Myeloid Differentiation Protein 2 Mediates Angiotensin II-Induced Liver Inflammation and Fibrosis in Mice

Abstract

Angiotensin II (Ang II) participates in the pathogenesis of liver injury. Our previous publications reported that myeloid differentiation protein 2 (MD2) mediates Ang II-induced cardiac and kidney inflammation by directly binding to Ang II. Thus, we hypothesize that MD2 is critical to Ang II-induced liver injury. Subcutaneous injections of Ang II for 8 weeks were adopted to build the liver injury model. With a specific MD2 inhibitor L6H21 and MD2 knockout mice, we reported that MD2 inhibition and knockout significantly mitigate liver inflammation and fibrosis in mice injected with Ang II. To be more specific, the functional and pathological damages induced by Ang II were mitigated by L6H21 or MD2 knockout. MD2 knockout or L6H21 administration inhibited the Ang II-induced upregulation of fibrosis markers, inflammatory cytokines, and adhesion molecules in gene or protein levels. The activation of NF-κB and Extracellular signal-regulated kinases (ERK) induced by Ang II was also reversed by L6H21 treatment or MD2 deficiency. Note that the co-immunoprecipitation study showed that L6H21 downregulated the ANG II-induced toll-like receptor 4 (TLR4)/MD2 complex in liver tissues while having no effects on MD2 expression. Our results reported the critical role of MD2 in the progress of liver injury and suggested that MD2 is a potential therapeutic target for liver injury.

Keywords: Angiotensin II; L6H21; inflammation; liver injury; myeloid differentiation 2.

Figure 1

Liver myeloid differentiation protein 2 (MD2) expression was upregulated by Angiotensin (Ang) II. C57BL/6 mice were injected with Ang II (1.4 mg/kg/day in phosphate buffer, pH 7.2) or PBS (Control) for 8 weeks (n = 7 in each group). Liver tissues were harvested. (A) MD2 expression was ascertained by Western blot (IB) analysis (representative of five independent determinations). (B) ANG II increased mouse liver mRNA levels of MD2. Real-time qPCR assay was used to examine the mRNA expression of MD2. The mRNA values were normalized to the housekeeping gene β-actin and reported as mean ± SEM (n ≥ 5, ^# p < 0.05, vs. Control group).

Figure 2

MD2 inhibition or MD2 knockout protected mice from Ang II-induced liver injury and dysfunction. (A) The structure of L6H21. (B,C) Liver function was ascertained by measuring (B) aspartate aminotransferase (AST) and (C) serum alanine aminotransferase (ALT) level in serum. (D) Representative histopathological variations in liver tissue ascertained with hematoxylin and eosin (H&E) staining (images captured at 200× magnification). Data are expressed as mean ± SEM (n ≥ 5, ^## p < 0.01, vs. Control group; NS, no significant difference vs. Control group; * p < 0.05, vs. Ang II group).

Figure 3

MD2 inhibition or MD2 knockout protected mice from Ang II-induced liver fibrosis. Mouse liver samples were prepared as described in materials and methods. (A–D) The mRNA levels of CTGF (A), α-SMA (B), COL-4 (C), and TGF-β (D) in liver tissues were ascertained by real-time qPCR. Representative light micrographs of the histochemical assessment of liver tissues: Sirius Red staining (E) and α-SMA immunohistochemistry (F) were employed for the detection of fibrosis (images captured at 200× magnification). (G) Quantification of the collagen area in panel E. (H) Quantification of the α-SMA-positive area in panel F. (I) The protein level of α-SMA in liver tissue was ascertained by Western blot. Data are presented as mean ± SEM (n ≥ 5, ^# p < 0.05, ^## p < 0.01, vs. Control group; NS, no significant difference, vs. Control group; * p < 0.05, ** p < 0.01, vs. Ang II group).

Figure 4

MD2 inhibition or MD2 knockout protected mice from Ang II-induced liver inflammation. Mouse liver samples were prepared as described in Materials and Methods. (A–D) The mRNA levels of IL-6 (A), TNF-α (B), VCAM-1 (C), and ICAM-1 (D) in liver tissues were ascertained by real-time qPCR. Data are presented as mean ± SEM (n ≥ 5, ^# p < 0.05, ^## p < 0.01, vs. Control group; NS, no significant difference vs. Control group; * p < 0.05, ** p < 0.01, vs. Ang II group). (E) The protein levels of ICAM-1 and VCAM-1 in liver tissues were ascertained by Western blot. (F) IκB-α degradation and ERK phosphorylation were ascertained by Western blot. (G) The complex of MD2/toll-like receptor 4 (TLR4) and input MD2 expression in liver tissue were determined by immunoprecipitation and Western blot, respectively.