The Regulatory Effects Of Itaconate On Neuroinflammation And SVZ Neurogenesis In The Mouse Model Of Ischemic Stroke

BackgroundIschemic stroke is the most common acute cerebral vascular disease.Cause the strict "time window" of thrombolysis or thrombectomy,and the ischemia/reperfusion injury,treatment for the majority of patients suffering ischemic stroke is largely unsatisfying.Therefore,it's necessary to investigate the novel methods and the associated cellular and molecular mechanisms.The death of nerve cells resulted from ischemia and hypoxia rapidly activates microglia/macrophages and astrocytes,triggering the neuroinflammation.The reactive microglia/macrophages plastically switch to pro-inflammatory M1 subtype and anti-inflammatory M2 subtype.Similarly,the reactive astrocytes plastically switch to pro-inflammatory A1 and anti-inflammatory A2 subtypes.The phenotypes of microglia/macrophages and astrocytes determine the protective or detrimental effects of neuroinflammation on stroke outcomes.Neurogenesis within the subventricular zone(SVZ)is notably enhanced after ischemic stroke,which promotes the functional recovery.Besides the neural stem cell intrinsic mechanisms and the local neuronal regulation,the inflammatory microenvironment is one of the critical factors influencing post-stroke SVZ neurogenesis.For example,tumor necrosis factor-a(TNF-?)and interleukin(IL)-1? have been shown to inhibit SVZ neurogenesis after ischemia,while IL-10 can exert enhancing effects.Recently,endogenous itaconate has been recognized as an important immunoregulatory metabolite.4-octyl itaconate(4-OI),the novel exogenous synthesized derivative of itaconate,is cell-permeable,and can be hydrolysed to itaconate in cells.In vitro study showed that 4-OI could elevate the levels of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)in lipopolysaccharide(LPS)-stimulated reactive macrophages,and inhibit the expression of pro-inflammatory factors like IL-1?.In the LPS-induced sepsis mouse model,4-OI could prolong survival and decrease the levels of serum IL-1? and TNF-?.4-PI could also activate neuronal Nrf2 signaling pathway,and inhibit cell apoptosis caused by hydrogen peroxide.Additionally,it has been identified that 4-OI could significantly restrict viral replication within neurons in the mouse model of Zika virus infection.To date,none of studies has revealed the roles of 4-OI in ischemic stroke,and how 4-OI affects post-stroke neuroinflammation and SVZ neurogenesis remains unknown.ObjectivesWe sought to investigate whether itaconate could have protective role in ischemic brain injury,and to further study the regulatory effects of itaconate on post-stroke neuroinflammation and SVZ neurogenesis,by using 4-OI.Methods1 Using adult male C57BL/6 mice,we obtained right middle cerebral artery occlusion(MCAO)and sham mice by introducing monofilament nylon suture with silicone-coated tip.MCAO group and sham group respectively included 4-OI-treated and vehicle-treated subgroups.2 2,3,5-triphenyltetrazolium chloride(TTC)staining,TUNEL staining,brain water content measurement,and western blot were performed to respectively assess infarct volume,peri-infarct cell apoptosis,brain edema,and protein levels of Nrf2 and HO-1 expressed in right hemisphere on day 3 post-operation.Neurologic deficits of 4-OI-treated and vehicle-treated MCAO mice were tested on days 1,3,7,10 and 14 after surgery.3 To identify the effects of 4-OI on the polarization of peri-infarct microglia/macrophages and astrocytes three days after operation,immunofluorescence was performed to observe toxic microglia/macrophages(M1)labeled by ionized calcium-binding adapter molecule 1(Ibal)and CD16/CD32,protective microglia/macrophages(M2)labeled by Ibal and CD206,toxic astrocytes(Al)labeled by glial fibrillary acidic protein(GFAP)and complement 3(C3),and protective astrocytes(A2)labeled by GFAP and S100A10.Further,western blot was used to measure protein levels of inducible nitric oxide synthase(iNOS),IL-1?TNF-?,C1q,IL-1?,CD206,arginase 1(Arg1),IL-10,C3,S100A10,lipocalin-2(LCN2),IL-15,IL-33,orosomucoid 2(ORM2),brain derived neurotrophic factor(BDNF)and glial cell line derived neurotrophic factor(GDNF)expressed in right hemisphere three days after surgery,identifying the influences of 4-OI on the expression of above pro-and anti-inflammatory factors..4 To recognize the influences of 4-OI on the proliferation and migration of SVZ neural stem cells fourteen days after operation,immunofluorescence was performed to oberve proliferating cells labeled by Ki67,proliferating neural stem cells labeled by 5-bromo-2'-deoxyuridine(BrdU)and Nestin,and migrating neural stem cells labeled by BrdU and DCX.On the basis of findings from immunofluorescence,western blot was further conducted to detect the protein levels of Nestin and DCX derived from the right SVZ fourteen days after surgery.5 Statistical analysis:GraphPad Prism software was used for statistical analysis and graphing.Results are expressed as mean ± SD.Two-sided Student's t-test was used to measure the differences between two selected groups.P<0.05 was considered statistically significant.Results1 The effects of 4-OI on brain ischemia.