Effects Of MiR-9-5p/CXC11 Axis In Hydrogen Sulfide Regulation Of Hypoxia-ischemia Brain Damage In Neonatal Mice

Objective:Neonatal hypoxia-ischemia(HI)brain damage is a common brain injury caused by hypoxia and ischemia in perinatal period.Hypoxia and ischemia could occur in perinatal period when blood oxygen concentration is decreased on account of the disturbance of circulation and gas exchange between the maternal and fetus.HI results in neonatal death in most cases,and even the symptom of long-term neurodevelopmental deficits is in perpetuity in surviving neonates.At present,mild hypothermia therapy has become a routine treatment for neonatal HI brain injury.However,there are still intractable problems to be processed such as short treatment time window and poor effect on severe children.Therefore,considering an alternative targeting approach for HI therapy is of great urgency.Hydrogen sulfide(H2S),one of the endogenous neuromodulators,is mainly produced by degradation of L-Cysteine(L-Cys)catalyzed by cystathionine β-synthase(CBS)mainly expressed in central nervous system and cystathionine y-lyase.The enzyme of 3-mercaptopyruvate sulfurtransferase could also catalyze the formation of H2S from the substrate.CBS is mainly expressed in central nervous system.Previous studies have found that H2S involved in regulating neuroinflammatory response after HI brain injury has a neuroprotective effect,but the specific mechanism have not been fully understood.MiR-9 is indispensable in brain development as one of the most expressed miRNAs in adult vertebrate brain.In the rat model of ischemic stroke,the expression of miR-9-5p is significantly decreased in ischemic tissue,and overexpression of miR-9-5p could ameliorate the cerebral infarction and neuronal function.However,the relationship between miR-9-5p and H2S in HI brain injury of newborn mice has not been reported.Therefore,the objective of this study is to explore the effect of miR-95p/CXCL11 axis in hydrogen sulfide regulation of HI brain damage in neonatal mice,so as to provide a new perspective for exploring the neuroprotective effect of H2S on HI brain injury.Results:1.The level of the miR-9-5p is decreased in HI miceFirstly,the HI brain injury model of neonatal mice was established according to the RiceVannucci method,and then the level of miR-9-5p in the cortex of the damaged brain area at 1 and 3 days after HI was detected by qRT-PCR.The results showed that the expression of miR9-5p in the damaged cerebral cortex at 1 and 3 days after HI was significantly decreased compared with the Sham group.BV2 cells were induced by lipopolysaccharide(LPS)to simulate neuroinflammation in vivo.The results showed that the expression of miR-9-5p was also significantly downregulated after LPS treatment for 2 and 4 hours compared with the Control group.2.The level of the miR-9-5p is restored by the L-Cys treatmentL-Cys and aminooxyacid(AOAA,inhibitors of H2S synthetase)were injected intraperitoneally at 1,2,and 3 days after HI.Then qRT-PCR was used to detect the level of miR-9-5p in the cortex of damaged brain areas of each group after 3 days of HI.The results showed that the expression of miR-9-5p was upregulated by L-Cys treatment and downregulated again by L-Cys combined with AOAA treatment.In addition,AOAA also reversed the upregulation effect of L-Cys on miR-9-5p in LPS induced BV2 cells.3.MiR-9-5p inhibitor partially block the neuroprotective effect of L-Cys on HI brain injury in neonatal miceFirstly,miR-9-5p inhibitor and negative control(NC)were injected into the lateral ventricle of neonatal mice 72 h before HI.L-Cys was injected by intraperitoneal injection at 1,2,and 3 days after HI.The results showed that L-Cys treatment could reduce the infarct size and the number of nerve cells apoptosis at 3 days after HI brain injury.Meanwhile,L-Cys treatment could also downregulate the expressions of pro-inflammatory cytokines IL-1β and TNF-α,and upregulate the levels of synapse related proteins(PSD95 and Syn),ARG-1,CBS and H2S.Besides,L-Cys treatment significantly improved the long-term spatial learning cognitive ability of HI mice.However,the neuroprotective effect of L-Cys was reversed by miR-9-5p inhibitor.In addition,L-Cys treatment can downregulate the levels of proinflammatory cytokines IL-1β and TNF-α in LPS-induced BV2 cells.MiR-9-5p inhibitor also reversed the anti-inflammatory effect of L-Cys.4.MiR-9-5p mimics could improve the brain injury induced by HI and upregulate the levels of CBS and H2SFirstly,miR-9-5p mimics and mimics NC were given to lateral ventricle of mice by intraventricular injection 72 h before HI.TTC staining showed that the brain infarct size of the HI+miR-9-5p mimics group decreased compared with the NC group.Western blot and immunohistochemical results showed that the expression of H2S synthetase CBS in HI+miR9-5p mimics group was upregulated.And the level of H2S in the damaged brain area of HI+miR9-5p mimcs group was significantly increased.5.MiR-9-5p targets the regulation of CXCL11mRNA expressionBioinformatics software was used to predict that CXCL11 mRNA was the downstream target gene of miR-9-5p,and the results of dual luciferase reporter genes confirmed that CXCL11 mRNA and miR-9-5p were linked by direct action.Then,compared with the Sham group the expression of CXCL11 mRNA in the damaged cortex of the HI group was significantly upregulated by qRT-PCR,and downregulated by L-Cys treatment.At the same time,CXCL11 levels in LPS-induced BV2 cells were also down regulated by L-Cys treatment.Conclusions:This study showed that the expression of miR-9-5p was downregulated after HI brain injury in neonatal mice.And H2S could ameliorate the neurological impairment after HI brain injury in neonatal mice by upregulating the expression of miR-9-5p.Meanwhile,overexpression of miR-9-5p could improve CBS and H2S levels.MiR-9-5p is involved in the neuroprotective effect of H2S after HI brain injury in neonatal mice through targeted binding to CXCL11 mRNA.