Effects Of Lactobacillus Plantarum-derived Extracellular Vesicles On Reducing Neuronal Apoptosis After Ischemic Stroke Via MiR-101a-3p And Its Mechanism

Part1:Study on the Role of Lactobacillus Plantarum-derived Extracellular Vesicles in Ischemia-reperfusion InjuryObjective:1.To explore whether Lactobacillus Plantarum-derived extracellular vesicles can cross the blood-brain barrier and be absorbed by ischemic neurons in vitro and in vivo;2.To explore the functional role of Lactobacillus Plantarum-derived extracellular vesicles in the t MCAO/R mouse model;3.To study the functional role of Lactobacillus Plantarum-derived extracellular vesicles in the primary neuronal OGD model.Methods:1.LEVs were Di R-labelled and injected into C57 mice via the tail vein and the IVIS system was applied to conduct imaging of C57 mice at 6,12,18 and 24 hours in vivo.LEVs were labeled with PHK26 and then injected into the t MCAO/R model via the tail vein,co-cultured with primary cortical neurons where neuronal uptake was detected by immunofluorescence staining.2.In t MCAO/R mice,the mice in test group were injected with LEVs via tail vein and the control group with PBS.m NSS score was used to assess neurological deficits,TTC staining was used to validate infarcts,HE staining was used for pathological observation of cortical ischemic semidark zones in mice,and Western blot technique was used to show apoptosis-associated proteins(Bax,Bcl-2,Total Caspase3 and Cleaved Caspase3),TUNEL staining for TUNEL~+and TUNEL~+Neu N~+cell rates.3.The neurons were co-cultured with LEVs(test group)or PBS(control group)with primary neurons for 24 hours,followed by OGD.The survival of neurons in each group was measured with CCK8,lactate dehydrogenase(LDH)for cell viability,Western blot for the expression of apoptosis-related proteins and the TUNEL staining for TUNEL+cell rate between the two groups.Results:1.Di R-labelled exocysts were observed in the skull of C57 mice by the IVIS system at 6,12,18 and 24 hours respectively,indicating the capability of LEVs injected through tail vein to cross the blood-brain barrier into brain tissue in C57 mice.Immunofluorescence assays suggested the existence of LEVs labeled by PHK26 in both cerebral ischemic cortical neurons and primary cortical neurons,indicating that LEVs can be taken up by ischemic neurons in vitro and in vivo.2.m NSS scores showed the significant reduction of neurological deficit LEVs at 3days post-stroke in t MCAO/R+LEVs treatment group compared to the t MCAO/R+PBS treatment group(p<0.05).Infarcts in t MCAO/R mice were examined by TTC staining,showing a reduced the infarct size after 3 days of t MCAO/R in t MCAO/R+LEVs treatment compared to the t MCAO/R+PBS treatment group(p<0.05).Pathological observation of the cortical ischemic semidark zone in mice was performed using HE staining.The results showed a proliferation of cortical glial cells in the t MCAO/R+PBS-treated group,in which cells were more swollen than before,mild degeneration was observed in the neuronal focal areas,and more obvious sparing&softening of the nerve felt.t MCAO/R+LEVs-treated group showed mild proliferation of cortical glial cells,where cells were basically normal and only in the focal areas appeared sparing&softening of the nerve felt.The expression of apoptosis-related proteins was detected using Western blot,reporting a reduced expression of Bax and Cleaved Caspase3 protein and an increase of Bcl-2 protein expression in the LEVs-treated group compared to the t MCAO/R+PBS-treated group(p<0.05).TUNEL~+and TUNEL~+Neu N~+cell rates were measured using TUNEL staining,and TUNEL+and TUNEL+Neu N+cell rates were reduced in the OGD+LEVs treated group compared to the PBS treated group(p<0.05).3.Neuronal survival in each group was detect by CCK8,LEVs treatment significantly elevated neuronal viability after OGD(p<0.05).Cell damage was aggravated 5-fold after OGD,while treatment with LEVs reduced cell death to 2-fold that of the