INTRODUCTION
Aneurysmal subarachnoid hemorrhage (aSAH), often occurring in people between 40 and 60 years of age, is the third most common type of stroke and has a worse clinical outcome.[1] The Hunt-Hess scale and modified Fisher scale, as the two conventional severity indicators, are preferred in clinical work to evaluate the clinical outcome of aSAH patients.[2,3] Delayed cerebral ischemia (DCI), mainly attributed to cerebral vasospasm, has been identified as a major clinical syndrome after aneurysm rupture, frequently leading to neurological impairments and poor outcome in aSAH patients.[4,5] Early brain injury is an important pathophysiological process following aSAH and clearly involves neuroinflammation.[6] In recent years, some inflammatory biomarkers have been broadly noted with respect to predicting DCI occurrence and poor outcomes after aSAH.[7,8]
A20, also known as tumor necrosis factor α-inducible protein 3, possesses inflammation-inhibitory effects by depressing the activation of nuclear factor-kappa B (NF-κB)[9] and may act as an endogenous protective factor in some inflammatory diseases, including acute myocardial infarction, chronic hepatitis B, and rheumatoid arthritis.[10⇓-12] A20 expression was dramatically up-regulated in damaged brain tissues following experimental head trauma or intracerebral hemorrhage.[13,14] Exogenous supplementation with A20 could significantly inhibit neuroinflammation, reduce brain edema, decrease blood-brain barrier disruption, and improve neurological function in rats after hemorrhagic stroke or traumatic brain injury, indicating that A20 may have a neuroprotective effect on acute brain injury (ABI). A20 expression by peripheral blood mononuclear cells was substantially increased in patients with intracerebral hemorrhage.[13] This study was designed to assess the prognostic role of serum A20 in aSAH.
METHODS
Study design and participant selection
We conducted a prospective cohort study from December 2020 to December 2022 at the Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine (Hangzhou, China). The main subjects of the study were consecutively recruited patients with first-time non-traumatic subarachnoid hemorrhage. All patients were enrolled according to the following inclusion criteria: (1) patients were over 18 years old; (2) rupture of a single intracranial aneurysm was confirmed by computed tomography angiography (CTA) or digital subtraction angiography (DSA); (3) patients were hospitalized within 24 h of symptom onset; and (4) ruptured intracranial aneurysms were secured by surgical clipping or endovascular intervention within 48 h after admission.
We further excluded patients with (1) other neurological disorders, such as ischemic or hemorrhagic stroke, moyamoya disease, arteriovenous malformation, intracranial tumors, or severe head trauma; (2) some special aneurysm conditions, such as rebleeding or pseudoaneurysm; and (3) other specific diseases, such as severe infection, malignant tumors, uremia, immune deficiency syndrome, liver cirrhosis, chronic heart or lung diseases. Alternatively, controls were recruited from healthy volunteers who were free of chronic diseases and had normal results in conventional tests.
All procedures of this study were completed in accordance with the Declaration of Helsinki, and the protocol of the study was approved by the Institutional Review Committee of Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine (Opinion No: 058-01). Written informed consent to join the study was obtained from relatives of patients and controls themselves.
Data collection
Demographic data, medical history, vital signs, and imaging information were recorded. The Hunt-Hess scale and modified Fisher scale were evaluated by at least two clinicians. When the two scores differed, they were re-evaluated by a third senior clinician. The location, shape, and diameter of the aneurysms were observed using CTA or DSA. Head computed tomography scans were performed at days 1, 7, 14 and 30 after surgery to investigate acute hydrocephalus and DCI.[15] Via a telephone inquiry, poststroke 90-day follow-up was performed by an uninformed physician. Modified Rankin Scale (MRS) scores ≥3 were defined as a poor outcome.
Sample collection and immune analysis
The collection time of fresh blood samples from aSAH patients was 1.5 to 25.5 h after stroke (median 10.0 h; 25th-75th percentiles 6.5-14.2 h). Blood samples from controls were obtained when they were enrolled in the current study. The blood samples were centrifuged within 30 min and promptly stored in a -80 °C refrigerator for subsequent determination. Using a commercially available enzyme-linked immunosorbent kit (Shanghai Kexing Trading Co., Ltd., Shanghai, China), serum A20 levels were determined following the manufacturer’s instructions. Detection limits ranged from 22 to 800 pg/mL, and coefficients of intra-assay and inter-assay variations were 8% and 10%, respectively. The average values of the two measurements were employed for the final analysis. A batch of samples was melted every six months, and then quantifications were performed by the same technician who was unfamiliar with the clinical information.
