In our study, it indicated that aberrant Wnt5a expression and JNK activation were detected in a mouse ALF model induced by D-Gal/LPS. In vivo, pretreatment of Box5, a Wnt5a antagonist, restored JNK activation, and exactly attenuated D-Gal/LPS-induced liver injury, as indicated by changes in liver pathology and ALT/AST levels, as well as decreased the mortality rate. In vitro, we demonstrated that downstream inflammatory cytokines expression and phagocytosis of THP-1 macrophages induced by rWnt5a were dependent on JNK activation, which could be reversed by Box5. In addition, rWnt5a-induced migration of THP-1 macrophages was also turned by Box5.
ALF is a deadly clinical disorder characterized by overwhelming hepatocyte death and rapid deterioration of normal liver function. Central to the pathogenesis of ALF is dysfunction of inflammation and immune response to various causes, which in turn exacerbates hepatocellular necrosis and apoptosis [24, 25]. ALF induced by D-Gal/LPS has been widely used as an animal model to elucidate pathogenesis and evaluate the efficiency of hepatoprotective agents [23, 26]. LPS, part of outer membrane of gram-negative bacteria, can greatly stimulate secretion of pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α . D-Gal, a specific hepatotoxic agent, can greatly increase hepatocellular death by enhancing their sensitivity to inflammatory injury . Resembling human ALF, co-injection of D-Gal and LPS into mice in our study resulted in large liver necrotic foci, increased hepatocellular apoptosis, loss of liver function and elevated expression of inflammatory cytokines.
Since it was first reported that cytokines such as IL-6, Interleukin-8 (IL-8), and Interleukin-15 (IL-15) were upregulated by Wnt5a in rheumatoid arthritis synovial fibroblasts , there has been huge interest in Wnt5a signaling in inflammatory process. A subsequent study indicated that Wnt5a promotes Interleukin-12 (IL-12) synthesis and enhances the inflammation of human mononuclear cells induced by microbial stimulation . Consistent with another research indicating that Wnt5a was upregulated in sera of patients with sepsis , our previous study showed that serum Wnt5a was increased in patients with ACHBLF compared with that in patients with CHB and healthy controls . In our present study, we also suggested that liver Wnt5a protein expression was significantly elevated in mice of D-Gal/LPS group contrast to that in Control group by immunohistochemistry. However, two forms of Wnt5a with distinct molecular weights were detected in liver tissues, when we further determined our results by Western blot. We observed an increase in the bigger molecular weight of Wnt5a (∼58 kDa) in mice with D-Gal/LPS administration. Similar findings that Wnt5a has two or more forms in one tissue have been observed in murine lung tissue  and neuron , although their Wnt5a were detected with different molecular weights from ours. Wnt proteins heavily rely on posttranslational modifications, such as glycosylation and palmitoylation, to secret and function, and they can also agglomerate into multimeric or oligomerized complexes [32, 33]. Perhaps, it partly explains our results, which undoubtedly needs further in-depth study to confirm. To get more insight of Wnt5a in ALF, we also evaluate the Wnt5a transcription by qPCR. Interestingly, Wnt5a mRNA expression in liver tissues of mice in D-Gal/LPS group was decreased compared with that in Control group, just opposite to its protein pattern. This contradictory expression of Wnt5a transcription and translation in liver tissue of D-Gal/LPS mice was similar to that observed in hepatocellular carcinoma and para-carcinoma liver tissues . Thus, we speculated that some unclear mechanisms remain in the regulation of Wnt5a protein expression except posttranslational modifications, which exactly requires us to explore in the future. Nevertheless, these data suggested that Wnt5a signaling was involved in the development of ALF.
To determine the potential role of Wnt5a in ALF, we evaluate the effect of Wnt5a inhibition on the development of ALF with its antagonist, Box5. Box5 has been described to be a competitive inhibitor of Wnt5a through binding to its receptor to inhibit the biological activity of Wnt5a signaling . Our results in the present study clearly showed that Wnt5a inhibition by Box5 alleviated pathologic severity, ameliorated liver function, and decreased mortality rate of ALF mice. In addition, Box5 pretreatment could also reduce the stimulation of inflammatory cytokines, like IL-1β, IL-6, TNF-α and IL-10 induced by D-Gal/LPS, although not significant. These results were consistent with those in diabetic nephropathy suggested by Li and et al . Taken together, it indicated that Wnt5a inhibition by Box5 could be a potential therapeutic strategy for ALF.
