Background: Age-related macular degeneration (AMD), a major eye degenerative disease, ultimately causes irreversible vision loss. Baicalin was identified to attenuate laser-induced chorodial neovascularization, indicating a therapeutic role in AMD. However, the exact mechanisms for baicalin in AMD remain unknown. Methods: MTT assay was performed to access the suitable concentration of baicalin or Aβ for treating ARPE-19 cells. CCK-8, morphology, and flow cytometry analysis were performed to evaluate cell viability and pyroptosis of baicalin in Aβ-envoked ARPE-19 cells. Quantitative real-time polymerase chain reaction and western blot analysis were subjected to measure the correlation between miR-223 and NLRP3. Luciferase reporter assay was performed to determine their direct relationship. Western blot analysis was subjected to determine pyroptosis-related proteins. Results: Baicalin inhibited Aβ-envoked pyroptosis in ARPE-19 cells. Mechanistically, baicalin significantly induced upregulation of miR-223 and downregulation of NLRP3, thus suppressing pyroptosis triggered by NLRP3 inﬂammasome signaling, yet such beneficial effects were reversed by miR-223 knockdown. Additionally, MCC950, a NLRP3 inhibitor, restored anti-pyroptosis activity of baicalin under miR-223 silencing. Conclusion: Baicalin alleviates intracellular pyroptosis and viability damage resulted from Aβ inducement in human retinal pigment epithelium cells via negative crosstalk of miR-223/NLRP3 inﬂammasome signaling, indicating that baicalin may be considered as a potential candidate for AMD therapy.
Age-related macular degeneration (AMD) is a major eye degenerative disease in aged individuals in the developed countries . Increased drusen deposition, retinal pigment epithelium (RPE), cell death, and secondary photoreceptor degeneration have been the main hallmarks of AMD . As it progresses, AMD ultimately causes irreversible vision loss. Clinically, AMD is divided into 2 main phenotypes, geographic atrophy and neovascular/exudative AMD. Unfortunately, there is no cure to both AMD forms, AMD has been regarded as the global eye health problem causing substantial economic and social burden . Thus, developing a potential candidate agent for treating AMD is urgent. Although AMD pathogenesis is complex and obscure, chronic oxidative stress and inﬂammation play key role in the progress of AMD [4-6]. Persistent inﬂammation activation has been reported to accelerate the formation of drusen. Various inﬂammatory molecules are also proposed as crucial biomarkers of AMD . Inﬂammation has been closely linked with the development of AMD. Therefore, pharmacological targeting inﬂammation inhibition might be an attractive strategy for AMD treatment.
Inﬂammasomes are a class of cytoplasmic protein complexes sensing endogenous or exogenous pathogen-associated molecular patterns or danger-associated molecular patterns that induce autoactivation of caspase-1 and the subsequently cleavage of proinﬂammatory cytokines, including pro-interleukin (IL)-1β and pro-IL-18 into their mature and secreted forms, which stimulate intracellular inflammation response . NOD-like receptor family pyrin domain-containing 3 (NLRP3), a widely studied inﬂammasome, was shown to be involved in various immune and inﬂammatory diseases including AMD . Previous studies have indicated that activated NLRP3 aggravates RPE death and potential visual loss. Meanwhile, the NLRP3 activation in the immune cells accumulating in the retinal area also contributes to the pathogenesis of AMD [9, 10]. Reversely, blocking the -NLRP3-mediated inﬂammasome signaling in the RPE slows down the AMD progress . It indicates that targeting NLRP3 is an important pharmacological mechanism for AMD therapy.
