| Intracerebral hemorrhage?ICH?,which results in high mortality and morbidity,accounts for 10–15%of all stroke cases;mortality is especially high in China,but effective therapies remain limited.Previous studies have indicated that inflammation plays a key role in ICH-induced secondary injury and that microglia activation,peripheral inflammatory cell infiltration,and the release of proinflammatory factors all participate in the pathogenesis of inflammation.Most previous studies have focused on the role of inflammatory factors.As a result,endogenous anti-inflammatory factors that can ameliorate ICH-induced inflammation have been overlooked.Thus,targeting endogenous anti-inflammatory factors could provide insights into the development of new ICH therapies.Zinc finger protein A20?TNFAIP3?is an endogenous antiinflammatory factor that can reduce the expression of destructive proinflammatory factors such as IL-1?and TNF-a by inhibiting NF-kB activation.A20 also interacts with IL-10 and TGF-?and reduces inflammation in diabetes and arthritis.Evidence has shown that A20 is essential for restricting TLR-induced TNF receptor–associated factor?TRAF6?ubiquitination.Our previous studies have shown that TLR4/NF-kB signaling plays an important role in ICH-induced inflammatory injury.Thus,we hypothesized that A20 may also ameliorate ICH-induced inflammation by negatively modulating TLR signaling.The present study aimed to investigate two hypotheses:1)A20 can ameliorate morphological?hematoma and brain edema?and behavioral outcomes after experimental ICH in mice;and 2)A20 can reduce NF-kB activation by regulating TRAF6 polyubiquitination.Part oneA20 expression in the PBMCs of ICH patients and the correlation with neurologic deficitsObjective:To explore the A20 expression in the PBMCs after ICH in patients,and clarify the correlation between A20 and the patients'neurologic deficits.Methods:A total of 49 patients with ICH were identified between December 2013 and June 2015.A total of 15 patients were excluded from the study,including 3 patients who died and 12 who were lost to follow-up.A total of 34 patients were enrolled in the study.Upon admission to our hospital,5 ml cubital vein blood was extracted for A20 detection using EDTA as an anticoagulant agent.PBMCs were isolated using a PBMC kit,according to the manufacturer's instructions,to determine A20 expression.Modified Rankin Scale?mRS?scores were used to evaluate neurologic deficits in the patients 3 mo after ICH.Correlation analysis was used to analyze the relationship between A20 mRNA expression and the patient mRS scores.All blood samples collected from the healthy controls and patients were used with their informed consent,and the study procedures were approved by the Ethics Committee of Xinqiao Hospital of the Third Military Medical University,China,and conducted in accordance with the Declaration of Helsinki and its amendments.Results:A20 expression in the ICH patients was significantly increased compared with that in the healthy controls.Correlation analysis indicated that A20 expression was negatively correlated with patient mRS scores 3 mo after ICH.Conclusions:A20 expression was increased in the PBMCs of ICH patients and negatively correlated with neurologic deficits.Part TwoA20 expression after ICH and its role in the hematoma absorption and the ICH-induced inflammation in mouseObjective:To investigate the A20 expression and the role in the hematoma absorption and the ICH-induced inflammation in mouse.Methods:Firstly,a ICH mouse model was established.The A20 expression at different time points after ICH were detected by qRT-PCR and Western-Blot.The expression of A20 in cells in perihematomal tissue was observed by immunofluorescence.The changes of Hematoma absorption,neurological deficits,inflammatory factors and neuronal apoptosis were observed by mRS score,ELSA,TUNEL in A20-/-mice.Results:A20 expression increased at 12 h after ICH,peaked at 1 d and then gradually declined.A20 expression increased at 12 h,peaked at 3 d,and then gradually declined.Seven days after ICH,A20 expression in the model was still higher than in the sham group,and A20expression was more prominent in microglia and neurons than in astrocytes.Compared with WT mice,the water content of the A20-/-mice was higher at 3 d after ICH.And the results showed that IL-?,IL-6,and TNF-a expression in the WT ICH mice was elevated compared with that in the WT sham group,and the expression levels in the A20-/-ICH mice were even higher than those in the WT ICH mice.The death rate was also significantly increased after ICH in the A20-/-mice.and compared with WT mice,the A20-/-mice exhibited an increase in TUNEL-positive cells in the perihematomal area.Conclusions:A20 expression increased after ICH in mice,especially in microglia and neurons,and A20 knockout increases inflammatory injury in perihematomal tissue after ICH.Part ThreeThe role of A20 in peripheral blood in the hematoma absorption and ICH-induced inflammation.Objective:To test the efficacy of peripheral A20 in alleviating ICH-induced