The Research On The Role And Mechanism Of CCL20 In Early Brain Injury After Subarachnoid Hemorrhage In Mice

BACKGROUND AND OBJECTIVES: Subarachnoid hemorrhage(SAH)is a very common clinical cerebrovascular syndrome with the characteristics of acute onset and high lethal and disability rate,accounting for about 5% of stroke-related diseases.Previous studies have suggested that the main cause of high mortality and disability rate is cerebrovascular spasm(CVS)secondary to hemorrhage.However,a large number of studies have found that drugs that inactivate the spastic substances or block the contraction of arterial smooth muscle have little clinical effect,and the mortality and disability rates have not decreased significantly.Recent studies have found that early brain injury(EBI)after subarachnoid hemorrhage is an important cause of its high mortality and disability rate,but the specific pathological mechanism of EBI is not yet fully clear.Previous studies have found that it mainly involves cerebral ischemia,brain edema,oxidative stress response,inflammatory response,cell apoptosis and so on.Therefore,it is very important to study the pathological mechanism of EBI and find appropriate treatment strategies and new targets for improving EBI after subarachnoid hemorrhage.CC chemokine ligand 20(CCL20)belongs to the subfamily of small cytokine CC,and CC chemokine receptor 6(CCR6)is its unique receptor.CCL20 can regulate cell chemotaxis,migration and inflammation via binding to CCR6,and play an important role in skin and mucosal surfaces,tumors and various autoimmune diseases under steady and inflammatory conditions.CCL20 has a direct toxic effect on primary neurons and oligodendrocytes in vitro.In ischemic brain injury,the expression of CCL20 and CCL6 were significantly up-regulated,while anti-CCL20 neutralizing antibody could significantly reduce the volume of cerebral infarction.In addition,CCL20 played a key role in inflammatory cascade reaction.In spinal cord injury,CCL20 neutralizing antibody is beneficial to the recovery of motor function,and can reduce inflammation by reducing IL-1beta,IL-6 and TNF-alpha.In addition,CCL20 can aggravate neuroinflammation after spinal cord injury by regulating the recruitment of Th17 cells and the secretion of IL-17 A.At the same time,some studies have shown that CCL20 expression is significantly up-regulated after SAH,but whether CCL20 is involved in the pathological process of early brain injury has not been reported.Therefore,in view of the important role of CCL20 in other physiological and pathological processes of the central nervous system,this study aims to explore the role and mechanism of CCL20 in early brain injury after subarachnoid hemorrhage by constructing SAH model in mice.METHODS:1.The model of subarachnoid hemorrhage in mice was established and randomly divided into seven groups: sham group,SAH 3 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group,SAH 72 h group and SAH 96 h group.CCL20 was detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.The expression of CCL20 in neurons,astrocytes and microglia after subarachnoid hemorrhage was further determined by immunofluorescence assay.2.The subarachnoid hemorrhage model of mice was established and randomly divided into six groups: sham group,SAH group,SAH + Ig G group,SAH + CCL20 blocking antibody concentration 1 μg/4 μl group(SAH + CCL20 1 μg),CCL20 blocking antibody concentration 5 μg/4 μl group(SAH + CCL20 5 μg),CCL20 blocking antibody concentration 7μg/4 μl group(SAH + CCL20 7 μg),SAH + CCL20 24 h and 72 h.After that,the neurological function score and the degree of brain edema were evaluated.3.The subarachnoid hemorrhage model of mice was established and randomly divided into five groups: sham group,SAH group,SAH+Ig G group,CCL20 blocking antibody concentration 5 μg/4 μl group(SAH+CCL20 5 μg),CCL20 blocking antibody concentration 7 μg/4 μl group(SAH+CCL20 7 μg).Neuronal apoptosis was detected by TUNEL assay.Immunofluorescence assay was used to detect the activation ofmicroglia in brain tissue of mice in each group.4.Microglia were cultured in vitro and divided into five groups:control group(con),oxyhemoglobin treatment group(Oxy Hb),oxyhemoglobin + interference control group(Oxy Hb + NC),oxyhemoglobin + CCL20 interference group(Oxy Hb + CCL20 si RNA),oxyhemoglobin + CCR6 interference group(Oxy Hb + CCR6 si RNA).RT-PCR and Western blot were used to detect the expression of CCL20 and CCR6 in in vitro SAH model and the interference effect of CCL20 si RNA and CCR6 si RNA on CCL20 and CCR6 expression were verified.Meanwhile,the effects of CCL20 and CCL6 knockdown on the activation of microglia and the expression of inflammatory factors IL-1β and TNF-alpha induced by oxyhemoglobin were detected by RT-PCR and Western blot.5.Microglia were cultured in vitro and divided into four groups:oxyhemoglobin treatment group,oxyhemoglobin + interference control group,oxyhemoglobin + CCL20 interference group,oxyhemoglobin +CCR6 interference group,and conditional medium was prepared.Neuronal cells were cultured in vitro and cultured in microglia conditioned medium.They were divided into five groups: non-conditioned medium(CM)group(non-CM),oxyhemoglobin conditioned medium group(Oxy Hb CM),oxyhemoglobin + interference control conditioned medium group(Oxy Hb+ NC