Pyroptosis is a form of programmed cell death triggered by perturbations of extracellular or intracellular homeostasis related to innate immunity . Due to leading to ruptures in the plasma membrane and subsequent releases of abundant inﬂammatory factors (e.g., IL-1β and IL-18) in a Caspase1-dependent manner, pyroptosis is identified as a novel proinflammatory regulated cell death . In general, pyroptosis acts as a defence mechanism protecting cells from infection by inducing pathological inflammation . However, persistent inflammation will result in multiple diseases take place, such as metabolic disorder, autoinflammatory diseases, AMD, and so on . Previously, it is reported that pyroptosis could increase RPE cells susceptibility to photooxidative damage-mediated cell death and contribute to RPE degeneration . Moreover, activation of pyroptosis mediated via NLRP3/Caspase1 signaling is a main mechanism of cellular death in AMD [8, 10, 14]. It suggests that inhibiting pyroptosis is an important insight of AMD intervention.
Scutellaria baicalensis Georgi, a common plant, is distributed in China, Korea, Mongolia, and Russia. In China, Scutellaria baicalensis Georgi has been regarded as the most widely used traditional Chinese medicine for treating pulmonary diseases, jaundice, hypertension, and so on . Baicalin, a main effective flavonoid compound, isolated from the raw root of Scutellaria baicalensis Georgi , similarly possesses various biological properties, including anti-inflammatory, antimicrobial, and antioxidant activities and targeting NLRP3 inflammasome and nuclear factor-kappa B (NF-κB) signaling is proposed as one of the main regulatory mechanisms [17-20]. Interestingly, it is demonstrated that baicalin also exerts active therapeutic in ocular diseases, such as glaucoma, retinopathy and AMD via antiangiogenesis, anti-apoptosis, anti-inflammatory, and antireactive oxygen species [16, 21-23]. However, the underlying molecular mechanism of baicalin in AMD is mainly unknown.
Here, we found that baicalin could alleviate Aβ-induced pyroptosis via increasing miR-223 expression while downregulating NLRP3 expression. Moreover, miR-223 overexpression suppressed NLRP3-triggered pyroptosis by targeting its 3′-untranslated region (3′-UTR). Therefore, we demonstrated that baicalin increased the expression of miR-223 and promoted miR-223/NLRP3/Caspase-1 feedback signaling, which mediated its anti-inflammation and anti-pyroptosis activity in Aβ-induced ARPE-19 cells. Our data clarified the protective role of baicalin in AMD in vitro and shed the possibility on baicalin treatment improving AMD in vivo.
Materials and Methods
Cell Culture and Treatment
The human RPE cell line ARPE-19 was obtained from -American Type Culture Collection (Manassas, VA, USA). Cells were seeded at a density of 20,000 cells/cm2 and cultured in high glucose Dulbecco’s Modified Eagle Medium containing with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies) at 37°C (95% air, 5% CO2). Cells were subjected to Aβ treatment, and then MTT, CCK-8, fluorescence activated cell sorter (FACS), gene expression, or reporter analysis were performed for further exploration in the absence or presence of baicalin (Yuanye Biotech, Shanghai, China) or MCC950 (Sigma-Aldrich, St. Louis, MO, USA) treatment (baicalin and MCC950 were dissolved in dimethylsulfoxide, Sigma-Aldrich) at indicated concentrates and time points.
ARPE-19 cells were seeded at a density of 20,000 cells/cm2 in 96-well plates and incubated with Aβ1–42 (Sigma-Aldrich) or baicalin at indicated concentrates. Then, MTT (10 µL of 5 mg/mL, Sigma-Aldrich) was added into cell. After another 3-h incubation, dimethylsulfoxide (200 µL) was added, and absorbance was measured on a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm and background absorbance measured at 630 nm. Background absorbance was subtracted from signal absorbance to obtain normalized absorbance values. The colorimetric signal obtained was proportional to the cell number.
ARPE-19 cells were seeded at a density of 20,000 cells/cm2 in 96-well plates and incubated with Aβ1–42 (10 µM) or baicalin (50 µg/mL) for 24, 48, and 72 h. Subsequently, cell was determined colorimetrically at 450 nm using the CCK-8 Kit (Dojindo, Tokyo, Japan).
