6-Shogaol Alleviates Endotoxin-Induced Uveitis via Activation of HIF-1α to Regulate Inflammatory Responses and Endoplasmic Reticulum Stress

Abstract

Purpose

6-Shogaol (6-S) has demonstrated anti-inflammatory effects in various disease models; however, its effects in uveitis have not been investigated. This research intends to investigate the therapeutic mechanisms of 6-S in acute uveitis.

Methods

An endotoxin-induced uveitis (EIU) mouse model was developed to assess the therapeutic potential of 6-S for uveitis. Disease severity was evaluated through anterior segment evaluation, clinical scoring, and pathology staining. RNA sequencing (RNA-seq) and network pharmacology explored the potential molecular mechanisms. In vitro, BV2 cells were stimulated by lipopolysaccharide. The mRNA and protein levels of pro-inflammatory cytokines, endoplasmic reticulum (ER) stress markers, and hypoxia-inducible factor 1-alpha (HIF-1α) in eyeballs and cells were detected. Transmission electron microscopy and Ca²⁺ levels were used to evaluate ER damage. The targeting effect of 6-S on HIF-1α was tested by HIF-1α siRNA.

Results

6-S alleviated EIU in mice. RNA-seq indicated that inflammatory pathways are potential mechanisms of 6-S treatment. Further results showed that 6-S inhibited the expression of pro-inflammatory cytokines in vivo and in vitro. Network pharmacology revealed that the effect of 6-S may related to the ER. 6-S downregulated GRP78, ATF4, and CHOP expression; suppressed PERK and IRE1α phosphorylation; and mitigated ER swelling and cellular Ca²⁺ overload. Moreover, 6-S upregulated the expression of HIF-1α in vivo and in vitro. Inhibiting HIF-1α largely eliminated the anti-inflammatory and relieving ER stress properties of 6-S.

Conclusions

6-S alleviates ER stress and inflammatory responses by modulating the expression of HIF-1α, thereby improving the prognosis of EIU.

Uveitis is a common intraocular inflammatory disease that impacts the uveal tract, retina, and other intraocular tissues. Epidemiological investigations have revealed that 10% to 25% of legal blindness in both developed and developing countries can be attributed to uveitis.^1,2 Acute uveitis (AU) induces substantial ocular discomfort and visual impairment. Severe or recurrent cases can result in structural complications—including cataracts, glaucoma, and cystoid macular edema—further worsening vision and complicating treatment. Microglia are pivotal immune cells in initiating and propagating AU. Upon activation by endogenous or exogenous stimuli, they release proinflammatory cytokines and chemokines, triggering inflammatory cascades and promoting leukocyte infiltration. This process exacerbates intraocular inflammation and disrupts the blood–ocular barrier.³ Reports show that the levels of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) in serum and aqueous are notably higher in acute phases of uveitis and that there is a significant negative correlation with visual acuity.^4,5 The endotoxin-induced uveitis (EIU) model, utilizing lipopolysaccharide (LPS) to rapidly elicit ocular inflammatory responses in both anterior segment and retina, serves as a well-established system for studying acute uveitis pathogenesis and therapeutic screening.⁶

Hypoxia-inducible factor 1-alpha (HIF-1α) widely participates in multiple physiological processes including cell proliferation, oxidative stress, and immunomodulation.⁷ Studies have shown that the HIF-1α activator suppressed M1-like polarization of RAW264.7 cells and decreased the ratio of M1/M2 macrophages in periodontitis tissues of mice.⁸ Moreover, HIF-1α can curtail pro-inflammatory factor levels and mitigate tissue damage by regulating autophagy.⁹ Nevertheless, some studies have shown that HIF-1α has pro-inflammatory effects under specific conditions. The glycolysis/HIF-1α axis affected IL-4–stimulated conversion of M2 macrophages to proinflammatory-prone M2.¹⁰ These findings suggest that HIF-1α potentially harbors complex mechanisms and plays multiple roles in inflammation and immune regulation.

Endoplasmic reticulum stress (ERS) is a cellular process that occurs in response to conditions such as misfolded and unfolded protein aggregation and disrupted Ca²⁺ homeostasis.^11,12 ERS correlates with the progression of diabetic retinopathy and fundus degenerative diseases,^13,14 and it can contribute to inflammatory diseases by affecting the function of immune cells and activating inflammatory signaling cascades.¹⁵ However, its role in uveitis remains elusive.

