Molecular Vision 2024; 30:1-17
<http://www.molvis.org/molvis/v30/1>
Received 17 January 2022 | Accepted 07 February 2024 | Published 10 February 2024
Molecular Vision 2024; 30:1-17
<http://www.molvis.org/molvis/v30/1>
Received 17 January 2022 |
Accepted 07 February 2024 |
Published 10 February 2024
Zhen Feng,1,2 Yang Yang,1,2 Cai-xing Shi,3 An-Qi Liu,1 Chuan-Ling Wu,4 Wen-Qiang Liu,1 Sheng-Xue Yu,1 Hong-Dan Yu,1 Zhong-Fu Zuo,1,5 Xue-Zheng Liu1
1Department of Anatomy, Histology and Embryology, Liaoning Key Laboratory of Diabetic Cognitive and Perceptive Dysfunction, Jinzhou Medical University, Jinzhou, Liaoning, China; 2Zhejiang Changzheng Vocational Technical College, Shenyang, China; 3China Medical University, Shenyang, China; 4Zhejiang Industrial Vocational and Technical College, Shaoxing, Zhejiang, China; 5Department of Anatomy, Histology and Embryology, Postdoctoral Research Station, Guangxi Medical University, Nanning, Guangxi, China
Correspondence to: Xue-Zheng Liu, Jinzhou Medical University, Department of Anatomy, Histology and Embryology, Jinzhou Medical University, Jinzhou, Liaoning, China 121000, Jinzhou, Liaoning, China: Phone: 13735860792; FAX: 86655; email: 951564552@qq.com
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.
Diabetic retinopathy (DR) is a main complication of diabetes and a cause of preventable blindness in adults [1]. Several researchers have shown that chronic inflammation plays a role in the development of DR [2]. In diabetes, the retina produces high levels of proinflammatory cytokines [3,4]. This research suggests that alterations in inflammation play an essential role in the development of DR and that specific cytokines may act as mediators.
Müller cells, which primarily regulate glutamate levels, are the primary glial cells that expand radially to cover nearly the entire width of the retina [5]. These cells are also the principal glial cells in the retina and are thought to be a significant source of inflammatory factors in DR [5-7]. To examine the expression of inflammatory markers in retinal hyperglycemia, we focused on specific inflammatory markers: IL-10, IL-6, tumor necrosis factor-α (TNF-α), IL-1β and nuclear factor-kappa B (NF-𝜅B). Some studies have reported increased levels of cytokines, including IL-1β and IL-6, in the vitreous fluid of patients with proliferative DR and the retinas of diabetic rats [8-10]. The inflammatory response in early diabetes involves cytokine release and activation of critical transcriptional regulatory factors, including NF-𝜅B [11].
Salidroside (SAL) is a bioactive extract from traditional herbal medicines, such as Robiola rose [12], which has the functions of preventing tumor growth, delaying aging and reducing oxidative stress [13-17]. Accumulating evidence has demonstrated that SAL exerts favorable anti-inflammatory and antiapoptotic effects [18,19]. In addition, evidence has shown that SAL has a positive impact on multiple complications of diabetes. For example, in the streptozotocin (STZ)-induced neuropathic pain rat model, SAL alleviates pain by inhibiting neuroinflammation in the spinal cord [20]. Previous studies have reported that SAL protects retinal endothelial cells from oxidative damage by enhancing the Bcl-2/Bax signaling pathway and activating endogenous antioxidant enzymes [21]. However, no studies have reported whether SAL alleviates the inflammatory response in DR.
Phosphatidylinositol-3-kinase (PI3K) is an essential regulator of multiple signal transduction pathways that mainly controls cell growth, apoptosis, and metabolism [22,23]. The serine/threonine kinase Akt is the downstream target of PI3K and plays a vital role in cell death and survival [24]. A major downstream effector of Akt is glycogen synthase kinase (GSK-3β), which activates the phosphorylation of the serine 9 residue [25]. Studies have shown that mesangial proliferative glomerulonephritis (MPGN) is affected by PI3K/Akt/GSK-3β, inhibiting mesangial cell inflammatory reactions [26]. Activation of the PI3K/Akt/GSK-3β signaling pathway is essential for improving neuroinflammatory responses [27]. Sulforaphene (SF) is one of the main isothiocyanates isolated from Raphani Semen, inhibited the activation of GSK-3β induced by LPS in BV-2 microglia cells and in the STZ-treated rats via increasing the protein expression of the ratio of p-GSK-3β(Ser9). To the best of our knowledge, it remains unclear whether SAL exerts an anti-inflammatory effect on rMC-1 cells by enhancing the PI3K/Akt/GSK-3β pathway.