Flow Cytometry and Pyroptosis Evaluation
A cell population with positive propidium iodide, PI (Abcam, Cambridge, UK) and caspase1 (Abcam) activities were enriched and detected by flow cytometry. The flow cytometry data were assessed using BD FACSDiva Software version 7.0 (Becton-Dickinson, USA).
For cell transfection, different expression plasmids (anti-miR, anti-miR-223, miR-NC, miR-223, wild or mutant 3′-UTR reporter of NLRP3) were transiently transfected into ARPE-19 cells or HEK293T cells (American Type Culture Collection) with FuGENE-HD (Roche) according to the ratio: 1 μg plasmid/5 μL FuGENE. After 48-h transfection, cells were used to following experiments. The anti-miR, anti-miR-223, miR-NC, and miR-223 were purchased from RiboBio Co., Ltd. (Guangzhou, China).
Total RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction
Total RNA from ARPE-19 cells was isolated using the TRIzol method (Invitrogen, Carlsbad, CA, USA). Then the RNA was reverse-transcribed to cDNA using reverse transcrptase (Takara -Dalian, China). First-strand cDNA was synthesized with a cDNA synthesis kit (Fermentas, Madison, WI, USA). Quantitative real-time polymerase chain reaction (PCR) was carried out using SYBR Green PCR Kit (ABI, USA). Individual transcripts in each sample were repeated at least 3 times and values were normalized to -GAPDH (glyceraldehyde 3-phosphate dehydrogenase).
Total Protein Extraction and Western Blot
The whole-tissue protein from ARPE-19 cells was extracted using RIPA buffer and separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies of NLRP3, Caspase 1, apoptosis-associated speck-like protein (ACS; 1:1,000; Cell Signaling Technology, USA), and GAPDH were used by 1:5,000 dilution in 5% bovine albumin at 4°C overnight followed by HRP-linked goat antirabbit antibody at room temperature for 2 h. Immunoreactive proteins were visualized using LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), according to the manufacturer’s instructions. The relative protein levels were normalized to GAPDH. All of the primary antibodies were purchased from Cell Signaling Technology.
Dual-Luciferase Reporter Assay
The predicted miR-223 binding site of the NLRP3 3′-UTR sequence or mutant sequence was cloned into the pMIR-REPORT vector (Ambion®; Thermo Fisher Scientific, Inc.) to construct the NLRP3-3′-UTR-WT plasmid or the NLRP3-3′-UTR-Mut plasmid. For NLRP3 3′-UTR reporter assay, the expression plasmids for miR-NC, miR-223, wild or mutant 3′-UTR reporter of NLRP3, and pREP7 (Renilla luciferase) were cotransfected into HEK293T cells with FuGENE-HD reporter, and Renilla was used to normalize transfection efficiencies. After 48 h, cells were collected for luciferase activity determination by using the dual luciferase reporter assay system (Promega Corp., Madison, WI, USA).
All values were expressed as means ± standard deviation (SD) and analyzed using the statistical package for the social science (SPSS, version 18.0). Paired or unpaired two-tailed t-tests were used to detect a difference in the mean values of treatment group and control and analysis of variance for the difference among more than 2 groups. Differences with p values <0.05 were considered to be statistically significant and denoted as (*).