6-Shogaol (6-S), one of the principal bioactive constituents in dried ginger rhizome, has been verified to possess anti-inflammatory, antioxidant, anticancer, and neuroprotective effects.^16–18 Several studies suggest that 6-S outperforms other ginger extracts in antioxidant and anti-inflammatory capabilities.^19,20 In a mouse model of arthritis, 6-S significantly reduced leukocyte infiltration into tissues, alleviated paw edema, and slowed arthritic symptom progression.²¹ In addition, 6-S exerted neuroprotective effects and attenuated ischemia/reperfusion-induced brain injury by regulating HIF-1α/hemoglobin oxygenase 1 (HO-1) expression.²² However, the molecular processes behind the involvement of 6-S in uveitis remain uncertain.

In this study, we established an EIU mouse model and a LPS-induced BV2 cell inflammation model. Results demonstrated that 6-S attenuated disease progression and intraocular inflammatory in EIU mice. Further studies suggest that 6-S may modulate ERS and inflammatory responses by targeting HIF-1α.

Materials and Methods

Establishment of EIU Model and 6-S Treatment

Female C57BL/6 mice (6–8 weeks of age) were purchased from Peng Yue (Jinan, China). They were acclimatized and fed for 1 week. LPS (Sigma-Aldrich, St. Louis, MO, USA) was made into a 1-mg/mL solution by being dissolved in phosphate-buffered saline (PBS). The control and EIU groups were administered 0.9% NaCl (0.5 mL) via intraperitoneal (IP) injection. The EIU+6-S group was treated with 6-S (30 mg/kg, 0.5 mL, IP; TargetMol, Boston, MA, USA), and the EIU+dexamethasone (DEX) group received DEX (1 mg/kg, 0.5 mL, IP). EIU was induced by subcutaneous injection of LPS solution (100 µL) into both thighs of mice 2 hours later, and PBS was injected into the control group. After 24 hours, slit-lamp observation of severity and clinical scoring according to a grading scale were conducted.²³ The mice were necropsied and their eyeballs were collected for subsequent experiments. This study was approved by the Research Ethics Committee of the Affiliated Hospital of Qingdao University and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Cell Culture and Stimulation

BV2 cells were sourced from Procell Life Science & Technology (Wuhan, China). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a biological incubator (5% CO_2, 37°C). The cells were pretreated with 6-S at different concentrations (3, 9, or 18 µM) or 9 µM for 2 hours; 0.1% dimethyl sulfoxide (DMSO) was used as a control. After that, cells were stimulated using LPS (1 µg/mL) or thapsigargin (Tg; 0.1 µg/mL) and cultured in a constant-temperature incubator for 24 hours.

RNA Sequencing

The uvea and retina from the control, EIU, and EIU+ 6-S groups of mice after 24 hours of LPS injection were used for RNA sequencing (RNA-seq). RNA-seq analysis was performed at Novogene (Beijing, China). Briefly, RNA-seq was carried out with the Illumina NovaSeq X system (Illumina, San Diego, CA, USA). Differential expression analysis was performed using the DESeq2 package in R 1.20.0 (R Foundation for Statistical Computing, Vienna, Austria). Corrected P ≤ 0.05 and |log₂(foldchange)| ≥ 1 were considered to be differentially expressed genes (DEGs). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs were performed using clusterProfiler 3.8.1. P < 0.05 and false discovery rate values < 0.25 were regarded as statistically significant gene sets.

Hematoxylin and Eosin Staining

The eyeballs were preserved in 4% paraformaldehyde for 2 days. They were embedded in paraffin and then sectioned, dewaxed, and hydrated as before.²⁴ After staining with a hematoxylin and eosin (H&E) solution, the sections were dehydration and sealed. Finally, a light microscope was used to view and take pictures of the sections.