In the present study, we investigated the anti-inflammatory effect of SAL on DR and explored its underlying mechanisms in vitro. SAL has potential as a treatment for DR, and its anti-inflammatory effect is associated with enhancing the PI3K/Akt/GSK-3β signaling pathway.
All experimental procedures were performed according to the National Institutes of Health guidelines and were approved by the Animal Use and Ethics Committee of Jinzhou Medical University. Adult male Sprague Dawley (SD) rats weighing 190–250 g were obtained from the Laboratory Animal Services Centre, Jinzhou Medical University (China, Liaoning). To induce diabetes, eight-week-old SD rats were given intraperitoneal injections of STZ (50 mg/kg, body, Sigma‒Aldrich). Blood glucose levels were measured 48 h after STZ injection, and rats with blood glucose levels above 16.7 mmol/l were considered diabetic and selected for research. The rats were randomly assigned to three groups: (1) the control group, (2) the DM group, with type 1 diabetes mellitus [28] (T1DM), and (3) the DM+SAL group, with type 1 diabetes mellitus and SAL treatment. SAL was dissolved in double-distilled water (ddHO) and administered by gavage at a dose of 3 g/kg daily for 12 consecutive weeks.
To analyze the number of ganglion cells in the retina, the eyes were enucleated, fixed in paraformaldehyde (PFA), and then sliced into 5-µm-thick sections. After being dewaxed in xylene and dehydrated with a graded series of alcohol, the slices were then transferred to hematoxylin for 1–2 min. Next, the sections were placed in an eosin solution for 5–6 min. Finally, images of the slices were taken using a simple microscope. The results were quantified using ImageJ.
The retinas were removed and fixed with 4% PFA, embedded in paraffin, and cut into 5-µm sections. After xylene dewaxing, the samples were dehydrated with a graded series of alcohol. Next, the antigen was repaired with an epifluorescence microscope (Olympus, Tokyo, Japan), and the sections were blocked in 10% goat serum at room temperature for 30 min. The cells were incubated with anti-p-NF-𝜅B p65 and anti-GS antibodies (mouse, 1:200) at 4 °C overnight. The next day, the specimens were treated with secondary antibodies (1:500, Abcam, anti-mouse, 488-conjugated, fluor647-conjugated anti-rabbit) at room temperature for two hours after being washed with PBS for five min and stained with 4',6-diamidino-2-phenylindole (DAPI; Abcam). Finally, the labeled cells and sections were visualized and processed using an epifluorescence microscope (Olympus, Tokyo, Japan).
Cell viability was determined using the Cell Counting Kit-8 assay (Saint-Bion, China) according to the manufacturer’s instructions. In brief, cells were cultured in 96-well plates (104 cells/well) in Dulbecco's modified eagle medium (DMEM) and treated with different concentrations of SAL. Subsequently, the cells were analyzed using an automatic microplate reader at 490 nm.
Cells were purchased from Cell Biolabs (Shanghai, China) and were cultured and passaged in DMEM (HyClone, Logan, UT) containing 10% fetal bovine serum (FBS, Sijiqing, China) with 1% penicillin‒streptomycin at 37 °C and 5% CO2. When the cells reached 70% confluence, they were incubated with high glucose (HG; 50 mM) for 48 h, and some cells were incubated with different concentrations of SAL (50, 100, 200, 400, 500, and 1000 µM) for 24 or 48 h. In addition, cells were pretreated with LY294002 (PI3K blocker) for one h and then incubated with HG and SAL for 48 h. The medium was changed every 24 h to maintain a constant glucose level.
An immunofluorescence analysis was conducted to examine the localization of NF-𝛋B and the expression of cytokines. In brief, rMC-1 cells on coverslips were fixed with 4% paraformaldehyde (PFA) for 30 min. The cells were blocked with a solution containing bovine serum albumin (BSA) and 0.1% Triton X-100 lysis buffer in PBS, which was followed by incubation at 4 °C with primary antibodies overnight. The primary antibodies were as follows: GS (mouse, Abcam 1:200), NF-kB p-65 (1:1000, CST, Cat# 3033), p-Akt (1:200, Affinity Biosciences, Cat# AF0016), p-GSK-3β (1:500, CST, Cat# 5558), IL-1β (1:200, Bioss, Cat# bs-6319R, China), IL-6 (1:200, Bioss, Cat# bs-6309R, China), and TNF-α (1:200, Immunoway, Cat# YT4689, China). The next day, the cells were treated with secondary antibodies (1:500, Abcam, anti-mouse, 488-conjugated, fluor647-conjugated anti-rabbit) at room temperature for two h after being washed with PBS three times for five min and stained with DAPI (Abcam). The slides were visualized and photographed under a fluorescence microscope (Olympus).