Noncanonical Wnt5a signaling comprises two main pathways: Wnt5a/Ca2+ pathway and the Wnt5a/JNK or planar cell polarity (PCP) pathway . Therein JNK signaling was widely demonstrated to be involved in inflammation [36, 38, 39]. Activated JNK participated in stimulation of many inflammatory cytokines by LPS [39, 40]. Phosphorylation of JNK mediated hepatoxicity in acetaminophen-induced ALF . Moreover, by attenuating JNK-mediated mitochondrial translocation, D-Gal/LPS-induced ALF could be ameliorated . JNK inhibition with two different JNK inhibitors in vivo markedly reduced hepatic necrosis and apoptosis in paracetamol-induced ALF . In this regard, we focused on JNK signaling as the downstream pathway of Wnt5a in our present research. As we expected, activated JNK was detected in liver tissues of mice in D-Gal/LPS group, and restored by Box5 pretreatment. In summary, results manifested that Wnt5a/JNK signaling play an important role in presence and progression of ALF.
Basal Wnt5a expression was observed in PBMC, PBMC-derived macrophage, alveolar macrophage, microglia and macrophage cell lines . We found that Wnt5a was overexpressed on liver macrophages rather than hepatocytes. Dysfunction of monocyte and macrophage is central to ALF development . As is widely known, it is necessary for the initiation and propagation of ALF that liver macrophages recognize and phagocyte pathogens or debris and stimulate inflammatory cytokines expression, as well as monocytes are recruited to differentiate into macrophages to expand the macrophage pool and promote tissue destruction. Considering above aspects, we explored the role of Wnt5a/JNK signaling in activation of THP-1 macrophages in vitro, which originally exist as one of monocyte cell lines, but was induced to differentiate into macrophage by PMA in our study. Results indicated that rWnt5a could induce increased mRNA expression of TNF-α and IL-6, and enhance the phagocytosis and migration abilities of THP-1 macrophages. These cellular regulations were dependent on JNK signaling except migration, as the JNK inhibitor, SP600125 totally abolished them. Box5 not only restored the above modulations of THP-1 macrophages but also blocked the activation of JNK induced by rWnt5a. To sum up, our findings supported that JNK signaling participated in the regulation of macrophages and Wnt5a was a regulator of JNK signaling. Furthermore, Wnt5a/JNK signaling participated in the development of ALF probably by regulating the activation of macrophages.
There are some limitations in our present study. One is that the mechanism under the contradictory expression of Wnt5a mRNA and protein expression remains unclear. Since our study focused more on the function of Wnt5a protein, more researches concerned on the regulation of transcription and translation are needed in the future. Another one is that the Wnt5a-knockout mice model is deserved to be the most accurate experimental tool to investigate its function. Unfortunately, homozygous Wnt5a-knockout mice exhibit perinatal lethality, due to developmental defects  and mice with Wnt5a-siRNA may not be blocked in liver Wnt5a protein expression, as suggested by our above results. Therefore, we selected Box5, stated as a Wnt5a antagonist, to inhibit Wnt5a protein in mice. If the more insight of Wnt5a is further determined, conditional Wnt5a-konck out mice model may be the best option in future researches.
In conclusion, our findings provide strong evidence that aberrant Wnt5a/JNK signaling mediated massive hepatocellular necrosis and apoptosis, increased serum ALT and AST, and elevated inflammatory cytokines expression in D-Gal/LPS-induced ALF mice. Box5, as wnt5a antagonist could efficiently abolish these mediations and eventually improved the outcomes of D-Gal/LPS-induced ALF mice. Moreover, Wnt5a, expressed primarily on liver macrophages, was demonstrated to induce the activation of THP-1 macrophages in a JNK-dependent manner, which were also reversed by Box5. Overall, our results supported that Wnt5a/JNK signaling was involved in the development of ALF, partly via the regulation of macrophages and Box5 may be a potential effective agent for the treatment of ALF.