| Part Ⅰ Protective Effect of Qingmangyihao Formula on Optic Nerve in Rats with Optic Nerve Tension InjuryObjective:Qingmangyihao Formula has achieved good optic nerve protection in clinical practice.Previous in vitro experiments showed that Qingmangyihao Formula can effectively inhibit the apoptosis of RGCs.This study aims to explore the optic nerve protection of Qingmangyihao Formula in rats with optic nerve traction injury.Methods:Some rats were injected with fluorescent gold retrogradely labeled RGCs by brain stereotaxic apparatus,and the optic nerve injury model of rats was established by the transverse traction method.The rats were randomly divided into the sham operation group,the model group,and the intervention groups with low,medium,and high doses of traditional Chinese medicine.According to the groups,the rats were treated with non-intervention,intragastric administration of normal saline,and intervention with different concentrations of Qingmangyihao decoction.(1)After 14 days of intervention,the fundus changes of rats with optic nerve injury were observed by fundus photography.(2)Visual electrophysiological changes in rats with optic nerve injury were detected by F-VEP.(3)The pathological changes of retinal tissue structure in rats with optic nerve injury were observed under the optical microscope.(4)Changes in the number of RGCs in rats with optic nerve injury induced by excitation fluorescence under the inverted fluorescence microscope.Results:(1)The intervention of low,middle,and high doses of Qingmangyihao prescription can improve the retinal vascular thinning and pale ischemia in rats with optic nerve injury,and the high dose is the best.(2)In the F-VEP test of optic nerve injury rats,the latency of P2 wave was prolonged and the amplitude of P2 wave was not significantly decreased(all p<0.05).The intervention of high-dose Qingmangyihao prescription could reduce the latency of P2 wave(p>0.05),and the intervention of low-dose and medium-dose Qingmangyihao prescription did not affect reducing the latency of P2 wave(all p>0.05).The intervention of low,medium,and high doses of Qingmangyihao Formula could not reduce the amplitude reduction of P2 wave(all p>0.05).(3)The intervention of low,medium,and high doses of Qingmangyihao Formula can effectively improve the disorder of retinal RGCs in rats with optic nerve injury,the swelling or shrinkage of the cell body,the concentrated staining of nuclear shrinkage,the change of intracellular vacuoles,the increase of intercellular space,and the decrease of the overall number of RGCs.(4)The density of retinal RGCs in rats with optic nerve injury decreased.The intervention of middle and high doses of Qingmangyihao can reduce the degree of RGCs density decrease(p<0.05).Conclusion:(1)Large number of apoptosis occurred in RGCs of rats 14 days after optic nerve traction injury.(2)The middle and high doses of Qingmangyihao Formula can inhibit the apoptosis of RGCs in rats with optic nerve injury.Part Ⅱ Effect of Qingmangyihao on Autophagy Activation of RGCs in Rats with Optic Nerve Tension InjuryObjective:Study 1 has verified through experiments that Qingmangyihao Formula has a certain inhibitory effect on the apoptosis of RGCs in rats with optic nerve traction injury.This study aims to explore the effect of Qingmangyihao Formula on the autophagy activation of RGCs in rats with optic nerve traction injury,and further clarify whether the protective effect of Qingmangyihao Formula on the optic nerve after optic nerve traction injury in rats is through the effect on the degree of autophagy.Methods:The optic nerve injury model of rats was established by the transverse traction method.The rats were randomly divided into sham operation group,model group,low-dose,medium-dose,and high-dose Chinese medicine intervention groups.According to the grouping,the rats were given non-intervention,normal saline gavage,and different concentrations of Qingmangyihao prescription liquid intervention.(1)After 14 days of intervention,the changes of various structures in the apoptosis process of RGCs after optic nerve injury in rats were observed by transmission electron microscope and autophagy bodies were found.(2)Western-blot molecular biological method was used to detect the expression of autophagy marker proteins LC-3 Ⅱ and p62.Results:(1)The structure of RGCs in the blank control group and the sham operation group was clear and complete.The ultrastructure of RGCs in the blank control group and the sham operation group was normal.The mitochondria,endoplasmic reticulum,Golgi complex,and other organelles in the cytoplasm were rich.There were occasionally a small number of autophagic vacuoles and almost no autophagosomes.In the model group and the intervention groups with different doses of traditional Chinese medicine,the structure of RGCs in rats was destroyed,the mitochondria swelled,the ultrastructure was incomplete,and the mitochondrial cristae disappeared.The rough endoplasmic reticulum swelled,the organelles decreased,the number of autophagolysosomes and lysosomes increased,and the vacuoles with uneven sizes appeared in the cytoplasm.In the model group and the intervention groups with low and medium doses of traditional Chinese medicine,the cell membrane disintegrated,and the cell contents were leaked.Compared with the model group,the autophagy in the high-dose TCM intervention group was alleviated to a certain extent,and the autophagosomes and lysosomes were reduced.