| Deep hypothermic circulatory arrest(DHCA)refers to the process of cardiac surgery and cardiopulmonary bypass(CPB),which can significantly reduce the body’s metabolic rate by lowering the body’s temperature in order to obtain a short and safe pause time and provide a clear vision and effective operation space for surgeons.Its research and application originated in the 1950 s.It is a common technique in cardiac surgery to deal with complex preceding heart disease and aortic arch diseases.DHCA process leads to abnormal metabolism of multiple organs and multiple organ damage.With the improvement of surgical techniques,the success rate of deep hypothermic circulatory arrest surgery and the occurrence of various complications have been significantly improved.Brain nervous system complications are still one of the main complications of this technique.The therapeutic effect of such neurological complications is limited and the treatment cycle is long,which seriously affects the surgical effect of cardiac surgery and the long-term quality of life of patients,increases the financial burden of patients and their families,and becomes a bottleneck to further improve the surgical effect.In the course of deep hypothermic circulatory arrest,selective anterograde cerebral perfusion and retrograde cerebral perfusion have been used.Compared with DHCA alone,perfusion brain protection can significantly reduce the incidence of neurological dysfunction [1,2].However,various perfusion techniques still have their limitations and can not achieve satisfactory clinical results.According to research reports,the incidence of new cerebrovascular events is 5.4% in patients undergoing hemiarthroplasty(non-complex aortic arch surgery)assisted by deep hypothermic circulatory arrest,and the incidence of temporary neurological dysfunction after deep hypothermic circulatory arrest is as high as 25% [3].The incidence of neuropsychological dysfunction within 12 weeks after deep hypothermic circulatory arrest cardiac surgery can be as high as 55% [4].And the incidence of neurological dysfunction in children is higher [5].With the maturity of surgery and perfusion technology,there is less and less space to improve brain protection by improving surgery and related assistant technology and shortening operation time.Therefore,it is urgent to develop new treatment methods to reduce the incidence of neurological complications after deep hypothermic circulatory arrest.Long non-coding RNA(lnc RNA)is a kind of RNA molecule whose length is more than 200 nt.It does not encode proteins,and its expression is tissue-specific and spacetime-specific.Lnc RNA is a set of regulatory sequences that play a role at transcriptional,post-transcriptional and epigenetic levels and participate in many physiological functions such as gene transcriptional regulation,epigenetic regulation,chromatin modification,ontogenetic regulation,cell programming and maintenance of stem cell pluripotency [10].Lnc RNA is highly expressed in the central nervous system and plays an important role in regulating the development of the central nervous system and neurodegenerative diseases [6,7].In recent years,many literatures have suggested that regulation of lnc RNA can enhance the ability of neurons to resist ischemia and hypoxia,revealing that lnc RNA plays an important role in stroke and ischemic brain damage [8-10].Lnc RNA growth arrest specific transcript 5(GAS5)is a tumor suppressive lnc RNA,which can interact with different target proteins and micro RNAs and regulate cell proliferation,differentiation and apoptosis[11-13].The expression of lnc RNA GAS5 is the highest in hippocampus of brain tissue[14].lnc RNA GAS5 inhibits glioma cell proliferation and promotes apoptosis[15,16].It is not clear whether lnc RNA GAS5 participates in the pathophysiological process of DHCA.In this study,we confirmed the relationship between the targeting regulation of lnc RNA GAS5 and micro RNA-23 a in rats by double luciferase assay,and found that the contents of lnc RNA GAS5 and micro RNA-23 a changed after DHCA,and further inhibited the apoptosis of brain neurons during deep hypothermic circulatory arrest by inhibiting the expression of lnc RNA GAS5 in the rat brain,thereby improving the brain damage after DHCA for future clinical application.Treatment provides research directions.Objective: To explore the downstream micro RNA gene that can be targeted by lnc RNA GAS5 direactly,and to explore the expression changes of lnc RNA GAS5 and its downstream micro RNA in brain tissue of rats after DHCA.By regulating the expression of Long noncoding RNA growth arrest specific 5(GAS5)in rat brain,the brain damage caused by deep hypothermic circulatory arrest in rats can be alleviated.METHODS: Molecular biology information technology was used to predict the Targetregulated micro RNAs of rat lnc RNA GAS5,and the correlation between predicted target and LNC GAS5 was confirmed by double luciferase assay.The rat model of deep hypothermic circulatory arrest was established and divided into four groups according to the time after DHCA.The expressions and changes of lnc RNA GAS5 and corresponding micro RNAs in hippocampus and cortex of rats were measured in Sham group,6 hours,12 hours and 24 hours after DHCA.Adeno-associated viruses containing small interfering RNA(si RNA)of lnc RNA were prepared for in vivo transfection and could inhibit the expression of GAS5 of lnc RNA.The rats were then randomly