat brains fixed with 4% paraformaldehyde were embedded in paraffin and cut into 3-µm coronal sections. These sections were then subjected to H&E staining. At room temperature, the paraffin sections were dewaxed using xylene followed by a descending ethanol gradient, rehydrated, stained with hematoxylin for 5 min at 25˚C, reacted with hydrochloric acid ethanol for 5 sec and stained with eosin for 1 min at 25˚C before being finally dehydrated. The slices were observed under a light microscope (Olympus, Japan) at a magnification of x400.
The dry 3-µm paraffin slices were subjected to antigen retrieval by heating in a microwave oven at 65˚C with pH 8.0 EDTA antigen repair buffer (Beijing Solarbio Science & Technology Co., Ltd.), then incubated in 0.01 M PBS (pH 7.4) containing 0.3% Triton X-100 (TBST) for 20 min at 25˚C prior to blocking in normal goat serum (OriGene Technologies, Inc.) for 30 min at 37˚C in BSA (OriGene Technologies, Inc.). The slices were incubated with anti-NLRP3 primary antibodies (cat. no. wl03379; 1:100; Wanleibio Co., Ltd.) overnight at 4˚C, followed by horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibodies (cat. nos. GB23303; 1:500; Wuhan Servicebio Technology Co., Ltd.) for 50 min at 25˚C. FITC-Tyramide (cat. no. G1225-1; 1:1,000; Wuhan Servicebio Technology Co., Ltd.) was added for 10 min at 25˚C. The sections were then placed into citric acid (pH 6.0) antigen repair solution (Wuhan Servicebio Technology Co., Ltd.) and heated in a microwave oven at 65˚C for 5 min. Anti-allograft inflammatory factor 1 (IBA-1) primary antibodies (cat. no. ab153696; 1:1,000; Abcam) were added to the sections, which were incubated overnight at 4˚C. A Cy3-conjugated fluorescent secondary antibody (cat. no. G1225-2; 1:1,000; Wuhan Servicebio Technology Co., Ltd.) was then added for 50 min at 25˚C, followed by an autofluorescence quenchant (cat. no. G1221; Wuhan Servicebio Technology Co., Ltd.) for 5 min. Immunoreactivity was visualized by the fluorescence of the dye to which the secondary antibodies were conjugated. DAPI dye (cat. no. G1012; Wuhan Servicebio Technology Co., Ltd.) was added and the slices were incubated for 10 min at 25˚C in the absence of light. The slices were sealed with an anti-fluorescence quenching sealant (cat. no. G1401; Wuhan Servicebio Technology Co., Ltd.), and images were captured using a fluorescence microscope (Olympus Corporation) with an excitation wavelength of 465-495 nm for NLRP3 (green), 510-560 nm for IBA-1 (red) and 330-380 nm (blue) for DAPI staining. Three magnification fields (x400) in the slices were randomly selected. The NLRP3-positive area (%), Iba-1 positive area (%) and number of microglia with co-localized NLRP3 and IBA-1 expression were determined using ImageJ 6.0 software (National Institutes of Health).
Brain tissue samples (50 mg) were lysed by RIPA Lysis Buffer (Wuhan Servicebio Technology Co, Ltd.) and centrifuged at 12,000 x g for 15 min at 4˚C and the supernatant was collected. The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein samples (40 µg/lane) were separated via SDS-PAGE (12 or 8% separation gel) and then transferred to a PVDF membrane (Merck KGaA). The membranes were incubated overnight at 4˚C with the following primary antibodies: NSE (cat. no. ab53025; 1:1,000; Abcam), NF-κBp105/p50 (cat. no. ab32360; 1:1,000; Abcam), NFκBp105/p50 (phospho S337; cat. no. ab194729; 1:1,000; Abcam), NLRP3 (cat. no. wl03379; 1:1,000; Wanleibio Co., Ltd.), ASC (cat. no. wl02462; 1:500; Wanleibio Co., Ltd.), caspase-1 (cat. no. wl03325; 1:500; Wanleibio Co., Ltd.), IL-1β (cat. no. ab9787; 1:1,000; Abcam) and GSDMD (cat. no. ab219800; 1:1,000; Abcam) and GAPDH (cat. no. 5174, 1:1,000; Cell Signaling Technologies, Inc.). After washing with TBST (0.1% tween), the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (cat. no. sc-2004; 1:10,000; Santa Cruz Biotechnology, Inc.) for 1 h at 25˚C. Proteins were detected using the Tanon™ High-sig ECL Western Blotting Substrate (Guangzhou Yuwei Biotechnology Instrument Co., Ltd.). Image J 6.0 software was used to analyze the intensities of the bands.
