Role And Mechanism Of Cardiac And Cerebral Protection Mediated By LOC339524 And UQCRC1

Objective:Basic diseases,surgical trauma,and intraoperative position changes have different effects on the physiological function of patients.The perioperative period is prone to ischemia-reperfusion injury of the fragile organs including the heart and brain.The stability of intraoperative respiratory and circulatory system directly affects the patient’s postoperative recovery,so how to protect patients and prevent perioperative adverse events is a major challenge for anesthesiologists.LOC339524 is a novel protein that was isolated and identified by the high-performance liquid chromatography-chip/mass spectrometry（HPLC-Chip/MS）technique in the proteomics of the human heart early arrest and perfusion in 2012,and then monoclonal antibody 5-d3 was prepared.Previous studies have found that LOC339524 may be involved in the development of embryonic rat heart and regulates the proliferation and proliferation of H9C2cardiomyocytes.In addition,our previous study also confirmed that LOC339524 is involved in the regulation of cerebral inflammation,and its high expression can significantly reduce the microglial cell inflammation caused by Lipopolysaccharide（LPS）.These all suggest that LOC339524 has important functions and may be involved in organ protection.However,the role and mechanism of LOC339524 in myocardial ischemia/reperfusion injury is unclear,and its possible signaling pathway for alleviating BV-2 inflammatory response in microglia has not been identified.Therefore,this study aims to investigate explore the protective effect and mechanism of LOC33524 over-expression in myocardial oxygen-glucose deprivation/reoxygenation injury,and the possible pathways to reduce inflammatory response by establishing LOC33524 overexpression in AC16 and bv-2 cell lines.It provides a new target for the development of specific cardiac and cerebral protective drugs.In addition,Ubiquinol-cytochrome-c reductase complex core protein 1（UQCRC1）is another protein that may protect myocardium.Previous studies suggested that changes in UQCRC1 expression directly affected mitochondrial function,which was characterized by mitochondrial dysfunction often accompanied by UQCRC1 down-regulation,and UQCRC1overexpression increased the respiration rate of mitochondrial complex III,indicating that UQCRC1 was important for mitochondrial function maintenance.Several studies have shown that mitochondria is a common target for multiple myocardial protection including pretreatment,but mitochondrial proteins involved in preconditioning to mediate endogenous protective effects of the myocardium are still unclear.The purpose of this study is to screen out the target proteins that may have protective effects in the mitochondrial inner membrane（MIM）and confirmed in vitro via constructing the myocardial protection model by long-term injection of Diazoxide（DZ）and isolating MIM protein.In order to further explore the mechanism of endogenous protective molecules generated by cardiomyocytes during pretreatment and to develop perioperative cardiomyocytes protective drugs.PartⅠ Protective effect and mechanism of LOC339524 on myocardial ischemia/reperfusion injury.Methods of Part Ⅰ:1.The possibility of LOC339524 protein expression and the influence of hypoxia on its expression were explored.1.1 To explore whether LOC339524 can exogenously express protein,the FLAG-tagged LOC339524 expression plasmid was constructed and transfected into 293T cells with lipofectamine^TM 3000.The protein was extracted after 48 hours and detected with the FLAG antibody.1.2 To explore whether LOC339524 can exogenously express protein,the heart,brain,spleen,kidney and other major organ protein of adult SD rats were extracted and the expression of LOC339524 protein in the above-mentioned tissues was analyzed by Western Blot after rat could express LOC339524 which was confirmed by fluorescence in situ hybridization（FISH）.2.To investigate the effect of hypoxia on LOC339524 expression.2.1 To investigate the effect of hypoxia on the expression and location of LOC339524 in vitro,Human myocardium AC16 cells were exposed to hypoxia（1%O₂）for24h or treated by 200μmol/L CoCl₂ for 10h to extract RNA and protein.RT-qPCR and Western Blot were used to detect the effect of hypoxia on the expression