control group.The expression of apoptosis-related proteins was examined using Western blot,and the expression of Bax and Cleaved caspase-3 proteins was significantly reduced in the OGD+LEVs-treated group compared to the OGD+PBS-treated group,and the expression of Bcl-2 protein was increased compared to the PBS-treated group(p<0.05).TUNEL staining was performed to detect TUNEL+cell rate in the two groups,and the LEVs-treated group showed significantly lower TUNEL+cell rate compared to the OGD+PBS-treated group(p<0.05).Conclusions:1.LEVs cross the blood-brain barrier and can be absorbed by neurons both in vitro and in vivo.2.LEVs treatment significantly reduced neurological impairment and brain infarct size in the t MCAO/R mouse model.3.LEVs treatment attenuated neuronal apoptosis after oxygen-glucose deprivation(OGD).Part2:Lactobacillus Plantarum-derived Extracellular Vesicles Regulate the c-Fos /TGF-β1 Pathway Through miR-101a-3p to Affect Post-ischemic stroke Neuronal ApoptosisObjectives: 1.To explore the miRNAs differentially expressed by primary neuronal OGD/R models treated with PBS and LEVs;2.To identify the miRNA(miR-101a-3p)and its downstream target proteins that are differentially expressed by LEVs of the most significance through altered ischemia models.3.To explore whether miR-101a-3p can manipulate neural function in the t MCAO/R mouse model;4.To investigate whether miR-101a-3p can regulate the c-Fos/TGF-β1 signaling pathway;5.To explore whether miR-101a-3p can manipulate neuronal apoptosis in tMCAO/R mice;6.To investigate whether miR-101a-3p can intervene neuronal OGD model survival through c-Fos and the downstream TGF-β1 pathway.Methods: 1.High-throughput sequencing(replicated 3 times)was applied to detect miRNA expression between the neuronal OGD model group and the LEVs treated + OGD model group.2.According to the sequencing results,the top 10 miRNAs with the most significant difference in up-regulated expression were selected.The four groups above were validated using q RT-PCR,in which the differentially expressed miRNA(miR-101a-3p)with the most significance was selected as the target of the study and its expression in LEVs was detected using q RT-PCR to determine that LEVs carried miR-101a-3p.Downstream target genes were predicted by bioinformatics analysis of miR-101a-3p,with the relationship further validated by luciferase reporter gene assay.3.miR-101a-3p overexpression group(miR-101a-3p agomir)and miR-101a-3p suppression group(miR-101a-3p antagomir)were injected in the lateral ventricle and negative controls(NC)miR-101a-3p agomir and miR-101a-3p antagomir defined as control group.C57BL/6 mice were randomly divided into sham-operated group,t MCAO/R model group,miR-101a-3p agomir + t MCAO/R model group,miR-101a-3p antagomir + t MCAO/R model group,NC miR-101a-3p agomir + t MCAO/R model group,NC miR-101a-3p antagomir + t MCAO/R model group,respectively.The m NSS score was applied to score the symptoms of neurological deficits in mice,TTC staining to determine the area of cortical brain infarction in mice.4.The mice were randomly divided into sham-operated group,t MCAO/R model group,miR-101a-3p agomir + t MCAO/R model group,miR-101a-3p antagomir + t MCAO/R model group,NC miR-101a-3p agomir + t MCAO/R model group,NC miR-101a-3p antagomir + t MCAO/R model group,respectively.The expression of miR-101a-3p in the cerebral ischemic cortex of each group was detected by q RT-PCR;the protein expression of c-Fos and TGF-β1 in the cerebral ischemic cortex of mice was detected by Western blot and the expression of c-Fos and TGF-β1 in the cerebral ischemic cortex of mice by immunofluorescence technique.5.Mice were randomly divided into sham-operated group,tMCAO/R model group,miR-101a-3p agomir + t MCAO/R model group,miR-101a-3p antagomir + t MCAO/R model group,NC miR-101a-3p agomir + t MCAO/R model group,NC miR-101a-3p antagomir + t MCAO/R model group,respectively.Western blot was applied to detect the