Protective Effectsand Mechanisms Of Heme Oxygenase-1 On Focal Cerebral Ischemia Model In Rats

BackgroundStroke is a serious medical and health event,which is manifested by poor blood supply to the brain and further leading to neuronal cell death.It seriously threatens human health and affects the quality of life.The effective treatments for this disease are still scarce.Tissue plasminogen activator(rt-PA)is currently the only effective drug for the treatment of acute ischemic stroke.However,its clinical application is hampered by the limitation of time window and thrombolytic contraindications.Therefore,it is great clinical and social significant to reveal the mechanism of stroke and find the new treatment methods.Heme oxygenase(HO)is a rate-limiting enzyme that degrades heme.Heme is oxidized and degraded by HO into the effective antioxidants carbon monoxide(CO),bilirubin,and free iron.HO has two isozymes,HO-1 and HO-2.HO-1 abundantly expressed in the central nervous system under the induction of ischemic injury,oxidative stress or other nociceptive stimuli.Some studies had found that HO-1 knockout mice were more sensitive to ischemia-reperfusion injury.In the transgenic rats,the overexpression of HO-1 could effectively reduce the affects of Ischemic stroke injury caused by middle cerebral artery occlusion(MCAO),which suggested that HO-1 had a protective effect in ischemic brain injury.However,there were still no effective means to over-express HO-1 in vivo.Adenovirus vector has been used in many study to carry the gene.It could be the better vector to deliver HO-1 in treatment of stroke.Regulatory T cells(Treg)and Th17 cells are two types of CD4+T lymphocyte subsets.Treg cells have been found to have negative regulatory effects on immune responses in recent years.They can be widely involved in allergic reactions,autoimmune reactions,and graft-versus-host response.Treg cells secrete the inhibitory cytokines,such as IL-10,TGF-β and other cytokines,to participate in the pathophysiological processes of many diseases.Th17 cells mainly secrete IL-17A,IL-6,and IL-23,which are related to pro-inflammatory reactions and autoimmune diseases.Previous studies have found that Th17 and Treg cells play an important role in the pathophysiology of cerebral infarction.Other studies have found that the protection of HO-1 against certain non-inflammatory diseases is related to the regulation of Th17 and Treg cells.HO-1,as an important protective protein,plays a key role in the pathogenesis of ischemic stroke.Its entry into the brain tissue can mediate immune and non-immune reactions and trigger oxidative stress in the body.Studies have suggested that T cell-mediated inflammation plays an important role in the pathological process of ischemic stroke,and HO-1 can reduce the inflammation in local tissues,and reducing brain damage.Recent studies have shown that different subtypes of CD4+T cells play different roles in brain injury after ischemic stroke.Treg cells can reduce inflammatory response and protect ischemic brain tissue.Studies have found that HO-1 can affect the differentiation of a variety of cells,such as osteoblasts,adipocytes,dendritic cells(DC),and Kupffer cells.HO-1 has also been reported to affect Th 17 cell differentiation in vitro.The cytokines TGF-β and IL-6 are necessary cytokines to promote the differentiation of naive T cells into Th17 cells.RORγt and signal transducer and activator of transcription(STAT3)are important to the differentiation of Th17 cells.Transcription factor TGF-P binds to serine or threonine kinase receptors on the surface of naive T cells,induces phosphorylation of transcription factors Smad2 and Smad3 to form heterodynes or trimmers into the nucleus,and synergistically promotes RORyt and Foxp3 expression.RORyt can promote the expression of IL-23R by naive T cells,so that differentiated Th17 cells can further mature and expand.In addition,Foxp3 is a characteristic transcription factor of Treg cells,which can promote the differentiation and proliferation of Treg cells.The IL-6 signaling pathway is the key to regulating the differentiation of naive T cells into Th17/Treg cells.Several studies also found that both HO-1 and the catalytic product CO can activate the intracellular STAT3 signaling pathway,and further protect against hyperoxic pulmonary endothelial injury and liver ischemia-reperfusion injury.However,other studies have found that CO-1,a product of HO-1,can significantly inhibit STAT3 overexpression induced by ischemia-reperfusion injury in rat liver transplantation models.In addition,inducing HO-1 expression can inhibit the STAT3 signaling pathway and block prostate cancer cells androgen receptor to restrict its tumor characteristics.The current research finds that the mechanism of action of HO-1 is different,but it is necessary to study the HO-1 action.This study intends to explore the function and mechanism of HO-1,mediated by the adenovirus vector,in tMCAO rat models.HO-1 may protect from further impair through the regulation of Th17/Treg in ischemic stroke models.It maybe provide a novel clinical treatment of ischemic stroke.Objectives1.To determine the protective effect of adenovirus vector-mediated HO-1 gene on focal cerebral ischemic injury in rats.2.To determine whether the protective effect of HO-1 gene on cerebral ischemic injury in rats is achieved by regulation of the Th17/Treg cells balance.3.To further explore