| Background and ObjectiveTraumatic brain injury(TBI)is a kind of trauma caused by a variety of serious damage to brain tissue diseases with the characteristics of high incidence and morbidity,which ranked first in all types of trauma according to statistics[1].In the United States alone,there are about 1 million 700 thousand new cases reported each year,with a total of about 5%of all deaths in the United States,with a mortality rate of more than 40%,and more than25%of the affected adults are unable to return to work within a period of up to 1 years after the injury[2-4].In developing countries,with the continuous improvement of social and economic level,the incidence of TBI is rising trend.TBI immediately after injury or a few days or weeks after symptoms can occur,causing widespread physical and mental disorders,including epilepsy,movement disorders,personality changes,memory disorder.TBI is a major health problem that affects both the public and the military.However,at present,there is no FDA approved treatment of traumatic brain injury.Disruption of the integrity of the blood-brain barrier(BBB)is increasingly recognized in a variety of diseases,including ischemia,multiple sclerosis,and neurodegenerative diseases such as AD?PD[5-8].The disruption of blood-brain barrier after traumatic brain injury was found by imaging studies in human[9].Loss of blood-brain barrier(BBB)integrity is a serious event caused by TBI,after which the BBB can no longer be a flawless protective barrier between the intravascular compartments and the brain,and its dysfunctions lead to leakage of fluid,proteins and immune cells[10].Therefore,it is very important to improve the outcome of TBI by improving the damage of blood-brain barrier.The blood-brain barrier is important for maintaining the stability of the central nervous system.The complete structure in the blood-brain barrier,brain microvascular endothelial cells by Occludin,Claudins and JAMS(junction Adhesion molecules)is formed by connecting three transmembrane protein,its directly or indirectly tight junction protein zonula occludens protein-1(ZO-1),connected to the cytoskeleton,stable connection of the whole structure.Liu et al.Found that ZO-1 and occludin are critical to the blood-brain barrier[11].Study found that in a rat model of cold injury cortex pure vasogenic edema,and increase in the expression of caveolin-1 found before the close connection loss,the expression of occludin and claudin-5 reduced[12].In addition,BBB integrity is also affected by certain physiological conditions,including inflammation and oxidative stress.Local inflammatory responses can result in BBB disruption.During the process,pro-inflammatory factors,including IL-1?,iNOS,MPO and MMP-9,are induced by brain injury.Meanwhile,anti-inflammation-associated factors arginase 1 and IL-10 are also stimulated for protection purpose.[13-20]In conclusion,TBI leads to disruption of the blood-brain barrier,and tight junction protein abnormalities and inflammation and oxidative stress may lead to increase permeability of the blood-brain barrier and vasogenic edema,but the corresponding treatment,currently no FDA approved traumatic brain injury.Previous studies have found that the protective effect of destruction of glibenclamide,saikosaponin,hydrogen sulfide and other drugs on blood brain barrier after traumatic brain injury,Yang etal on the previous Chinese herbal medicine in treatment of traumatic brain injury in the literature meta analysis found that the results of Meta analysis showed that traditional Chinese medicine is beneficial to the treatment of TBI was obvious effect.But there are limitations that animal studies more carefully designed and the report is necessary[21-24].Cordycepin,3'-deoxyadenosine,was first isolated from fungal in nucleoside antibiotic,anti-inflammatory,free radical scavenging antioxidant,anti-tumor and anti leukemia,antibacterial and immune regulation,reducing blood glucose and blood lipid[25].A study found that Tokina cordycepin could significantly reduce proinflammatory cytokine levels,including NO,PGE2,TNF-aand IL-1,and increase the expression of anti-inflammatory cytokines,such as interleukin-10[26].In addition,cordycepin can reduce age-related oxidative stress and improve the antioxidant capacity of rats[27].Cordycepin protects hippocampal cells against neurotoxicity through its anti-oxidant properties,and inhibits apoptosis of the cells[28].Therefore,it is necessary to investigate whether cordycepin can improve the damage of blood-brain barrier after traumatic brain injury.Methods1.TBI Sprague-Dawley rats with cordycepin treatment:TBI rats were established via controlled cortical impact(CCI)injury,according to the previous description.2.Stroke severity evaluation:Stroke severity evaluation was performed 24 h post-injury.Neurological severity scores used a 10-point scale for 10 functional tasks,0 point indicates normal function,while 10 points suggest the most severe neurological dysfunctions[29].3.For measurements of brain water content:Brain water contents were calculated based on Hatashita's wet-dry method[30].4.Evans blue assessment:BBB disruption was evaluated by Evans blue leakage following surgical procedures.5.Quantitative real-time PCR:Twenty-four hours after injury,in order to investigate the mRNA of ZO-1,occluding,IL-1?,iNOS,MMP-9,arginase-1,NOX-1,NOX-2,NOX-4.tissues were homogenized in lysis buffer for RNA isolation.Qiagen kits(Qiagen,Valencia,CA,USA)were used to isolate RNA(RNeasy mini kit),synthesis of cDNA(QuantiTect reverse transcription kit RNA)and the subsequent qPCR(SYBR Green Master Mix).6.Western blotting:Tissues were collected and western blotting method was performed as previously described for observing the expression