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. 2026 Apr 24;10(5):e0954. doi: 10.1097/HC9.0000000000000954

CRISPLD2 protects against liver inflammation and fibrosis via GRP78 to repress HMGB1/TLR4 axis–mediated STING palmitoylation

Haoye Zhang 1, Juan Wang 1, Hui Yang 1, Yu Yan 1, Jiafeng Zou 1, Zhenguo Liu 1,2,✉
PMCID: PMC13120513  PMID: 42043873

Abstract

Background:

Liver fibrosis caused by chronic inflammation remains the major driver of various liver diseases. However, limited effective therapies have been identified for liver fibrosis. Herein, we elucidated the complicated molecular mechanisms underlying liver fibrosis.

Methods:

Primary hepatocytes were co-cultured with JS-1 cells. Inflammatory cytokine levels were assessed by ELISA. Liver fibrosis markers and target molecular levels were detected by western blotting and immunohistochemical staining. Molecular mechanisms were analyzed by Acyl-biotin exchange (ABE) assay, Co-IP, proximity ligation assay, biotin pull-down, and GST pull-down assays. Co-localization and subcellular localization of molecules were observed by immunofluorescent staining. Liver fibrosis was induced by CCl4 in mice and determined by Masson Trichrome and Sirius Red staining. Liver injury was evaluated by HE staining, serum ALT, and AST levels.

Results:

High mobility group box 1 (HMGB1) bound to toll-like receptor 4 (TLR4) to facilitate palmitoylation of stimulator of interferon genes (STING), which led to hepatocyte inflammation and JS-1 cell activation in vitro. Furthermore, cysteine-rich secreted protein LCCL domain protein 2 (CRISPLD2) blocked HMGB1/TLR4 axis–mediated palmitoylation of STING, and subsequent liver fibrosis. Mechanistically, CRISPLD2 recruited 78 kDa glucose-regulated protein (GRP78) to trigger TLR4 degradation via an autophagic–lysosomal pathway. CRISPLD2 treatment alleviated CCl4-induced inflammation and liver fibrosis in mice via inactivation of the HMGB1/TLR4/STING pathway.

Conclusions:

CRISPLD2 mitigated hepatocyte inflammatory response and fibrosis via interaction with GRP78 to inactivate the HMGB1/TLR4 pathway and consequently restrain STING palmitoylation.

Keywords: cysteine-rich secreted protein LCCL domain protein 2, HMGB1/TLR4, inflammation, liver fibrosis, STING

INTRODUCTION

Liver fibrosis is recognized as the terminal stage of multiple chronic hepatic disorders. 1 Without effective control, liver fibrosis may progress into cirrhosis that ranks the 11th leading reason for death worldwide. 2 Under liver injury conditions, hepatic stellate cells are activated and release various pro-inflammatory cytokines, thereby leading to continuous inflammation. 3 This uncontrolled inflammation may result in irreversible injury and eventual liver fibrosis. 4 Therefore, inhibition of inflammation represents an effective strategy to mitigate liver fibrosis.

High mobility group box 1 (HMGB1) acts as a damage-associated molecular pattern (DAMP) with cytokine-like activity and can be released from cells under pathological conditions. 5 The released HMGB1 subsequently interacts with the cell surface receptors, including toll-like receptor 4 (TLR4). 6 Activation of the HMGB1/TLR4 pathway may facilitate the production of inflammatory cytokines and amplify inflammatory response, which accelerates the progression of liver fibrosis. 7 Thus, the HMGB1/TLR4 axis might be a key modulator for liver fibrosis, and inactivation of this pathway is considered an anti-inflammatory approach during liver fibrosis.

Stimulator of interferon genes (STING) is a pivotal regulator of innate immunity. 8 Petrasek et al. 9 firstly reported that STING activation led to liver inflammation and fibrosis. Another study documented that STING deficiency protected against carbon tetrachloride–induced liver fibrosis. 10 TLR4 has been suggested to be an upstream activator of STING in hepatocellular carcinoma. 11 Palmitoylation is an important kind of post-translational modification. A recent study revealed that palmitoylation of STING was responsible for the release of pro-inflammatory and pro-fibrotic cytokines in steatohepatitis. 12 However, whether the HMGB1/TLR4 axis was involved in the modulation of STING palmitoylation in liver fibrosis has not been identified.

Cysteine-rich secreted protein LCCL domain protein 2 (CRISPLD2) is an LPS (lipopolysaccharide)-binding protein that can block the binding of LPS to its target TLR4 to restrain inflammation. 13 Zhang et al. 14 documented that CRISPLD2 severed as an inhibitor of HMGB1/TLR4 pathway–mediated hepatic fibrogenesis. GRP78 (78 kDa glucose-regulated protein) is an endoplasmic reticulum chaperone. Liver-specific GRP78 deficiency could aggravate various hepatic disorders via disturbing endoplasmic reticulum homeostasis. 15 Of note, GRP78 was demonstrated to promote the selective autophagy and degradation of TLR4, which repressed inflammation in myeloid cells. 16 Interestingly, CRISPLD2 was predicted to interplay with GRP78. Therefore, we speculated that CRISPLD2 might recruit GRP78 to degrade TLR4, and subsequently inactivated HMGB1/TLR4 pathway to affect STING palmitoylation, thereby restraining inflammation and liver fibrosis.

In this study, we found that HMGB1 treatment aggravated inflammation and liver fibrosis by inducing STING palmitoylation at the C64 site via TLR4, which was abolished by CRISPLD2 via interaction with GRP78 to facilitate TLR4 degradation. Our findings uncovered the complicated mechanisms underlying inflammation and liver fibrosis, which provided promising intervening measures for liver fibrosis.

METHODS

Human liver samples

We collected non-fibrotic liver tissues from adjacent tissues during liver cancer resection (n=5) and liver fibrosis tissues from post-cirrhotic liver biopsies (n=18). The liver tissues were frozen in liquid nitrogen immediately after removal. All experiments was performed in compliance with both the Declarations of Helsinki and Istanbul and had been approved by the Ethics Committee of the Third Xiangya Hospital of Central South University (Approval No.: 2021-S278). All patients signed their written informed consent before surgery.

