Neuroprotective Effects Of Sonic Hedgehog Signaling Pathway In Ischemic Injury

Research BackgroundCerebral ischemia is a common cerebrovascular disease and the mechanism is complex.It often induces neuronal apoptosis,leading to brain damage.Stroke is one of the most causes of disability and death worldwide.The recovery situation of the patients is dependent on the ischemia duration,brain location,lesion size,and ischemia-evoked endogenous protection.Insufficient delivery of oxygen and glucose is unable to maintain homeostasis in ischemic stroke and leads to cell death by multiple processes including oxidative stress,inflammation,excitotoxicity,ionic imbalance,and apoptosis.Oxygen or sugar imbalances are vital factors for cerebral ischemic injury and improving oxygen supply/consumption balance are able to alleviate the injury.It remains a challenge to improve therapeutic procedures and life quality of the patients with stroke.To find effective therapeutic targets,many studies have been conducted into the possible mechanism and signaling pathway in stroke.Cerebral ischemia induces the formation of reactive oxygen species(ROS)in brain tissue.Excess generation of ROS can activate diverse signaling pathways and regulate the expression of genes encoding a variety of proinflammatory proteins.Meanwhile,some beneficial factors were induced to combat ischemia-induced damage when ischemia occurred.The sonic hedgehog(Shh)signaling pathway in ischemic stroke have been reported in previous studies.Shh is the most widely characterized of the three vertebrate Hedgehog homologs,and is essential for proper embryonic development.Binding of Shh to its receptor Patched(Ptch1)results in the de-repression of Smoothened(Smo).This leads to the activation of Gli2,which regulates the transcription of target genes that include Glil and Ptch1 that control cell growth,survival,and tissues.differentiation.Shh-signaling antagonists that bind to Smo include cyclopamine,SANT1,and Cur-61414.Shh signaling agonists that bind to Smo include the synthetic small molecules purmorphamine and SAG.Recent studies have indicated that Shh pathway is activated in many neurological disease,and the pathway has been gaining recognition as a key contributor to the cellular response to neuronal injury in vitro and in vivo.It has been reported that shh is upregulated in multiple cell types after stroke and the function of the subacute phase is improved via s agonist treatment.Shh with anti-oxidative,anti-apoptotic,and pro-angiogenic effects is reported to improve the function after focal cerebral ischemia.The shh signal pathway as a regulator presents a vital function in self-renewal and functional recovery after brain injury,especially for ischemia.Activation of Shh pathway can be signaling to the downstream target genes ROS and neuronal apoptosis to exert neuroprotective function.But the mechanisms underlying are poorly understood.Our research focused on the expression level and effects of Shh signaling pathway during oxygen-glucose deprivation(OGD)injury in rat adrenal pheochromocytoma cells(PC 12)and primary rat cortical neurons.Objective1.Rat adrenal pheochromocytoma cells(PC 12)and primary rat cortical neurons were exposed to oxygen-glucose deprivation(OGD)injury.To observe the effects of oxygen-glucose deprivation injury on Shh,Gli-1 and Ptch expression,the effect of Shh agonist PUR on glil transfer and the effect of Cyc pretreatment on Shh expression.2.Primary rat cortical neurons were exposed to oxygen-glucose deprivation(OGD)injury.To observe the effects of PUR on OGD-induced apoptosis and ROS levels.3.Primary rat cortical neurons were exposed to oxygen-glucose deprivation(OGD)injury.To observe the effects of PUR and Cyc on OGD-induced expression of neuroligin and neurexin,CREB and BDNF.4.Primary rat cortical neurons were exposed to oxygen-glucose deprivation(OGD)injury.To observe the effects of PUR and Cyc on OGD-induced expression of p-Akt and p-NF-?B.5.To establish the