The Role And Mechanism Of NLRP3-mediated Neuroinflammatory Response In Traumatic Brain Injury

Part Ⅰ The effect of mitophagy on neuroinflammatory response and neuronal death after traumatic brain injuryObject:To study the occurrence and specific mechanism of mitophagy after traumatic brain injury(TBI),and to explore the relationship between mitophagy and neuroinflammatory response.Methold:The brain tissues of patients with or without TBI were applied to assess the autophagy and mitophagy via electron microscopy and western blotting.Controlled cortical impact,an in vivo TBI model,was done using WT and GFP-LC3 transgenic Sprague Dawley rats.The occurrence of mitophagy was detected by immunofluorescence,western blotting and electron microscopy.Autophagic Inhibitors of BAF and 3-MA were used to assess TBI-induced mitophagy.To assess mitophagy and release of Cyt C from damaged mitochondria,brain tissues were respectively abstracted 1,3,6 and 24 hours after trauma.We detected the distribution of cardiolipin on mitochondria under normal state or after TBI and used PLS3 or CLS to inhibit the TBI-induced redistribution of cardiolipin in order to assess the relation between cardiolipin and mitophagy.Mdivi-1 was used to inhibit TBI-induced mitophagy.Both TUNEL and western blotting were used to detect the cell death.Cortical lesion volume,bean balance,and morris water maze were to done to assess the post-traumatic neurological function recovery.Results:1,In comparison to sham,the result of electron microscopy showed a significant increase of damaged mitochondria engulfed by autophagic vacuolar in TBI tissues.2,western blotting showed autophagy marker protein LC3-II was significantly increased after TBI.Both p62 decreased and mitochondrial marker protein(COXIV,MnSOD,TOM40)markedly decreased.3,immunofluorescence suggested an increased co-localization of GFP-LC3 and mitochondrial;4,mitophagy was earlier than the release of cyt c from mitochondria.5,Cardiolipin was transferred from mitochondrial inner membrane to the outer membrane after TBI.The externalized cardiolipin interacted with LC3 to mediate TBI-induced mitophagy.6,inhibition of mitophagy significantly enhanced TBI-induced neuroinflammation and markedly worsen neurological function.Conclusion:1,Mitophagy was significantly increased after TBI.2,Mitophagy was earlier than the occurrence of cell death.3,Cardiolipin,as a role in the identification of damaged mitochondria,was involved in TBI-induced mitophagy;4,Mitophagy played a neuroprotective effect.The inhibition of mitophagy could enhance the neroinflammation and exacerbate nerve dysfunction.Part Ⅱ The key role of NLRP3 inflammasome after traumatic brain injuryObject:To study the occurrence of neuroinflammatory response after TBI,and to explore the important regulatory effect of NLRP3 and its relationship with prognosis.Methold:The brain tissues of epilepsy patients were used as the control group.The inflammation in brain tissues of TBI patients was detected by immunohistochemistry and western blotting.We assessed the function of NLRP3 knockdown in TBI-induced neuroinflammation.The NLRP3 knockout,caspase-1 knockout,IL-1βR knockout transgenic mice were provided by Jackson.The model of controlled cortical impact was established.The motor,learning,memory and cognition recovery were studied using bean balance and morris water maze.Results:1,immunohistochemistry showed that the expressions of NLRP3,IL-1βand IL-18 markedly increased after TBI,suggesting a significant increase in neuroinflammatory response.2,Compared to sham,western blotting results showed that the levels of cleaved-caspase-1(P20)and secretory IL-1β were significantly increased after TBI;3,The expressions of caspase-1(P20)and secretory IL-1β were significantly decreased after NLRP3 knockdown.4,In comparison to WT mice,NLRP3-/-,caspase-1-/-,IL-1βR-/-transgenic mice performed better in both bean balance and mirros water maze.Conclusion:1,After TBI,the expression of NLRP3 was increased.Neuroinflammatory response was significantly enhanced.2,TBI-induced neuroinflammation was significantly reduced by NLRP3 knockdown.3,NLRP3 knockout,or caspase-1 knockout,or IL-1βR knockout were beneficial to recover neurological function after TBI.Part Ⅲ Omega-3 fatty acids regulate NLRP3 inflammasome activation and prevent behavior deficits after traumatic brain injuryObject:To investigate how omega-3 inhibits neuroinflammation and prevents neurological deficits induced by TBI.Methold:On Day 1 of pregnancy,mothers were randomly allocated to receive either standard(EPA 20:5 n-3<0.02%and DHA 22:6 n-3<0.02%of total free fatty acids),or high ω-3 FAs diet(EPA 5.3%and DHA 23.8%of total free fatty acids)The ω-3(DHA,22:6,#D2534),ω-6(oleic acid,18:2,#62160),and ω-9(linolenic acid,18:1,#01008)for the gavages(100 mg/kg,twice a week for 6 weeks before surgery)were supplied by sigma(ALDRICH,USA).The anti-inflammatory effects of ω-3 Fas were inhibited by GPR40 receptor antagonist GW1100 or siRNA GPR40,which was assessed by ELISA and western blotting.The combination of ARRB2 and NLRP3 was detected by immunoprecipitation Western blotting for cleaved-caspase-3 and TNUNEL staining were done to assess cell death.Brain water content and cortical lesion volume were performed.Beam balance and morris water maze were done to assess behavior deficits TBI-induced TBI.Results:ω-3 FAs had the potential to eliminate the caspase-1 cleavage and IL-1βsecretion induced by TBI but that ω-6Fas and ω-9Fas did not.ARRB2 abrogated TBI-induced inflammation activation via directly binding with NLRP3 in theω-3Fas-mediated pathway.ω-3Fas prevented NLRP3 mitochondrial localization,which is required for optimal NLRP3 inflammasome activity.The treatment withω-3Fas reduced the number of TUNEL positive cells in comparison to rats treated with vehicle.To confirm these results,we employed western blot to evaluate the level of cleaved caspase-3,commonly used to detect neuronal death regardless of the specific cell death pathway.Consistent with the data of TUNEL,individuals treated with ω-3 FAs showed less TBI-induced cell death than those treated with vehicle.After TBI,ω-3 FA-treated rats showed much better beam balance test results than rats treated with vehicle.The results of morris water maze showed that both TBI+ω-3Fas and control groups performed progressively better than the TBI + vehicle group during the hidden platform test.GW1100 reduced the inhibitory effect ofω-3Fas on TBI-induced cell death and behavior deficits,suggesting that the protective effect of ω-3Fas on TBI is partially dependent on inflammatory inhibition.Conclusion:1,ω-3Fas inhibited neuroinflammatory response and prevented neurological dysfunction caused by TBI.2,ω-3Fas could promote ARRB2 binding to NLRP3 through GPR40 and inhibite NLRP3 binding to damaged mitochondria.3,ω-3Fas had the potential to reduce cortical lesion volume and brain edema,and promote behavior function recovery via GPR40/ARRB2/NLRP3 signaling pathway.