Immunofluorescence Staining

Mice eyeballs were fixed with 4% paraformaldehyde. Next, the retinas were prepared into four-leaf clover shapes. Following permeabilization with 0.3% Triton X-100 and blocking with goat serum, the retinas were incubated with IBa-1 antibody (1:200) overnight and then incubated with Alexa Fluor 488 (green)-conjugated goat anti-rabbit IgG (1:100; Elabscience, Wuhan, China) for 1 hour. Then, 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, Beijing, China) to label the cell nucleus. Images were taken by fluorescence microscopy (Zeiss Axio Vert, Jena, Germany).

Cell Viability Assay

BV2 cells were treated with different concentrations of 6-S (9, 12, 15, 18, 22, or 30 µM) or 1‰ DMSO cell culture medium for 24 hours. Cell Counting Kit-8 (MedChemExpress, Shanghai, China) was added to each well and incubated for 1 hour, after which the absorbance at 450 nm was measured.

Real-Time Quantitative PCR

Using RNeasy Isolation Reagent (Vazyme, Nanjing, China), total RNA was extracted and then reverse transcribed to cDNA. The cDNA solution was mixed with SYBR Green quantitative polymerase chain reaction (qPCR) premix (Biosharp, Hefei, China). The PCR amplification and real-time fluorescence detection were then performed. Table 1 illustrates the real-time qPCR primer sequences.

Table 1.

Primers Sequences for Real-Time qPCR

	Nucleotide Sequence	Primer
β-actin	5′-ACGGCCAGGTCATCACTATTG-3′	F
	5′-AGAGGTCTTTACGGATGTCAACGT-3′	R
IL-1β	5′-CTTTCCCGTGGACCTTCCA-3′	F
	5′-CTCGGAGCCTGTAGTGCAGTT-3′	R
TNF-α	5′-ACAAGGCTGCCCCGACTAC-3′	F
	5′-TGGGCTCATACCAGGGTTTG-3′	R
IL-6	5′-ACCACTCCCAACAGACCTGTCT-3′	F
	5′-CAGATTGTTTTCTGCAAGTGCAT-3′	R
iNOS	5′-TGTCTGCAGCACTTGGATCAG-3′	F
	5′-AAACTTCGGAAGGGAGCAATG-3′	R
GRP78	5′-CTCCGGCGTGAGGTAGAAAA-3′	F
	5′-AGAGCGGAACAGGTCCATGT-3′	R
ATF4	5′-GGTGGCCAAGCACTTGAAAC-3′	F
	5′-TCCATTTTCTCCAACATCCAATCT-3′	R
CHOP	5′-AGGAGGTCCTGTCCTCAGATGA-3′	F
	5′-ATGTGCGTGTGACCTCTGTTG-3′	R
HIF-1α	5′-TTTTGGCAGCGATGACACA-3	F
	5′-CGAAGTGGCTTTGGAGTTTCC-3′	R

Western Blot

Using radioimmunoprecipitation assay (RIPA) solution (Solarbio) with 1% protease inhibitor, the proteins of tissues or cells were thoroughly lysed. Protein concentration was measured. Following denaturation, the proteins were separation by electrophoresis and then transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk for 2 hours, the membrane was then incubated overnight with primary antibodies (Table 2) and incubated with anti-rabbit or mouse IgG (1:10,000, Affinity Biosciences, Cincinnati, OH, USA) for 2 hours. Enhanced chemiluminescence (ECL; Affinity Biosciences) was used to reveal the blot.

Table 2.

Antibodies for Western Blot and Immunofluorescence

Antibody	Cat. No.	RRID	Manufacturer
β-actin	AF7018	AB_2839420	Affinity Biosciences
GRP78	M1506-2	AB_3073113	HuaBio
ATF4	60035-1-IG	AB_2058598	Proteintech
CHOP	ET1703-05	AB_3070363	HuaBio
HIF-1α	HA722778	AB_3683539	HuaBio
PERK	ER64553	AB_3683561	HuaBio
p-PERK	29546-1-AP	AB_2935416	Proteintech
IRE1α	HA723225	AB_3683560	HuaBio
p-IRE1α	AP1442	AB_3683584	ABclonal
IBa-1	ET1705-78	AB_3070606	HuaBio

Enzyme-Linked Immunosorbent Assay

Protein levels of TNF-α, IL-6 and IL-1β were determined using an enzyme-linked immunosorbent assay (ELISA) kit (BioLegend, San Diego, CA, USA). Supernatants of BV2 cells were harvested. Regarding tissue samples, uveas and retinas of the EIU mice were placed in PBS to make a tissue homogenate. The manufacturer's instructions were followed when conducting the experimental procedures.