expression of apoptosis-related proteins(Bax,Bcl-2,Total Caspase3 and Cleaved Caspase3)in ischemic cortex where TUNEL and Neu N double-positive cell staining techniques to detect apoptosis of neurons.6.After treated with OGD/R,cells in each group of were followed up,with divided into OGD/R model group,miR-101 a-3p mimi + OGD/R model group,miR-101a-3p inhibitor + OGD/R model group,NC miR-101a-3p mimi + OGD/R model group,NC miR-101a-3p inhibitor + OGD/R model group,respectively.LDH was adopted to determine cell survival;q RT-PCR was used to detect the expression of miR-101a-3p in each group;Western blot was applied to detect the expression of apoptosis-related proteins(Bax,Bcl-2,Total Caspase3 and Cleaved Caspase3)and c-Fos and TGF-β1 in each group;TUNE TUNE staining was conducted to detect neuronal apoptosis.Results: 1.A total of 441 miRNAs were detected differentially expressed in LEVs-treated versus PBS-treated ischemic neuronal specimens,of which 117 were up-regulated and 324 down-regulated.2.The top 10 upregulated miRNAs were selected for q RT-PCR to validate the outcomes of miRNA microarray analysis.In OGD-induced neurons,showed the differential expression of miR-101a-3p,miR-148b-3p,and miR-186-5p in the LEV group compared to the PBS group(p<0.05),which was consistent with the outcomes in t MCAO/R mice(p<0.05).In both OGD-induced neurons and t MCAO/R mice,only miR-101a-3p expression differed,showing the more pronounced differences.Therefore,we hypothesized that LEVs exert neuroprotective effects by altering miR-101a-3p expression in ischemic neurons in vivo and in vitro.Afterwards,93 target genes of miR-101a-3p were predicted by database of miRmap,miRanda,micro T,Pic Tar,and PITA.Target Scan was carried out to obtain the binding sites between miR-101a-3p and c-Fos in humans,rats,and mice.A dual-luciferase reporter gene assay was implemented to confirm whether miR-101a-3p targets c-Fos,which showed that in contrast to the mimic-NC + c-Fos-3’UTR-WT group,reporting the evident decline of the luciferase activity in the miR-101a-3p mimic + c-Fos-3’UTR-WT group,which,however,showed no remarkable difference between the mimic-NC+ c-Fos-3’UTR-MUT group and the miR-101a-3pmimic + c-Fos-3’UTR-MUT group.3.The infarct volume in t MCAO/R mice increased led by the injection of miR-101a-3p antagomir(p<0.05),whilst dramatically declined 3 days post-ischemia treated with miR-101a-3p agomir(p < 0.05).The m NSS score display,neurological deficits of mice 3 days following t MCAO/R were signally enhanced by miR-101a-3p antagomir treatment(p < 0.05)while significantly ameliorated by miR-101a-3p agomir treatment(p<0.05).4.As verified by q RT-PCR results,miR-101a-3p antagomir treatment remarkably reduced miR-101a-3p in comparison to treatment with PBS or antagomir-NC(p<0.05),whereas miR-101a-3p agomir treatment markedly augmented miR-101a-3p expression in t MCAO/R mice in comparison to treatment with PBS or agomir-NC(p<0.05).Furthermore,western blot analysis also documented the elevation of c-Fos and TGF-β1 expression in t MCAO/R mice after miR-101a-3p antagomir treatment(p < 0.05)while the reduced expression subsequent to miR-101a-3p agomir treatment(p<0.05).Afterwards,immunofluorescence was performed to detect cFos expression,reporting that the proportion of c-Fos+ Neu N+ cells were noticeably elevated by miR-101a-3p antagomir treatment while observably decreased by miR-101a-3p agomir treatment in the cortical peri-ischemic brain tissues of t MCAO/R mice.5.Western blot was used to detect the expression of apoptosis-related proteins.Compared to treatment with PBS or inhibitor-NC,treatment with miR-101a-3p antagomir significantly elevated Bax and Cleaved Caspase3 protein expression(p<0.05)while significantly decreased Bcl-2 protein expression(p<0.05).And the comparison between treatment with miR-101a-3p agomir and treatment with PBS or agomir-NC(p<0.05)showed the opposite results.TUNEL staining showed that treatment with miR-101a-3p