| Objective By using Shi-da-la-zhi-wan and its separate prescript-tions for preventing and treating cerebral ischemia reperfusion injury model rats,to explore the mechanism of shi-da-la-zhi-wan in the treatment of cerebral ischemia-reperfusion injury rats and its possible effective components.Methods 204 male SPF-grade Sprague?Dawley rats were randomly divided into blank control group(group A),sham operation group(group B),model group(group C),Shi-da-la-zhi-wan group(group D),croton group(group E),croton+three-drugs group(Group F),and croton+extra-drugs group(group G),and the last 5 groups were used to prepare focal cerebral ischemia reperfusion models.And according to the different time points of material collection,it is divided into 1d,3d and 7d groups.The neurological scores of the rats in each group were scored by Garcia JH method.HE staining was used to observe the pathological changes of brain tissue at various time points in rats;TUNEL was used to observe the apoptosis of cerebral ischemia-reperfusion;Immunofluorescence,RT-PCR and Western Blot were used to observe the protein expression levels of ERK1/2 and ERK5 in the MAPK-ERK signaling pathway.Results1.Neural function score results(1)Compared with the blank control group and the sham operation group,thescores of each group were reduced(P < 0.01);compared with the model group,the scores of each drug intervention group were increased(P < 0.05 or P <0.01);(2)Among the drug groups,the neurological score of the shi-da-la-zhi-wan group was higher than that of the separate prescriptions groups,and the score of the croton+three-drugs group was the highest in each separate prescriptions groups(P < 0.05 or P < 0.01);(3)The neurological scores of the model group and each drug groups increased with time.2.Western-bolt results(1)Compared with the blank control group and the sham operation group,the activation levels of p-ERK1/2 and p-ERK5 in the hippocampus of each group of rats were increased(P < 0.01);Compared with the model group,the activation levels of p-ERK1/2 and p-ERK5 were increased in each drug group(P< 0.05 or P<0.01);(2)In each drug groups,the activation levels of p-ERK1/2 and p-ERK5 in the shi-da-la-zhi-wan group were better than those in the separate prescriptions groups.The activation degree of p-ERK1/2 and p-ERK5 in croton + three drugs group was the most significant in each separate prescriptions groups(P<0.05 or P<0.01).(3)The activation degree of p-ERK1/2 and p-ERK5 in the model group and each drug group increased with time.3.RT-PCR results(1)Compared with the blank control group and sham operation group,the relative expression levels of mRNA of ERK1/2 and ERK5 in the hippocampus of rats in each groups were significantly increased(P < 0.01);Compared with the model group,the expression levels of ERK1/2 and ERK5 in each drug group were increased(P < 0.05 or P < 0.01);(2)Among the drug groups,the relative mRNA levels of ERK1/2 and ERK5 in the shi-da-la-zhi-wan group are better than those in the separate prescriptions groups.The relative mRNA levels of ERK1/2 and ERK5 in the croton+three-drugs group was the highest in each separate prescriptions groups(P< 0.05).(3)The relative mRNA levels of ERK1/2 and ERK5 in the model group and each drug groups increased with time.4.HE staining results in hippocampus of rat brain(1)Compared with the blank control group and the sham operation group,the neurons in the hippocampal CA1 area of the model group were loosely arranged,disordered,and fuzzy,and the nucleus and cytoplasmic boundaries were not clear.Compared with the model group,the neuron morphology of each drug group improved significantly;(2)The improvement effect was most obvious in the shi-da-la-zhi-wan group among the drug groups;the neuron cell morphology improvement in the croton+three-drugs group was more obvious in each separate prescriptions groups.5.TUNEL results in hippocampus of rat brain(1)Compared with the blank control group and the sham operation group,the number of apoptotic cells in the hippocampal region of the brain tissue of the model group was significantly increased(P < 0.01);Compared with the model group,the number of apoptotic cells in each drug groups was reduced(P< 0.05 or P < 0.01);(2)The number of apoptotic cells in the shi-da-la-zhi-wan group was lower in each drug groups than it in the separate prescriptions groups.The croton+three-drugs group had the lowest apoptotic cells count in each separate prescriptions groups(P<0.05);(3)The counts of apoptotic cells in the model group and each drug groups decreased with time.6.Immunofluorescence results(1)Compared with the blank control group and the sham operation group,the positive cell counts of p-ERK1/2 and p-ERK5 in the hippocampus of rats were significantly increased(P < 0.01);Compared with the model group,the p-ERK1/2and p-ERK5 positive cell counts of each drug groups were increased(P<0.05 or P< 0.01);(3)Among the drug groups,the positive cell counts of p-ERK1/2 and p-ERK5 in the shi-da-la-zhi-wan group were the highest.And the croton+three-drugs group had the most positive cell counts in each separate prescriptions groups(P < 0.05 or P < 0.01);(3)The number of p-ERK1/2 and p-ERK5 positive cells in the model group and each drug group increased with time..Conclusions1.Shi-da-la-zhi-wan and its separate prescriptions groups can improve neurological damage and inhibit neuronal apoptosis after cerebral ischemia reperfusion in rats,which may be related to its involvement in regulating the expression of ERK1/2 and ERK5 in MAPK-ERK pathway.2.Among the separate prescriptions groups,the four-drug combination of croton,bupleurum,black gardenia,and aloe vera has the best treatment effect.So we speculate that this group of drugs may be the main drug composition of Shi-da-la-zhi-wan that can effectively inhibit focal cerebral ischemia-reperfusion injury;and croton may be one of the main drugs that play a brain protective role in the four drugs. |
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