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NF-κB pathway
International Immunopharmacology 79 (2020) 106175
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Ghrelin alleviates traumatic brain injury-induced acute lung injury through pyroptosis/NF-κB pathway Xue-Fei Shaoa, ⁎ Di Qiangc,
⁎,1
T
, Bo Lib,1, Jun Shena, Qi-Fu Wanga, San-Song Chena, Xiao-Chun Jianga,
a
Department of Neurosurgery, Yi Ji Shan Hospital of Wannan Medical College, Wuhu, China Tianjin University of Traditional Chinese Medicine, Tianjin, China c Department of Dermatology and STD, Yi-Ji Shan Hospital of Wannan Medical College, Wuhu, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Traumatic brain injury Acute lung injury Pyroptosis Inflammation NF-κB pathway
Acute lung injury (ALI) is one of the severe complications in patients with traumatic brain injury (TBI), contributing to the high mortality. Ghrelin has protective effects against various inflammatory diseases, but the effects of Ghrelin on TBI-induced ALI and its mechanisms remain unknown. In this study, Ghrelin administration was performed on the mice with TBI, then histological change in cortex and lung tissues, lung vascular permeability and macrophage number in bronchoalveolar lavage fluid (BALF) were examined, respectively. Simultaneously, the alterations of proinflammatory factors and pyroptosis-related proteins in lung tissues were detected. As a result, TBI-induced ALI was ameliorated after Ghrelin treatment, which was demonstrated by improved histology, reduced lung vascular permeability, and peripheral macrophage number. Furthermore, Ghrelin decreased the mRNA levels of proinflammatory factors (IL-1β, IL-6, TNF-α and IL-18), the protein levels of pyroptosis-related proteins (NLRP3, Caspase1-P20, HMGB1 and Gasdermin D), and the phosphorylation levels of NF-κB in lung tissues. These results showed that Ghrelin attenuating TBI-induced ALI might be via ameliorating inflammasome-induced pyroptosis by blocking NF-κB signal, which are important for the prevention and treatment of TBI-induced ALI.
1. Introduction Traumatic brain injury (TBI) is a major public health concern and is a major cause of high mortality and morbidity worldwide [1]. People suffering a brain injury often have chronic health problems, such as physical, emotional and behavioral problems, which obviously lower life quality and increase care cost. What is worse, many extracranial complications frequently occur after TBI and cause poor clinical outcomes [2]. Acute lung injury (ALI), as the most common complication, is an independent predictor of poor outcomes in patients with TBI and strongly increases the mortality [3]. The incidence of ALI in patients with severe brain injury is between 5 and 30% [4]. ALI is considered to be a manifestation of a systemic inflammatory response caused by severe TBI [5]. Rogers et al. found a significantly increased lung weight and the presence of pulmonary edema, congestion and hemorrhage but not of other organs in 50% of patients who died immediately or within
96 h after severe head injury [6]. Therefore, seeking an effective therapy against TBI-induced ALI is very important to improve the poor clinical outcomes and lower the mortality of patients with TBI. Ghrelin, a 28-amino acid peptide mainly secreted in the stomach, is a neuroendocrine hormone and a novel gastrointestinal hormone. Ghrelin promotes appetite, regulates energy expenditure and metabolism, stimulates gastric acid secretion, and controls pancreatic endocrine function largely through its actions on the growth hormone secretagogue receptor (GHS-R). Previous studies have shown that Ghrelin had protective effects against neuronal injury, focal ischemia/reperfusion, stroke, Parkinson’s (PD) and Alzheimer’s (AD) diseases [7–10]. Ghrelin can cross blood-brain barrier to modulate systemic inflammation, and protect against neuronal injury by reducing apoptosis, alleviating inflammation and oxidative stress, and stimulating vagus nerve [11–13]. Additionally, Ghrelin is involved in the regulation of gastrointestinal motility, ameliorates intestinal dysfunction resulting from TBI
⁎ Corresponding authors at: Department of Neurosurgery, Yi Ji Shan Hospital of Wannan Medical College, No. 2 ZheShan West Road, Wuhu, Anhui 241000, China. (X.-F. Shao). Department of Dermatology and STD, Yi-Ji Shan Hospital of Wannan Medical College, No. 2 ZheShan West Road, Wuhu, Anhui 241000, China (D. Qiang). E-mail addresses: [email protected] (X.-F. Shao), [email protected] (D. Qiang). 1 Xue-Fei Shao and Bo Li contributed equally to this work and should be considered co-first authors.
https://doi.org/10.1016/j.intimp.2019.106175 Received 24 October 2019; Received in revised form 22 December 2019; Accepted 30 December 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
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0.1–0.2 = mild damage, 0.2–0.3 = moderate damage, 0.3–0.4 = severe damage. Thus, the range of scores was from 0 to 1.6, respectively.
by decreasing TNF-α-induced intestinal inflammation [14]. Ghrelin reduces intracerebral hemorrhage-induced intestinal barrier dysfunction by upregulating Zonula occludens-1 and claudin-5, and downregulating intestinal ICAM-1 [15]. Furthermore, Ghrelin also ameliorates ALI induced by various causes including sepsis, lipopolysaccharide, bleomycin, pancreatitis, and oleic acid [16–20]. However, the effects of Ghrelin on TBI-induced ALI and its mechanisms are not clear. In the present study, we investigated the effects of Ghrelin against TBI-induced ALI, and hypothesized its mechanism by the inflammasome-induced pyroptosis and NF-κB signal.
2.4. Detection of proinflammatory factor levels in lung tissues Total RNA was extracted from lung tissues using TRIzol reagents (Invitrogen). The obtained RNA was reverse transcribed into cDNA using Reverse Transcription Kit (TransGen, China). The mRNA expression levels of IL-1β, IL-6, TNF-α, and IL-18 were measured with quantitative real-time PCR (qPCR) using SYBR Green Master Mix (TransGen, China)) and ABI 7500 FAST. The primers were synthesized as follows: IL-6 (F primer: 5′-AGT TGC CTT CTT GGG ACT GA-3′; R primer: 5′-TCC ACG ATT TCC CAG AGA AC-3′), TNF-α (F primer: 5′CGT CAG CCG ATT TGC TAT CT-3′; R primer: 5′-CGG ACT CCG CAA AGT CTA AG-3′), IL-1β (F primer: 5′-CTA TGT CTT GCC CGT GGA G-3′; R primer: 5′-CAT CAT CCC ACG AGT CAC A-3′), IL-18 (F primer: 5′AAG GAC ACT TTC TTG CTT GCC-3′; R primer: 5′-AAA TCA TGC AGC CTC GGG TAT TCT G-3′), and GAPDH (F primer: 5′-GCC TCG TCT CAT AGA CAA GAT G-3′; R primer: 5′-CAG TAG ACT CCA CGA CAT AC-3′). Relative gene expression was calculated using the 2−ΔΔCt relative quantification method.
