Tea Polyphenols Reduce Inflammation of Orbital Fibroblasts in Graves’ Ophthalmopathy via the NF-κB/NLRP3 Pathway

This study aimed to explore the effects of tea polyphenols (TP) on inflammation of orbital fibroblasts in Graves’ ophthalmopathy (GO) and to provide new ideas for GO treatment. Primary orbital fibroblasts were extracted from orbital adipose/connective tissues of patients with and without GO. Real-time quantitative PCR (RT-qPCR) was used to detect the expression of interleukin (IL)-6, IL-1β, and monocyte chemotactic protein (MCP)-1 in non-GO and GO orbital fibroblasts. The CCK-8 assay was used to determine the appropriate concentration of TP for subsequent experiments. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the effects of TP on lipopolysaccharide (LPS)-induced production of inflammatory cytokines. Nuclear factor-κB (NF-κB) expression was measured using Western blotting analysis. NOD-like receptor 3 (NLRP3) expression was detected using both Western blotting analysis and immunofluorescence staining. The mRNA levels of IL-6, IL-1β, and MCP-1 in GO orbital fibroblasts were significantly higher than those in non-GO cells. TP treatment significantly inhibited LPS-induced production of inflammatory factors, including IL-6, IL-1β, and MCP-1. TP also inhibited the expression levels of NF-κB and NLRP3. Inflammation in the GO orbital fibroblasts was higher than that in non-GO cells. TP inhibited the production of inflammatory cytokines in GO orbital fibroblasts in vitro through the NF-κB/NLRP3 pathway. These findings suggest that TP may have a potential role in GO treatment.

Graves' ophthalmopathy (GO) is an autoimmune disease that has been recognized as the most common manifestation of Graves' disease outside the thyroid gland. The main clinical manifestations of GO include exophthalmos, exposure keratitis, eye movement disorders, and optic neuritis. GO severely affects visual function and appearance and may lead to blindness in severe cases. The pathological mechanism of GO has not been fully elucidated. Previous studies suggest that inflammation, adipogenesis, and glycosaminoglycan (GAG) accumulation are involved in the pathogenesis of GO [1] . Inflammation is a hallmark of GO. Histological analysis has shown that inflammation promotes collagen and GAG deposition and eventually aggravates the progression of GO [2] . Moreover, at the onset of GO, the orbital inflammatory environment induces the release of inflammatory factors, such as interleukin (IL)-6, from orbital fibroblasts, which further amplifies inflammatory responses [3] . Therefore, orbital inflammation plays a key role in the pathogenesis of GO [4] , and inhibiting inflammation may suppress the development of GO.
A number of cytokines are involved in the inflammation stage of GO pathogenesis. IL-6, IL-1β, and monocyte chemotactant protein (MCP)-1 expression are significantly upregulated in GO tissues compared with normal tissues [5,6] . IL-6 contributes to the pathogenesis of GO by stimulating the expression of thyroid stimulating hormone receptor (TSH-R) and increasing fat content during the differentiation of orbital adipose tissues [7,8] . It has also been reported that orbital volume is positively correlated with mRNA levels of IL-6 in orbital tissues [9] . Moreover, IL-6 has been shown to stimulate B cell differentiation and release functional autoantibodies [10] . IL-1β also induces the production of cytokines from orbital fibroblasts, thereby activating monocytes, T lymphocytes, and B lymphocytes [11] .
In recent years, many traditional Chinese medicine extracts, such as astragaloside Ⅳ, quercetin, curcumin, have shown therapeutic potential in the treatment of GO, owing to their anti-inflammatory properties [4,5,12] . Tea polyphenol (TP) refer to polyphenols extracted from tea leaves. They are the main functional substance of the tea plant and have various health effects, such as immunity enhancement, anticarcinogenic, antibacterial, and antioxidant effects [13,14] . TP also shows beneficial effects on cancers and cardiovascular diseases [15] . The antiinflammatory effect of TP has been widely reported in vitro and in animal models [16,17] . However, whether TP can exert an anti-inflammatory effect in GO remains unknown. In the present study, we investigated the effects of TP on inflammation of orbital fibroblasts isolated from GO patients.

