| ObjectiveThis study intends to clarify the relationship between miR-494 and RIPK1 expression levels and neuronal damage in epileptic hippocampus,and to prove that miR-494 down-regulates the expression of RIPK1,inhibits the activity of the NF-?B signaling pathway,promotes neuronal cell proliferation and suppresses cells apoptosis,delays the progress of Ep,and then plays a protective role in the damage of hippocampal neurons in epilepsy.Method1.Temporal lobe hippocampus of epilepsy patients was collected,and quantitative PCR was used to detect the expression of miR-494 and RIPK1 mRNA.The correlation between miR-494 and RIPK1 was analyzed and the relationship between miR-494 and clinical characteristics including disease course,age of first attack,etiology,whether other diseases were analyzed.2.2Rat model of lithium chloride-pilocarpine epilepsy was established.Rat model was treated with miR-494 agomir,RIPK1 siRNA and miR-494 agomir+oe-RIPK1 to observe the behavioral and computer changes of the rat.HE staining,electron microscopy,Nessler staining,FJC staining was performed to observe hippocampal neuronal cell damage.Hoechst 33342 staining,Tunnel staining was to observe hippocampal neuron apoptosis.Quantitative PCR and Western Blot was to detect hippocampal neuron NF-?B signaling pathway activation.3.The primary neuronal cells of newborn SD rats were isolated in vitro,and the EP-like neuron model was induced with magnesium-free extracellular fluid.Dual luciferase reporter gene demonstrates the interaction of miR-494 and RIPK1.EP-like metacells were treated with miR-494 agomir,RIPK1 siRNA,and miR-494 agomir+oe-RIPK1,MTT was to detect cell proliferation,Hoechst 33342 staining and flow cytometry was used to detect apoptosis,quantitative PCR and Western Blot was used to detect the activation of NF-?B signaling pathway.The effects of miR-494 and RIPK1 expression levels on the proliferation,apoptosis and activation of NF-?B signaling pathway in Ep-like neurons were observed through in vitro studies.Results 1.The miR-494 level in the temporal lobe of epilepsy patients was(0.71±0.22)(p<0.05),which was(1.89±0.28)in the control group(p<0.05).There was a negative correlation between miR-494 and RIPK1 mRNA levels in the temporal lobe brain tissue of patients with epilepsy,r=0.6578,p<0.001.2.The relative expression of miR-494 mRNA in brain tissue of patients with disease course ?5 years,5-10 years(included)and>10 years was(0.82±0.19),(0.61±0.15)and(0.43±0.11)respectively.The level of miR-494 in patients with long disease duration was lower than that in patients with short disease duration(p<0.05).3.The relative expression of miR-494 was(0.65±0.10)in patients with the first seizure age<18 years old,which was(0.82±0.12)in patients with the first seizure age ? 18 years old.The miR-494 level of the patients with younger first seizure age was lower than that of patients with older first seizure age(p<0.05).4.The success rate of modeling epilepsy induced by lithium chloride-pilocarpine in rats was 95%.The number of slow waves in the spinal column of the EEG in the Ep group was significantly higher than that in the normal control group(p<0.05);The number of spine slow waves in the agomir-miR-494 group and RIPK1-siRNA group was significantly lower than that in the agomir-NC group and siRNA-NC group(both<0.05);the number of spine slow waves in the agomir-miR-494+oe-RIPK1 group increased significantly compared with the agomir-miR-494 group(p<0.05).5.HE staining showed that the neurons in the normal group were neatly arranged,structurally complete and normal in shape;the neurons in the Ep group,agomir NC group,and siRNA-NC group were triangular,the cytoplasm was concentrated,and there were mitotic divisions and recessive nucleoli.The degree of injury to hippocampal neurons in agomir-miR-494 group and RIPK1-siRNA group was less than that in Ep group;the degree of injury in agomir-miR-494+ oe-RIPK1 group was more serious than that in agomir-miR-494 group.6.Electron microscopy showed that the neurons in the normal group had normal ultrastructure,oval nuclei,even chromatin distribution,and clear nucleoli.Neurons in Ep group,agomir NC group and siRNA-NC group had a large amount of heterochromatin,without rough endoplasmic reticulum and free ribosomes.The ultrastructure of neurons in agomir-miR-494 group and RIPK1-siRNA group was normal,with rough endoplasmic reticulum,ribosome and mitochondria.The damage of hippocampal neurons in the agomir-miR-494+oe-RIPK1 group was more serious than in the agomir-miR-494 group.7.The Nissl staining showed that the number of neurons in Ep group decreased compared with normal group(p<0.05).The number of neurons in agomir-miR-494 group and RIPK1-siRNA group was higher than that in agomir-NC group and siRNA-NC group(p all<0.05);the number of neurons in agomir-miR-494+oe-RIPK1 group was higher than that of agomir-miR-494 group decreased(p<0.05).8.FJC staining showed that the number of FJC-positive degenerative neurons in Ep group was higher than that in normal group(p<0.05);The number of FJC positive cells in agomir-miR-494 group and RIPK1-siRNA group was lower than that in agomir NC group and siRNA-NC group(both<0.05).The FJC positive cells in the agomir-miR-494+oe-RIPK1 group increased compared with the agomir-miR-494 group(all p<0.05).9.TUNEL staining results showed that the number of apoptotic neurons in Ep group increased compared with normal group(p<0.05).The positive cells in agomir-miR-494 group and RIPK1-siRNA group were lower than those in agomir NC group and siRNA-NC group(both<0.05).The positive cells in the