| Objective: In recent years,the popularity of intravenous thrombolysis and the development of intravascular therapy,bring hope to ischemic stroke patients.However,reperfusion injury secondary to vascular recanalization could lead to more servious brain damage and functional impairment.To seek treatment intervention that protect against ischemic/reperfusion(I/R)injury is of great significance to ischemic stroke.Vagus nerve stimulation(VNS)has been a safe and effective adjunct treatment for intractable epilepsy,since its approval for clinical use by the Food and Drug Administration(FDA)in 1997.Recent studies have shown that VNS is neuroprotective in cerebral I/R rat models.A recent clinical trial has shown that VNS paired with rehabilitation training is feasible to treat patients with upper-limb weakness after ischemic stroke.Although the molecular mechanisms involved in VNS-mediated neuroprotection are partially known,more detailed mechanisms need further exploration.L-PGDS is a multi-functional protein acting as an extracellular transport protein of lipophilic molecules and an enzyme responsible for catalyzing the conversion of prostaglandin H2(PGH2)to PGD2.L-PGDS is abundant in the central nervous system(CNS),and would be beneicial against cerebral Ischemia.Nonetheless,the function of L-PGDS in the CNS,especially in cerebral ischemia,has not been fully clarified.The aim of the present study was to investigate the effects and possible mechanisms of VNS on the apoptosis,inflammation and blood-brain barrier after cerebral I/R injury in rats,and whether L-PGDS was involved in these processes.The results might provide new ideas for the treatment of ischemic stroke.Methods: Part Ⅰ : The right middle cerebral artery occlusion/reperfusion(MCAO/R)models were established with an intraluminal ilament technique.The expression of L-PGDS was examined by western blot and ELISA at 12 h,24 h,3 days and 7 days after reperfusion.Double immunostaining was performed to determine the cellular distribution of L-PGDS on the peri-infarct cortex.We employed lentivirus shRNA-L-PGDS to silence L-PGDS expression.The expression of green fluorescent protein(GFP)was observed with laser scanning confocal microscope.The mRNA level of L-PGDS was examined by q-PCR to confirm the inhibition efficiency of lentiviral vector.The MCAO/R models were prepared after lentivirus transfection for 7 days;VNS initiated 30 min after MCAO.During the experimental process,the blood pressure(BP),blood gas levels,heart rate(HR)and cerebral blood flow(CBF)of the right MCA were measured.The protein and mRNA expression levels of L-PGDS were detected by western blot and q-PCR at 24 h after reperfusion.Neurological evaluations were performed and infarct volume was assessed at 24 h following reperfusion.The neuronal apoptosis was assessed using TUNEL staining.We examined the levels of Bcl-2,Bax and cleaved caspase-3 in the peri-infarct cortex by western blotting.We detected the interaction between L-PGDS and PPARγ by Co-Immunoprecipitation(COIP).The protein and mRNA expression levels of PPARγ were examined by western blot and q-PCR.Part Ⅱ: We employed lentivirus shRNA-L-PGDS to silence L-PGDS expression.The MCAO/R models were prepared after lentivirus transfection for 7 days;VNS initiated 30 min after MCAO.At 24 h after reperfusion,we detected evans’ blue(EB)extravasation and brain water content in the rats.The expression of occludin,MMP-9 and AQP4 were measured by western blotting.We detected the levels of tumor necrosis factor α(TNF-α)and interleukin 6(IL-6)in the ischemia penumbra by ELISA.Results: Part Ⅰ:(1)The expression of L-PGDS was increased after cerebral I/R injury,this increasing trend lasted up to 3 days after reperfusion,reached a peak at 24 h.L-PGDS was colocalized with NeuN,a marker for neurons,and APC,a marker for oligodendrocytes but was not coexpressed with GFAP,a marker for astrocytes.(2)The lentiviral vector could be effectively transfected into the rat brain tissue.Compared with the normal and empty virus groups,lentiviral shRNA-L-PGDS significantly inhibited L-PGDS mRNA expression(p<0.05).(3)During the experimental period,there were no significant changes in the physiological parameters(HR,BP,blood gases and CBF of the right MCA)after VNS treatment.