injuryMethods:Firstly,the WT-A20-/-mouse Parabiosis model was constructed.The expression of A20 in PBMCs of A20-/-receptor mice was detected by immunofluorescence after 3 weeks in parallel.The changes of hematoma volume,brain water content,inflammatory factor expression and neuronal apoptosis in A20-/-receptor mice were observed on the third day after ICH.Results:Immunofluorescence staining revealed that the same amount of A20-positive cells in A20-/-parabiont PBMCs 3 d after ICH compared with WT mice and WT parabionts.However,A20 positive cells were not detected in the PBMCs of A20-/-mice 3 d after ICH.The hematoma volume,hemoglobin content,brain water content,inflammation factors expression,and neuronal apoptosis were all obviously decreased in the A20-/-parabiont group compared with the A20-/-group.However,the hematoma volume,brain water content,and IL-1??TNF-a levels in perihematomal tissue still were higher 3 d after ICH compared with the WT and WT parabiont groups.Conclusions:A20 in peripheral blood may also be involved in regulating ICH-induced inflammation?Part FourThe effects of A20 overexpression on Hematoma Absorption and Secondary Inflammatory Injury in Mice after ICHObjective:To evaluate the role of A20 in ICH-induced inflammatory injury in mice and to evaluate the therapeutic effect of A20 on ICH.Methods:The A20 lentiviral overexpression vector was constructed,and the expression of A20were detected by RT-PCR,Western-Blot and immunofluorescence.The changes of hematoma absorption,inflammatory factor expression,neurological function score,mortality and neuronal apoptosis in A20 overexpression group were observed by ELSA,mRS score and TUNEL staining.Results:A20 expression was higher 7 d after LV-A20 injection compared with the sham and vector groups.And A20 was mainly expressed in microglia and neurons,which is consistent with the cell types showing A20 expression after ICH.At 3 d after ICH,hematoma volume and hemoglobin content in the A20-overexpressing mice were significantly lower than those in the WT mice.The brain water content in the ICH ipsilateral hemisphere of the LV-A20mice was decreased at 3 d after ICH compared with the WT and LV-GFP groups.NDS and IL-b,IL-6,and TNF-a expression were lower in the A20-overexpressing mice than in the WT mice.TUNEL staining demonstrated that A20.overexpression prevented ICH-induced apoptosis 3 d after ICH.Compared with the WT and LV-GFP groups,the number of.TUNEL-positive cells was significantly decreased in the LV-A20 group.Conclusions:A20 overexpression can reduce post-ICH inflammatory injury in micePart FiveThe role of A20 in hematoma absorption and secondary inflammatory response after ICH by regulating the TRAF6-mediated polyubiquitinationObjective:To observe the role of E3 ubiquitin ligase TFAF6 in hematoma absorption and inflammatory response after ICH,and to clarify A20 regulating ICH-indced inflammatory response by inhibiting TFAF6-mediated polyubiquitination.Methods:We firsted constructed the TRAF6 shRNA.The mice were injected with TRAF6 shRNA in the lateral ventricle of the mice.The expression of NF-?Bp65 and inflammatory factors were detected by Western-blot and ELISA at 1d,3d and 7d after ICH in TRAF6 shRNA group.The changes of TRAR6-mediated ubiquitination in A20-/-group,WT group,A20overexpression group and empty vector group were observed by Western-blot,CO-IP,EMSA and ELSA.Results:We found that TRAF6 expression was obviously increased after ICH,and the expression trend was consistent with NF-kB p65 expression.Inhibiting the expression of TRAF6 using TRAF6-targeted shRNA relieved the cerebral edema and neurologic deficits,clearly downregulated NF-kB expression,and inhibited the subsequent secretion of inflammatory cytokines.TRAF6-Ubc13 and TRAF6-UbcH5c interactions were increased in A20-/-mice compared with WT mice.In contrast,these interactions were reduced in the A20overexpression group compared with the WT group.In A20-/-mice,TRAF6 did not interact with Itch,RNF11,or TAX1BP1.However,these interactions were increased in the A20-overexpressing mice In A20-/-mice,Ubc13 and UbcH5c expression was increased,whereas RNF11 and TAX1BP1 expression was decreased.In the LV-A20 group,Ubc13 and UbcH5c expression was decreased,whereas RNF11 and TAX1BP1 expression was increased.No change in Itch expression was observed in the A20-/-or LV-A20 groups.Moreover,we detected changes in the level of ubiquitinated TRAF6 at 3 d after ICH in the A20-positive,negative,and overexpression groups using Co-IP experiments.The results showed that inhibiting A20 promoted TRAF6 ubiquitination,whereas overexpressing A20 downregulated TRAF6 ubiquitination levels.Additionally,IkBa and NF-kB p65 degradation was increased in A20-/-mice,and an increase in NF-kB activation was also detected in A20-/-group;however,the IkBa degradation,NF-kBp65,and NF-kB activity were reversed in the LV-A20 group.Conclusions:A20 suppressed ICH-induced inflammatory injury by regulating the polyubiquitination of the E3 ubiquitin ligaseTRAF6. |
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