Transient Transfection With siRNA

HIF-1α small interfering RNA (siHIF-1α) and negative control siRNA (siNC) were designed by GenePharma (Shanghai, China). The siRNA transfection complex was prepared according to the manufacturer's protocol using the CALNPTM RNAi in vitro transfection kit (DNano, Beijing, China). Cells were treated with the transfection complex to silence HIF-1α. After 24 hours, the cells were used for further experiments.

Ca²⁺ Concentration Assay

A Fluo-4 AM probe (Beyotime, Shanghai, China) was used to determine cytoplasmic Ca²⁺ concentration. After incubation with Fluo4-AM solution for half an hour in the dark, BV2 cells underwent three rinses with PBS. Fluorescence intensity (488/520 nm) was measured by flow cytometry.

Network Pharmacology

Potential pharmacologic targets of 6-S were retrieved using PharmMapper, SwissTargetPrediction, and SuperPred databases. Pathologic targets associated with uveitis were acquired from the DisGeNET, GeneCards, and OMIM databases. The intersecting targets of uveitis and 6-S were identified using the Venny website: https://bioinfogp.cnb.csic.es/tools/venny/ and plotted in a Venn diagram. Interactions between these overlapping targets were assessed using the STRING database (confidence score ≥ 0.90), and then protein–protein interaction (PPI) networks were constructed using Cytoscape 3.6.1. The DAVID database was employed to conduct GO analysis on the intersecting targets, with the cutoff value adjusted to P < 0.05.

Reactive Oxygen Species Assay

The intracellular reactive oxygen species (ROS) content was tested using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe (Beyotime). BV2 cells were treated with 2′,7′-DCFH-DA solution in the dark for 30 minutes and then washed three times to remove unbound probes. A fluorescence microscope was used to take pictures, and fluorescence zymography was used to gauge the intensity of the fluorescence.

Nitric Oxide Assay

The nitric oxide concentration was tested using a Beyotime NO assay kit. The assay reagents were added to cell supernatant. The absorbance was measured at 540 nm.

Transmission Electron Microscopy

BV2 cells were fixed with 2.5% glutaraldehyde. Ultrathin sections were prepared and the morphology of the endoplasmic reticulum (ER) was observed by transmission electron microscopy (JEOL, Ltd., Tokyo, Japan).

Statistical Analysis

Data are expressed as mean ± SD and were analyzed and plotted using Prism 8 (GraphPad, Boston, MA, USA). Differences between the two groups were examined with the unpaired two-tailed Student’s t-test. Multiple group differences were evaluated using the one-way analysis of variance (ANOVA) test, and Bonferroni analysis was used to compare the two groups further. P < 0.05 was regarded as significant. All experiments were repeated independently three times.

Results

6-S Improved the Prognosis of EIU Mice

In order to explore the therapeutic potential of 6-S for uveitis, EIU mice were treated with 6-S, and DEX served as a positive control. Anterior segment photomicrographs showed significant conjunctival and ciliary vascular congestion in EIU mice compared with normal mice, along with miosis, keratic precipitates, and localized iris adhesions. 6-S and DEX significantly reduced these symptoms (Fig. 1A), and, consistently, both 6-S and DEX decreased the clinical scores of EIU mice (Fig. 1B). Pathology photographs revealed fibrin exudation and inflammatory cell infiltration in the ciliary body and anterior segment of the eye in EIU mice. There was only a small amount of immune cell infiltration in 6-S and DEX treatment (Fig. 1C). Figure 1D shows that microglia in the EIU group exhibited both significantly enhanced fluorescence intensity compared with the control group and characteristic amoeboid morphological transformation. 6-S and DEX both inhibited these changes. Proinflammatory cytokines in uveal and retinal were further examined, the mRNA and protein levels of IL-6, IL-1β, and TNF-α were significantly elevated in the EIU group compared with the control group. Both 6-S and DEX treatment suppressed their expression, and the effects were consistent (Figs. 1E–J). In conclusion, 6-S ameliorated symptoms and suppressed inflammation in EIU mice to a degree comparable to DEX treatment.

