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. 2023 Feb 1;107(2):392-404.
doi: 10.1097/TP.0000000000004317. Epub 2022 Oct 12.

The Novel MyD88 Inhibitor TJ-M2010-5 Protects Against Hepatic Ischemia-reperfusion Injury by Suppressing Pyroptosis in Mice

Zhimiao Zou  1   2   3   4 , Runshi Shang  1   2   3   4 , Liang Zhou  1   2   3   4 , Dunfeng Du  1   2   3   4 , Yang Yang  1   2   3   4 , Yalong Xie  1   2   3   4 , Zeyang Li  1   2   3   4 , Minghui Zhao  1   2   3   4 , Fengchao Jiang  5 , Limin Zhang  1   2   3   4 , Ping Zhou  1   2   3   4
Affiliations
Free PMC article

The Novel MyD88 Inhibitor TJ-M2010-5 Protects Against Hepatic Ischemia-reperfusion Injury by Suppressing Pyroptosis in Mice

Zhimiao Zou et al. Transplantation. .
Free PMC article

Abstract

Background: . With the development of medical technology and increased surgical experience, the number of patients receiving liver transplants has increased. However, restoration of liver function in patients is limited by the occurrence of hepatic ischemia-reperfusion injury (IRI). Previous studies have reported that the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway and pyroptosis play critical roles in the development of hepatic IRI.

Methods: . A mouse model of segmental (70%) warm hepatic IRI was established using BALB/c mice in vivo. The mechanism underlying inflammation in mouse models of hepatic IRI was explored in vitro using lipopolysaccharide- and ATP-treated bone marrow-derived macrophages. This in vitro inflammation model was used to simulate inflammation and pyroptosis in hepatic IRI.

Results: . We found that a MyD88 inhibitor conferred protection against partial warm hepatic IRI in mouse models by downregulating the TLR4/MyD88 signaling pathway. Moreover, TJ-M2010-5 (a novel MyD88 inhibitor, hereafter named TJ-5) reduced hepatic macrophage depletion and pyroptosis induction by hepatic IRI. TJ-5 treatment inhibited pyroptosis in bone marrow-derived macrophages by reducing the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells, decreasing the release of high-mobility group box-1, and promoting endocytosis of lipopolysaccharide-high-mobility group box-1 complexes.