of LOC339524.At the same time,the cytoplasmic and nuclear proteins of AC16 cells in normoxia and hypoxia were extracted respectively,then the expression position of LOC339524 protein under hypoxia was analyzed by Western Blot.2.2 To investigate the effect of hypoxia on the LOC339524 expression of myocardium and serum exosomes in vivo,adult SD rats were placed in an artificial experimental chamber with a simulated altitude of 5000m in the high-altitude environment for0-72h.Then rats were sacrificed,and serum exosomes and myocardial tissues were collected.The expression of LOC339524 mRNA in the myocardium was detected by RT-qPCR.The expression of LOC339524 protein in exosomes and myocardium was evaluated by Western Blot.This explores the effect of hypoxia on the expression of LOC339524 in vivo.3.Whether Nonsense-Mediated mRNA Decay（NMD）played a regulatory role in LOC339524 were investigated.3.1 LOC339524 gene and protein levels were detected after activation or inhibition of the NMD pathway.high expressed lentivirus（LV-TOPO-UPF1/RENT1）of key factor up-frameshift protein 1（UPF1/RENT1）in the NMD pathway was constructed and transfected into AC16,H9C2,HMO6 and LO2 cells,and these cell lines with UPF1/RENT1 high expression were constructed.LOC339524 gene and protein levels were detected by RT-PCR and Western Blot respectively after siRNA silencing UPF1/RENT1 expression in the above four cells.3.2 The possible mechanism of LOC339524 escape NMD regulation was deliberated.LOC339524 high expressed lentivirus（LV-TOPO-LOC339524）was transfected into AC16cells to establish a stable cell line with LOC339524 high expression.The expression of phosphorylated eukaryotic translation initiation factor 2α（p-eIF2α）was analyzed by Western Blot to identify the possible mechanism of LOC339524 escape from NMD regulation.4.The following methods were used to investigate the protective effect and possible mechanism of LOC339524-mediated oxygen and glucose deprivation/Reoxygenation（OGD/R）.4.1 The optimal time point of OGD/R was screened.AC16 and AC16-LOC339524^+/+cells were separately deprived of oxygen and glucose for 0/3/6/9/12/24/48/72h then reoxygenated for 12h,and cells were harvested and stained by Annexin V-FITC/PI or mitochondrial membrane potential JC-1 to evaluate the apoptosis of oxygen-glucose deprivation at a different time/reoxygenation for 12h through flow cytometry.In subsequent studies,the most significant OGD/R time points were detected by the above two methods.For the selected time point,the expressions of apoptosis-related proteins including cleaved caspase3,poly ADP-ribose polymerase（PARP1）and bcl-2 were detected by Western blot.4.2 Transcriptome sequencing（RNA-seq）analyzed the differential gene and signaling pathways（focusing on the PI3K and MAPK pathways）after LOC339524 overexpression,and explored the interaction network between differential genes initially,which provided references for the screening of subsequent signaling pathways.The reliability of RNA-seq was identified by RT-qPCR and Western Blot.4.3 The effect of LOC339524 high expression on PDGFR/PI3K/Akt pathway protein was investigated.Under the guidance of RNA-seq,the cells were subjected to OGD/R injury,and the protein was extracted.Western blot was used to analyze the total and phosphorylated proteins of PDGFRα（Tyr754）,PDGFRβ（Tyr751）,PIK3C2B（Y228）,Akt（Thr308）,Akt（Ser473）and GSK3β（Ser9）in PDGFR/PI3K/Akt pathway.4.4 The possible mechanism of LOC339524’s myocardial protective effect was inversely confirmed with using PDGFR and PI3K inhibitors.Prior to cell OGD/R injury,1umol/L Crenolanib（PDGFR inhibitor）and 10umol/L LY294002 or 100nmol/L Wortmannin（both were PI3K specific inhibitors）were used to inhibit the activation of the corresponding proteins.The effects of the above inhibitors on apoptosis were evaluated by flow cytometry.Western Blot was used to detect changes in phosphorylation levels of PDGFRα（Tyr754）,PDGFRβ（Tyr751）,PIK3C2B（Y228）,Akt（Thr308 and Ser473）and GSK3β（Ser9）as well as changes in cleaved caspase3,PARP1-83kd,and bcl-2.It reconfirmed the possible mechanism on myocardial protection of LOC339524.Results of Part Ⅰ:1.LOC339524 could encode the protein.Under normal circumstances,the endogenous expression of LOC339524 protein could be detected in the main tissues of rats with 5-D3 antibody,and it is mostly expressed in the