Role And Mechanism Of High Mobility Group Box-B1 And Its Acetylation Modification In Hypoxic-Ischemic Brain Injury In Neonatal Mice

Part Ⅰ Role and mechanism of High Mobility Group Box-1 in hypoxic-ischemic brain injury in neonatal miceBackground: Neonatal hypoxic-ischemic encephalopathy(HIE)tends to leave a variety of neurological sequelae.High Mobility Group Box-1(HMGB1)can be passively released by necrotic cells or to actively secreted by stimulated immune cells to trigger inflammatory response as a kind of damage associated molecular pattern.Previous studies have found that peripheral blood HMGB1 levels are increased in children with HIE,and animal models of hypoxic-ischemic brain injury(HIBI)have found that HMGB1 is released from neurocyte in a time-dependent manner.Since inflammation is one of the important contributors to HIBI,HMGB1 may play a crucial role in the damage caused by the neuroinflammatory response in HIBI.Objectives: To determinating the potential role and mechanism of HMGB1 in HIBI by measuring the level of inflammatory cytokines,the expression level of HMGB1 and its downstream signaling pathway-related proteins,the degree of brain damage,and the long-term neurobehavioral function via using different doses of the HMGB1 inhibitor Glycyrrhizin after HI insult.Methods: Rice-Vannucci classic method was used to make HIBI model of postnatal day 7 mice.The first phase of the experiment was set up in five groups,including a sham operation control group(Sham group),HI + PBS group,and HI +GLY(low-dose group(25mg / Kg),medium-dose group(50mg / Kg),and high-dose group(100mg / kg)),intraperitoneal injection,qd × 3d.Neurobehavioral tests were performed at 4-5 weeks of age in mice.Histomorphological changes of brain were observed by HE staining after sacrifice,and myelin sheaths and neurons of hippocampus were observed by immunofluorescence staining.In the second phase,the optimal drug dose was determined according to the experimental results of the first phase.Three groups were set up,including Sham group,HI + PBS and HI + GLY groups,the method of administration was the same as that in phase one.Three days after HI,qRT-PCR and ELISA were used to detect hippocampal inflammatory factor levels;western blot(WB)was used to detect the levels of apoptosis-related proteins,HMGB1 and its downstream signaling pathway-related proteins of hippocampus;immunofluorescence staining was used to observe microglia and astrocytes and immunohistochemistry and nucleoplasmic protein separation were used to examine the spatial localization of HMGB1.Results: The results of the experiment phase I: 1.The results of neurobehavioral tests showed that compared with the Sham group,the HI + PBS group had significantly longer escape latency and a significant reduction in the number of platform crossing and the percentage of target quadrant residence time in the morris water maze test,the residence time and the number of entries into in open arm were significantly reduced in the elevated plus maze test,the number of entries into and the time spent in the central zone but not the distance travelled throughout the entire open-field arena or the average speed were decreased in the open-field test,the abovementioned neurobehavioral defects were significantly improved after the use of the HMGB1 inhibitor,especially in the medium-dose GLY group.2.Brain morphology,HE staining results showed that compared with the Sham group,the brain of HI + PBS group was significantly damaged,the hippocampal and cortical structural integrity was damaged,the cells were arranged loosely,and vacuole degeneration appeared and a large number of nuclear dense inflammatory cells infiltrated.The immunofluorescence results showed the MBP signal intensity around the hippocampus is significantly weakened,the neuron fluorescence signal is interrupted,and the intensity is weakened,after using the HMGB1 inhibitor,the degree of damage was reduced,and the MBP and NeuN fluorescence intensity were increased,especially in the medium-dose GLY group.In summary,it is determined that the 50mg/kg is the optimal drug dose.The results of the experiment phase Ⅱ: 3.Apoptosis test results showed that the number of tunel-positive cells increased significantly in the HI+PBS group compared with the Sham group,while they decreased in the HI+GLY group compared with the HI+PBS group.The expression of hippocampal bcl-2 protein decreased and cleaved caspase-3 protein increased significantly after HI