(2)Compared with the blank control group,the expression level of LC3-Ⅱprotein in the sham operation group had no significant change(p>0.05).The expression levels of LC3-Ⅱ protein in the model group and the low,medium,and high dose Chinese medicine intervention groups were significantly increased(all p<0.05).Compared with the blank control group,there was no significant change in the expression level of p62 protein in the sham operation group(p>0.05).Compared with the blank control group,the expression levels of p62 protein in the model group and the low,medium,and high dose Chinese medicine intervention groups were significantly decreased(all p<0.05).Compared with the model group,the decrease of p62 protein expression level after the intervention of low,medium,and high doses of Qingmangyihao Formula was reduced(all p<0.05).Conclusion:(1)Autophagy was activated in RGCs of rats after optic nerve injury.(2)The intervention of middle and high doses of Qing Blind No.1 prescription can inhibit excessive autophagy activation to achieve the optic nerve protection of model rats.Part Ⅲ The regulation of Qingmangyihao on Bcl-2/Beclin-1 autophagy pathway in RGCs of rats with optic nerve traction injuryObjective:The second study has been verified by experiments that autophagy was activated in RGCs after optic nerve traction injury,and the intervention of middle and high doses of Qingmangyihao prescription can reduce RGCs apoptosis by reducing autophagy activity.This study aims to further explore the regulatory effect of Qingmangyihao Formula on Bcl-2/Beclin-1 autophagy pathway in RGCs of rats with optic nerve traction injury,and clarify the specific target of Qingmangyihao Formula for the protection of optic nerve after optic nerve traction injury in rats.Methods:The optic nerve injury model of rats was established by the transverse traction method.The rats were randomly divided into sham operation group,model group,low-dose,medium-dose,and high-dose Chinese medicine intervention groups.According to the grouping,the rats were treated with non-intervention,normal saline gavage,and different concentrations of Qingmangyihao prescription.(1)After 14 days of intervention,the apoptosis of RGCs was observed by immunofluorescence Tunel staining and NeuN staining under laser confocal microscope.(2)Western-blot molecular biological method was used to detect the expression of JNK1,Bcl-2,p-Bcl-2,Beclin-1,and PI3KC3 proteins related to Bcl-2/Beclin-1 autophagy pathway.Results:(1)TUNEL staining stained the apoptotic RGCs nuclei green,NeuN staining could mark the RGCs cells red,DAPI labeled the RGCs nuclei blue,and TUNEL/NeuN/DAPI was superimposed to obtain ’Merge’.There was a regular arrangement of RGCs cell layer in the blank control group,and the number of TUNEL positive cells was less.There was no significant difference between the sham operation group and the blank control group.Compared with the blank control group,a large number of TUNEL-positive cells were observed in the model group and the low,medium,and high doses of Chinese medicine intervention groups after optic nerve injury,which were coincident with the fluorescence of NeuN-positive cells.Compared with the model group,the number of apoptotic cells and NeuN positive cells in the high-dose Chinese medicine intervention group was relatively reduced.(2)Compared with the blank control group,the expression level of JNK1 protein in the sham operation group had no significant change(p>0.05).The expression level of JNK1 protein in the model group and the low,medium,and high dose Chinese medicine intervention groups was significantly increased(all p<0.05).The expression level of Bcl-2 protein in the model group and the low,medium,and high dose Chinese medicine intervention groups were significantly decreased(all p>0.05).Compared with the blank control group,the expression level of pBcl-2 protein in the sham operation group had no significant change(p>0.05).The expression level of pBcl-2 protein in the model group and the low,medium,and high dose Chinese medicine intervention groups were significantly decreased(all p<0.05).The expression level of Beclin-1 protein in the model group and the low,medium,and high dose Chinese medicine intervention groups was significantly increased(all p<0.05).The expression level of PI3KC3 protein in the model group and the low,medium,and high dose Chinese medicine intervention groups was significantly increased(all p<0.05).Compared with the model group,the expression of PI3KC3 protein in the low,medium,and high dose Chinese medicine intervention groups was decreased(all p<0.05).Conclusion:(1)The activation of Bcl-2/Beclin-1 autophagy pathway was observed in RGCs of rats 14 days after optic nerve injury.(2)High-dose Qingmangyihao Recipe intervention can inhibit the apoptosis of RGCs in rats with optic nerve injury by regulating Bcl-2/Beclin-1 autophagy pathway. |
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