Figure 1.

6-S treatment improved the progression of EIU. (A) Anterior segment of the eye. Yellow arrows indicate pupil narrowing; white arrows, iris adhesions; red arrows, ciliary congestion. (B) Clinical scores (n = 6). (C) H&E-stained images. (D) Fluorescence images of microglia in retinal spreads stained with IBA1 (green) or DAPI (blue) and merged images. (E–G) Proinflammatory cytokine mRNA levels in mouse uvea and retina (n = 3). (H–J) Proinflammatory cytokine protein levels (n = 3). Values are expressed as mean ± SD. ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 denote significant differences from the EIU group.

6-S Restored Transcriptional Profiles and Modified Inflammatory Changes in EIU Mice

RNA-seq was performed to investigate transcriptional profile alterations and the action mechanisms of 6-S in EIU mice. Figures 2A and 2B present the number and expression trends of DEGs highly associated with the inflammatory response. These genes were upregulated in the EIU group and downregulated in the EIU+6-S group, including IL-6, Cxcl1, Mmp3, and Nfkbia. There were 955 common DEGs in the EIU versus control comparison and the EIU+6-S versus EIU comparison (Fig. 2C). As is evident from the heatmap, gene expression in the EIU+6-S group was opposite that in the EIU group but similar to that in the control group based on clustering analysis (Fig. 2D). We further analyzed DEGs between the EIU+6-S and EIU groups. GO analysis revealed that biological processes and molecular functions associated with inflammation, such as defense response, immune response, and cytokine activity, were significantly downregulated in the EIU+6-S group (Fig. 2E, Supplementary Table S1). KEGG enrichment analysis indicated highly enriched cytokine–cytokine receptor interaction (MMU04062) and TNF signaling pathway (MMU04668) (Fig. 2F, Supplementary Table S2). Collectively, 6-S treatment attenuated pro-inflammatory gene expression and restored transcriptional homeostasis in EIU mice.

Figure 2.

Effect of 6-S on EIU transcriptional profiles. (A, B) DEGs volcano plots. (C) Venn diagram of DEGs. (D) Heatmaps of shared DEGs. (E) GO analysis. (F) KEGG enrichment analysis of DEGs in EIU+6-S versus EIU.

6-S Attenuated Oxidative Stress and Inflammation In Vitro

Microglia are pivotal targets for AU inflammatory mechanisms and therapeutic strategies. First, the appropriate concentration of 6-S in BV2 cell was assayed. Data suggested that 6-S concentration at 18 µM and below did not impair cells viability (Fig. 3A). Next, LPS was used to trigger oxidative stress and inflammation in BV2 cells. 6-S treatment decreased the mRNA levels of iNOS and NO concentrations (Figs. 3B, 3C). The ROS fluorescence intensity was also attenuated in cells treated with 6-S. Surprisingly, the ROS level decreased with increasing concentration of 6-S (Figs. 3D, 3E). We further checked the expression of cellular pro-inflammatory cytokines. 6-S inhibited the mRNA and protein levels of IL-6 and IL-1β and the protein levels of TNF-α elicited by LPS in a concentration-dependent manner, and extremely suppressed mRNA expression of TNF-α at low concentrations (Figs. 3F–K). Based on these findings, 6-S attenuated LPS-induced oxidative stress and inflammation in microglia.

Figure 3.

6-S exerts anti-inflammatory and anti-oxidative stress effects in BV2 cells. (A) The viability of BV2 cells after treatment with 6-S in different concentrations for 24 hours. (B) RT-qPCR to analyze mRNA level of Nos2. (C) The NO content of cell supernatant. (D, E) 2′,7′-DCFH-DA probe to test intracellular ROS content. (F–H) Proinflammatory cytokine mRNA levels in BV2 cells. (I–K) Proinflammatory cytokine protein levels. Values are expressed as mean ± SD (n = 3). ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 denote significant differences from the LPS group.

