Salidroside ameliorates diabetic retinopathy and Müller cell inflammation via the PI3K/Akt/GSK-3β/NF-𝜅B pathway

Abstract

Purpose: To determine whether salidroside (SAL) modulates inflammatory cytokines in rat retinal Müller cells (rMC-1) in a hyperglycemic environment by investigating the anti-inflammatory mechanisms of SAL in vitro and in vivo.

Methods: A streptozotocin (STZ)-induced diabetic rat model was established to examine the effects of SAL using hematoxylin and eosin (H&E) staining and immunohistochemistry. rMC-1 cells were grown in 50 mM of high-glucose medium. These simulated diabetic conditions were used to evaluate the anti-inflammatory effects of SAL using a Cell Counting Kit-8 (CCK-8) assay, immunofluorescence staining, western blotting, and real-time polymerase chain reaction (qRT‒PCR). H&E staining was used to analyze the number of ganglion cells in the retina. rMC-1 lysates were processed for qRT‒PCR to measure the steady-state mRNA expression levels of inflammatory markers, such as interleukin 6 (IL-6), interleukin 10 (IL-10), and interleukin 1β (IL-1β). Western blot analysis and immunofluorescence staining were performed to determine the levels of these inflammatory markers.

Results: Our study showed that SAL reversed retinal ganglion cell loss and attenuated nuclear factor kappa B (NF-𝜅B) p65 translocation to the nucleus in STZ-induced diabetic rats. Incubating rMC-1 in different concentrations of SAL for 24 to 48 h affected cell viability. Furthermore, SAL treatment significantly decreased the protein levels of IL-6, TNF-α, and IL-1β compared with those in cells cultured in high glucose (HG). The mRNA expression levels of IL-6 and IL-1β were considerably reduced after SAL treatment, whereas the mRNA expression levels of IL-10 were significantly increased. Interestingly, the beneficial effects of SAL on HG-treated rMC-1 cells were abolished by the PI3K inhibitor LY294002.

Conclusions: These results indicate that SAL treatment reduces cytokine activation in cultured rMC-1. Furthermore, SAL prevents diabetic retinopathy (DR), in part, by modulating the PI3K/Akt/GSK-3β/NF-kB pathway to inhibit Müller cell activation. Thus, SAL is expected to be a potential agent for ameliorating the progression of DR.

Figure 1

SAL prevents ganglion cell loss due to STZ-induced injury. A: H&E staining showing that SAL protects ganglion cells in diabetic rats. The arrowheads represent retinal ganglion cells. B: Quantitative analysis of the number of ganglion cells. SAL-treated retinas showed a significantly higher number of ganglion cells than the DM group. (^#p<0.05). Values are presented as mean ± SD (n=3). Scale bar=100 μm. ***p<0.001, versus CON group. ^#p<0.05, versus DM group.

Figure 2

SAL affects the distribution of phosphorylated NF-𝜅B p65 in the retinas of STZ-induced diabetic rats. Detection of the distribution of NF-𝜅B p65 in the retina by immunofluorescence analysis. NF-𝜅B p65 is mainly expressed in the cytoplasm. The DM group showed increased expression in the ONL and INL. SAL-treated mice showed relatively lower expression in the ONL and INL.

Figure 3

Immunocytochemical analysis of rMC-1 cell cultures. Red: rMC-1 stained for GS. Blue: nuclear staining with DAPI. Merged labeling of GS. Scale bar=100 μm.

Figure 4

SAL had no cytotoxic effects on rMC-1 viability, as measured using the CCK-8 kit. A: rMC-1 cells were incubated with increasing concentrations of SAL for 24 h at 37 °C. B: rMC-1 cells were incubated with increasing concentrations of SAL for 48 h at 37 °C. There was no significant reduction in viable cell numbers after treatment with SAL at concentrations up to 1000 µM.

