| Objective1.In this study,Eerdun-wurile was observed to protect the retinal ganglion cell in rats with retinal ischemia and reperfusion injury,expounds Eerdun-wurile retinal Ischemia reperfusion injury of disease prevention and cure function and its possible mechanism.2.To establish the model of the retinal ischemia reperfusion injury and ovserved Eerdun-wurile for the retinal ischemia reperfusion injury pathology change,calcium content of retinal tissue,neurotrophic factors and retinal tissue mRNA and protein expression of related genes,and the thus to explore the mechanism of action of the retinal ischemia reperfusion injury disease,provide experimental basis for clinical application and research.Methods1.Animal screening:Buy 127 healthy male and female half SD rats of clean grade,weight 280±20g.According to random number table method,the rats were normal group,model group,Eerdun-wurile group(Low concentration,medium concentration,high concentration),procyanidins control group.The mechanism of eerdun-wurile group was selected after cell apoptosis.2.Replication of the retinal ischemia reperfusion injury rat model:with 10%Chloral hydrate 0.35ml/100g success after intraperitoneal injection of anesthesia,The rats were fixed on a fixed plate and the eyes were molded.Tropicamide Eye Drops drops of mydriasis and Proparacaine Hydrochloride Eye Drops eye surface anesthesia,the children’s scalp acupuncture of 0.9%sodium chloride solution was inserted into the anterior chamber of the rat to avoid damage to the iris and lens,and then the infusion bottle slowly increased 150cm,and the intraocular pressure of 110mmHg(14.67kpa)(lkpa=7.5mmHg)was formed in the eye,which was close to the systolic blood pressure of the rat.It can cause the retinal blood flow to block,and then slowly reduce the height of the infusion bottle after 1h,lowering the intraocular pressure,the color of the iris and the ball conjunctivitis quickly returned tonormal,and the retina of the rat was orange,indicating that the model of ischemia reperfusion injury was successfully established.3.Methods of administration:The Eerdun-wurile is soaked in boiling water and mashed to make the suspension.Divide normal group,model group,Eerdun-wurile group(high dose 0.81g/kg,medium dose 0.54g/kg,Low dose 0.27g/kg)and procyanidins group(0.81g/kg).Monfolin Eerdun-wurile and procyanidins group 1 times/d,continuous series of 7d,the last time,after the 6h,the mold was made.The drug group was given the same dosage during the reperfusion period.It’s a mechanism that has been studied in the Eerdun-wurile to kill the rats at 0.54 g/kg.4.Materials drawing and index detection:① After reperfusion,24h,3D,5D and 7d were injected into the abdominal cavity by injection of anesthetic(10%chloral hydrate).After the rats were killed,the eyeball was removed immediately,and the optic nerve was retained for about 1-2mm;In 4%paraformaldehyde phosphate buffer(PH = 7.4)within 4 degrees fixed,dehydrated,conventional paraffin embedding,HE staining to observe retinal tissue pathology change detection of ganglion cell apoptosis with TUNEL method.② On the 7 day after modeling,with 10%chloral hydrate anesthesia depth in rats after celiac artery blood 3～4 ml,3000 r/min centrifugated 15min,serum was collected,using the method of double antibody sandwich enzyme-linked immunosorbent determination of serum level of BDNF and bFGF.③ Model 24h and 7d,the anesthetized rats wait for the rat to die,the eyeball was removed immediately,under the microscope,the retinal tissue was stripped and put into the EP tube and put into the liquid nitrogen tank and then transferred to the refrigerator at-80 ℃.The content of calcium in retinal tissue was detected by spectrophotometer.The expression of GFAP protein was detected by Western Blot and GFAP mRNA was detected by RT-PCR method.5.Statistical treatment:using the SPSS20.00 software for statistical treatment.And the data is showed mean ±standard deviation.Comparison between groups using F test.LSD test comparison between the two groups.P<0.05 has statistical significance.Results1.The influence of pathological changes of retinal tissue in the retinal ischemia reperfusion injury ratsIn the normal group,the retinal structure was clear,the ganglion cells were arranged closely,the nucleus margin was clear,the cell volume was large,round or elliptic,and the cells in the inner and outer nuclear layer were arranged uniformly.No inflammatory cells were invaded in the ganglion cell layer.After retinal ischemia reperfusion 24h,retinal edema,loose tissue,disordered arrangement,nucleus concentration and large inflammatory cell infiltration ganglion cell layer.After retinal ischemia reperfusion 72h,the retinal edema was reduced and the cells were closely arranged,and the inflammatory cells in ganglion cells increased significantly.After retinal ischemia reperfusion 5d and 7d,the retinal edema subsided significantly,the thickness of the retina became thinner,the thickness of inner nuclear layer and inner plexiform layer decreased most obviously,and the cells presented vacuoles and nuclear