Molecular Mechanism Of Astragaloside IV Regulating MMP-9-mediated NLRP3/Caspase-1 Signaling Pathway To Improve Hypoxic-ischemic Brain Injury

Purpose:The pathogenesis of neonatal hypoxic-ischemic encephalopathy(HIE)may be related to activation of the NOD-like receptor family pyrrole domain-3(NLRP3)and matrix metalloproteinase-9(MMP-9).The effect of inflammation on the development of nervous system is highly dependent on the timing of treatment,and early assessment of the inflammatory severity of HIE is very important to fine-tune interventions.The purpose of this study was to observe the Law of time variation of MMP-9,NLRP3 inflammasome and its downstream effectors caspase-1 and interleukin IL-1?in the early stage of hypoxic ischemic brain injury in neonatal rats,and to clarify the role of NLRP3/MMP9 signaling pathway in the HIE process.At the same time,in vivo and in vitro cell experiments were conducted to prove the treatment effect and mechanism of Astragaloside IV(AS-IV)on HIE,providing experimental basis for the selection of treatment timing after hypoxic ischemic brain injury,and providing basis for clinical application of Astragaloside IV in the treatment of neonatal brain injury.Material and method:7-day-old rats with body weight of 10 to 14g were randomly divided into hypoxic-ischemia group(HI group,n=40)and sham group(Sham group,n=40).Specimens were collected at 0 h,4 h,8 h,12 h and 24 h after surgery(8 specimens were randomly selected at each time point).In the HI group,rats were double-ligated with the right common carotid artery and then exposed to hypoxia(8%O₂and 92%N₂)for 2 h.In the Sham group,the skin was cut and the right common carotid artery was bluntly separated,and the skin suture was conducted directly without ligation.Before and after surgery,cranial ultrasound was used to measure the maximum end systolic flow velocity(ESV),end diastolic flow velocity(EDV),and resistance index(RI)proximal to bilateral middle cerebral arteries(MCA).The degree of brain injury was evaluated by acoustic radiation force pulse imaging(ARFI).At different time points within 24 h,pathological changes of hippocampus were observed by HE staining.NLRP3,Caspase-1 and IL-1?in brain tissues were detected by immunohistochemistry.MMP-9,NLRP3,Caspase-1 and IL-1?m RNA and protein expressions in brain tissues were detected by RT-q PCR and Western blot.The oxygen-glucose(OGD)deprivation model of HT22 cells was established,and the mouse hippocampal neurons were divided into 4 groups:control group,OGD group,OGD+MMP-9 inhibitor group,OGD+MMP-9 activator group.HT22 cells viability was measured by CCK-8 method at different time points within 48 h after hypoxia,and the changes of cell morphology were observed.MMP-9,TIMP-1,NLRP3,Caspase-1,GSDMD and IL-1?protein and m RNA expressions in HT22 cells were detected by Western blot and RT-q PCR at different time points within 24 h.The expression changes of NLRP3 and MMP-9 in hypoxic HT22 cells were detected by immunofluorescence assay at different time points within 24 h.According to the above experimental results,the induction conditions of oxygen and glucose deprivation for 8 h were selected in the follow-up experiments.The effect of activating or blocking MMP-9 on the expression of NLRP3 was observed by immunofluorescence assay.The expression of NLRP3 and its downstream effectors Caspase-1,GSDMD,IL-1?protein and m RNA were detected by Western blot and RT-q PCR.24 7-day-old rats with body weight ranging from 10 to 14g were randomly divided into sham group,HI group and HI+AS-IV group,with 8 rats in each group.The modeling methods were the same as before.In HI+AS-IV group,AS-IV(10mg/kg)was dissolved in DMSO solution(10m L/kg)immediately after HI modeling.After calculation according to the weight of the offspring,the rats were given gavage once every 8 h,for a total of 3 times.24 h later,brain tissue was sampled after isoflurane anesthesia.The effects of AS-IV on hippocampal brain tissue of HIE neonatal rats were observed by HE staining.Caspase-1 and TUNEL immunofluorescence staining were used to detect the effect of AS-IV on the occurrence of pyroptosis in hippocampal CA1 region