Minocycline inhibits PARP‑1 expression and decreases apoptosis in diabetic retinopathy

Ying Wu, Yongdong Chen, Qiang Wu, Lili Jia, Xinhua Du, Ying Wu, Yongdong Chen, Qiang Wu, Lili Jia, Xinhua Du

Abstract

The present study aimed to investigate the mechanism underlying the effects of minocycline on diabetic retinopathy‑associated cellular apoptosis. A total of 40 Sprague Dawley (SD) rats were used as a diabetic retinopathy model following injection with streptozotocin. Among the 34 rats in which the diabetes model was successfully established, 24 rats were divided into two experimental groups: I and II (T1 and T2, respectively), and orally administered with various doses of minocycline. The remaining 10 rats served as the diabetic retinopathy control group. An additional group of 10 healthy SD rats with comparable weight served as normal controls. The rats in T1 and T2 groups were treated daily for eight consecutive weeks with minocycline at a dose of 2.5 mg/kg and 5 mg/kg, respectively. The mRNA expression levels of poly (ADP‑ribose) polymerase‑1 (PARP‑1) were subsequently measured by reverse transcription‑quantitative polymerase chain reaction, and the protein expression levels of poly‑ADP‑ribose were measured by western blot analysis and immunohistochemistry. Retinal morphology was observed following hematoxylin and eosin staining, and retinal cell apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase‑3 activity assays. The amplitudes of the electroretinogram (ERG) b‑wave and oscillary potentials (OPs) were measured using visual electrophysiology, and compared among the four groups. The results of the present study demonstrated that in the diabetic rats, retinal PARP‑1 gene expression was markedly upregulated, the number of apoptotic cells and the activity levels of caspase‑3 were increased, and the amplitude of the ERG b‑wave and the OPs were markedly lower as compared with the normal rats. Following treatment with minocycline, the abnormal expression of PARP‑1 in the retina was inhibited, and cellular apoptosis was decreased. In conclusion, the results of the present study suggest that PARP‑1 is involved in the development of diabetic retinopathy, and minocycline is able to inhibit PARP‑1 expression and decrease cellular apoptosis, suggesting that minocycline may prove to be a promising drug for the treatment of diabetic retinopathy.

