| Objective Based on ERK5 Signal Pathway,to investigate the effect of Shidalazhi Pill on anti-apoptosis of brain cells in rats with cerebral ischemia-reperfusion injury,and its protective effect on ICVD and its mechanism.Methods 238 SD rats were divided into blank group,sham operation group,model group,Nimodipine group,Shidalazhi Pill group.According to the concentration of the drug,the Shidalazhi Pill group was further divided into high,middle and low dose groups;So the group had a total of 7 groups.According to the time of extracting brain tissue samples,each group was divided into 1d,3d,and 7d group,and each group had a total of 14 rats;except for the blank and sham-operated groups,the other groups established cerebral ischemiareperfusion model.The above groups were intragastrically administered at the same time every day,twice a day;the improvement of neurological deficits in rats was comprehensively evaluated by reference to Garcia JH scoring rules at 1d,3d,and 7d after model establishment.The positive counts of ERK5 were observed by immunofluorescence method;Observation of the apoptosis of nerve cells by TUNEL method;Western blot analysis of phosphorylation of P-ERK5;The relative expression of ERK5 gene in the ischemic side of brain was detected by RT-PCR.Results1.Neurological deficit assessment test results(1)The neurological function of the blank group and the sham operation group had no obvious defects at all time points,and the diet and activities were good(P>0.05).(2)There were significant differences between the model group and the drug groups at each time point compared with the blank group and the sham operation group(P<0.01).(3)There was a statistically significant difference in each drug group compared with the model group(P<0.01 orP<0.05).(4)There was no significant difference in the improvement of neurological function between the Nimodipine group and low,middle and high doses of Shidalazhi Pill groups(P>0.05).(5)Shidalazhi Pill groups comparison: there was no significant difference in the improvement of neurological function between the low dose group and the middle dose group,between the middle dose group and the high dose group(P>0.05),but the significant difference between the low dose group and the high dose group(P<0.01).(6)The neurological deficits of the 3d and 7d groups were improved,but the difference was not significant(P>0.05).but compared with the 1d group,there was a statistically significant difference between the two groups(P<0.01 or P<0.05).2.ERK5 immunofluorescence results(1)There was no significant difference between the blank group and the sham operation group(P>0.05).The number of positive cells in the other groups was higher than that in the blank group and the sham operation group,and the difference was significant(P<0.01).(2)The number of positive cells at each time point of each drug group was significantly different compared with the model group(P<0.01).(3)There was no significant difference in the number of positive cells between the Nimodipine group and the middle doses of Shidalazhi Pill group(P>0.05),but the difference between the low and high doses of Shidalazhi Pill groups was statistically significant(P<0.05).(4)Shidalazhi Pill groups comparison: compared with the middle and high dose groups,the low dose group was significantly different(P<0.01).There was no significant difference between the middle dose and high dose groups(P>0.05).Among them,the high dose group has the highest expression.(5)There was no significant difference between the 1d group and the 3d group(P>0.05),but compared with the 1d group,the 7d group was significantly different(P<0.01);compared with the 3d group,the 7d group was statistically significant(P<0.05).The number of positive cells in the 7d group was the highest.3.Results of apoptosis in rats after cerebral ischemia and reperfusion(1)Compared with the blank group and the sham operation group,the rats on the ischemic side showed obvious apoptotic cells(P<0.01 or P<0.05).(2)Compared with the model group,the number of apoptotic cells in each drug group was significantly decreased(P<0.01).(3)There was no significant difference between the time points of the Nimodipine group and the middle dose of Shidalazhi Pill groups(P>0.05),which was statistically significant compared with the low and high dose groups of Shidalazhi Pill(P<0.01 or P<0.05).(4)Shidalazhi Pill groups comparison: the difference in the number of apoptotic cells between the different dose groups was significantly different(P<0.01),and the decrease in the high dose group was the most obvious.(5)The apoptotic cells were the most in the 1d group and the least in the 7d group(P<0.01).4.Western Blot test results(1)There was no significant difference between the blank group and the sham operation group(P>0.05).(2)There was significant difference between the model group and each drug group compared with the blank group and the sham operation group(P<0.01).(3)The phosphorylation levels of each drug group were significantly higher than those of the model group(P<0.01 or P<0.05).(4)There was no significant difference between the time points of Nimodipine group and the middle dose group of Shidalazhi Pill(P>0.05),but compared with the low and high dose groups of Shidalazhi Pill,there was difference significant(P<0.01).(5)Shidalazhi Pill groups comparison: There was a statistically significant difference in phosphorylation level between the dose groups(P<0.01 or P<0.05),and the high dose group was the most obvious(P<0.01).(6)The phosphorylation level was not significantly increased in the 1d group of each drug group,and the up-regulation was significantly increased in the 7d group(P<0.05).5.RT-PCR detection of ERK5 relative expression results(1)There was no significant difference in the relative expression between the blank group and the sham operation group(P>0.05).(2)The relative expression of the model group and each drug group was significantly higher than that of the blank group and the sham operation group(P<0.01 or P<0.05).(3)The relative expression of each drug group was significantly higher than that of the model group(P<0.01).(4)There was no significant difference in the expression of the Nimodipine group and the middle dose group of the Shidalazhi Pill(P>0.05),but compared with the low and high dose groups of Shidalazhi Pill,there was statistically significant difference(P< 0.01 or P<0.05).(5)Shidalazhi Pill groups comparison:there was a difference in the expression level between each dose group(P<0.01 or P<0.05),and the high dose expression was the highest(P<0.01).(6)The expression of the drug group was the least in the 1d group and the highest in the 7d group(P<0.01 or P<0.05).Conclusions1.After cerebral ischemia-reperfusion in rats,the neurological deficit was restored to a certain degree by the intervention of Shidalazhi Pill,and the improvement was most obvious in the 7d groups.2.The apoptotic cells in the ischemic side of rats with cerebral ischemia-reperfusion showed a downward trend with the prolongation of the intervention time of Shidalazhi Pill,and the number of apoptosis was the highest in the 1 d groups and the least in the 7d groups.3.Shidalazhi Pill can up-regulate the expression of ERK5 protein kinase in ischemic brain tissue of rats with cerebral ischemia-reperfusion,which may be related to the mechanism of activation of ERK5 signaling pathway to play an anti-apoptosis and protect neurons. |
PDF Full Text Request