PPAR? Ligand-fenofibrate Increase Heme Oxygenase 1 Expression While Limits Neuronal Injury During Intracerebral Hemorrhage

BackgroundSpontaneously ICH is one of the devastating diseases with high rates of mortality and disability.Peroxisome proliferator-activated receptors alpha(PPARa)is a therapy target in atherosclerosis and cardiovascular diseases.However,anti-inflammatory effects of PPARa in intracerebral hemorrhage(ICH)remain unknown.So we investigated the anti-inflammatory effects of fenofibrate,a ligand of PPARa,in ICH rat model.Then we administrated fenofibrate to ICH rats to determine whether PPARa plays a role in ameliorating brain injury and promoting restoration of the neurons after ICH.Secondary brain injury is a complicated process that involves the space occupying effect of hematoma,cascade of blood coagulation system,broken of red blood cells,the formation of brain edema,participation of inflammatory response and the complement system,and excessive produce of active oxygen radicals,etc.One factor was found to be involved in all of these process,namely heme oxygenase 1(HO-1).In addition,NF?B is involved in the pathological and physiological process of brain injury after ICH,and plays a central role in this process.Therefore,after clarifying the neuroprotective effects of PPARa,we selected HO-1 and NF?B as study objects,and evaluated the effects of fenofibrate on HO-1 and NF?B.Then we investigate whether fenofibrate could regulate the expression of HO-1 and NF?B to alleviate brain injury in PPARa-dependent manner.The whole thesis includes three parts:Part1:Fenofibrate Increases Heme Oxygenase 1 Expression and Astrocyte Proli-feration While Limits Neuronal Inj ury During Intracerebral HemorrhagePart2:The study of mechanism of fenofibrate to relieve brain injury after cerebral hemorrhagePart3:Fenofibrate alleviate brain damage after cerebral hemorrhage in PPARa-dependent manner.Part1:Fenofibrate Increases Heme Oxygenase 1 Expression and Astrocyte ProliferationObjectives1.To explore whether PPARa was involved in the pathophysiological process of ICH to provide the basis for the next study.2.To determine whether PPAR? ligand-fenofibrate could reduce brain tissue damage after ICH.Materials and Methods1.Establishing hypertensive cerebral hemorrhage rat model Intracerebral hemorrhage(ICH)was induced using the autologous blood perfusion model.Male Sprague-Dawley rats undersodiumpentobarbital(1%)anesthesia(0.06g/kg;i.p.)were immobilized in a stereotactic apparatus frame(RWD Life Science Co.,Ltd.Shenzhen,China).A 1-mm-diameter burr hole was made in the skull(1 mm anterior and 3 mm lateral to bregma).Seventy microliter fresh autologous whole blood was drawn using the microsyringe from the caudal vein and was inserted into the right striatum for blood injection into the caudate putamen(5.5 mm deep to bregma).2.Rat brain astrocytes isolationAdult rat brain astrocytes were isolated according to Astrocytes methods and protocols(Tracy F.Uliasz et al,2012)9.Briefly,the rat was euthanizing by deeply anesthetized with sodium pentobarbital.The brain was removed from the skull and placed in Dissection medium(DM).Cerebral hemispheres were removed from the rest of the brain.After pull off the meninges,cortices were rinsed with PBS and immersed in dissection trypsin.Sterile scissors and forceps were used to cut cortices into small pieces.Incubated for 20-30 minutes at 37?,cortices pieces were pipetted up and down slightly several times to dissociate cells.Centrifuged at 720 g for 3 min at room temperature,the pellet were resuspened in DMEM containing 10%FBS and antibiotics.The cell suspension was transferred to 10 cm dish and incubated in a humidified incubator containing 95%air and 5%C02 at 37?.Medium was changed two days after cells seeded.Cell growth was monitored daily after one week in culture.Astrocytes should reach confluence between days 7 and 14.3.Preparation of paraffin sections of brain tissue4.Nissl stainingThree days after surgery,rats were deeply anesthetized with sodium pentobarbital and perfused intracardially with 4%paraformaldehyde in PBS.Brains were removed and were fixed in 10%neutral buffered formalin and then embedded in paraffin.Serial section was carried out to produce 5 microns slices.Slides were deparaffinized,rehydrated,and stained with Toluidine Blue(sc-253710)to indicate nissl body.5.immunofluorescence stainingFor immunostaning,astrocytes were cultured on cover slips in 35 mm dishes and were fixed with 3.7%paraformaldehyde for 15 min and then permeabilized with 0.3%Triton X-100 for 5 min on ice.Cells were incubated with anti-GFAP and anti-S100B antibodies overnight at 4?,washed,and stained for an hour with FITC-labeled secondary antibody at room temperature.Cover slips were mounted with Mowoil containing 1?g/ml 4',6-diamidino-2-phenylindole(DAPI)for DNA staining.Images were captured on an Olympus fluorescence microscope(Olympus