middle cerebral artery occlusion(MCAO)model and observe the effects of PUR on nerve function recovery after MACO injury in rats.Materials and Methods1.Production of primary neurons oxygen-glucose deprivation(OGD)modelPND1 mice were used to harvest cortical neurons which were then cultured in serum-free neurobasal medium with 2%B27 and 1%penicillin-streptomycin.Cells were cultured in poly-D-lysine-coated twelve-well plates for 7 days.Cells were cultured at 37? under 95%nitrogen and 5%CO2 for 6 h in glucose-free DMEM to be subjected to OGD(125 mM NaCl,2.8 mM KCl,1.5 mM MgCl2,0.05 mM MgSO4,2 mM CaCl2,0.83 mM NaH2PO4,24 mM NaHCO3,2 mM HEPES).After OGD,primary neurons were placed in the original neurobasal medium with PUR(20?M)with/without Cyc(10 ?M)for the times indicated.2.Production of PC 12 cell oxygen-glucose deprivation(OGD)modelPC 12 cells were cultured in DMEM containing 5%FBS and 1%penicillin/streptomycin.For MSCs-exosomes treatment,PC 12 cells were seeded with FBS-free culture medium in 12-well plates and treated with exosomes(100 ?g/mL)from transfected and untransfected MSCs for the times indicated.To mimic the ischemic condition in vitro,PC 12 cells were exposed to OGD for 6 h.PC 12 cells were then placed in the original medium with or without PUR(20 ?M)with/without Cyc(10 ?M)for the times indicated.3.Preparation of Middle cerebral artery occlusion(MCAO)MCAO was performed according to the protocols previously described.Rats were anesthetized with isoflurane.The right common carotid artery(CCA),external carotid artery(ECA),and internal carotid artery(ICA)were isolated followed by clamping of the ICA and CCA with microartery clips.The proximal portion of the ECA was ligated using a 5-0 polyester suture and severed at 3.0 mm from the bifurcation of the CCA.The ICA was then completely dissociated,and microsurgical scissors were used to incise a small opening in the arterial wall at 3 mm from the arterial bifurcation at the proximal end of the ECA.A thread embolus was inserted into the ECA parallel with that of the ICA,and the clamp on the ICA was then removed.After achieving microresistance,advancement of the embolus was stopped and the ECA was then tightened with a 5-0 polyester suture.The sham group animals underwent similar surgical procedures without applying the occlusion.The brain infarct was assessed with use of 2,3,5,-triphenyltetrazolium chloride(TTC)staining at 2 days following injury.Neurological functions and brain water content were assessed at 2 days following injury4.Determination of Apoptosis by Flow Cytometric AnalysisApoptosis of PC12 cells was assessed by using the Annexin V-FITC/PI Double Labeling Apoptosis Detection Kit.The percentage of annexin V-positive cells was analyzed by a FACS flow cytometer C6.All assays were performed in triplicate and each experiment was repeated three times.5.Tunel stainingCellular death was determined with use of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(Tunel)staining.The number of Tunel-positive cells were measured in six randomly selected microscopic fields at 200×magnification within the lesion area of each group described above(N=4 mice/group).6.Determinations of ROS productionThe production of ROS in primary neurons was detected by the DHE reactive oxygen species detection kit.Cells were stained with 10 ?M DHE for 30 min.The fluorescence images were obtained after cells rinsing and mounting with use of fluorescent microscopy(BX51;Olympus,Tokyo,Japan).The DHE-staining results were procured with use of the Image-pro plus image analysis system.7.Western BlotThe tissues were homogenized with RIPA containing PMSF and protease/phosphatase inhibitors following centrifuged at 4? at 13,800 × g for 10 min.Then 5 × loading buffer was added to the protein supernatant,and the total protein was quantified using a BCA assay kit CWBIO.Equal amounts of protein were separated by SDS-PAGE,and then transferred to PVDF membranes.Following blocked in 5%non-fat milk for 2 h.Western Blot was used to detect Shh,Gli-1,Patch,p-CREB,CREB,BDNF,p-Akt,Akt,NF-?B,p-NF-?B,?