Methods
Patients were recruited according to the Bartley diagnostic criteria and clinical activity scores (CAS) [18] , where ≥4 points indicated active GO lesions and <4 points indicated inactive GO. The orbital adipose/ connective tissue explants were obtained from GO patients (3 females, 3 males; average age: 55±6.3 years) who underwent decompression operation. Normal control tissues were harvested from patients with no history or clinical evidence of any thyroid disease (2 females, 2 males; average age: 34.8±11.9 years) during eye evisceration or upper lid blepharoplasty. This study was approved by the Institutional Review Board of The First Affiliated Hospital of Guangxi Medical University [approval number: 2019 (KY-E-092)] and adhered to the tenets of the Declaration of Helsinki. All patients provided written informed consent. All GO patients had achieved a stable euthyroid state for at least 3 months and their clinical activity scores at the time of surgery were <4. No GO patients received steroid or radiotherapy treatment for at least 3 months prior to surgery.
Primary cells were extracted from orbital adipose tissues and cultured in DMEM containing 20% fetal bovine serum and 2% penicillin/gentamycin in a 5% CO 2 /37°C incubator. After spreading, cells were maintained in DMEM supplemented with 10% fetal bovine serum and 2% penicillin/gentamycin. Cells at passage 3-8 were used for subsequent experiments. The extracted cells were identified as orbital fibroblasts using immunohistochemistry (IHC). The CCK-8 assay was performed to examine the effect of TP at different concentrations on the viability of orbital fibroblasts. Orbital fibroblasts were divided into three groups: GO, GO + LPS (10 μg/mL), and GO + LPS (10 μg/mL) + TP (100 μmol/L), and cultured in low-serum medium for 24 h.

IHC
An appropriate concentration of primary cells was seeded on a glass slide in a culture dish. The supernatant was discarded after cells adhered to the wall. The cells were washed three times with PBS and incubated with 4% cell fixation solution at 4°C overnight. The cell slide was removed and washed three times with PBS. Subsequently, cells were incubated in 3% H 2 O 2 deionized water for 20 min at room temperature and washed three times with PBS. Then cells were incubated with normal goat blocking serum at 37°C for 30 min, and then incubated with a primary antibody (1:400) at 4°C overnight. After washing three times with PBS, an enzyme-labeled secondary antibody diluted in working solution was added to the cells and incubated at 37°C for 30-60 min. After washing in PBS three times, a DAB color developer solution was added. After counterstaining with hematoxylin for 4-10 min, the dye solution was washed, and the slide was placed in water for 5 min. After dehydration and transparency, the slides were coverslipped and sealed with neutral gum.

CCK-8 Assay
The extracted primary orbital fibroblasts were used in the CCK-8 assay. Cells were resuspended in medium and plated into a 96-well plate (100 μL per well). PBS was added to the peripheral wells to avoid edge effects. The wells filled with culture medium were used as blank controls. Five duplicate wells were set for each concentration group. Cells were maintained in a 37°C/5% CO 2 incubator for 24 h until cells adhered to the wall. The culture medium was aspirated from each well and TP at various concentrations (0, 12.5, 25, 50, 100, 200, and 400 μmol/L in medium) was added to cells. After 24 h of incubation, the original culture medium was replaced with DMEM/F12 medium containing a 10% CCK-8 solution. Cells were maintained in a 37°C/5% CO 2 incubator in the dark for 2 h. A multifunctional microplate reader was used to detect the absorbance (A) at 450 nm. Cell viability was calculated using the following formula: Cell viability = (A value in experimental group − Reference A value)/ (A value in control group -Reference A value). The viability of orbital fibroblasts treated with different concentrations of TP was plotted.

RT-qPCR
The concentration and quality of RNA were determined using an ultraspectrophotometer (model: NANODROP 2000, Thermo Fisher Scientific, USA). The RNA reverse transcriptase kit (Takara; 37°C for 60 min, 85°C for 5 min) was used. The following program was then applied according to the instructions of the expansion kit (Takara, China): 10 s at 95°C, 5 s at 95°C, 20 s at 60°C, 60 s at 95°C, 30 s at 55°C, 30 s at 95°C for 40 cycles. The 2 -ΔΔCT method was used to determine mRNA levels. The primer sequences of IL-6, IL-1β, and MCP-1 are shown in table 1.

ELISA
The supernatants of orbital fibroblasts from each group were harvested and centrifuged at 1000 g for 10 min. The supernatant was collected for analysis. The ELISA kits were brought to room temperature (21±5°C) prior to use. The reagents were mixed by vortexing and the supernatant was diluted to an appropriate concentration. The expression levels of inflammatory factors IL-6, IL-1β, and MCP-1 were detected following the manufacturer's instructions.