Total RNA was extracted using an RNA simple total RNA kit (Tiangen Biotech Co. Ltd, Beijing, China). cDNA was synthesized from 2 𝜇g of total RNA using a PrimeScript RT reagent kit (Invitrogen; Thermo Fisher Scientific, Inc.). PCR was performed using SYBR Premix Ex Taq and specific primers for the target genes, and the results are presented as 2-∆∆ctTable 1.
The cells were collected, washed, and lysed in radioimmunoprecipitation assay (RIPA) buffer. A bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China) was used to measure the protein concentrations of the lysates. Denaturing sample buffer was added to 40–60 𝜇g of protein and loaded onto precast 10% to 12% sodium dodecyl sulfate-PAGE (SDS‒PAGE) gels for separation. The protein samples were transferred onto polyvinylidene fluoride (PVDF) membranes and then blocked for two h in 0.5% BSA. The PVDF membranes were incubated overnight at 4 °C with the following antibodies: GAPDH (1:2500, CUSABIO, Cat# CSB-MA000071M0m, China), Akt (1:2000, Affinity, Cat# AF0836), p-Akt (1:2000), GSK-3β (1:2000, CST, Cat# 12,456), p-GSK-3β (1:2000), IL-1β (1:500), IL-6 (1:500), and TNF-α (1:500). The next day, the membranes were washed with tris buffered saline (TBST) three times for five min and incubated with goat-rabbit/mouse IgG (1:5000, Proteintech, Wuhan, China) at room temperature for two h. An electrochemiluminescence (ECL) kit (Solarbio, Beijing, China) was used to observe the immunoreactive bands, and an image analyzer (Bio-Rad) was used for imaging. The relative protein levels were quantified by ImageJ. In addition, ImageJ was used to measure the area and gray value of each band and the gray ratio for each group.
Analysis was performed using GraphPad Prism (version 8.0, GraphPad Software Inc., San Diego, CA). The data are expressed as the mean ± structural equation modeling (SEM). A one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (multiple comparisons) and a t test (paired data) were used, and p-values less than 0.05 were considered statistically significant in all tests (Figure 1).
H&E-stained retinal sections showed a loss of ganglion cells in the diabetic retina compared to the control group. The SAL-treated retinas (3 g/kg bodyweight) showed markedly higher ganglion cell numbers.
Normally, NF-𝜅B p65 is mainly expressed in the cytoplasm. However, the DR group showed the strongest expression of NF-𝜅B in the outer nuclear layer (ONL) and outer plexiform layer (OPL) compared to the control group. Surprisingly, SAL-treated retinas showed significantly decreased expression of NF-𝜅B p65 in the ONL. As shown in Figure 2, these results indicate the nuclear translocation of NF-𝜅B p65, but SAL treatment reduced the intranuclear expression of NF-𝜅B p65.
The identities of the cultured rMC-1 cells were confirmed by immunocytochemistry using antibodies against the rMC-1 marker GS. Nuclei were stained with DAPI. Almost all cells were positive for GS (Figure 3).
To investigate the effect of SAL on the viability of rMC-1 cells, CCK-8 assays were performed. As shown in Figure 4A,B, the number of viable rMC-1 cells was not significantly different compared to the control group. This result indicates that SAL (0–1000 µM) had no cytotoxic effects on rMC-1 cells.
IL-1β and IL-6 are important inflammatory cytokine markers. As shown in Figure 5, there were significantly increased protein levels of IL-1β, IL-6, and TNF-α when rMC-1 cells were under HG conditions compared to the cells treated with different concentrations of SAL. In addition, in rMC-1 cells treated with different concentrations of SAL (100 µM, 200 µM, 500 µM), the expression of proinflammatory cytokines was significantly inhibited in a dose-dependent manner. Thus, we chose 500 µM as our experimental concentration. To determine cellular localization, each treatment (CON, HG, HG+SAL) was performed in triplicate, and 15 images were captured per well using a fluorescence microscope. Figure 6 shows that pretreatment with SAL significantly reduced the nuclear translocation of NF-kB p65 compared with that in the HG group.
