Deficiency of CD147 Attenuated Non-alcoholic Steatohepatitis Progression in an NLRP3-Dependent Manner

Figures

• 

  FIGURE 1. CD147 was increased during NASH progression in MCD diet-induced C57/BL6 mice. (A) Representative H&E staining images of tissues from NASH model mice. (B) Serum ALT and AST levels in NASH model mice (n = 6). (C) The mRNA levels of inflammatory cytokines in liver tissues from mice with NASH (n = 6). (D) Serum IL-1β and IL-18 levels in NASH model mice (n = 6). (E) Protein levels of CD147 in liver tissues from mice with NASH (n = 2). (F) The mRNA levels of CD147 in liver tissues from mice with NASH (n = 6). (G) IHC staining for CD147 in liver tissues of NASH model mice. One-way ANOVA followed by Dunnett’s test was used to compare NASH model mice with normal group. *p < 0.05, **p < 0.01, and ***p < 0.001.

• 

  FIGURE 2. Increased expression of CD147 in liver tissues from NAFLD patients. (A) IHC staining for CD147 in human liver tissues from NAFLD patients and healthy donors. (B) Protein levels of CD147 in liver tissues from normal, steatosis, and NASH patients. Band relative intensity was normalized by β-actin, and unpaired t test was used for statistical analysis. *p < 0.05.

• 

  FIGURE 3. CD147 hepatocyte-specific deletion impeded NASH progression in MCD diet-fed mice. Bsgflx/flx and Alb;Bsgflx/flx mice were fed an MCD or control diet for 2 weeks. (A) Representative images of H&E and Oil Red O staining of liver sections. (B) Serum ALT and AST levels (n = 6). One-way ANOVA followed by Bonferroni’s multiple comparison test was used to compare indicated group. **p < 0.01, ***p < 0.001. (C) Representative TUNEL images of liver sections and numbers of apoptotic TUNEL-positive cells (n = 6). Unpaired t test was used to compare apoptotic cell numbers. *p < 0.05. (D) Hepatic protein levels of Bax and Bcl-2. Band relative intensity was normalized by α-tubulin, and One-way ANOVA followed by Bonferroni’s multiple comparison test was used for statistical analysis. *p < 0.05, **p < 0.01, and ***p < 0.001. (E) mRNA levels of IL-1β and IL-18 (n = 5). One-way ANOVA followed by Bonferroni’s multiple comparison test was used to compare each group. *p < 0.05, **p < 0.01. (F) Serum levels of IL-1β and IL-18 (n = 5). One-way ANOVA followed by Bonferroni’s multiple comparison test was used to compare each group. *p < 0.05.

• 

  FIGURE 4. CD147 deficiency in hepatocytes inhibited activation of the NF-κB/NLRP3 signaling pathway. (A) Protein levels of NLRP3 in liver tissues of C57/BL6 NASH model mice (n = 2). Band relative intensity was normalized by α-tubulin, and one-way ANOVA followed by Dunnett’s test was used for statistical analysis. *p < 0.05. (B) mRNA levels of NLRP3 in liver tissues of C57/BL6 NASH model mice (n = 6). One-way ANOVA followed by Dunnett’s test was used for statistical analysis. *p < 0.05 and **p < 0.01. (C) Protein levels of NLRP3 in Bsgflx/flx and Alb;Bsgflx/flx mice fed control or MCD diet (n = 2). (D) mRNA levels of NLRP3 in Bsgflx/flx and Alb;Bsgflx/flx mice fed control or MCD diet (n = 5). One-way ANOVA followed by Bonferroni’s multiple comparison test was used to compare each group. ***p < 0.001. (E) Protein levels of p65 and p-p65 in Bsgflx/flx and Alb;Bsgflx/flx mice fed control or MCD diet (n = 3). One-way ANOVA followed by Bonferroni’s multiple comparison test was used to compare each group. *p < 0.05 and **p < 0.01. (F) Protein levels of nuclear p65 in Bsgflx/flx and Alb;Bsgflx/flx mice fed control or MCD diet (n = 3). One-way ANOVA followed by Bonferroni’s multiple comparison test…

• 

  FIGURE 5. CD147 deficiency in hepatocytes regulated NLRP3 inflammasome activation due to the induction of CypA. (A) Serum levels of CypA in C57/BL6 NASH model mice (n = 6). One-way ANOVA followed by Dunnett’s test was used for statistical analysis. *p < 0.05. (B) Representative images of Oil Red O staining of primary hepatocytes isolated from C57/BL6 mice. (C) Protein levels of CD147 in primary hepatocytes isolated from C57/BL6 mice. (D) mRNA levels of CD147 in primary hepatocytes isolated from C57/BL6 mice. Unpaired t test was used for statistical analysis. **p < 0.01. (E) Primary hepatocytes were isolated from Bsgflx/flx or Alb;Bsgflx/flx mice and treated with CypA (2 μg/ml) and/or MCD medium for 24 h. Protein levels of p-p65, p65, NLRP3, and CD147 were detected. (F) MIHA cells were transfected with pcDNA3.1 or pcDNA3.1-CD147 and treated with DMSO or PDTC (100 μM) for 24 h. Protein levels of p-p65, p65, NLRP3, and CD147 were detected.

• 

  FIGURE 6. The CypA inhibitor TMN355 attenuated the severity of MCD diet-induced NASH in mice. Starting 7 days after MCD feeding, C57/BL6 mice were treated with TMN355 (10 and 20 mg/kg) or vehicle (DMSO) three times weekly before sacrifice. (A) Representative images of H&E and Oil Red O staining of liver sections. (B) Serum ALT and AST levels (n = 6). (C) Liver TG content (n = 6). One-way ANOVA followed by Dunnett’s test was used to compare each group with MCD + DMSO group. *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Protein levels of NLRP3, p65 and p-p65 in MCD diet-induced C57/BL6 mice that were treated with TMN355 or DMSO.

• 

  FIGURE 7. Molecular mechanisms by which CypA and CD147 contribute to NASH progression. Under MCD diet feeding, damaged hepatocytes release CypA, and CD147 expression is enhanced. The formation of the CypA/CD147 complex results in an inflammatory response through the NF-κB/NLRP3 signaling pathway.