Statistical analysis
SPSS 25.0 and MedCalc 9.6.4.0 were applied for statistical analysis, and graphics were made using GraphPad Prism version 9.0. The Kolmogorov-Smirnov test or Shapiro-Wilk test was selected to determine the normal distribution of quantitative data. Quantitative data are reported as the mean±standard deviation if normally distributed and as the median (25th-75th percentile) if non-normally distributed. Qualitative data were expressed in the form of number (percentage). Multiple-group comparisons were performed for nonnormally distributed quantitative data via the Kruskal-Wallis H test. Data were compared between two groups using the Chi-square test, Fisher’s exact test, t test or Mann-Whitney U test as appropriate. Using the Spearman correlation coefficient, bivariate correlations were assessed. Multiple linear regression models were established to determine whether serum A20 levels were correlated with Hunt-Hess scores or modified Fisher scores. To determine the factors independently associated with 90-day clinical outcomes and DCI, we established a binary logistic regression model that included variables with P<0.05 in univariate analysis. Receiver operating characteristic (ROC) curve analysis was performed to calculate the area under the curve. A P-value <0.05 was considered statistically significant.
RESULTS
Participant selection and patient characteristics
In this study, 148 aSAH patients were initially evaluated based on the inclusion criteria. We then excluded 36 patients for the reasons shown in supplementary Figure 1. Finally, 112 aSAH patients and 112 healthy controls were further analyzed. There were no significant differences in age, sex, smoking or alcohol consumption between patients and controls (all P>0.05; Table 1).
Table 1. Comparisons of demographic data and vascular risk factors between controls and patients with aneurysmal subarachnoid hemorrhage
| Parameters | Patients (n=112) | Control (n=112) | P-value |
|---|---|---|---|
| Age, years Sex (male/female) Current smoking Alcohol consumption | 50.2±9.2 46/66 33 (29.5) 26 (23.2) | 51.8±11.3 58/54 21 (18.8) 29 (25.9) | 0.249 0.108 0.061 0.641 |
Data are reported as mean±standard deviation or numbers (percentages). Intergroup comparisons of various variables were performed using the χ2 test or Fisher’s exact test for qualitative data and the Mann-Whitney U test for quantitative data.
Patients were admitted from 0.5 to 24.0 h after symptom onset (median 9.4 h, 25th-75th percentiles 5.6-13.9 h). Hunt-Hess scores and modified Fisher scores ranged from 1 to 5 (median 4, percentiles 25th-75th 3-4) and 1 to 4 (median 3, percentiles 25th-75th 3-4), respectively. MRS scores of 0, 1, 2, 3, 4, 5 and 6 were found in 9, 13, 44, 13, 16, 9 and 8 patients, respectively (median 2; 25th-75th percentiles 2-4).
A total of 90 intracranial aneurysms were located in the anterior circulation and 22 in the posterior circulation. There were 95 patients with cystic aneurysms and 17 patients with other types of aneurysms. There were 65 patients with aneurysms <10 mm in diameter and 47 patients with aneurysms ≥10 mm in diameter. The number of patients who chose surgical clipping and endovascular embolization to fix the aneurysms was 42 and 70, respectively. Twelve patients had acute hydrocephalus, 14 patients underwent external ventricular drainage, and 15 patients and 29 patients suffered from intraventricular hemorrhage or DCI, respectively. There were 23 hypertensive patients and 7 diabetic patients. A total of 46 patients had poor outcomes (MRS scores ≥3).
Changes of serum A20 levels
In the aSAH patients, serum A20 levels ranged from 39.0 to 204.2 pg/mL with a median of 123.7 pg/mL (25th-75th percentiles 108.0-161.9 pg/mL). Among the 112 controls, 25 controls had serum A20 levels <22 pg/mL (detection limits of assay). The median serum A20 level in the remaining 87 controls was 25.8 pg/mL (range 22.1-42.8 pg/mL, 25th-75th percentiles 23.5 -32.4 pg/mL). Using the Mann-Whitney U test, it was clear that serum A20 levels in aSAH patients were significantly higher than those in healthy controls (P<0.001; supplementary Figure 2).
In supplementary Table 1, close correlations were found between serum A20 levels and Hunt-Hess scores (P<0.001), modified Fisher scores (P<0.001), blood glucose levels (P=0.021), and intraventricular hemorrhage (P=0.002).
As shown in Table 2, serum A20 levels were independently correlated with Hunt-Hess scores and modified Fisher scores when the above significantly correlated variables were entered in a multiple linear regression model. In addition, the serum A20 levels in patients were significantly elevated with increasing Hunt-Hess scores or modified Fisher scores (P<0.001; supplementary Figure 3).
Table 2. Multivariate linear regression analysis between elevated serum A20 levels and other variables
| Variables | β (95% confidence interval) | t | P-value |
|---|---|---|---|
| Intraventricular hemorrhage Hunt-Hess scores Modified Fisher scores Serum glucose levels | 13.402 (-3.356-30.160) 9.854 (2.481-17.227) 10.349 (1.273-19.424) 1.082 (-0.373-2.537) | 1.585 2.649 2.261 1.474 | 0.116 0.009 0.026 0.143 |
Correlations are presented using a multivariate linear regression model. *P<0.05.