To further demonstrate the role of the PI3K/Akt/GSK-3β signaling pathway in the anti-inflammatory effects of SAL, rMC-1 cells were preincubated with 500 µM SAL for six h and then treated with or without 50 mmol/L glucose for 48 h. The phosphorylation of Akt and GSK-3β was detected by western blotting and immunofluorescence analysis. SAL had a protective effect, potentially by activating the PI3K/Akt/GSK-3β signaling pathway. As shown in Figure 7 and Figure 8, the protein levels of p-Akt and P-GSK-3β were markedly decreased in rMC-1 cells that were treated with HG, whereas treatment with SAL caused a marked increase in p-Akt and p-GSK-3β levels compared with those in the HG groups. To further explore the effect of SAL on this pathway, rMC-1 cells were pretreated with LY294002 (20 µM). The levels of p-Akt and p-GSK-3β/GSK-3β were significantly decreased. These results demonstrate that treatment with SAL enhances the activation of p-Akt and p-GSK-3β.
HG can induce an inflammatory response in rMC-1 cells. To clarify the anti-inflammatory effects of SAL on rMC-1 cells, the mRNA expression levels of IL-6, IL-1β, and IL-10 were determined by q-PCR. As shown in Figure 9A-C, SAL significantly upregulated the level of IL-10 mRNA induced by HG and reduced the mRNA expression levels of IL-6 and IL-1β. However, LY294002 increased the mRNA expression of inflammatory factors and reduced the anti-inflammatory effect of SAL. These results indicate that SAL inhibits the HG-mediated inflammatory response in rMC-1 cells through the Akt/GSK-3β signaling pathway.
To explore SAL-mediated effects on PI3K/Akt/GSK-3β in rMC-1, 20 µM of LY204002 was incubated with rMC-1 cells. As shown in Figure 10 and Figure 11, the expression levels of the inflammation-associated proteins IL-1β, IL-6, and TNF-α were detected by western blotting and immunofluorescence analysis. Compared to the control group, HG markedly increased the protein expression of IL-1β, IL-6, and TNF-α. SAL treatment reduced the expression levels of inflammation-related proteins compared to the HG group. Treatment with LY294002 significantly increased the protein expression of IL-1β, IL-6, and TNF-α compared to the SAL-treated group and decreased the anti-inflammatory effect of SAL. These results show that suppression of the PI3K/Akt/GSK-3β signaling pathway attenuated the anti-inflammatory effect of SAL.
As shown in Figure 12, NF-𝜅B p65 was markedly translocated to the nucleus after HG treatment compared to the control group. However, rMC-1 cells treated with SAL exhibited decreased NF-𝜅B p65 translocation to the nucleus compared to cells treated with HG. However, LY294002 promoted NF-𝜅B p65 translocation to the nucleus. These results suggest that treatment with SAL can reduce NF-𝜅B p65 translocation to the nucleus induced by HG through the activation of PI3K/Akt/GSK/3β.
SAL is a common traditional Chinese medicine (TCM) that has been widely used to enhance tumor growth and protect the cardiovascular system [29]. The favorable effects of SAL on various chronic diseases have been described previously. Moreover, emerging evidence has indicated that SAL has great therapeutic potential for treating diabetic encephalopathy [12,30], and it has been shown to effectively inhibit oxidative stress through the Akt/GSK-3β/NrF2 pathway in an ischemia/reperfusion SD rat model [31]. Studies have also shown that SAL improves lower limb motor function and neuronal tissue damage in rats with spinal cord injuries by reducing the gene expression and production of IL-1β, IL-6, and TNF-α [32]. As previously described, Müller cells are the principal glial cell type in the retina [5] and are essential for maintaining normal retinal function.
To the best of our knowledge, no evidence has yet been presented to suggest that SAL has anti-inflammatory effects in vivo or in vitro through the PI3K/Akt/GSK-3β signaling pathway. Therefore, we studied the anti-inflammatory effects of SAL on rMC-1 cells under HG conditions using an STZ-induced diabetes rat model.
