| BackgroundCerebral infarction is characterized with high incidence among the cerebral vascular diseases.Patients suffered from cerebral infarction often show various degrees of neurologic deficits without timely and effective therapies such as thrombolysis and interventional thrombectomy.The sequelae seriously influence the patients’ life quality and add to the economic burden and care burden of their families.The main priority of current research in neurology is exploring promising therapy for cerebral infarction and underlying mechanisms of the protective effects.Blood brain barrier is physiologically a functional barrier between the brain and peripheral circulation system.The barrier is involved in regulating the transportation of nutrients and ions,inhibiting the access of peripheral toxicants and immune cells to the brain,and maintaining the brain microenvironment homeostasis.Tight junction proteins occludin and zonula occludens-1(ZO-1)are main components of the blood brain barrier and regulate the blood brain barrier permeability.Previous research has shown that the expression of matrix metalloproteinase-9(MMP9)is increased after cerebral infarction,resulting in the degradation of occludin and ZO-1,and the increased permeability of blood brain barrier.Blood brain barrier dysfunction can incur imbalance of water and ions,promote brain edema and cell death,and aggravate brain injury.Promoting blood brain barrier integrity may be important for limiting ischemic injury.Neurogenesis persists in the entire life of mammals and is characterized with the generation of neurons from neural stem cells/neural precursor cells.The adult neurogenesis,which is enhanced after cerebral infarction,mainly occurs in the subventricular zone(SVZ)and the hippocampal dentate gyrus(DG).In the SVZ,neuroblasts migrate to peripheral ischemic area and differentiate into neurons.However,newborn cells in the hippocampal DG migrate to hippocampal granular layer and undergo further differentiation.These newborn neurons can replace dead or injured cells,integrate into the existing neural circuits and promote the neurologic functional recovery.Therefore,endogenous neurogenesis may be one of the key targets in therapy for cerebral infarction.There are various and abundant microbes in the gut,which are obtained from the mother at birth and influenced by diet and environment.Absence of gut microbiota can incur the increased permeability of blood brain barrier and downregulate the expression of tight junction proteins in mice.Additionally,enteric microbiota can promote the expression of synaptic plasticity related genes and alleviate diabetes induced cognitive impairment.The microbiota-gut-brain axis links gut microbiota with the brain and the microbial metabolites play key roles in the axis.Indole-3-propionic acid(IPA)belongs to microbial metabolites.It’s reported that IPA can inhibit neuroinflammation and promote neurologic recovery in the classical experimental model of multiple sclerosis.IPA can also reduce neurons death in a cell culture model of Parkinson’s disease.Additionally,IPA alleviates the toxic effects of Aβ and inhibits the death of cells.In the rat model of bilateral common carotid arteries occlusion,IPA can inhibit oxidative stress and protect hippocampal neurons.However,the roles of IPA in cerebral infarction remain unknown.Therefore,the research aims to investigate whether IPA shows protective effects after ischemic stroke and explore its regulatory effects on blood brain barrier integrity and neurogenesis.Part Ⅰ The protective effects of IPA in a mouse model of cerebral infarctionObjectiveTo investigate whether IPA shows beneficial roles in a mouse model of cerebral infarction.Methods1 Adult male C57/BL6 mice were performed with right transient middle cerebral artery occlusion(MCAO)or corresponding Sham surgery by suture method and randomly divided into four groups:Sham+vehicle group,Sham + IPA group,MCAO+vehicle group,MCAO+IPA group.2 Neurologic deficits were assessed by five-point scale on days 3 and 7 after cerebral infarction to evaluate the beneficial effects of IPA on neurologic functional recovery.3 Brain water content was measured on day 3 post-operation to assess the effects of IPA on brain edema.4 TUNEL staining and western blot of Bcl2 protein were performed on day 3 after cerebral infarction to explore the effects of IPA on apoptosis.5 The infarct volume was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining on day 3 after cerebral infarction and by Nissl staining on day 7 post cerebral infarction to identify the effects of IPA on the brain injury.Results1 Mice from MCAO+ IPA group displayed significantly lower scores than those from MCAO+vehicle group on days 3 and 7 after cerebral infarction(P<0.05).2 There was no significant difference in brain water content between vehicle-treated and IPA-treated Sham mice(P>0.05).However,IPA-treated MCAO mice showed lower brain water content compared to vehicle-treated MCAO mice(P<0.05).3 IPA significantly decreased the number of TUNEL-positive cells in peri-infarct area(P<0.05).4 IPA did not influence the expression of apoptotic inhibitor Bcl2 in Sham mice,but significantly upregulated the protein level of Bcl2 in MCAO mice(P<0.05).5 TTC staining showed that the infarct volume in mice from MCAO+IPA group was lower than that from MCAO+vehicle group(P<0.05).6 Nissl staining revealed