| Stroke is a medical condition in which poor blood flow to the brain results in brain damage.There were two types of stroke:ischemic stroke and bleeding stroke.Ischemic stroke,accounting for approx.90%of all stroke cases,typically occurs in the middle cerebral artery(MCA).Currently,recanalization by intravenous(i.v)thrombolysis and thrombectomy is first-line treatments for ischemic stroke patients.However,one of the major criteria for i.v.thrombolysis is the 4.5 h therapeutic time window.Considering the low number of stroke patients eligible for treatment using thrombolysis,novel treatment options are critically needed.New therapy targeting post-ischemic inflammation is highly being emphasized experimentally and clinically.Such treatment might be beneficial to stroke patients with good collateral blood supply who suffer permanent ischemia.Inflammation is integral to the pathophysiology of ischemic stroke.The first immune cells to sense a stroke are the brain-resident microglial cells.In the acute phase of ischemic stroke,microglia are activated and then circulating immune cells invade the peri-infarct and infarct core.Resident and infiltrating cells together orchestrate the post-stroke inflammatory response through soluble and membrane-bound signaling molecules,including cytokines.Inflammation can be both detrimental and beneficial at particular stages after a stroke.On one hand it can contribute to expansion of the infarct,on the other hand it is also responsible for infarct resolution.Microglia appear to display classical M1 and M2 phenotypes after ischemic stroke.Ml phenotypes microglia aggravate brain injury though produce a series of pro-inflammatory cytokines,such as TNF-a,IL-1β,IL-6,MCP-1,COX-2 and NO.Inhibiting inflammatory response caused by excessive activation of microglia could ameliorates secondary injury in stroke models.As one of the preferred drugs for the clinical treatment of stroke,Naoxintong capsule(NXTC)could alleviate neurological injury and inhibit inflammatory response in patients with ischemic stroke.Pharmacological studies demonstrated that NXTC protected the mouse brain against ischaemia injury by ameliorating the infarct volume,brain oedema,and inflammatory cytokines.Even though various studies have shown that anti-inflammatory actions might play an important role in treatment of stroke by NXTC.However further investigations are still required to confirm the specific mechanisms,which will be beneficial for its effective clinical application.Hence,in order to solve the problem of unclear mechanism of NXTC.In this research,LPS induced microglia BV-2 inflammation model was adopted to investigate the expression of inflammatory cytokines by NXTC.Then,an integrative strategy combined with with high-depth sequencing and co-expression network analysis was used to identify the expression profiles of lncRNAs and mRNAs in BV-2 microglia cells after pretreatment with NXTC intestinal absorption(NXTCIA).Finally,the expressions of lncRNAs and mRNAs in vitro and in vivo were determined by quantitative reverse transcription polymerase chain reaction(qRT-PCR).By combined the sequencing results,co-expression network,bioinformatics analysis and quantitative RT-PCR,we aimed to prove NXTC-associated mechanism and provide valuable information for its clinical application.Content1.In this part,the effect of different concentration of NXTC intestinal absorption(NXTCIA)on BV-2 was assessed.Then,LPS induced microglia BV-2 inflammatory model was adopted.Then,the levels of inflammatory cytokines TNF-α,Il-1β,Il-6,MCP-1,and COX-2 and NO production in BV-2 microglia cell culture supernatants after pretreatment with NXTCIA followed by LPS stimulation were measured by ELISA kits and Griess reagent.2.An RNA-Seq method was performed to identify the differentially expressed lncRNAs and mRNAs in microglia BV-2 after pretreatment with NXTCIA.Then,a lncRNA-mRNA co-expression network was constructed to explore the regulatory functions of the lncRNAs through the annotation of their co-expressed mRNAs.The completed network,might play a