The Effects Of NLRP6 On Inflammatory Response And Pyroptosis In OGD/R Model

Background: Inflammation plays an important role in the mechanism of cerebral ischemia-reperfusion injury.During cerebral ischemia/reperfusion,inflammasome is activated,then promoting the release of inflammatory cytokines,amplification of the inflammatory process and mediating pyroptosis.These leads to aggravate cerebral ischemia/reperfusion injury.NLRP6 is a member of the NLRs family,which is a protein with unique functions in the NLRs family.Early studies have found that NLRP6 could be activated upon stimulation by endogenous and exogenous factors,and assembled to inflammasomes,then promoting the inflammatory process.However,in recent studies have shown that NLRP6 could inhibit the inflammatory response and promote healing of intestinal damage in bacterial intestinal infections.In addition,NLRP6 has been reported to inhibit inflammatory responses after acute peripheral nerve injury,and contribute to the recovery of peripheral nerve injury.The function of NLRP6 in cerebral ischemia/reperfusion injury is largely unknown,and its further mechanism needs full investigation.Objective: To investigate the distribution of NLRP6 and its effect on neurons in the cerebral ischemia reperfusion vitro model,and further explore the mechanism of NLRP6 in inflammatory response and neurons pyroptosis.Methods:(1)Primary neurons,primary astrocytes and hapi microglias were cultured,and establishing a stable OGD/R model to identify the localization of NLRP6 by cell immunofluorescence.(2)Primary astrocytes were cultured and the OGD/R model was established.After OGD for 1,2,4 and 6 hours,reoxygenated for 24 hours.Western blot and qRT-PCR were used to detect the expression of NLRP6.(3)To establish the non-contact neuron-astrocyte co-cultures model,the astrocytes were infected with NLRP6 siRNA or NLRP6 overexpressing lentivirus.After OGD for 4 hours,reoxygenated for 24 hours.the viability and apoptosis of neurons were detected by CCK8 assay and flow cytometry.(4)The astrocytes were treated with NLRP6 siRNA or NLRP6 overexpressing lentivirus.After OGD for 4 hours,reoxygenated for 24 hours.The protein expressions of NLRP6?Cleaved-Caspase-1?IL-1??IL-18 were detected by Western blot,and the expression levels of IL-1? and IL-18 in the cell supernatants were detected by ELISA Kit.(5)The astrocytes were treated with NLRP6 siRNA or NLRP6 overexpressing lentivirus.After OGD for 4 hours,reoxygenated for 24 hours.The combination of NLRP6 and ASC was detected by co-immunoprecipitation assay.(6)The astrocytes were treated with caspase-1 inhibitor Ac-YVAD-cmk and NLRP6 overexpressing lentivirus.After OGD for 4 hours,reoxygenated for 24 hours,the protein expressions of NLRP6?pro-caspase-1? Cleaved caspase-1? IL-1??IL-18 were detected by Western blot.(7)To establish the non-contact neuron-astrocyte co-cultures model,the astrocytes were infected with NLRP6 siRNA or NLRP6 overexpressing lentivirus.After OGD for 4 hours,reoxygenated for 24 hours.Western blot assay was used to detect the expression of NLRP6 in astrocytes and the expression of GSDMD-NT in neurons.(8)To establish the non-contact neuron-astrocyte co-cultures model,the astrocytes were infected with caspase-1 inhibitor Ac-YVAD-cmk and NLRP6 overexpressing lentivirus.After OGD for 4 hours,reoxygenated for 24 hours.The expressions of NLRP6 in astrocytes and the expression of Cleaved-Caspase-1 and GSDMD-NT in neurons were detected by Western blot,the pyroptosis of neurons were detected by flow cytometry.Results:(1)NLRP6 was mainly expressed in astrocytes after OGD/R,but little expressed in neurons and microglias.(2)The expression of NLRP6 was increased after OGD/R as compared to the sham group,and peaked at 4 h after OGD.(3)Compared with si-NC group,the viability of neurons was increased and the apoptosis of neurons was decreased in NLRP6 si-RNA group,while the viability of neurons was decreased and the apoptosis of neurons was increased in NLRP6 overexpression group in comparison with those in LV-NC group.(4)The expression level of NLRP6?Cleaved-Caspase-1?IL-1??IL-18 were significantly elevated after OGD/R.NLRP6 si-RNA inhibits the elevation of Cleaved-Caspase-1?IL-1??IL-18 were apparently decreased in NLRP6 si-RNA group in comparison with those in OGD/R group.But those proteins level were dramatically up-regulated in NLRP6 overexpressing group.(5)Compared with OGD/R group,NLRP6-ASC was more effective in co-precipitating of astrocytes infected by NLRP6 overexpression lentivirus while NLRP6-ASC was less co-precipitated in astrocytes infected by NLRP6 siRNA.Meanwhile,lentiviral overexpression of NLRP6 elevated the protein expression levels of IL-18 and IL-1?,which was blocked by caspase-1 inhibitor Ac-YVAD-cmk.(6)After OGD/R,the protein level of GSDMD-NT was obviously decreased following NLRP6 knockdown.In contrast,NLRP6 overexpression induced a significantly incresed expression of GSDMD-NT.(7)Ac-YVAD-CMK could block the increase level of Cleaved-caspase-1 and GSDMD-NT due to NLRP6 overexpression.In addition,it also can rescue NLRP6-mediated pyroptosis in neurons.Conclusions:(1)NLRP6 expression increased predominantly in astrocytes subjected to OGD/R,which aggravating the neuron injury.(2)After OGD/R,NLRP6 could bind to ASC,then activate caspase-1 form into inflammasome,which promoted inflammatory response and induced pyroptosis,leading to aggravate the neuron injury.