| Background and Objectives:Cerebral ischemia reperfusion(CIR)injury is a severe brain injury caused by the restoration of blood flow after an ischemic stroke.Injuries from CIR often deteriorate a patient’s neurological functions,cause great physical and mental harm to the patient,and place burdens of treatment on their family and country.However,at present,the prevention and treatment of CIR injury is very limited,and more research is urgently needed to develop new and effective drugs or intervention methods.Biliverdin(BV)has been shown to be a potent antioxidant with anti-apoptotic and anti-inflammatory cytoprotective effects.As shown in a rat model of CIR injury,BV reduces oxidative stress,inhibits inflammation,and reduces cerebellar infarct volume.However,there is a lack of knowledge about the mechanism underlying this protection.Long non-coding RNA(LncRNA)and microRNA(miRNA)are two of the most commonly found non-coding RNAs(ncRNAs)that play regulatory roles.LncRNA/miRNA/mRNA play an important role in the regulation of CIR injury in a variety of ways.However,to date,the roles and mechanisms of BV-regulated ncRNAs and their corresponding molecular networks in CIR injury are not well understood.Through in vitro and in vivo experiments,this study will investigate the protective effect of biliverdin on CIR injury and explore the interaction between relevant molecules in the regulatory axis of LncRNA H19/miR-181b-5p/Esml and the mechanism of this regulatory axis in the treatment of CIR injury by biliverdin for the purpose of providing a theoretical and research basis for the prevention and treatment of CIR injury.Methods:Part Ⅰ.Suture-occluded rat models of middle cerebral artery occlusion/reperfusion(MCAO/R)were established in vivo,and reperfusion was performed 2 h after ischemia and treated with 35 mg/kg BV.24 h after reperfusion,TTC staining was used to assess cerebral infarct volume,Nissl staining and TUNEL staining were used to evaluate cell damage and apoptosis in the ischemic penumbra area,respectively.After 6 h of reperfusion,we detected LncRNA H19 and miR-181b-5p expression in the ischemic penumbra of the sham group and the MCAO/R group using the FISH assay,and expression of Esml and neuron-specific markers(NSE)using immunofluorescence.Expression of LncRNA H19,miR-181b-5p and Esm1 mRNA,as well as Esm1 protein and apoptosis related proteins Cleaved caspase-3(C-caspase-3),Bax and Bcl-2 in the ischemic penumbra of each group were detected by RT-qPCR or Western blot.In vitro,the oxygen and glucose deprivation/reoxygenation(OGD/R)model of neonatal rat cortical neuron was established.After 2 h hypoxia and glucose deficiency culture,the neuron was reoxygenated and treated with 2 μg/ml BV.Cell proliferation was assessed by EdU staining,cell viability was determined with CCK-8,cell apoptosis was detected by flow cytometry.The expression of LncRNA H19,miR-181b-5p and Esm1 mRNA and the expression of Esm1 protein and apoptosis-related proteins C-caspase-3,Bax and Bcl-2 were detected by RT-qPCR and Western blot,respectively.Part Ⅱ.In vivo,overexpressed LncRNA H19 and its negative control adenovirus were injected into the lateral ventricle of rats to up-regulated LncRNA H19.MCAO/R model was constructed and treated with BV.Cerebral infarct volume,cell damage,and cell apoptosis were assessed by TTC staining,Nissl staining,and TUNEL staining,respectively,24 h after reperfusion.6 h after reperfusion,the expression of LncRNA H19,miR-181b-5p and Esm1 mRNA and the protein expressions of Esm1 and apoptosis-related molecules C-caspase-3,Bax and Bcl-2 in ischemic penumbra tissues were detected by RT-qPCR and Western blot,respectively.In vitro,first,the binding relationship between LncRNA H19 and miR-181b-5p was verified by an RNA immunoprecipitation(RIP)assay in neurons.Second,LncRNA H19 and miR-181 b-5p,as well as miR-181b-5p and Esm1 were tested for their targeting regulation in 293 T cells using a dual reporter gene assay.Finally,according to the different group treatments,the overexpressed LncRNA H19,the overexpressed or knockdown