2. Materials and methods 2.1. Animal experiments Thirty adult male C57BL mice (10–12 weeks old and weight 20–25 g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China. SCXX (Jing) 2018-0005), and were randomly divided into Sham group, TBI group and Ghrelin group, ten mice each group. Before a cortical contusion impact (CCI), Sham and TBI groups were intraperitoneally injected with normal saline, and Ghrelin group was intraperitoneally administrated with Ghrelin (Phoenix Pharmaceuticals, USA), 10 μg dose each individual. 1 h later, after intraperitoneal injection of 50 g/L chloral hydrate solution (7 ml/kg), all mice were fixed in the stereotaxic brain locator. The scalp incision was performed along the midline of the skull to expose the right parietal bone. Then the cranial bone window with a diameter of 3.5 mm was made at the junction 5 mm behind the anterior fontanel and 2 mm beside the right of the midline, and the complete dura mater was exposed. Mice from TBI group and Ghrelin group were subjected to a strike on the dura mater at a 1.5 mm depth and 5 m/s rate for 120 ms by the electron cortical contusion impactor (eCCI 6.3, Custom Design and Fabrication, Richmond, VA, USA). The incisions in all mice were sutured. 1 h later, once again Sham and TBI groups were treated with normal saline, and Ghrelin group was administrated with Ghrelin at 10 μg dose each individual. 48 h later, the animals were abdominally anesthetized, abdominal aortic blood, bronchoalveolar lavage fluid (BALF) by bronchoalveolar lavage method, lung and cortex tissues were successively collected. Experimental procedures were approved by the Animal Ethics Committee of the Academy of Military Medical Sciences.
2.5. Flow cytometry analysis BALF was centrifuged at 3000 rpm for 10 min at 4 °C. Cell pellets were stained with CD11C PE-cy7 and F4/80-PE antibodies (Tianjin Sungene Biotech Co., Ltd.) for 40 min at 4 °C in the dark. After washing with PBS twice, detection was performed using a flow cytometer (Beckman CytoFLEX, USA). 2.6. Western blotting Total proteins from lung tissues were extracted using RIPA buffer containing a protease inhibitor cocktail. The protein concentrations were determined using BCA protein assay kits (Thermo Fisher Scientific, MA, USA). After denatured, 50ug total proteins were separated using 10% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% (w/v) fat-free milk for 1 h at room temperature and were incubated at 4 °C overnight with the primary antibodies: p-NF-κB (Cell Signaling Technology, MA, USA), GAPDH (Abcam, USA), NLRP3 (Abcam, USA), Caspase1-p20 (Santa Cruz Biochemicals, Santa Cruz, USA), HMGB1 (Abcam, USA), Gasdermin D (Cell Signaling Technology, MA, USA). HRP-conjugated secondary antibodies were used for further incubation with the membranes for 1 h at room temperature. After the addition of chemiluminescent HRP substrate (Millipore, MA, USA), the signals of the membranes were visualized using a chemiluminescence imager (Bio-Rad).
2.2. Albumin assay in BALF The albumin level in BALF was detected using ELISA kit (USCN Life Science Inc., Hubei, Wuhan, China). Briefly, BALF samples were incubated in 96-well plates coated with albumin capture antibody for 2 h at room temperature. After washing, biotin-conjugated antibody was added into the wells for another 2 h incubation, then HRP-conjugated streptavidin was added to catalyze the following TMB reagent. Finally, the catalytic reaction was stopped by the addition of sulfuric acid, and the OD values were measured at 450 nm using a microplate reader (ELx800NB, Biotek, Winooski, CT, USA).
2.7. Statistical analysis Statistical analyses were performed using one-way ANOVA followed by multiple comparisons performed with post hoc Bonferroni test to analyze significances between two groups using SPSS v.17.0 (SPSS Inc., Chicago, IL, USA), P values < 0.05 were considered significantly differential. All data are presented as mean ± standard deviation (SD).
2.3. Histological examination The cortex and lung tissues were fixed in 4% paraformaldehyde. The samples were embedded in paraffin, then were cut into 5 μm sections and stained with hematoxylin and eosin (H&E). Histology and pathology were observed in a blinded manner using a BX53 light microscope (Olympus Corporation, Japan) at 200x magnification. Lung injury was scored by a blinded observer according to the following four pathologic scoring items: edema, hemorrhage, erythrocyte leakage in the alveolar wall, and thickness of the alveolar wall. Each item was graded according to a four-point scale: 0–0.1 = minimal damage,
3. Results 3.1. TBI establishment and effect of Ghrelin against TBI As shown in Fig. 1, after a cortical contusion impact performance, the histological observation of brain cortices showed that in TBI group patchy hemorrhage, edema, varied cell morphology around the contusion lesion were clearly visible, and vacuole-like changes, neuronal degeneration and apoptosis were obviously increased, while the 2
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Fig. 1. Morphological observation of cortex tissues. HE staining of brain cortices from three groups was performed, and the morphologies were photographed at 200 times enlargement. The histological results show TBI caused cortex tissues impaired, while Ghrelin treatment improved damaged cortex tissues.
Ghrelin treatment could reduce the invasion of peripheral macrophages into lung after TBI.
morphological structure of nerve cells was normal in Sham group, suggesting TBI was successfully established. Furthermore, Ghrelin treatment obviously reduced neuronal degeneration, the extent of dark nuclear staining, the number of shrunken cells, the formation of intercellular gaps and lessened edema, suggesting that Ghrelin could alleviate TBI.