Cell culture and treatment

Primary hepatocytes were extracted from male C57BL/6J mice (8 weeks old; SLAC Laboratory Animal Center, Shanghai, China) and maintained with RPMI 1640 medium (Gibco, NY, USA) with 10% fetal bovine serum (FBS; Gibco) as previously described. 17 JS-1 and HEK293T cells were purchased from Procell (Wuhan, China) and cultured with DMEM (Gibco) containing 10% FBS. The primary hepatocytes were treated with 5 μg/mL recombinant mouse HMGB1 protein (50913-M01H; Sino Biological, Beijing, China) for 3, 9, 18, 24, and 48 hours or 1, 5, 10, and 20 μg/mL recombinant mouse CRISPLD2 protein (CSB-YP805372MO; Cusabio, Hubei, Wuhan) for 24 hours. The control primary hepatocytes were treated with 20 μg/mL recombinant mouse IgG3 protein (51096-MNAH; Sino Biological). CRISPLD2 was resuspended and dissolved in phosphate-buffered saline (PBS). The purified CRISPLD2 protein activity was assessed by migration assay. For the migration assay, serum-free medium containing 1.25×104 primary hepatocytes was placed on transwell membranes. The transwell inserts were placed in a 24-well plate filled with medium containing 1, 5, 10, and 20 μg/mL of CRISPLD2 for 24 hours. Medium with IgG3 was used as a negative control. Cells that passed through the membranes were fixed, stained, and then assessed and counted under light microscopy. For each experiment, cell viability was assessed by trypan blue exclusion before use in assays. The primary hepatocytes were treated with 10 nM TLR4 inhibitor (TAK-242, HY-11109; MCE, NJ, USA) for 24 hours; 100 μM palmitoylation inhibitor, 2-bromopalmitate (2-BP, HY-111770; MCE) for 2 and 4 hours; 10 μM palmitoylation enhancer, palmostatin B (HY-120911; MCE) for 2, 4, and 8 hours; 20 μM proteasome inhibitor MG132 (HY-13259; MCE); and 10 mM autophagy–lysosome inhibitor chloroquine (CQ, HY-17589A; MCE) for 4 hours.

Cell transfection

Wild-type-STING (STING-WT) and mutant-STING C64 (STING-Mut or STING-C64A) were provided by GeneChem (Shanghai, China) and transfected into HEK293T cells (5×105 cells per well) using Lipofectamine 2000 (Thermo Fisher Scientific). Lentiviruses carrying shNC, shGRP78, STING-WT, and STING-C64A were packaged by GeneChem (Shanghai, China). The primary hepatocytes were infected with the lentiviruses at a multiplicity of infection of 30, followed by selection with 2 μg/mL puromycin (MCE).

Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-6, IL-1β, and TNF-α in the supernatant of primary hepatocytes and mouse serum were detected using the Mouse IL-6 ELISA Kit (SEKM-0007), Mouse IL-1β ELISA Kit (SEKM-0002; Solarbio, Beijing, China), and Mouse TNF-α ELISA Kit (SEKM-0034) from Solarbio (Beijing, China).

Western blotting

Total protein was extracted from primary hepatocytes and JS-1 cells using the protein extraction kit (KGB5303; KeyGEN, Nanjing, China) and quantified using a BCA kit (KGB2101; KeyGEN). The protein samples were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotted to polyvinylidene fluoride membranes, blocked in 5% skim milk for 1 hour, and incubated with primary antibodies against α-SMA (ab7817, 1:1000; Abcam), FN (ab2413, 1:1000; Abcam), col1a1 (ab279711, 1:1000; Abcam), CRISPLD2 (orb478011, 1:500; Biorbyt, NC, USA), TLR4 (MA5-16216, 1:1000; Thermo Fisher Scientific, MA, USA), ATG3 (720056, 1:1000; Thermo Fisher Scientific), ATG7 (ab133528, 1:10000; Abcam), ATG12 (ab155589, 1:1000; Abcam), ATG16L1 (ab233796, 1:500; Abcam), LC3 (ab192890, 1:2000; Abcam), p62 (ab109012, 1:10000; Abcam), STING (PA5-83404, 1:1000; Thermo Fisher Scientific), GRP78 (MA5-27686, 1:2000, Thermo Fisher Scientific), PCNA (ab29, 1:1000, Abcam), Calnexin (ab22595, 1:1000; Abcam), and β-actin (ab6276, 1:5000; Abcam) overnight at 4°C. Goat anti-rabbit IgG (ab6721, 1:2000; Abcam) or Goat anti-mouse IgG (ab205719, 1:2000; Abcam) was applied for 30 minutes. The protein bands were developed using the New Super ECL Detection Kit (KGC4602; KeyGEN). The blots were quantified using the ImageJ software.

Acyl-resin-assisted capture (Acyl-RAC)

The primary hepatocytes were lysed to obtain total protein, and free thiols were blocked with NEM at 50°C for 1 hour. After acetone precipitation, the samples were rinsed with 70% acetone and resuspended in 1% SDS, 1 mM EDTA, 100 mM HEPES. S-palmitoylated proteins were bound with Thiopropyl Sepharose (Sigma-Aldrich) supplemented with 2 M neutral hydroxylamine for 4 hours at room temperature with rotation. After rinsing with 1% SDS, 1 mM EDTA, 100 mM HEPES, the proteins were eluted with SDS-PAGE loading buffer and detected by Western blotting.

Acyl-biotin exchange (ABE)

The ABE assay was used to assess STING palmitoylation as described previously. 18 The protein lysates were precipitated with anti-STING antibody (MA562089, Thermo Fisher Scientific) and Protein G magnetic beads to extract STING palmitoylated protein. The samples were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). After incubation with rotation at room temperature for 40 minutes, Biotin-BMCC buffer was added to the samples and then incubated with rotation at 4°C for 40 minutes. The immunoprecipitated protein was collected from the eluted proteins after Protein G magnetic beads were boiled at 80–90°C. STING protein expression was quantified by Western blotting. The blots were quantified using the ImageJ software.

Co-immunoprecipitation (Co-IP)

Primary hepatocytes were lysed with the Membrane and Cytosol Protein Extraction Kit (P0033; Beyotime, Haimen, China). The protein A/G magnetic beads were pre-conjugated with anti-LC3 (ab229327; Abcam), anti-p62 (ab109012; Abcam), anti-TLR4 (48-2300; Thermo Fisher Scientific), or anti-GRP78 (PA5-85169; Thermo Fisher Scientific) for 2 hours. The protein extracts were immunoprecipitated with the magnetic beads overnight at 4°C. Finally, the protein was eluted from the magnetic beads and detected by western blotting.

Biotin pull-down assay

Primary hepatocytes were incubated with biotin-labeled CRISPLD2 or a scrambled peptide for 2 hours. Cell lysates were incubated with streptavidin-agarose beads overnight 4°C. Then, the bound proteins were eluted from the magnetic beads using 8 M urea. The pulled-down proteins were measured by western blotting.

GST pull-down assay

The GRP78 fragment was inserted into the pGEX-4T-1 vector. The recombinant plasmids and the pGEX-4T-1 vector were transformed into Escherichia coli BL21, and recombinant protein expression was induced by 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 20°C for overnight. The bacteria were harvested, resuspended, sonicated, and centrifuged at 12,000g for 30 minutes. The recombinant proteins were purified using glutathione magnetic beads for 2 hours at 4°C. After 4 times washing, the lysates from 2×106 primary hepatocytes overexpressing His-CRISPLD2 were added with recombinant GST-GRP78 or GST protein (5 μg). After incubation in anti-GST-tag magnetic beads overnight at 4°C, the proteins were eluted and detected by western blotting.

