Expression Of Sphk1 In Spontaneous Intracerebral Hemorrhage And Its Related Influencing Mechanism

Objectives:1.To assess the diagnostic value of sphingosine kinase 1(Sphk1)expression levels in spontaneous intracerebral hemorrhage(SICH);2.To study the association between Sphk1 expression levels in the serum of patients with SICH and inflammatory factors,severity of the patient’s condition and prognosis;3.To analyse the effect of Sphk1 on SICH through the regulation of apoptosis-related factors.Methods:This study is divided into three parts.1:Gene screening: We selected Sphk1 for study by analysis of gene microarray sequences(GSE24265 microarray)in perihematomal tissue and corresponding contralateral white and grey matter brain tissue from patients with spontaneous cerebral haemorrhage(4 males and 7 females)in the Gene Expression Omnibus database(GEO),as well as relevant literature reports.2:Clinical study:1.A case-control study was used to select 90 patients with SICH who attended Taizhou people’s hospital from October 2021 to March 2022 as the case group and 40 patients with health check-ups during the same period as the control group.General clinical data included age,sex,smoking history,hypertension,diabetes,and coronary heart disease were recorded for the case group and control group,respectively.2.Glasgow Coma Scale(GCS score)and Glasgow Outcome Scale(GOS score)were recorded for SICH patients at admission and 90 days after illness.3.Real-time fluorescence quantitative PCR(Real Time Quantitative,RTq PCR)was used to detect the expression level of serum Sphk1 in case group and control group,and ROC(Receiver Operating Characteristic Curve)curve was drawn to evaluate the diagnostic value of Sphk1 in SICH.4.ELISA was performed to detect Tumor necrosis factor(TNF-α),C-reactive protein(C-Reactive Protein(CRP),and Sphk1 in the serum of case and control patients,respectively.Reactive Protein(CRP)and oncogene p53 expression levels were measured in the serum of patients with SICH,and the relationship between serum Sphk1 expression levels and CRP,p53,TNF-α,GCS score and GOS score was analyzed by Pearson correlation.3:Cell experiment: 1.Human umbilical vein endothelial cells(HUVECs)cultured in vitro were transfected and divided into three groups: Sphk1-si RNA transfection group,transfection simulant negative control group(NC group)and blank control group(Control group).At the same time,the expression level of Sphk1 in each group was measured.Cell apoptosis was detected by flow cytometry.2.The expression of caspase-3(Cysteinyl Aspatate Speciific PRoteinase-3,Ca spase-3),B-lymphomatous tumor-2(B-Cell Lymphoma-2,Bcl-2)and p53 in m RNA and protein were detected by RT-q PCR and Weste Rn Blot,WB,respectively.Results:1.Clinical findings: 1)The expression level of serum Sphk1 in the case group was significantly higher than that in the control group(1.964±0.762 vs.1.051±0.823,P<0.001).analysis of the ROC curve results showed that the area under the Sphk1 curve was 0.806,with a 95% confidence interval of 0.721-0.890 The sensitivity was 0.644,the specificity was 0.875,and the best cut-off value was 0.519,P<0.05,the difference was statistically significant.2)The expression level of p53 in the serum of SICH patients was significantly higher than that of the control group(0.826±0.049 vs.0.799±0.047,P<0.001),CRP and TNF-α were not seen in the case and control groups(r=0.649,P<0.001);no significant correlation with CRP(r=-0.016,P>0.05);no significant correlation with TNF-α(r=0.020,P>0.05);3)The expression level of Sphk1 in serum of patients in case group was positively correlated with p53,(r=0.649,P<0.001);no significant correlation with CRP(r=-0.016,P>0.05);no significant correlation with TNF-α(r=0.020,P>0.05).3)Sphk1 expression level was negatively correlated with the GCS score(r=-0.524,P<0.001);and with the GOS score(r=-0.676,P<0.001).2.Cellular assay results: 1)The expression level of Sphk1 in the si RNA transfected group was significantly downregulated compared to the NC group compared to the Control group(0.344±0.085 vs.1.002±0.108 vs.0.987±0.103,P<0.05).2)Flow cytometry analysis showed that the apoptosis rate in the Sphk1-si RNA transfected group was significantly lower compared to the NC group compared with Control group(5.034%vs.12.563% vs.10.132%,P<0.05).3)Expression levels of apoptotic factors in m RNA:the expression level of anti-apoptotic factor Bcl-2 was significantly upregulated in the Sphk1-si RNA transfected group compared with NC group compared with Control group(2.039±0.142 vs.0.142 vs.0.948±0.125 vs.0.996±0.101,P<0.05);the expression level of the pro-apoptotic factor p53 was significantly down-regulated in the Sphk1-si RNA transfected group compared with the NC group and the Control group(0.559±0.889 vs.1.040±0.113 vs.0.991±0.091,P<0.05);the expression level of the pro apoptotic factor Caspase-3 expression level was significantly down-regulated in the Sphk1-si RNA transfected group compared to the NC group compared to the Control group(0.599±0.031VS0.992±0.111VS0.978±0.120,P<0.05).4)Expression of apoptotic factors at the protein level: anti-apoptotic factor Bcl-2 expression level was significantly lower in the Sphk1-si RNA transfected group was significantly up-regulated in the expression level compared with the NC group compared with the Control group(1.919±0.146 vs.0.982±0.158 vs.0.881±0.200,P<0.05);the expression level of the pro-apoptotic factor p53 was significantly down-regulated in the Sphk1-si RNA transfected group compared with the NC group compared with the Control group(0.454± 0.085 vs.1.071±0.109 vs.1.013±0.123,P<0.05);the expression level of pro-apoptotic factor caspase-3 was significantly down-regulated in the Sphk1-si RNA transfected group compared with the NC group and the Control group(0.445±0.081 vs.0.994±0.101 vs.0.956±0.115,P< 0.05).Conclusions:1.The expression level of Sphk1 was significantly higher in the serum of SICH patients compared with the control group,suggesting that Sphk1 can be used as an auxiliary diagnostic marker for SICH;2.p53 was highly expressed in the serum of SICH patients,while the expression level of Sphk1 in the serum of SICH patients was positively correlated with p53 expression;3.Sphk1 expression levels correlated with disease severity and prognosis in SICH patients;4.The role of Sphk1 in the pathophysiological process of SICH may be related to its regulation of apoptotic cells,which can help us to further understand the mechanism of SICH.