Baicalin Alleviates Aβ-Induced Pyroptosis in RPE Cell
Aβ, a major constituent of drusen, has been proved to induce the AMD phenotype . Therefore, Aβ-induced ARPE-19 (human RPE cell line) was chosen as a model to explore the pharmacology effects of baicalin on regulating AMD progress. First, we optimized the concentration of Aβ in ARPE-19 cells for further experiments. According to MTT assay, Aβ (10 μmol/L) might be the most suitable dose, which could successfully cause approximately 50% cells viability loss in ARPE-19 cells (Fig. 1a). Additionally, the result of CCK-8 assay showed that cell viability was significantly suppressed by Aβ treatment (Fig. 1b). Morphologically, after stimulating Aβ (10 μmol/L), more number of dead cells were observed in ARPE-19 cells as compared with untreated control group (Fig. 1c). Recently, the pyroptosis triggered by Aβ could cause RPE dysfunction in the pathogenesis of AMD . Similarly, FACS analysis revealed that Aβ significantly increased positive cell proportion with PI/Caspase1 dual staining in ARPE-19 cells (Fig. 1d). Interestingly, such dysfunction in ARPE-19 cells induced by Aβ was reversed by 50 μg/mL baicalin (the suitable dose was determined by MTT assay in Fig. 1e) treatment. As shown in Figure 1f–h, baicalin protected cell damage and pyroptosis from Aβ insult. Thus, these data indicated that baicalin could alleviate Aβ-induced pyroptosis in ARPE-19 cells in vitro.
Baicalin Inhibits Pyroptosis via Upregulating miR-223 Expression in ARPE-19 Cells
miRNA-223, a myeloid-enriched microRNA, has been regarded as a negative regulator of inflammation in various conditions involving in many inﬂammatory disorders, infections, and cancers [26-28]. Meanwhile, previous studies have reported that NLRP3 transcription and activity are negatively regulated by miR-223 in numerous cells line, which contributes to anti-pyroptosis . However, whether miRNA-223/NLRP3 interplay mediates baicalin attenuating pyroptosis remains unclear. Excitingly, in Aβ-challenged ARPE-19 cells, baicalin markedly increased miRNA-223 expression, while it reduced pyroptosis markers (NLRP3, Caspase1 and ACS) and proinflammation cytokines (IL-1β and IL-18) expression (Fig. 2a, b). To further clarify the correlation between miRNA-223 expression and pyroptosis in ARPE-19 cell, the transfection of miR-223 inhibitor (anti-miR-223) was applied. Results showed that miRNA-223 silencing largely decreased miRNA-223 expression (Fig. 2c), but induced NLRP3, -IL-1β, and IL-18 mRNA levels in condition of Aβ incubation (Fig. 2d). FACS and western blot analysis also indicated that downregulation of miRNA-223 promoted NLRP3, Caspase1, and ACS pyroptosis-related protein levels and block cell pyroptosis in Aβ-insulted ARPE-19 cells (Fig. 2e, f). Our data suggested baicalin might exert anti-pyroptosis activity via upregulating miR-223.
Identifying NLRP3 as a miR-223 Target Gene
It is predicted that miR-223 could interact with the 3′-UTR of NLRP3 according to mRNATargetScanHuman (http://www.targetscan.org; Fig. 3a). To further confirm the direct crosstalk between the 2 molecules, a dual-luciferase reporter assay was performed. As shown in Figure 3b, cotransfection with miR-223 markedly reduced the luciferase activity of the plasmid expressing the wild type of the respective fragment of NLRP3 3′-UTR. However, the luciferase activity of mutant NLRP3 3′-UTR reporter was not affected by cotransfection with miR-223 mimics or negative control. Gene expression analysis similarly presented that miR-223 significantly decreased the expression of NLRP3, while miR-223 silencing showed the opposite result (Fig. 3c, d). These results indicated that miR-223 could directly bind to the 3′-UTR of NLRP3 and negatively regulate NLRP3 expression.
