The Role Of NLRP3 Inflammasome In 2,5-hexanedione-induced Neurotoxicity And The Protective Effects Of Gly

Background:n-Hexane is an organic solvent and is widely used in industrial production process,such as paint,electronic cleaning,printing and shoemaking.If it is not used properly,n-hexane exposure easily causes occupational poisoning,and induces peripheral neuropathy characterized by sensory and motor dysfunction.2,5-hexanedione(HD)is the toxic metabolite of n-hexane,which is the terminal poision that mediates n-hexane-induced neurotoxicity.We previously found that HD can induce neuroinflammatory response and release of proinflammatory factors to damage neurons in rats,indicating a critical role of neuroinflammation in HD-induced neurotoxicity.However,the potential mechanisms of HD-induced neuroinflammation remain unclear.In recent years,inflammasome becomes the focus in the field of neuroinflammation,which can produce mature cytokines and regulate the inflammatory response.Nucleotide-binding oligomerization domain-like receptor protein 3(NLRPP3)inflammasome is the most widely studied inflammasome and can be activated by a wide rage of pathogens and dangerous molecules.Once activation,NLRP3 inflammasome causes the activation of caspase-1.On the one hand,activated caspase-1 promotes the maturation and release of IL-1? and IL-18,and then drives the inflammatory response through a variety of downstream signaling pathways,leading to neuronal damage.On the other hand,activated caspase-1 can cleave Gasdermin D protein(GSDMD)togenerate N-terminal cleavage product(GSDMD-N).GSDMD-N induces formation of channels on the cell membrane and subsequent the release of inflammatory cytokines,amplifying the inflammatory response and related nerve damage,which is called pyroptosis.Strong evidence suggested that the activation of NLRP3 inflammasome has been implicated in the pathogenesis of multiple neurological disorders,including Alzheimer's disease(AD),Parkinson's disease(PD)and other neurodegenerative diseases.However,whether HD could induce neurotoxicity through NLRP3 inflammasome still remains unclear.Aims:We therefore performed this study to investigate the effects of HD on the activation of NLRP3 inflammasome by using HD-induced rat neuropathy model to explore the mechanism of HD-induced nerve damage.Furthermore,the protective effects of glybenclamide(Gly),an inhibitor of NLRP3 inflammasome,on HD-induced neurotoxicity were further investigated to provide theoretical basis for the molecular mechanism and clinical research of neuropathy in response to HD.Methods:1.HD-induced neuropathy rat model: Forty-five adult male SD rats in SPF grade were randomly divided into 3 groups after 7 days of adaptive feeding: control,200mg/kg HD and 400 mg/kg HD groups(n = 15 for each group).HD was administrated by intraperitoneal injection once a day(5 days per week for consecutive 8 weeks).Rats in Con group were given equal amount of 0.9% normal saline.2.Gly protection model: Sixty adult male SD rats in SPF grade with 7 days of adaptive feeding were randomly divided into 4 groups: control,HD,HD + Gly,and Gly alone groups(n = 15 for each group).HD was administrated to rats at a dosage of 400mg/kg as described above.Gly was injected into rats 30 minutes prior to HD for 5 times a week for consecutive 8 weeks.Rats in Con and Gly groups were given equal amount of 0.9% saline and Gly,respectively.3.Western blot analysis: The expression and activation of NLRP3 inflammasome in rat sciatic nerve were detected.The expressions of GSDMD ? MBP,NF-L,and macrophages markers CD11 b and Iba-1 were detected.4.Immunofluorescence: The frozen sections of rat sciatic nerve were prepared.The effects of HD on the infiltration of immune cells were investigated by using anti-Iba-1 antibody.Immunofluorescence staining with anti-NLRP3 and caspase-1antibodies was performed to investigate the effects of HD on NLRP3 activation in macrophages.5.Electron microscopic observation: The pathological changes of axons of sciatic nerve in rats were seen by transmission electron microscopy.6.q RT-PCR assay: The m RNA levels of M1(i NOS,TNF-?)and M2(Arg-1,MRC-1)markers were detected.The gene expressions of NLRP3 inflammasome(ASC,NLRP3,caspase-1,IL-1 ?)were analysized by quantitative RT-PCR assay.7.Enzyme linked immunosorbent assay(ELISA): The content of IL-1? in the sciatic nerve tissue was detected by ELISA kit.8.Oxidative stress test: The changes of Malondialdehyde(MDA),Glutathione Peroxidase(GSH)and total antioxidant capacity in sciatic nerve of rats were detected by commercial assay kits.Results:1.The establishment of HD-induced rat neuropathy model and the change of gait score: Rats with 200 mg/kg HD intoxication displayed mild gait abnormality with toes on the ground.Rats in 400 mg/kg HD group had typical characteristics of toxic peripheral neuropathy by showing gait instability,hind limbs powerless,dragging,and finally unable to stand until paralysis.Gait score analysis showed that the gait score of rats increased gradually after HD exposure.At 8 weeks of exposure,the average gait score of 200 mg/kg HD group rats was 1.50 ± 0.55,which was not statistically significant with that of Con group.The average gait score of 400 mg/kg HD group rats was 4.00 ± 0.00,which was significant higher than that of Con group(P < 0.01).2.The effects of HD on the activation of NLRP3 inflammasome in the sciatic nerve of rats: Western blot showed that compared with the Con group,the expression levels of ASC,NLRP3,cleaved caspase-1 and IL-1? remained