Regulation Of CBS On Microglial NLRP3 Inflammasome Activation And Its Role In LPS-induced Parkinson’s Disease Related Neuroinflammation

Objective:Chronic neuroinflammation plays an important role in the pathogenesis of Parkinson’s disease(PD).Activation of microglial NLRP3 inflammasome acts as a central event in PD-related inflammatory cascade.This project is designed to study the role and underlying mechanisms of cystathionine β-synthase(CBS)-H2S axis in NLRP3 inflammasome activation and PD-related neuroinflammation.Methods:Male C57BL/6J mice(about 8 weeks old)were used in vivo study.PD mouse model was established by injection with LPS(7.5 μg/1.5 μl per site)into bilateral substantia nigra using the stereotaxic apparatus.To explore the potential effect of CBS overexpression on NLRP3 inflammasome and PD-related neuroinflammation,the mouse substantia nigra was bilaterally injected with recombinant adeno-associated-virus encoding cbs(rAAV-Cbs)or its vector(rAAV-Vec)at two weeks before LPS or saline administration.One week after LPS injection,the morphology and number of microglia in substantia nigra were studied by immunohistochemistry.The expression of NLRP3 inflammasome related proteins in the ventral midbrain and striatum were examined by western blotting.Quantitative PCR was used to detect thepro-IL-1β mRNA level.Both primary cultured microglia and BV2 cells were used for in vitro study.Cells were primed with(500 ng/ml)LPS for 3 hours,and then cultured overnight in fresh culture medium,followed by addition with ATP(2.5 mM)for 30 min to induce NLRP3 inflammasome activation.SAM and H2S donors were added and incubated before ATP treatment.To establish CBS overexpression in BV2,cells were infected with Lenti-Cbs or Lenti-Vec.BV2 cells was transfected with si-cbs to induce cbs silence.The expression of CBS and NLRP3 inflammasome related proteins in both the culturc supernatant and whole cell lysate was studied by western blotting.cbs and pro-IL-1βmRNA levels were examined by quantitative PCR.The content of IL-1β in the supernatant was analyzed by ELISA.The total level of intracellular reactive oxygen species(ROS)was assessed using the molecular probe DCFH-DA.Results:1.Substantia nigra injection with LPS downregulated the expression of CBS but upregulated the expression of NLRP3 inflammasome related proteins in the substantia nigra and corpus striatum in PD mouse model.Compared to saline injection,the expression of NLRP3 inflammasome related proteins(NLRP3、ASC、pro-IL-1β)were increased,associated with the decrease of CBS expression in the ventral midbrain and striatum following LPS injection into substantia nigra.2.LPS enhanced the mRNA level of pro-IL-1β but reduced that of cbs mRNA in primary cultured microglia.Compared with the untreated group(0 h),the level of cbs mRNA was decreased,associated with the increase of pro-IL-1β mRNA level in LPS-treated microglia.3.CBS overexpression attenuated LPS-induced activation of microglia and NLRP3 inflammsome in vivo.In rAAV-Vector+SALINE group,the microglia in the reticular part of substantia nigra exhibited as branched rod-like.In rAAV-Vector+LPS group,the number of Iba-1 positive microglia in the reticular part of substantia nigra increased,and the Iba-1 fluorescence intensity was significantly higher than that in rAAV-Vector+SALINE group.And the inserted enlarged images showed that the microglia morphology change into amoeboid-like,with enlarged cell bodies and shortened processes.Moreover,compared with rAAV-Vector+SALINE group,the inflammasome-related proteins such as NLRP3,ASC,mature IL-1β and caspase-1 in the ventral midbrain of rAAV-Vector+LPS group were significantly higher than those in the rAAV-Vector+SALINE group.The level of pro-IL-1β mRNA in the striatum of LPS-injected mice was also higher than that of saline-injected rAAV-Vector mice.Compared with rAAV-Vector+LPS group,the morphological change of microglia and Iba-1 fluorescence intensity were attenuated in rAAV-Cbs+LPS group.This was accompanied with the decrease of NLRP3 inflammasome-related proteins expression and pro-IL-1β mRNA level.This indicated that CBS overexpression inhibited LPS-induced microglia activation,pro-inflammatory factor transcription and NLRP3 inflammasome activation.4.CBS activator SAM and CBS overexpression inhibited the NLRP3 inflammasome activation in microglia.Compared with control,the levels of mature IL-1β and caspase-1 protein in the culture supernatant from ATP-treated primed microglia were higher than those from ATP-untreated microglia.Pretreatment with CBS agonist SAM significantly attenuated the increase of mature IL-1β and caspase-1 protein in the culture supernatant induced by ATP treatment.Similar results were obtained in cbs-overexpressing BV2 cells that infected with Lenti-Cbs.5.H2S donor inhibited the activation of NLRP3 inflammasome in micoglia.Compared with control,the levels of mature IL-1 β and caspase-1 protein in the culture supernatant from ATP-treated primed microglia were higher than those from ATP-untreated microglia.Pretreatment with H2S-releasing compounds GYY4137 or NaHS consistently reduced the increase of mature IL-1β and caspase-1 protein in the culture supernatant induced by ATP treatment..6.cbs knockdown activated NLRP3 inflammasome in BV2 cells.cbs silencing by RNA interferencing in BV2 microglia enhanced the expressions of NLRP3 and the mature forms of IL-1β and easpase-1 in cells while the levels of pro-IL-1β and pro-caspase-1 did not change significantly.7.CBS agonist reduced the yield of intracellular total ROS in microglia.In LPS primed BV2,the level of total ROS increased remarkably after ATP treatment,and pre-incubation with SAM significantly reduced the production of intracellular ROS induced by ATP.Conclusion:CBS serves as an important inhibitor of microglial NLRP3 inflammasome,which may be related to its inhibition on intracellular ROS formation.LPS and other pathological stimuli downregulate CBS expression in microglia,which dampens the suppression of CBS on NLRP3 inflammasome activation and subsequent release of mature IL-1β.This leads to aggravation of neuroinflammatory cascades.Targeted modulation of CBS by its specific agonist or via viral-mediated CBS overexpression may inhibit NLRP3 inflammasome and PD-related neuroinflammation.