of neonatal HIE rats.Western blot was used to detect the effects of AS-IV on the protein expression levels of MMP-9,NLRP3,Caspase-1,GSDMD,IL-1?protein in neonatal rats.Oxygen and glycogen deprivation model of HT22 cells was established and randomly divided into control group,simple OGD group and OGD+AS-IV group.As-IV concentration gradient(50-400?mol/L)was set,and CCK8 was used to detect the effects of different concentrations of As-IV on the activity and morphology of hypoxic HT22 cells.The expressions of MMP-9,NLRP3,Caspase-1,GSDMD,IL-1?protein were detected by Western blot.The effects of AS-IV on NLRP3 and MMP-9 after 8 h hypoxia were detected by immunofluorescence assay.Caspase-1 and TUNEL immunofluorescence staining were used to observe the effect of AS-IV on hypoxic HT22 cell pyroptosis.Results:1.Craniocerebral ultrasound results showed that the blood flow velocity of the right vertebrobasilar artery in the ischemia group increased at 4 h after the modeling,and the echo of brain parenchyma enhanced at 24 h.3 young rats vertebrobasilar artery blood theft occurred at 24 h after HI.Compared with MCA on the left,ESV significantly decreased at 4and 24 h,EDV significantly decreased at 24 h,and RI increased at 4,12 and 24 h in the HI group(P<0.05).Compared with Sham group,ESV of right MCA in HI group decreased and RI increased at 24 h(P<0.05),EDV of left MCA showed a progressive increase.4h after hypoxia ischemia,acute ischemia hiopathological features began to appear in the right hippocampal pyramidal cell layer,including cell density decreased,nuclear pyknosis and fragmentation,and a few nerve cells were eosinophilic degeneration which showed red neuron.At 8 h,some cells formed more vacuoles,and the cells were loosely arranged and disordered.The eosinophilic degeneration of nerve cells increased significantly at 12 h,while the volume and number of neurons decreased at 24 h,and eosinophilic changes were somewhat reduced.Immunohistochemistry results were as below.Compared with 0h,NLRP3and IL-1?significantly increased at 4 h,8 h,12 h and 24 h(P<0.01),Caspase-1 increased at 8h and 12 h(P<0.01).NLRP3 protein and m RNA expression at 4 h,8 h,24 h in HI group was significantly higher than sham group(P<0.05),Caspase-1 was significantly higher than sham group at 12 h(P<0.001),IL-1?was significantly higher than sham group at 8 h(P<0.01).The change trend of MMP-9 is the same as NLRP3.2.After oxygen and glucose deprivation(OGD)exposure for 8 h,the volume of HT22 cells began to shrink,and the cells were surrounded by detritus,and nucleus showed fragmentation.With the prolongation of OGD time,cells lost their original shape and the number of cells decreased obviously.CCK-8 showed that,after OGD for 8 h,HT22 cell viability decreased compared with that of 0 h(P<0.001),and cell viability decreased to 9.9±1.1%for 48 h.Compared with 0 h,NLRP3,MMP-9,GSDMD,Pro-Caspase-1,cleaved Caspase-1 and IL-1?in HT22 cells began to increase after 4 h OGD,at 8 h and 12 h,which increased most significantly,and slightly decreased at 24 h.The above proteins were increased at 4 h,8 h,12h and 24 h after OGD(P<0.01).The variation trend of NLRP3 and MMP-9 was consistent at all time points during 24 h of OGD,while TIMP-1 was opposite.It was observed by fluorescence microscopy that under OGD stimulation,NLRP3 and MMP-9 co-localization increased.Compared with Control group,HT22 cells co-localization phenomenon was the most obvious at 8 h and 12 h after OGD,and slightly decreased at 24 h.Pearson correlation coefficients(PCC)of NLRP3 and MMP-9 at each time point were all greater than 0.8,and PCC comparison showed no statistical significance(P>0.05).NLRP3 decreased rapidly by adding MMP-9 inhibitor,and the co-localization decreased significantly compared with OGD group(P<0.001).After MMP-9 agonist added,there was no obvious increase of NLRP3,compared with OGD group(P>0.05).After activation or blocking MMP-9,PCC of NLRP3and MMP-9 at each time point were all higher than 0.8,and PCC comparison showed no statistical significance(P>0.05).Compared with Control group,the protein and m RNA of NLRP3,IL-1?,Pro-Caspase-1 and Cleaved Caspase-1 didn't obviously