unchanged in the sciatic nerve of rats in the 200 mg/kg HD group.The expressions of ASC,NLRP3,cleaved caspase-1 and IL-1? were increased in the sciatic nerve of rats in 400mg/kg HD group compared with Con group(P < 0.05).q RT-PCR showed that compared with Con,there was no significant change in NLRP3,ASC,caspase-1 and IL-1? m RNA content in200mg/kg HD-intoxicated rats;while the m RNA contents of NLRP3,ASC,caspase-1and IL-1? were significantly higher in 400 mg/kg HD group(P < 0.05).ELISA assay showed that there was no significant increase in IL-1? content in the 200 mg/kg HD group,but significant increase of IL-1? content was observed in the 400 mg/kg HD rats compared with the Con group(P < 0.01).These results suggested that 400 mg/kg HD could induce the activation of NLRP3 inflammasome in the sciatic nerve of rats.3.The effects of HD on the infiltration of macrophages in the sciatic nerve of rats: Western blot showed that compared to Con group,there was no significant change in the expression of CD11 b and Iba-1 in the 200 mg/kg HD group,while the expressions of CD11 b and Iba-1 was increased in the 400 mg/kg HD group(P < 0.01).Immunofluorescence staining showed that compared with Con group,the number of Iba-1-labeled macrophages was increased in 400 mg/kg HD group rats.The results indicated that 400 mg/kg HD could induce macrophage infiltration.4.The effects of HD on the polarization of macrophages in the sciatic nerve of rats: q RT-PCR showed that the m RNA levels of M1 and M2 markers in the 200 mg/kg HD group had no significant change compared with the Con group.Compared with the Con,the m RNA levels of M1 markers(i NOS,TNF-?)in 400 mg/kg HD group were increased,simutenuously,and the m RNA levels of M2 markers(Arg-1,MRC-1)were decreased,indicating that 400mg/kg HD could induce macrophages to M1 polarization.5.The effects of Gly on the activation of NLRP3 inflammasome in the sciatic nerve of HD-intoxicated rats: Western blot showed that compared with Con group,the expression levels of NLRP3,cleaved caspase-1 and IL-1? in 400mg/kg HD group rats.Gly markedly decreased 400 mg/kg HD-induced expressions of NLRP3,caspase-1 and IL-1? in rats(P < 0.05).Consistent with that of Western blot,ELISA assay showed that compared with Con group,the content of IL-1? in 400 mg/kg HD group rats was increased,which was significantly reduced by Gly treatment(P < 0.01).6.The effects of Gly on the infiltration and polarization of macrophages in the sciatic nerve of HD-intoxicated rats: Western blot indicated compared with Con group,the expressions of CD11 b and Iba-1 in 400 mg/kg HD group rats were increased.Gly can reduced 400mg/kg HD-induced expressions of CD11 b and Iba-1 in rats(P <0.05).Consistent with that of Western blot,immunofluorescence staining showed that compared with Con group,the number of Iba-1 positive cells in 400 mg/kg HD group rats was increased.q RT-PCR showed that compared with Con group,the m RNA levels of i NOS and TNF-? in 400 mg/kg HD group rats were increased,while the m RNA levels of Arg-1 and MRC-1 were decreased.Gly reduced the m RNA levels of i NOS and TNF-? and simutenuously recovered the m RNA levels of M2 markers Arg-1 and MRC-1 in the sciatic nerve of rats(P<0.05).7.Effects of Gly on oxidative stress in sciatic nerve of HD-intoxicated rats:Compared with Con group,400 mg/kg HD increased MDA content and reduced GSH level in the scatic nerve of rats.Gly obviously decreased 400mg/kg HD-induced increase of MDA content and decrease of GSH level(P < 0.05).Total antioxidant capacity assay showed that compared with Con group,total antioxidant capacity was reduced in the sciatic nerve of 400 mg/kg HD group rats,which was significantly recovered by Gly(P < 0.05).8.The effects of Gly on the sciatic nerve toxicity in HD-intoxicated rats:Western blot indicated that compared with Con group,the content of GSDMD-N was increased in 400 mg/kg HD group rats,while the expressions of NF-L and MBP were decreased.Gly decreased 400 mg/kg HD-induced increase GSDMD-N expression and decrease of NF-L and MBP expressions in rats(P < 0.01).Consistent with that of Western blot,immunofluorescence staining showed that compared with Con group,the expression of MBP was decreased in the sciatic nerve of 400mg/kg HD group rats,which was markedly increased by Gly(P < 0.05).Electron microscopy showed that compared with Con group,the structure of myelin sheath was obviously changed,and the myelin sheath was significantly thin in 400 mg/kg HD group rats.Gly significantly reduced 400mg/kg HD-induced injury of myelin sheath in the sciatic nerve of rats.9.The effects of Gly on the body weight and gait in HD-intoxicated rats:Compared with Con group,rats in 400 mg/kg HD group displayed reduced body weight and abnormal gait.Gly significantly increased the body weight and improved gait performance in 400 mg/kg HD-intoxicated rats.Quantitative analysis revealed a 13.7%increase of body weight of rats exposed to combined Gly and 400mg/kg HD compared with 400mg/kg HD group(P < 0.01).The average gait score of the rats exposed to combined Gly and 400 mg/kg HD was 2.00±0.76,which was significantly lower than that of the 400 mg/kg HD alone group(P < 0.01).Conclusion:1.HD can induce the activation of NLRP3 inflammasome in sciatic nerve of rats.2.The involvement of NLRP3 in HD-induced neurotoxicity may be related to the regulation of macrophage infiltration,M1 polarization and oxidative stress.3.Gly,an inhibitor of NLRP3 inflammasome,can alleviate HD-induced neurotoxicity and motor dysfunction in rats.