CKX41,Japan)coupled to a cooled charge-coupled device camera(QICAM,Japan)and processed by using the QCapture Pro 6.0 program.6.immunofluorescence histochemical stainingGFAP immunofluorescence staining was performed on brain tissue sections in ICH model rats and fenofibrate group to reflect the activation of astrocytes.7.Western blot PPARa protein expression level was detected.8.Total RNA extraction and reverse transcription-PCR quantification9.Statistical analysisResults1.Autologous blood perfusion ICH rat model was established.Total RNA of brain tissue was extracted and mRNA level of PPARa was measured using real-time PCR.Our results showed that mRNA level of PPARa was upregulated within 24 hours,then decline from 36 hours,and continuously decreased in 72 hours in ICH rats brain.2.Increased Nissl body and astrocytes after gastric perfusion of fenofibrate three days.3.Fenofibrate promoted astrocytes proliferation in cultured adult rat brain astrocytes.Conclusion1.The changed and regularity of PPARa mRNA and protein expression after ICH proved that PPARa was involved in the pathophysiological process of the lesion site of patients with cerebral hemorrhage.2.Fenofibrate could reduce brain tissue damage after ICH both in vitro and in vivo.Part2:The study of mechanism of fenofibrate to relieve brain injury after cerebral hemorrhageObjectivesSelected HO-1 and NF?B as study objects,to evaluate the effects of fenofibrate on HO-1 and NF?B in ICH.Materials and Methods1.Model of intracerebral hemorrhage in LN-18 cellsTo mimic ICH in LN-18 cells,hemin was supplemented to cell culture medium at a final concentration of 5?M for 24 hours.2.State of inflammatory response after ICHTo induce inflammation in LN-18 cells,LPS was introduced into culture medium at 1?g/mL.3.Western blotHO-1 and NF?B protein expression level was detected after fenofibrate intervention.Results1.Fenofibrate resulted in increase of HO-1 expression and decrease of NF?B protein expression in LN-18 cells.2.Hemin induced upregulation of both NF?B and HO-1.3.Decrease of HO-1 protein level induce by LPS was rescued by fenofibrate,while increase of MMP9 induced by LPS was blocked by fenofibrate.ConclusionOur data indicated that in ICH injury,the protective effects of fenoifibrate may due to downregulate NF?B and upregulate HO-1.Part3:Fenofibrate alleviate brain damage after cerebral hemorrhage in PPARa-dependent manner..Objectives1.To explore whether the effect of fenofibrate on the expression of HO-1 and NF?B was mediated by PPARa path.2.To Clarify the relationship between HO-1 and NF?B.Materials and Methods1.siRNAAccession Number of the gene of interest was found on the NCBI.This thesis used siRNA in website(http://maidesigner.thermofisher.com/rnaiexpress/design.do)design.For small RNA interference assay,LN-18 cells were transfected with 50 nM siRNAs for 48 hours.2.Western blotAfter transfected with 50nM PPAR? siRNAs for 48 hours,LN-18 cells were harvested for Western blot to check the expression of HO-1 and NF?B protein expression respectively.Results1.Treatment with PPARa siRNA could upregulate HO-1 but downregulate NF?B protein and mRNA expression in LN-18 cells.2.Treatment with PPARa siRNA could upregulate HO-1 but downregulate NF?B protein and mRNA expression in rat brain astrocytes.3.Silence of PPARa not only decreased HO-1expression significantly but also inhibited fenofibrate-induced increase of HO-1 in rat brain astrocytes.4.The protein level of NF?B did not increase although HO-1 was abolished.ConclusionOur results indicated that the protective effects of fenofibrate in ICH rat were dependent on PPARa and mediated by upregulating HO-1 and down regulating NF?B.Summary1.Our results indicated that:1.PPARa was involved in the pathophysiological process of the lesion site of patients with cerebral hemorrhage.2.Increased Nissl body and astrocytes after gastric perfusion of fenofibrate 3.Fenofibrate promoted astrocytes proliferation in cultured adult rat brain astrocytes.So furthmore shows fenofibrate could reduce brain tissue damage after ICH both in vitro and in vivo.4.Our data indicated that in ICH injury,the protective effects of fenoifibrate may due to downregulate NF?B and upregulate HO-1.5.Our results indicated that the protective effects of fenofibrate in ICH rat were dependent on PPARa and mediated by upregulating HO-1 and down regulating NF?B.2.The significance of this study is as follows:1.It proves that fenofibrate can reduce brain damage and protect nerve function after ICH through non-lipid-lowering effect;2.The new pathways of PPARa were revealed,suggesting that PPARa and HO-1 could be important targets for the treatment of ICH.3.The protective mechanism of fenofibrate in secondary brain injury afterICH is clarified,which provides a new exploration direction for the treatment of ICH,and provides the theoretical basis of physiology and pathophysiology for the development of new drugs.