6-S Attenuated ERS In Vivo

Network pharmacology was conducted to further understand the potential mechanism of the impact of 6-S in uveitis. A total of 1021 6-S–related genes (after removal of duplicates) and 1653 genes related to uveitis (after removing duplicates) were integrated from databases. We identified 88 intersecting therapeutic targets in the two genesets. Figure 4A shows the PPI networks for key pharmaceutical targets. GO analysis of the shared genes suggested that the effect mechanism of 6-S may involve the ER lumen (Fig. 4B, Supplementary Table S3). In addition, GO analysis of RNA-seq data revealed significant enrichment of integral component of ER membrane and cellular response to unfolded protein (Supplementary Table S1). Findings indicate that there was a significant increase in GRP78, ATF4, and CHOP mRNA and protein levels in the uvea and retina of EIU mice. Conversely, the levels of these factors decreased in the 6-S–treated mice (Figs. 4C–F). In addition, PERK and IRE1α phosphorylation levels were increased in EIU mice, and 6-S treatment effectively normalized p-PERK and p-IRE1α levels (Fig. 4G). These results suggest that 6-S attenuated endoplasmic reticulum stress in the uvea and retina of EIU mice.

Figure 4.

Network pharmacological analysis revealed that the ER is the effect site of 6-S. (A) Venn diagram and PPI network of common targets between 6-S and uveitis. (B) GO analysis of overlapping genes. (C–E) The mRNA levels of ERS in mouse uvea and retina (n = 4). (F, G) Images of western blotting results for ERS and corresponding statistics (n = 3). Values are expressed as mean ± SD. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 denote significant differences from the EIU group.

6-S Alleviated ERS In Vitro

To delve deeper into how 6-S combats ERS, we investigated the expression levels of ERS markers in BV2 cells. LPS upregulated the Hspa5, Atf4, and Ddit3 mRNA levels, which was reversed by 6-S treatment (Figs. 5A–C). Protein levels of these markers showed a similar trend. Additionally, 6-S also inhibited the phosphorylation of PERK and IRE1α induced by LPS (Fig. 5D). Transmission electron microscopy images showed that the ER appeared significantly swollen and dilated, and the number of ribosomes was reduced after LPS treatment. However, no significant swelling of the ER was observed under 6-S treatment (Fig. 5E). Because ERS can affect intracellular Ca²⁺ levels, we measured these levels in BV2 cells. Flow cytometry results showed that the mean fluorescence intensity in the 6-S group was lower than that in the LPS group (Fig. 5F). To further elucidate the the therapeutic mechanism of 6-S, we treated BV2 cells with Tg, an ERS inducer. Tg markedly upregulated IL-6, IL-1β, and TNF-α levels, but 6-S treatment effectively attenuated this trend (Figs. 5G–L). Based on these findings, 6-S mitigated LPS-triggered ERS and subsequently blocked the ERS-mediated inflammatory cascade.

Figure 5.

6-S inhibited ER damage in BV2 cells. (A–C) The mRNA levels of ERS. (D) Images of western blotting results for ERS and corresponding statistics. (E) ER ultrastructure in BV2 cells. Yellow arrow indicates swollen ER; blue arrow, normal endoplasmic reticulum. Magnification 10,000×. (F) Flow cytometry to detected intracellular Ca²⁺ levels. (G–I) Proinflammatory cytokine mRNA levels in BV2 cells. (J–L) Proinflammatory cytokine protein levels. Values are expressed as mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 denote significant differences from the LPS group.

HIF-1α Was a Target of 6-S

We further explored the possible targets of 6-S. Results demonstrated a decline in HIF-1α mRNA and protein levels in the uveas and retinas of EIU mice, and 6-S treatment upregulated the expression of HIF-1α (Figs. 6A, 6B). Similar results were also observed in BV2 cells, where 6-S treatment promoted the expression of HIF-1α, although LPS stimulation had no significant effect on HIF-1α in BV2 cells (Figs. 6C, 6D). We transfected BV2 cells with siNC or siHif-1α. Treatment of siHIF-1α significantly inhibited the upregulation of HIF-1α by 6-S (Figs. 6E, 6F). Moreover, compared to the siNC+LPS+6-S group, IL-6, IL-1β, and TNF-α mRNA and protein expression was increased in the siHIF-1α+LPS+6-S group (Figs. 6G–L). These data suggest that HIF-1α may be a target of 6-S and that silencing HIF-1α eliminates the anti-inflammatory effects of 6-S.