Correlation of serum A20 levels with 90-day poor outcome
In supplementary Table 2, patients with poor outcomes had a significantly increased percentage of intraventricular hemorrhage and showed significant elevations in blood glucose levels, serum A20 levels, Hunt-Hess scores, and modified Fisher scores compared to patients with good outcomes (all P<0.05). Using multivariate analysis, Hunt-Hess scores, modified Fisher scores, and serum A20 levels independently predicted the development of poor outcomes at 90 d after hemorrhagic stroke (all P<0.05; Table 3).
Table 3. Multivariate logistic regression analysis for risk factors for poor outcome in aneurysmal subarachnoid hemorrhage
| Variables | Odds ratio (95% confidence interval) | P-value |
|---|---|---|
| Hunt-Hess scores Modified Fisher scores Intraventricular hemorrhage Serum A20 levels Serum glucose levels | 2.907 (1.326-6.375) 3.640 (1.516-8.743) 0.755 (0.140-4.070) 1.015 (1.000-1.031) 1.040 (0.925-1.168) | 0.008* 0.004* 0.744 0.047* 0.512 |
The results are shown as odds ratios (95% confidence intervals) according to binary logistic regression analysis. *P<0.05.
As shown in Figure 1, using ROC curve analysis, serum A20 levels effectively distinguished patients at risk of poor outcomes (AUC 0.771, 95% CI 0.682-0.845). The best serum A20 concentration (128.15 pg/mL) was selected using the maximum Youden index, which predicted poor outcomes with medium-high sensitivity (73.9%) and specificity (74.2%) values. Furthermore, in Figure 2, its prognostic ability was similar to those of Hunt-Hess scores and modified Fisher scores (all P>0.05).
Figure 1.
Figure 1.
Predictive ability of serum A20 levels for poor outcome at 90 d after aneurysmal subarachnoid hemorrhage under receiver-operating characteristic curve.
Figure 2.
Figure 2.
Comparison of discriminatory capability with respect to serum A20 levels, Hunt-Hess scores and modified Fisher scores for 90-day poor outcome following aneurysmal subarachnoid hemorrhage under receiver operating characteristic curve.
Relationship between serum A20 levels and DCI
In supplementary Table 3, the proportion of intraventricular hemorrhage, serum A20 levels, blood glucose levels, Hunt-Hess scores, and modified Fisher scores were higher in patients with DCI than in patients without DCI (all P<0.05). In addition, as shown in Table 4, multivariate logistic regression analysis showed that Hunt-Hess scores, modified Fisher scores, and serum A20 levels were independently associated with DCI risk after aSAH (all P<0.05).
Table 4. Multivariate logistic regression analysis for risk factors for DCI in aneurysmal subarachnoid hemorrhage
| Variables | Odds ratio (95% confidence interval) | P-value |
|---|---|---|
| Hunt-Hess scores Modified Fisher scores Intraventricular hemorrhage Serum A20 levels Serum glucose levels | 2.818 (1.200-6.622) 2.799 (1.126-6.956) 0.546 (0.105-2.826) 1.018 (1.001-1.035) 1.042 (0.918-1.181) | 0.017* 0.027* 0.471 0.042* 0.527 |
The results are shown as odds ratios (95% confidence intervals) according to binary logistic regression analysis. *P<0.05.
As shown in Figure 3, serum A20 levels effectively differentiated the risk of DCI under the ROC curve (AUC 0.777, 95% CI 0.688-0.850). Using the Youden method, serum A20 concentrations higher than 160.55 pg/mL distinguished the development of DCI with a sensitivity of 65.5% and a specificity of 89.2%. As shown in Figure 4, its ability to discriminate the occurrence of DCI was equivalent to those of Hunt-Hess scores and modified Fisher scores (all P>0.05).
Figure 3.
Figure 3.
Predictive ability of serum A20 levels for delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage under receiver-operating characteristic curve.
Figure 4.
Figure 4.
Comparison of discriminatory capability with respect to serum A20 levels, Hunt-Hess scores and modified Fisher scores for delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage under receiver operating characteristic curve.
DISCUSSION
In this study, we confirmed that elevated serum A20 levels after aSAH were significantly correlated with Hunt-Hess scores and modified Fisher scores and were independently associated with DCI and poor outcomes. In addition, when compared with Hunt-Hess scores and modified Fisher scores, serum A20 levels showed a similar ability to predict DCI and poor outcomes at 90 days post-injury. Such results suggest that serum A20 may be a prognostic biomarker of aSAH.
The tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB signaling pathway is involved in the inflammatory process of ABI diseases, including SAH.[16,17] Mechanistically, A20 could inhibit the TRAF6/NF-κB signaling pathway to down-regulate the expression of some destructive proinflammatory factors, such as interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha, thereby reducing inflammatory responses.[18⇓-20] Specifically, using in vivo and in vitro models of SAH, A20 silencing could obviously increase TRAF6, NF-κB, and inflammatory cytokine levels, aggravate neuronal apoptosis, brain edema, and blood-brain barrier damage, and therefore worsen neurological impairments;[6,17,21] nonetheless, A20 overexpression could significantly reverse those effects. Another experimental study showed that supplementation with A20 could significantly reduce mouse neuroinflammation induced by intracerebral hemorrhage through the suppression of TRAF6 polyubiquitination.[13] Similarly, silencing A20 obviously increased neuronal death and neuroinflammation in rats with traumatic brain injury.[14] Thus, A20 may act as an endogenous anti-inflammatory factor and therefore exert a neuroprotective effect.
Under normal conditions, A20 is predominantly expressed in neurons of the human or animal brain.[13,22,23] After ABI, A20 expression is increased in astrocytes, microglia and neurons of animals with subarachnoid hemorrhage,[18] ischemic stroke,[24] traumatic brain injury[14] and intracerebral hemorrhage.[25,13] The mRNA expression of A20 was increased in the peripheral blood cells of patients with intracerebral hemorrhage.[13] Our study showed similar results: serum A20 levels were significantly higher in patients with aSAH than in healthy controls. In consideration of its neuroprotective effects, it is inferred that an elevation of A20 expression may be a compensatory reaction to ABI.
Compelling evidence has confirmed that A20 can be released from brain tissues after ABI.[14] Thus, A20 may leak into peripheral blood via a disrupted blood-brain barrier, thereby leading to an enhancement of A20 levels in the peripheral blood. Our study found a significant elevation in serum A20 levels in patients with aSAH compared to healthy controls. Therefore, it is deduced that A20 in serum from aSAH patients may be at least partially derived from injured brain tissues. However, the mRNA expression of A20 was increased in the peripheral blood cells of patients with intracerebral hemorrhage, implying that A20 could be directly derived from peripheral blood cells. A systemic inflammatory response after ABI could be induced,[26] and hence, an increase in A20 mRNA expression by peripheral blood cells may compensate for systemic injury after ABI. Thus, it is presumed that a portion of serum A20 among patients with aSAH in the current study may originate from peripheral blood cells.
It is unclear whether there is a strong association of serum A20 with a poor long-term outcome in patients with aSAH. However, A20 has anti-inflammatory, antioxidative and antiapoptotic effects, therefore displaying neuroprotective functions.[27] Herein, we speculate that serum A20 may be a predictor of poor outcomes 90 d after aSAH. In this study, poor results were defined as MRS scores ≥3. The final results showed that in addition to Hunt-Hess scores and modified Fisher scores, serum A20 levels were independently associated with poor outcome. In addition, serum A20 levels, Hunt-Hess scores and modified Fisher scores had similar prognostic abilities under the ROC curve. Our study provides further evidence to support the hypothesis that serum A20 may be a useful biomarker for assessing disease severity after aSAH.
As is known to all, Hunt-Hess scores and modified Fisher scores are commonly used clinically to evaluate the poor outcome of aSAH patients.[2,3] In this study, our results suggest that A20 levels are also a prognostic determinant of aSAH. In addition, serum A20 levels were independently associated with DCI, which occurs after aSAH and leads to a poor outcome in such patients. The results indicated that serum A20 levels were of certain value in predicting the outcome of aSAH compared with Hunt-Hess scores and modified Fisher scores. These results may strongly support the postulation that serum A20 may represent a promising prognostic biomarker for aSAH. However, the current study included only a medium sample of aSAH patients. Perhaps this is a preliminary study regarding the role of serum A20 as a prognostic biomarker of aSAH. In the future, a larger cohort study is needed to verify this conclusion.
CONCLUSIONS
Our study found that disease severity reflected by the Hunt-Hess scores and modified Fisher scores is closely related to serum A20 levels, which independently predict a poor outcome and DCI after aSAH. Therefore, we speculate that A20 may be a potential biomarker of outcome in aSAH.
Funding: This work is financially supported by grants from Key Research and Development Projects of Zhejiang Province (2020C03071) and the Construction Fund of Medical Key Disciplines of Hangzhou (OO20200485, OO20200055).
Ethical approval: This study was approved by the Institutional Review Committee of the Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine (058-01).
Conflicts of interest: The authors have no conflicts of interest.
Contributors: TY, ZYC, and SDZ contributed equally to this work. TY, ZYC, SDZ, XQD, and SYD conceived, designed the study, analyzed the data, and wrote the paper. ZFW, QD, WHY, WH, YKZ, and KYW contributed to data acquisition and analysis.
