| Background:Intracerebral hemorrhage(ICH)refers to non-traumatic rupture of blood vessels in the brain parenchyma,accounting for about 15%of all strokes,and the mortality is as high as 40%.It is one of the diseases that seriously threaten human life for high morbidity,lethality and disability,it brings heavy mental and economic burden to patients and their families.Within 24 hours after intracerebral hemorrhage,the erythrocytes from the blood vessel break into the brain parenchyma beginning to lyse,hemoglobin is released and will be further broken down into heme.Next Heme is degraded by heme oxygenase-1(HO-1)to biliverdin,carbon monoxide.and iron.All the by-products from hematoma will increase the inflammatory response,promote the peroxidation of protein,nucleic acid,carbohydrate and lipid,disrupt the cell signaling pathway and cause cell death eventually.Microglia,known as brain macrophages,have the functions of immune monitoring,defense,antigen presentation,phagocytosis and secretion.With appropriate stimulation,microglia can be activated into two states:the classically activated phenotype M1 and the activated phenotype M2.After nuclear factor E2-related factor 2(Nrf2)is activated,it can promote M2 polarization of microglia and up-regulate the expression of Cluster of differentiation 163(CD 163)on M2 microglia.The scavenger receptor CD 163 can recognize hemoglobin,increases the ability of microglia to engulf hemoglobin,and promote the absorption of hematoma.In the theory of traditional Chinese medicine,intracerebral hemorrhage is the blood of deviating from meridians,if the blood stasis stops outside the brain vessels,it will oppress the brains and the compression of the brains is the earliest pathogenesis of the acute stage of cerebral hemorrhage.In traditional Chinese medicine,the main treatment methods for extravasated blood are improving blood circulation and dispersing stasis.Naoxueshu oral liquid is a traditional Chinese medicine preparation with the effect of benefiting qi,improving blood circulation and dispersing stasis,it is widely used in clinic,but it is unknown whether it will cause rebleeding in the acute stage of intracerebral hemorrhage,for safety,the drug is given 3 days after intracerebral hemorrhage.A large number of clinical studies have confirmed that the curative effect is significant,which can promote the absorption of hematoma,reduce the volume of hematoma after intracerebral hemorrhage and improve the prognosis significantly.Therefore,it is an great significance to evaluate the safety of Naoxueshu oral liquid in the treatment of cerebral hemorrhage and to study its mechanism of promoting the absorption of hematoma.Objective:1.To explore the safety evaluation of Naoxueshu oral liquid at the time of administration,so as to provide theoretical and experimental basis for the clinical treatment of hemorrhagic stroke under the guidance of the theory " invigorating qi and activating blood".2.To explore the mechanism of Naoxueshu oral liquid promoting the M2 polarization of microglia through Nrf2-Hb-Hp-CD 163 pathway to affect the absorption of hematoma in intracerebral hemorrhage,so as to provide theoretical and experimental basis for the clinical treatment of hemorrhagic stroke under the guidance of the theory "invigorating qi and activating blood".Methods:1.A model of intracerebral hemorrhage was produced by injection of collagenase VII into the caudatum of healthy male SD rats of SPF grade.The rats were divided into 4 groups with 20 rats in each group by random number table method:the model group,the Naoxueshu oral liquid group 1,the Naoxueshu oral liquid group 2 and the edaravone group.The sham group and the sham administration group were entered the injection needle into the caudatum of rats.Two hours after operation for intracerebral hemorrhage in rats,the sham operation group and the model group were given 0.27ml/100g of normal saline by gavage,the Naoxueshu oral liquid group 1 were given 0.27ml/100g of Naoxueshu oral liquid by gavage,the edaravone group were given 0.54mg/100g of edaravone injection by intraperitoneal injection.One day after successful modeling of cerebral hemorrhage in rats,the Naoxueshu oral liquid group 2 were given 0.2 7ml/100g of Naoxueshu oral liquid by gavage.Rats in each group were administered once every 24 hours for a total of 3 days.One day and three days after operation,20 rats in each group were evaluated by modified neurological severity score(mNSS)score.Three days after surgery,6 rats in each group were evaluated by open-field experiments.Snapping the beheading of rats brain with 3 rats in each group,respectively,by HE staining,Niss’l staining and tunel staining were used to detect pathologic structure and nerve cells apoptosis after three days treatment.2.After three days of medication,3 rats in each group were used to prepare pathological frozen sections,and 5 rats in each group were used to take fresh frozen brain tissue.Immunofluorescence was used to detect the expression of Nrf2,Arginase-1(Arg-1),CD 163 and HO-1.Western blot was used to detect the expression of Nrf2,Arg-1,CD 163 and HO-1.Results:1.MNSS score and open field experiment showed that the neurological function score of rats in the model group was significantly higher than that in the sham group,the walking distance and standing times were significantly less than those of the sham group,and the neurological function was obviously impaired.Compared with the model group,the neural function scores of the Naoxueshu oral liquid group 1 was significantly decreased,the walking distance and standing times increased obviously,and the neurologic deficit were significantly improved.Compared with the Naoxueshu oral liquid group 2,the neural function scores of the Naoxueshu oral liquid group 1 have a decreasing trend,and the walking distance and standing times have an increasing trend.2.HE stanning,Niss’l stanning and tunel staining showed that compared with the sham group,the rats in the model group had an obvious increase of the edema of brain,the morphological changes of nerve cells was obvious and the number of apoptotic neurons increased significantly.Compare to the model group,rats in the Naoxueshu oral liquid group 1 showed a low edema of brain and apoptotic neurons.Compare to the Naoxueshu oral liquid group 2,the edema of brain and apoptotic neurons of the Naoxueshu oral liquid group 1 have a decreasing trend3.Immunofluorescence results showed the expressions of Nrf2,Arg-1,CD 163 and HO-1 in the model group were significantly higher than those in the sham group.Compared with the model group,the expressions of Nrf2,Arg-1,CD 163 and HO-1 in brain tissues of rats after cerebral hemorrhage in the Naoxueshu oral liquid group 1 were significantly increased.Compared with the Naoxueshu oral liquid group 2,the expressions of Nrf2,Arg-1,CD163 and HO-1 in brain tissues of rats after cerebral hemorrhage in the Naoxueshu oral liquid group 1 have an increasing trend.4.Western blot showed the expressions of Nrf2,Arg-1,CD 163 and HO-1 in the model group were significantly higher than those in the sham group.Compared with the model group,the expressions of Nrf2,Arg-1,CD163 and HO-1 in brain tissues of rats after cerebral hemorrhage in the Naoxueshu oral liquid group 1 were significantly increased.Compared with the Naoxueshu oral liquid group 2,the expressions of Nrf2,Arg-1,CD163 and HO-1 in brain tissues of rats after cerebral hemorrhage in the Naoxueshu oral liquid group 1 have an increasing trendConclusion:After operation,Naoxueshu oral liquid can significantly alleviate nerve function defect,reduce the pathological damage of brain tissue,alleviate nerve cell damage,protect the normal function of neurons.Naoxueshu oral liquid can obviously promote the clearance and decomposition of hemoglobin in hematoma,by Nrf2/CD163 pathway promoting M2 type polarization of microglia.And the experimental results show that the rats after cerebral hemorrhage treated with Naoxueshu oral liquid after cerebral hemorrhage did not show any adverse reactions,and the curative effect of postoperative administration have an increasing trend than that after 1day. |
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