Objective To investigate the effect and underlying mechanism of Qingguang’an Granules(青光安颗粒剂,QGAG)on mitochondrial autophagy(mitophagy)of retinal ganglion cells(RGCs)in rats with chronic ocular hypertension(COH...Objective To investigate the effect and underlying mechanism of Qingguang’an Granules(青光安颗粒剂,QGAG)on mitochondrial autophagy(mitophagy)of retinal ganglion cells(RGCs)in rats with chronic ocular hypertension(COH).Methods Sixty Sprague Dawley(SD)rats,half males and half females,were randomly assigned to three groups:the control,model,and QGAG(2.5 g/kg)groups,with 20 rats in each group.Rats’model of COH was established by cauterizing episcleral veins in the model group and QGAG group.Three weeks after successful modeling,rats in the QGAG group were intra-gastrically administered with QGAG,while rats in the control group and the model group received an equal dose of normal saline.After three months of intragastric administration,intraocular pressure(IOP)of all rats was measured.The mitophagy was monitored by the immunofluorescence method,the mitochondrial membrane potential was measured using the JC-1 method,and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy.Meanwhile,rat RGCs were labeled using the fluorescent gold method,and RGCs density in each group was calculated.Moreover,RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling(TUNEL)assay.Finally,the expression levels of Parkin,optineurin,microtubule-associated protein 1 light chain 3-Ⅱ/microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅱ/LC3-Ⅰ),recombinant lysosomal associated membrane protein 1(LAMP1),and B-cell lymphoma-2(Bcl-2)in RGCs were determined by Western blot assay.The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction(qRT-PCR).Results The QGAG reduced IOP in COH rats,and inhibited mitophagy and apoptosis of RGCs(P<0.05).Besides,the QGAG significantly increased the expression levels of Parkin and Bcl-2(P<0.05),and inhibited the expression levels of optineurin,LAMP1,and LC3-Ⅱ/LC3-Ⅰ(P<0.05)in RGCs of COH rats.Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage caused by glaucoma,which may be mediated through the mitophagy ubiquitination via the Parkin/PINK1-related pathway.展开更多
基金Regional Fund Project of National Natural Science Foundation of China(81860870)China Postdoctoral Science Foundation(2018M640754)+3 种基金Hunan Natural Science Foundation Project(2020JJ5436)Program of Chinese Medicine Innovative-Backbone Talents of China(Xiang CM[2019]67)Hunan Province“225”Program for Cultivation of High-level Health Talents(Xiang CM[2019]196)Open Fund Project of Hunan Provincial Engineering Technology Research Center for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases and Visual Function Protection with Chinese Medicine(2018YZD02).
文摘Objective To investigate the effect and underlying mechanism of Qingguang’an Granules(青光安颗粒剂,QGAG)on mitochondrial autophagy(mitophagy)of retinal ganglion cells(RGCs)in rats with chronic ocular hypertension(COH).Methods Sixty Sprague Dawley(SD)rats,half males and half females,were randomly assigned to three groups:the control,model,and QGAG(2.5 g/kg)groups,with 20 rats in each group.Rats’model of COH was established by cauterizing episcleral veins in the model group and QGAG group.Three weeks after successful modeling,rats in the QGAG group were intra-gastrically administered with QGAG,while rats in the control group and the model group received an equal dose of normal saline.After three months of intragastric administration,intraocular pressure(IOP)of all rats was measured.The mitophagy was monitored by the immunofluorescence method,the mitochondrial membrane potential was measured using the JC-1 method,and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy.Meanwhile,rat RGCs were labeled using the fluorescent gold method,and RGCs density in each group was calculated.Moreover,RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling(TUNEL)assay.Finally,the expression levels of Parkin,optineurin,microtubule-associated protein 1 light chain 3-Ⅱ/microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅱ/LC3-Ⅰ),recombinant lysosomal associated membrane protein 1(LAMP1),and B-cell lymphoma-2(Bcl-2)in RGCs were determined by Western blot assay.The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction(qRT-PCR).Results The QGAG reduced IOP in COH rats,and inhibited mitophagy and apoptosis of RGCs(P<0.05).Besides,the QGAG significantly increased the expression levels of Parkin and Bcl-2(P<0.05),and inhibited the expression levels of optineurin,LAMP1,and LC3-Ⅱ/LC3-Ⅰ(P<0.05)in RGCs of COH rats.Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage caused by glaucoma,which may be mediated through the mitophagy ubiquitination via the Parkin/PINK1-related pathway.