key role in the core regulation of post-ischaemia inflammation,was further analyzed.3.A middle cerebral artery occlusion(MCAO)model was adopted to investigate the expression level of TNF-α,Il-1β,Il-6,MCP-1,COX-2 and NO in hippocampus after pretreatment with NXTC.The potential IncRNA and mRNA were verified in MCAO and LPS induced microglia.Results1.Evaluation of the Protection Effects of NXTC Against LPS induced BV-2 inflammatory response.There are no obvious cytotoxic effects on BV-2 microglia cells at different doses of NXTCIA.Further,NXTCIA significantly increased the viability of BV-2 microglia cells at the 62.5 μg·mL-1 and 31.25 μg·mL-1 doses.In LPS induced BV-2 inflammation model,the levels of TNF-α,II-1β,Il-6,MCP-1,COX-2 and NO were markedly elevated,whereas four NXTCIA concentrations(7.81,15.63,31.25,and 62.5 μg·mL-1)could significantly lower these levels in a dose-dependent manner.2.Systematic Investigation of Differentially Expressed Genes via RNA SequencingThere were 87 lncRNA and 519 mRNA transcripts differentially expressed upon stimulation of BV-2 cells with LPS.However,an important portion of these transcripts(34 lncRNA and 392 mRNA)was reversed following pretreatment with NXTCIA.GO enrichment analysis showed that the transcripts reversed by NXTC were mainly involved in the biological process of inflammatory response,cellular response to interleukin-1,cellular response to tumur necrosis factor,immune response,and chemokine signalling pathway.Among the differentially expressed IncRNA and mRNA,Gas5,Meg,Ccl2,Ccl5,Ccl7,Cxcll,Cxcl10,Cx3cl1,Tnfaip3,Tnfrsf9,1118,Il1b,Il6,Rarres2,Tlr3,and Tlr7 might participated in NXTC against LPS induced inflammatory response.34 lncRNAs and 1662 mRNAs were used to construct a lncRNA-mRNA co-expression network.The results showed that the co-expression network contained 1698 nodes and 4400 connections.lncRNA Gas5 was co-expressed with 62 NXTCIA-rescued mRNAs may play an important role in NXTC against inflammation through act on inflammatory response,chemokine signalling pathway,cellular response to tumur necrosis factor,and immune response.3.Further Validation of Gas5 and Its Co-expression mRNAs in vitro and in vivoThe expression levels of TNF-α,Il-1β,Il-6,MCP-1,COX-2 and NO were significantly increased in the hippocampus 24h after MCAO,whereas 110 mg·kg-1 NXTC markedly suppressed these inflammatory mediators.The qRT-PCR results showed that NXTC could significantly suppress the expression level of Gas5,Ccl5, IL-1b,IL-6 and promote the expression level of Tnfaip3 in the hippocampus 24 h after MCAO and LPS induced BV-2 cell.Taken together,our findings showed that NXTC treatment provides protective effects against inflammatory response induced by cerebral ischaemia in vivo and in vitro.A comprehensive method combining RNA-Seq technology,IncRNA and mRNA co-expression network analysis,and qRT-PCR revealed that Gas5,Ccl5,Il-lb,Il-6,and Tnfaip3 are closely associated with inflammatory response in NXTC-pre-treated BV-2 microglia cells and the MCAO hippocampus.These findings provide a novel understanding of NXTC mechanisms which may be a potential target in treatment of stroke.The innovation point of the study1.RNA-Seq high-throughput sequencing technology was applied to investigate the mechanism of NXTC on post-ischemic inflammation.It is showed that the differentially expressed lncRNA and mRNA reversed by NXTC might effect on the development of post-ischemic inflammation through participate in inflammatory response,cellular response to interleukin-1,cellular response to tumur necrosis factor,immune response,and chemokine signalling pathway.2.A high-depth method and lncRNA-mRNA co-expression network analysis showed that lncRNA Gas5 was co-expressed with 62 NXTCIA-rescued mRNAs may participated in NXTC against post-ischemic inflammation.3.A comprehensive method combining RNA-Seq technology,IncRNA and mRNA co-expression network analysis,and qRT-PCR revealed that Gas5,Ccl5,Il-lb,Il-6,and Tnfaip3 are linked with the post-ischemic inflammation. |
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