3.5. Ghrelin suppresses the expression of proinflammatory factors and pyroptosis-related proteins in the lung tissues In order to reveal the molecular mechanism of Ghrelin alleviating TBI-induced ALI, the levels of proinflammatory factors and pyroptosisrelated proteins in lung tissues were detected. As shown in Fig. 5 and Fig. 6, the mRNA levels of IL-1β, IL-6, TNF-α and IL-18, the proteins expression of LRP3, Caspase1-P20, HMGB1 and Gasdermin D, and the phosphorylation level of NF-κB in lung tissues were significantly increased after TBI, while those were obviously decreased after Ghrelin treating TBI.
3.2. Ghrelin ameliorates TBI-induced ALI As shown in Fig. 2, Sham group showed clear structure of alveolar wall and no secretions in respiratory bronchioles, while TBI group showed that there were telangiectasis in thickened alveolar wall, erythrocyte leakage in alveoli, obvious hyperplasia in alveolar epithelium, secretions in respiratory bronchioles. However, Ghrelin group showed thinner alveolar wall, less erythrocytes and secretions in respiratory bronchiolar compared to TBI group.
4. Discussion
3.3. Ghrelin reduces lung vascular permeability
TBI is at high risk for the development of various medical complications, in particular non-neurological organ failures including cardiovascular and respiratory dysfunctions are of major clinical importance in critical care medicine, cause high mortality of TBI patients [21]. ALI is an independent predictor of unfavorable outcomes in TBI patients. However, there have not been effective therapeutic drugs for TBI-induced ALI, in particular simultaneously effective for the treatments of TBI and ALI. In this study, we showed exogenous -Ghrelin administration could ameliorate TBI and TBI-induced ALI by improving the histopathology of brain cortices and lung tissues, reducing lung vascular permeability and the invasion of peripheral macrophages into lung. Increased pulmonary vascular permeability is a critical factor in the formation of pulmonary edema which is one of the hallmarks of ALI, and is associated with endothelial dysfunction [22,23]. Increased
In order to measure the change of lung vascular permeability after TBI and Ghrelin treating TBI, the albumin levels in BALF were detected. As a result, the albumin level in TBI group was significantly higher than that in Sham group. However, the albumin level was obviously decreased after Ghrelin treating TBI (Fig. 3). 3.4. Ghrelin reduces the invasion of peripheral macrophages As shown in Fig. 4, the number of CD11c-F4/80-positive cell in BALF was significantly increased in TBI group compared to Sham group. However, Ghrelin treatment further decreased the number of CD11c-F4/80-positive cell in BALF after TBI, which suggested that 3
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Fig. 2. Morphological observation of lung tissues. The HE results were photographed at 200 times enlargement. The arrowheads indicate that TBI group shows damaged histology with telangiectasis, erythrocyte leakage and secretions in lung tissues compared to Sham group, while Ghrelin group shows improved histology with thinner alveolar wall, less erythrocytes and secretions in respiratory bronchiolar compared to TBI group. The arrows indicate that the alveolar wall is normal in Sham group and is edematous in TBI group, while Ghrelin administration ameliorates edema.
hormones into endothelial cells, and modulate the capillary permeability [25,26]. Low-level urinary albumin excretion reflects the changes of vascular permeability in a variety of acute injuries. Additionally, we also showed Ghrelin reduced the number of macrophages in BALF. It was reported that alveolar macrophages played an important role during the development of ALI [24]. The primary role of alveolar macrophages is to protect the lung from inhaled substances. Beck-Schimmer et al. [27] reported that alveolar macrophages prevented neutrophil influx by controlling MCP-1 expression through alveolar epithelial cells, declaring that alveolar macrophages play an anti-inflammatory role. Therefore, decreased albumin level and macrophage number in BALF demonstrated the protective effect of Ghrelin against TBI-induced ALI. More importantly, Ghrelin treatment suppressed the expressions of proinflammatory factors IL-1β, IL-6, TNF-α and IL-18 mRNAs, pyroptosis-related proteins NLRP3, Caspase1-P20, HMGB1 and Gasdermin D, and the phosphorylation level of NF-κB in lung tissues. Previous studies have revealed that elevated IL-1β, IL-6, TNF-α and IL-18 promoted ALI [28–31]. IL-1β and IL-18 belong to IL-1 gene family which is a group of cytokines involved in a variety of acute and chronic lung diseases including ALI. Overexpression of IL-1 family members in the lungs induces local increases of the proinflammatory cytokines including IL-6 and TNF-α, resulting in severe lung injury [28], suggesting TBI might elevate IL-1β and IL-18 levels to induce the expressions of IL6 and TNF-α, which was one of pathogenesis of TBI-induced ALI. Furthermore, the phosphorylation level of NF-κB was decreased as well after Ghrelin administration. The phosphorylation of NF-κB can activate of NF-κB pathway, which further stimulates inflammation by inducing
Fig. 3. Albumin levels in BALF. The albumin levels in BALF from three groups were detected to evaluate lung vascular permeability. The albumin level in BALF is significantly increased in TBI compared to Sham, while that is obviously decreased in Ghrelin compared to TBI. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
pulmonary vascular permeability persists throughout the course of adult respiratory distress syndrome and is related to a clinical score of lung injury severity [24]. In this study, we showed the albumin level in BALF was obviously decreased after Ghrelin treating TBI. Albumin is an important carrier protein to transport fatty acids, ions, drugs, and
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Fig. 4. Flow cytometry analysis for the invasion of peripheral macrophages in lung. (A) Representative flow cytograms. (B) Histograms showing the percentage of macrophages in BALF from three groups. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
protective effect of Ghrelin against TBI-induced ALI via pyroptosis pathway. Interestingly, the current study showed pyroptosis-related proteins NLRP3, Caspase1-P20, HMGB1 and Gasdermin D were decreased when Ghrelin ameliorated TBI-induced ALI. Pyroptosis is a proinflammatory form of cell death, which is mediated by inflammasomes. The activation of NF-κB pathway is an inflammatory response by the production of proinflammatory factors. Both pyroptosis and NF-κB pathways participate in the pathogenesis of many inflammatory diseases. Inflammasomes are responsible of activating inflammation responses due to a diverse set of inflammationinducing stimuli through the production of proinflammatory factors [47–49]. In this study, we showed Ghrelin attenuated TBI-induced ALI and decreased IL-1β, IL-6, TNF-α and IL-18 mRNA levels, NF-κB phosphorylation level, and pyroptosis-related proteins NLRP3, Caspase1P20, HMGB1 and Gasdermin D in mouse lung tissues, suggesting Ghrelin might attenuate TBI-induced ALI via ameliorating inflammasome-induced pyroptosis by blocking NF-κB signal. In conclusion, it was found that Ghrelin ameliorated TBI-induced ALI, suggesting a new potential therapy for Ghrelin to prevent and treat ALI. Simultaneously, our data demonstrated the mechanism of Ghrelin attenuating TBI-induced ALI might be via ameliorating inflammasomeinduced pyroptosis by blocking NF-κB signal, modulating the pyroptosis/NF-κB pathway may be valuable in the treatment of TBI-induced ALI.