(1)Among MCAO mice,those received 4-OI showed significantly reduced infarct volume compared with those received vehicle(P<0.05).(2)4-OI-treated MCAO mice showed the significantly decreased number of peri-infarct TUNEL positive cells compared with vehicle-treated MCAO mice(P<0.05).(3)4-OI didn't affect the brain water content of sham mice(P>0.05),but significantly decreased that of MCAO mice(P<0.05).(4)On day 1 post stroke,there was no significant change in the neurologic deficit scores between 4-OI-treated and vehicle-treated groups(P>0.05).However,on days 3,7,10 and 14,4-OI-treated group had significantly lower scores than did the vehicle-treated group(P<0.05).(5)The protein levels of Nrf2 and HO-1 didn't have significant differences between 4-OI-treated and vehicle-treated sham groups(P>0.05).However,the expression of the above two proteins was significantly elevated in 4-OI-treated MCAO group compared with that in vehicle-treated MCAO group(P<0.05).2 The effects of 4-OI on neuroinflammation after ischemic stroke.(1)Animals in 4-OI-treated group showed the significantly decreased number of peri-infarct Ibal and CD16/CD32 double positive cells compared with those in vehicle-treated group(P<0.05).(2)4-OI didn't significantly change the protein levels of iNOS,IL-1?,TNF-?,C1q and IL-1? expressed in sham mice(P>0.05).The protein levels of above five pro-inflammatory factors were significantly reduced in 4-OI-treated MCAO group compared with those in vehicle-treated MCAO group(P<0.05).(3)Mice injected with 4-OI had the significantly increased number of peri-infarct Ibal and CD206 double positive cells compared with those injected with vehicle(P<0.05).(4)The expression of CD206,Arg1 and IL-10 proteins didn't have significant changes between 4-OI-administrated and vehicle-administrated sham groups(P>0.05),but significantly increased in 4-OI-administrated MCAO group compared with that in vehicle-administrated MCAO group(P<0.05).(5)4-OI-treated MCAO mice showed significantly less peri-infarct GFAP and C3 double positive cells than did the vehicle-treated MCAO animals(P<0.05).(6)4-OI didn't significantly change C3 protein level in sham group(P>0.05),but significantly reduced that in MCAO group(P<0.05).(7)Compared with vehicle-delivered MCAO mice,4-OI-delivered MCAO mice had the significantly increased number of peri-infarct GFAP and S100A10 double positive cells(P<0.05).(8)4-OI didn't significantly change S100A10 protein level in sham group(P>0.05),but significantly elevated that in MCAO group(P<0.05).(9)The expression of LCN2,IL-15,IL?33,ORM2,BDNF and GDNF didn't have significant differences between 4-OI-administrated and vehicle-administrated sham groups(P>0.05).But among MCAO mice,4-OI-treated group displayed the significantly downregulated protein levels of LCN2 and IL-15,and the elevated expression of IL-33,ORM2,BDNF and GDNF compared with vehicle-treated group(P<0.05).3 The effects of 4-OI on SVZ neurogenesis after cerebral infarction.(1)4-OI-treated and vehicle-treated sham mice didn't have the significantly different number of Ki67 positive cells:in SVZ(P>0.05),but 4-OI-treated mice showed the significantly increased number of ipsilateral Ki67 positive cells in SVZ compared with vehicle-treated mice(P<0.05).(2)4-OI didn't significantly change the number of SVZ BrdU and Nestin double positive cells in sham group(P>0.05),but could significantly increase the number of ipsilateral SVZ BrdU and Nestin double positive cells in MCAO group(P<0.05).(3)4-OI didn't significantly affect SVZ-derived Nestin protein level in sham group(P>0.05),but significantly enhanced that in MCAO group(P<0.05).(4)The number of BrdU and DCX double positive cells in SVZ was not significantly different between 4-OI-treated and vehicle-treated sham groups(P>0.05).However,among MCAO mice,those injected with 4-OI showed the significantly increased number of ipsilateral SVZ BrdU and DCX double positive cells compared with those injected with vehicle(P<0.05).(5)The expression of SVZ-derived DCX in 4-OI-treated sham group didn't significantly differ with that in vehicle-treated sham group(P>0.05),however,that in 4-OI-delivered MCAO group was significantly elevated compared with that in vehicle-delivered MCAO group(P<0.05).Conclusions1 Itaconate protects against ischemic brain injury in mice,and this effect is probably related to the elevated expression of Nrf2 and HO-1.2 Itaconate alleviates toxic neuroinflammation post stroke.3 Itaconate promotes SVZ neurogenesis after brain ischemia.4 Itaconate may provide a therapeutic target for ischemic stroke.