Reference
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Subarachnoid haemorrhage (SAH) is the third most common subtype of stroke. Incidence has decreased over past decades, possibly in part related to lifestyle changes such as smoking cessation and management of hypertension. Approximately a quarter of patients with SAH die before hospital admission; overall outcomes are improved in those admitted to hospital, but with elevated risk of long-term neuropsychiatric sequelae such as depression. The disease continues to have a major public health impact as the mean age of onset is in the mid-fifties, leading to many years of reduced quality of life. The clinical presentation varies, but severe, sudden onset of headache is the most common symptom, variably associated with meningismus, transient or prolonged unconsciousness, and focal neurological deficits including cranial nerve palsies and paresis. Diagnosis is made by CT scan of the head possibly followed by lumbar puncture. Aneurysms are commonly the underlying vascular cause of spontaneous SAH and are diagnosed by angiography. Emergent therapeutic interventions are focused on decreasing the risk of rebleeding (ie, preventing hypertension and correcting coagulopathies) and, most crucially, early aneurysm treatment using coil embolisation or clipping. Management of the disease is best delivered in specialised intensive care units and high-volume centres by a multidisciplinary team. Increasingly, early brain injury presenting as global cerebral oedema is recognised as a potential treatment target but, currently, disease management is largely focused on addressing secondary complications such as hydrocephalus, delayed cerebral ischaemia related to microvascular dysfunction and large vessel vasospasm, and medical complications such as stunned myocardium and hospital acquired infections.Copyright © 2022 Elsevier Ltd. All rights reserved.
Comparison of aneurysmal subarachnoid hemorrhage grading scores in patients with aneurysm clipping and coiling
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Past studies revealed the prognosis differed between aneurysmal subarachnoid hemorrhage (aSAH) patients with surgical clipping and endovascular coiling. We retrospectively reviewed aSAH patients in our institution to investigate the effectiveness of grading scores between two groups. In the surgical clipping group (n = 349), VASOGRADE had a favorable performance for predicting delayed cerebral ischemia (DCI) (area under curve (AUC) > 0.750), and had better results than clinical (World Federation of Neurosurgical Societies (WFNS), Hunt & Hess (HH) and radiological scores (modified Fisher Scale (mFS), Subarachnoid Hemorrhage Early Brain Edema Score) (P < 0.05). Clinical and combined scores (VASOGRADE, HAIR) had favorable performance for predicting poor outcome (AUC > 0.750), and had better results than radiological scores (P < 0.05). In the coiling group (n = 320), none of the grading scores demonstrated favorable predictive accuracy for DCI (AUC < 0.750). Only WFNS and VASOGRADE had AUC > 0.700, with better performance than mFS (P < 0.05). The clinical and combined scores showed favorable performance for predicting a poor outcome (AUC > 0.750), and were better than the radiological scores (P < 0.05). Radiological scores appeared inferior to the clinical and combined scores in clipping and coiling groups. VASOGRADE can be an effective grading score in patients with clipping or coiling for predicting DCI and poor outcome.
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Delayed cerebral ischemia (DCI) is common after subarachnoid hemorrhage (SAH) and represents a significant cause of poor functional outcome. DCI was mainly thought to be caused by cerebral vasospasm; however, recent clinical trials have been unable to confirm this hypothesis. Studies in humans and animal models have since supported the notion of a multifactorial pathophysiology of DCI. This review summarizes some of the main mechanisms under investigation including cerebral vascular dysregulation, microthrombosis, cortical spreading depolarizations, and neuroinflammation.Recent guidelines have differentiated between DCI and angiographic vasospasm and have highlighted roles of the microvasculature, coagulation and fibrinolytic systems, cortical spreading depressions, and the contribution of the immune system to DCI. Many therapeutic interventions are underway in both preclinical and clinical studies to target these novel mechanisms as well as studies connecting these mechanisms to one another. Clinical trials to date have been largely unsuccessful at preventing or treating DCI after SAH. The only successful pharmacologic intervention is the calcium channel antagonist, nimodipine. Recent studies have provided evidence that cerebral vasospasm is not the sole contributor to DCI and that additional mechanisms may play equal if not more important roles.
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Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a member of the TRAF family and an important multifunctional intracellular adaptin of the tumor necrosis factor superfamily and toll/IL-1 receptor (TIR) superfamily. TRAF6 has been studied in several central nervous system diseases, including ischemic stroke, traumatic brain injury, and neurodegenerative diseases, but its role in subarachnoid hemorrhage (SAH) has not been fully illustrated. This study was designed to explore changes of expression level and potential roles and mechanisms of TRAF6 in early brain injury (EBI) after SAH using a Sprague-Dawley rat model of SAH induced in 0.3 mL non-heparinized autologous arterial blood injected into the pre-chiasmatic cistern. First, compared with the sham group, we found that the expression levels of TRAF6 increased gradually and peaked at 24 h after SAH. Second, the results showed that application of TRAF6 over-expression plasmid and genetic silencing siRNA could increase or decrease expression of TRAF6, respectively, and severely exacerbate or relieve EBI after SAH, including neuronal death, brain edema, and blood-brain barrier injury. Meanwhile, the levels of autophagy and oxidative stress were reduced and increased separately. Finally, GFP-TRAF6-C70A, which is a TRAF6 mutant that lacks E3 ubiquitin ligase activity, was used to explore the mechanism of TRAF6 in SAH, and the results showed that EBI and oxidative stress were reduced, but the levels of autophagy were increased under this condition. Collectively, these results indicated that TRAF6 affected the degree of EBI after SAH by inhibiting autophagy and promoting oxidative stress.© 2017 International Society for Neurochemistry.