All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, Inc.). The Shapiro-Wilk test was used to validate assumptions of normality. One-way ANOVA followed by a Tukey's multiple comparison test was employed to analyze differences among groups. The Kruskal-Wallis test was used to evaluate non-normally distributed data, with Dunn's test for intergroup comparisons. P<0.05 was considered to indicate a statistically significant differences.
The extraction yield of PPO was 0.2%. The PPO was obtained as a faint yellow, transparent liquid. The total ion chromatogram obtained from the GC-MS analysis of the PPO is shown in
The antioxidant activities of PPO in the cerebral cortex tissues of the CIRI model rats are shown in
To investigate the effects of PPO on CIRI, cerebral cell morphology was evaluated with H&E staining (
When NF-κBp105 is lysed, it forms NF-κBp50 and thereby serves pro-inflammatory functions (
To determine whether PPO is able to inhibit NLRP3 activation in the microglia, the expression and co-expression of NLRP3 and IBA-1 were detected using immunofluorescence double staining. The expression of NLRP3 and IBA-1, and the co-expression of NLRP3 + IBA-1 increased markedly in the microglia following CR/CPR (
To investigate the effects of PPO on the inflammatory response and pyroptosis-associated proteins in the CA/CPR model rats, the levels of NLRP3, ASC, caspase-1, IL-1β and GSDMD were analyzed using western blotting (
In the present study, PPO was obtained from fresh and mature Shatian pomelo peels. The main ingredient of PPO was found to be limonene (88.683%), followed by nootkatone (5.732%) and myrcene (1.027%). The results of the
The proportions of limonene and other components detected in the present study differ from those in other studies (
The generation of excessive ROS leads to cellular damage and triggers the activation of microglia and immune pathways (
NLRP3, a specific pattern recognition receptor, initiates cell death processes (pyroptosis) as soon as it is activated by factors induced by pathological damage. It promotes the pyroptosis-associated protein cascade and causes inflammation and pyroptosis (
The control of NLRP3 activity can be divided into two processes, namely priming and activation. Priming prepares NLRP3 for subsequent activation (
IL-1β is produced during CIRI; it induces cerebrovascular endothelial cells to express adhesion molecules, which mediate interaction between the endothelial cells and leukocytes, thereby promoting the infiltration of leukocytes into the brain tissues. Leukocyte infiltration activates microglial cells, which causes them to secrete inflammatory factors and aggravates brain injury. In addition, leukocytes adhere and accumulate in microvessels, causing blockages and reducing cerebral blood flow, which also aggravates brain injury. These pathological changes occur at a late stage after CIRI (
An increase in ROS levels can stimulate the phosphorylation of IκB by casein kinase II or tyrosine kinases, and thereby induce NF-κB gene transcription (
In conclusion, the present study indicated that the NLRP3 inflammasome, which is associated with pyroptosis, is involved in CA/CPR-induced CIRI. Furthermore, PPO treatment downregulated the expression of multiple factors in the NLRP3 inflammasome and ameliorated brain cell death. PPO, as a mixture, may inhibit the inflammation and pyroptosis caused by activation of the NLRP3 inflammasome through its antioxidant capacity. However, further research is required to identify the active components and the underlying mechanism.
Not applicable.
The present study was supported by the National Natural Science Foundation of China (grant nos. 81660312 and 81360286).
All data generated and/or analyzed during this study are included in this published article.
XSZ and LX contributed equally to this work, in terms of developing the concept and study design, acquiring, analyzing and interpreting the data, authenticate the raw data and drafting the manuscript. WYW, GYZ and XYT performed the experiments and analyzed the data. MHC designed and led the study. All authors read and approved the final manuscript.
The study was approved by the Animal Care and Use Committee of Guangxi Medical University (Nanning, China).
Not applicable.
The authors declare that they have no competing interests.
PPO constituents detected by GC-MS and identified by comparison with purified compound standards. (A) GC spectrum of PPO. The main components are indicated by arrows. (B) GC-MS spectrum of PPO. PPO, pomelo peel oil; GC, gas chromatography; MS, mass spectrometry.