Figure 6.

HIF-1α was a target of 6-S. (A) Hif1a mRNA levels in mouse uvea and retina. (B) Images of western blotting results for HIF-1α in mouse uvea and retina and corresponding statistics. (C) Hif1a mRNA levels in BV2 cells. (D) Images of western blotting results for HIF-1α in BV2 cells and corresponding statistics. (E) Hif1a mRNA levels in BV2 cells. (F) Images of western blotting results for HIF-1α in BV2 cells. (G–I) Proinflammatory cytokine mRNA levels in BV2 cells. (J–L) Proinflammatory cytokine protein levels. Values are expressed as mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 denote significant differences from the LPS group.

6-S Suppressed ERS by Targeting HIF-1α

To determine whether 6-S exerts protective effects against ERS through HIF-1α, we assessed ER stress marker expression and intracellular Ca²⁺ levels following HIF-1α knockdown. Treatment with siHIF-1α almost completely eliminated the inhibitory effect of 6-S on ERS markers. Compared to the siNC+LPS+6-S group, the siHIF-1α+LPS+6-S group showed increased mRNA and protein levels of GRP78, ATF4, and CHOP (Figs. 7A–D). In addition, siHIF-1α treatment significantly reversed the inhibitory effect of 6-S on the phosphorylation of PERK and IRE1α (Fig. 7D). Similarly, HIF-1α silencing significantly diminished the effect of 6-S against intracellular Ca²⁺ overload (Figs. 7L, 7M). These findings demonstrate that HIF-1α knockdown reversed the protective effect of 6-S against ERS in BV2 cells.

Figure 7.

HIF-1α knockdown reversed the effects of 6-S in ERS. (A–C) The mRNA levels of ERS in BV2 cells. (D) Western blotting results for ERS and corresponding statistics. (E) Flow cytometry to detected intracellular Ca²⁺ levels. Values are expressed as mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Discussion

The impact of natural foods has received increasing attention in health care and disease treatment.²⁵ Ginger, a common and readily available natural food, has been used medicinally for millennia to treat various diseases such as pain and arthritis.^26,27 6-S is a prominent ginger product that shows therapeutic potential in inflammatory disease. The present study reveals the protective influence of 6-S in EIU. 6-S suppressed intraocular inflammation in EIU mice and decreased the release of oxidative stress products and proinflammatory cytokines in microglia. HIF-1α and ERS may be partially involved in the molecular mechanism by which 6-S inhibits microglia proinflammation.

During an acute uveitis attack, patients experience severe ocular pain and congestion, usually mediated by immune cells and inflammatory mediators.²⁸ Pro-inflammatory cytokines can increase vascular permeability, activate and chemotax immune cells to infiltrate the eye, and amplify the inflammatory response.²⁹ Our study demonstrated that 6-S effectively alleviated ocular symptoms and restored the normal structure of the ciliary body in EIU mice, inhibiting the exudation and infiltration of inflammatory cells. Notably, 6-S treatment also significantly suppressed the expression of IL-6, TNF-α, and IL-1β. RNA-seq further revealed the anti-inflammatory actions of 6-S, as inflammatory pathways such as cytokine–cytokine receptor interaction and the TNF signaling pathway were highly enriched. These findings suggest that 6-S improves the prognosis of EIU by mediating attenuation of the intraocular inflammatory response.

Microglia are resident immune cells in the eye that maintain intraocular microhomeostasis and respond to pathological changes.³⁰ Activation of microglia is closely associated with the onset and progression of uveitis.³¹ Our results showed that 6-S treatment significantly suppressed microglia activation in the retina of EIU mice. Consistent with other macrophages, when exogenous or endogenous signals are recognized, microglia undergo M1 polarization and secrete M1 proinflammatory mediators to enhance inflammatory responses.³² We constructed a model of microglia inflammation. Notably, 6-S inhibited the levels of M1 polarization markers IL-6, TNF-α, IL-1β, and iNOS in a concentration-dependent manner, reversing the reprogramming of microglia to M1 type. Microglial activation also leads to overproduction of ROS and NO. ROS can spread immune activation by serving as second messengers. ROS accumulation in cells also promotes macrophage M1-type polarization.³³ 6-S treatment significantly suppressed the cellular ROS content and NO levels. NO has a powerful and complex regulatory role in the immune response. Overproduction of NO mediated by iNOS has been reported to cause retinal damage and vascular complications under pathological conditions.³⁴ These findings suggest that 6-S may treat EIU by inhibiting microglia polarization toward M1 and reducing oxidative stress products.