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Reducing excessive inflammation is beneficial for the recovery from intracerebral hemorrhage (ICH). Here, the roles and mechanisms of A20 (TNFAIP3), an important endogenous anti-inflammatory factor, are examined in ICH. A20 expression in the PBMCs of ICH patients and an ICH mouse model was detected, and the correlation between A20 expression and neurologic deficits was analyzed. A20 expression was increased in PBMCs and was negatively related to the modified Rankin Scale score. A20 expression was also increased in mouse perihematomal tissues. A20 and A20-overexpressing mice were generated to further analyze A20 function. Compared with wild-type (WT) mice, A20 and A20-overexpressing mice showed significant increases and decreases, respectively, in hematoma volume, neurologic deficit score, mortality, neuronal degeneration, and proinflammatory factors. Moreover, WT-A20 parabiosis was established to explore the role of A20 in peripheral blood in ICH injury. ICH-induced damage, including brain edema, neurologic deficit score, proinflammatory factors, and neuronal apoptosis, was reduced in A20 parabionts compared with A20 mice. Finally, the interactions between TRAF6 and Ubc13 and UbcH5c were increased in A20 mice compared with WT mice; the opposite occurred in A20-overexpressing mice. Enhanced IκBα degradation and NF-κB activation were observed in A20 mice, but the results were reversed in A20-overexpressing mice. These results suggested that A20 is involved in regulating ICH-induced inflammatory injury in both the central and peripheral system and that A20 reduces ICH-induced inflammation by regulating TRAF6 polyubiquitination. Targeting A20 may thus be a promising therapeutic strategy for ICH.Copyright © 2017 by The American Association of Immunologists, Inc.
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PMID:31607859
[Cited within: 4]
Programmed cell death is an important biological process that plays an indispensable role in traumatic brain injury (TBI). Inhibition of necroptosis, a type of programmed cell death, is pivotal in neuroprotection and in preventing associated inflammatory responses. Our results showed that necroptosis occurred in human brain tissues after TBI. Necroptosis was also induced by controlled cortical impact (CCI) injury in a rat model of TBI and was accompanied by high translocation of high-mobility group box-1 (HMGB1) to the cytoplasm. HMGB1 was then passed through the impaired cell membrane to upregulate the receptor for advanced glycation end-products (RAGE), nuclear factor (NF)-κB, and inflammatory factors such as interleukin-6 (IL-6), interleukin-1 (IL-1β), as well as NACHT, LRR and PYD domains-containing protein 3 (NLRP3). Necroptosis was alleviated by necrostatin-1 and melatonin but not Z-VAD (a caspase inhibitor), which is consistent with the characteristic of caspase-independent signaling. This study also demonstrated that tumor necrosis factor, alpha-induced protein 3 (TNFAIP3, also known as A20) was indispensable for regulating and controlling necroptosis and inflammation after CCI. We found that a lack of A20 in a CCI model led to aggressive necroptosis and attenuated the anti-necroptotic effects of necrostatin-1 and melatonin.Copyright © 2019 Bao, Fan, Zhao, Xu, Liu, Chao, Liu, You, Liu, Wang and Ji.
Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group
DOI:10.1161/STROKEAHA.110.589275
PMID:20798370
[Cited within: 1]
In clinical trials and observational studies there is considerable inconsistency in the use of definitions to describe delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage. A major cause for this inconsistency is the combining of radiographic evidence of vasospasm with clinical features of cerebral ischemia, although multiple factors may contribute to DCI. The second issue is the variability and overlap of terms used to describe each phenomenon. This makes comparisons among studies difficult.An international ad hoc panel of experts involved in subarachnoid hemorrhage research developed and proposed a definition of DCI to be used as an outcome measure in clinical trials and observational studies. We used a consensus-building approach.It is proposed that in observational studies and clinical trials aiming to investigate strategies to prevent DCI, the 2 main outcome measures should be: (1) cerebral infarction identified on CT or MRI or proven at autopsy, after exclusion of procedure-related infarctions; and (2) functional outcome. Secondary outcome measure should be clinical deterioration caused by DCI, after exclusion of other potential causes of clinical deterioration. Vasospasm on angiography or transcranial Doppler can also be used as an outcome measure to investigate proof of concept but should be interpreted in conjunction with DCI or functional outcome.The proposed measures reflect the most relevant morphological and clinical features of DCI without regard to pathogenesis to be used as an outcome measure in clinical trials and observational studies.
