| Background:Spinal cord injury(SCI)is a serious neurological disorder and one of the major causes of disability in young adults.The pathogenesis of SCI is very complex,including primary and secondary injuries.After primary injuries caused by fractures,compression,etc.,a series of pathological and physiological changes such as inflammation,tissue hypoxia,neuronal necrosis and apoptosis,and local inhibitory microenvironment further aggravate SCI,leading to severe sensory and motor dysfunction.In clinical practice,despite many treatments,a large portion of SCI patients’ symptoms continue to persist or worsen.SNHG7 is a type of Lnc RNA(long non-coding RNA)that plays an important role in various cellular events after SCI,but its expression and functional effects in spinal cord injury are still unclear.Purpose:To explore the expression level of SNHG7 in SCI and its regulatory effects on apoptosis,inflammation and oxidative stress during SCI,and determine whether the regulatory effects of SNHG7 on SCI are related to mi R-199b-5p and TRAF3 through in vivo and in vitro experiments.Finding new molecular targets has a certain protective effect on SCI and elucidating their mechanisms of action.Method:(1)Expression of SNHG7 in LPS-induced BV-2 MCs in vitroUsing LPS intervention microglial cells to construct a neural injury model,q RT-PCR is adopted to detect the expression changes of SNHG7 in LPS-induced BV-2 MCs.Then,si-SNHG7 is transfected into LPS-induced BV-2 MCs cells,and divided into Control group,LPS-induced group,LPS+si-NC group,and LPS+si-SNHG7 group.Flow cytometry is employed to detect the apoptosis rate of cells,and Western blotting is used to detect the expression of apoptosis-related proteins Bax,Bcl-2,and cleaved caspase 3.ELISA is utilized to detect the levels of inflammatory factors TNF-α,IL-1β,and IL-6.finally,glutathione(GSH),superoxide dismutase(SOD)enzyme,and malondialdehyde(MDA)reagent kits are used to detect the expression of oxidative stress markers MDA,SOD,and GSH-Px.(2)Expression of SNHG7 regulating TRAF3 through targeting mi R-199b-5pFirst,the possible downstream target genes of SNHG7 in the database and validate the targeting effect of SNHG7 on mi R-199b-5p is retrieved using luciferase reporter,RNA pull-down,and RNA immunoprecipitation(RIP).The expression changes of mi R-199b-5p in the SCI cell model is detected using q RT-PCR.Then LPS and transfect BV2 MCs cells are induced and divided into Control group,LPS-induced group,LPS+si-NC group,and LPS+si-SNHG7 group.The effect of SNHG7 on the expression of mi R-199b-5p in the LPS-induced SCI cell model is detected using q RT-PCR.To explore the regulatory mechanism and function of mi R-199b-5p in SCI,the bioinformatics analysis software is used to predict its downstream targets and validate them through luciferase reporter gene experiments.To clarify the regulatory role of mi R-199b-5 in SCI,BV-2 MCs cells induced by LPS are transfected with mi R-199b-5p mimics,and divided into Control group,LPS-induced group,LPS+mi R-NC group,and LPS+mi R-199b-5p mimics group.The apoptosis rate of cells is detected by flow cytometry,and the expression of apoptosis-related proteins is detected by Western blotting.The levels of inflammatory factors are detected by ELISA.The expression of oxidative stress markers is detected using GSH,SOD,and MDA assay kits.