Proximity ligation assay

The interaction between GRP78 and TLR4 proteins was determined using the Duolink proximity ligation assay kit (DUO92007; Sigma-Aldrich). In short, the primary hepatocytes were fixed, permeabilized, blocked, and probed with primary antibodies against GRP78 (MA5-27686, 1:100; Thermo Fisher Scientific) and TLR4 (MA5-16216, 1:50; Thermo Fisher Scientific). Duolink probes were added, followed by ligation and amplification. DAPI was adopted for nuclear counterstaining, and protein interaction was examined under a confocal microscope (Zeiss, Oberkochen, Germany).

Animal model

Male C57BL/6J mice (8 wk old) were randomly divided into sham, CCl4, CCl4+CRISPLD2, CCl4+shNC, CCl4+shSTING, CCl4+shSTING+ STING-WT, and CCl4+shSTING+ STING-C64A groups (n=6 per group). For liver fibrosis induction, the mice were intraperitoneally injected with 0.5 mL/g CCl4 (488488; Sigma-Aldrich, MO, USA) twice a week for 5 weeks. 19 Recombinant CRISPLD2 protein (100 ng/g) was intravenously injected into the mice three times per week for 5 weeks. The sham mice were intravenously injected with 0.9% saline, accordingly. Adeno-associated virus serotype 8 (AAV8) can efficiently facilitate liver-specific expression of exogenous genes. 20 AAV8 carrying shNC, shSTING, STING-WT, or STING-C64A were purchased from Genomeditech (Shanghai, China) and administered into each mouse by tail vein injection at a dose of 1.5×1011 v.g. 21 The mice were euthanized 2 days after the last CCl4 administration, and blood and liver tissues were harvested.

Histopathological examination

The liver tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 4 μm sections. Liver tissue morphology was observed using the Hematoxylin and Eosin (HE) stain kit (G1120; Solarbio). According to the previous study, the liver lobular inflammation was semi-quantified using a scale of 0–3. 22 Liver fibrosis was evaluated using the Masson Trichrome Stain Kit (G1340; Solarbio) and Modified Sirius Red Stain Kit (G1472; Solarbio) as the manufacturer’s instructions, respectively. The fibrosis area was analyzed using the ImageJ software.

Serum biochemistry

Peripheral blood samples were collected and centrifuged at 3000 rpm for 20 minutes at 4°C to separate serum. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were assessed by an automatic biochemistry analyzer (Sysmex BX-3010, Kobe, Japan).

Immunofluorescent staining

The pre-prepared liver sections as described above were subjected to antigen retrieval in sodium citrate buffer, followed by permeabilization with 0.2% Triton X-100 and blocking with 2% bovine serum albumin (BSA) for 1 hour. The primary hepatocytes were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 2% BSA for 1 hour. Subsequently, the tissue sections or primary hepatocytes were incubated with primary antibodies against LC3 (ab192890, 1:50; Abcam), p62 (ab109012, 1:50; Abcam), TLR4 (MA5-16216, 1:50; Thermo Fisher Scientific), GRP78 (MA5-27686, 1:100; Thermo Fisher Scientific), CRISPLD2 (orb478011, 1:100; Biorbyt, NC, USA) overnight at 4°C. After washing, the sections or cells were probed with Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (ab150077, 1:200; Abcam) or Goat Anti-Mouse IgG H&L (Cy5) (ab6563, 1:100; Abcam) for 1 hour. After nuclear staining with DAPI, the fluorescence was observed under a laser confocal microscope (Thermo Fisher Scientific).

Immunohistochemical staining

The liver sections were subjected to deparaffinization, rehydration, and antigen retrieval in sodium citrate buffer. After recovery to room temperature, the sections were exposed to 3% H2O2 for 30 minutes. Subsequently, the sections were blocked in normal horse serum and incubated with primary antibodies against α-SMA (ab7817, 1:50; Abcam), FN (ab2413, 1:50; Abcam), and col1a1 (ab279711, 1:50; Abcam) at 4°C overnight. After application with biotinylated secondary antibodies, the sections were treated with DAB chromogen. Images were obtained by a light microscope (Olympus).

Statistical analysis

Results are presented as mean±standard deviation (SD) of 3 independent experiments. The Student t test (2 groups) or 1-way analysis of variance (ANOVA) followed by the Tukey post hoc test (3 or more groups) was conducted using GraphPad Prism 8.0 software. p-values <0.05 were considered significant.

RESULTS

HMGB1-induced hepatocyte inflammatory response and fibrosis via TLR4 activation

To evaluate the role of HMGB1 in liver fibrosis, the primary hepatocytes treated with the HMGB1 recombinant protein were co-cultured with JS-1 cells. The release of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α from primary hepatocytes was time-dependently enhanced by HMGB1 recombinant protein (Figure 1A). Besides, HMGB1 treatment raised the protein levels of liver fibrosis markers α-SMA, FN, and col1a1 in JS-1 cells in a time-dependent manner (Figure 1B). To validate the involvement of TLR4 in the pro-fibrosis function of HMGB1, TLR4 inhibitor TAK-242, together with the HMGB1 recombinant protein were added into primary hepatocytes. We found that HMGB1-mediated release of IL-6, IL-1β, and TNF-α was partially suppressed by TAK-242 (Figure 1C). Moreover, TAK-242 partly reversed HMGB1-induced up-regulation of α-SMA, FN, and col1a1 in JS-1 cells (Figure 1D). These results indicated that HMGB1/TLR4 pathway activation led to hepatocyte inflammatory response and fibrosis.

FIGURE 1.

FIGURE 1

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Activation of the HMGB1/TLR4 axis induced hepatocyte inflammatory response and fibrosis. (A) Primary hepatocytes were treated with 5 μg/mL HMGB1 for 3, 9, 18, 24, and 48 hours. IL-6, IL-1β, and TNF-α levels in the culture medium were assessed by ELISA. (B) JS-1 cells were co-cultured with primary hepatocytes treated with 5 μg/mL HMGB1 for 3, 9, 18, 24, and 48 hours, and α-SMA, FN, and col1a1 protein abundance in JS-1 cells was determined by western blotting. (C) Primary hepatocytes were added with 5 μg/mL HMGB1 together with or without 10 nM TAK-242 for 24 hours, and IL-6, IL-1β, and TNF-α levels in the culture medium were detected by ELISA. (D) Primary hepatocytes with various treatments were co-cultured with JS-1 cells, and western blotting analysis of α-SMA, FN, and col1a1 protein levels in JS-1 cells. n=3. One-way ANOVA was performed for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; COL1A1, collagen type I alpha 1 chain; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; HMGB1, high mobility group box 1; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

HMGB1/TLR4 axis facilitated STING palmitoylation at the C64 site to trigger hepatocyte inflammatory response and fibrosis