the expression of proinflammatory factors including IL-1β, IL-6, TNF-α and IL-18 [32–35]. Previous studies have shown the protective effect of Ghrelin against ALI is mediated through inhibition of NF-κB to reduce IL-1β, IL-6, TNF-α [16,36,37]. Although Ghrelin administration strikingly reduced IL-18 in mice with 2,4,6-trinitrobenzene sulfonic acidinduced colitis [38], this study firstly found Ghrelin attenuated TBIinduced ALI by down-regulating the production of IL-18, suggesting a novel anti-inflammatory action of Ghrelin in pulmonary tract. In this study, in addition to act an inflammatory mediator by downregulating the production of proinflammatory factors, Ghrelin also strongly reduced pyroptosis-related proteins NLRP3 (NOD-like receptor family, pyrin domain-containing-3), Caspase1-P20, HMGB1 and Gasdermin D. Pyroptosis is an inflammasome-mediated programmed cell death pathway. Previous studies have shown that NLRP3, Caspase1-P20 (Active Caspase1), HMGB1 (high-mobility group box 1) and Gasdermin D are the critical activators of pyroptotic cell death [39–42]. Some studies demonstrated that Ghrelin reduced TNF-α-induced human hepatocyte apoptosis, autophagy, and pyroptosis to prevent obesity-associated NAFLD [43], improved muscle function in dystrophin-deficient mdx mice by inhibiting NLRP3 inflammasome activation [44], protected the heart against ischemia/reperfusion injury via inhibition of NLRP3 and Caspase1 [45], exerted protective effects against oxidative stress and inflammation in a mouse model of myocardial ischemia/reperfusion injury via the HMGB1 and TLR4/NF-κB pathway [46]. However, there have not been reports about the 5
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Fig. 5. Proinflammatory factor mRNA levels in lung tissues. The expressions of proinflammatory factors IL-1β, IL-6, TNF-α and IL-18 mRNAs were measured by qPCR. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
Funding statement
CRediT authorship contribution statement
This study was funded by the priority of research funds of Wannan Medical College (WK2017ZF04), the new project of Yi-Ji Shan Hospital (Y1822), the teaching quality and teaching reform project of Wannan Medical College (2018jyxm58), the Collegiate Major Natural Science Research Projects (KJ2018ZD027, KJ2017A267) of Anhui Province, and Natural Science foundation of Anhui province (1908085QH362).
Xue-Fei Shao: Conceptualization, Methodology, Writing - original draft, Funding acquisition. Bo Li: Data curation. Jun Shen: Visualization, Investigation. Qi-Fu Wang: Formal analysis. San-Song Chen: Validation. Xiao-Chun Jiang: Project administration. Di Qiang: Writing - review & editing.
Fig. 6. Pyroptosis-related protein and NF-κB phosphorylation levels in lung tissues. The levels of pyroptosis-related proteins (NLRP3, Caspase1-P20, HMGB1 and Gasdermin D), as well as NF-κB phosphorylation in lung tissues were detected by western blot. (A) Representative western blot images of two samples from each group. (B) Histogram of Gray values. The gray levels of protein bands were read and showed in the form of histograms. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
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Declaration of Competing Interest
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No potential conflicts of interest were disclosed. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.intimp.2019.106175. References [1] C.R. Summers, B. Ivins, K.A. Schwab, Traumatic brain injury in the United States: an epidemiologic overview, Mt Sinai J. Med. 76 (2) (2009) 105–110. [2] C.D. Kemp, J.C. Johnson, W.P. Riordan, et al., How we die: the impact of nonneurologic organ dysfunction after severe traumatic brain injury, Am. Surg. 74 (9) (2008) 866–872. [3] L. Mascia, E. Zavala, K. Bosma, et al., High tidal volume is associated with the development of acute lung injury after severe brain injury: an international observational study, Crit. Care Med. 35 (8) (2007) 1815–1820. [4] A. Salim, M. Martin, C. Brown, et al., The presence of the adult respiratory distress syndrome does not worsen mortality or discharge disability in blunt trauma patients with severe traumatic brain injury, Injury. 39 (1) (2008) 30–35. [5] L.W. Eberhard, D.J. Morabito, M.A. Matthay, et al., Initial severity of metabolic acidosis predicts the development of acute lung injury in severely traumatized patients, Crit. Care Med. 28 (2000) 125–131. [6] F.B. Rogers, S.R. Shackford, G.T. Trevisani, et al., Neurogenic pulmonary edema in fatal and nonfatal head injuries, J. Trauma 39 (1995) 860–866. [7] L.M. Frago, E. Baquedano, J. Argente, et al., Neuroprotective actions of ghrelin and growth hormone secretagogues, Front. Mol. Neurosci. 4 (2011) 23. [8] N.E. Lopez, L. Gaston, K.R. Lopez, et al., Early ghrelin treatment attenuates disruption of the blood brain barrier and apoptosis after traumatic brain injury through a UCP-2 mechanism, Brain Res. 1489 (2012) 140–148. [9] L. Qi, X. Cui, W. Dong, et al., Ghrelin protects rats against traumatic brain injury and hemorrhagic shock through upregulation of UCP2, Ann. Surg. 260 (1) (2014) 169–178. [10] Z.B. Andrews, The extra-hypothalamic actions of ghrelin on neuronal function, Trends Neurosci. 34 (1) (2011) 31–40. [11] Q. Jiao, X. Du, Y. Li, et al., The neurological effects of ghrelin in brain diseases: Beyond metabolic functions, Neurosci. Biobehav. Rev. 73 (2017) 98–111. [12] N. Sato, S. Kanai, S. Takano, et al., Central administration of ghrelin stimulates pancreatic exocrine secretion via the vagus in conscious rats, Jpn. J. Physiol. 