Roles of TRAF6 in central nervous system
DOI:10.2174/1570159X16666180412094655
PMID:29651950
[Cited within: 1]
Tumor necrosis factor receptor-associated factor (TRAF) is an important binding protein of tumor necrosis factor (TNF) superfamily and the toll/IL-1 receptor (TIR) superfamily, which play an important role in innate immunity and acquired immunity. TRAFs family have 7 members (TRAF1-7), and TRAF6 has its special facture and biological function. TRAF6 has two special domains: C-terminal domain and N-terminal domain, which could integrate with multiple kinases and regulate signaling pathway function as an E3 ubiquitin ligase. Studies have increasingly found that TRAF6 is closely related to central nervous system diseases, such as stroke, Traumatic brain injury, neurodegenerative diseases and neuropathic pain. Further research on the pathophysiological mechanism may be expected to become the new targets for the treatment of central nervous system diseases.Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
Tumor necrosis factor receptor-associated factor regulation of nuclear factor κB and mitogen-activated protein kinase pathways
DOI:10.3389/fimmu.2018.01849 URL [Cited within: 2]
A20 establishes negative feedback with TRAF6/NF-κB and attenuates early brain injury after experimental subarachnoid hemorrhage
DOI:10.3389/fimmu.2021.623256
URL
[Cited within: 2]
Nuclear factor (NF)-κB–ty -50mediated neuroinflammation plays a crucial role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). As an important negative feedback regulator of NF-κB, A20 is essential for inflammatory homeostasis. Herein, we tested the hypothesis that A20 attenuates EBI by establishing NF-κB–associated negative feedback after experimental SAH. In vivo and in vitro models of SAH were established. TPCA-1 and lentivirus were used for NF-κB inhibition and A20 silencing/overexpression, respectively. Cellular localization of A20 in the brain was determined via immunofluorescence. Western blotting and enzyme-linked immunosorbent assays were applied to observe the expression of members of the A20/tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB pathway and inflammatory cytokines (IL-6, IL-1β, TNF-α). Evans blue staining, TUNEL staining, Nissl staining, brain water content, and modified Garcia score were performed to evaluate the neuroprotective effect of A20. A20 expression by astrocytes, microglia, and neurons was increased at 24 h after SAH. A20 and inflammatory cytokine levels were decreased while TRAF6 expression was elevated after NF-κB inhibition. TRAF6, NF-κB, and inflammatory cytokine levels were increased after A20 silencing but suppressed with A20 overexpression. Also, Bcl-2, Bax, MMP-9, ZO-1 protein levels; Evans blue, TUNEL, and Nissl staining; brain water content; and modified Garcia score showed that A20 exerted a neuroprotective effect after SAH. A20 expression was regulated by NF-κB. In turn, increased A20 expression inhibited TRAF6 and NF-κB to reduce the subsequent inflammatory response. Our data also suggest that negative feedback regulation mechanism of the A20/TRAF6/NF-κB pathway and the neuroprotective role of A20 to attenuate EBI after SAH.
A20 suppresses vascular inflammation by recruiting proinflammatory signaling molecules to intracellular aggresomes
DOI:10.1096/fj.14-258533
PMID:25667218
[Cited within: 1]
A20 protects against pathologic vascular remodeling by inhibiting the inflammatory transcription factor NF-κB. A20's function has been attributed to ubiquitin editing of receptor-interacting protein 1 (RIP1) to influence activity/stability. The validity of this mechanism was tested using a murine model of transplant vasculopathy and human cells. Mouse C57BL/6 aortae transduced with adenoviruses containing A20 (or β-galactosidase as a control) were allografted into major histocompatibility complex-mismatched BALB/c mice. Primary endothelial cells, smooth muscle cells, or transformed epithelial cells (all human) were transfected with wild-type A20 or with catalytically inactive mutants as a control. NF-κB activity and intracellular localization of RIP1 was monitored by reporter gene assay, immunofluorescent staining, and Western blotting. Native and catalytically inactive versions of A20 had similar inhibitory effects on NF-κB activity (-70% vs. -76%; P > 0.05). A20 promoted localization of RIP1 to insoluble aggresomes in murine vascular allografts and in human cells (53% vs. 0%) without altering RIP1 expression, and this process was increased by the assembly of polyubiquitin chains (87% vs. 28%; P < 0.05). A20 captures polyubiquitinated signaling intermediaries in insoluble aggresomes, thus reducing their bioavailability for downstream NF-κB signaling. This novel mechanism contributes to protection from vasculopathy in transplanted organs treated with exogenous A20.© FASEB.