(4)Compared with the I/R group,VNS treatment enhanced the protein and mRNA expression of L-PGDS following ischemic stroke(p<0.05);the L-PGDS expression was inhibited and the enhancement effect induced by VNS was weakened after L-PGDS shRNA treatment(p<0.05).(5)VNS treatment prevented neurological impairment and reduced the infarct volume after cerebral I/R(p<0.05);silencing L-PGDS expression could worsen neurological deicits and brain damage induced by cerebral I/R,and weakened the VNS-induced neuroprotetion(p<0.05),indicate that L-PGDS may mediate the neuroprotective efects induced by VNS in rats subjected to ischemic stroke.(6)In comparison to the I/R group,the number of TUNEL-positive neurons was signiicantly decreased after VNS treatment(p<0.05);while it was dramatically increased after L-PGDS shRNA treatment(p<0.05).(7)Compared with the sham I/R group,the expression level of Bax and caspase-3 activity in the I/R group were signiicantly increased(p<0.05),the protein levels of Bcl-2 in the I/R group were remarkably decreased(p<0.05).Compared with the I/R group,the levels of Bax and cleaved caspase-3 were down-regulated(p<0.05)and the level of Bcl-2 was up-regulated after VNS treatment(p<0.05);the Bax and cleaved caspase-3 expression were increased(p<0.05)and Bcl-2 level was decreased after silencing L-PGDS(p<0.05).The anti-apoptotic effect induced by VNS was diminished following down-regulation of L-PGDS(p<0.05),indicate that L-PGDS could mediate the suppression of apoptosis induced by VNS after I/R injury in rats.(8)The CO-IP results exhibited that there is an interaction between L-PGDS and PPARγ.(9)Compared with the I/R group,VNS treatment enhanced the protein and mRNA levels of PPARγ following ischemic stroke(p<0.05);this enhancement effect induced by VNS was inhibited after silencing L-PGDS expression(p<0.05).Part Ⅱ:(1)At 24 h after reperfusion,compared with the sham I/R group,the evans’ blue extravasation and brain water content were increased after cerebral I/R injury(p<0.05).Compared with the I/R group,the leakage of EB and brain water content were decreased by VNS treatment(p<0.05);after silencing L-PGDS expression,the leakage of EB and brain water content were significantly increased(p<0.05),and attenuated the protective effect on the blood-brain barrier(BBB)induced by VNS(p<0.05).(2)Compared with the sham I/R group,the expression of AQP4 and MMP-9 were increased(p<0.05)and the expression of occludin was decreased after cerebral I/R injury(p<0.05).Compared with the I/R group,VNS treatment decreased the expression of AQP4 and MMP-9(p<0.05)and increased the occludin expression(p<0.05);After L-PGDS shRNA treatment,the expression of AQP4 and MMP-9 were further upregulated(p<0.05)and the occludin expression was further downregulated(p<0.05),and the protection on BBB induced by VNS was attenuated(p<0.05),indicating that L-PGDS may mediate the protective effect of VNS on the BBB after cerebral I/R injury.(3)VNS decreased inflammatory response induced by cerebral I/R injury,suppressed the levels of TNF-α and IL-6 in the ischemia penumbra(p<0.05).After L-PGDS shRNA treatment,VNS-induced anti-inflammatory effect was significantly decreased,and the levels of inflammatory factors were increased(p<0.05),suggesting that L-PGDS may be involved in the anti-inflammatory effect induced by VNS after ischemic stroke.Conclusions:(1)VNS treatment reduces the infarct volume,improves the neurological deficits in the rats subjected to acute cerebral ischemia/reperfusion injury.(2)VNS decreases the number of neuronal apoptosis in the ischemic penumbra,inhibits the expression of Bax and cleaved caspase-3,increases the expression of anti-apoptotic protein Bcl-2,suppresses the apoptotic response indued by acute cerebral ischemia/reperfusion.(3)VNS down-regulates the expression of MMP-9 and AQP4,decreases the degradation of occludin,relieves the BBB injury and brain edema after ischemia/reperfusion injury.(4)VNS reduces the inflammatory response in the peri-infarct cortex in the actue cerebral ischemia/reperfusion rats.(5)VNS treatment increases the expression of L-PGDS in the ischemic penumbra;the increase in L-PGDS expression promotes PPARγ expression,and plays an important role in the VNS-induced anti-apoptotic and anti-inflammatory effects as well as BBB protection after cerebral ischemia-reperfusion injury. |
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