liver,heart,and uterus.In addition,the FLAG antibody detected a protein band at²9kDa,indicating that LOC339524 could be encoded protein exogenously.2.Hypoxia induces LOC339524 expression and nucleus to cytoplasmic transfer.In vitro,both physical hypoxia（1%O₂）and chemical hypoxia-induced by CoCl₂ could induce LOC339524 high expression in AC16 cells and transferred from nucleus to cytoplasm.In vivo,LOC339524 expression in rat myocardial tissue was increased with the prolongation of hypoxia.When hypoxia reached 72h,its expression in serum exosomes was increased significantly.3.LOC33924 may escape nonsense-mediated mRNA degradation（NMD）by regulating p-eIF2αexpression.LOC33924 gene and protein levels did not conform to the theoretical decrease or increase in NMD regulation theory,whether UPF1/RENT1 was highly expressed by lentivirus or silenced by siRNA.When LOC339524 was highly expressed,p-eIF2αexpression was significantly higher than the control group.4.LOC339524 could significantly reduce the OGD/R injury in AC16 cells by regulating the PDGFR/PI3K/Akt pathway.4.1 Oxygen glucose deprivation 24h/reoxygenation 12h was the most appropriate time point for LOC339524 to protect AC16 cardiomyocytes.Annexin V-FITC/PI and JC-1 detected that apoptosis rates increased slightly with the prolongation of oxygen-glucose deprivation time from 0 to 9h/reoxygenation 12h,and apoptosis rates in LOC339524 overexpression group were slightly lower than those in the control group（no statistical significance）.When oxygen and glucose were deprived of 12²4h/reoxygenated for 12h,the apoptosis rate was significantly increased.Currently,the apoptosis rate of the LOC339524 over-expression group was significantly lower than control group and was most significant in the oxygen-glucose deprivation 24h/reoxygenation 12h.Although the LOC339524 overexpression group showed obviously protective effect when the oxygen-glucose deprivation 48⁷2h/reoxygenation 12h,considering that the apoptotic rate of the whole cell at this time was extremely high,the oxygen-glucose deprivation24h/reoxygenation 12h was finally taken as the time point of subsequent cells injury.At this time point,the expression of cleaved caspase3 and its downstream protein PARP1-83kd in LOC339524 high expression group were significantly lower than that of control group,and the protective protein bcl-2 was significantly increased.4.2 LOC339524 protected AC16 cells from OGD/R via the PDGFR/PI3K/Akt pathway.4.2.1 RNA-seq results indicated that 16 genes of the PI3K pathway and 15 genes of the MAPK pathway were highly expressed after LOC339524 over-expression.4.2.2 Western blotting confirmed that LOC339524 overexpression significantly increased the phosphorylation levels of PDGFR alpha（Tyr754）,PDGFR beta（Tyr751）,PIK3C2B（Y228）,Akt（Thr308 and Ser473）and GSK3β（Ser9）in the PI3K pathway,resulting in significantly less Annexin V-FITC/PI positive cells and decreased mitochondrial membrane potential JC-1 after OGD/R injury than those of the control group.4.2.3 Although the overall apoptosis increased with Crenolanib pretreatment for 1 h before OGD/R inhibited the activation of PDGFRαand PDGFRβ,the LOC339524overexpression group still had the effect of alleviating apoptosis,ie compared with the Control+Crenolanib group,p-PDGFRα（Tyr754）was still significantly increased,the expression of activated caspase3 and PARP1-83kd was significantly decreased,bcl-2protein was significantly increased,and the proportion of cells with decreased mitochondrial membrane potential was also significantly lower than that of the control group.4.2.4.Prior to OGD/R,LY294002 or Wortmannin was used to inhibit Akt activation.Although p-Akt（Thr308 and Ser473）and p-GSK3β（Ser9）were importantly decreased,the LOC339524 overexpression group was significantly higher than the control group.The expression of activated caspase3 and parp1-83kd were less expressed than the control group,bcl-2 was more expressed than the control group,and Annexin V-FITC/PI positive cells were significantly lower than the control group.The expression of bcl-2 was higher than that of the control group.Part Ⅱ Mechanism of LOC339524 inhibiting lipopolysaccharide-induced BV-2microglia inflammatory response.Methods of Part Ⅱ:5.Based on bioinformatics analysis and transcriptomics,the mechanisms of LOC339524 to alleviate BV-2 inflammation in