Establishment Of Hypoxia Stress Mouse Model And Alternative Splicing Event Analysis

Hypoxia refers to the low level of oxygen in the physical sense or the lack of oxygen demand in the sense of physiological function.Neurons are the basic units of the structure and function of the nervous system.As an important structure of the brain responsible for learning and storing memory,hippocampal tissue is extremely sensitive to hypoxia.Cerebral hypoxia can lead to the degeneration of neurons and glial cells,cause local neurological dysfunction and brain damage,and lead to stroke,Parkinson’s and other diseases.Alternative splicing is very important in eukaryotes and is an important cause of biological functional complexity and protein diversity.Abnormal alternative splicing will lead to many diseases.However,most of the studies on the regulatory mechanism of hypoxia-mediated alternative splicing are based on animal or cell models.In recent years,it has been reported that hypoxia experimental models are established in many ways,such as high pressure,atmospheric pressure,intermittence and persistence,but the model biology and treatment conditions of these models are different,and the model is still in the stage of exploration and improvement.Therefore,the establishment of hypoxia model as the basic work is extremely important.The purpose of this study was to establish an effective animal model and cell model of mice under intermittent hypoxia stress,and to explore the changes of alternative splicing patterns of pre-mRNA in hippocampal tissue and neurons of mice under intermittent hypoxia stress.The details are as follows:1.Establishment of intermittent hypoxia stress mouse model: SPF male BALB/c inbred strain mice were selected to establish a mouse model of intermittent hypoxia,with21% oxygen and 7% oxygen alternating for 10 minutes,4 hours a day for 6 weeks.The animal model was evaluated by observing the pathological changes of hippocampal tissue under hypoxia stress by HE staining,detecting the activity of oxidation factors by ELISA method,and detecting the expression level of hypoxia marker gene mRNA and protein by RT-q PCR and Western Blot.Using the normoxic group as control,with the prolongation of hypoxia stress cycle,the morphology of pyramidal cells in CA1 region of hippocampal tissue became irregular,the cells arranged sparsely,and showed varying degrees of damage;The activity of SOD in serum and hippocampal tissue of mice in different hypoxia treatment groups decreased;Compared with the normoxic control group,the mRNA expression levels of HIF-1α,HO-1,Vegfa and Tpi1 in the hippocampus of intermittent hypoxia mice showed an upward trend;the protein expression levels of HIF-1α,Ca9,HO-1 and EPO in the hippocampal tissue of intermittent hypoxia mice showed an upward trend compared with the normoxic control group,indicating that hypoxia caused oxidative damage to hippocampal tissue.2.The mouse hippocampal neuron cell line HT22 was selected as the research object to establish the hypoxia cell model.In the three-gas incubator,the HT22 cells were treated with 1% hypoxia for 12 h(H12),24 h(H24),36 h(H36)and 48 h(H48),respectively,and corresponding normoxic group was used as control for each treated group.The cell viability was measured by MTT and CCK-8 methods,the changes of ROS content were observed by DCFH-DA probe,and the hypoxia-related gene mRNA and protein expression levels were detected by RT-q PCR and Western Blot methods to evaluate the cell model.Using normoxic group as control,the results showed that the cells conform to the basic law of cell growth;the morphology of H12 and H24 cells had no obvious change,but the synapses of H36 cells,especially H48,began to elongate,the shape was irregular,and the number of dead cells increased,the survival rate of H12 and H24 increased,the survival rate of H36 cells decreased,and the survival rate of H48 decreased significantly;The fluorescence brightness of H12,H24 and H36 had no significant change,but that of H48 was significantly enhanced,indicating that the content of ROS increased;The mRNA expression levels of HIF-1α,HO-1,Vegfa,Mrps17,Pgk1,Tpi1,Trp53,Fosl1,Il-6 and Bax in different hypoxia groups showed an upward trend;The protein levels of HIF-1α,Ca9,HO-1 and Bax showed an upward trend.3.Verification of the differential pre-mRNA alternative splicing events: In order to further determine the effectiveness of the intermittent hypoxia animal model and hypoxia cell model,semi-quantitative RT-PCR was used to detect the changes of exon inclusion of Rbm41,Lrrc45,Edrf1 and NF1 genes.The results showed that hypoxia could promote the exon skipping in splicing of Rbm41,Lrrc45 and Edrf1 variable exons and promote the inclusion of NF1 exons in the hippocampus of mice.and the same results were observed in HT22 neurons,indicating that hypoxia may be involved in the regulation mechanism of alternative splicing.The mentioned results show that this experiment successfully established an intermittent hypoxia animal and cell model,and hypoxia may be involved in the regulation of alternative splicing in the brain.This work provides an experimental platform for elucidating of hypoxia-related regulation mechanism,and is helpful to further understand the effect of hypoxia on alternative splicing,and lays a foundation for subsequent transcriptome sequencing to explore the regulation mechanism of alternative splicing in brain hypoxia injury.