insult.After GLY treatment,the expression of Bax and cleaved caspase-3 decreased significantly compared with the HI+PBS group,while the expression of bcl-2increased significantly.4.Compared with the Sham group,the fluorescence intensity of GFAP and Iba1 in the ipsilateral hippocampus and the surrounding area of the HI +PBS group was enhanced,while the fluorescence intensity of the HI + GLY group were reduced compared to the HI + PBS group.5.The levels of inflammatory cytokines IL-1β,IL-6 and TNF-α were significantly increased in the ipsilateral hippocampus after HI insult,and the inhibition of HMGB1 could reduce the abnormally elevated levels.After HI insult,the protein expression of HMGB1 and TLR4,TLR2,My D88,RAGE and p-NF-κB in the ipsilateral hippocampus increased significantly,and the above protein levels in the HI + GLY group decreased significantly compared with the HI + PBS group.6.The HMGB1 mRNA level in the ipsilateral hippocampus of the HI + PBS group was significantly higher than that of the Sham group,and the use of GLY reversed this change.The results of immunohistochemistry and nucleoplasmic protein separation showed that HMGB1 of hippocampus in Sham group was mainly located in the nucleus,while cytoplasmic localization was obviously appeared in the HI + PBS group and this relocation phenomenon was weakened in the HI + GLY group.Conclusions: HMGB1 may participate in the pathogenesis of HIBI in neonatal mice.Inhibition of HMGB1 can reduce the HI-induced neuroinflammatory response,apoptosis,myelin sheath and neuron damage and brain destruction,thereby improving long-term neurobehavioral function in in neonatal HIBI mice.Part Ⅱ Role and mechanism of microglial SIRT1-mediated HMGB1 acetylation modification in hypoxic-ischemic brain injury in neonatal miceBackground: Microglia are responsible for eliciting early and pronounced inflammatory reactions in the immature brain after HI insult.Damaged neurons after HI insult induce microglial activation and promote the release of inflammatory cytokines,leading to ongoing secondary neuronal and oligodendroglial injury.The results of the part I of this study demonstrated that inhibition of HMGB1 can effectively attenuate neuroinflammation and alleviate brain damage.The acetylated modification of HMGB1 in immune cells affects its spatial localization and secretion.It is unclear whether HMGB1 secretion and acetylated modification exist in microglial cells in HIBI.SIRT1 can modify HMGB1 in other disease models.Therefore,we speculate that the dynamic changes of HMGB1 in microglia after HI insult affect the occurrence and development of neuroinflammation,and SIRT1 may participate in the acetylation modification process of HMGB1.Objectives: To determinate the role of SIRT1 in the pathogenesis of HIBI involved in HMGB1 by detecting the level of inflammation,brain damage,neurobehavioral changes and HMGB1 in microglia via regulating SIRT1 after HI insult in vivo,and to explore the molecular mechanism of this effect through in vitro experiments.Methods: In in vivo experiment part: HIBI models of P7 mice were made and set as Sham group,HI + PBS group,HI + SIRT1 agonist(RES)group,and HI + SIRT1 inhibitor(EX527)+ SIRT1 agonist group,intraperitoneal injections were performed immediately and at 12 h after HI insult.The mice were subjected to cylinder test,forelimb suspension experiment,and open-field test at 4-5 weeks of age;the mice were sacrificed 24 h after HI,and the histomorphological changes were observed by HE staining;inflammatory cytokines levels and HMGB1 mRNA changes were detected by qRT-PCR or ELISA;the protein expression levels of SIRT1 and TLR4 / My D88 / NF-κB were examined by WB;the spatial localization of HMGB1 in microglia was observed by immunofluorescence.In in vitro experiment part: The mouse microglial cell line BV2 was subjected to oxygen glucose deprivation(OGD)to simulate the HI process and was set to the Control group,OGD+DMSO group,OGD+RES group and OGD+EX527+RES group.Inflammatory cytokines and HMGB1 levels were detected by q RT-PCR and ELISA;the protein expression levels of TLR4 / MyD88 / NF-κB were examined by WB;immunofluorescence and nuclear/cytoplasmic fractions detection were used to determine HMGB1 distribution in the nucleus and cytoplasm;modification of HMGB1 by SIRT1 was detected by SIRT1 deacetylase activity test and co-immunoprecipitation;the level of LDH release from the supernatant after co-culture of BV2 conditioned medium and primary neurons