| Background and ObjectiveIntracerebral hemorrhage(ICH)is a devastating subtype of stroke,accounting for 12%～20%of all strokes,with a mortality rate of 43%～51%within 30 days,and a poor prognosis.Most survivors retain a neurological functional handicap related to the specific site of hemorrhage.Currently,effective specific treatment measures are still lacking.Although surgery has made significant progress in primary brain damage after ICH,little progress has been made in inflammatory cascade after ICH.More and more evidences show that inflammation is the key factor leading to secondary brain injury after ICH.Microglia/macrophages are the main cell types leading to secondary brain damage after ICH,and the related inflammatory factors include cytokines,chemokines,matrix metalloproteinases(MMPs),thrombin,etc.Extracellular matrix metalloproteinase inducer(EMMPRIN,CD 147),a highly glycosylated type Ⅰ transmembrane protein,belongs to the immunoglobulin(Ig)superfamily,which includes two extracellular(EC)Ig like domains,EC Ⅰ and EC Ⅱ.Each domain of EMMPRIN can bind to different proteins,thus showing different functions.Among them,the EC Ⅰ domain of EMMPRIN has been identified as the stimulation inducing site of MMP,which is up regulated by regulating the expression of MMPs in ICH.The main components of Naoxueshu oral liquid include leech,astragalus root,rhizoma chuanxiong,etc.The study shows that its effective components can pass through the blood-brain barrier,mainly used for hemorrhagic stroke.Clinical studies have confirmed that conventional treatment combined with Naoxueshu oral liquid can reduce the inflammatory response of ICH patients,accelerate the absorption of intracerebral hematoma,reduce brain edema and improve the neurological dysfunction of patients.Related studies have shown that Naoxueshu oral liquid can improve the secondary brain damage after ICH by reducing the level of MMP-9.MMPs are related to the inflammatory processes of the central nervous system.It is of special concern that MMPs can transform apoptotic molecules into activated forms,which leads to the myelin degradation,destruction of the blood-brain barrier and tissue injury,aggravates the inflammatory response,and further aggravates the brain damage after ICH.However,EMMPRIN,as the upstream regulator of MMPs,has not yet been studied whether Naoxueshu oral liquid can improve brain damage after ICH by suppressing the expression of EMMPRIN.Through the construction of ICH model in rats,the expression of EMMPRIN at different time points in brain injury after ICH was determined,and the histopathological evaluation of brain damage after ICH at different time points was made,so as to explore whether Naoxueshu oral liquid can suppress the expression of EMMPRIN this new therapeutic target to improve the brain damage after ICH,and provide a new scientific basis for the clinical application of Naoxueshu oral liquid in ICH.Methods(1)96 adult male SD rats were randomly divided into ICH control group(32 rats),sham surgery group(32 rats),Naoxueshu oral liquid treatment group(NXS treatment group,32 rats).Each group was further assigned into four subgroups according to the administration time(1d,2d,3d,7d),with 8 rats in each subgroup.(2)Sham surgery group was injected with the same dose of sterile saline,the other two groups were injected with 0.5μL type Ⅶ collagenase into the right striated rat model.(3)NXS treatment group:24 hours after ICH induction,the rats were given Naoxueshu oral liquid 1 ml/kg,once every 8 hours,until they were executed at the corresponding time point.The sham surgery group and ICH control group were given the same dose of Sterile saline.(4)Neurobehavioral evaluation to clarify the improvement of neurological function of ICH rats by Naoxueshu oral liquid.(5)Rats in each group were killed at the set time points(1d,2d,3d,7d).Brain tissues were taken and paraffin sections were made.(6)Changes in the expression of EMMPRIN in the brain tissues of rats in each group at different time points were detected,and histopathological evaluation was conducted on the brain damage after ICH in each group.The brain damage area at different time points after ICH was evaluated by HE staining.Fluoro-jade B staining was used to observe the degeneration of nerve cells at different time points.Iba1 immunofluorescence staining was used to observe the activation of microglia at different time points.Death cells at different time points was detected by TUNEL fluorescence staining.(7)Through the statistical analysis of the above indicators,we can verify whether Naoxueshu oral liquid can improve the brain injury after ICH by suppressing the expression of EMMPRIN.Results(1)After 7 days of treatment with Naoxueshu oral liquid,the degree of neurological deficit in ICH rats was improved(P<0.05).(2)Statistical analysis of EMMPRIN immunohistochemistry showed that the expression of EMMPRIN increased after ICH,the highest expression was 2-3 days after ICH,then decreased gradually,and it was still expressed at 7 days.On 1d,2d,3d and 7d after ICH,compared with sham surgery group,the number of EMMPRIN positive cells in NXS treatment group and ICH control group increased significantly(P value<0.001),and the number of EMMPRIN positive cells in NXS treatment group was significantly lower than that in ICH control group(P<0.01).(3)The statistical analysis of HE staining results showed that the brain tissue was damaged after ICH,and the brain damage area was the largest in 2-3 days after ICH,and recovered to 7 days.On 1d,2d,3d and 7d after ICH,there was no obvious bleeding injury except for the injury of acupuncture channel in sham surgery group.Compared with sham surgery group,the area of brain damage in both the NXS treatment group and the ICH control group increased significantly(P value<0.001).Only 7 days after ICH,compared with ICH control group,NXS treatment group can significantly reduce the area of brain damage after ICH(P<0.01).(4)Fluoro Jade B(FJB)staining results statistical analysis showed that neuronal degeneration death increased gradually after ICH,peaked at 3 days,then decreased gradually,and was still expressed at 7 days.On 1d,2d and 3d after ICH,the number of FJB positive cells in NXS treatment group and ICH control group was significantly higher than that in sham surgery group(P value<0.001).The number of FJB positive cells in the NXS treatment group was significantly lower than that in the ICH control group 3 days and 7 days after ICH(P<0.01).(5)The results of immunofluorescence of Ibal showed that the number of Ibal positive cells increased significantly 24 hours after ICH,reached a peak in 2-3 days,and then decreased gradually,and was still expressed at 7 days.On 1d,2d,3d and 7d after ICH,the number of Ibal positive cells in the brain damage area in NXS group and ICH control group was significantly higher than that in sham surgery group(P value<0.001),and on 3d and 7d after ICH,the number of Iba1 positive cells in NXS treatment group was significantly lower than that in ICH control group(P<0.001).(6)TUNEL staining showed that there were more death cells in the brain damage area after ICH,and the number of death cells peaked at the 3d after ICH,and decreased significantly at the 7th day after ICH.On 1d,2d,3d and 7d after ICH,The number of death cells in NXS treatment group and ICH control group was significantly higher than that in sham surgery group(P value<0.001),and the number of TUNEL positive cells in NXS treatment group was significantly lower than that in ICH control group(P<0.01).conclusionNaoxueshu oral liquid may play the role of brain protection by suppressing the expression of EMMPRIN,which can reduce the area of brain damage,the activation of microglia,the degeneration and death of neurons and the number of death brain cells after ICH,so as to improve the brain injury after intracerebral hemorrhage. |
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