| Objective:Through the establishment of reproducible and stable continuous high intraocular pressure animal model,to observe the effect of yijingqijudihuang formula on retinal ganglion cells apoptosis of chronic high intraocular pressure rats,and discuss its mechanism of action,and provide scientific basis for the clinical application of traditional Chinese medicine.Method:1.In a study of establishment of high intraocular pressure model of SD rats.The model of chronic ocular hypertension was established by the method of cauterization of the superior scleral vein.The rats with high intraocular pressure were sacrificed on the 7 days?14 days?21 days?28 days?10 rats of Blank control group was no special treatment.The changes of intraocular pressure were observed in rats by tonometry.The pathological changes of rats retinal tissues after operation of 7 days,14 days,21 days,28 days were observed by hematoxylin and eosin(HE)staining.The RGCs cell apoptosis rates of rats after operation of 7 days,14 days,21 days,28 days were observed by Tunel detection.2.In a study of the mechanism of yijingqijudihuang formula.60 male Sprague-Dawley rats were divided into 5 groups randomly:blank group,model group,traditional Chinese medicine high dose group,medium dose group and low dose group,with 12 rats in each group.In addition to the blank group,the other 4 groups were used to establish the model of chronic ocular hypertension.All groups were given intervention for one month.We used tonometry to observe the changes of intraocular pressure of every groups rats.The pathological changes of retinal tissues Of every groups rats were researched by hematoxylin and eosin(HE)staining.The RGCs cell apoptosis rates of every groups rats were observed by Tunel detection.The protein expression of Bcl-2?bax and Caspase-3 of every groups rats RGCs tissues were detected by immunohistochemical staining.The relative expression of Bcl-2mRNA,BaxmRNA,Caspase-3mRNA of every groups rats were detected by RT-PCR.Results:1.In a study of establishment of high intraocular pressure model of SD rats.(1)tonometry:immediately after modeling,the IOP of the model group was significantly higher than that of the blank group(P<0.01).Rats after operation of 7 days,14 days,21 days,28 days was significantly higher than that of the blank group(P<0.01)and no significant difference than the immediately after modeling(P>0.05),there is no significant difference between each time point(P>0.05).(2).Under light microscope observation after HE,there were differences between monel and blank group.with prolonged time of high intraocular pressure,Monel group are not intact,irregular,have the RGCs layer arranged in disorder.By acounting RGCs cells,we found that Rats after operation of each time point was significantly lower than the blank group(P<0.01).With the extension of model,the number of RGCs cells at each time point were diminishing(P<0.01).(3)Tunel showed that the RGCs cell apoptosis rates of each time point was significantly higher than the blank group(P<0.01).With the extension of model,each time point were increasing(P<0.01).2.In a study of the mechanism of yijingqijudihuang formula.(1)tonometry:immediately after modeling,the model group and medication group were significantly different from the blank group(P<0.01),and the two groups have no significant difference(P>0.05).After the intervention of one month,the model group and medication group have no significantly difference from immediately after modeling(P>0.05).there is no significant difference between each medication group(P>0.05).(2)HE showed that Monel group are not intact,irregular,have the RGCs layer arranged in disorder.Chinese medicine middle dose group were better than monel group(P<0.01).(3).Tunel showed that the RGCs cell apoptosis rates of monel group was significantly higher than the blank group(P<0.01).Chinese medicine middle dose group were better than monel group(P<0.01).High and low dose group was not statistically significant than monel group(P>0.05).(4)Compared with the normal group,there were significant differences with the model group and medication group(P<0.01);Compared with the model group,middle dose group have higher expression of Bcl-2(P<0.01)and lower expression of Bax and Caspase-3(P<0.01);high and low dose group have no statistical significance(P>0.05).(5)RT-PCR showed that,The relative expression of Bcl-2mRNA of middle and low dose group were higher than the model group(P<0.01),the high dose group had no statistical significance than the model group(P>0.05).The BaxmRNA and Caspase-3mRNA of middle dose group were lower than the model group(P<0.01).The BaxmRNA of low dose group were a bit lower than the model group(P<0.05).The BaxmRNA of high group and Caspase-3mRNA of high and low group had no statistical significance than the model group(P>0.05).Conclusions:1.By the method of cauterization of the superior scleral vein,a stable intraocular hypertension animal model was established.After the model was established,the intraocular pressure was significantly increased.2.Yijingqijudihuang formula have obvious protective effect on retinal ganglion cells apoptosis of chronic high intraocular pressure rats.3.The mechanism of yijingqijudihuang formula controling RGCs apoptosis is linked with the high expression of Bcl-2 and the low expression of Bax and Caspase-3. |
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