Cyclophilin Inhibitor NV556 Reduces Fibrosis and Hepatocellular Carcinoma Development in Mice With Non-Alcoholic Steatohepatitis

Abstract

Hepatocellular carcinoma (HCC), the third major cause of cancer mortality, can result from non-alcoholic steatohepatitis (NASH). Due to limited efficacy of drugs approved for HCC and no drug available yet for NASH, identification of new effective treatments is crucial. Here, we investigated whether NV556, a cyclophilin inhibitor derived from sanglifehrins, would decrease the development of NASH and HCC in a preclinical mouse model. For our experiment, male mice were administered streptozotocin to disrupt pancreatic cells and nourished with high-fat diet since 3 weeks of age. Afterward, NV556 or vehicle was orally administered daily for 6 weeks before the 14-week-old time point for the development of NASH, or 10 weeks before the 30-week-old time point for the establishment of HCC. Body weight, blood glucose level, and liver weight were recorded. Moreover, for NASH, livers were histologically examined for inflammation and steatosis. Collagen was measured by Sirius Red staining of hepatic tissues. Systemic cytokine levels in serum were detected by multiplex assays. For HCC, nodules of livers were measured and scored according to a developed system with number and size of nodules as criteria. NV556 significantly decreased collagen deposition (p = 0.0281), but did not alter inflammation, steatosis, body and liver weight, and systemic cytokine production compared to control mice with NASH symptoms. For HCC, NV556 statistically reduced the number (p = 0.0091) and diameter of tumorous nodules (p = 0.0264), along with liver weight (p = 0.0026) of mice.Our study suggests NV556 as a promising candidate for treatment of NASH-derived fibrosis and HCC.

Keywords: cytokines; histology; inflammation; steatosis; tumor burden.

Figure 1

NV556 reduced liver fibrosis at NASH timepoint. (A) Flowchart for studies on NASH and fibrosis. Fibrosis was induced in male C57BL/6J mice by intraperitoneal injection of 200 µg of STZ 2 days after birth followed by a free-feeding high-fat diet (60% kcal fat) after 3 weeks of age for 11 weeks. 50mg/kg NV556 or vehicle control was administered via oral gavage daily to mice for 42 days before sacrifice at 14 weeks of age. (B) Body weight (left), blood glucose level (middle), and liver weight (right) of mice. (C) Hematoxylin and eosin (H&E) staining on paraffin-embedded liver sections. Bar indicates 50μm. (D) Ballooning, inflammation, and steatosis scoring on H&E stained tissues. (E) Liver fibrosis identified by Sirius red staining (left) and quantified by ImageJ (right). Bar indicates 100μm. (F) Percentage of Sirius Red staining. All error bars indicate ± standard error from the mean, with p-values from two-tailed Mann-Whitney test.

Figure 2

Representations of nodular livers with tumor burden scoring. Diameter and numbers of detectable nodules in entire livers of mice were considered for scoring from 0 to 7, as seen in Table 1. (A) No detectable nodules; score of 0. (B) Only and no more than 4 small nodules; score of 1. (C) More than 4 small detectable nodules, with no larger nodules; score of 2. (D) Less than 3 medium-sized nodules and no larger nodules; score of 3. (E) 3 or more medium-sized nodules with no larger nodules; score of 4. (F) No more than 1 large nodule; score of 5. (G) No more than 2 large nodules; score of 6. (H) 3 or more large nodules; score of 7. Nodules in yellow circles indicate those fitting each scoring criterium.

Figure 3

NV556 decreased body weight, liver weight, tumor burden, inflammation, and fibrosis in mice with NASH-induced HCC. (A) Flowchart for studies on HCC. HCC was induced in male C57BL/6J mice by intraperitoneal injection of 200 µg of STZ 2 days after birth followed by a free-feeding high-fat diet (60% kcal fat) after 3 weeks of age for 27 weeks. Fifty mg/kg NV556 or vehicle control was administered via oral gavage daily to mice for 70 days before sacrifice at 30 weeks of age. (B) Body weight (left), blood glucose level (middle), and liver weight (right) of mice. (C) Number of detectable nodules (left) and nodular scoring for tumor burden (right) in livers of mice. (D) H&E staining on paraffin-embedded liver sections. Bar indicates 50μm. (E) Inflammation, steatosis, and ballooning scoring on H&E stained tissues. (F) Liver fibrosis identified by Sirius red staining (left) and quantified by ImageJ (right). Bar indicates 100μm. (G) Percentage of Sirius Red staining. All error bars indicate ± standard error from the mean, with p-values from two-tailed Mann-Whitney test.