| Objective: 1.To observe the protective effect of mi R-193b-3p on focal cerebral ischemia-reperfusion(I/R)injury in rats.2.To observe the protective effect of mi R-193b-3p on OGD/R-treated neuron injury.3.To explore the mechanism of mi R-193b-3p neuroprotection.Methods: 1.Protective effect of mi R-193b-3p on focal I/R injury in rats.(1)Adult male SD rats(250-280g)were subjected to transient middle cerebral artery occlusion(MCAO)injury.On the 3rd,6th,12 th,24th,48 th,72th hours after reperfusion,the levels of mi R-193b-3p and 5-LOX m RNA expression were detected in the cerebral cortex of rats after focal cerebral I/R injury by q PCR.(2)Animals were randomly divided into 6 groups: 1)sham-operated group(which received only a neck incision,no I/R injury or any treatment,n =11);2)vehicle group(which received I/R injury and DEPC-treated dd H2 O,n= 11);3)angomir group(which received I/R injury and mi R-193b-3p agomir,n=11);4)angomir control group(which received I/R injury and mi R-193b-3p agomir control,n=11);5)antagomir group(which received I/R injury and mi R-193b-3p antagomir,n=11);6)antagomir control group(which received I/R injury and mi R-193b-3p antagomir control,n=11).(3)Neurological deficit scores,infarct volumes,neuron damage were measured.The content of LTB4 and Cys LTs(including LTC4,LTD4 and LTE4)was detected by ELISA;q PCR was used to determine the expression of 5-LOX-m RNA;WB was used to detect the expression of 5-LOX protein in rat brain tissue.2.Protective effect of mi R-193b-3p on OGD/R-induced neuronal injury.(1)In an in vitro experiment,cultured PC12 cells were exposed to oxygen–glucose deprivation and reperfusion(OGD/R).(2)Neuron-like rat pheochromocytoma cells PC12 were randomly divided into the following groups: 1)normoxic group(which was cultured in normal oxygen conditions without any treatment),2)OGD/R group(which was cultured in OGD/R conditions without any treatment),3)mim-mi R-193b-3p group(which was cultured in OGD/R conditions and treated with mi R-193b-3p mimic),4)mim-mi R-NC group(which was cultured in OGD/R conditions and treated with mimic control),5)inh-mi R-193b-3p group(which was cultured in OGD/R conditions and treated with mi R-193b-3p inhibitor),6)inh-mi R-NC group(which was cultured in OGD/R conditions and treated with inhibitor control).(3)The viability of PC12 cells was evaluated by MTT assay;the lactate dehydrogenase release(LDH)was evaluated by biochemical kit;the apoptosis rate was determined by flow cytometry(FCM);the expression of 5-LOX-m RNA was detected by q PCR;the expression of 5-LOX protein was detected by WB.3.Effect of mi R-193b-3p was mediated by 5-LOX.(1)Bioinformatics analysis was used to predict the binding sites of mi R-193b-3p.(2)The dual-luciferase reporter gene assay was applied to verify the potential interaction between 5-LOX m RNA and mi R-193b-3p.(3)Effect of mi R-193b-3p and 5-LOX-si RNA on OGD/R-induced neuronal injury.PC12 were randomly divided into the following groups: 1)normoxic group(which was cultured in normal oxygen conditions without any treatment),2)OGD/R group(which was cultured in OGD/R conditions without any treatment),3)mim-mi R-NC group(which was cultured in OGD/R conditions and treated with mimic control),4)mim-mi R-193b-3p group(which was cultured in OGD/R conditions and treated with mi R-193b-3p mimic),5)mim-mi R-193b-3p+si-5-LOX group(which was cultured in OGD/R conditions and additional mi R-193b-3p mimic and 5-LOX-si RNA treatment),6)inh-mi R-NC group(which was cultured in OGD/R conditions and treated with inhibitor control),7)inh-mi R-193b-3p group(which was cultured in OGD/R conditions and treated with mi R-193b-3p inhibitor),8)inh-mi R-193b-3p +si-5-LOX group(which was cultured in OGD/R conditions and additional mi R-193b-3p inhibitor and 5-LOX-si RNA treatment).The viability of PC12 cells was evaluated by MTT assay;the lactate dehydrogenase release(LDH)was evaluated by biochemical kit;the apoptosis rate was determined by flow cytometry(FCM).Results: 1.Protective effect of mi R-193b-3p on focal I/R injury in rats.(1)Compared to that of the sham group,the levels of 5-LOX m RNA in the ischemic cortex was significantly increased in I/R group at 12 h and 24 h after reperfusion(P<0.05).Similarly,the mi R-193b-3p level of I/R group was significantly increased at 3-72 h after reperfusion(P<0.05)when compared with that of sham group.