Conclusions: . Inhibition of MyD88 may protect the liver from partial warm hepatic IRI by reducing pyroptosis in hepatic innate immune cells. These results reveal the mechanism underlying the development of inflammation in partially warm hepatic IRI and the induction of cell pyroptosis.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1.
FIGURE 1.
Inhibition of MyD88 dimerization extenuates hepatic IRI-induced liver injury in mice. A, The effects of various concentrations of TJ-5 (25, 50, and 75 mg/kg) on liver function of normal wild-type mice. Blood samples were collected from following a single intraperitoneal injection of TJ-5 for 3 consecutive days. Blood samples were also collected from mice not administered with TJ-5. B, Serology tests of liver enzymes in all groups. The IRI + TJ-5 group had significantly lower serum levels of the indicated enzymes compared with the IRI/Sham group. C, Histopathological examination (H&E) of the liver sections in all groups; representative sections are shown. D, The severity of pathological liver injury based on Suzuki’s scores. E, Immunohistochemical staining images showing MPO-positive cells in liver tissues. Representative staining images are shown. F, A bar graph displaying the quantitative results of MPO-positive cells/mm2 per group. G, The TUNEL assay and DAPI staining results illustrating hepatocyte apoptosis levels in each group. Data are expressed as mean ± SD (n = 3~6, per group). ***P < 0.001. Data shown are representative of 3 replicate experiments. ALT, alanine transaminase; AST, aspartate aminotransferase; DAPI, 4',6-diamidino-2-phenylindole; ddH2O, double-distilled water; H&E, hematoxylin and eosin; IRI, ischemia-reperfusion injury; LDH, lactate dehydrogenase; MPO, myeloperoxidase; MyD88, myeloid differentiation factor 88; TJ-5, TJ-M2010-5; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling.
FIGURE 2.
FIGURE 2.
TJ-5 suppresses pyroptosis and reduces the secretion of pro-inflammatory factors in mice models of hepatic IRI. A, Staining results of MyD88 (red) and DAPI (blue) in liver tissues. Average IOD values of MyD88 are shown. B, The MyD88 expression level in Kupffer cells in each group. C, Western blotting analysis showing proteins levels of GSDMD and cleaved GSDMD (GSDMD-N) in liver tissues. D, Quantification of Western blot results of GSDMD and GSDMD-N. E, RT-PCR analysis of mRNA expression levels of Il1b and Il18 mRNA in liver tissues. F and G, Levels of serum inflammatory factors as determined by ELISA. H, The representative immunofluorescence images of stained liver sections with F4/80-specific antibody and DAPI indicating the number of macrophages. Quantitative analysis of the number of F4/80-positive cells/mm2. Data results are expressed as mean ± SD (n = 3~6, each group). *P < 0.05, **P < 0.01, and ***P < 0.001. All data are representative of 3 replicates experiments. ELISA, enzyme-linked immunoassay; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSDMD, gasdermin D; IL, interleukin; IOD, integral optical density; IRI, ischemia-reperfusion injury; mRNA, messenger RNA; MyD88, myeloid differentiation factor 88; RT-PCR, real-time polymerase chain reaction; TJ-5, TJ-M2010-5; TNF-α, tumor necrosis factor-α.
FIGURE 3.
FIGURE 3.
TJ-5 may suppress pyroptosis by inhibiting the TLR4/MyD88/NF-κB signaling pathway in mice models of hepatic IRI. A and B, Western blot analysis showing TLR4 and MyD88 expression levels in liver tissues. C, The mRNA expression levels of TLR4 and MyD88 in liver tissue. A histogram showing the relative expression levels. D, Western blotting analysis indicating the expression level of NF-κB p65 in the cytosolic and nuclear domains. E and F, Western blotting results illustrating expression level of NLRP3, pro-caspase-1, cleaved caspase-1, pro-caspase-11, and cleaved caspase-11 in liver tissue samples in the indicated groups. G–J, Quantification of Western blotting bands normalized to proteins bands of GAPDH. Data are expressed as mean ± SD (n = 3~6, each group). *P < 0.05, **P < 0.01, and ***P < 0.001. All data are representative of 3 replicate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRI, ischemia-reperfusion injury; mRNA, messenger RNA; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TJ-5, TJ-M2010-5; TLR4, Toll-like receptor 4.
FIGURE 4.
FIGURE 4.
TJ-5 attenuates LPS- and ATP-induced pyroptosis of BMDMs. The cells and cell culture supernatants were collected 2 h following ATP treatment. A, Flow cytometry analysis was performed to determine the purity of the BMDM population-based on CD11b and F4/80 antibodies. B, The effects of various concentrations of TJ-5 (1–70 μM) on BMDM viability as determined by the CCK8 assay. C, The PI staining illustrating the proportion of nonviable/dying cells per group; representative results are shown. D, The LDH levels in cell culture supernatants of BMDMs were quantified by an automatic biochemical analyzer. E, Western blotting analysis showing protein expression levels of GSDMD and GSDMD-N in BMDMs in all groups. F, Quantification of Western blot results of GSDMD and GSDMD-N. Data are expressed as mean ± SD (n = 3 samples each group). *P < 0.05, **P < 0.01, and ***P < 0.001. All results are from at least 3 independent repeat experiments. ATP, adenosine triphosphate; ddH2O, double-distilled water; ddH2O, double-distilled water; BMDM, treated bone marrow-derived macrophage; CCK8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; FSC-A, forward-scatter area; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSDMD, gasdermin D; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; ns, not significant; PE, phycoerythrin; PI, propidium iodide; SSC-A, side scatter area; TJ-5, TJ-M2010-5.