Figure 5

SAL reduced the HG-induced expression of proinflammatory cytokines. rMC-1 cells were pretreated with different concentrations of SAL (100, 200, 500 µM) for 6 h and then stimulated with HG (50 mM) for 48 h. A: The levels of IL-1β, IL-6, and TNF-α were determined by western blotting. B–D: Quantitative analyses of the protein expression of IL-1β, IL-6, and TNF-α.

Figure 6

SAL suppresses the nuclear translocation of NF-𝜅B p65 in rMC-1 cells. A: The nuclear translocation of NF-𝜅B p65 was determined using microscopy. B: Fifteen fields were imaged per well, and cells showing nuclear translocation of NF-𝜅B were counted. These data are expressed as a ratio to the total number of cells in the field. The experiments were performed in triplicate, and the values are expressed as the percentage of cells per field that localized NF-𝜅B to the nucleus. Scale bar=50 µm. *p<0.05, **p<0.01, ***p<0.001, versus CON group. ^##p<0.01, versus HG group.

Figure 7

SAL influenced the PI3K/Akt/GSK-3β signaling pathway under HG conditions. A: western blot analyses were used to determine the protein expression of p-Akt and p-GSK-3β. The protein levels of p-Akt and P-GSK-3β were markedly decreased in rMC-1 cells treated with HG, whereas treatment with SAL caused a marked increase in p-Akt and p-GSK-3β compared with that in the HG groups. B–C: Quantitative results of protein density determination. *p<0.05, versus CON group. ^#p<0.05, versus HG group. ^△p<0.05, versus HG+SAL group.

Figure 8

SAL influenced the PI3K/Akt/GSK-3β signaling pathway under HG conditions. Immunofluorescence detection of the protein expression p-Akt and p-GSK-3β. The results were consistent with the western blot results. Values are presented as mean ± SD (n=3). Scale bar=100 µm.

Figure 9

SAL influenced the mRNA expression of inflammatory factors in rMC-1 cells. The mRNA expression of IL-1β, IL-6, and IL-10 was determined by qRT‒PCR. A–B: The mRNA levels of IL-1β and IL-6 were significantly decreased in rMC-1 cells cultured with SAL compared with those in the HG group (^##p<0.01). C: The mRNA expression levels of IL-10. In the SAL group, the mRNA levels of IL-10 were significantly increased compared with those in the HG group. Values are presented as mean ± SD (n=3). *p<0.05, versus CON group. ^#p<0.05, ^##p<0.01, versus HG+SAL group. ^△p<0.05, versus HG+SAL group.

Figure 10

SAL reduced the HG-induced expression of pro-inflammatory cytokines in rMC-1 through the PI3K/Akt/GSK-3β signaling pathway. Western blot analysis of cytokine expression. A–D: western blot analysis of protein expression and quantitative analyses of IL-1β, IL-6, and TNF-α. **p<0.01, versus CON group. #p<0.05, ^##p<0.01, versus HG group. ^△p<0.05, ^△△p<0.01 versus HG+SAL group.

Figure 11

SAL reduced the HG-induced expression of pro-inflammatory cytokines in rMC-1 through the PI3K/Akt/GSK-3β signaling pathway. The protein expression of IL-1β, IL-6, and TNF-α was determined by immunofluorescence analysis. The results were consistent with the western blot results. Values are presented as mean ± SD (n=3). Scale bar=100 μm.

Figure 12

SAL attenuated phosphorylation of NF-𝜅B p65 translocation to the nucleus by enhancing the PI3K/Akt/GSK-3β signaling pathway. Immunofluorescence analysis of NF-𝜅B p65 translocation to the nucleus in rMC-1 cells. A: The nuclear translocation of NF-𝜅B p65 was determined by microscopy. B: Fifteen fields were imaged per well, and cells in which NF-𝜅B was located in the nucleus were counted. These data are expressed as a ratio to the total number of cells in the field. The experiments were performed in triplicate, and the values are expressed as the percentage of cells per field that localized NF-𝜅B to the nucleus. Values are presented as mean ± SD (n=3). Scale bar=50 μm. **p<0.01, versus CON group. ^#p<0.05, versus HG group. ^△p<0.05, versus HG+SAL group.