53 (6) (2003) 443–449. [13] Y. Date, N. Murakami, K. Toshinai, et al., The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats, Gastroenterology 123 (4) (2002) 1120–1128. [14] V. Bansal, S.Y. Ryu, C. Blow, et al., The hormone ghrelin prevents traumatic brain injury induced intestinal dysfunction, J. Neurotrauma 27 (12) (2010) 2255–2260. [15] Y. Cheng, Y. Wei, W. Yang, et al., Ghrelin attenuates intestinal barrier dysfunction following intracerebral hemorrhage in mice, Int. J. Mol. Sci. 17(12). pii (2016) E2032. [16] R. Wu, W. Dong, M. Zhou, et al., Ghrelin attenuates sepsis-induced acute lung injury and mortality in rats, Am. J. Respir. Crit. Care Med. 176 (8) (2007) 805–813. [17] J. Chen, X. Liu, Q. Shu, et al., Ghrelin attenuates lipopolysaccharide-induced acute lung injury through NO pathway, Med. Sci. Monit. 14 (7) (2008) BR141-146. [18] Y. Imazu, S. Yanagi, K. Miyoshi, et al., Ghrelin ameliorates bleomycin-induced acute lung injury by protecting alveolar epithelial cells and suppressing lung inflammation, Eur. J. Pharmacol. 672 (1–3) (2011) 153–158. [19] X. Zhou, C. Xue, Ghrelin attenuates acute pancreatitis-induced lung injury and inhibits substance P expression, Am. J. Med. Sci. 339 (1) (2010) 49–54. [20] X. Tian, Z. Liu, T. Yu, et al., Ghrelin ameliorates acute lung injury induced by oleic acid via inhibition of endoplasmic reticulum stress, Life Sci. 196 (2018) 1–8. [21] S. Ramtinfar, S.Y. Chabok, A.J. Chari, et al., Early detection of nonneurologic organ failure in patients with severe traumatic brain injury: Multiple organ dysfunction score or sequential organ failure assessment? Indian J. Crit. Care Med. 20 (10) (2016) 575–580. [22] M.A. Cercone, W. Schroeder, S. Schomberg, et al., EphA2 receptor mediates increased vascular permeability in lung injury due to viral infection and hypoxia, Am.
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Ghrelin alleviates traumatic brain injury-induced acute lung injury through pyroptosis/NF-κB pathway Xue-Fei Shaoa, ⁎ Di Qiangc,
⁎,1
T
, Bo Lib,1, Jun Shena, Qi-Fu Wanga, San-Song Chena, Xiao-Chun Jianga,
a
Department of Neurosurgery, Yi Ji Shan Hospital of Wannan Medical College, Wuhu, China Tianjin University of Traditional Chinese Medicine, Tianjin, China c Department of Dermatology and STD, Yi-Ji Shan Hospital of Wannan Medical College, Wuhu, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Traumatic brain injury Acute lung injury Pyroptosis Inflammation NF-κB pathway
Acute lung injury (ALI) is one of the severe complications in patients with traumatic brain injury (TBI), contributing to the high mortality. Ghrelin has protective effects against various inflammatory diseases, but the effects of Ghrelin on TBI-induced ALI and its mechanisms remain unknown. In this study, Ghrelin administration was performed on the mice with TBI, then histological change in cortex and lung tissues, lung vascular permeability and macrophage number in bronchoalveolar lavage fluid (BALF) were examined, respectively. Simultaneously, the alterations of proinflammatory factors and pyroptosis-related proteins in lung tissues were detected. As a result, TBI-induced ALI was ameliorated after Ghrelin treatment, which was demonstrated by improved histology, reduced lung vascular permeability, and peripheral macrophage number. Furthermore, Ghrelin decreased the mRNA levels of proinflammatory factors (IL-1β, IL-6, TNF-α and IL-18), the protein levels of pyroptosis-related proteins (NLRP3, Caspase1-P20, HMGB1 and Gasdermin D), and the phosphorylation levels of NF-κB in lung tissues. These results showed that Ghrelin attenuating TBI-induced ALI might be via ameliorating inflammasome-induced pyroptosis by blocking NF-κB signal, which are important for the prevention and treatment of TBI-induced ALI.
1. Introduction Traumatic brain injury (TBI) is a major public health concern and is a major cause of high mortality and morbidity worldwide [1]. People suffering a brain injury often have chronic health problems, such as physical, emotional and behavioral problems, which obviously lower life quality and increase care cost. What is worse, many extracranial complications frequently occur after TBI and cause poor clinical outcomes [2]. Acute lung injury (ALI), as the most common complication, is an independent predictor of poor outcomes in patients with TBI and strongly increases the mortality [3]. The incidence of ALI in patients with severe brain injury is between 5 and 30% [4]. ALI is considered to be a manifestation of a systemic inflammatory response caused by severe TBI [5]. Rogers et al. found a significantly increased lung weight and the presence of pulmonary edema, congestion and hemorrhage but not of other organs in 50% of patients who died immediately or within
96 h after severe head injury [6]. Therefore, seeking an effective therapy against TBI-induced ALI is very important to improve the poor clinical outcomes and lower the mortality of patients with TBI. Ghrelin, a 28-amino acid peptide mainly secreted in the stomach, is a neuroendocrine hormone and a novel gastrointestinal hormone. Ghrelin promotes appetite, regulates energy expenditure and metabolism, stimulates gastric acid secretion, and controls pancreatic endocrine function largely through its actions on the growth hormone secretagogue receptor (GHS-R). Previous studies have shown that Ghrelin had protective effects against neuronal injury, focal ischemia/reperfusion, stroke, Parkinson’s (PD) and Alzheimer’s (AD) diseases [7–10]. Ghrelin can cross blood-brain barrier to modulate systemic inflammation, and protect against neuronal injury by reducing apoptosis, alleviating inflammation and oxidative stress, and stimulating vagus nerve [11–13]. Additionally, Ghrelin is involved in the regulation of gastrointestinal motility, ameliorates intestinal dysfunction resulting from TBI
⁎ Corresponding authors at: Department of Neurosurgery, Yi Ji Shan Hospital of Wannan Medical College, No. 2 ZheShan West Road, Wuhu, Anhui 241000, China. (X.-F. Shao). Department of Dermatology and STD, Yi-Ji Shan Hospital of Wannan Medical College, No. 2 ZheShan West Road, Wuhu, Anhui 241000, China (D. Qiang). E-mail addresses: [email protected] (X.-F. Shao), [email protected] (D. Qiang). 1 Xue-Fei Shao and Bo Li contributed equally to this work and should be considered co-first authors.
