| Objective:The Middle Cerebral Artery Occlusion Reperfusion(MCAO/R)model in rats was established and treated with Huoxue Rongluo decoction,to explore the protective mechanism of Huoxue Rongluo decoction on Cerebral ischemia Reperfusion injury.Methods:72 healthy male SD rats were divided into sham group,model group(I/R),Huoxue Rongluo Low-dose group(HXRLF-L),Huoxue Rongluo medium-dose group(HXRLF-M),Huoxue Rongluo high-dose group(HXRLF-H)and Nimodpine group randomly,with 12 rats in each group.Among each group,9 rats were guaranteed to be successfully modeled.In sham group,we only separated the common carotid artery.In the other groups,we used thread plug to establish the middle cerebral artery Occlusion reperfusion model(MCAO/R).According to the human and animal body surface area conversion method,the crude drug concentration of 1.17g/ml in HXRLF-M group was given ingastric administration,while the low dose group was given half the dose(0.585g/ml)and the high lose group was given twice the dose(2.34g/ml).Meanwhile the concentration of Nimodiping group was 1.1mg/ml.The sham group and model group were perfused with equivalent distilled water.The intragastric dose of rats was calculated as lml/100g.The first administration time was 36h before modeling,followed by every 24h,a total of 3 times.Two hours after modeling and 24 hours after reperfusion,to grade the neurological function of the rats and to test the contents of IL-18 and IL-1β in serum by ELISA.TTC staining was used for surveying the volume of cerebral infarction,and HE staining was used for observing the pathological changes of hippocampus and cortex.The protein expression levels of NLRP3 and Caspase-1/P20 in hippocampus were tested by Western blotting.Immunohistochemistry was put to use to gauge the expression of Caspase-1 in hippocampus and cortex.Immunofluorescence double staining(Caspase-1 and TUNEL)was used for detecting pyroptosis.Results:1.Neurological deficit score:Compared with Sham group,the neurological deficit score of I/R rats was significantly higher(P<0.01);Compared with the I/R group,the neurological deficit scores of rats in HXRLF-M group,HXRLF-H group and Nimodipine group were declined,and the differences were statistically significant(P<0.05 or P<0.01).2.Determination of cerebral infarct volume:Compared with Sham group,the area of cerebral infarction in I/R group was significantly increased(P<0.01),and different degrees of infarct formation were observed in the brain tissue of rats in each drug group;Compared with I/R group,the volume of cerebral infarction in HXRLF-M group,HXRLF-H group and Nimodipine group decreased significantly(P<0.01);Compared with the HXRLF-M group,the infarct volume increased in the HXRLF-H group(P<0.01),Nimodipine group had no significant change.3.HE staining:By observing the pathological morphology of the hippocampus and cortex in rats,it was found that compared with sham group,nerve nucleus pyrosis,nucleoli disappearance,cell body deformation and brain tissue damage were serious in the model group,while all drug groups could alleviate nerve cell damage in the model group to varying degrees.4.Enzyme linked immunosorbent assay(ELISA):Compared with Sham group,the levels of inflammatory factors IL-1β and IL-18 in the serum of I/R group was went up remarkablely(P<0.01).Compared with I/R group,The contents of IL-1β and IL-18 in the serum of HXRLF-M group,HXRLF-H group and Nimodipine group were significantly falled off,with statistically significantly(P<0.01),and HXRLF-M group and Nimodipine had more obvious effects on regulating serum inflammatory factors IL-1βand IL-18,with no statistical difference between the two groups.5.Western blotting:Compared with Sham group,higher expression of NLRP3,Pro-Caspase-1,Cleaved-Caspase-1 in the hippocampus of rats in I/R group were significantly increased(P<0.01).Compared with I/R group,the expressions of NLRP3,Pro-Caspase-1,and Cleaved-Caspase-1 were down-regulated in the HXRLF-M group,HXRLF-H group,and Nimodipine group.The decreases were most significant in the HXRLF-M group and Nimodipine group,with statistical significance(P<0.05).6.Immunohistochemical staining:Compared with Sham group,the level of Caspase-1 in hippocampus CA1 and cortex of I/R group was noticeably rise,with statistically significant(P<0.01).Compared with model group,HXRLF-M group,HXRLF-H group and Nimodipine group reduced the expression of caspase-1 protein in hippocampus and cortex,with statistical significance(P<0.05),HXRLF-M group and Nimodipine group were the most prominent,and the efficacy of the two groups was basically the same.7.Immunofluorescence staining:Compared with the Sham group,the pyroptosis rate of nerve cells in the cortical area of I/R group was significantly and statistically increased(P<0.01).Compared with I/R group,all drug groups could reduce the pyroptosis rate of nerve cells to varying degrees,among which HXRLF-M group,HXRLF-H group and Nimodipine group had statistical significance(P<0.01).Compared with HXRLF-M group,the pyroptosis rate of HXRLF-H group was slightly higher(P<0.05).Conclusion:1.Huoxue Rongluofang can improve the neurological function and reduce the volume of cerebral infarction in MCAO/R rats.The mechanism may be related to inhibiting the pyroptosis mediated by NLRP3 inflammasome,inhibiting the maturation and release of pro-inflammatory factors,alleviating the inflammatory cascade and thus playing a neuroprotective role.2.In MCAO/R rat model rats,the neuroprotective effect of Huoxue Rongluo decoction was dose-related,in which medium and high doses were effective therapeutic doses,and the efficacy of medium dose was superior to high dose,comparable to nimodipine. |
PDF Full Text Request