Baicalin Alleviates Aβ-Induced Pyroptosis through Regulating miR-223/NLRP3 Axis
To further understand whether miR-223/NLRP3 regulatory network plays an essential role in baicalin-mediated pyroptosis in RPE cell, flow cytometry, quantitative real-time-PCR, and western blot analysis were subjected to evaluate the anti-pyroptosis and anti-inflammation activity of baicalin in the presence or absence of anti-miR-223 in Aβ-challenged ARPE-19 cells. As illustrated, baicalin protected the cells from Aβ-induced pyroptosis (Fig. 4a) and upegulated miR-223 expression (Fig. 4b). Meanwhile, baicalin reduced pyroptosis markers (NLRP3, Caspase1, and ACS) and proinflammation cytokines (IL-1β and IL-18) expression (Fig. 4b, c). Expectedly, such beneficial effects were reversed with miR-223 knockdown by anti-miR-223 transfection (Fig. 4a–c), indicating that miR-223 might be a crucial participant in baicalin-mediated pyroptosis attenuation. Finally, to clarify whether NLRP3 is a key downstream mediator of miR-223, MCC950, a NLRP3 antagonist, was coincubated with Aβ, anti-miR-223, and baicalin in ARPE-19 cells. As presented in FACS analysis (Fig. 4d), MCC950 treatment obviously reduced pyroptosis caused by miR-223 knockdown in condition that Aβ and baicalin coincubation. Consistently, in the same condition, MCC950 exactly reduced miR-223-mediated pyroptosis markers (NLRP3, Caspase1, and ACS) expression in protein levels (Fig. 4e). Thus, these findings identified that miR-223/NLRP3 axis is involved in baicalin-mediated pyroptosis suppression in Aβ-induced ARPE-19 cells.
Previous researches have demonstrated that baicalin plays a therapeutic role in metabolic diseases, cancer, fibrosis diseases, especially in inflammatory and degenerative diseases, via its antioxidant, anti-inﬂammatory, antiapoptotic, and antitoxicity properties [19, 20, 30, 31]. Meanwhile, many flavonoids have been reported to reduce the risk of ocular diseases such as cataract, glaucoma, and AMD . However, the protective effects and pharmacological mechanisms of baicalin in AMD are still limited. Our current study provides the evidence that baicalin exerts protective roles in improving AMD via its anti-pyroptosis and anti-inflammatory functions mediated by modulating miR-223/NLRP3 axis in Aβ-induced ARPE-19 cells. It suggested baicalin might be considered for the treatment of AMD in vivo.
Although the pathogenesis in AMD development is not fully understood, pyroptosis, a highly inflammatory form of programmed cell death in Caspase-1-dependent, is still regarded as a main risk factor in the pathogenesis of AMD. Aβ, the main component of drusen in AMD patients, can trigger inﬂammation and pyroptosis in RPE cells via Caspase-1 activation [12, 14]. To evaluate the activity of baicalin in anti-AMD, we subjected ARPE-19 cells to baicalin treatment with Aβ stimulation. As observed in our studies, baicalin treatment inhibited Aβ-induced damage and caspase-1 activation in ARPE-19 cell. Such results suggested us baicalin could alleviate AMD development via blocking pyroptosis in ARPE-19 cell.
NLRP3 inﬂammasome is indicated be involved in promoting inflammation and pyroptosis in AMD progress [9, 14]. Increased mRNA level of NLRP3 in AMD lesions of the RPE cells has been observed . Oxidative stress could induce pyroptosis in AMD via the NLRP3 inﬂammasome activation . Inversely, NLRP3 inﬂammasome blockade inhibits VEGF-A-induced AMD, and small molecule NLRP3 inhibitors have been shown to be effective on treating AMD in vivo . Due to the important role of NLRP3 inﬂammasome pathway in the pyroptosis and pathogenesis of AMD, targeting NLRP3 inﬂammasome signaling is regarded as a novel and promising therapy for AMD treatment. Previously, the anti-inﬂammatory property of baicalin via NF-κB signaling inhibition has been observed . Additionally, it is reported that baicalin suppress LPS-stimulated NLRP3 inammasome signaling . Thus, we speculated baicalin would reduce pyroptosis via hindering NLRP3-mediated inflammatory response in RPE cell. Expectedly, in gene expression analysis, we found baicalin markedly decreased NLRP3 inflammasome signaling genes, NLRP3, Caspase1, and ACS expression levels in ARPE-19 cell, it reflected baicalin inhibited NLRP3 inammasome signaling. IL-1β and IL-18 are major inﬂammatory effector cytokines of inﬂammasome activation [25, 33]. These chronic proinﬂammatory cytokines would induce persistent stress to RPE cells and result in pyroptosis in RPE cells . It is established NLRP3 silencing in human RPE greatly decrease the expression of inﬂammatory IL-1β and IL-18 and subsequent pyroptosis in RPE cells . Similarly, baicalin indeed decreased the IL-1β and IL-18 expression. Our results proved that baicalin blocked NLRP3 inflammasome signaling and thereby protected the cells from Aβ-evoked pyroptosis and inflammation in RPE cells.
