| Inflammation is critical for focal cerebral ischemia/reperfusion and can aggravate ischemic and anoxic injury.Increasing over-actived microglia secrete pro-inflammatory cytokines TNF-? and IL-1? in the peri-ischemic areas during the acute period,which inducing neuronal apoptosis and worsening patient prognosis.Therefore,reducing inflammatory damage of the peri-ischemic areas during acute stage of ischemic stroke is critical.The nuclear factor-kappa B(NF-?B)signaling pathway is critical for regulation of inflammation after ischemic stroke.Deubiquitinases(DUBs)negatively regulates the NF-?B signaling pathway.Cylindromatosis(CYLD)is one of subclasses of the deubiquitinase(DUB)family.The function of CYLD is related to deubiquitinating K63-linked chains to suppress negative regulation of NF-?B over-activation,reducing the inflammatory injury.Thus,CYLD may be a potential endogenous regulator against inflammatory injury after the ischemic stroke.However,CYLD studies are limited that explore temporal and spatial expression and describe the effects on inflammatory injuryElectroacupuncture(EA),a non-drug treatment,has been often studiedduring ischemic stroke in rats.in China.EA is also used as an adjuvant therapy for treating ischemic stroke and inflammatory diseases in China.Some studies have reported that EA could reduce the inflammatory injury via suppressing NF-?B signaling pathway.Thus,EA may inhibit NF-?B signaling pathway via regulating CYLD.However,this is associated with CYLD expression is not clear.Therefore,we explored temporal and spatial expression of CYLD,a negative feedback inhibitor of the NF-?B signaling pathway,to learn whether CYLD is essential for EA and reduction of inflammatory injury after focal cerebral ischemia/reperfusion.Objective:1.To explore temporal and spatial expression of CYLD and the effect of EA on CYLD in the peri-ischemic areas after the ischemic stroke.2.To investigate the neuroprotective and anti-inflammation effects of CYLD in the peri-ischemic areas after the ischemic stroke.3.To explore whether CYLD is reqiured for the effect of EA on anti-inflammatory injury via inhibiting the NF-?B signaling pathway in the peri-ischemic areas after focal cerebral ischemia/reperfusion.Methods:1.SD Rats were randomly divided into three groups:sham,MCAO/R and MCAO/R+EA.The level of CYLD mRNA and protein were tested by RT-qPCR and western blot respectively after 6,12,24,48 and 72 h reperfusion.The main cell type expressing CYLD and the spatial expression in the peri-ischemia areas after the ischemic stroke using double-immunofluorescence labeling staining.2.SD Rats were randomly divided into four groups:MCAO/R,MCAO/R+LV-shCYLD,MCAO/R+LV-CYLD,MCAO/R+LV-control.We used neurological scores and TTC staining to explore the neuroprotective effect of CYLD and used ELISA to test the levels of TNF-? and IL-1? at the indicated time.3.Rats were randomly divided into five groups:sham,MCAO/R,MCAO/R+EA,MCAO/R+LV-shCYLD+EA,MCAO/R+ LV-control+EA.Neurological scores and TTC staining was used to explore that CYLD is required for the neuroprotective effect of EA.The level of Bcl2ala mRNA which is the marker of microglia activation was detected by RT-qPCR.The morphology of microglia and neuronal chemokine CX3 CL 1 expression were investigated by using immunofluorescence labeling staining after 24 h reperfusion.The levels of TNF-? and IL-1? in the border region of the ischemic cortex were tested after 24 h reperfusion using ELISA.Western blot was used to test the ratio of p-I?B?/I?B? and nuclear translocation ratio of NF-?B p65.Double-immunofluorescent staining was used to observe co-expression between CYLD and NF-?B p65.Results:1.CYLD mRNA began to fall in after 6 h reperfusion in the MCAO/R group.CYLD mRNA was lowest after 24 h reperfusion in the MCAO/R group and this increased at 48 h and peaked after 72 h reperfusion.CYLD mRNA increased from 12 to 72 h in the MCAO/R+EA group compared with the MCAO/R group(each time point P<0.05).Western blot showed that CYLD protein was expressed in sham group.CYLD protein expression was lowest after 24 h reperfusion and increased after 48 h reperfusion in the MCAO/R group.CYLD protein peaked after 72 h reperfusion in the MCAO/R group compared with the sham group(each time point P<0.05).CYLD protein significantly increased from 24 to 72 h after reperfusion in the MCAO/R+EA group compared with the MCAO/R group(each time point P<0.05).CYLD protein was mainly expressed in cortical neurons of peri-ischemic areas after ischemic stroke,but there was little co-expression with Iba1 or GFAP2.After 72 h reperfusion,neurological function for the MCAO/R+LV-CYLD group was improved compared with the MCAO/R group(P<0.05).However,compared with the MCAO/R group,rat neurological function was significantly reduced in the MCAO/R+LV-shCYLD group(P<0.05).The results of TTC staining was consistent with Neurological function.ELISA showed that TNF-a and IL-1? in the border region of ischemic areas of rats after MCAO/R+LV-CYLD were significantly decreased compared with MCAO/R 72 h after reperfusion(P<0.05).TNF-? and IL-1? with MCAO/R+LV-shCYLD were increased compared with MCAO/R(P<0.05).3.Poor neurologic function and a larger infarct volume was observed with MCAO/R+LV-shCYLD+EA compared to MCAO/R+EA.Bcl2ala mRNA was significantly higher in the MCAO/R+LV-shCYLD+EA group compared with MCAO/R+EA group.Immunofluorescent staining showed that there was less neuronal CX3CL1 positive cells in the MCAO/R+EA group than in the MCAO/R group(P<0.05).Compared with MCAO/R+EA treatment,more neuronal CX3 CL 1 positive cells were found in the peri-ischemic cortices of the MCAO/R+LV-shCYLD+EA group(P<0.05).There were fewer activated microglia in the MCAO/R+EA group than in the MCAO/R group.However,compared with the MCAO/R+EA group,there were more activated microglia cells with larger cell bodies and retracted,thickened protuberances in the ischemic border region in the MCAO/R+LV-shCYLD+EA group.ELISA showed that the levels of TNF-? and IL-1? were increased in the MCAO/R+LV-shCYLD+EA group compared with the MCAO/R+EA group(P<0.05,P<0.05).Western blot showed that compared with MCAO/R+EA,CYLD protein decreased and the ratio of p-I?B?/I?B increased and more nuclear translocation of NF-?B p65 in MCAO/R+LV-shCYLD+EA(P<0.05,P<0.05,P<0.05).Double-immunofluorescent staining showed that there were fewer CYLD immunopositive cells and more NF-?B p65 nuclear translocation with MCAO/R+LV-shCYLD+EA than in the MCAO/R+EA group(P<0.05,P<0.05).Conclusion:1.CYLD mRNA and protein began to fall in after 6 h reperfusion.CYLD mRNA and protein were lowest after 24 h reperfusion in the MCAO/R group and this increased at 48 h and peaked after 72 h reperfusion.EA could upregulated CYLD expression of peri-ischemic areas after ischemic stroke.CYLD protein was mainly expressed in cortical neurons of peri-ischemic areas after focal cerebral ischemia/reperfusion.2.CYLD overexpression may play a significant neuroprotective and anti-inflammatory role in peri-ischemic areas after focal cerebral ischemia/reperfusion.3.Upregulating neuronal CYLD expression by EA may prevent NF-kB nuclear translocation and decrease neuronal CX3CL1 expression,inducing subsequent inhibition of microglial activation and pro-inflammatory cytokines in peri-ischemic areas. |
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