https://doi.org/10.1016/j.intimp.2019.106175 Received 24 October 2019; Received in revised form 22 December 2019; Accepted 30 December 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
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0.1–0.2 = mild damage, 0.2–0.3 = moderate damage, 0.3–0.4 = severe damage. Thus, the range of scores was from 0 to 1.6, respectively.
by decreasing TNF-α-induced intestinal inflammation [14]. Ghrelin reduces intracerebral hemorrhage-induced intestinal barrier dysfunction by upregulating Zonula occludens-1 and claudin-5, and downregulating intestinal ICAM-1 [15]. Furthermore, Ghrelin also ameliorates ALI induced by various causes including sepsis, lipopolysaccharide, bleomycin, pancreatitis, and oleic acid [16–20]. However, the effects of Ghrelin on TBI-induced ALI and its mechanisms are not clear. In the present study, we investigated the effects of Ghrelin against TBI-induced ALI, and hypothesized its mechanism by the inflammasome-induced pyroptosis and NF-κB signal.
2.4. Detection of proinflammatory factor levels in lung tissues Total RNA was extracted from lung tissues using TRIzol reagents (Invitrogen). The obtained RNA was reverse transcribed into cDNA using Reverse Transcription Kit (TransGen, China). The mRNA expression levels of IL-1β, IL-6, TNF-α, and IL-18 were measured with quantitative real-time PCR (qPCR) using SYBR Green Master Mix (TransGen, China)) and ABI 7500 FAST. The primers were synthesized as follows: IL-6 (F primer: 5′-AGT TGC CTT CTT GGG ACT GA-3′; R primer: 5′-TCC ACG ATT TCC CAG AGA AC-3′), TNF-α (F primer: 5′CGT CAG CCG ATT TGC TAT CT-3′; R primer: 5′-CGG ACT CCG CAA AGT CTA AG-3′), IL-1β (F primer: 5′-CTA TGT CTT GCC CGT GGA G-3′; R primer: 5′-CAT CAT CCC ACG AGT CAC A-3′), IL-18 (F primer: 5′AAG GAC ACT TTC TTG CTT GCC-3′; R primer: 5′-AAA TCA TGC AGC CTC GGG TAT TCT G-3′), and GAPDH (F primer: 5′-GCC TCG TCT CAT AGA CAA GAT G-3′; R primer: 5′-CAG TAG ACT CCA CGA CAT AC-3′). Relative gene expression was calculated using the 2−ΔΔCt relative quantification method.
2. Materials and methods 2.1. Animal experiments Thirty adult male C57BL mice (10–12 weeks old and weight 20–25 g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China. SCXX (Jing) 2018-0005), and were randomly divided into Sham group, TBI group and Ghrelin group, ten mice each group. Before a cortical contusion impact (CCI), Sham and TBI groups were intraperitoneally injected with normal saline, and Ghrelin group was intraperitoneally administrated with Ghrelin (Phoenix Pharmaceuticals, USA), 10 μg dose each individual. 1 h later, after intraperitoneal injection of 50 g/L chloral hydrate solution (7 ml/kg), all mice were fixed in the stereotaxic brain locator. The scalp incision was performed along the midline of the skull to expose the right parietal bone. Then the cranial bone window with a diameter of 3.5 mm was made at the junction 5 mm behind the anterior fontanel and 2 mm beside the right of the midline, and the complete dura mater was exposed. Mice from TBI group and Ghrelin group were subjected to a strike on the dura mater at a 1.5 mm depth and 5 m/s rate for 120 ms by the electron cortical contusion impactor (eCCI 6.3, Custom Design and Fabrication, Richmond, VA, USA). The incisions in all mice were sutured. 1 h later, once again Sham and TBI groups were treated with normal saline, and Ghrelin group was administrated with Ghrelin at 10 μg dose each individual. 48 h later, the animals were abdominally anesthetized, abdominal aortic blood, bronchoalveolar lavage fluid (BALF) by bronchoalveolar lavage method, lung and cortex tissues were successively collected. Experimental procedures were approved by the Animal Ethics Committee of the Academy of Military Medical Sciences.
2.5. Flow cytometry analysis BALF was centrifuged at 3000 rpm for 10 min at 4 °C. Cell pellets were stained with CD11C PE-cy7 and F4/80-PE antibodies (Tianjin Sungene Biotech Co., Ltd.) for 40 min at 4 °C in the dark. After washing with PBS twice, detection was performed using a flow cytometer (Beckman CytoFLEX, USA). 2.6. Western blotting Total proteins from lung tissues were extracted using RIPA buffer containing a protease inhibitor cocktail. The protein concentrations were determined using BCA protein assay kits (Thermo Fisher Scientific, MA, USA). After denatured, 50ug total proteins were separated using 10% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% (w/v) fat-free milk for 1 h at room temperature and were incubated at 4 °C overnight with the primary antibodies: p-NF-κB (Cell Signaling Technology, MA, USA), GAPDH (Abcam, USA), NLRP3 (Abcam, USA), Caspase1-p20 (Santa Cruz Biochemicals, Santa Cruz, USA), HMGB1 (Abcam, USA), Gasdermin D (Cell Signaling Technology, MA, USA). HRP-conjugated secondary antibodies were used for further incubation with the membranes for 1 h at room temperature. After the addition of chemiluminescent HRP substrate (Millipore, MA, USA), the signals of the membranes were visualized using a chemiluminescence imager (Bio-Rad).
2.2. Albumin assay in BALF The albumin level in BALF was detected using ELISA kit (USCN Life Science Inc., Hubei, Wuhan, China). Briefly, BALF samples were incubated in 96-well plates coated with albumin capture antibody for 2 h at room temperature. After washing, biotin-conjugated antibody was added into the wells for another 2 h incubation, then HRP-conjugated streptavidin was added to catalyze the following TMB reagent. Finally, the catalytic reaction was stopped by the addition of sulfuric acid, and the OD values were measured at 450 nm using a microplate reader (ELx800NB, Biotek, Winooski, CT, USA).
