NLRP-3/caspase-1/GSDMD Axis Mediated Microglia Pyroptosis

Hypoxic-ischemic encephalopathy(HIE),one of the most severe complications of neonatal asphyxia,is one of the prime causes of neonatal death and severe post-injury neurological deficits.The survivors of severe HIE often suffer from irreversible neurological impairments.However,the therapeutic interventions of HIE remain limited,mainly due to cognition deficiency of HIE pathophysiology.So it is crucial to reveal the underlying mechanisms for potential therapeutic target after HIE.Recent studies showed that microglia inflammatory activation and the following inflammation act in the pathogenesis of hypoxic-ischemic brain damage(HIBD).However,the mechanism of microglia inflammatory activation still remains unclear.Pyroptosis is a recently found programmed cell death pattern accompanied with inflammation.And in the meantime,it is distinguished by both necrosis and apoptosis morphologically.The NLRP-3 inflammasome is important not only in microglia inflammatory activation,but also in canonical pyroptosis as an upstream factor,indicating that pyroptosis may be related with microglia inflammatory activation.So in the present research,we aim to find out the role of NLRP-3/caspase-1/GSDMD axis mediated microglia pyroptosis in HIBD microglia inflammatory activation by means of clinical trials,in vivo and in vitro experiments.Part Ⅰ The role of NLRP-3 mediated pyroptosis in microglia inflammatory activation in neonatal HIBD ratsPurpose To find out the role of NLRP-3 mediated pyroptosis in early and sustained microglia inflammatory activation in HIBD.Methods HIBD model was established with rats 7 days of age and the expression of pyroptosis regulating molecules including NLRP-3,caspase-1,GSDMD and IL-1β in the cortex was tested 0h,2h,6h,12h and 24h after HIBD with qRT-PCR and western blot to get a knowledge of pyroptosis level in cortex early after HIBD.MCC950,the specific inhibitor of NLRP-3,was also used for intervention.Besides the measurement of NLRP-3,caspase-1,GSDMD and IL-1β levels,the expression of CD68,one of the specific indicators of microglia inflammatory activation,was also tested.At the same time,TTC staining,HE staining and TEM were also used for the observation of tissue injury,microglia pyroptosis and microglia activation 24h after HIBD.1 month after HIBD,protein levels of NLRP-3,caspase-1,GSDMD,IL-1β and CD68 were also tested to find out whether pyroptosis also played a sustained role in HIBD.Results Both mRNA and protein levels of NLRP-3,caspase-1,GSDMD and IL-1βin cerebral cortex were elevated at the very early stage of HIBD(since 0h after HIBD),while MCC950 intervention after HIBD suppressed the rising trend of the above molecules as well as microglia inflammatory activation indicator CD68.Signs of cortex injury,microglia pyroptosis and microglia inflammatory activation were also found 24h after HIBD.MCC950 intervention alleviated not only the expression of the molecules above but also the tissure injury,microglia pyroptosis and microglia inflammatory activation.1 month after HIBD,protein levels of pyroptosis regulating molecules including NLRP-3,caspase-1,GSDMD,IL-1β and microglia inflammatory activation indicator CD68 were still upregulated compared with Sham group while MCC950 down-regulated them.Conclusions NLRP-3 mediated pyroptosis is closely related with rapid and sustained microglia inflammatory activation in HIBD.The inhibition of NLRP-3,which is the upstream of pyroptosis,can prevent microglia inflammatory activation.Part Ⅱ Mechanism of NLRP-3/caspase-1/GSDMD axis mediated microglia pyroptosis in HIBD related microglia inflammatory activation in vitroPurpose To furtherly explore the effect of NLRP-3/caspase-1/GSDMD axis mediated pyroptosis in microglia inflammatory activation in HIBD and the mechanism behind.Methods HIBD model was established with HAPI microglia cell line and expression of pyroptosis regulating molecules including NLRP-3,caspase-1,GSDMD,IL-1β and CD68 was tested 0h,2h,6h,12h and 24h after oxygen-glucose deprivation(OGD)by means of western blot,and evidence of microglia pyroptosis and microglia inflammatory activation were searched with inverted microscope and transmission electron microscope(TEM)to find the relationship between microglia pyroptosis and its activation after hypoxic-ischemic injury.Also OGD model of microglia transfected with NLRP-3 overexpressed,NLRP-3 interfered and GSDMD interfered lentivirus was built to furtherly explore the effect of pyroptosis on microglia inflammatory activation early after OGD by the measurement of NLRP-3,caspase-1,GSDMD,IL-1βand CD68 expression,the manifestation of pyroptosis(LDH+TEM)and microglia inflammatory activation(TEM).Results In non-infected microglia OGD model,protein levels of NLRP-3,caspase-1,GSDMD,IL-1β and CD68 were all elevated ever since 0h/2h after OGD and histological signs of microglia pyroptosis and activation were also found.While NLRP-3 overexpressed microglia showed more severe injury and higher levels of pyroptosis regulating molecules and microglia inflammatory activation.NLRP-3 and GSDMD silent microglia showed the opposite trend.Conclusions NLRP-3/caspase-1/GSDMD axis mediated canonical pyroptosis of microglia results in rapid microglia inflammatory activation in HIBD by increased inflammatory cytokines releasing.Inhibition of canonical pyroptosis in HIBD could alleviate brain damage by inhibiting microglia inflammatory activation.Part Ⅲ Expression of key pyroptosis molecules and their relationship with the severity of hypoxic-ischemic injury in HIE neonatesPurpose To investigate the expression of key molecules of NLRP-3/caspase-1/GSDMD axis mediated pyroptosis in the peripheral blood of HIE babies and their relationship with severity of hypoxic-ischemic injury.Methods mRNA levels of NLRP-3/caspase-1/GSDMD axis mediated pyroptosis regulating molecules including NLRP-3,caspase-1,GSDMD and IL-1β in peripheral blood samples of HIE babies and the normal term newborns of similar gestational age were tested within 72h after birth.Also the relationship between expression levels of the above molecules and 5min Apgar score of the patients were analyzed.Results In peripheral blood samples of HIE babies,mRNA levels of NLRP-3,caspase-1,GSDMD and IL-1β were all elevated compared with control group measured with qRT-PCR within 72h after birth.And expression levels of the above molecules related positively with the severity of hypxic-ischemic injury,which was represented by 5 min Apgar score of the patients.Conclusions Expression of pyroptosis regulating molecules in peripheral blood samples of HIE babies was elevated early and it related positively with the severity of hypxic-ischemic injury.Summary1.The experiments with model animals and cells indicated that NLRP-3/caspase-1/GSDMD axis mediated canonical pyroptosis of microglia results in rapid and sustained microglia inflammatory activation in HIBD by releasing more inflammatory cytokines.2.Inhibition of NLRP-3/caspase-1/GSDMD axis mediated canonical pyroptosis in HIBD could alleviate brain damage by inhibiting microglia inflammatory activation.3.Clinical trials indicated that pyroptosis played a part at the early stage of HIE and the levels of key pyroptosis molecules related positively with the severity of hypxic-ischemic injury.