Study On The Neuroprotective Effects And Potential Mechanisms Of Nicotiflorin On Traumatic Brain Injury

Traumatic brain injury（TBI）is a neurosurgical traumatic disease caused by external forces,which leads to brain tissue indirectly or directly exposed to the outside and high mortality rate,treating difficulties and poor prognosis.Patients surviving from TBI are always suffering from cognitive impairment,depression,anxiety and other emotional disorders in the long term,resulting in the deterioration of quality of life and consumption of medical resources.The shortage of knowledge on TBI’s pathological mechanisms and the lack of brain protective drugs to treat it have long troubled global public health and the international military medicine.Flos Carthami,a kind of traditional Chinese medicine which can promote blood circulation and remove stasis,has been widely used in the treatment of ischemic encephalopathy and its sequelae.One of the flavonol compounds with excellent pharmacological activities extracted from Flos Carthami is nicotiflorin.The nicotiflorin injection has entered the stage of clinical trial as a new classⅠtraditional Chinese medicine for the treatment of acute ischemic stroke.However,whether nicotiflorin can play a neuroprotective role in the treatment of traumatic brain injury and the specific mechanisms are not yet reported.Based on clinical needs and military medical demands,in this study,Wistar rats were used to establish in vivo TBI models by weight-drop and fluid percussion injury methods to comprehensively evaluate the efficacy of nicotiflorin in the acute stage of TBI and to investigate the effects of nicotiflorin on alleviating cognitive and emotional dysfunction in the mid-long-term.Based on transcriptomics,proteomics and their combination analysis,the differentially expressed genes,proteins and important pathways related to nicotiflorin’s neuroprotective effects were screened.On this basis,BV2 microglia cells were used to establish mechanical injury model and LPS inflammatory injury model in vitro,on which the potential molecular mechanism of nicotiflorin’s neuroprotective effect was discussed around the prime PI3K/Akt signaling pathway.In general,this assay can be divided into five parts:1.Therapeutic effect of nicotiflorin on closed diffuse weight-drop TBI in ratsMale Wistar rats were divided into sham,TBI,low-dose,medium-dose and high-dose（2.5,5.0,10.0 mg/kg）of nicotiflorin groups to study on the closed diffuse weight-drop TBI model.The m NSS score evaluated by balance beam test and water content of brain tissue were measured within 3 days after injury.The results showed that TBI caused brain edema in at least 72 h and 24 h after TBI was confirmed as the peak time point of brain swelling,when the water content of rat brain tissue increased significantly from（78.23±0.26）%to（79.68±0.58）%.Nicotiflorin could significantly reduce the m NSS score and brain edema after TBI in rats with a dose-effect relationship.2.Therapeutic effect of nicotiflorin on lateral fluid percussion injury TBI in ratsThe fluid percussion TBI model was established to farther evaluate the therapeutic effect of nicotiflorin on TBI.Water content,Evans blue content and levels of MDA,GPx and IL-1βof brain tissue were measured at 24 h after injury.The paraffin sections of brain tissue were made for HE,Nissl and FJB staining to observe pathological changes of brain tissue.Besides,cell apoptosis was detected by TUNEL method,and microglia cells were observed by Iba-1immunohistochemistry.The results showed that treating with nicotiflorin of low-dose,medium-dose and high-dose could significantly reduce brain edema in rats with a certain dose-effect relationship.10.0 mg/kg nicotiflorin significantly reduced the blood brain barrier leakage after TBI（P<0.01）,significantly brought down the levels of MDA and IL-1βand maintained the activity of GPx enzyme（P<0.01）in the brain of TBI rats.Pathological findings indicated that nicotiflorin protected the morphology and neuronal function of brain tissue,slowed down neuronal degeneration,reduced microglia activation,and significantly reduced neuronal apoptosis index from 34.17%to 21.61%（P<0.01）.3.Nicotiflorin reduced cognitive dysfunction and mood disorders of TBI ratsMorris Water maze tests were performed on the 7^th day and the 14^thday after TBI,open field tests and elevated plus maze tests were performed on the 3^rd,7^th,14^th,and 28^th day after TBI respectively.Compared with model group,treating with nicotiflorin 10.0 mg/kg/d significantly