To explore the downstream mechanism of the HMGB1/TLR4 axis in liver fibrosis, we focused on STING, a regulator of liver fibrosis. As analyzed by ABE, the palmitoylation level of STING in primary hepatocytes was enhanced by HMGB1, and this change was weakened by TAK-242 (Figure 2A). In addition, the promotive effect of HMGB1 on STING palmitoylation was time-dependently repressed by palmitoylation inhibitor 2-BP (Figure 2B). As expected, palmitoylation enhancer, palmostatin B increased the palmitoylation level of STING in primary hepatocytes with the prolonging of time (Figure 2C). As shown in Figure 2D, both mouse and rat STING proteins were predicted to be palmitoylated at the C64 site. Transfection with STING-WT evidently raised the palmitoylation level of STING in HEK293T cells, which was abolished after mutation at the C64 site (Figure 2E). Furthermore, as compared with the mutant-STING C64 (STING-C64A) group, STING-WT transfection strikingly elevated the release of IL-6, IL-1β, and TNF-α and levels of α-SMA, FN, and col1a1 in the presence or absence of HMGB1 administration (Figures 2F, G). Moreover, STING knockdown-mediated reduction in the levels of TNF-α, IL-6, and IL-1β in the supernatant of primary hepatocytes was reversed by STING-WT, but not affected by STING-C64A (Supplemental Figure S1A, http://links.lww.com/HC9/C330). In addition, α-SMA, FN, and col1a1 expression in JS-1 cells was reduced by STING silencing, whereas this change was counteracted by STING-WT, rather than STING-C64A (Supplemental Figure S1B, http://links.lww.com/HC9/C330). For in vivo results, CCl4 exposure-induced obvious pathological changes and fibrosis in the liver tissues (Supplemental Figure S2A, http://links.lww.com/HC9/C330), enhanced levels of serum ALT and AST (Supplemental Figure S2B, http://links.lww.com/HC9/C330), fibrosis markers (α-SMA, FN, and col1a1) (Supplemental Figure S2C, http://links.lww.com/HC9/C330), and inflammatory cytokines (IL-6, IL-1β, and TNF-α) (Supplemental Figure S2D, http://links.lww.com/HC9/C330) could be attenuated by STING depletion; however, the inhibitory effect of shSTING was neutralized by STING-WT, but not STING-C64A. To sum up, activation of the HMGB1/TLR4 pathway palmitoylated STING at the C64 site, which resulted in an inflammatory response and liver fibrosis.

FIGURE 2.

FIGURE 2

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STING palmitoylation at the C64 site was involved in HMGB1/TLR4 axis–mediated hepatocyte inflammatory response and fibrosis. (A) Primary hepatocytes were treated with 5 μg/mL HMGB1 combined with or without 10 nM TAK-242 for 24 hours, and the ABE assay evaluated STING palmitoylation level. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (B) Primary hepatocytes were treated with 100 μM 2-BP (a palmitoylation inhibitor) for 2–4 hours, and ABE assay analysis of STING palmitoylation level. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (C) STING palmitoylation level in primary hepatocytes exposed to 10 μM palmostatin B (a palmitoylation enhancer) for 2, 4, and 8 hours was analyzed by ABE assay. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (D) Conservative analysis of STING protein sequences among different species. (E) HEK293T cells were transfected with STING-WT or STING-C64A, and the Acyl-RAC assay determined STING palmitoylation level. Primary hepatocytes infected with lentiviruses carrying STING-WT or STING-C64A and treated with 5 μg/mL HMGB1, followed by co-culture with JS-1 cells. (F) ELISA measured IL-6, IL-1β, and TNF-α levels in the supernatant of primary hepatocytes. (G) Western blotting analysis of α-SMA, FN, and col1a1 protein abundance in JS-1 cells. n=3. One-way ANOVA was performed for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: ABE, Acyl-Biotin Exchange; α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; COL1A1, collagen type I alpha 1 chain; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; HAM, hydroxylamine; HMGB1, high mobility group box 1; 2-BP, 2-bromopalmitate; STING, stimulator of interferon genes; STING-WT, wild-type STING.

CRISPLD2 blocked HMGB1/TLR4 axis–mediated hepatocyte inflammatory response and fibrosis via inhibiting STING palmitoylation

CRISPLD2 has been identified as an inhibitor of the HMGB1/TLR4 pathway. 14 We further verified whether CRISPLD2 could influence HMGB1/TLR4-mediated STING palmitoylation during liver fibrosis. First, we validated CRISPLD2 protein activity by transwell migration assay. As shown in Supplemental Figure S3, http://links.lww.com/HC9/C330, compared with IgG3-treated cells, 1, 5, 10, and 20 μg/mL of CRISPLD2 promotes migrated cells through the transwell membrane, supporting the preservation of protein function or activity in CRISPLD2 protein. As illustrated in Figure 3A, co-treatment with CRISPLD2 recombinant protein concentration-dependently reduced HMGB1-induced production of IL-6, IL-1β, and TNF-α from primary hepatocytes. In addition, the increased α-SMA, FN, and col1a1 expression in JS-1 cells co-cultured with HMGB1-treated primary hepatocytes was gradually lowered by CRISPLD2 recombinant protein with the concentration increasing (Figure 3B). Notably, HMGB1-mediated STING palmitoylation in primary hepatocytes was suppressed by CRISPLD2 recombinant protein in a dose-dependent manner (Figure 3C). Therefore, CRISPLD2 protected against hepatocyte inflammatory response and fibrosis through repressing HMGB1/TLR4-mediated STING palmitoylation.

FIGURE 3.

FIGURE 3

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CRISPLD2 inactivated the HMGB1/TLR4 axis to repress STING palmitoylation, thus suppressing hepatocyte inflammatory response and fibrosis. HMGB1-treated primary hepatocytes were further stimulated with 1, 5, 10, and 20 μg/mL CRISPLD2 for 24 hours, followed by co-culture with JS-1 cells. (A) ELISA determined IL-6, IL-1β, and TNF-α levels in the supernatant of primary hepatocytes. (B) α-SMA, FN, and col1a1 protein abundance in JS-1 cells was assessed by western blotting. (C) STING palmitoylation levels were evaluated by the ABE assay. The cells were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. n=3. One-way ANOVA was performed for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. ABE, Acyl-Biotin Exchange; α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; COL1A1, collagen type I alpha 1 chain; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; HAM, hydroxylamine; HMGB1, high mobility group box 1; STING, stimulator of interferon genes.

CRISPLD2 promoted TLR4 degradation via an autophagic–lysosomal pathway

We further elucidated the detailed modulatory mechanism of CRISPLD2 in the HMGB1/TLR4 pathway. CRISPLD2 recombinant protein obviously reduced TLR4 protein level in primary hepatocytes, and counteracted HMGB1-induced up-regulation of TLR4 (Figure 4A). In addition, CRISPLD2-mediated down-regulation of TLR4 protein was inhibited by autophagy–lysosome inhibitor CQ, rather than proteasome inhibitor MG132 (Figure 4B), suggesting that CRISPLD2 degraded TLR4 via the autophagic–lysosomal pathway. Furthermore, ATG7 level and LC3 II/I ratio were upregulated, and p62 was downregulated by CRISPLD2 in primary hepatocytes (Figure 4C). However, HMGB1 exhibited the opposite effect, which was reversed by CRISPLD2 (Figure 4C). Moreover, the interaction between TLR4 and LC3 or p62 was intensified by CRISPLD2 but attenuated by HMGB1, and the inhibitory action of HMGB1 on the interaction between TLR4 and LC3 or p62 was neutralized by CRISPLD2 (Figure 4D). Accordingly, CRISPLD2 strengthened the co-localization of TLR4 and LC3 or p62 in primary hepatocytes and abrogated HMGB1-mediated inhibition in the co-localization of TLR4 (Supplemental Figure S4, http://links.lww.com/HC9/C330). These findings proved that TLR4 was degraded by CRISPLD2 through the autophagic–lysosomal pathway.