PPO improves cell morphology, reduces ROS levels and attenuates the expression of NSE and NF-κB in a cardiopulmonary resuscitation rat model. (A) Hematoxylin and eosin staining of brain tissue from rats in the (A-a) sham, (A-b) NS, (A-c) Gly, (A-d) PPO-L, (A-e) PPO-M and (A-f) PPO-H groups. A large number of cerebral cortex cells with normal morphology were observed in the sham group (indicated by black arrows). However, numerous abnormal cerebral cortex cells exhibiting nuclear pyknosis, intense staining, vacuolation, swelling and necrosis (indicated by red triangles) were visible in the NS and Gly groups. Cell morphology was ameliorated in the PPO groups. Magnification, x400. (B) Antioxidant activity of PPO determined by an ROS assay. Representative western blots of NSE (C) and (D) quantified results. (E) Representative western blots of NF-κB and (F-I) quantified results. Band intensities were quantified using densitometric software. The band intensities of (F) NF-κBp105, (G) NF-κBp50 and (H) NF-κBp-p105 were normalized to that of GAPDH, and (I) the NF-κBp-p105/NF-κBp105 ratio was calculated. Data are presented as the mean ± SEM of two independent experiments (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. the sham group; #P<0.05 and ##P<0.01 vs. the NS group; &P<0.05 and &&P<0.01 vs. the Gly group; @@P<0.01 vs. the PPO-L group (n=5). PPO, pomelo peel oil; ROS, reactive oxygen species; NSE, neuroenolase; NS, physiological saline; Gly, glycerin; PPO-L, 10 mg/kg PPO; PPO-M, 20 mg/kg PPO; PPO-H, 40 mg/kg PPO; NF-κBp-p105, phosphorylated NF-κBp105.
PPO decreases the expression of NLRP3 in the microglia. (A) Immunofluorescence staining of rat brain slices. Green staining indicates NLRP3 protein expression, red staining indicates IBA-1 protein expression and DAPI staining indicates nuclei (blue). Magnification, x400 and x2,000. (B) Column charts showing the (B-a) NLRP3-positive area (%), (B-b) IBA-1-positive area (%) and (B-c) the amount of NLRP3 expression co-localized with IBA-1-positive microglia in each group quantified using image analysis software. All data are presented as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. the sham group; #P<0.05 and ##P<0.01 vs. the NS group; &&P<0.01 vs. the Gly group. PPO, pomelo peel oil; NLRP3, NLR family pyrin domain-containing 3; IBA-1, allograft inflammatory factor 1; Gly, glycerin; PPO-L, 10 mg/kg PPO; PPO-M, 20 mg/kg PPO; PPO-H, 40 mg/kg PPO.
Effect of PPO on NLRP3, ASC, caspase-1, IL-1β, and GSDMD in rat brain tissue. Representative western blots of (A) NLRP3, ASC, caspase-1, IL-1β and (B-a) GSDMD. The column charts shows the levels of (B-b) NLRP3, (B-c) ASC, (B-d) caspase-1, (B-e) caspase-1p20, (B-f) pro-IL-1β, (B-g) IL-1βp17, (B-h) GSDMD and (B-i) GSDMD-N in the various treatment groups. Band intensity was normalized to GAPDH and quantified using image analysis software. Data are presented as the mean ± SEM (n=5). *P<0.05, **P<0.01 and ***P<0.001 vs. the sham group; #P<0.05 and ##P<0.01 vs. the NS group; &&P<0.01 vs. the Gly group; @P<0.05 and @@P<0.01 vs. the PPO-L group; $P<0.05 vs. the PPO-M group. PPO, pomelo peel oil; NLRP3, NLR family pyrin domain-containing 3; ASC, apoptosis-associated speck-like protein containing a CARD; IL-1b, interleukin-1b; GSDMD, gasdermin D; GSDMD-N, GSDMD N-domain; Gly, glycerin; PPO-L, 10 mg/kg PPO; PPO-M, 20 mg/kg PPO; PPO-H, 40 mg/kg PPO.
Chemical components of pomelo peel oil.
No. | Compound | Molecular formula | Retention time (min) | Peak area (%) |
---|---|---|---|---|
1 | α-pinene | C10H16 | 5.309 | 0.127 |
2 | Myrcene | C10H16 | 6.677 | 1.027 |
3 | Limonene | C10H16 | 8.079 | 88.683 |
4 | γ-terpinene | C10H16 | 10.356 | 0.250 |
5 | Limonene oxide | C10H16O | 11.207 | 0.270 |
6 | α-terpineol | C10H18O | 14.223 | 0.149 |
7 | Carvone | C10H14O | 15.273 | 0.403 |
8 | Geranyl acetate | C12H20O2 | 15.837 | 0.234 |
9 | Carophyllene | C15H24 | 16.332 | 0.505 |
10 | α-caryophyllene | C15H24 | 21.530 | 0.193 |
11 | α-gurjunene | C15H24 | 22.393 | 0.351 |
12 | 8-cedren-13-ol | C15H24O | 23.939 | 0.183 |
13 | Nerolidol | C15H26O | 29.380 | 0.208 |
14 | Globulol | C15H26O | 31.674 | 0.184 |
15 | Nootkatone | C15H22O | 35.392 | 5.732 |
16 | Osthole | C15H16O3 | 41.095 | 0.196 |
17 | Eicosane | C20H42 | 43.278 | 0.197 |
- | Total | - | - | 98.89 |