Western Blotting Analysis
Western blotting analysis was performed to detect the expressions of NF-κB and NLRP3. After treatment, the culture medium was discarded, and cells were washed three times with PBS. About 160 μL of lysis buffer (RIPA) containing 1% PMSF was added to each dish. Cells at the bottom of the dish were scraped off, transferred to 1.5 mL tubes, and lysed on ice. Then cells were centrifuged at 12 000 g/min for 25 min at 4°C. The supernatant was obtained and transferred to a new tube. The protein concentration was determined using BCA assay. The protein samples were mixed with 5× protein loading buffer and denatured at 100°C. An equal amount of protein sample was loaded onto SDS polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes after electrophoresis. After blocking in 5% skimmed milk powder at room temperature for 1 h, the blots were washed three times with TBST (0.1% Tween 20), followed by overnight incubation with a primary antibody solution at 4°C. After three washes with TBST, the blots were incubated with a fluorescent secondary antibody for 1 h at room temperature in the dark. Image scanning and analysis were performed using the Odyssey Fc imaging system (LI-COR, USA).

Immunofluorescence Staining
NLRP3 expression was detected using immunofluorescence staining. Orbital fibroblasts were seeded on cell slides and treated with or without TP at different concentrations for 24 h. After three washes with PBS, cells were fixed in 4% paraformaldehyde for 30 min. The cells were thoroughly washed and penetrated with 3% Triton X-100. After 3 min, cells were washed and 50 μL of goat serum was added. The above procedures were performed at room temperature. After incubating in the blocking solution, 50 μL of primary antibody (1:100 dilution) was added and the slide was placed in a wet box at 4°C overnight. The slide was removed and washed three times with PBS. Then 50 μL of secondary antibody (1:200 dilution) was added and incubated for 1 h in the dark at room temperature. After three washes with PBS, cells were stained with DAPI for 5 min. After thorough washing, cells were observed under an inverted fluorescence phase-contrast microscope.

Statistical Analysis
mRNA levels and protein expressions of target genes were normalized to the internal control. Data are shown as mean ± standard deviation (SD) of at least three independent experiments. All data analyses were performed using SPSS 22.0. Data between two groups or among multiple groups were analyzed using either the Student's t-test or one-way analysis of variance. P<0.05 was considered significant.

Primary Cell Identification
As shown in fig. 1, cells isolated from orbital adipose/connective tissues showed positive vimentin staining and strong cytoplasm staining (brown),  indicating that cells were derived from mesenchymal cells. The staining for desmin, myoglobin, keratin, and S100B was negative. Cell nuclei were stained blue-purple and no brown staining in the cytoplasm was observed. Negative desmin staining indicated that cells were not derived from smooth muscle cells or cardiomyocytes. Negative myoglobin staining indicated that muscle and striated muscle were not the sources of cells. The absence of keratin expression indicated that no cells were derived from the skin. Negative S100B staining suggested that these cells were not derived from nerve cells, skin melanocytes, or other sources. Taken together, IHC confirmed that cells isolated from orbital tissues were fibroblasts.

Expression of Inflammatory Cytokines in GO and Non-GO Orbital Fibroblasts
RT-qPCR analysis showed that MCP-1 expression in GO orbital fibroblasts was approximately 18-fold higher than that in non-GO orbital fibroblasts (P<0.05; fig. 2A). The IL-6 levels in the GO orbital fibroblasts was 4.7-fold higher than those in non-GO orbital fibroblasts (P<0.05; fig. 2B). IL-1β levels in the GO orbital fibroblasts were also significantly higher than those in normal control cells (P<0.05; fig. 2C). These results indicate that the higher the concentration of TP, the lower the viability of orbital fibroblasts ( fig. 3). Therefore, TP at a concentration of 100 μmol/L was used for subsequent experiments.

TP Inhibits LPS induced Production of Inflammatory Cytokines in Orbital Fibroblasts
As shown in fig. 4, the GO+LPS group showed significantly higher mRNA levels of IL-6, IL-1β, and MCP-1 than the GO group (P<0.05), suggesting that LPS induced inflammation in GO orbital fibroblasts. After the addition of TP, the levels of inflammatory cytokines were significantly reduced (P<0.05), indicating that TP suppressed LPS-induced inflammation in GO orbital fibroblasts ( fig. 4A-4C). The results of ELISA were consistent with those of the qRT-PCR data ( fig. 4D-4F).