-actin proteins expression,and then secondary antibodies were incubated to membrane at 37? for 60 min.The chemiluminescent signal was developed with use of ECL kit reagents and then detected with use of the Tanon Imaging System.The density of protein bands was semi-quantified using ImageJ.8.Determinations of mRNA levelsTotal RNA of tissue was isolated using TRIzol reagent CWBIO according to the manufacturer.Total RNA of EVs and H2S-EVs was extracted to use Sera Mir EVs RNA Extraction Kit after isolation of EVs using ExoQuick-TCTM.Complementary DNA(cDNA)was synthesized using a reverse transcription system with ReverTra Ace Qpcr RT Kit.Reverse transcriptase quantitative real-time PCR(qRT-PCR)was used to detect the mRNA expression of factors after OGD treatment9.Immunofluorescent stainingThe cells were incubated with primary antibody(Gli-1,1:100)overnight at 4?.The next day,the slices were incubated with secondary antibody at 37? for 30 min.The nucleus was stained with DAPI for 10 min.Images were obtained with fluorescent microscopy.Immunofluorescent staining was used to detect Gli-1 nuclear translocation.Analyses of the images were captured using the Image-Pro Plus 6.0 software(Media Cybernetics,MD,USA).10.Statistical AnalysesValues are expressed as mean ± SD.Statistical analysis was performed by one-way ANOVAs followed by Tukey's post hoc comparisons,using prism software.p value<0.05 was considered statistically significant.Results1.Compared with the Control group,the OGD exposure demonstrated a higher expression of Shh at 4 h and 8 h,whereas decreased at 1 h and 24 h.the expression Gli-1 and Ptch were down-regulated after OGD exposure at 24 h.PUR treatment promoted Gli-1 nuclear translocation.These effects of PUR on the Shh pathway following HI exposure were blocked by Cyc pre-treatment.2.OGD group increased the percentages of apoptotic cells,compared with the Control group Treatment with 20 ?M PUR significantly reduced the proportion of TUNEL-positive cells.Cyc blocked the effect of PUR on the number of TUNEL-positive cells.3.Compared to the control group,OGD increased ROS levels following exposure for 24 h and PUR treatment attenuated OGD-induced ROS levels.The effect of PUR on ROS generation was blocked by Cyc.4.The expression of neuroligin and neurexin decreased at 8 h and 24 h after OGD exposure.PUR treatment upregulated OGD-reduced neuroligin and neurexin at 8 h after OGD.The effect of PUR on neuroligim and neurexin expression was blocked by Cyc.5.Compared to the Control group,OGD significantly decreased p-CREB and BDNF expression following OGD exposure.PUR treatment increased p-CREB expression and BDNF expression.The effect of PUR on p-CREB and BDNF was blocked by Cyc.6.Compared with the Control group,OGD group decreased the expression of p-Akt and increased the expression of p-NF-?B after 4 h post-injury.PUR treatment increased p-Akt expression and decreased p-NF-?B expression.7.PUR promoted the recovery of nerve function after injury in MACO rats and reduced the apoptosis of nerve cells.Conclusion1.OGD decreased the expression of Shh,Ptch and Gli-1,increased the number of apoptotic cells and reactive oxygen species generation.2.OGD decreased the expression of synaptic proteins(neuroligin and neurexin).3.Pretreatment with Purmorphamine,a Shh agonist,increased Gli-1 in the nucleus of neurons,protecting against OGD injury,Shh inhibitor,cyclopamine,showed the opposite effect.4.PUR activation of Shh signals promoted the phosphorylation of CREB,and upregulated the expression BDNF,neuroligin and neurexin following OGD.5.PUR activation of Shh signals increased the phosphorylation of Akt and decreased the phosphorylation of NF-?B following OGD.These results demonstrate that activation of Shh signaling pathway protects from OGD-induced neuronal cells apoptosis and synaptic injury by reinforcing the activation of Akt signaling and decreased NF-?B signaling.