Experimental study on the effect of PINK1-mediated mitophagy on diabetic retinal neuroglial network lesions

Title : Experimental study on the effect of PINK1-mediated mitophagy on diabetic retinal neuroglial network lesions

Abstract:

Objective: To explore the role of mitophagy in diabetic retinal neuroglial network lesions and analyze its mechanisms. Methods Experimental study. SD rat retinal müller cells (rMCs) were cultured in vitro, and the cells were divided into control (Control), hyperosmosis (HP), high glucose (HG) and carbonyl cyanide m-chlorophenylhydrazone groups (CCCP). Intracellular reactive oxygen species (ROS) expression was detected using a 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe; cellular mitochondrial membrane potential was detected by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbonyl cyanide iodide (JC-1) live-cell staining; and western blot was performed to detect PTEN-induced presumptive kinase 1 (PINK1), PARK2 gene-encoded protein (Parkin), recombinant human autophagy effector protein 1 (Beclin-1), microtubule-associated protein light chain 3 (LC3), and autophagy junction protein (p62) expression. The PINK1 silencing model was constructed by transfection of small interfering RNA (siRNA) with cationic liposome Lipofectamine 3000, and the cells were divided into Control, HG, HG+si-NC and HG+si-PINK1 groups. Mitochondrial red fluorescent probe (Mito-Tracker Red CMXRos) and lysosomal green fluorescent probe (Lyso-Tracker Green) staining was used to evaluate the effect of silencing PINK1 on mitophagy in cells under high glucose; Western blot and real-time fluorescence quantitative PCR (q-PCR) were used to detect the effect of silencing PINK1 on Parkin, Beclin-1, LC3, and p62 expression. The diabetic retinal neuroglial network lesion (DRN) model was constructed in SD rats, which was divided into Control, DRN, and DRN+3-(2,4-dichloro-5-methoxyphenyl)-2- thio-2,3-dihydro-4(1H)-quinazolinone (Mdivi-1) groups using the random number table method, with a total of 18 rats and 6 rats in each group. Optical coherence tomography (OCT) was used to detect the thickness of the retinal nerve fibre layer (RNFL) in rats, electroretinography was used to assess retinal function.

Results:  DCFH-DA fluorescent probe results showed that the values of ROS fluorescence intensity in Control and HG groups were 3.2±2.07 and 20.73±2.42 (t=-9.55, P<0.01), respectively; JC-1 assay results showed that the JC-1 Aggregates/Monomer ratio in Control and HG groups were 1.23±0.15 and 0.37±0.09 (t = 8.53, P < 0.01); Western blot assay showed that the relative expression of PINK1, Parkin, and Beclin-1 proteins in Control and HG groups were 0.88±0.15 and 1.14±0.05, 0.67±0.05 and 1.00± 0.08, 0.75±0.07 and 1.00±0.13 (t_PINK1 = -2.89, P < 0.05; t_Parkin = -6.19, P < 0.01; t_Beclin-1 = -2.99, P < 0.05), and 0.27±0.07 and 0.65±0.07 (t = -6.75, P < 0.01) for LC3II/LC3I, respectively, and the relative expression of p62 protein was 1.24±0.10 and 0.92±0.09 (t=4.18, P<0.05). After silencing PINK1, mitochondrial and lysosomal co-localisation was reduced in the HG+si-PINK1 group compared with the HG group; Western blot assay showed that the relative expression of Parkin and Beclin-1 proteins in the HG group and the HG+si-PINK1 group was 0.70±0.06 and 0.43±0.08, 0.84±0.16 and 0.28±0.11 (t_Parkin = 4.59, P < 0.05; t_Beclin-1 = 4.99, P < 0.01), 1.32±0.16 and 0.19± 0.06 for LC3II/LC3I, respectively (t = 11.22, P < 0.01), and the relative expression of p62 protein was 0.52±0.08 and 1.03±0.10 (t = -6.87, P < 0.01); q-PCR showed that the relative expression of Parkin, Beclin-1, LC3, and p62 mRNA in the HG group versus the HG+si-PINK1 group was 1.72±0.15 and 0.73±0.08, 1.52±0.27 and 0.56±0.08, 3.33±0.26 and 1.61±0.11, 0.79±0.10 and 1.63±0.15 (t_Parkin=10.28, P<0.01; t_Beclin-1=5.79, P<0.01; t_LC3=10.67, P<0.01; t_p62=-8.10, P<0.01). In vivo studies revealed that OCT showed that the thickness of the RNFL layer in the retinal tissue of rats in the Control and DRN groups was 98.33±1.53 and 84.67±1.53, respectively (t=10.96, P<0.01); electroretinograms showed that compared with the control group, rat optic rod cells in the DRN group showed reduced amplitude of the a-wave and b-wave responses, prolonged a-wave latency, and maximum mixed response a-wave and b-wave amplitudes were reduced, the average amplitude of oscillatory potentials was reduced, the cone cell response a-wave and b-wave amplitudes were reduced, the a-wave latency was prolonged, and the amplitude and phase of the scintillation light response were reduced and prolonged (P < 0.05). Mdivi-1 treatment ameliorated these changes. 

Conclusion: High glucose induced an increase in ROS expression in retinal müller cells, a decrease in mitochondrial membrane potential, and an activation of mitophagy, the mechanism of which was related to the overactivation of the PINK1/Parkin signalling axis. Inhibiting mitophagy reduces glial network lesions in DRN rats.

Biography:

Xiangxia Luo, Doctor of Medicine, Chief Physician, Doctoral Supervisor, and Deputy Director of Gansu Provincial Hospital of Traditional Chinese Medicine, is recognized as one of the fourth batch of National Outstanding Clinical Talents in Traditional Chinese Medicine, a leading talent in Gansu Province, a famous TCM doctor of Gansu, and a sixth batch Western Light (Tongren Hospital) visiting scholar. She specializes in diagnosing and treating hemorrhagic eye diseases such as diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration. She is skilled in the integrated treatment of retinitis pigmentosa, uveitis, glaucoma, optic neuropathy, and dry eye syndrome through a combination of traditional Chinese and Western medicine.