agomir-miR-494+oe-RIPK1 group increased compared with the agomir-miR-494 group(all p<0.05).10.Hoechst 33258 staining results showed that the apoptosis rate in Ep group was higher than that in normal group(p<0.05).The apoptosis rate of Agomir-miR-494 group and RIPK1-siRNA1 group was lower than that of agomir-NC group and siRNA-NC group(all p<0.05).The apoptosis rate of Agomir-miR-494+oe-RIPK1 group was higher than that of agomir-miR-494 group(all p<0.05).11.Quantitative PCR and Western blot results showed that compared with the normal group,the expression of Bcl-2 mRNA and protein in the Ep group decreased,while the expression of Bax mRNA and protein increased(p<0.05).The mRNA and protein expressions of Bcl-2 in the agomir-miR-494 group and RIPK1-siRNA group were higher than those in the agomir-NC group and siRNA-NC group,while the mRNA and protein expressions of Bax decreased(p<0.05).The mRNA and protein expressions of Bcl-2 in the agomir-miR-494+oe-RIPK1 group were lower than those in the agomir-miR-494 group,while the mRNA and protein expressions of Bax were increased(p<0.05).12.Quantitative PCR and Western Blot results showed that compared with the normal group,the expression of miR-494 in hippocampus of rats in Ep group decreased significantly(p<0.01),while the expression of RIPK1 and NF-? Bp65 increased(p<0.01).The expression of miR-494 in the agomir-miR-494 group was higher than that in the agomir-NC group(p<0.01),and the expression of RIPK1 and NF-? Bp65 decreased(p<0.01).The expression of RIPK1 and NF-?Bp65 in hippocampus of RIPK1-siRNA group was lower than that of siRNA-NC group(p<0.01).The expressions of RIPK1 and NF-? Bp65 in the agomir-miR-494+oe-RIPK1 group were higher than those in the agomir-miR-494 group(p<0.05).13.Rat hippocampal neuron cells isolated and cultured in vitro adhered on the first day of culture;the cell volume began to increase on the third day;the cell body became longer and the number increased on the seventh day.The purity identification results of neurons indicate that the purity of hippocampal neuron cells isolated in vitro exceeds 90%.14.MTT analysis showed that the viability of neurons in Ep group decreased compared with the control group(p<0.05).The cell viability of miR-494 mimic group and RIPK1-siRNA group was higher than that of mimic NC group and siRNA-NC group(both<0.05),and the cell viability of miR-494mimic+oe-RIPK1 group was lower than that of miR-494 mimic group(p both<0.05).15.Hoechst 33342 staining showed that the nuclei of the control group were uniformly stained in a round or oval shape with regular morphology.Neurons in the Ep group,simulated NC group,and siRNA-NC group all exhibited keratosis,enhanced apoptotic fluorescence signals,and nuclei.Fragmentation and apoptotic bodies(p<0.05).Apoptosis in miR-494 mimic group and RIPK1-siRNA group was lower than that in Ep group;apoptotic cells in miR-494 mimic+oe-RIPK1 group increased compared to miR-494 mimic group(both p<0.05).16.The results of Annexin V-FITC/PI double staining showed that the apoptosis rate of neurons in Ep group was significantly increased compared with the control group.The apoptosis rate of neurons in miR-494 mimic group and RIPK1-siRNA group was lower than that in mimic NC group and siRNA-NC group(both p<0.05).The apoptosis rate of neurons in miR-494 mimic group+oe-RIPK1 group was higher than that of miR-494 mimic group(p<0.05).17.Quantitative PCR and Western blot results showed that the expression level of Bcl-2 in neurons of Ep group decreased compared with the control group,while the expression level of Bax increased(p all<0.05).The expression of Bcl-2 in neurons of miR-494 mimic group and RIPK1-siRNA group was higher than that of mimic NC group and siRNA-NC group,and Bax expression was decreased(p<0.05).The expression of Bcl-2 in miR-494 mimic group+oe-RIPK1 group was lower than that of miR-494 mimic group,while the expression of Bax increased(p<0.05).18.Dual luciferase reporter gene detection showed that the luciferase activity in hippocampal neuron cells co-transfected with wt-miR-494 reporter gene vector and oe-RIPK1 was lower than that of wt-miR-494 reporter gene vector and mimic NC group(P<0.05).The luciferase activity in hippocampal neuron cells co-transfected with mut-miR-494 reporter gene vector and oe-RIPK1 was higher than that of luciferase in hippocampal neuron cells co-transfected with mut-miR-494 reporter gene vector and oe-NC There was no significant difference in enzyme activity(p>0.05).19.Quantitative PCR and Western blot results showed that the expression of miR-494 in Ep group was lower than that in control group,and the expression of RIPK1 and NF-? Bp65 increased(p<0.05).MiR-494 mimic group miR-494 expression level was higher than mimic NC group,RIPK1 and NF-? Bp65 expression decreased(p<0.05).Compared with the siRNA-NC group,the expression of RIPK1 and NF-?B p65 in the RIPK1-siRNA group was reduced(p<0.05).The expression of RIPK1 and NF-? B p65 in the miR-494 mimic+oe-RIPK1 group increased compared with the miR-494 mimic group(p<0.05).Conclusions 1.The expression of miR-494 is reduced in the brain tissue of patients with epilepsy,and the expression of RIPK1 is increased.The level of miR-494 is related to the patient's disease course and age at first seizure,suggesting that miR-494 is an epilepsy-related factor.2.MiR-494 can inhibit the expression of RIPK1,cause inactivation of the NF-?B signaling pathway,promote the proliferation of hippocampal neuronal cells in Ep rats,inhibit apoptosis,reduce neuronal damage and Ep progress. |
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