FIGURE 4.

FIGURE 4

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CRISPLD2 degraded TLR4 via an autophagic–lysosomal pathway. (A) Primary hepatocytes were treated with 5 μg/mL HMGB1, 10 μg/mL CRISPLD2, or a combination of them. Western blotting analysis of TLR4 protein level. (B) Primary hepatocytes were treated with 10 μg/mL CRISPLD2, or combined with 20 μM MG132 or 10 mM CQ, and TLR4 protein level was assessed by western blotting. Primary hepatocytes treated with 5 μg/mL HMGB1, 10 μg/mL CRISPLD2, or a combination of them. (C) Western blotting analysis of the protein levels of ATG3, ATG7, ATG12, ATG16L1, LC3 II/I, and p62. (D) The protein interaction between TLR4 and LC3 or p62 was confirmed by Co-IP. n=3. One-way ANOVA was performed for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: ANOVA, analysis of variance; ATG, autophagy-related protein; CQ, chloroquine; Co-IP, co-immunoprecipitation; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; HMGB1, high mobility group box 1; LC3, microtubule-associated protein 1 light chain 3; MG132, proteasome inhibitor; TLR4, toll-like receptor 4.

CRISPLD2 contributed to the translocation of GRP78 from the cytoplasm to the cytomembrane

Bioinformatics predicted that CRISPLD2 might interact with GRP78. Thus, we aimed to investigate the regulation of CRISPLD2 in GRP78 in primary hepatocytes. Western blotting indicated that GRP78 protein abundance was not changed in hepatocytes after treatment with CRISPLD2, HMGB1, or a combination of them (Figure 5A). CRISPLD2 treatment facilitated the interaction between CRISPLD2 and GRP78 proteins in hepatocytes, which was weakened by HMGB1 co-administration (Figure 5B). Furthermore, the biotin pull-down assay revealed that CRISPLD2 directly bound to GRP78 in the cytomembrane of primary hepatocytes (Figure 5C). Consistently, the GST pull-down assay further verified the direct interaction between CRISPLD2 and GRP78 (Supplemental Figure S5A, http://links.lww.com/HC9/C330). Besides, the interplay between cytomembrane CRISPLD2 and GRP78 proteins was promoted by CRISPLD2, and this effect was attenuated by HMGB1 (Figure 5D). In addition, the co-localization of CRISPLD2 and GRP78 proteins in the cytomembrane of CRISPLD2-treated primary hepatocytes was observed by immunofluorescence staining (Figure 5E). Moreover, GRP78 was mainly expressed in the cytoplasm of hepatocytes, and CRISPLD2 treatment facilitated the translocation from cytoplasm to cytomembrane; however, HMGB1 co-treatment partly restrained CRISPLD2-mediated cytomembrane translocation of GRP78 (Figure 5F). Collectively, the CRISPLD2/HMGB1 axis regulated the cytomembrane translocation of GRP78 in primary hepatocytes.

FIGURE 5.

FIGURE 5

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CRISPLD2 facilitated the cytomembrane translocation of GRP78. Primary hepatocytes were exposed to 5 μg/mL HMGB1, 10 μg/mL CRISPLD2, or a combination of them. (A) GRP78 protein level was analyzed by western blotting. (B) Interplay between CRISPLD2 and GRP78 proteins was evaluated by Co-IP. (C) The binding of CRISPLD2 to GRP78 protein in the cytomembrane of primary hepatocytes was validated by a biotin pull-down assay. (D) Co-IP validated the interaction between CRISPLD2 and GRP78 proteins in the cytomembrane. (E) The co-localization of CRISPLD2 and GRP78 in CRISPLD2-treated primary hepatocytes was observed by immunofluorescent staining (scale bar=25 μm). (F) The subcellular localization of GRP78 in primary hepatocytes was evaluated by immunofluorescent staining, and the cytomembrane was labeled by WGA staining (scale bar=25 μm). n=3. One-way ANOVA was performed for statistical analysis. Abbreviations: ANOVA, analysis of variance; Co-IP, co-immunoprecipitation; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; GRP78, 78 kDa glucose-regulated protein; HMGB1, high mobility group box 1; WGA, wheat germ agglutinin.

CRISPLD2 recruited GRP78 to degrade TLR4, thereby repressing STING palmitoylation–mediated hepatocyte inflammatory response and fibrosis

To verify the involvement of GRP78 in CRISPLD2-mediated protection against liver fibrosis, GRP78 was knocked down in primary hepatocytes. The silencing efficiency of shGRP78 was validated by western blotting (Figure 6A). Co-IP assay demonstrated that CRISPLD2-induced interaction between GRP78 and CRISPLD2 in hepatocytes was mitigated by GRP78 knockdown (Figure 6B). Besides, GRP78 deficiency weakened the interplay between cytomembrane CRISPLD2 and GRP78 proteins in the presence of CRISPLD2 (Figures 6C, D). In addition, GRP78 was translocated from the cytoplasm to the cytomembrane after CRISPLD2 treatment, whereas this translocation was reduced by GRP78 silencing (Figure 6E). Proximity ligation assay validated that CRISPLD2 recruited GRP78 to TLR4 in hepatocytes (Supplemental Figures S5B, C, http://links.lww.com/HC9/C330). Besides, Co-IP assay proved that CRISPLD2 recombinant protein treatment enhanced the binding of GRP78 to TLR4 in hepatocytes (Supplemental Figure S5D, http://links.lww.com/HC9/C330). Furthermore, CRISPLD2-induced up-regulation of ATG7 and LC3 II/I ratio, and down-regulation of TLR4 and p62 were abolished by GRP78 depletion (Figure 6F). CRISPLD2-mediated enhancement in binding of TLR4 to LC3 or p62 was suppressed by GRP78 deficiency (Figure 6G and Supplemental Figure S6A, http://links.lww.com/HC9/C330). The inhibition of CRISPLD2 in STING palmitoylation in HMGB1-treated hepatocytes was partially reversed by shGRP78 (Supplemental Figure S6B, http://links.lww.com/HC9/C330). Accordingly, GRP78 knockdown counteracted CRISPLD2-mediated repression in IL-6, IL-1β, and TNF-α release from primary hepatocytes in the presence of HMGB1 (Supplemental Figure S6C, http://links.lww.com/HC9/C330), as well as α-SMA, FN, and col1a1 expression in JS-1 cells (Supplemental Figure S6D, http://links.lww.com/HC9/C330). Next, we detected the expression of CRISPLD2, GRP78, and STING palmitoylation in human liver biopsy samples and evaluated their correlation. We found that in comparison with normal non-fibrosis liver samples, CRISPLD2 and GRP78 levels were lower, while STING protein level and STING palmitoylation level were higher in liver fibrosis samples (Supplemental Figure S7A, http://links.lww.com/HC9/C330). Besides, both CRISPLD2 and GRP78 levels were negatively correlated with STING palmitoylation level in the liver fibrosis samples (Supplemental Figure S7B, http://links.lww.com/HC9/C330). The above observations revealed that CRISPLD2 interacted with GRP78 in the cytomembrane to cause TLR4 degradation, and consequently restrained STING palmitoylation to alleviate hepatocyte inflammatory response and fibrosis.