Previous studies have reported that DR promotes apoptosis in retinal ganglion cells and decreases retinal thickness and GS expression [33,34]. In the present study, we observed the protective effects of SAL against DR and explored the underlying mechanism. We used in vitro and in vivo systems to determine the following:
1. In vivo, we first verified the anti-inflammatory and protective effects of salidroside against STZ-induced DR. Compared with the control group, the DR group of rats exhibited significantly increased nuclear translocation of NF-𝜅B p65 and the loss of ganglion cells. However, these effects were ameliorated by SAL treatment.
2. In vitro, we observed that salidroside suppressed the release of IL-1β, IL-6, and TNF-α and inhibited NF-𝜅B p65 nuclear translocation by enhancing the PI3K/Akt/GSK-3β signaling pathway induced by HG. These results indicate that PI3K/Akt/GSK-3β might be the anti-inflammatory signaling pathway mediated by SAL in DR. Our findings strongly suggest that SAL may possess protective anti-inflammatory potential in DR.
Inflammation plays an important role in the development of DR, and Müller cells are major factors in the secretion of inflammatory factors [2,35]. Generally, NF-𝜅B p65 is located within the cytoplasm and is kept inactive by interactions with its inhibitor. Once activated, phosphorylated NF-𝜅B dimers are released and translocate to the nucleus, which induces the production of cytokines [36-38]. A prior study reported that NF-𝜅B p65 nuclear localization occurs in Müller cells exposed to LPS [39]. In our study, we demonstrated that NF-𝜅B p65 significantly translocated to the nucleus in DR. Furthermore, we determined whether SAL induced the expression of IL-1β, IL-6, and TNF-α under HG conditions in rMC-1 cells. After these cytokines were detected by western blotting and immunofluorescence, the results showed that the expression of IL-1β, IL-6, and TNF-α was increased, but SAL treatment reduced the expression of cytokines and NF-𝜅B p65 translocation to the nucleus. These results indicate that Müller cell secretion of cytokines is affected by SAL-mediated anti-inflammatory effects under HG conditions.
The Akt/GSK-3β signaling pathway plays a pivotal role in neuroinflammation and neuroprotection [40]. The inactivation of GSK-3β, which is mainly regulated through the phosphorylation of Ser-9 (p-GSK-3β), participates in the inhibition of inflammatory reactions and pain [41,42]. Therefore, we examined the PI3K/Akt/GSK-3β signaling pathway to elucidate the mechanisms through which SAL modulates the inflammatory response and attenuates NF-𝜅B p65 translocation to the nucleus in rMC-1 cells. Our data indicated that SAL preconditioning significantly enhanced the phosphorylation of Akt and GSK-3β at Ser-9.
Although our findings demonstrate the importance of the anti-inflammatory effects of SAL on DR, there are some caveats and limitations that need to be addressed in future studies. We focused on the anti-inflammatory effect of SAL on rMC-1 cells. However, inflammation is regulated by multiple types of retinal cells in addition to rMC-1 cells, such as microglial and vascular endothelial cells [43,44]. Previous studies have reported that the injury responses of activated Müller cells and microglial cells in the retina are not independent but rather involve bidirectional feedback signals [45]. Therefore, in future experiments, the study of Müller–microglial coculture systems would be useful to further examine the anti-inflammatory role of SAL in DR. Taken together, these data demonstrate that SAL alleviates rMC-1 cell inflammation induced by HG through the activation of PI3K/Akt/GSK-3β signaling. Moreover, PI3K/Akt/GSK-3β phosphorylation was associated with NF-kB translocation to the nucleus and HG-induced rMC-1 cell secretion of cytokines.
In conclusion, our study demonstrates that SAL treatment attenuates HG-induced inflammation in rMC-1 cells by activating the PI3K/Akt/GSK-3β signaling pathway. The findings provide additional evidence supporting the clinical use of SAL for its anti-inflammatory effect and to protect retinal function in DR. However, further experiments are needed to elucidate the intrinsic mechanism of SAL.
Acknowledgments
Authors’ contributions: XZL and ZFZ designed the experiments. YY and CXS conducted the experiments and animal study. ZF analyzed the experiment data and prepared all figures. AQL, CLW, WQL, SXY and HDY provided technical support. ZF wrote the manuscript. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the, research, authorship, and publication of this article. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and publication of this article: This work was supported by the Foundation of Education Department of Liaoning Province (N0s. LJKMZ20221241). Dr. Xue-Zheng Liu (951564552@qq.com) and Dr. Zhong-Fu Zuo (164352674@qq.com) are co-corresponding authors for this paper.