TP Inhibits LPS induced Production of Inflammatory Cytokines in Orbital Fibroblast by Suppressing the NF-κB/NLRP3 Pathway
Western blotting analysis demonstrated that LPS exposure significantly induced the expression of NF-κB in GO orbital fibroblasts compared with the

DISCUSSION
In this study, we measured the expression of inflammatory cytokines in GO orbital fibroblasts stimulated with LPS. Our results showed that TP inhibited LPS-triggered inflammatory responses in GO orbital fibroblasts by suppressing the NF-κB/NLRP3 pathway. GO is a common orbital disease. Orbital fibroblasts are both target and effector cells during the pathogenesis of GO. Therefore, in the current study, we investigated the effects of TP on orbital fibroblasts. The primary cells extracted from orbital tissues were identified as orbital fibroblasts using IHC staining. We further compared the mRNA levels of IL-6, IL-1β, and MCP-1 between GO and non-GO orbital fibroblasts. These inflammatory cytokines were highly expressed in GO orbital fibroblasts. LPS is a strong inducer of inflammation, and studies have shown that it activates NF-κB and mitogen-activated protein kinase signaling pathways through the transmembrane protein Toll-like receptor 4 (TLR4), inducing inflammatory responses in various cells such as macrophages [19] . In recent years, LPS has been used by researchers to induce inflammation in GO [20,21] . In this study, LPS stimulation significantly elevated the mRNA levels of IL-6, IL-1β, and MCP-1 in GO orbital fibroblasts, suggesting that LPS induces the production of inflammatory cytokines in orbital fibroblasts.
Polyphenols exhibit a wide range of biological activities, such as anti-inflammatory and anti-oxidant properties. They also exert an inhibitory effect on platelet aggregation and the proliferation and migration of vascular smooth muscle cells [22] . Our results showed that LPS-induced production of inflammatory factors, including IL-6, IL-1β, and MCP-1, in orbital fibroblasts was significantly reduced in the presence of TP. Thus, we concluded that TP suppressed LPSmediated inflammation in GO orbital fibroblasts. The anti-inflammatory effect of TP has also been reported in other diseases. Psoriasis is an autoimmunerelated inflammatory disease. Previous experiments have shown that EGCG, an important component of TP, inhibits imiquimod-induced psoriasis-like inflammation in BALB/c mice. Topical application of EGCG was shown to reduce psoriasis-like dermatitis, improves the pathological structure of the skin, reduces T-cell infiltration, and decreases plasma levels of IL-17A, IL-17F, IL-22, and IL-23 [23] . These data indicate that TP has an anti-inflammatory effect, which is consistent with our findings.
The TLR4, NF-κB, and NLRP3 pathways are involved in cell inflammation and apoptosis [24,25] . NF-κB plays a key role in regulating the transcription of pro-inflammatory genes. Under LPS induction, TLR4 can be specifically activated to induce NF-κB-mediated secretion of inflammatory cytokines, such as IL-6 and MCP-1 [26,27] . Activation of NF-κB upregulates the expression of the NLRP3 inflammasome and promotes expression of pro-inflammatory cytokines IL-1β and IL-18 [28,29] . In the present study, NF-κB and NLRP3 expression were determined using Western blotting analysis. We found that NF-κB and NLRP3 expression was related to levels of inflammatory factors, indicating that TP may inhibit LPS-mediated inflammation in GO orbital fibroblasts by suppressing the NF-κB/NLRP3 pathway. Wang et al showed that green TP plays an anti-inflammatory role in mice with liver injury by inhibiting NF-κB signaling and activation of NLRP3 inflammatory bodies, thereby preventing LPS-induced inflammation in the liver [30] . Previous studies and our present findings highlight the anti-inflammatory role of TP in human diseases.
In summary, LPS induced the expression of inflammatory cytokines in GO orbital fibroblasts in vitro. TP attenuated LPS-triggered inflammatory responses through the NF-κB/NLRP3 pathway. Further animal studies and clinical trials are needed to examine whether TP has a therapeutic effect on GO. Our findings provide new ideas for the development of adjuvant treatment methods for GO patients.

Open Access
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