change by using MMP-9 inhibitor(P>0.05).Compared with OGD group,the protein and m RNA expressions of NLRP3,Pro-caspase-1,cleaved caspase-1,GSDMD and IL-1?were significantly decreased(P<0.001).By introducing MMP-9 agonist,NLRP3,Pro-caspase-1,cleaved caspase-1,GSDMD and IL-1?expression were significantly increased compared with control group(P<0.01),but only pro-caspase-1 and Cleaved caspase-1 were increased compared with OGD group(P<0.01).3.After 24 h of modeling,HE staining of brain tissue showed that the density of pyramidal cell layer in hippocampus of HI model group decreased,arrangement was loose,and hierarchy was disordered.Some cells showed vacuolar degeneration and typical red neurons.The degree of degeneration of hippocampal neurons in AS-IV group was reduced,and acute ischemic changes were significantly reduced.Caspase-1 and TUNEL were used to label pyroptosis cells.Compared with Sham group,more pyroptosis cells were observed in the hippocampus of neonatal rats after HI.At the same time,the rate decreased significantly after AS-IV treatment.Compared with sham operation group,the expression levels of NLRP3,GSDMD,Caspase-1 and IL-1?in brain tissue of HIE model group were significantly increased,while the expression levels of NLRP3,Caspase-1 and GSDMD in brain tissue of AS-IV treatment group were significantly lower than those of HI model group.4.When the HT22 cells were OGD for 8 h,different concentrations AS-IV was applied to intervene.The results of CCK8 experiment showed that compared with OGD group the activity increased after 100?400?mol/L AS-IV intervention(P<0.05).When the concentration was 200?mol/L,hypoxia damage was significantly reduced.Compared with Control group,cell viability was increased most obviously.Meanwhile,we studied the effects of AS-IV 200?mol/L on the cell viability of HT22 cells at different time points of OGD as well.The experiment also found that AS-IV was the best treatment for 8 h.Compared with control group,the protein of MMP-9,NLRP3,Pro-caspase-1,cleaved-caspase-1,GSDMD and IL-1?in OGD group were significantly increased(P<0.001).The protein of MMP-9,NLRP3,Pro-caspase-1,cleaved-caspase-1,GSDMD and IL-1?in the OGD group were significantly decreased(P<0.01)after the intervention of 100-400?mol/L AS-IV.There was no significant difference of NLRP3,pro-caspase-1 and cleaved-caspase-1,GSDMD and IL-1?between200?mol/L and 400?mol/L groups(P>0.05).In order to explore the effect of As-IV on NLRP3 and MMP-9,co-localization of NLRP3 and MMP-9 was significantly increased under hypoxia stimulation compared with control group by fluorescence microscopy(P<0.001).Compared with OGD group the decrease of NLRP3 and MMP-9 double positive cells which was the most obvious phenomenon after AS-IV treatment with three concentrations of100?400?mol/L(P<0.01).There was no significant difference between 200?mol/L and400?mol/L groups(P>0.05).Caspase-1 and TUNEL were used to label pyroptosis cells.Compared with Control group,more pyroptosis cells were observed and the rate of pyroptosis decreased significantly after AS-IV treatment.Conclusion:1.m RNA and protein expressions of NLRP3,Caspase-1 and IL-1?were all increased at 4-8 h after hypoxic ischemic brain injury in neonatal rats,suggesting that NLRP3 inflammators may be involved in the pathogenesis of hypoxic ischemic brain injury in neonates and the up-regulation time might be earlier than 4 hours.NLRP3/caspase-1/IL-1?signaling pathway might be a new therapeutic target for the treatment of neonatal hypoxic-ischemic brain injury.2.There was a certain correlation between the expression of MMP-9 and NLRP3 during the development of hypoxic ischemic brain injury in neonatal rats.Inhibition of MMP-9 could significantly reduce the expression of NLRP3,Caspase-1,IL-1?and GSDMD.The NLRP3/MMP9 signaling pathway might be an important convergence point affecting occurrence of cell pyroptosis.3.AS-IV therapy can alleviate hypoxic-ischemia-induced brain injury in neonatal rats and inhibit the inflammatory response of hypoxic-ischemia brain tissue and HT22 hippocampal neurons in neonatal rats,which might be mediated by the NLRP3/MMP-9 inflammatory body axis.