the mechanism of HO-1 regulating the differentiation of Th17 and Treg cells.Materials and MethodsSD rats were used in the study,and they were randomly divided into four groups,which were sham operation group(sham,SH group),empty vector group(vehicle,V group),adenovirus vector group(Ad group),and adenovirus HO-1 group(Ad-HO-1).Before ischemic treatment,rats in V group,Ad group and Ad-HO-1 group were injected with saline,adenovirus and recombinant adenovirus carried the HO-1 respectively through lateral ventricle for 3 days.A transient middle cerebral artery occlusion(tMCAO)method was used to construct a focal cerebral ischemic infarction model.24 hours after tMCAO,GFP in the vector was used to assess adenovirus infection.Western blot was used to detect HO-1 expression in each group of rats.TCC were used to detect the volume of cerebral infarction in rats.Neurological deficit score was used to evaluate the neurological deficit of rats.The neuronal apoptosis was evaluated by measuring the activity of Caspase-3 and the TUNEL assay.IL-6,IL-17A,and IL-10 in the serum and brain tissue homogenate were detacted by Elisa.IL-17A、IL-10and IL-6 in the supernatant of the spleen cell culture were also detected with Elisa.The expression of p-STAT3 and RORγt in brain tissue was determined by Western blot.The mRNA expression of IL-10 and IL-17A in brain tissue was measured by Real-time PCR.The number and the expression intensity of IL-10R in Th17 cells and Treg cells were detected by FCM.After the above tests,the effects of HO-1 overexpression on Th17 and Treg key cytokines and receptor was analysis.CD4+ T cells were isolated from the spleen of healthy SD rats and divided into four groups:Th0 group(anti-rat CD3 antibody was added to the culture system),Th17 group(on the basis of Th0 group,rat TGF-β1,IL-6,IL-23,IL-2,and anti-rat IL-4 antibody,anti-rat IFN-y antibody were added to the culture system),Th17+Hemin group(hemin,HO-1 inducer),and Th17+SnPP group(SnPP,HO-1 inhibitor).Cells were treated for-5 days,and then cells were collected.The ratio of Th17 cells was detected by FCM.The cytokines(IL-6,IL-17A,IL-10 and TGF-β)were detected by Elisa.The mRNA levels of the factors(RORγt,Foxp3 and STAT3)were detected by Realtime PCR.IL-6,IL-10,TGF-β and IL-17A in the cell culture supernatant were detected with ELISA.FCM data analysis uses software FCM express v3.0.The statistical analysis was performed using the software GraphPad Prism.v 7.0,and the expression comparison of each index between groups was analyzed by single factor analysis of variance,with a test level of α=0.05.In the statistical analysis,continuous variables with normal distribution are all expressed as mean ±SD(standard deviation).Quantitative data were analyzed and compared between groups using t-test or ANOVA.The Kruskal-Wallis ANOVA method was used to analyze the data of abnormal distribution between groups.SPSS software(v18.0)was used for statistical analysis and P<0.05 was considered statistically significant.Results1.24 h after tMCAO,HO-1 in the brain tissue of the Ad-HO-1 group was significantly increased compared with the control groups.The total cerebral infarction volume in the Ad-HO-1 group was significantly lower than the control group.The apoptosis index of the Ad-HO-1 group was significantly lower than that of the Ad group.The activation of caspase-3 and TUNEL-positive apoptotic cells was significantly reduced.in the Ad-HO-1 group.Tthe neurological scores of the Ad-HO-1 group were significantly higher than those of the Ad group.2.In the serum,IL-17A significantly decreased in the Ad-HO-1 group.the IL-6 was not significantly different with the control group.In the brain tissue,IL-17A decreased at the gene transcription level and protein level,and IL-10 significantly increased,.but IL-6 had no changed.For spleen cells,IL-17A was lower than that of the Ad group;meanwhile,the levels of IL-6 were not significantly different.3.RORyt in the brain tissue was reduced,and p-STAT3 was not significantly different.Compared with the control group,the proportion of Th17 cells in the spleen cells of the rats was significantly reduced,and the proportion of Treg cells was higher in the spleen or brain tissue.4.The expression of IL-10R in CD4+T cells in Ad-HO-1 group was significantly higher.Th17 cells induced by Hemin could unregulated HO-1 mRNA expression in vitro.SnPP inhibited their HO-1 activity,but there was no difference significance.IL-17A mRNA in Th17+Hemin group was reduced IL-6 mRNA and protein in the culture supernatants were reduced.The IL-10 mRNA and protein were not changed.TGF-βmRNA was increased significantly,but protein decreased.The mRNA expression of RORγt STAT3,and Foxp3 was inhibited(compared with Th0 or Th17).The expression of STAT3 mRNA in the Th0+Snpp group was reduced.Conclusions1.Ad-HO-1 group had significantly higher neurological score than Ad group or V group.After HO-1 overexpression,neuronal cell death was significantly reduced after cerebral ischemia-reperfusion.HO-1 has effective neuroprotective effect on brain injury after transient cerebral ischemia.2.HO-1 inhibited the expression of Th17 cell-specific transcription factor RORyt,which inhibited the differentiation of Th17 cells,down-regulate the expression of IL-17A in Th17 cells,and resumed the MCAO ischemic injury.3.HO-1 up-regulated Treg cell differentiation and IL-10R expression,and resets the immune balance of the Th cell.