that IPA significantly reduced the infarct volume of MCAO mice(P<0.05).ConclusionsIPA has beneficial roles in a mouse model of cerebral infarction,including alleviating neurologic deficits,inhibiting brain edema and apoptosis and reducing infarct volume.Part Ⅱ The regulatory effects of IPA on blood brain barrier permeabilityObjectiveTo explore the regulatory effects of IPA on blood brain barrier permeability.Methods1 The models and groups were made as described in Part Ⅰ.2 Mice were treated with exogenous tracer Evans blue dye to evaluate the permeability of blood brain barrier on day 3 after cerebral infarction.Frozen sections of mice brains were prepared and the leakage of Evans blue dye was observed under fluorescence microscope.3 Western blot was used to measure the expression levels of occludin,ZO-1,EMMPRIN,MMP9,nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)in the ischemic hemisphere.Immunofluorescence staining of ZO-1 and MMP9 was also performed on day 3 after cerebral infarction.Results1 Quantification of Evans blue leakage showed that IPA-treated MCAO mice had lower leakage than vehicle-treated MCAO mice(P<0.05).2 Immunofluorescence of Evans blue also revealed that IPA reduced the peri-infarct leakage of Evans blue.3 IPA slightly upregulated the ZO-1 area in Sham mice(P>0.05),but significantly increased the ZO-1 area in peri-infarct zone(P<0.05).4 The protein levels of occludin and ZO-1 were not significantly different between Sham+vehicle group and Sham+IPA group(P>0.05).IPA-treated MCAO mice had significantly higher protein levels of occludin and ZO-1 compared to vehicle-treated MCAO mice(P<0.05).5 IPA-treated MCAO mice showed less peri-infarct MMP9-positive cells than vehicle-treated MCAO mice(P<0.05).6 IPA did not affect the protein levels of EMMPRIN and MMP9 in Sham mice(P>0.05),but significantly downregulated the expression levels of EMMPRIN and MMP9 in MCAO mice(P<0.05).7 There was no significant difference in the protein levels of Nrf2 and HO-1 between vehicle-treated and IPA-treated Sham mice(P>0.05).Mice from MCAO+IPA group had significantly higher protein levels of Nrf2 and HO-1 than that from MCAO+vehicle group(P<0.05).Conclusions1 IPA upregulates the expression of tight junction proteins and alleviates blood brain barrier disruption after cerebral infarction.2 IPA inhibits EMMPRIN/MMP9 dependent tight junction protein degradation.3 IPA upregulates the protein levels of Nrf2 and HO-1,which may partially participate in the protective effects of IPA on blood brain barrier after cerebral infarction.Part Ⅲ The regulatory effects of IPA on SVZ and hippocampal neurogenesisObjectiveTo investigate the effects of IPA on SVZ and hippocampal neurogenesis.Methods1 The models and groups were performed as described in Part Ⅰ.2 Immunofluorescence staining was used to observe Ki67-positive proliferative cells in SVZ and hippocampal DG on day 7 after cerebral infarction.The results could indicate the effects of IPA on cell proliferation in SVZ and hippocampal DG.3 To identify the effects of IPA on the generation of neuroblasts,immunofluorescence staining was used to evaluate 5-bromo-2’-deoxyuridine(BrdU)and doublecortex(DCX)double positive neuroblasts in SVZ and hippocampal DG on day 7 after cerebral infarction.Additionally,the protein level of DCX was also measured by western blot.Results1 In the SVZ,there was no significant difference in the number of Ki67-positive cells between Sham+vehicle group and Sham+IPA group(P>0.05).Mice from MCAO+IPA group had more Ki67-positive cells than those from MCAO+vehicle group(P<0.05).2 IPA had no significant effects on the number of SVZ BrdU and DCX double positive cells in Sham mice(P>0.05),but significantly upregulated the number of BrdU and DCX double positive cells in MCAO mice(P<0.05).3 No significant change was found in the protein level of SVZ DCX after IPA treatment for Sham mice(P>0.05).IPA-treated MCAO mice showed higher DCX level in the SVZ than vehicle-treated MCAO mice(P<0.05).4 IPA did not change the number of DG Ki67-positive cells in Sham mice(P>0.05),but significantly increased the number of Ki67-positive cells in MCAO mice(P<0.05).5 In the hippocampal DG,there was no significant difference in the number of BrdU and DCX double positive cells between vehicle-treated Sham mice and IPAtreated Sham mice(P>0.05).MCAO mice had more BrdU and DCX double positive cells after IPA treatment(P<0.05).6 Mice from Sham+ vehicle group and Sham+IPA group showed similar DCX level in the hippocampus(P>0.05).However,DCX level in MCAO mice was significantly increased after IPA treatment(P<0.05).Conclusions1 IPA promotes cell proliferation,increases the number of neuroblasts,and enhances SVZ neurogenesis after cerebral infarction.2 IPA facilitates cell proliferation,upregulates the number of neuroblasts,and accelerates hippocampal neurogenesis after cerebral infarction.Final Conclusions1 IPA shows protective effects in a mouse model of cerebral infarction.2 IPA upregulates the expression of tight junction proteins and promotes blood brain barrier integrity.The inhibition of EMMPRIN/MMP9 and promotion of Nrf2/HO-1 may partially participate in the regulatory effects of IPA on blood brain barrier.3 IPA enhances the cell proliferation and the generation of neuroblasts,promoting SVZ and hippocampus neurogenesis after cerebral infarction. |
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