2.7. Statistical analysis Statistical analyses were performed using one-way ANOVA followed by multiple comparisons performed with post hoc Bonferroni test to analyze significances between two groups using SPSS v.17.0 (SPSS Inc., Chicago, IL, USA), P values < 0.05 were considered significantly differential. All data are presented as mean ± standard deviation (SD).
2.3. Histological examination The cortex and lung tissues were fixed in 4% paraformaldehyde. The samples were embedded in paraffin, then were cut into 5 μm sections and stained with hematoxylin and eosin (H&E). Histology and pathology were observed in a blinded manner using a BX53 light microscope (Olympus Corporation, Japan) at 200x magnification. Lung injury was scored by a blinded observer according to the following four pathologic scoring items: edema, hemorrhage, erythrocyte leakage in the alveolar wall, and thickness of the alveolar wall. Each item was graded according to a four-point scale: 0–0.1 = minimal damage,
3. Results 3.1. TBI establishment and effect of Ghrelin against TBI As shown in Fig. 1, after a cortical contusion impact performance, the histological observation of brain cortices showed that in TBI group patchy hemorrhage, edema, varied cell morphology around the contusion lesion were clearly visible, and vacuole-like changes, neuronal degeneration and apoptosis were obviously increased, while the 2
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Fig. 1. Morphological observation of cortex tissues. HE staining of brain cortices from three groups was performed, and the morphologies were photographed at 200 times enlargement. The histological results show TBI caused cortex tissues impaired, while Ghrelin treatment improved damaged cortex tissues.
Ghrelin treatment could reduce the invasion of peripheral macrophages into lung after TBI.
morphological structure of nerve cells was normal in Sham group, suggesting TBI was successfully established. Furthermore, Ghrelin treatment obviously reduced neuronal degeneration, the extent of dark nuclear staining, the number of shrunken cells, the formation of intercellular gaps and lessened edema, suggesting that Ghrelin could alleviate TBI.
3.5. Ghrelin suppresses the expression of proinflammatory factors and pyroptosis-related proteins in the lung tissues In order to reveal the molecular mechanism of Ghrelin alleviating TBI-induced ALI, the levels of proinflammatory factors and pyroptosisrelated proteins in lung tissues were detected. As shown in Fig. 5 and Fig. 6, the mRNA levels of IL-1β, IL-6, TNF-α and IL-18, the proteins expression of LRP3, Caspase1-P20, HMGB1 and Gasdermin D, and the phosphorylation level of NF-κB in lung tissues were significantly increased after TBI, while those were obviously decreased after Ghrelin treating TBI.
3.2. Ghrelin ameliorates TBI-induced ALI As shown in Fig. 2, Sham group showed clear structure of alveolar wall and no secretions in respiratory bronchioles, while TBI group showed that there were telangiectasis in thickened alveolar wall, erythrocyte leakage in alveoli, obvious hyperplasia in alveolar epithelium, secretions in respiratory bronchioles. However, Ghrelin group showed thinner alveolar wall, less erythrocytes and secretions in respiratory bronchiolar compared to TBI group.
4. Discussion
3.3. Ghrelin reduces lung vascular permeability
TBI is at high risk for the development of various medical complications, in particular non-neurological organ failures including cardiovascular and respiratory dysfunctions are of major clinical importance in critical care medicine, cause high mortality of TBI patients [21]. ALI is an independent predictor of unfavorable outcomes in TBI patients. However, there have not been effective therapeutic drugs for TBI-induced ALI, in particular simultaneously effective for the treatments of TBI and ALI. In this study, we showed exogenous -Ghrelin administration could ameliorate TBI and TBI-induced ALI by improving the histopathology of brain cortices and lung tissues, reducing lung vascular permeability and the invasion of peripheral macrophages into lung. Increased pulmonary vascular permeability is a critical factor in the formation of pulmonary edema which is one of the hallmarks of ALI, and is associated with endothelial dysfunction [22,23]. Increased
In order to measure the change of lung vascular permeability after TBI and Ghrelin treating TBI, the albumin levels in BALF were detected. As a result, the albumin level in TBI group was significantly higher than that in Sham group. However, the albumin level was obviously decreased after Ghrelin treating TBI (Fig. 3). 3.4. Ghrelin reduces the invasion of peripheral macrophages As shown in Fig. 4, the number of CD11c-F4/80-positive cell in BALF was significantly increased in TBI group compared to Sham group. However, Ghrelin treatment further decreased the number of CD11c-F4/80-positive cell in BALF after TBI, which suggested that 3
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Fig. 2. Morphological observation of lung tissues. The HE results were photographed at 200 times enlargement. The arrowheads indicate that TBI group shows damaged histology with telangiectasis, erythrocyte leakage and secretions in lung tissues compared to Sham group, while Ghrelin group shows improved histology with thinner alveolar wall, less erythrocytes and secretions in respiratory bronchiolar compared to TBI group. The arrows indicate that the alveolar wall is normal in Sham group and is edematous in TBI group, while Ghrelin administration ameliorates edema.