of ZO-1 and occludin.7.ELISA:Total proteins were extracted(see western blotting method)24 h post-injury.20?g total proteins were applied for IL-10 detection according to the manufacturer's instruction(Wuhan Boster Biological Technology LT,Wuhan,China).Final results were reported as pg/mg.8.Evaluation of the activities of MPO and NOX:The MPO activity was measured according to the manufacturer's instruction(Nanjing institute of biological engineering,Nanjing,China)in the tissues 24 h post-injury.The NOX activity was detected by lucigenin-enhanced chemiluminescence.9.Statistics:All data were statistically analyzed by GraphPad Prism software(GraphPad Software,USA),using one-way ANOVA analysis followed by a Tukey's post hoc test.P-values less than 0.05 should be considered as statistical significant.At least three independent repeats were performed for each experiment.Results1.Cordycepin treatment attenuates TBI-induced impairments dose-dependently Rats from sham group underwent identical surgical procedures,in the absence of a CCI.TBI rats were treated with either saline(vehicle control group)or cordycepin(1,5,10 and 20 mg/kg).Stroke severity in the animals of different experimental groups was characterized by neurological severity scores,brain infarct volumes and water contents.Our results indicated that TBI rats had higher neurological severity scores(Fig.la),larger brain infarct volumes(Fig.lb)and more brain water contents(Fig.1c)than the sham group.Moreover,cordycepin-treated TBI rats showed lower neurological severity scores(Fig.la),smaller brain infarct volumes(Fig.1b)and less brain water contents(Fig.1c)than the vehicle control group,suggesting cordycepin treatment attenuated TBI-induced impairments.In addition,cordycepin treatment acted in a dose-dependent manner(Fig.la-lc).We applied 20 mg/kg cordycepin treatment in our following experiments due to its best efficacy of protection.2.Cordycepin treatment defends BBB integrity following TBI BBB integrity was evaluated by Evans blue assessment,where Evans blue leakage was positively correlated with loss of BBB integrity.Our findings revealed that BBB integrity was compromised in the vehicle control group,while 20 mg/kg cordycepin treatment group partially rescued the TBI-induced loss of BBB integrity(Fig.2a).Statistical analyses of the amount of Evans blue content(Fig.2b)further confirmed that cordycepin treatment defended BBB integrity following TBI.In addition,contralateral sham rats without TBI served as negative controls,and showed no differences in the Evans blue content between the vehicle-and cordycepin-treated groups.3.Cordycepin alleviates TBI-induced loss of tight junction proteins in the ipsilateral cortex of experimental animals Relative mRNA expression levels of ZO-1 and occludin were analyzed by qRT-PCR.Compared with sham rats(sham and sham+cordycepin),TBI rats(TBI+vehicle and TBI+cordycepin)had lower ZO-1 and occludin at the transcriptional level(Fig.3 a).In addition,the mRNA levels of ZO-1 and occludin were similar between sham group and sham+cordycepin group,and between TBI+vehicle group and TBI+cordycepin group,respectively.Next,the protein expression of ZO-1 and occludin was examined by Western blot.Consistent with the mRNA results,TBI rats(TBI+vehicle and TBI+cordycepin)had relatively lower protein levels of both ZO-1 and occludin(Fig.3b,3 c),while sham group and sham+cordycepin group had similar protein levels of ZO-1 and occludin(Fig.3b,3 c).However,statistical analyses of the intensity of protein bands demonstrated that TBI+cordycepin group had significantly higher ZO-1 and occludin than TBI+vehicle group(Fig.3c),indicating cordycepin was able to alleviate TBI-induced loss of tight junction proteins,but not loss of mRNAs.Taken together,cordycepin probably exerted neuroprotective effects at the post-transcriptional level.4.Cordycepin suppresses local inflammation in the ipsilateral brains following TBI Local inflammation is known to play an important role in BBB integrity,we therefore analyzed the levels of pro-inflammatory factors,including mRNA levels of IL-1?(Fig.4a),iNOS(Fig.4b)and MMP-9(Fig.4f)and MPO activity(Fig.4e).We also examined the levels of anti-inflammation-associated factors,including mRNA level of arginase 1(Fig.4c)and protein expression level of IL-10(Fig.4d).As expected,local inflammation was induced in the ipsilateral brains following TBI,but inhibited after cordycepin treatment(Fig.4a,4b,4e,4f).Meanwhile,TBI-induced brain damage stimulated anti-inflammation substances for protection purpose,and cordycepin further enhanced the production of anti-inflammation substances(Fig.4c,4d).5.Cordycepin inhibits NADPH oxidase(NOX)expression and activity in the ipsilateral brains following TBI Excessive reactive oxygen species(ROS)after stroke contributes to BBB disruption,and NADPH oxidases(NOXs)are the main sources of ROS.Our results confirmed that TBI rats displayed higher NOX activity than the sham control rat(Fig.5a),while cordycepin treated TBI rats showed lower NOX activity than the TBI+vehicle rats(Fig.4a).NOX-1,NOX-2 and NOX-4 are three types of NOXs present in the central nerve system[31].We further examined the mRNA levels of the three NOXs,and found that only NOX-1 was induced in TBI rats,which was inhibited in cordycepin-treated TBI rats(Fig.5b).NOX-2 and NOX-4 were affected by neither TBI-induced injury nor cordycepin treatment(Fig.5c,5d).Conclusion1.Cordycepin has neuroprotective effects against TBI.2.Cordycepin attenuates blood-brain barrier disruption caused by TBI.3.These effects may be linked to inhibition of inflammation and oxidative stress.4.We propose that Cordycepin is a potential target of gene therpy for TBI... |
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