Figures

Figure 1
Figure 1
mRNA expression levels of poly (ADP-ribose) polymerase-1 (PARP-1) in the retinal tissue samples of the CON, DM, T1, and T2 groups. The mRNA expression levels of PARP-1 in the retinal tissue samples of the DM rats were significantly higher, as compared with the CON group. Following minocycline treatment, the abnormally elevated mRNA expression levels of PARP-1 in the retinal tissue samples of the DM rats were suppressed (F=12.56, P=0.0021), as compared with the DM group. No statistically significant difference was observed in PARP-1 gene expression between the T1 and T2 treatment groups (P>0.05). *P<0.01 vs. the other groups; **P<0.05 vs. CON. CON, normal control group; DM, diabetic retinopathy group; T1, 2.5 mg/kg minocycline treatment group; T2, 5 mg/kg minocycline treatment group.
Figure 2
Figure 2
Expression levels of PAR proteins in the retinal tissue samples of DM rats were significantly higher, as comapred with the CON group. Following minocycline treatment, the expression levels of the PAR proteins were downregulated in the DM rats, as compared with the DM group. (A and B) Western blot analysis demonstrated a significant increase in the protein expression levels of the PAR proteins of the DM group, as compared with the other three groups, however no significant difference was observed between the CON, T1 and T2 groups (F=39.02). *P<0.01 vs. CON; **P<0.01 vs. DM. (C) PAR expression was observed in the retinal ganglion cell layer, inner nuclear layer, and outer nuclear layer, as determined by immunohistochemistry. The PAR protein staining in the retinal tissue samples of the CON group was weakly positive. (D) Positive PAR expression increased significantly in the DM rats. (E and F) Following minocycline treatment, the expression levels of the PAR proteins were downregulated in the T1 and T2 groups. Scale bars, 50 µm. CON, normal control group; DM, diabetic retinopathy group; T1, 2.5 mg/kg minocycline treatment group; T2, 5 mg/kg minocycline treatment group; PAR, poly (ADP-ribose).
Figure 3
Figure 3
The number of apoptotic cells increased significantly following 10 weeks in the DM rats, and decreased following minocycline treatment. (A) A small number of apoptotic cells were present in the rat retinal tissue samples of the CON group. (B) The number of apoptotic cells increased significantly following 10 weeks in the DM rats. The apoptotic cells were located in the GCL, INL and ONL. (C and D) Following minocycline treatment, significantly less apoptotic cells were present in the retinal tissue samples of the DM rats in the T1 and T2 groups. (E) The apoptotic index in the GCL of the DM rats was significantly higher, as compared with the CON group. Following minocycline treatment, the elevated apoptotic index in the GCL of the retinal tissue samples of the DM rats was suppressed (F=53.57; *P<0.01 vs. CON). No significant difference was observed between the GCL apoptotic index of the T1 and T2 treatment groups (**P>0.05). (F) Apoptotic index of the INL of the DM rats was significantly elevated, as compared with the CON group. Following minocycline treatment, the elevated apoptotic index in the INL of the retinal tissue samples of the DM rats was unchanged (F=28.32; *P<0.01 vs. CON). No significant difference was observed between the INL apoptotic index of the T1 and T2 treatment groups (P>0.05). (Scale bars, 25 µm). CON, control group; DM, diabetic retinopathy group; T1, 2.5 mg/kg minocycline treatment group; T2, 5 mg/kg minocycline treatment group; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer.
Figure 4
Figure 4
The activity levels of caspase-3 in the retinal tissue samples of the CON, DM, T1 and T2 groups. The activity levels of caspase-3 were significantly elevated in the retinal tissue samples of the DM rats, as compared with the CON group. Following minocycline treatment, the elevated activity levels of caspase-3 in the retinal tissue samples of the DM rats was suppressed (F=27.24; *P<0.01 vs. CON and **P<0.01 vs. DM). No statistically significant difference in the activity levels of caspase-3 was observed between the T1 and T2 treatment groups (P>0.05) CON, normal control group; DM, diabetic retinopathy group; T1, 2.5 mg/kg minocycline treatment group; T2, 5 mg/kg minocycline treatment group.
Figure 5
Figure 5
OPs of the retina in the CON, DM, T1 and T2 groups. (A) OPs of the rats in the CON, DM, T1 and T2 groups. (B) Amplitude analysis of the rat OPs in the CON, DM, T1 and T2 groups. The OP amplitudes in the DM group was significantly lower, as compared with the CON group (F=10.46; P=0.0011). *P<0.01 vs. CON. Following minocycline treatment, the OP amplitude increased, but remained lower as compared with the CON group. No statistically significant difference was observed between the OP amplitudes of the T1 and T2 groups (**P>0.05). CON, normal control group; DM, diabetic retinopathy group; T1, 2.5 mg/kg minocycline treatment group; T2, 5 mg/kg minocycline treatment group; OP, oscillary potential.
Figure 6
Figure 6
B-waves of the rats in the CON, DM, T1 and T2 groups, as demonstrated by ERG. (A) B-waves of the rats in the CON, DM, T1 and T2 groups. (B) Amplitude analysis of the ERG b-waves of the rats in all four groups. The b-wave amplitude in the DM group was significantly lower, as compared with the CON group (F=32.52; *P<0.0001 vs. CON), and following minocycline treatment, the b-wave amplitude increased, but remained lower than that of the CON group. The b-waves in the T2 groups were significantly greater, as compared with the T1 group (#P<0.001 vs. T1). CON, normal control group; DM, diabetic retinopathy group; T1, 2.5 mg/kg minocycline treatment group; T2, 5 mg/kg minocycline treatment group; ERG, electroretinogram.

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