microglia were investigated.5.1 The effect of LOC339524 on LPS-induced inflammatory mediators was tested.Immunofluorescence detected inflammatory mediator cyclooxygenase-2（COX-2）and inducible nitric oxide synthase（iNOS）with 100ng/mL LPS treatment in BV-2 cells after highly expressed LOC339524 or silenced LOC339524 by siRNA.5.2 The binding of NF-κB to the promoter of LOC339524 and its effect on LOC339524 expression were discussed.The wild types of 1509-1520,2000-2009,2758-2769 and 2971-2981 regions of LOC339524 promoter and their corresponding mutant vectors were constructed,then transfected into 293 cells and stimulated with 25 ng/mL TNF-αfor 24h or pre-treated for 1h with 0.5μmol/L PDTC（IκBαspecific inhibitor）for 1h before stimulation.Dual-luciferase reporter assay evaluated the binding of NF-κB to the promoter region of LOC339524 and its effect on LOC339524 expression.5.3 The possible mechanisms of LOC339524 in reducing LPS-induced inflammatory response were explored in vitro.5.3.1 To explicit the effect of LOC339524 on nuclear displacement of NF-κB/p65,BV-2 cells were stimulated by 100ng/mL LPS for 3h after changing LOC339524 expression,and the nuclear factor-κB/p65（NF-κB/p65）expression were observed by immunofluorescence.Then cytosolic and nuclear protein was extracted,Western Blot analyzed the NF-κB/p65 expression in cytoplasm and nucleus.5.3.2 To clarify whether the anti-inflammatory effects of LOC339524 were related to Src/NF-κB and p38MAPK,BV-2 cells were stimulated by 100ng/mL LPS for 30min after changing LOC339524 expression,Western Blot detected the phosphorylation of Src and inhibitor of nuclear factor-κB kinase（Ikk）,nuclear factor-κB inhibitor alpha（IκBα）and NF-κB/p65 in NF-κB pathway.Prior to LPS stimulation,IκBαspecific inhibitor PDTC（0.5μmol/L）or p38 MAPK specific inhibitor SB203580（20μmol/L）was pretreated for 1h,and phosphorylation of IκBα,NF-κB/p65 and p38 MAPK were analyzed by Western Blot.Results of Part Ⅱ:5.LOC339524 reduced inflammatory factors via Src/NF-κB and p38 MAPK pathways.5.1 LOC339524 reduced the expression of inflammatory mediators.LOC339524overexpression visibly reduced LPS-induced COX-2 and iNOS expression in BV-2 cells and nuclear translocation of NF-κB/p65,while LOC339524 silenced the opposite.5.2 NF-κB bound to the 1509-1520 region of LOC339524 promoter and activated the expression of this gene.After TNF-αstimulation,The OD value of LOC339524-1520promoter vector（LOC-1520+TNF-αgroup）was significantly higher than that of non-stimulation group（LOC-1520+normal group）and mutant group（LOC-MUT-1520+TNF-αgroup）,while the OD value of other promoter regions did not change obviously.5.3 Src/NF-κB and p38MAPK pathways were possible mechanisms for LOC339524 to reduce inflammation5.3.1 LOC339524 distinctly inhibited Src activity.Phosphorylation of Src（Tyr 527）（inhibited Src after phosphorylation）by LPS stimulation was significantly increased,while no effect on phosphorylation of Src（Tyr 416）（activated Src after phosphorylation）.After LOC339524 silence,Src（Tyr 527）phosphorylation was reduced,and Src（Tyr 416）phosphorylation remained unchanged.5.3.2 LOC339524 evidently decreased the activation of NF-κB and p38MAPK pathway induced by LPS.Ikkα/β,IκBα,and NF-κB/p65 phosphorylation were significantly reduced compared with the con+LPS group;when LOC339524 was silenced,Ikkα/β,IκBα,and NF-κB/p65 phosphorylation were significantly higher than the NC+LPS group.Prior to LPS stimulation,PDTC-specific inhibition of IκB activation significantly reduced IκB and NF-κB/p65 phosphorylation by LOC339524 silence.Whereas SB203580specifically inhibited p38MAPK activation prior to LPS stimulation,it notably reduced phosphorylation of p38MAPK by LOC339524 silence.Part Ⅲ Study on the role of UQCRC1 in myocardial protection of rats pretreated with drugsMethods of Part Ⅲ:6.Screening and preliminary validation of target proteins in the mitochondrial inner membrane that may be involved in preconditioning-mediated myocardial protection.6.1 Myocardial protection model in rats was established.A single intraperitoneal injection of 50mg/kg isoproterenol hydrochloride（ISO）was used to establish rat myocardial injury model,or continuous intraperitoneal injection of 20 mg·kg^-1·d^-1 diazoxide（DZ）for different time（3d⁸W）before constructing rat myocardial injury model,or in combination with 80 mg·kg^-1·d^-1 