(2)Compared to that of the sham group,focal cerebral I/R injury induced a significant increase in the neurological deficit score of the vehicle group(P<0.01).Compared to that of the agomir control group,the neurological deficit score was significantly lower in the agomir group(P< 0.01).However,the neurological deficit score in the antagomir group was significantly higher than that of the antagomir control group(P < 0.01).(3)Compared to that of the sham group,focal cerebral I/R injury induced an obvious infarction in the rat brain(P<0.01).Compared to those of the corresponding control groups,the infarct size was significantly smaller in the agomir group(P < 0.01)and significantly larger in the antagomir group(P < 0.05).When rats were subjected to focal cerebral I/R injury,neurons were condensed,and the cytoplasm disappeared,resulting in an obvious pericellular space.Additionally,nuclear staining was intensified,and the size of nuclei expanded,which might have been the result of chromatin condensation.When rats were treated with the mi R-193b-3p agomir,focal cerebral I/R-induced soma condensation and nuclei karyopyknosis were alleviated.However,focal cerebral I/R-induced morphological changes were further aggravated by mi R-193b-3p antagomir treatment.(4)In the sham group,LTB4 and Cys LTs were maintained at a relatively low level.When subjected to focal cerebral I/R injury,the levels of LTB4 and Cys LTs were significantly higher in the ischemic penumbra(P<0.01).Compared to those in the agomir control group,LTB4 and Cys LTs levels were significantly lower in the agomir group(P < 0.01).However,compared to those in the antagomir control group,the levels of LTB4 and Cys LTs were significantly higher in the antagomir group(P < 0.05).(5)Compared to those in the sham group,both 5-LOX m RNA and protein levels were significantly higher in the cortex of the focal cerebral I/R injury group(P<0.01).Furthermore,the levels of both 5-LOX m RNA and protein were significantly lower when rats were treated with the mi R-193b-3p agomir(P < 0.01)but significantly higher when they were treated with the mi R-193b-3p antagomir(P < 0.01).2.Protective effect of mi R-193b-3p on OGD/R-induced neuronal injury.(1)Compared to that in the normoxia group,the cell survival rate was significantly lower in the OGD/R group(P < 0.01),LDH release was significantly higher in the OGD/R group(P < 0.01),the apoptosis rate of OGD/R-treated cells was significantly higher in the OGD/R group.Compared to that in the mim-mi R-NC group,the cell survival rate was significantly higher in the mim-mi R-193b-3p group(P < 0.01).However,compared to that in the inh-mi R-NC group,the cell survival rate was significantly lower in the inh-mi R-193b-3p group(P < 0.01).Compared to that in the mim-mi R-NC group,LDH release was significantly lower in the mim-mi R-193b-3p group(P < 0.01),However,compared to that in the inh-mi R-NC group,LDH release was significantly higher in the inh-mi R-193b-3p group(P < 0.01).The apoptosis rate of OGD/R-treated cells was significantly decreased by mim-mi R-193b-3p,but inh-mi R-193b-3p promoted further apoptosis.(2)Both 5-LOX m RNA and protein levels were significantly higher in OGD/R groups than in the normoxia group(P < 0.01).Compared to those of the mim-mi R-NC group,the levels of both 5-LOX m RNA and protein were significantly lower in the mim-mi R-193b-3p group(P < 0.01).However,compared to those in the inh-mi R-NC group,the levels of both 5-LOX m RNA and protein were significantly higher in the inh-mi R-193b-3p group(P < 0.01).3.Effect of mi R-193b-3p was mediated by 5-LOX.(1)mi RNA-193b-3p was predicted to bind the 3’UTR of 5-LOX-m RNA according to bioinformatics calculations.(2)The results of the dual-luciferase reporter gene assay showed that mi R-193b-3p was located on the 3’ untranslated region(3’UTR)of 5-LOX m RNA.(3)Cell injury that was aggravated by the mi R-193b-3p inhibitor.Additionally,the effects of the mi R-193b-3p inhibitor on OGD/R were partially reversed by treatment with 5-LOX si RNA in vitro.Conclusions: 1.mi R-193b-3p agomir has a potentially neuroprotective effect on cerebral focal I/R-induced injury.2.mi R-193b-3p mimic has a neuroprotective effect on OGD/R-induced neuronal injury.3.Bioinformatics and dual-luciferase reporter gene assays showed that mi R-193b-3p was located on the 3’ untranslated region(3’UTR)of 5-LOX m RNA.4.The effects of the mi R-193b-3p inhibitor on OGD/R were partially reversed by treatment with 5-LOX si RNA in vitro.5.mi R-193b-3p alleviates focal cerebral I/R-induced injury in rats by regulating 5-LOX expression. |
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