divided into four groups(n=10): Sham group,DHCA group,DHCA+vector(DHCA+AAV NC)group,DHCA+si RNA GAS5 group.Fourteen days before operation,rats were injected into lateral ventricle.The hippocampus tissues of rats were collected 12 hours after DHCA.The contents of mi R-23 a,lnc RNA GAS5,phosphate and tension homology(PTEN),protein kinase B(Akt),phospho-Akt(p-Akt),B-cell lymphoma-2(Bcl-2),Bcl-2-associated protein(Bax)and cleaved-Caspase-3 were determined by Western Blot and q RT-PCR.This is the case.In another parallel experiment,rats in each group(n=10)completed the water maze test 7 days after operation.After 7 days after the end of the water maze experiment,rats were killed under anesthesia and the hippocampal brain tissues were obtained for histological HE staining,Nissil staining and TUNEL staining.In this study,we confirmed the relationship between the targeting regulation of lnc RNA GAS5 and micro RNA-23 a in rats by double luciferase assay,and found that the contents of lnc RNA GAS5 and micro RNA-23 a changed after DHCA,and further inhibited the apoptosis of brain neurons during deep hypothermic circulatory arrest by inhibiting the expression of lnc RNA GAS5 in the rat brain,thereby improving the brain damage after DHCA for future clinical application.Treatment provides research directions.Results: In the double luciferase assay,the luciferase value of pre-micro RNA-23 a cotransfected with lnc RNA GAS5-WT decreased significantly(Mi-23+lnc RNA GAS5 WT vs NC-micro RNA-23+lnc RNA GAS5 WT,P < 0.05),but not in the micro RNA-137 group(P = 0.835)and the micro RNA-21 group(P = 0.707).In hippocampus,the expression of lnc RNA GAS5 increased significantly after DHCA and reached its peak at 12 hours(P<.001,at 6,12 and 24 hours after DHCA vs the sham group,respectively),while in cortex,the expression of lnc RNA GAS5 increased relatively slowly,and showed statistical significance at 12 and 24 hours after DHCA(P= 238,P<.001,and P<.001,at 6,12 and 24 hours after DHCA vs the sham group,respectively).Group).The expression of lnc RNA GAS5 in rat hippocampus at 6,12 and 24 hours after DHCA was significantly higher than that in rat cortex(P<.001,at 6,12 and 24 hours after DHCA,respectively).Compared with sham group,the expression of micro RNA-23 a in hippocampus decreased significantly after DHCA(P<.001,at 6,12 and 24 hours after DHCA);and compared with the content of micro RNA-23 a in cortex,the expression of micro RNA-23 a in hippocampus was much lower than that in cortex(P=.01,P<.001,and P=.012,at 6,12 and 24 hours after DHCA).The blank adeno-associated virus(vector)did not affect the expression of lnc RNA GAS5 and micro RNA-23a(P= 995,the DHCA group vs the DHCA + vector group).Compared with Sham group,PTEN increased significantly in DHCA group and DHCA + vector group(P <.001 the DHCA group vs the sham group,P= 002 the DHCA + vector group vs the sham group),but decreased significantly in DHCA + si RNA GAS5 group compared with DHCA group(P <.001),and the expression of p-Akt/Akt and Bcl-2 increased significantly in DHCA + si RNA GAS5 group compared with DHCA group(P < 0.001),while the expression of Bax increased significantly in DHCA + si RNA GAS5 group compared with DHCA group(P < 0.001).It decreased significantly(P < 0.001).Compared with Sham group,cleaved-Caspase-3 was highly expressed in DHCA group and DHCA+vector(DHCA+AAV NC)s group(P< 0.001),while cleaved-Caspase-3 in DHCA+si RNA GAS5 group was significantly lower than that in DHCA group(P< 0.001).In TUNEL staining,more apoptotic cells were found in DHCA group and DHCA + vector group than in Sham group(P < 0.001),and fewer apoptotic cells were found in DHCA + si RNA GAS5 group than in DHCA group(P < 0.001).HE staining showed that the pathological damage scores of DHCA group and DHCA + vector group were significantly higher than those of Sham group(P < 0.001),while those of DHCA + si RNA GAS5 group were significantly lower than those of DHCA group(P < 0.001),and more neurons survived in DHCA + si RNA GAS5 group than that of DHCA group(P < 0.001).In water maze test,DHCA group and DHCA + si RNA GAS5 group compared with Sham group,the rats of DHCA + si RNA GAS5 group were significantly lower than that of DHCA + vector group(P < 0.001).The escape latency increased significantly from the first day to the fourth day(P<.001,P<.001,P= 008 and P= 001)compared with DHCA group,DHCA + si RNA group 5 was significantly lower than DHCA group due to the down-regulation of lnc RNA GAS5(P<.001,P<.001,P= 008 and P= 001,the DHCA + vector group vs the sham group at day 1,2,3 and 4,respectively).The escape latency from the first day to the fourth day was reduced(P<.001,P<.001,P= 016,and P= 003).Compared with sham group,the number of last platform crossing in DHCA group was significantly reduced(P <.001).Compared with DHCA + si RNA GAS5 group,the number of last platform crossing in DHCA group was also significantly reduced(P <.001).There was no significant difference between DHCA + vector group and DHCA group(P > 0.5).CONCLUSION: We inhibited the expression of lnc RNA GAS5 and regulated the expression of micro RNA-23a/PTEN pathway and related proteins,which can increase the number of surviving neurons,improve the cognitive and memory function of rats after DHCA.Ultimately attenuate cerebral injury induced by deep hypothermic circulatory arrest.It also opens up a new direction for clinical treatment in the future. |
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