antagomir significantly increased TUNEL+ and TUNEL+Neu N+ cell rates in comparison to treatment with PBS or inhibitor-NC(p<0.05).And the comparison between treatment with miR-101a-3p agomir and treatment with PBS or agomir-NC(p<0.05)showed the opposite results(p<0.05).6.Cell viability was evaluated depending on the LDH assay,manifesting that miR-101a-3p inhibitor treatment considerably diminished the viability of OGD-induced neurons(p < 0.05),which was contrary to outcomes of miR-101a-3p mimic treatment(p<0.05).In addition,q RT-PCR analysis also demonstrated that miR-101a-3p inhibitor treatment contributed to the prominent decline of miR-101a-3p expression in OGD-induced neurons compared to treatment with PBS or inhibitor-NC,while miR-101a-3p mimic treatment reversely contributed to the prominent rise in comparison to treatment with PBS or mimic-NC(p < 0.05).As reflected by western blot analysis,c-Fos and TGF-β1 expression intervened by miR-101a-3p inhibitor down-regulation was increased after treatment with miR-101a-3p inhibitor compared to treatment with PBS or inhibitor-NC,which was reversed in miR-101a-3p mimic c-Fos and TGF-β1 expression(p<0.05).Protein blotting and TUNEL staining analyses were conducted to detect the neuroprotective effects of miR-101a-3p in vitro.Treatment with miR-101a-3p inhibitor significantly increased Bax,Cleaved Caspase3 protein expression and TUNEL+ cell rate(p<0.05)and significantly decreased Bcl-2 protein expression(p<0.05)in comparison to treatment with PBS or inhibitor-NC.And the comparison between treatment with miR-101a-3p mimic and treatment with PBS or mimi-NC reported the opposite.Conclusions: 1.Treatment with LEVs alters the expression of miRNAs in ischemic neurons.2.LEVs manipulate their downstream target c-Fos by altering the expression of miR-101a-3p in an ischemia model.3.miR-101a-3p regulates neural function in t MCAO/R mice.4.miR-101a-3p regulates c-Fos and downstream TGF-β1.5.miR-101a-3p regulates neuronal apoptosis in t MCAO/R mice.6.miR-101a-3p inhibits c-Fos and downstream TGF-β1 pathways to increase neuronal OGD/R model survival.Part3:has-miR-101-3p Expression in Peripheral Blood of Patients with Acute Ischemic StrokeObjectives:1.To investigate the plasma expression of has-miR-101-3p in normal patients and in patients with acute ischemic stroke.2.To explore whether the plasma expression of has-miR-101-3p in normal patients and in patients with acute ischemic stroke shows differences in gender.3.To explore the plasma expression of has-miR-101-3p in acute ischemic stroke patients before and after transvenous thrombolysis.Methods:1.Peripheral blood were sampled and collected from normal patients and patients with acute ischemic stroke,plasma was extracted and stored at-80°C,accompanied with the collection of clinical case data.2.The expression of has-miR-101-3p in peripheral plasma from normal patients and acute ischemic stroke patients was measured with q RT-PCR.3.Statistical analysis was carried out for calculating the clinical correlation between has-miR-101-3p and acute ischemic stroke.Results:1.Compared with age-and sex-matched cognitively healthy controls,patients with acute ischemic stroke showed significantly lower plasma levels of has-miR-101-3p(p < 0.05).2.has-miR-101-3p expression was reduced in the plasma of both male and female acute ischemic stroke patients,without gender-based differences in has-miR-101-3p levels(p < 0.05)3.has-miR-101-3p expression in plasma of acute ischemic stroke patients was upregulated 24 hours after receiving intravenous thrombolysis(p < 0.05).Conclusions:1.has-miR-101-3p expression was decreased in the plasma of acute ischemic stroke patients;2.has-miR-101-3p expression was reduced in the plasma of both male and female patients with acute ischemic stroke;3.has-miR-101-3p expression was increased in plasma from patients with acute ischemic stroke after recovery of neurological function treated with intravenous thrombolysis.