Dimerization and ubiquitin mediated recruitment of A20, a complex deubiquitinating enzyme
DOI:10.1016/j.immuni.2013.03.008
PMID:23602765
[Cited within: 1]
A20 is an anti-inflammatory protein linked to multiple human autoimmune diseases and lymphomas. A20 possesses a deubiquitinating motif and a zinc finger, ZF4, that binds ubiquitin and supports its E3 ubiquitin ligase activity. To understand how these activities mediate A20's physiological functions, we generated two lines of gene-targeted mice, abrogating either A20's deubiquitinating activity (Tnfaip3(OTU) mice) or A20's ZF4 (Tnfaip3(ZF4) mice). Both Tnfaip3(OTU) and Tnfaip3(ZF4) mice exhibited increased responses to TNF and sensitivity to colitis. A20's C103 deubiquitinating motif restricted both K48- and K63-linked ubiquitination of receptor interacting protein 1 (RIP1). A20's ZF4 was required for recruiting A20 to ubiquitinated RIP1. A20(OTU) proteins and A20(ZF4) proteins complemented each other to regulate RIP1 ubiquitination and NFκB signaling normally in compound mutant Tnfaip3(OTU/ZF4) cells. This complementation involved homodimerization of A20 proteins, and we have defined an extensive dimerization interface in A20. These studies reveal how A20 proteins collaborate to restrict TNF signaling.Copyright © 2013 Elsevier Inc. All rights reserved.
NF-κB and disease
DOI:10.3390/ijms21239181
URL
[Cited within: 1]
The role of NF-κB in all diseases characterized by an inflammatory process, from cancer to autoimmune diseases, is known, but—precisely because it is involved in many diseases—this transcriptional factor continues to attract scientific research and the new knowledge that emerges is fundamental in highlighting the therapeutic potential that this factor can have in the various diseases in which it is involved [...]
Down-regulation of A20 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus
DOI:10.1007/s10875-012-9764-2
PMID:22898832
[Cited within: 1]
A20 gene functions in negative immunoregulation and its SNP is related to SLE disease. But its expression level in immune cells from SLE patients is still unclear. The aim of this study is to investigate whether the expression of A20 is associated with pathogenesis of SLE.Real-time transcription-polymerase chain reaction analysis (RT-PCR) was used to determine expression of A20 mRNA in peripheral blood mononuclear cells (PBMC) from 37 patients with SLE and 32 healthy controls.A20 expression was decreased in SLE patients compared with healthy controls (p = 0.0133). The expression level of A20 gene negatively correlated with the SLE disease activity index (SLEDAI) (r =-0.4661, p = 0.0036) and erythrocyte sedimentation rate (ESR) (r =-0.5222, p = 0.0009). Furthermore, SLE patients with nephritis had a lower expression of A20 than those without nephritis (p = 0.0188).Our results suggest that the insufficient expression of A20 gene in PBMC may take part in the pathogenesis of SLE disease.
Comparative distribution of protein components of the A 20 ubiquitin-editing complex in normal human brain
DOI:10.1016/j.neulet.2012.05.043
PMID:22634524
[Cited within: 1]
Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration.Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
Upregulation of neuronal zinc finger protein A 20 expression is required for electroacupuncture to attenuate the cerebral inflammatory injury mediated by the nuclear factor-kB signaling pathway in cerebral ischemia/reperfusion rats
DOI:10.1186/s12974-016-0731-3 URL [Cited within: 1]
Melatonin suppresses microglial necroptosis by regulating deubiquitinating enzyme A 20 after intracerebral hemorrhage
DOI:10.3389/fimmu.2019.01360 URL [Cited within: 1]
Occurrence of early epilepsy in children with traumatic brain injury: a retrospective study
DOI:10.1007/s12519-021-00502-4
PMID:35150398
[Cited within: 1]
Early post-traumatic seizures (EPTS) refer to epileptic seizures occurring within one week after brain injury. This study aimed to define the risk factors of EPTS and the protective factors that could prevent its occurrence.This is a single-center retrospective study in the PICU, Beijing Children's Hospital. Patients diagnosed with traumatic brain injury (TBI), admitted with and without EPTS between January 2016 and December 2020 were included in the study.We included 108 patients diagnosed with TBI. The overall EPTS incidence was 33.98% (35/108). The correlation between EPTS and depressed fractures is positive (P = 0.023). Positive correlations between EPTS and intracranial hemorrhage and subarachnoid hemorrhage had been established (P = 0.011and P = 0.004, respectively). The detection rates of EPTS in the electroencephalogram (EEG) monitoring was 80.00%. There was a significant difference in the EEG monitoring rate between the two groups (P = 0.041). Forty-one (37.86%, 41/108) post-neurosurgical patients were treated with prophylactic antiepileptic drugs (AEDs), and eight (19.51%, 8/41) still had seizures. No statistical significance was noted between the two groups in terms of prophylactic AEDs use (P = 0.519). Logistic regression analysis revealed that open craniocerebral injury and fever on admission were risk factors for EPTS, whereas, surgical intervention and use of hypertonic saline were associated with not developing EPTS.Breakthrough EPTS occurred after severe TBI in 33.98% of pediatric cases in our cohort. This is a higher seizure incidence than that reported previously. Patients with fever on admission and open craniocerebral injuries are more likely to develop EPTS.© 2022. Children's Hospital, Zhejiang University School of Medicine.
A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics
DOI:10.1007/s00011-022-01570-6 [Cited within: 1]