was evaluated;and immunofluorescence was used to detect HMGB1 localization in neurons.Results: The results of in vivo experiments: 1.Compared with the HI + PBS group,the range of brain damage in the HI + RES group was reduced.The HE staining results confirmed this improvement,while EX527 co-treatment counteracted this repair effect.2.The results of neurobehavioral tests showed that SIRT1 agonists increased the HI insult-induced reduced number of central zone entries and dwell time,improved impaired forelimb muscle strength and asymmetry of limb activity,and the improvement was partially destroyed by inhibiting SIRT1.3.Activation of SIRT1 decreased the levels of IL-1β,IL-6,and TNF-α induced by HI,and co-treatment with inhibitors counteracted this inhibitory effect;wb results showed that the expression of SIRT1 decreased after HI,the activation of SIRT1 up-regulated the expression of SIRT1,down-regulated the elevated levels of TLR4,My D88 and nuclear NF-κB,in contrast,compared to the HI + PBS group,the SIRT1 inhibitor co-treatment group did not exhibit any protective effect;there was no significant difference in HMGB1 mRNA levels between groups.4.Immunofluorescence results showed that the intensity of Iba1 in the Sham group was weak,and HMGB1 was located in the microglia nuclei.After HI insult,the fluorescence intensity of Iba1 dramatically surged,and HMGB1 was partially localized outside the nucleus;SIRT1 agonists attenuated HI-induced Iba1 intensity and decreased cytoplasmic localization of HMGB1,while SIRT1 inhibitor co-treatment of HMGB1 showed an increased extranuclear distribution.The results of in vitro experiments: 1.The optimal experimental drug concentrations of RES or EX527 were determined to be 25 μM and 100 μM,respectively,and no significant effect on cell viability was observed upon OGD treatment for 3 h.2.Compared with the control group,the levels of IL-1β,IL-6 and TNF-α elevated significantly after OGD,and the expressions of TLR4,MyD88 and nuclear NF-κB protein increased significantly,which was inhibited by SIRT1 agonist treatment.Howerve,pre-administration of SIRT1 inhibitor treatment destroyed the inhibitory effect of the agonist.3.The results of immunofluorescence showed that compared with the control group,the extranuclear localization of HMGB1 significantly increased after OGD;the content of HMGB1 in the supernatant increased,and WB showed that HMGB1 was transferred from the nucleus to the cytoplasm after nucleoplasmic protein separation;SIRT1 agonists increased the retention of HMGB1 in the nucleus,while the pretreatment with SIRT1 inhibitors increased the secretion;there was no significant difference in mRNA levels of HMGB1 at 0h or 12 h after OGD/R.4.Co-IP and WB results showed that after OGD,the expression of SIRT1 decreased,the level of acetylated HMGB1 increased,and the interaction between HMGB1 and SIRT1 weakened.SIRT1 agonist increased the expression of SIRT1 and its deacetylase activity,enhanced the interaction between SIRT1 and HMGB1,reduced the acetylation level of HMGB1 and inhibited HMGB1 from translocating to cytoplasm,although SIRT1 inhibitor pretreatment had no significant effect on the expression of SIRT1 after OGD,it can inhibit SIRT1 deacetylase activity,and aggravate the active secretion of HMGB1.5.Co-culture of BV2 CM and neurons showed that LDH release levels increased significantly after OGD,and SIRT1 agonist treatment inhibited LDH release;immunofluorescent co-standard staining showed that CM from the OGD group caused a significant extranuclear localization of neurons HMGB1,and In the OGD + RES group,this positioning phenomenon was weakened.5.Co-culture of BV2 CM and neurons showed a significant increase in LDH release after OGD,and SIRT1 agonist treatment could inhibit this abnormality;double immunofluorescent labeling showed that CM from the OGD group caused a significant extranuclear localization of neurons HMGB1,while such localization was weakened in the OGD+RES group.Conclusions: After HI insult,microglia can actively secrete HMGB1 to participate in neuroinflammation.SIRT1 participates in the acetylation modification of HMGB1.Increasing the expression or activity of SIRT1 can reduce the acetylation of HMGB1,inhibit the secretion of HMGB1,weaken the downstream inflammatory cascade response,and ultimately improve HI insult-induced brain damage and neurobehavioral impairment.By confining HMGB1 to the nucleus,a new window may be opened for the treatment of neonate HIE.