FIGURE 6.

FIGURE 6

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CRISPLD2 interacted with GRP78 to cause TLR4 degradation, and consequently restrained STING palmitoylation–mediated hepatocyte inflammatory response and fibrosis. (A) Western blotting analysis of GRP78 protein level in primary hepatocytes infected with lentiviruses carrying shNC or shGRP78. CRISPLD2-treated primary hepatocytes were infected with lentiviruses carrying shNC or shGRP78. (B) The binding of CRISPLD2 to GRP78 protein was analyzed by Co-IP. (C, D) The interaction between CRISPLD2 and GRP78 proteins in the cytomembrane was validated by biotin pull-down and Co-IP assay. (E) Immunofluorescent staining observed the subcellular localization of GRP78 in primary hepatocytes (scale bar=25 μm). (F) Western blotting analysis of TLR4, ATG7, LC3 II/I, and p62 protein levels. (G) Co-IP evaluated the binding of TLR4 to LC3 or p62. n=3. The Student t test (for A) and one-way ANOVA (for B–G) were performed for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: ANOVA, analysis of variance; ATG7, autophagy-related protein 7; Co-IP, co-immunoprecipitation; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; GRP78, 78 kDa glucose-regulated protein; LC3, microtubule-associated protein 1 light chain 3; shNC, non-targeting short hairpin RNA control; shGRP78, short hairpin RNA targeting GRP78; STING, stimulator of interferon genes; TLR4, toll-like receptor 4.

CRISPLD2 suppressed the inflammatory response and liver fibrosis in vivo

Finally, the influence of CRISPLD2 on liver fibrosis was evaluated in CCl4-exposed mice in vivo. Histopathological examination showed that CCl4 exposure resulted in obvious pathological changes and fibrosis in the livers, which could be relieved by treatment with CRISPLD2 (Figures 7A, B). The enhanced serum ALT and AST levels of CCl4-challenged mice were abolished in the CRISPLD2 treatment group (Figure 7C). In addition, the co-localization of cytomembrane CRISPLD2 and GRP78 proteins, TLR4, and LC3 or p62 proteins was reduced in CCl4-stimulated livers, and this change was partly reversed by CRISPLD2 (Figures 7D, E). Moreover, STING palmitoylation level was elevated in the liver tissues of CCl4-exposed mice, which was inhibited by CRISPLD2 (Figure 7F). As expected, the enhanced levels of inflammatory cytokines (IL-6, IL-1β, and TNF-α) and fibrosis markers (α-SMA, FN, and col1a1) in response to CCl4 were suppressed by CRISPLD2 (Figures 7G, H). Taken together, CRISPLD2 mitigated in vivo inflammatory response and liver fibrosis.

FIGURE 7.

FIGURE 7

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CRISPLD2 relieved inflammation and liver fibrosis in vivo. CCl4-challenged mice were treated with recombinant protein CRISPLD2. (A) Pathological changes in livers were evaluated by HE staining (scale bar=50 μm). (B) Liver fibrosis was examined by the Masson Trichrome and Sirius Red staining (scale bar=50 μm). (C) Serum ALT and AST levels were assessed. (D, E) Immunofluorescent staining evaluated the co-localization of CRISPLD2 and GRP78, TLR4, and LC3, or p62 in liver tissues (scale bar=50 μm). (F) STING palmitoylation level in livers was analyzed by ABE assay. The samples were added with hydroxylamine (+HAM) buffer or lysis buffer (−HAM). Palm-STING, STING palmitoylation. (G) Serum IL-6, IL-1β, and TNF-α levels were determined by ELISA. (H) α-SMA, FN, and col1a1 expression in liver tissues were examined by immunohistochemical staining (scale bar=50 μm). n=6. One-way ANOVA was performed for statistical analysis. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: ABE, Acyl-Biotin Exchange; α-SMA, alpha-smooth muscle actin; ANOVA, analysis of variance; ALT, alanine aminotransferase; AST, aspartate aminotransferase; COL1A1, collagen type I alpha 1 chain; CRISPLD2, cysteine-rich secreted protein LCCL domain 2; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; GRP78, 78 kDa glucose-regulated protein; HAM, hydroxylamine; HE, hematoxylin and eosin; LC3, microtubule-associated protein 1 light chain 3; Palm-STING, STING palmitoylation; STING, stimulator of interferon genes; TLR4, toll-like receptor 4.

DISCUSSION

Liver fibrosis remains a worldwide health issue, potentially resulting in end-stage liver diseases, including liver cirrhosis and cancer. 23 Currently, effective therapeutic agents for liver fibrosis are still lacking. Inflammation has been considered a vital contributing factor to liver fibrosis. 24 Repression of inflammation offers a promising approach to delay the progression of liver fibrosis. Our data demonstrated that CRISPLD2 recruited GRP78 to block HMGB1/TLR4 pathway–mediated STING palmitoylation, which suppressed inflammation to alleviate liver fibrosis. These results highlight the beneficial effect of CRISPLD2 on liver fibrosis, suggesting its therapeutic possibility in end-stage liver diseases.

As a crucial inflammatory mediator, HMGB1 is activated during chronic inflammation and is a major driver for liver fibrosis. 25 TLR4 is one of the pattern recognition receptors of HMGB1. 26 It has been proven that HMGB1 binds to TLR4 to trigger inflammation and consequently exacerbate liver fibrosis. 27 Consistently, our findings indicated that TLR4 inhibitor TAK-242 could relieved HMGB1-induced hepatocyte inflammatory response and fibrosis. However, the detailed mechanism through which the HMGB1/TLR4 pathway facilitates liver fibrosis remains unclear. Studies have identified STING as a crucial contributor to liver inflammation.28,29 Palmitoylation plays a key role in the activation of STING. 30 Thus, STING palmitoylation has been a potential target for the treatment of STING-dependent disorders. 31 In this study, we found that the HMGB1/TLR4 pathway led to STING palmitoylation at the C64 site, which activated STING to drive inflammatory response and liver fibrosis. Our findings provided novel evidence for STING palmitoylation as a downstream mechanism of the HMGB1/TLR4 axis in liver fibrosis.

