The Regulation Of Microglia NLRP3 Inflammasome Activation By Cbs-H₂S Signaling Axis And Its Role In The Pathogenesis Of Parkinson’s Disease

Objective:To explore the impact of cystathionine-β-synthase(Cbs)-hydrogen sulfide(H2S)axis on microglia NLRP3 inflammasome and the potential mechanisms underlying it,and to further investigate the effect of microglia Cbs on Parkinson’s disease(PD)related neuroinflammation and losses of dopamine neurons.Methods:For in vitro study,lipopolysaccharides(LPS)was used to stimulate the murine BV2 microglia cell line to construct an inflammation model,and Western blotting was used to detect Cbs expression.In order to explore the effect of Cbs knockdown on the activation of microglial NLRP3 inflammasomes,small RNA interference method was applied to silence the Cbs gene.In brief,primary microglia cells were transfected with small siRNA targeting Cbs for 48 hours,follow by priming with LPS for 3 hours and then incubated with fresh medium overnight,and finally stimulated with ATP(Adenosine triphosphate),Nigericin or MSU(monosodium urate)to induce NLRP3 inflammasome activation.In order to explore the effect of Cbs activation or endogenous H2S on inflammasome activation,similar protocols were applied except that Cbs agonist SAM or H2S slow-releasing compound GYY4137 was given before ATP/Nigericin/MSU stimulation for 12 h.Western blot was used to detect the expression of Cbs,pro-IL-1β and its mature form IL-1β.In addition,the IL-1βcontent in the cell supernatant were also detected by ELISA(Enzyme Linked Immune Sorbent Assay).Immunofluorescence was used to detect the formation and number of ASC specks.A great amount of studies report that the mitochondrial ROS serve as a mediator of NLRP3 inflammasome activation.In order to clarify the mechanism of Cbs-H2S regulating NLRP3 inflammasome,the fluorescent probe MitoSOX was used to detect the level of mitochondrial ROS.In a pilot in vivo study,LPS(500 μg/kg)was intraperitoneally injected into male C57BL/6 mice of different ages,the expression and cellular distribution of Cbs in the substantia nigra were detected by Western blotting and immunofluorescence to explore the potential effect of neuroinflammation and aging on Cbs expression.Second,7.5 μg LPS(5 mg/ml)were injected into the substantia nigra(AP:-3.00 mm,ML:±1.25 mm,DV:+4.5 mm)using the stereotaxic apparatus to construct an inflammation-associated PD mouse model.In order to explore the effect of Cbs knockout on PD-related neuroinflammation and dopamine neuron losses,the Cre-loxP gene knockout system was used to construct a genetically engineered mouse with microglial Cbs knockout.cKO mice were intraperitoneally injected with Tamoxifen for 5 consecutive days to induce the expression of Cre recombinase at 21 days post born.This study uses the same breeding method to obtain non-inducible microglia Cbs-specific knockout mice,namely Cbsflox/flox;Cx3cr1Cre.both male cKO and WT mice were injected with LPS or saline into the substantia nigra.In order to explore the effect of Cbs overexpression on PD-related inflammation,the mice were bilaterally injected with recombinant adeno-associated-virus encoding Cbs(AAV-Cbs)or its Vector(AAV-Vec)into the substantia nigra at two weeks before LPS or saline administration.In parallel,this study also used the Cbs agonist SAM via intraperitoneal injection with SAM(50 mg/kg)to observe its effect of Cbs activation on PD-related inflammation and dopaminergic neuron loss induced by LPS.Western blot and ELISA were used to detect the expression of inflammasome-related proteins(pro-IL-1β and its mature form,ASC)and the content of IL-1β in the substantia nigra.And the immunofluorescence study was applied to detect the morphology and quantity of microglia,the formation of ASC specks,and the changes of NLRP3 proteins expression.DAB staining was used to detect the damage/loss of dopaminergic neurons in the substantia nigra.Results:(1)Compared with neonatal(P1)mice,the expression of Cbs in the striatum was increased gradually with aging,reaching a peak at 6 months,and then gradually decreased with aging,and the expression of Cbs at 20 months is lower than that of neonatal mice,and the changes in Cbs expression with aging are mainly in microglia.(2)Microglia Cbs expression rapidly increased followed by a decrease in response to inflammatory stimulation.Compared with the basal level(0 h),Cbs expression increased rapidly after LPS stimulation and maintained at a high level for a period of time(0.5-3 h).Cbs expression gradually decreased afterward 3 h;For in vivo study,Microglia showed a typical amoebic morphology at 3 h after LPS injection compared with control group(0 h),and the expression of Cbs increased.Microglia morphology gradually returned to the basal level at 24 h and 7 days after LPS injection,accompanied by the decrease of Cbs expression in microglia.(3)Cbs knockdown/knockout in microglia promoted NLRP3 inflammasome activation and IL-1β generation.In vitro study,IL-1β generation and Caspase-1 activity increase in Cbs knockdown group compared with nonsense siRNA transfected cells(si-Control group);In vivo study,the inflammasome-related protein(such as IL-1β、NLRP3)in cKO mice was higher than WT mice on neuroinflammation induced by LPS.(4)Cbs activation/overexpression and H2S donor inhibited NLRP3 inflammasome activation and IL-1β production in microglia.In vitro study,Compared with ATP,Nigericin or MSU stimulation,pretreatment with SAM or GYY4137 before stimulation with ATP,Nigericin or MSU reduced the formation of ASC specks.Moreover,the IL-1β content in the supernatants of the cells co-treated with SAM or GYY or NaHS decreased consistently compared with ATP-stimulated group;In vivo study,the mice were bilaterally injected with recombinant adeno-associated-virus encoding Cbs(AAV-Cbs)and its agonist SAM consistently reduced the formation of ASC specks and IL-1βproduction.(5)Cbs-H2S axis had an impact on mitochondrial ROS production.Cbs knockdown promotes ATP-induced LPS pretreatment mitochondrial ROS levels.Conversely,Cbs agonists or H2S donors significantly reduced mitochondrial ROS levels in LPS-pretreated cells after ATP stimulation.(6)Conditional knockout of Cbs gene in microglia aggravated the losses of dopaminergic neuron induced by LPS.Conversely,Cbs overexpression and its agonist attenuated LPS-induced loss of dopaminergic neuron.Conclusion:The Cbs expression in microglia is closely related to aging and inflammatory stimulation.Cbs serves a"braking molecule" of inflammation,inhibiting the activation of NLRP3 inflammasome and IL-1β production and secretion induced by inflammasome stimulators.And its molecular mechanism may be related to the inhibition of mitochondrial ROS production.Cbs knockout in microglia enhances the activation of NLRP3 inflammasomes and IL-1 β generation,aggravating the loss of dopaminergic neurons caused by inflammatory stimuli.On the contrary,Cbs overexpression or its agonists reduces the activation of NLRP3 inflammasomes and IL-1 β production,and thus alleviates the loss of dopaminergic neurons caused by inflammatory stimuli.