miR181b-5p,the overexpressed or knockdown Esml and their corresponding negative control adenovirus were transfected or co-transfected into neurons to regulate the expression of the target molecule.OGD/R model was constructed and then treated with BV.EdU staining,CCK-8 assay and flow cytometry were used to evaluate the cell proliferation,cell viability and apoptosis of neurons,respectively.Moreover,the expression of miR-181b-5p and Esm1 mRNA and the expression of Esm1 protein and apoptosis-related proteins C-caspase-3,Bax and Bcl-2 were detected by RT-qPCR and Western blot,respectively.Results:Part Ⅰ.In vivo,BV reduced the cerebral infarct volume,alleviated the injury and apoptosis of nerve cells in the ischemic penumbra,inhibited the expression of the proapoptotic proteins C-caspase-3 and Bax and promoted the expression of the antiapoptosis protein Bcl-2 in the ischemic penumbra.FISH assay confirmed that LncRNA H19 and miR-181b-5p could be expressed in the ischemic penumbra cortex,and the MC AO/R group showed increased levels of LncRNA H19 and decreased levels of miR181b-5p.Immunofluorescence staining showed that Esm1 could be co-expressed with NSE and expression of Esm1 increased in the ischemic penumbra cortex of the MCAO/R group.In ischemic penumbra tissue,BV upregulated miR-181b-5p and downregulated LncRNA H19 and Esm1 mRNA and protein expressions.In vivo,BV promoted the cell proliferation,increased cell viability,decreased the rate of apoptosis,inhibited the expression of the pro-apoptotic proteins C-caspase-3 and Bax and promoted the expression of the anti-apoptotic protein Bcl-2 in neurons that were injured by OGD/R.Further,BV upregulated miR-181b-5p expression and downregulated LncRNA H19 and Esm1 mRNA and protein expression.Part Ⅱ.In vivo,the effect of LncRNA H19 overexpression was opposite to that of biliverdin and was manifested as increased infarct size,increased injury and apoptosis of neural cells in the ischemic penumbra,upregulated expression of pro-apoptotic protein,C-caspase-3 and Bax and down-regulated expression of the anti-apoptotic protein Bcl-2 in ischemic penumbra tissue.Furthermore,miR-181b-5p was downregulated and Esm1 mRNA and protein expression were upregulated in ischemic penumbra tissues.In vitro,RIP experiments confirmed that LncRNA H19 could sponge and interact with miR-181b-5p.Dual luciferase reporter gene assay confirmed that LncRNA H19 can target miR-181b-5p,and Esm1 is the direct target of miR-181b-5p.Overexpression of LncRNA H19 and Esm1,knockdown of miR-181b-5p reversed the protective effects of BV,resulting in reduced cell proliferation,decreased cell viability,increased apoptosis rate,and upregulated expression of the pro-apoptotic protein Ccaspase-3 and Bax and downregulated the expression of the antiapoptotic protein Bcl2.Overexpression of LncRNA H19 and overexpression of miR-181b-5p adenovirus were co-transfected into cells,and the adverse effects of overexpression of LncRNA H19 could be reversed by overexpression of miR-181b-5p.Knockdown of miR-181b5p and knockdown of Esm1 adenovirus were co-transfected into cells,and the adverse effects of knockdown of miR-181b-5p could be reversed by knockdown of Esm1.All showed increased cell proliferation,increased viability,decreased apoptosis rates,down-regulated expression of pro-apoptotic proteins C-caspase-3 and Bax,and upregulated expression of anti-apoptotic protein Bcl-2.In addition,LncRNA H19 negatively regulated the expression of miR-181b-5p and positively regulated Esm1.miR-181b-5p can negatively regulate the expression of Esm1.Conclusions:1.Biliverdin could inhibit apoptosis and alleviate reperfusion injury from cerebral ischemia.Biliverdin could upregulate the expression of miR-181b-5p and downregulate the expression of LncRNA H19 and Esm1.2.The mechanism by which biliverdin ameliorates cerebral ischemia reperfusion injury could be the upregulation of miR-181b-5p expression through downregulating LncRNA H19 and then targeting downregulation of Esm1 to inhibit apoptosis. |
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