CRISPLD2 has 2 LCCL domains with LPS-binding affinity, which have been documented to protect against endotoxin shock. 32 Besides, CRISPLD2 also possessed an anti-inflammatory capacity without binding to LPS in multiple inflammation-related disorders, such as liver fibrosis, 14 asthma, 33 and lung fibrosis. 34 Of note, CRISPLD2 could mitigate HMGB1-triggered inflammation during hepatic fibrosis. 14 Herein, our results were consistent with previous researchers and further discovered that CRISPLD2 inhibited STING palmitoylation through inactivation of the HMGB1/TLR4 pathway, thereby suppressing hepatocyte inflammatory response and fibrosis. Therefore, our findings for the first time proved that CRISPLD2 exhibited an anti-inflammatory effect via inactivation of the HMGB1/TLR4/STING axis in liver fibrosis.

Although our observations demonstrated that CRISPLD2 repressed HMGB1/TLR4/STING-mediated liver inflammatory response, the potential mechanism underlying this process remains obscure. GRP78 is an endoplasmic reticulum stress-related factor, which is expressed in cell surface and released into the extracellular compartment upon activation. 35 A previous study documented that GRP78 could contribute to the transfer of TLR4 to the lysosome for degradation. 36 Autophagy has been recognized as a modulatory mechanism for lysosomal degradation. 37 Wu et al. 16 reported that secreted GRP78 favored the degradation of TLR4 via the selective autophagy in myeloid cells. In this work, CRISPLD2 recruited and facilitated the cytomembrane translocation of GRP78 and induced selective autophagy to degrade TLR4 in primary hepatocytes, which subsequently restrained STING palmitoylation to protect against hepatocyte inflammatory response and fibrosis. It has been reported that CRISPLD2 can exert its antifibrotic effect via various pathways. For example, CRISPLD2 administration repressed HMGB1-induced inflammation during hepatic fibrogenesis via miR-155. 14 Another work documented that CRISPLD2 affected fetal lung fibroblast expansion and migration via 44/42MAPK pathway. 38 So far, whether other extracellular interactions can participate in CRISPLD2-mediated inhibition in liver inflammation and fibrosis remains unclear. In our future study, we will focus on this issue.

In summary, our data demonstrated that CRISPLD2 relieved inflammation to mitigate liver fibrosis via autophagy-mediated TLR4 degradation with assistance of GRP78, and subsequent inhibition of STING palmitoylation. Our observations identify the CRISPLD2/TLR4/STING axis as a pivotal modulator in hepatocyte inflammatory response and fibrosis and provide a theoretical foundation for the development of novel therapeutics for liver fibrosis. However, the clinic correlation of our findings needs to be evaluated and validated in clinical samples in the future.

Supplementary Material

hc9-10-e0954-s001.pdf (1.5MB, pdf)
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Acknowledgments

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (No. 82570741), the Natural Science Foundation of Hunan Province (No. 2022JJ70070, 2023JJ30831), the National Natural Science Foundation of China Youth Fund (No. 82200671), and the Hunan Provincial Natural Science Foundation Youth Fund (No. 2023JJ40913).

CONFLICTS OF INTEREST

The authors have no conflicts to report.

Footnotes

Abbreviations: 2-BP, 2-bromopalmitate; AAV8, adeno-associated virus serotype 8; ABE, Acyl-Biotin Exchange; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; BSA, bovine serum albumin; Co-IP, co-immunoprecipitation; CQ, chloroquine; CRISPLD2, cysteine-rich secreted protein LCCL domain protein 2; DAMP, damage-associated molecular pattern; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GRP78, 78 kDa glucose-regulated protein; HE, hematoxylin–eosin; HMGB1, high mobility group box 1; ITPG, isopropyl-β-D-1-thiogalactopyranoside; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; STING, stimulator of interferon genes; STING-Mut, mutant-STING; STING-WT, wild type-STING; TLR4, toll-like receptor 4.

Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal’s website, www.hepcommjournal.com.

Contributor Information

Haoye Zhang, Email: 1325382907@qq.com.

Juan Wang, Email: waju1203@hotmail.com.

Hui Yang, Email: 1119884109@qq.com.

Yu Yan, Email: xyyanyu0516@163.com.

Jiafeng Zou, Email: zjf_2001@126.com.

Zhenguo Liu, Email: liuzg729@163.com.

