Down-regulation of common NFκB-iNOS pathway by chronic Thalidomide treatment improves Hepatopulmonary Syndrome and Muscle Wasting in rats with Biliary Cirrhosis

Abstract

Thalidomide can modulate the TNFα-NFκB and iNOS pathway, which involve in the pathogenesis of hepatopulmonary syndrome (HPS) and muscle wasting in cirrhosis. In bile duct ligated-cirrhotic rats, the increased circulating CD16⁺ (inflammatory) monocytes and its intracellular TNFα, NFκB, monocyte chemotactic protein (MCP-1) and iNOS levels were associated with increased circulating MCP-1/soluable intercellular cell adehesion molecule-1 (sICAM-1), pulmonary TNFα/NOx, up-regulated M1 polarization, exacerbated angiogenesis and hypoxemia (increased AaPO₂) in bronchoalveolar lavage (BAL) fluid and pulmonary homogenates. Meanwhile, a significant correlation was noted between circulating CD16⁺ monocyte/M1 (%) macrophages in BAL; M1 (%) macrophages in BAL/pulmonary iNOS mRNA expression; pulmonary iNOS mRNA expression/relative pulmonary MVD; pulmonary NOx level/AaPO₂; circulating CD16⁺ monocyte/M1 (%) macrophages in muscle homogenates; 3-nitrotyrosine (representative of peroxynitrite) concentration/M1 (%) macrophages in muscle homogenates. The in vitro data demonstrated an iNOS-dependent inhibition of thalidomide on the TNFα-stimulated angiogenesis and myogenesis in human pulmonary artery endothelial cells (HPAECs) and C2C12 myoblasts. Significantly, the co-culture of CD16⁺ monocyte from different rats with HPAECs, or co-culture of supernatant of above mixed cultures with HPAECs or C2C12 myoblasts stimulated angiogenesis, migration and myogenesis. Our findings demonstrate that TNFα inhibitor thalidomide markedly diminishes the severity of experimental HPS and muscle wasting by down-regulation of common peripheral and local NFκB-iNOS pathway.

Figure 1. Effects of thalidomide on rat circulating monocyte and pulmonary macrophages.

(A–D) Intracellular cytokines levels in circulating CD16⁺ monocytes; (E) number of M1/M2 macrophages [F4/80(+)/CD11c(+)cells]/[F4/80(+)/CD206(+) cells] and M1/M2 genes expression (G) in BAL fluid; (F) pulmonary relative MVD (microvascular density); (H) pulmonary TNFα levels; (I) Arterial blood gas (ABG) data-A-a gradient (AaPO₂); ^#,##p < 0.05, 0.01 vs. S-V; *p < 0.05 vs. BDL-V.

Figure 2. Effects of thalidomide on cirrhotic lungs.

(A,B) mRNA/protein expressions. (C) correlation between M1 macrophages (%) in BAL fluid and circulating CD16⁺ monocyte (%); (D) correlation between M1 macrophages (%) in BAL fluid and iNOS mRNA level in lung; (E) correlation between iNOS mRNA level in lung and relative pulmonary MVD; (F) total pulmonary nitric oxide (NOx) level; (G) correlation between pulmonary NOx level and AaPO₂ (mmHg); (H) HPAECs tube formation after various treatments. ^#,##p < 0.05, 0.01 vs. S-V (buffer only group); *p < 0.05 vs. BDL-V; ^δp < 0.05 vs. TNFα groups; ^†p < 0.05 vs. TNFα + thalidomide (thal) groups.

Figure 3. In vitro effects of thalidomide on HPAECs.

(A) HPAECs migration assays after various treatments; (B) various mRNA expression in HPAECs’ cell lysates; (C) angiogenic, (D) migration index in co-cultivation of CD16⁺ monocyte from different rats with HPAECs or system of co-incubated HPAECs with supernatant of co-cultured CD16⁺ monocyte + HPAECs; (E) ratio of mRNA expressions in co-cultured CD16⁺ monocyte+HPAECs to those in mono-cultured HPAECs. ^#,##p < 0.05, 0.01 vs. S-V; *p < 0.05 vs. BDL-V; ^δp < 0.05 vs. TNFα groups; ^†p < 0.05 vs. TNFα+thalidomide (thal) groups.

Figure 4. Effect of thalidomide treatment on the cirrhotic muscles.

IHC images for measurement of (A) cross-section area of muscle fibers with α-sarcometric actin antibody and (D) macrophages infiltration with CD68 antibody; (B,C) various mRNAs/proteins expression; (E) numbers of M1/M2 macrophages in muscle homogenates; (F) correlation between muscular M1 macrophages (%) in BAL fluid and circulating CD16⁺ monocyte (%); (G) 3-nitrotyrosin (representative of peroxynitrite) concentration in anterior tibialis muscle homogenates. ^#,##p < 0.05, 0.01 vs. S-V; *p < 0.05 vs. BDL-V.

Figure 5. Effects of thalidomide treatment on myogenic profiles.

(A) Correlation between muscular 3-nitrotyrosine concentration and M1 macrophages (%); correlation between muscular (B) MyoD and (C) MHC II mRNA expression with 3-nitrotyrosine concentration in anterior tibialis muscle homogenates. (D) Representative immunofluorensence (IF) image and bar graphs of acute effects of thalidomide on fusion index and myotube diameter of C2C12 cells; (E) various mRNA expression in C2C12s’s cell lysates; (F) relative fusion index and (G) myotube diameter in co-culture of CD16⁺ monocyte collected from different rats with C2C12 cells or co-incubation of supernatant of co-cultured CD16⁺ monocyte + C2C12 cells with C2C12 cells; (H) ratio of mRNA expressions in co-cultured CD16⁺ monocyte+C2C12 cells to those in mono-cultured C2C12 cells. ^#,##p < 0.05, 0.01 vs. S-V; ^*,**p < 0.05, 0.01 vs. BDL-V; ^δp < 0.05, 0.01 vs. TNFα groups; ^†p < 0.05 vs. TNFα+thalidomide (thal) groups.

Figure 6. Schematic representative hypothesis for anti-sarcopenia and anti-angiogenesis effect of chronic thalidomide treatment on cirrhotic rats of our study.

Abbreviations: BDL: bile duct ligation; TNFα: tumor necrosis factor-alpha; MCP-1: monocyte chemoattractic protein-1; VEGF: vascular endothelial growth factor; iNOS: inducible nitric oxide synthase; NFκB: nuclear factor kappa B; HPAEC: human pulmonary artery endothelial cells; BAL: bronchoalveolar lavage; MVD: microvascular density; MHC: myosin heavy chain.