JQ-1 ameliorates schistosomiasis liver fibrosis by suppressing JAK2 and STAT3 activation

Schistosomiasis is a serious parasitic infection caused by Schistosoma. The parasite deposits eggs in the host liver, causing inflammation that activates hepatic stellate cells (HSCs), which leads to liver fibrosis. Currently, there is no effective therapy for liver fibrosis; thus, treatments are urgently needed. Therefore, in the present study, mice infected with Schistosoma japonicum were treated with JQ-1, a small-molecule bromodomain inhibitor with reliable anti-tumor and anti-inflammatory activities. The fibrotic area of the liver measured by computer-assisted morphometric analysis and the expression levels of the cytoskeletal protein alpha smooth muscle actin (α-SMA) and of collagen assessed by quantitative PCR and immunohistochemistry were significantly decreased in the liver following JQ-1 treatment compared with vehicle-treated controls. Total RNA was extracted from the liver of JQ-1–treated Schistosoma-infected mice for RNA-sequencing analysis. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that JQ-1 affected biological processes and the expression of cellular components known to play key roles in HSC transdifferentiation into myofibroblasts. In vitro treatment with JQ-1 of JS-1 cells, a mouse HSC line, indicated that JQ-1 significantly inhibited JS-1 proliferation but had no effect on JS-1 activity, senescence, or apoptosis. Western blot results showed that JQ-1 inhibited the expression of phosphorylated JAK2 and phosphorylated STAT3 without altering expression levels of these non-phosphorylated proteins. Taken together, these findings suggested that JQ-1 treatment ameliorated S. japonicum egg–induced liver fibrosis, at least in part, by suppressing HSC activation and proliferation through the inhibition of JAK2/STAT3 signaling. These results lay a foundation for the development of novel approaches to treat and control liver fibrosis caused by S. japonicum. Author summary When a host is infected with Schistosoma, a common parasite that affects more than a million people and hundreds of thousands of livestock, the parasite deposits eggs in the liver of the host. The eggs lead to liver inflammation, which activates stellate cells in the liver. These stellate cells generate most of the excessive extracellular matrix that replaces healthy liver parenchyma with fibrous tissue, causing liver fibrosis. Schistosoma japonicum causes the most severe liver damage of all the schistosome parasites. Therefore, inhibiting the activation of the liver stellate cells and removing the activated stellate cells are key strategies for treating liver fibrosis caused by this parasite. JQ-1 is a potent inhibitor of the BET family of bromodomain proteins and is structurally similar to inhibitors being tested in clinical trials for various types of cancers. Here we found that administering JQ-1 to mice infected with S. japonicum decreased the degree of liver fibrosis. JQ-1 inhibited the activation and proliferation of liver stellate cells by blocking the phosphorylation and thus the activation of JAK2 and STAT3 to achieve its therapeutic effects. Thus, this study provides insights into the development of new therapeutic strategies for Schistosoma-induced liver fibrosis.


Introduction
Among the three major schistosomes known to infect humans (Schistosoma japonicum, 69 Schistosoma mansoni, and Schistosoma haematobium), S. japonicum causes the most 70 severe pathological damage. In China, S. japonicum is a common parasite associated 71 with chronic liver fibrosis [1]. Currently, zoonotic schistosomiasis caused by S. 72 japonicum is major public health threat affecting more than a million people and 73 hundreds of thousands of livestock in China [1]. Egg-induced lesions in the liver and 74 intestinal wall of the host are important complications of infection with S. japonicum. 75 The immune response of the host to the eggs results in granuloma and fibrosis [2]. 76 Because the mechanism of fibrosis is not fully understood, there is no effective therapy 77 for liver fibrosis. Although the U.S. Food and Drug Administration (FDA) has recently 78 approved pirfenidone and nintedanib as treatments, additional new therapies are needed 79 for treating liver fibrosis [3]. of JQ-1 on hepatic fibrosis caused by S. japonicum. 94 Therefore, the aim of the present study was to explore the mechanisms of action 95 underpinning the therapeutic effect of JQ-1 by using a mouse model of hepatic fibrosis 96 naturally caused by S. japonicum. We found that the degree of liver fibrosis in mice 97 treated with JQ-1 was significantly decreased, mainly through inhibiting the activation 98 and proliferation of hepatic stellate cells (HSCs). The activation and proliferation of  Oncomelania hupensis snails infected with S. japonicum (provided by Hunan 119 Provincial Institute of Parasitic Diseases in China) were exposed to light for 3-4 h (25-120 28 °C) to induce the release of cercariae. Hepatic fibrosis was experimentally induced 121 in 18 mice by infecting them with 18 ± 2 of these cercariae through the skin of abdomen.

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The infected mice were randomly divided into two groups, experimental and control, 123 with nine mice in each group.     Fisher's exact test, and P ≤ 0.05 was considered statistically significant.      The degree of liver cell damage was evaluated by measuring the enzyme levels of AST 289 and ALT in the serum of the mice. As shown in Figure 1F, serum transaminase activity 290 in the JQ-1 group was significantly lower than that in the HP-β-CD group (P < 0.05).

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In the JQ-1 group, the level of hydroxyproline in the liver tissue of mice was also 13 292 significantly lower than that in the HP-β-CD group ( Figure 1G, P < 0.05). Taken 293 together, these results indicated that the degree of liver fibrosis in the JQ-1 group was 294 significantly decreased. The immunohistochemical results showed that the expression levels of α-SMA and 299 Col1a1 in mouse liver significantly decreased after JQ-1 treatment ( Fig. 2A and 2B).    Table   324 2). Based on the KEGG pathway database, a pathway analysis was performed to predict  Table   330 3).

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In cytology studies, we found that although JQ-1 had no effect on cell viability (Fig.   334 4A) or senescence-associated β-galactosidase activity (Fig. 4B), it significantly 335 inhibited the proliferation of JS-1 cells (Fig. 4C). The results of a TUNEL assay (Fig.   336 4D and 4E) provided further evidence that this inhibition of JS-1 cells was specific to 337 proliferation and was not caused by apoptosis.  with JQ-1 in S. japonicum egg-induced liver fibrosis. We found that JQ-1 treatment 363 significantly attenuated S. japonicum egg-induced fibrosis and significantly improved 364 the gross appearance of the liver (Fig. 1). We also observed that JQ-1 treatment  The JAK/STAT signaling pathway transmits extracellular information from the cell 395 membrane to the nucleus to modulate the transcription of target genes, including genes 396 whose protein products are crucial for defense against pathogens, differentiation, 397 proliferation, and apoptosis [15]. The JAK2/STAT3 signaling pathway is directly To further investigate the mechanism by which JQ-1 inhibits HSC activation in the 417 present study, the mouse HSC line (JS-1 cells) was cultured and JQ-1 administered in 418 subsequent in vitro experiments. We found that the expression levels of p-JAK2 and p-419 STAT3 were significantly reduced in the JQ-1 treated JS-1 cells (Fig. 6), suggesting BET to acetylated lysine, which can regulate numerous targets to affect hepatic fibrosis.

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Suppressing JAK2 and STAT3 activation may be a JQ-1 mechanism that inhibits 426 schistosomal hepatic fibrosis. However, our study did not determine whether one of the 427 BET proteins is responsible for the transcription of genes involved in the activation and 428 proliferation of HSCs; thus, the underlying mechanism requires future investigation.