REFERENCES

  • 1.Dano LB, Rao KP, Keno TD. Fractional order differential equations for chronic liver cirrhosis with frequent hospitalization. BMC Res Notes. 2022;15:332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Asrani SK, Devarbhavi H, Eaton J, Kamath PS. Burden of liver diseases in the world. J Hepatol. 2019;70:151–171. [DOI] [PubMed] [Google Scholar]
  • 3.Hammerich L, Tacke F. Hepatic inflammatory responses in liver fibrosis. Nat Rev Gastroenterol Hepatol. 2023;20:633–646. [DOI] [PubMed] [Google Scholar]
  • 4.Li J, Yuan Y, Fu Q, Chen M, Liang H, Chen X, et al. Novel insights into the role of immunomodulatory extracellular vesicles in the pathogenesis of liver fibrosis. Biomark Res. 2024;12:119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Sims GP, Rowe DC, Rietdijk ST, Herbst R, Coyle AJ. HMGB1 and RAGE in inflammation and cancer. Annu Rev Immunol. 2010;28:367–388. [DOI] [PubMed] [Google Scholar]
  • 6.Ren W, Zhao L, Sun Y, Wang X, Shi X. HMGB1 and Toll-like receptors: Potential therapeutic targets in autoimmune diseases. Mol Med. 2023;29:117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Yao Y, Zuo X, Shao F, Yu K, Liang Q. Jaceosidin attenuates the progression of hepatic fibrosis by inhibiting the VGLL3/HMGB1/TLR4 signaling pathway. Phytomedicine. 2024;128:155502. [DOI] [PubMed] [Google Scholar]
  • 8.Li H, Guo X, Aquino E, Wu C. Mini review: STING activation during non-alcoholic fatty liver disease. Front Nutr. 2023;10:1139339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Petrasek J, Iracheta-Vellve A, Csak T, Satishchandran A, Kodys K, Kurt-Jones EA, et al. STING–IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease. Proc Natl Acad Sci U S A. 2013;110:16544–16549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Iracheta-Vellve A, Petrasek J, Gyongyosi B, Satishchandran A, Lowe P, Kodys K, et al. Endoplasmic reticulum stress-induced hepatocellular death pathways mediate liver injury and fibrosis via stimulator of interferon genes. J Biol Chem. 2016;291:26794–26805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sun B, Zhang Q, Sun T, Liu J, Cao Y, Liang B, et al. Radiofrequency hyperthermia enhances the effect of OK-432 for hepatocellular carcinoma by activating of TLR4–cGAS–STING pathway. Int Immunopharmacol. 2024;130:111769. [DOI] [PubMed] [Google Scholar]
  • 12.Wang Q, Bu Q, Xu Z, Liang Y, Zhou J, Pan Y, et al. Macrophage ATG16L1 expression suppresses metabolic dysfunction–associated steatohepatitis progression by promoting lipophagy. Clin Mol Hepatol. 2024;30:515–538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang S, Pei L, Qu J, Sun L, Jiang W, Li W, et al. CRISPLD2 attenuates pro-inflammatory cytokines production in HMGB1-stimulated monocytes and septic mice. Am J Transl Res. 2021;13:4080–4091. [PMC free article] [PubMed] [Google Scholar]
  • 14.Zhang H, Liu Z, Liu S. HMGB1 induced inflammatory effect is blocked by CRISPLD2 via MiR155 in hepatic fibrogenesis. Mol Immunol. 2016;69:1–6. [DOI] [PubMed] [Google Scholar]
  • 15.Ji C, Kaplowitz N, Lau MY, Kao E, Petrovic LM, Lee AS. Liver-specific loss of glucose-regulated protein 78 perturbs the unfolded protein response and exacerbates a spectrum of liver diseases in mice. Hepatology. 2011;54:229–239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Wu Z, Xu Z, Zhou X, Li H, Zhao L, Lv Y, et al. sGRP78 enhances selective autophagy of monomeric TLR4 to regulate myeloid cell death. Cell Death Dis. 2022;13:587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Matsumoto M, Ogawa W, Teshigawara K, Inoue H, Miyake K, Sakaue H, et al. Role of the insulin receptor substrate 1 and phosphatidylinositol 3-kinase signaling pathway in insulin-induced expression of sterol regulatory element binding protein 1c and glucokinase genes in rat hepatocytes. Diabetes. 2002;51:1672–1680. [DOI] [PubMed] [Google Scholar]
  • 18.Fu Y, Zhang F, Lang L, Liang S, Wang J, Zhang S, et al. Inhibition of DHHC9-mediated CD36 palmitoylation lessens high-fat diet (HFD)-induced impairment of pubertal mammary gland development through the JNK–ERK pathway. Cell Mol Biol Lett. 2025;30:117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Cloutier M, Variya B, Akbari SA, Rexhepi F, Ilangumaran S, Ramanathan S. Profibrogenic role of IL-15 through IL-15 receptor alpha-mediated trans-presentation in the carbon tetrachloride-induced liver fibrosis model. Front Immunol. 2024;15:1404891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Dou D, Lu J, Li D, He F, Zhu X, Zhang X, et al. Preclinical evaluation of AAV8-R338L gene therapy for hemophilia: Efficacy, pharmacokinetics, distribution, excretion and toxicity in mouse models and non-human primates. Pharm Res 2025;42, 1717–1732. [DOI] [PubMed] [Google Scholar]
  • 21.Zhao F, Niu X, Song G, Wang L, Fu Y, Li S, et al. Hepatic stellate cell-specific miR-214 expression alleviates liver fibrosis without boosting steatosis and inflammation. J Transl Med. 2025;23:810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zhao L, Zhang Y, Qiao Y, Jia L, Luo S. 17(R)-RvD1 ameliorates liver injury in hyperuricemia through inhibiting pyroptosis via NF-kappaB signaling pathway. Drug Des Devel Ther. 2025;19:6573–6585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Szternel L, Sobucki B, Wieprzycka L, Krintus M, Panteghini M. Golgi protein 73 in liver fibrosis. Clin Chim Acta. 2024;565:119999. [DOI] [PubMed] [Google Scholar]
  • 24.Zhu L, Tong H, Ren C, Chen K, Luo S, Wang Q, et al. Inflammation unleashed: The role of pyroptosis in chronic liver diseases. Int Immunopharmacol. 2024;141:113006. [DOI] [PubMed] [Google Scholar]
  • 25.Zhong H, Gui X, Hou L, Lv R, Jin Y. From inflammation to fibrosis: Novel insights into the roles of high mobility group protein box 1 in schistosome-induced liver damage. Pathogens. 2022;11:289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Mattke J, Darden CM, Lawrence MC, Kuncha J, Shah YA, Kane RR, et al. Toll-like receptor 4 in pancreatic damage and immune infiltration in acute pancreatitis. Front Immunol. 2024;15:1362727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Jing X, Zhou G, Zhu A, Jin C, Li M, Ding K. RG-I pectin-like polysaccharide from Rosa chinensis inhibits inflammation and fibrosis associated to HMGB1/TLR4/NF-kappaB signaling pathway to improve non-alcoholic steatohepatitis. Carbohydr Polym. 2024;337:122139. [DOI] [PubMed] [Google Scholar]
  • 28.Xu D, Qu X, Yang T, Sheng M, Bian X, Zhan Y, et al. The Foxo1–YAP–Notch1 axis reprograms STING-mediated innate immunity in NASH progression. Exp Mol Med. 2024;56:1843–1855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wu Q, Leng X, Zhang Q, Zhu YZ, Zhou R, Liu Y, et al. IRF3 activates RB to authorize cGAS-STING–induced senescence and mitigate liver fibrosis. Sci Adv. 2024;10:eadj2102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mukai K, Konno H, Akiba T, Uemura T, Waguri S, Kobayashi T, et al. Activation of STING requires palmitoylation at the Golgi. Nat Commun. 2016;7:11932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Hansen AL, Mukai K, Schopfer FJ, Taguchi T, Holm CK. STING palmitoylation as a therapeutic target. Cell Mol Immunol. 2019;16:236–241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wang ZQ, Xing WM, Fan HH, Wang KS, Zhang HK, Wang QW, et al. The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function. J Immunol. 2009;183:6646–6656. [DOI] [PubMed] [Google Scholar]
  • 33.Yan D, Hamed O, Joshi T, Mostafa MM, Jamieson KC, Joshi R, et al. Analysis of the indacaterol-regulated transcriptome in human airway epithelial cells implicates gene expression changes in the adverse and therapeutic effects of beta(2)-adrenoceptor agonists. J Pharmacol Exp Ther. 2018;366:220–236. [DOI] [PubMed] [Google Scholar]
  • 34.Zhang H, Kho AT, Wu Q, Halayko AJ, Limbert Rempel K, Chase RP, et al. CRISPLD2 (LGL1) inhibits proinflammatory mediators in human fetal, adult, and COPD lung fibroblasts and epithelial cells. Physiol Rep. 2016;4:e12942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Delpino A, Castelli M. The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane, is released into cell culture medium and is also present in human peripheral circulation. Biosci Rep. 2002;22(3–4):407–420. [DOI] [PubMed] [Google Scholar]
  • 36.Qin K, Ma S, Li H, Wu M, Sun Y, Fu M, et al. GRP78 impairs production of lipopolysaccharide-induced cytokines by interaction with CD14. Front Immunol. 2017;8:579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.He Z, Li F, Yan J, Liu M, Chen Y, Guo C. The dual role of autophagy during porcine reproductive and respiratory syndrome virus infection: A review. Int J Biol Macromol. 2024;282(pt 3):136978. [DOI] [PubMed] [Google Scholar]
  • 38.Zhang H, Sweezey NB, Kaplan F. LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts. Am J Physiol Lung Cell Mol Physiol. 2015;308:L391–L402. [DOI] [PubMed] [Google Scholar]

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