Background
Progressive neuronal loss is responsible for symptom onset in retinal degeneration diseases, the most common of which is retinitis pigmentosa (RP), which is the leading cause of inherited retinal degeneration-associated blindness and affects approximately 1.5 million people worldwide [
1]. With the advent of next-generation sequencing and recent advances in gene therapy, an increasing number of gene mutations responsible for RP have been identified [
2]. Gene therapy for RP is limited by the heterogeneous genetic basis of this disease, highlighting the importance of developing therapies that act independently of mutation status [
3]. Drugs targeting the broad pathological processes that are common to different kinds of RP caused by various mutations may be beneficial for this complex disorder [
4].
Neuroinflammation is now widely recognized to participate in many chronic neurodegenerative diseases, including multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease, and attenuation of neuroinflammation is an effective therapeutic approach for these diseases [
5]. There is increasing evidence that microglia, the resident immune cells in the retina, induce immune responses and create a chronic inflammatory environment in the retinae of RP patients [
6]. The anterior vitreous cavities of RP patients were found to contain many inflammatory cells, and the levels of various pro-inflammatory chemokines and cytokines are upregulated in the aqueous humour and vitreous fluid in individuals with this disease [
7], suggesting a role for inflammation in the process of retinal degeneration. Research findings provide strong support for the concept that microglia-mediated neuroinflammation contributes to the overall apoptosis of photoreceptors in the retinae of rd10 mice [
8].
Ferulic acid (FA), a phenolic compound present in the plant wall, is a major active ingredient of some traditional Chinese medicines (TCMs), such as
Ferula asafetida,
Angelica, and
Ligusticum wallichii, which are TCM prescriptions used to improve microcirculation in ischaemic diseases [
9,
10]. Increasing research has demonstrated that FA suppress detrimental immunoreactions under various conditions. FA is easily obtainable and has good application prospects for the treatment of Alzheimer’s disease (AD) based on its potent immunomodulatory properties [
11,
12]. In an ovalbumin-induced model of respiratory allergies, FA administration was shown to dampen Th2-mediated immunity [
13]. These research results indicate that FA may be a novel immunosuppressive agent. As microglia-mediated neuroinflammation is believed to be one of the key processes responsible for neurodegeneration in RP and related diseases, immunosuppression targeting microglia is a promising strategy for the treatment of these diseases. We therefore speculate that FA may be capable of slowing or arresting the retinal degeneration process owing to its ability to suppress microglia-mediated neuroinflammation.
In our study, the effect of FA immunomodulation on the pathological process of retinal degeneration in the rd10 mouse model of RP was evaluated. The results showed that FA suppressed the transformation of microglia into a reactive phenotype and rescued retinal degeneration in rd10 mice. The underlying mechanism may have been FA-induced suppression of the expression of interferon regulatory factor 8 (IRF8), a key factor that promotes microglial activation, and thus a reduction in the production of inflammatory cytokines.
Methods
Rd10 mice and FA treatment
Rd10 mice (The Jackson Laboratory) were housed in a specific pathogen-free facility in the Animal Laboratories of Yantai Yuhuangding Hospital. All animal studies adhered to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. It has been reported that in rd10 mice, retinal abnormalities begin on postnatal day (P)7 [
14,
15] and that photoreceptor death peaks around P25 and is nearly complete by P35 [
16,
17]. For treatment, rd10 animals were intragastrically administered 25 mg/kg, 50 mg/kg or 100 mg/kg FA (1,270,311, Sigma) every day from P4 to P24. Before intragastric administration, the mice were anaesthetized with isoflurane in animal anaesthesia ventilator system (Matrx, America); the mice experienced no pain and awoke quickly. The animals were sacrificed via sodium pentobarbital injection (200 mg/kg; intraperitoneal) on P25, and their eyeballs were enucleated for further investigation.
Electroretinogram (ERG) recordings
Before ERG recordings, the mice were adapted to the dark overnight. The mice were anaesthetized by intraperitoneal pentobarbital sodium (50 mg/kg) prior to pupil dilation using 1% tropicamide. ERGs were recorded with a Ganzfeld stimulator (Roland Consult, Germany) that generated and controlled light stimuli. Scotopic ERGs were recorded following a 1.3-ms single flash with an intensity of-1.52, − 0.52, 0.48 or 1.0 log cd s/m2. A total of 5 responses per intensity were averaged for each flash stimulus. Intraperitoneal sodium pentobarbital (200 mg/kg) was then used to euthanize the mice. The amplitudes of the major ERG components (a- and b-waves) were measured (RETI System software) using automated and manual methods.
Hematoxylin and eosin (HE) staining
Eyeballs were fixed in formalin overnight prior to being embedded in and sectioned into 3-μm slices. Before staining, xylene was used for deparaffinization, and the sections were rehydrated in an ethanol gradient prior to being washed in PBS. Then, the sections were stained with HE. A microscope (Leica DM4000, Germany) was used to analyse retinal histology and count nuclei in the outer nuclear layer (ONL).
Cell culture and FA treatment
BV2 murine microglial cells (purchased from cell bank, Kunming Institute of Zoology) were cultured as previously described [
18]. In brief, the cells were maintained in DMEM (high-glucose) containing 10% FBS and penicillin/streptomycin. The microglia were activated by lipopolysaccharide (LPS, 50 ng/ml, L6529, Sigma) for 1 h and then treated with FA (0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/ml, 1 mg/ml, 2 mg/ml or 5 mg/ml, 6529, Sigma). After 24 h, the cells were collected for downstream analyses.