(2)The mechanism of SNHG7 targeting mi R-199b-5p/TRAF3 in regulating mouse spinal cord injuryCells are grouped according to the intervention condition: Control group,LPS group,LPS+si-NC group,LPS+si-SNHG7 group,LPS+si-SNHG7+mi R-199b-5p inhibitor group,and LPS+si-SNHG7+TRAF3 group.Western blot is employed to detect whether SNHG7 regulates the expression of TRAF3 protein through mi R-199b-5p.Flow cytometry is used to detect the apoptosis rate of cells in each group,and Western blotting is used to detect the expression of apoptosis-related proteins in each group of cells.Then ELISA is used to detect the expression levels of inflammatory factors in cells in each group and GSH,SOD,and MDA assay kits are to detect the expression of oxidative stress markers in cells in each group.In order to confirm the effect of SNHG7 on the progression of SCI disease through mi R-199b-5p regulation of TRAF3 protein in the body,an SCI mouse model is established and the standardized Basso Mouse Scale(BMS)and cresyl violet staining are used to quantitatively determine whether the SCI model is successfully established.Then q RT-PCR is used to detect the expression of SNHG7 in the SCI mouse model and the level of TRAF3 m RNA in SCI tissue.Next,the mice are grouped according to the intervention conditions: Sham group,SCI group,SCI+LV-sh-NC group,SCI+LV-sh-SNHG7 group,and SCI+LV-sh-SNHG7+TRAF3group.The motor function of mice in each group is measured on the 1st,3rd,7th,and14 th days after injury,and cresyl violet staining is used to quantitatively determine the effect of SNHG7 silencing on spinal cord injury function recovery.TUNEL is used to detect changes in cell apoptosis in spinal cord tissue of mice in each group and ELISA is used to detect the levels of inflammatory factors TNF-α,IL-1β,and IL-6 in mice in each group.GSH,SOD,and MDA assay kits are used to detect the expression of oxidative stress markers in spinal cord tissue of mice in each group.Result:(1)Expression of SNHG7 in LPS-induced BV-2 MCs in vitroq RT-PCR results showed that SNHG7 is abnormally highly expressed in LPS-induced BV-2 MCs cells.Silencing SNHG7 significantly reverses apoptosis in LPS-induced BV-2 MCs cells.ELISA detection results show that silencing SNHG7 significantly reverses the secretion of inflammatory factors TNF-α,IL-1β,and IL-6 in LPS-induced BV-2 MCs.Oxidative stress detection shows that silencing SNHG7 significantly reverses the upregulation of oxidative stress markers MDA,SOD,and GSH-Px in LPS-induced BV-2 MCs.(2)Expression of SNHG7 regulating TRAF3 through targeting mi R-199b-5pThe target of SNHG7 in the experimental verification of bioinformatics analysis and luciferase reporter gene is mi R-199b-5p.The q RT-PCR shows that mi R-199b-5p is significantly downregulated in the in vitro SCI model,and SNHG7 silencing can significantly reverse the decrease in mi R-199b-5p levels induced by LPS.The downstream gene of mi R-199b-5p is TRAF3,and Western blot results show that overexpression of mi R-199b-5p can significantly inhibit TRAF3 protein expression,while silencing of mi R-199b-5p can significantly promote TRAF3 protein expression.The q RT-PCR detection results show that TRAF3 m RNA is significantly upregulated in LPS-induced BV-2 MCs.Western blot detection shows whether SNHG7 can regulate TRAF3 protein expression through mi R-199b-5p,the results show that compared with the Control group,the expression of TRAF3 protein in BV-2 MCs cells of the LPS group and LPS+si-NC group is significantly upregulated.Compared with the LPS+si-NC group,the expression level of TRAF3 protein in BV-2 MCs cells of the LPS+si-SNHG7 group is significantly downregulated.When co-transfected with mi R-199b-5p inhibitor or TRAF3 in cells,the expression level of TRAF3 protein in BV-2 MCs cells of the LPS+si-SNHG7+mi R-199b-5p inhibitor group and LPS+si-SNHG7+TRAF3 group is significantly upregulated.The mi R-199b-5p overexpression has a significant inhibitory effect on the levels of inflammatory factors and oxidative stress response in SCI cells.