The ER is the main site of protein synthesis and folding in cells and is also involved in physiological responses such as lipid metabolism and Ca²⁺ regulation. Network pharmacology suggests that the ER lumen may be a major cellular component of 6-S action. Oxidative stress, immune disorders, and DNA damage can cause gathering of misfolded or unfolded proteins in the ER, subsequently triggering ERS.³⁵ When the levels of unfolded proteins increase, GRP78 dissociates from the ER transmembrane receptors PERK and IRE1α, which phosphorylate and activate the unfolded protein response (UPR).³⁶ ATF4 is a key factor in the UPR signaling pathway. In primary open-angle glaucoma, ATF4 activation induces inflammatory cytokine production, trabecular meshwork cell dysfunction, and apoptosis.³⁷ ATF4 can directly upregulate CHOP, which regulates multiple gene transcription and induces apoptosis through mitochondria-dependent and death receptor pathways.³⁸ In this study, 6-S inhibited the increase of ERS markers in LPS-stimulated cells and ocular tissues and alleviated ER swelling. The ER is a major intracellular Ca²⁺ reservoir, and ERS can further lead to intracellular Ca²⁺ imbalance.³⁹ Studies have shown that calcium overload can increase the production of free radicals and ROS, ultimately leading to inflammatory response exacerbation and metabolic disorders.⁴⁰ As expected, LPS induced significant intracellular Ca²⁺ overload, which was ameliorated by 6-S treatment. ERS mediates expansion of inflammation and cell death.⁴¹ 6-S inhibits the downstream inflammatory response triggered by ERS; therefore, it is hypothesized that 6-S inhibits ERS and protects Ca²⁺ homeostasis, thereby suppressing the inflammatory response in the EIU.

We demonstrated that 6-S significantly promoted HIF-1α expression in EIU mice and LPS-stimulated BV2 cells. HIF-1α exhibits complex bidirectional crosstalk with ERS. Emerging evidence demonstrates that HIF-1α knockdown potentiates the activation of ERS-sensing molecules.⁴² It has been reported that the effect of 6-S on ERS and Ca²⁺ homeostasis was eliminated due to HIF-1α silence. A study similarly demonstrated that roxadustat significantly reduced microglia hyperactivation and attenuated neuroinflammation in the hippocampus of acutely brain-injured mice by promoting the HIF-1α signaling pathway.⁴³ Furthermore, silencing HIF-1α promoted nuclear factor kappa B (NF-κB), TNF-α, and IL-6 protein expression in BV2 cells, suggesting that HIF-1α may be engaged in microglial cell immunoregulation.⁴⁴ Consistently, we found that the ability of 6-S to inhibit pro-inflammatory cytokines was eliminated through silencing HIF-1α. However, reports show that HIF-1α binds directly to the IL-1β promoter and promotes IL-1β expression in macrophages.⁴⁵ This phenomenon may stem from the diverse regulatory mechanisms exerted by HIF-1α on IL-1β across different cell types and conditions. In this study, the inhibition of IL-1β by HIF-1α may be indirectly regulated through the alleviation of ERS. Given these findings, we concluded that the anti-inflammatory and ER homeostatic maintenance effects of 6-S are mediated by HIF-1α.

This study does have some limitations. Multiple immune cells, including neutrophils and macrophages, play important roles in the pathological process of uveitis. Investigating the regulatory effects of 6-S on different cell types is crucial for understanding its mechanisms in AU. Furthermore, the exact effects of 6-S on HIF-1α require additional in vivo studies for further validation.

In conclusion, we demonstrated that 6-S ameliorates EIU through HIF-1α–mediated regulation of inflammatory and ERS. This research offers novel perspectives on the pathogenesis of AU, and we anticipate that 6-S will be a promising strategy for the treatment of AU.