Immunofluorescence staining
For retinal wholemounts, eyes were immersed in 4% paraformaldehyde (PFA) fixative for 30 min, and the retinal cups were separated carefully from the eyeballs. Both retinal wholemounts and cell slides were stained using primary and secondary antibodies, washed extensively and flat-mounted. The following primary antibodies were used: anti-iba1 (019–19,471, Wako Chemicals) and anti-iNOS (sc-7271, Santa Cruz). The following secondary antibodies were used: Alexa Fluor 488-conjugated donkey anti-rabbit IgG H&L and Alexa Fluor 555-conjugated donkey anti-goat IgG H&L. The retinal wholemounts and cell slides were visualized via confocal microscopy (Carl Zeiss LSM710, Germany).
RNA sequence
RNA was isolated from retinae using TRIzol Reagent (Invitrogen), and a Bioanalyzer 2100(Agilent) was used to gauge the quality of the resultant nucleic acid. RNA preparation, library construction, and sequencing were conducted using a BGISEQ-500 instrument at the Beijing Genomics Institute (BGI, Shenzhen, China).
RT-PCR
Total RNA was isolated from the retina of rd10 using the RNAiso Plus kit (TAKARA Bio Inc., Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio Inc., Japan) was then used to prepare cDNA following the instructions. Rea-time PCR was performed in a 20-μL reaction system, containing 10 μL of 2 × SYBR Premix Ex Taq, 2 μL of cDNA, and 10 μmol/L primer pairs. The thermocycler settings were 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 34 s.
Western blot analysis
RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, MA, USA) was used to lyse homogenized retinal tissue. The samples were then centrifuged for 10 min at 10,000 rpm, after which a BCA assay was used for protein quantification. Equivalent amounts of protein were utilized for western blotting. The blots were incubated overnight in primary antibodies, including anti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8(sc-365,042, SANTA) and β-actin (ab28696, Abcam, Cambridge, MA). After being washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
Statistics
Each experiment, including the immunostaining, qPCR and western blotting experiments, was replicated 3 times. All quantitative data was analysed using 2-tailed Student’s t test or one-way ANOVA by SPSS 21.0. The data are the means ± standard errors of the mean (SEMs). P < 0.05 indicated significance.
Discussion
The progression of RP is largely driven by neuroinflammatory processes. Herein, we demonstrated the efficacy of FA, a Chinese herbal monomer with immunomodulatory potential. Specifically, we demonstrated that FA was able to suppress neuroinflammation and thereby slow the degenerative progression of RP in rd10 mice. Together, these findings highlight the potential value of FA or similar immunomodulatory treatments in slowing retinal degeneration in RP patients.
FA has been recognized as an important chemical with several biological activities, including direct and indirect anti-inflammatory, antioxidant, antiviral, antiallergic, antimicrobial, antithrombotic, anticarcinogenic, and hepatoprotective actions [
21]. Increasing attention has been paid to the ability of FA to suppress inflammation by regulating the immune response [
22‐
24]. FA can modulate immune activity in many cell types. For example, Cho et al. found that sustained FA treatment of mice can suppress Aβ-induced astrocyte activation, thereby preventing the production of associated inflammatory cytokines and free radicals that can drive AD-associated inflammation [
25]. FA treatment attenuates dextran sulfate sodium-induced colitis in model mice and induces Treg differentiation [
26]. FA can also suppress LPS-mediated IL-1β and IL-6 secretion from macrophages [
27]. In this study, the results indicate that FA is able to suppress microglial activation and reduce the expression of IL-1β, IL-6 and CCL2 in rd10 mice.
Microglia are the only immune cells that normally reside in the retina in significant numbers. In the retinae of humans with RP, rod apoptotic death is associated with the migration of these microglia from the inner to the outer retina. In the rd10 mouse model of RP, infiltration of activated microglia into the subretinal space can be detected by P16 even though apoptotic death of photoreceptors is only evident at P19, indicating that microglia themselves are capable of driving the apoptosis of these cells [
8]. Microglial activation contributes to inflammation through the secretion of inflammatory cytokines and chemokines that accelerate photoreceptor apoptosis, including TNF-α, IL-1β, IL-6, and CCL2 [
28]. Furthermore, these cells can also phagocytose both dead apoptotic and live stressed photoreceptor cells, aggravating retinal degeneration [
29‐
31].
Activation-induced gene expression in microglia is tightly regulated by many transcription factors [
32]. IRF family proteins are thought to be essential regulators of immune cell activation and responsiveness [
33]. IRF8 is almost exclusively expressed in myeloid and lymphoid cells of the immune system, with retinal IRF8 is found only in microglia [
34], which share features with cells of the myeloid lineage. Changes in retinal IRF8 levels are therefore thought to contribute to changes in microglial activity and/or infiltration. IRF8 is known to regulate the expression of IFN-β, IL-12, iNOS, and related genes [
35,
36]. In this study, high IRF8 levels were observed in the retina of rd10 mice. In vitro, we found that LPS insult induced a marked elevation of IRF8 in BV2 cells as well as an increase in the number of iNOS
+ microglia, suggesting a key role for IRF8 in microglial activation. Strikingly, FA reduced IRF8 and p-STAT1 levels, suggesting that FA suppresses microglial activation partly by regulating IRF8 expression. Additional research on the mechanistic basis of microglial activation and the regulatory role of FA in this process has the potential to offer further insight into the process and prevention of neuroinflammation and to further elucidate the therapeutic utility of FA as an agent for the treatment of neurodegenerative diseases.
Conclusion
In conclusion, our findings herein suggest that treatment with FA is sufficient to suppress microglial activation in a murine model of RP and thereby markedly attenuates associated neurodegeneration and disease progression. At the mechanistic level, the effect of FA seems to be at least partially linked to its ability to suppress STAT1 activation and IRF8 expression. These results thus highlight the immunomodulatory and anti-inflammatory properties of FA, suggesting that this compound or its derivatives may have value for the treatment of retinal degeneration.
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