CM),oxyhemoglobin + CCL20 interference group(Oxy Hb+CCL20si RNA CM)and oxyhemoglobin + CCR6 interference group(Oxy Hb+CCR6 si RNA CM).The apoptosis of neurons was detected by TUNEL assay.6.Neurons were cultured in vitro and divided into four groups:control group(con),oxyhemoglobin treatment group(Oxy Hb),oxyhemoglobin + interference control group(Oxy Hb + NC),oxyhemoglobin + CCL20 interference group(Oxy Hb + CCL20 si RNA).The expression of CCL20 was detected by Western blot.The apoptosis of neurons was detected by TUNEL assay.7.Neurons cultured in vitro were divided into three groups:oxyhemoglobin treatment group(Oxy Hb),oxyhemoglobin + interference control group(Oxy Hb + NC),oxyhemoglobin + CCL20 interference group(Oxy Hb + CCL20 si RNA),and conditioned medium was prepared.Microglia were cultured in vitro and cultured in neuron conditioned medium.They were divided into four groups: non-conditional medium group(CM[-]),oxyhemoglobin conditioned medium group(CM[+]),oxyhemoglobin + interference control conditioned medium group(NC CM[+]),oxyhemoglobin + CCL20 interference conditioned medium group(CCL20 si RNA CM[+]).Western blot was used to detect the expression of CCR6 and Iba1 in microglia,and RT-PCR and Western blot were used to detect the expression of IL-1β and TNF-alpha in microglia.8.Neurons were cultured in vitro and oxyhemoglobin conditionedmedium was prepared.Microglia cultured in vitro were divided into four groups: CM[-],CM[+],Oxy Hb conditioned medium-cultured microglia after interference control transfection group(CM[+] + NC),Oxy Hb conditioned medium-cultured microglia after CCR6 interference transfection group(CM[+] + CCR6 si RNA).Western blot was used to detect the expression of CCR6 and Iba1 in microglia,and RT-PCR and Western blot were used to detect the expression of IL-1β and TNF-alpha in microglia.RESULTS:1.CCL20 gene and protein expression were significantly up-regulated after SAH.The peak values were at 3 h,48 h and 72 h after SAH.CCL20 gene and protein were significantly expressed and increased in neurons and microglia after SAH,but not in astrocytes.2.The neurological deficits of SAH mice were impaired.After CCL20 neutralizing antibody was given,The neurological deficits of SAH mice at the time windows of 24 h and 72 h were significantly improve.Brain water content of SAH mice was significantly increased after SAH.After CCL20 neutralizing antibody was given,Brain water content at the time windows of 24 h and 72 h were significantly reduced.3.At 24 hours after SAH,the percentage of apoptotic neurons and the number of activated microglia were significantly increased.After CCL20 neutralizing antibody was given,the percentage of apoptotic neurons andthe number of activated microglia were significantly reduced.4.Oxyhemoglobin can significantly induce the expression of CCL20,CCR6 and Iba1 proteins in microglia,CCL20 si RNA can significantly inhibit the effects of oxyhemoglobin on the expression of CCL20,CCR6 and Iba1 proteins,and CCR6 si RNA can also significantly inhibit the effects of oxyhemoglobin on the expression of CCR6 and Iba1 proteins in microglia.The expression of IL-1β and TNF-alpha in microglia was inhibited by CCL20 si RNA and CCR6 si RNA,and the effects of oxyhemoglobin on the expression of IL-1β and TNF-alpha in microglia were significantly inhibited.5.Conditioned medium from oxyhemoglobin-treated microglia could significantly induce neuronal apoptosis,and the percentage of apoptotic neurons in the conditioned medium of microglia transfected with CCL20 si RNA and CCR6 si RNA was significantly decreased.6.Oxyhemoglobin can significantly induce the expression of CCL20 protein in neurons,CCL20 si RNA can significantly inhibit the effect of oxyhemoglobin on the expression of CCL20 protein in neurons;oxyhemoglobin can significantly induce neuronal apoptosis,but CCL20 si RNA has no significant effect on the percentage of apoptotic neurons induced by oxyhemoglobin.。7.The expression of CCR6 and Iba1 proteins in microglia was significantly induced by oxyhemoglobin-treated neuron conditionedmedium,and the expression of CCR6 and Iba1 proteins in microglia was significantly restored by CCL20 knockdown in neurons.The expression of IL-1β and TNF-alpha in microglia could be significantly induced by oxyhemoglobin-treated neuron conditioned medium,while the expression of IL-1β and TNF-alpha in microglia could be significantly restored by CCL20 knockdown in neurons.8.CCR6 knockdown in microglia could significantly restore the expression of CCR6 and Iba1 induced by oxyhemoglobin-treated neuron conditioned medium.Meanwhile,CCR6 knockdown in microglia could significantly restore the expression of IL-1β and TNF-alpha induced by oxyhemoglobin-treated neuron conditioned medium.CONCLUSION: CCL20 is up-regulated in neurons and microglia after SAH,and participates in the pathological process of early brain injury such as inflammatory reaction and neuronal apoptosis.Inhibition of CCL20 activity can significantly inhibit inflammatory reaction,neuronal apoptosis and improve neurological dysfunction after EBI.At the same time,we found that the activation of CCL20/CCR6 signal in microglia can induce microglia activation and neuron apoptosis,and neuron-derived CCL20 can regulate microglia activation by regulating the expression of CCR6 in microglia.