enrichment.After treatment,the rat retinal histological change trend similar to the model group,edema,loose plexiform layer to a lesser degree,nuclear enrichment cells less,the number of Apoptosis and the thinning degree of retinal thickness in 7d cells were all lighter than those in the model group.2.The influence of apoptosis of retinal tissue in the retinal ischemia reperfusion injury ratsThe apoptosis of retinal cells was detected by TUNEL.After retinal ischemia reperfusion injury,and the cells of TUNEL markers in the experimental group were mostly found in the ganglion cell layer and the core layer of the inner retina.Compared with the model group,the positive rate of apoptotic cells decreased in the four treatment groups at 24h,3d,5d and 7d,among them,after treatment of retinal ischemia reperfusion 24h,compared with model group the positive rate of apoptotic cells of Eerdun-wurile low dose group,Eerdun-wurile medium dose group and proanthocyanidin group were significant decreased(P<0.05).After treatment of retinal ischemia reperfusion 3d,compared with model group the positive rate of apoptotic cells of Eerdun-wurile medium dose group and proanthocyanidin group were significant decreased(P<0.05).After treatment of retinal ischemia reperfusion 5d,compared with model group the positive rate of apoptotic cells of Eerdun-wurile low dose group and proanthocyanidin group were significant decreased(P<0.05).After treatment of retinal ischemia reperfusion 7d,compared with model group the positive rate of apoptotic cells of Eerdun-wurile medium dose group was significant decreased(P<0.05).There was no statistically significant difference between Eerdun-wurile three group and proanthocyanidin group(P>0.05).3.The effect of Ca 2+content in retinal tissue of retinal ischemia reperfusion injury ratsAfter 24h injury in rats,compared with the normal group,the calcium content in the retina of model group was significantly increased(P<0.01).Compared with the model group,the calcium content of proanthocyanidin group was significantly decreased(P<0.05).There was no statistically significant difference between Eerdun-wurile group and proanthocyanidin group(P>0.05).4.The effect of serum neurotrophic factor BDNF and bFGF in retinal ischemia reperfusion injury rats4.1 The effect of bFGFAfter 7d injury in rats,compared with the normal group,serum bFGF content in model group was significantly increased(P<0.05).Compared with the model group,serum bFGF content of Eerdun-wurile group and proanthocyanidin group were significantly increased(P<0.05).Compared with of Eerdun-wurile group,serum bFGF content of proanthocyanidin group were significantly increased(P<0.05).4.2 The effect of BDNFAfter 7d injury in rats,compared with the normal group,serum BDNF content in model group was significantly decreased(P<0.01).Compared with the model group,serum BDNF content of Eerdun-wurile group and proanthocyanidin group were significantly increased(P<0.05).There was no statistically significant difference between Eerdun-wurile group and proanthocyanidin group(P>0.05).5.The effect of GFAP on the retinal tissue of the retinal ischemia reperfusion injury rats5.1 RT-PCR results:After 7d injury in rats,compared with the normal group,GFAP mRNA expression of the model group was significantly increased(P<0.01).Compared with the model group,GFAP mRNA expression of Eerdun-wurile group and proanthocyanidin group were significantly decreased(P<0.01).Compared with the of Eerdun-wurile group,GFAP mRNA expression of proanthocyanidin group was significantly decreased(P<0.05).5.2 Western blot result:After 7d injury in rats,compared with the normal group,GFAP protein expression level of the model group was significantly increased(P<0.01).Compared with the model group,GFAP portein expression level of Eerdun-wurile group and proanthocyanidin group were significantly decreased(P<0.01).Compared with the of Eerdun-wurile group,GFAP protein expression level of proanthocyanidin group was significantly decreased(P<0.05).Conclusion1.The results of TUNEL apoptosis showed that the effect of the Eerdun-wurile medium dose on the apoptosis of retinal ganglion cells was obvious,which could effectively inhibit cell apoptosis and provide the Eerdun-wurile medium dose group for retinal ischemia reperfusion injury mechanism research.2.Eerdun-wurile can significantly increase the calcium content of the retinal tissue in the retinal ischemia reperfusion injury rat,and protect the function of retinal ganglion cells.3.Eerdun-wurile can improve the activity of serum neurotrophic factor BDNF and bFGF in the retinal ischemia reperfusion injury rats,and it has protective function of the ganglion cells from the retinal ischemia reperfusion injury.4.Eerdun-wurile could decrease expression level of GFAP protein and expression of GFAP mRNA in the retinal ischemia reperfusion injury tissue in rats,protect the structure and function of the retina,stimulate the synthesis and secretion of neurotrophic factor,so as to protect the retinal ischemia reperfusion injury. |
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