FIGURE 5.
FIGURE 5.
Inhibition of MyD88 reduces the secretion of inflammatory factors in BMDMs treated with LPS and ATP. A and B, The real-time PCR results showing mRNA levels of Il1b, Il18, Il6, and Tnfa in BMDMs. C and D, The expression level of specific inflammatory cytokines (IL-1β, IL-18, IL-6, and TNF-α) in cell culture supernatants of BMDMs based on ELISA. A histogram showing summarized results. Data are expressed as mean ± SD (n = 3 samples each group). *P < 0.05, **P < 0.01, and ***P < 0.001. All the experimental results shown in the Figures are from at least 3 independent repeat experiments. BMDM, bone marrow-derived macrophage; ELISA, enzyme-linked immunoassay; IL, interleukin; LPS, lipopolysaccharide; mRNA, messenger RNA; MyD88, myeloid differentiation factor 88; PCR, polymerase chain reaction; TNF-α, tumor necrosis factor-α.
FIGURE 6.
FIGURE 6.
TJ-5 reduces LPS- and ATP-induced canonical pyroptosis in BMDMs by suppressing the TLR4/MyD88/NF-κB signaling pathway. A and D, Western blotting results showing protein levels of NLRP3, pro-caspase-1, cleaved caspase-1, TLR4, and MyD88 in BMDMs. E, Western blotting analysis indicating the expression levels of NF-κB p65 in cytosolic and nuclear domains of BMDMs. B, C, F, and G, A histogram showing Western blot bands. Data are expressed as mean ± SD (n=3 samples each group). *P < 0.05, **P < 0.01, and ***P < 0.001. Each independent experiment included 3 replicates. BMDM, bone marrow-derived macrophage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, nucleotide-binding oligomerization domain-like receptors family pyrin domain containing 3; TJ-5, TJ-M2010-5; TLR4, Toll-like receptor 4.
FIGURE 7.
FIGURE 7.
TJ-5 may alleviate noncanonical pyroptosis in BMDMs by inhibiting HMGB1 secretion and reducing endocytosis of LPS-HMGB1 complexes. A and B, Western blotting results showing protein expression levels of pro-caspase-11 and cleaved caspase-11. C, The expression level of HMGB1 in cell culture supernatants of BMDMs as determined by ELISA. D and E, Western blotting results indicating the protein expression level of RAGE. F, BMDMs were treated with LPS-FITC (green) and ATP with or without TJ-5. Immunofluorescence microscopic images showing cellular localization of HMGB1 (red) and LPS. The nuclear region was stained with Hoechst (blue). Representative confocal immunofluorescence microscopic images are shown. G, BMDMs were pretreated with or without TJ-5 for 2 h and then stimulated with LPS (500 ng/mL) and HMGB1 (400 μg/mL) for 8 h. Immunofluorescence microscopic images showing cellular localization of HMGB1 (red) and LPS; the nuclear region was stained with Hoechst (blue). Representative confocal immunofluorescence microscopic images are shown. Data are expressed as mean ± SD (n = 3 samples each group). *P < 0.05, **P < 0.01, and ***P < 0.001. Each independent experiment included 3 replicates. BMDMs, bone marrow-derived macrophages; ELISA, enzyme-linked immunoassay; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMGB1, high-mobility group box-1; LPS, lipopolysaccharide; RAGE, receptor for advanced glycation end products; TJ-5, TJ-M2010-5.
FIGURE 8.
FIGURE 8.
A diagram illustrating the potential signaling pathway underlying development of pyroptosis in macrophages. Previous studies have shown that pyroptosis occurs exclusively in myeloid innate immune cells during hepatic IRI. LPS or other endogenous factors activate TLR4/MyD88/NF-κB, which induces the nuclear translocation of NF-κB. The activated NF-κB signaling pathway increases cellular expression of inflammatory cytokines and activation of NLRP3. The activated NLRP3 forms a complex termed NLRP3 inflammasome with ASC and pro-caspase-1. The inflammasome complex transforms pro-caspase-1 into mature form (caspase-1). Activated caspase-1 cleaves pro-IL-1β and pro-IL-18 into active IL-1β and IL-18, respectively, and GSDMD into GSDMD-N, resulting in the induction of pyroptosis because of the formation of pores on the cellular membrane. This process is referred to as canonical pyroptosis. In the present study, results showed that pyroptosis triggered the release of inflammatory cytokines and HMGB1. LPS-HMGB1 complex enters the cytoplasm through the endolysosome pathway stimulating endocytosis. The intracytoplasmic LPS released from the endolysosome directly activates caspase-11, which then cleaves GSDMD into GSDMD-N resulting in occurrence of pyroptosis. This pathway is known as the noncanonical pathway of pyroptosis. The diagram was created using the BioRender website (https://biorender.com/). ASC, apoptosis-associated speck-like protein; GSDMD, gasdermin D; HMGB1, high-mobility group box-1; IL, interleukin; IRI, ischemia-reperfusion injury; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, nucleotide-binding oligomerization domain-like receptors family pyrin domain containing 3; RAGE, receptor for advanced glycation end products; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α.
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