shortened the latency of rats in the water maze positioning cruise tests（P<0.01）,and increased the number of platform crossing times in the space exploration tests（P<0.05）.The results of open field experiment and elevated plus maze experiment showed that nicotiflorin could improve the spontaneous activity of rats within 14 days after TBI,significantly increase the total movement distance and central area movement distance in the open field of brain injury rats,and increased the dwell time ratio and movement distance ratio in open arms in elevated plus maze tests.These results suggested that nicotiflorin could alleviate cognitive impairment and lightened the anxiety and depression emotions after TBI in rats.4.Selection of regulatory factors and pathways related to the neuroprotective effects of nicotiflorin in TBI models based on transcriptomics and proteomicsThe injured brain tissues of rats of the sham,TBI and nicotiflorin groups was obtained at24 h after lateral fluid percussion injury TBI.125 DEGs（Differentially expressed genes）were screened by RNA-seq analysis.GO analysis showed that DEGs was mainly involved in neuronal regeneration and development,neurotransmitter transmission,immunophagocytosis and other biological processes,and KEGG analysis revealed that DEGs were enriched in MAPK,m TOR,phosphatidylinositol and other signaling pathways（P<0.05）.Transcriptome analysis and proteomics were conducted on the brain tissue samples of rats obtained 7 days after TBI.392 DEGs between nicotiflorin group and TBI group were screened out,which were mainly involved in the production and regulation of IL-8,activation of microglia,chemotaxis of granulocytes and other inflammatory immune processes by GO analysis.KEGG analysis revealed that DEGs were significantly enriched in complement system,chemokine signaling pathway,Toll-like receptor and TNF signaling pathway.Proteomics analysis announced 6400 trusted proteins were identified and 92 DEPs among them were enriched to Wnt and Toll-like receptor signaling pathways by GO analysis,and phosphatidylinositol signaling pathway and NF-κB signaling pathway by KEGG analysis.Transcriptome-proteome association analysis results showed that the expression of key factors CD64,ENOG410YNB1,PTX3 and COL1A showed significant expressing difference at both transcription and protein levels between TBI group and nicotiflorin group（P<0.05）.And DEGs and DEPs were enriched in PI3K/Akt and some other signaling pathways with extreme significance（P<0.05）and high abundance（protein number≥10,gene number≥29）.5.Nicotiflorin plays a neuroprotective role by regulating PI3K/Akt signaling pathwayBV2 microglia cells were used as vectors to construct mechanical injury and LPS inflammatory injury TBI models in vitro,and the survival rate and LDH outflow of BV2 cells were detected after intervention of different concentrations of nicotiflorin.It was found that nicotiflorin in the range of 1-100μM significantly reduced the death rate（P<0.05）and LDH release（P<0.01）of BV2 cells in mechanical injury and chemical injury TBI models.PI3K inhibitor LY294002 was used to block the PI3K/Akt pathway,and the survival rate and LDH outflow of BV2 cells in different treatment groups were detected.The m RNA expression levels of IL-1β,TNF-αand Casepase3 in BV2 cells were detected by q RT-PCR.The protein expression levels of PI3K,p-PI3K,Akt,p-Akt,Bcl-2 and PTEN were detected by Western-blot.The results showed that 100μM nicotiflorin could significantly reduce the survival rate and increase LDH outflow of BV2 cells induced by LPS injury,significantly reduce the m RNA expressions of IL-1β,TNF-αand Casepas E3,and up-regulate the protein expressions of p-Akt/Akt,p-PI3K/PI3K and Bcl-2.Compared with nicotiflorin treatment group,add of LY294002 reduced the viability of BV2 cells,increased LDH release,the expression of IL-1β,TNF-α,casepase3 at m RNA level,and decreased p-Akt/Akt,P-PI3K/PI3K,and the expression of PTEN.These results indicated that up-regulating the activation of PI3K/Akt signaling pathway is one of the underlying mechanisms how nicotiflorin plays the neuroprotective role in TBI models.In conclusion,nicotiflorin could play a protective role in the acute period after TBI in rats,and has the potential to alleviate the cognitive and emotional disorders after TBI in the middle and long term.Nicotiflorin can also protect BV2 microglia cells from the mechanical damage and LPS damage in vitro.The neuroprotective effect of nicotiflorin may be related to the activation of PI3K/Akt signaling pathway.