5-hydroxydecanoic acid（5-HD）,the specific grouping were as follows:1)Group 1 was the control group.2)Group 2 was ISO group:only subcutaneous injection of ISO（50mg/kg dissolved in saline）.3)Group 3 was the DZ+ISO group:before ISO injection,rats were intraperitoneally injected with 20 mg·kg^-1·d^-1DZ every day for 3 consecutive days.4)In groups 4,5,6,7 and 8,rats were intraperitoneally injected with DZ(20 mg·kg^-1·d^-1,once daily)for 1,2,4,6 and 8 weeks respectively.An equal volume of saline was injected during all other groups of administration.5)In groups 9,5-HD was given together with DZ for 8 weeks.6.2 The rats were sacrificed,collected hearts,and screened the target protein in MIM.1)Partial myocardium was embedded in paraffin and stained with TUNEL to observe the apoptosis of myocardial tissues in the above groups.2)Partial myocardium was used to extract MIM protein and analyzed differential MIM proteins in DZ 0w,4w,and 6w by two-dimensional fluorescence differential gel electrophoresis（2D-DIGE）combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS）.3)Western Blot detected UQCRC1 expression after DZ treatment at 0w,4w,and 6w.6.3 To further clarify the myocardial protection of UQCRC1 in vitro,H9C2 cells were treated with 100μmol/L DZ,or 100μmol/L DZ and 100μmol/L 5-hydroxydecanoic acid（5-HD）for 24h.Western Blot evaluated t UQCRC1 expression in the above group.Then the cells were subjected to OGD/R injury,Hoechst33342 staining was used to detect the apoptosis of cells.Before H9C2 cells were subjected to OGD/R injury,UQCRC1 expression was silenced by siRNA,and Hoechst33342 staining was used to further evaluate the apoptosis of cells.Results of Part Ⅲ:6.UQCRC1 may be an endogenous protective target protein produced by cardiomyocytes during pretreatment.6.1 The rat myocardial protection model was successfully established.The percentage of TUNEL-positive cells in the ISO group was markedly higher than that in the control group.When DZ was injected for a short time（<4w）,a significant protective effect was shown,i.e.the proportion of TUNEL positive cells was substantially reduced compared to the ISO group.When the injection time of DZ was more than 4 weeks,the protective effect of DZ not only disappeared but also showed the damaging effect,that is,the proportion of TUNEL positive cells increased dramatically.Combining 5-HD with DZ partially reversed the loss of protection caused by the long-term effect of DZ.6.2 In the differential protein of myocardial MIM after long-term treatment with DZ,UQCRC1 expression was consistent with the change of myocardial protection after DZ treatment.When the duration of DZ injection<4w,UQCRC1 expression in myocardial MIM protein was memorably increased,while it was significantly reduced when the time was>4w.6.3 UQCRC1 protected H9C2 cells from OGD/R injury.Compared with the control group,the DZ 24h group significantly increased UQCRC1 expression,while in the combined use of 5-HD,this effect disappeared.Correspondingly,after OGD/R injury,the proportion of cells stained with strong blue by Hoechst33342 in DZ 24h group was markedly lower than the control group,and increased significantly after 5-HD.,The cells were subjected to OGD/R injury after silencing UQCRC1 expression by siRNA and the proportion of cells stained with strong blue by Hoechst33342 in siUQCRC1 group was apparently higher than that in the NC group.Conclusions:1.It is the first time to confirm that LOC339524 annotated as LncRNA can encode a protein,and this protein which is expressed in major tissues and organs has important functions.2.Hypoxia induces high expression of LOC339524 in the myocardium of SD rat and AC16 cells.LOC339524 high expression attenuates the OGD/R injury and increases the survival rate of cardiomyocytes by activating the PDGFR/PI3K/Akt pathway.3.LOC339524 alleviates LPS induced inflammatory response of BV-2 microglial cells by inhibiting Src activity,and then inhibiting two downstream signaling pathways.One is the NF-κB pathway and the other is the p38MAPK pathway,which were ultimately achieved by reducing the expression of the inflammatory mediators iNOS and COX-2.4.UQCRC1,a differential protein screened from rat cardiac mitochondrial inner membrane proteins,may be a target protein that myocardial cells actively produce endogenous protection during drug pretreatment.