Vitamin D3 Protects against Diabetic Retinopathy by Inhibiting High-Glucose-Induced Activation of the ROS/TXNIP/NLRP3 Inflammasome Pathway

Li Lu, Qianyi Lu, Wei Chen, Jingwen Li, Chunxia Li, Zhi Zheng, Li Lu, Qianyi Lu, Wei Chen, Jingwen Li, Chunxia Li, Zhi Zheng

Abstract

Purpose: This study aimed to evaluate the mechanisms underlying the effects of 1,25-dihydroxyvitamin D (vitamin D3) on diabetes-induced retinal vascular damage and retinal vascular endothelial cell apoptosis.

Methods: Diabetic and control rats were randomly assigned to receive vitamin D3 or vehicle for 6 months. Additionally, human retinal microvascular endothelial cells (HRMECs) were incubated in normal or high-glucose medium with or without vitamin D3. Morphological changes in retinal tissues and retinal vascular permeability were examined, and cellular apoptosis was detected by fluorescence staining. Intracellular reactive oxygen species (ROS) levels were determined using fluorescent probes. Proteins were examined by Western blotting.

Results: Vitamin D3 significantly downregulated intracellular ROS and inhibited TRX-interacting protein (TXNIP)/NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome pathway activation. Additionally, vitamin D3 reduced vascular endothelial growth factor (VEGF) expression and the Bax/Bcl-2 ratio. These changes were associated with retinal recovery and with decreases in retinal vascular permeability and retinal capillary cell apoptosis.

Conclusions: Vitamin D3 decreases diabetes-induced ROS and exerts protective effects against retinal vascular damage and cell apoptosis in association with inhibition of the ROS/TXNIP/NLRP3 inflammasome pathway. Understanding the mechanisms of action of vitamin D3 has important implications for preventing and treating inflammatory-related illnesses such as diabetic retinopathy.

Figures

Figure 1
Figure 1
Vitamin D3 inhibits the high-glucose-induced death of HRMECs by inhibiting expression of proteins in the NLRP3 inflammasome pathway. (a) Western blot analysis of proteins in the NLRP3 inflammasome pathway in the NG, NG + VD, HG, HG + VD, and NG + NLRP3-overexpressing and HG + NLRP3-knockdown groups. (b) Quantitative analysis of the bands. (c) Transfection of HRMECs with plasmid DNA (CTR: control group; scramble: scrambled shRNA control group; vector: empty vector control group). (d) Co-staining of Hoechst33238 and propidium iodide was performed to assess apoptosis of HRMECs. (e) Quantification of apoptotic cells. The results are expressed as the mean percentage of apoptotic cells ± SD. n = 6 (∗∗: NG versus HG, P < 0.05; ##: HG versus HG + VD, P < 0.05; ∗: NG + NLRP3 Op versus NG, P < 0.05; #: HG + NLRP3 KD versus HG, P < 0.05).
Figure 2
Figure 2
(a) Representative images of retinas at month 6. (b-c) The retinal thickness and cell numbers in the GCL (cells per 100 μm). At month 6, the GCL and IPL were obvious and well organized in the NC group. The retinal thickness in diabetic rats was reduced and the number of GCL cells decreased (DM), whereas in diabetic rats treated with vitamin D3, the retinas were more normal in structure and thicker than those in diabetic rats (NC: normal control group; NC + VD: normal control group + vitamin D3; DM: diabetes mellitus group; IPL: inner plexiform layer; GCL: ganglion cell layer). Data represent mean ± SD (n = 10). ∗∗DM versus NC, P < 0.05; ##DM + VD versus DM, P < 0.05. Data are expressed as the mean ± standard deviation. Scale bar, 30 μm.
Figure 3
Figure 3
Protective effect of vitamin D3 against apoptosis of retinal cells at month 6. (a) No TUNEL-positive cells were seen in the NC or NC + VD groups; abundant red fluorescent nuclei were observed in the DM group. Sparse red fluorescent nuclei were found in the DM + VD group. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. (b) Western blot of retinal extracts using anti-Bcl-2 and anti-Bax antibodies; β-tubulin was used as the loading control. (c) Bax/Bcl-2 ratios (data represent mean ± SD (n = 10) ∗∗: NC versus DM, P < 0.05, ##: DM versus DM + VD, P < 0.05. NC: normal control group; NC + VD: normal control group + vitamin D3; DM: diabetes mellitus group; DM + VD: diabetes mellitus group + vitamin D3. Scale bar, 30 μm.
Figure 4
Figure 4
Vitamin D3 attenuates the elevated BRB breakdown in diabetic rats by inhibiting VEGF expression and the TXNIP/NLRP3 pathway. (a) Retinal vascular permeability was quantified by detecting the retinal Evans blue leakage (data are the means ± SD, n = 10∗∗: DM versus NC, P < 0.05 and ##: DM + VD versus DM, P < 0.05). (b, c) Retinal VEGF protein expression was inhibited by vitamin D3 (data are the means ± SD, n = 10∗∗: DM versus NC, P < 0.05 and ##: DM + VD versus DM, P < 0.05). (d, e) Quantitative Western blot analyses of TXNIP and the NLRP3 inflammasome in whole retinal extracts. Expression was normalized to that of β-tubulin. Data are the means ± SD, n = 10 (∗∗: NC versus DM, P < 0.05; ##: DM + VD versus DM, P < 0.05).
Figure 5
Figure 5
Vitamin D3 inhibits glucose-induced ROS production in HRMECs. (a) To measure ROS production, cells were labelled with H2DCF-DA. The figures show representative data from three independent experiments. (b) Quantitative analysis of the measured fluorescence intensity was performed. Values are the means ± SD of three independent experiments (n = 6, ∗∗: HG versus NG, P < 0.05, ##: HG + VD and HG + NAC versus HG, P < 0.05). (c) Western blot and quantitative analyses of TXNIP from whole retinal extracts. Expression was normalized to that of β-tubulin. Data are the means ± SD, n = 6 (∗∗: HG versus NG, P < 0.05, ##: HG + VD and HG + NAC versus HG, P < 0.05).

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