Inhibition Effect Of Xingnaojing On Parthanatos In Intracerebral Hemorrhage Mice

Objective:(1)To define the role of Parthanatos in neuronal death after cerebral hemorrhage.(2)To explore the inhibition effect of Xingnaojing on Parthanatos in neuronal cells of intracerebral hemorrhage miceMethod: The experimental mice were randomly divided into sham operation group(sham),cerebral hemorrhage group(ICH),and Xingnaojing treatment group(ICH+XNJ),and the mice were treated as follows:(1)Administration: The mice in the ICH+XNJ group were intraperitoneally injected with Xingnaojing 3 days before surgery(10 ml/kg 1/day),2 hours after surgery,and after surgery(10 ml/kg 1/day)until executed.The mice in the Sham group and ICH group were intraperitoneally injected with the same amount of saline at the same time point.(2)Neurobehavioral measures and brain water content: Sham group(n=6),ICH group(n=6),ICH+XNJ group(n=6),Neurobehavioral measures were evaluated at 24 h and 72 h after surgery,and mice were sacrificed after neurobehavioral measures at 72 h and the brain water content was measured.(3)HE staining: Mice in sham group(n=3),ICH group(n=3)and ICH+XNJ group(n=3)were sacrificed at 24 h and 72 h after surgery.HE staining was performed to observe the pathological changes.(4)FJB staining to observe the number of peripheral neuronal death in mouse hematoma: The sham group(n=5),the ICH group(n=5),and the ICH+XNJ group(n=5)were sacrificed 24 h after the surgery,and FJB staining was performed to observe the number of peripheral neuronal death in mouse hematoma.(5)Immunofluorescence staining: To observe the expression of PAR and AIF around the hematoma of mice: Sham group(n=5),ICH group(n=5),ICH+XNJ group(n=5)were sacrificed 24 h after surgery,and immunofluorescence staining was performed to observe the expression of PAR and AIF around the hematoma of mice.(6)Western blot: The sham group(n=6),the ICH group(n=6),and the ICH+XNJ group(n=6)were sacrificed 24 h after the surgery,and Western blot was used to detect the expression levels of PARP1,PAR and AIF in mice brains.Results:(1)Compared with the sham operation group,all neurological function scores in ICH group were lower(P<0.01);Xingnaojing treatment improved the neuroscores at 24 h and 72 h after surgery(P>0.05).At 24 h and 72 h,the results of corner turn test were not improved by the treatment(P>0.05).The result of the forelimb placement test was not improved at 24h(P>0.05),but the treatment improved the result of the forelimb placement test at 72 h after surgery(P<0.05).(2)The water content of the brain tissue in the hemorrhagic side was significantly increased at 72 h after surgery(P<0.01).Xingnaojing treatment reduced the water content of the hemorrhagic brain tissue after cerebral hemorrhage(P<0.01).(3)HE staining results: ICH group showed necrotic neuron cells,inflammatory cell infiltration,loose tissue arrangement,disordered cell arrangement compared with sham group,Xingnaojjing treatment showed reduced neuronal cell necrosis and inflammatory cell infiltration,attenuated loose tissue and interstitial edema compared with ICH group.(4)In FJB staining,FJB staining positive cells increased significantly in ICH group compared with sham group(P<0.01);FJB staining positive cells in the Xingnaojing treatment group decreased significantly(P<0.01).(5)Western blot results showed that the level of PAR increased in ICH(P<0.01)and the proportion of nuclear AIF/plasma AIF increased(P<0.01) compared with the sham group.The level of PAR(P<0.05)and the ratio of nuclear AIF/plasma AIF(P<0.05)decreased significantly compared with ICH group.There was no significant difference in the expression of PARP1 between sham group and ICH group,ICH group and Xingnaojing group(P>0.05).Similar to the result of western blot,the results of immunofluorescence staining showed that after ICH,the expression of PAR around the hematoma increased significantly,and AIF translocation to nuclear increased.The expression of PAR and AIF translocation to nuclear in the Xingnaojing treatment group were all lower than that in ICH group.Conclusion:(1)Xingnaojing alleviated neural injury after ICH in mice;(2)Parthanatos is one of the mechanisms of neuronal necrosis after ICH in mice;(3)Xingnaojing exerted neuroprotective effects by inhibiting Parthanatos after ICH.