hormones into endothelial cells, and modulate the capillary permeability [25,26]. Low-level urinary albumin excretion reflects the changes of vascular permeability in a variety of acute injuries. Additionally, we also showed Ghrelin reduced the number of macrophages in BALF. It was reported that alveolar macrophages played an important role during the development of ALI [24]. The primary role of alveolar macrophages is to protect the lung from inhaled substances. Beck-Schimmer et al. [27] reported that alveolar macrophages prevented neutrophil influx by controlling MCP-1 expression through alveolar epithelial cells, declaring that alveolar macrophages play an anti-inflammatory role. Therefore, decreased albumin level and macrophage number in BALF demonstrated the protective effect of Ghrelin against TBI-induced ALI. More importantly, Ghrelin treatment suppressed the expressions of proinflammatory factors IL-1β, IL-6, TNF-α and IL-18 mRNAs, pyroptosis-related proteins NLRP3, Caspase1-P20, HMGB1 and Gasdermin D, and the phosphorylation level of NF-κB in lung tissues. Previous studies have revealed that elevated IL-1β, IL-6, TNF-α and IL-18 promoted ALI [28–31]. IL-1β and IL-18 belong to IL-1 gene family which is a group of cytokines involved in a variety of acute and chronic lung diseases including ALI. Overexpression of IL-1 family members in the lungs induces local increases of the proinflammatory cytokines including IL-6 and TNF-α, resulting in severe lung injury [28], suggesting TBI might elevate IL-1β and IL-18 levels to induce the expressions of IL6 and TNF-α, which was one of pathogenesis of TBI-induced ALI. Furthermore, the phosphorylation level of NF-κB was decreased as well after Ghrelin administration. The phosphorylation of NF-κB can activate of NF-κB pathway, which further stimulates inflammation by inducing
Fig. 3. Albumin levels in BALF. The albumin levels in BALF from three groups were detected to evaluate lung vascular permeability. The albumin level in BALF is significantly increased in TBI compared to Sham, while that is obviously decreased in Ghrelin compared to TBI. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
pulmonary vascular permeability persists throughout the course of adult respiratory distress syndrome and is related to a clinical score of lung injury severity [24]. In this study, we showed the albumin level in BALF was obviously decreased after Ghrelin treating TBI. Albumin is an important carrier protein to transport fatty acids, ions, drugs, and
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Fig. 4. Flow cytometry analysis for the invasion of peripheral macrophages in lung. (A) Representative flow cytograms. (B) Histograms showing the percentage of macrophages in BALF from three groups. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
protective effect of Ghrelin against TBI-induced ALI via pyroptosis pathway. Interestingly, the current study showed pyroptosis-related proteins NLRP3, Caspase1-P20, HMGB1 and Gasdermin D were decreased when Ghrelin ameliorated TBI-induced ALI. Pyroptosis is a proinflammatory form of cell death, which is mediated by inflammasomes. The activation of NF-κB pathway is an inflammatory response by the production of proinflammatory factors. Both pyroptosis and NF-κB pathways participate in the pathogenesis of many inflammatory diseases. Inflammasomes are responsible of activating inflammation responses due to a diverse set of inflammationinducing stimuli through the production of proinflammatory factors [47–49]. In this study, we showed Ghrelin attenuated TBI-induced ALI and decreased IL-1β, IL-6, TNF-α and IL-18 mRNA levels, NF-κB phosphorylation level, and pyroptosis-related proteins NLRP3, Caspase1P20, HMGB1 and Gasdermin D in mouse lung tissues, suggesting Ghrelin might attenuate TBI-induced ALI via ameliorating inflammasome-induced pyroptosis by blocking NF-κB signal. In conclusion, it was found that Ghrelin ameliorated TBI-induced ALI, suggesting a new potential therapy for Ghrelin to prevent and treat ALI. Simultaneously, our data demonstrated the mechanism of Ghrelin attenuating TBI-induced ALI might be via ameliorating inflammasomeinduced pyroptosis by blocking NF-κB signal, modulating the pyroptosis/NF-κB pathway may be valuable in the treatment of TBI-induced ALI.
the expression of proinflammatory factors including IL-1β, IL-6, TNF-α and IL-18 [32–35]. Previous studies have shown the protective effect of Ghrelin against ALI is mediated through inhibition of NF-κB to reduce IL-1β, IL-6, TNF-α [16,36,37]. Although Ghrelin administration strikingly reduced IL-18 in mice with 2,4,6-trinitrobenzene sulfonic acidinduced colitis [38], this study firstly found Ghrelin attenuated TBIinduced ALI by down-regulating the production of IL-18, suggesting a novel anti-inflammatory action of Ghrelin in pulmonary tract. In this study, in addition to act an inflammatory mediator by downregulating the production of proinflammatory factors, Ghrelin also strongly reduced pyroptosis-related proteins NLRP3 (NOD-like receptor family, pyrin domain-containing-3), Caspase1-P20, HMGB1 and Gasdermin D. Pyroptosis is an inflammasome-mediated programmed cell death pathway. Previous studies have shown that NLRP3, Caspase1-P20 (Active Caspase1), HMGB1 (high-mobility group box 1) and Gasdermin D are the critical activators of pyroptotic cell death [39–42]. Some studies demonstrated that Ghrelin reduced TNF-α-induced human hepatocyte apoptosis, autophagy, and pyroptosis to prevent obesity-associated NAFLD [43], improved muscle function in dystrophin-deficient mdx mice by inhibiting NLRP3 inflammasome activation [44], protected the heart against ischemia/reperfusion injury via inhibition of NLRP3 and Caspase1 [45], exerted protective effects against oxidative stress and inflammation in a mouse model of myocardial ischemia/reperfusion injury via the HMGB1 and TLR4/NF-κB pathway [46]. However, there have not been reports about the 5
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Fig. 5. Proinflammatory factor mRNA levels in lung tissues. The expressions of proinflammatory factors IL-1β, IL-6, TNF-α and IL-18 mRNAs were measured by qPCR. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
Funding statement
CRediT authorship contribution statement
This study was funded by the priority of research funds of Wannan Medical College (WK2017ZF04), the new project of Yi-Ji Shan Hospital (Y1822), the teaching quality and teaching reform project of Wannan Medical College (2018jyxm58), the Collegiate Major Natural Science Research Projects (KJ2018ZD027, KJ2017A267) of Anhui Province, and Natural Science foundation of Anhui province (1908085QH362).
Xue-Fei Shao: Conceptualization, Methodology, Writing - original draft, Funding acquisition. Bo Li: Data curation. Jun Shen: Visualization, Investigation. Qi-Fu Wang: Formal analysis. San-Song Chen: Validation. Xiao-Chun Jiang: Project administration. Di Qiang: Writing - review & editing.
Fig. 6. Pyroptosis-related protein and NF-κB phosphorylation levels in lung tissues. The levels of pyroptosis-related proteins (NLRP3, Caspase1-P20, HMGB1 and Gasdermin D), as well as NF-κB phosphorylation in lung tissues were detected by western blot. (A) Representative western blot images of two samples from each group. (B) Histogram of Gray values. The gray levels of protein bands were read and showed in the form of histograms. Data are presented as mean ± SD. * p < 0.05 vs. Sham group. # p < 0.05 vs. TBI group. n = 10 in each group.
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International Immunopharmacology 79 (2020) 106175
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Declaration of Competing Interest
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