miRNAs are small noncoding RNA molecules that typically suppress the translation and the stability of transcripts through binding to complementary sequences in the 3′-UTR of their target mRNAs. miRNAs are implicated in playing essential roles in almost all biological functions [34, 35]. miR-223, a myeloid-enriched microRNA, has been connected with a large number of inﬂammatory conditions [26, 36]. miR-223 has been shown to directly suppress the canonical NF-kB Pathway in basal keratinocytes . miR-223 loss results in continuous neutrophil recruitment and the excessive neutrophilic inﬂammation . miR-223 responds to stimuli to control the production of IL-6 and IL-1β in immune cells . Moreover, NLRP3 inﬂammasome activation is found to be largely miR-223 dependent . Bioinformatics analysis and previous studies have highly suggested the negative regulation of NLRP3 expression by miR-223 that exists in many cell lines. However, a directly regulatory between miR-223/NLRP3 cross talk and their role in inﬂammasome activation in AMD are not well established in RPE cells. To do so, we firstly used NLRP3 3′-UTR reporter and NLRP3 gene expression analysis to confirm the causal link between the 2 molecules. Consistently, miR-223 decreased the NLRP3 3′-UTR reporter activity and inhibited NLRP3 expression, while such effects were abolished by miR-223 knockdown. Next, we analyzed the miR-223 functional role in regulating NLRP3 inﬂammasome signaling and pyroptosis in Aβ-evoked ARPE-19 cells. Interestingly, miR-223 knockdown promoted NLRP3 inﬂammasome-related pyroptosis. Our data provided us that miR-223 exerted a negative regulation for NLRP3 inﬂammasome and pyroptosis in RPE cells. Meanwhile, we found miR-223 expression levels were increased by baicalin treatment in ARPE-19 cells. It hinted us that miR-223 may be involved in pharmacological mechanisms of baicalin. To investigate it, miR-223 was repressed in ARPE-19 cells, we observed miR-223 knockdown block the beneficial effects of baicalin on protecting ARPE-19 cells from Aβ-induced activation of NLRP3 inﬂammasome and pyroptosis, but such adverse manifestations were rescued by NLRP3 inhibitor, MCC950 cotreatment. It provided us the proof that the miR-223/NLRP3 axis could mediate the protection of on Aβ-induced damage in RPE cells.
In summary, we explored the activities and mechanisms of anti-apoptosis and anti-inflammation of baicalin in Aβ-induced ARPE-19 cells and demonstrated that baicalin exerted protective effects on improving inflammation and pyroptosis through miR-223/NLRP3 axis. Although considering the poor bioavailability of baicalin, it still puts forward a possibility of improving AMD by using baicalin or baicalin-contained traditional medicines in clinic.
This work was supported by Zhejiang Provincial Science and Technology program of Traditional Chinese Medicine (No. 2019ZA072).
Statement of Ethics
No humans were involved in this research.
The authors declare that they have no conflicts of interest to disclose.
H.-J.S.: conceptualization; H.-J.S.: design; X.-M.J.: clinical study; J.X.: intellectual content; X.-M.J.: literature research; Q.X.: data analysis; Q.X.: statistical analysis; H.-J.S.: writing review and editing.