(3)The mechanism of SNHG7 targeting mi R-199b-5p/TRAF3 in regulating mouse spinal cord injuryIn vitro experiments: Flow cytometry shows that compared with the LPS+si-SNHG7 group,the apoptosis rate of cells is downregulated again when co-transfected with mi R-199b-5p inhibitor or TRAF3,indicating that the introduction of mi R-199b-5p inhibitor or TRAF3 inhibits cell apoptosis.Western blot analysis reveal that compared with the LPS+si-SNHG7 group,the expression of Bax and cleaved caspase3 proteins in cells is upregulated,while the expression of Bcl-2 is downregulated when co-transfected with mi R-199b-5p inhibitor or TRAF3.The level of cell inflammatory factors is partially upregulated.Furthermore,compared with the LPS+si-SNHG7 group,the levels of SOD and GSH in cells significantly decrease,while the level of MDA significantly increase.Therefore,it is confirmed that SNHG7 can regulate the levels of inflammation and oxidative stress in SCI cells through the mi R-199b-5p/TRAF3 pathway,thereby regulating the damage of SCI cells.In vivo experiments: The results show a significant decrease in motor function and preserved tissue area in SCI mice,confirming the successful modeling.SNHG7 is significantly upregulated in SCI mouse models.BMS detection results show that on day 14,the SCI+LV-sh-SNHG7 group had significantly higher scores than the SCI+LV-sh-NC group,while the SCI+LV-sh-SNHG7+TRAF3 group had significantly lower scores than the SCI+LV-sh-NC group,suggesting that SNHG7 regulates neural function recovery in mice after SCI through mi R-199-5p/TRAF3.Quantitative analysis of cresyl violet staining shows that compared to the Sham group,the tissue area of mice in the SCI+LV-sh-NC group is significantly reduced at different distances from the lesion center,both on the tail side and the nose side.Compared to the LV-sh-NC group,the SCI+LV-sh-SNHG7 group of mice treated with sh-SNHG7 show a significant increase in the number of preserved tissues.Compared to the SCI+LV-sh-SNHG7 group,the tissue area of mice in the SCI+LV-sh-SNHG7+TRAF3group is significantly reduced at different distances from the lesion center,both on the tail side and the nose side.TUNEL test results show that compared with the sham group,the apoptosis rate is upregulated in the SCI group;compared with the SCI group and SCI+LV-sh-NC group,the apoptosis rate is decreased in the SCI+LV-sh-SNHG7 group;compared with the SCI+LV-sh-SNHG7 group,the apoptosis rate is upregulated in the SCI+LV-sh-SNHG7+TRAF3 group,indicating that SNHG7 affects the apoptosis of SCI spinal cord tissue cells through the mi R-199b-5p/TRAF3 axis.ELISA test results show that compared with the sham group,the expression levels of TNF-α,IL-1β,and IL-6 in the serum of the SCI group are upregulated;compared with the SCI group and SCI+LV-sh-NC group,the expression levels of TNF-α,IL-1β,and IL-6 were decreased;compared with the SCI+LV-sh-SNHG7 group,the levels of the three inflammatory factors are upregulated in the SCI+LV-sh-SNHG7+TRAF3 group.The oxidative stress index test results show that compared with the sham group,the expression levels of ROS and MDA in the mouse tissues of the SCI group are significantly upregulated,while the levels of SOD and GSH-Px are significantly decreased.Compared with the SCI group,the levels of ROS and MDA decrease,while the levels of SOD and GSH-Px increase in the mouse tissues of the SCI+LV-sh-SNHG7 group.Compared with the SCI+LV-sh-SNHG7 group,the levels of ROS and MDA increase,while the levels of SOD and GSH-Px decrease in the SCI+LV-sh-SNHG7+TRAF3 group.Conclusion:(1)In vitro experiments showed that SNHG7 is abnormally highly expressed in the LPS-induced BV-2 MCs injury model,and silencing SNHG7 can significantly inhibit apoptosis,inflammation,and oxidative stress response in BV-2MCs cells,suggesting that SNHG7 may have important pathophysiological functions in SCI.(2)In the disease process of SCI,SNHG7 may regulate apoptosis rate,inflammatory response,and oxidative stress response by targeting mi R-199b-5p/TRAF3.(3)The downstream gene of mi R-199b-5p is TRAF3 and the expression level of TRAF3 is significantly upregulated in SCI mouse models and cell models.In vivo experiments confirm that SNHG7 regulates SCI cell apoptosis,inflammation,and oxidative stress through the mi R-199b-5p/TRAF3 pathway. |
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