Hepatectomy is a feasible and relatively safe procedure for managing different liver diseases, such as liver cancer, liver abscess, hepatic cyst and liver trauma, even used in living donor liver transplantation(Anugwom, Leventhal, & Debes, 2022; X. Li, et al., 2018). Clinical outcomes of patients received hepatectomy are strongly dependent on the proliferative ability of remaining hepatocytes(Michalopoulos & Bhushan, 2021). Deficiency of liver regeneration will lead to serious complications, for example, hepatectomy liver failure, which further causes severe clinical problem. Improving the liver regeneration comes to be a promising and available therapy to prevent hepatectomy liver failure and could reduce the degree of morbidity and mortality after hepatectomy(Golse, et al., 2013; Ray, Mehta, Golhar, & Nundy, 2018). Although a growing number of researches have been conducted to reveal this complex regulatory process, the mechanism of liver regeneration still keeps obscure and no clinically available therapeutic agents exist, which all contribute to a high degree of morbidity and mortality resulted from impaired/dysfunctional liver regeneration.
Although CIRP plays a positive role through up-regulation gene related to hepatocyte proliferation during certain carcinoma development, no researches have been performed to explore whether CIRP can regulate liver regeneration after hepatectomy(Pibiri, 2018). In this study, we have shown that CIRP protein level in the liver of WT mice was increased during regeneration, with a peak at 24 ~ 48 h after hepatectomy, which suggested an important role of CIRP during the progression into G1/S phase of the cell cycle. Further analysis is necessary to determine how and by which mechanisms CIRP is regulated during regeneration. We also conducted two-thirds partial hepatectomy on WT and CIRP KO mice to investigate the relation of CIRP and liver regeneration. The ratio of liver to body weight and Ki6 staining level are commonly used to evaluate the ability of liver regeneration(B. Zhang, et al., 2022). Our research results demonstrated that the ratio of liver to body weight and Ki67-positive staining level peaked at the 3rd day, while those levels were lower in CIRP KO group compared to control group, indicating that CIRP deficiency inhibited liver regeneration. A wide variety of cytokines, growth factors, and hormones and their downstream signaling pathways are involved in liver regeneration(Ozaki, 2020). Among these regulatory factors, the STAT3 pathway, as a principle signaling pathway, has been extensively studied(Fazel Modares, et al., 2019; Hu, et al., 2020). Meanwhile, previous studies demonstrated that CIRP expression is positively correlated with the activation of STAT3, further promoting cell proliferation and survival in tumor(Sakurai, et al., 2015). Therefore, we focused on investigating whether CIRP promoted hepatocyte proliferation through regulating STAT3 after hepatectomy. Results from loss- and gain-of-function experiments confirmed the effect of CIRP on STAT3 signaling, in which CIRP overexpression significantly up-regulated p-STAT3 level and promoted cells proliferation. Moreover, the effect of CIRP overexpression on hepatocyte proliferation was restored by the p-STAT3 antagonist Stattic. Conversely, CIRP knockdown dramatically reduced p-STAT3 level and inhibited cells proliferation, but the inhibition of cell proliferation was reversed with the STAT3 activator Colivelin. These results indicated that CIRP positively regulated hepatocyte proliferation via STAT3 signaling.
Paradoxically, CIRP deficiency restrained hepatocyte proliferation, but alleviated liver injury and oxidation stress. As shown in previous researches, CIRP can perform different functions depending on its location inside or outside the cells(Liao, et al., 2017). In response to stress, through regulating its targets, iCIRP is implicated in multiple cellular processes such as cell proliferation, cell survival, circadian modulation, telomere maintenance, and tumor formation and progression(Corre & Lebreton, 2023). After released, eCIRP induces inflammatory responses, causing tissue injury(Godwin, et al., 2015). The mechanism underlying the pro-inflammatory effects of eCIRP has been previously revealed as a DAMP to activate TLR4/MD to trigger inflammation, which can be blocked by C23(Denning, Yang, Hansen, Prince, & Wang, 2019; F. Zhang, Yang, Brenner, & Wang, 2017). Therefore, we hypothesized that CIRP played an intracellular role in promoting hepatocyte proliferation. However, once secreted, CIRP acts as a pro-inflammatory factor to promote inflammation, leading to liver injury after hepatectomy. To better understand the underlying mechanisms, the mice were treated with C23 after hepatectomy. The results showed that C23 administration did not affect the ability of liver regeneration, but it could alleviate liver damage. Meanwhile, in vitro experiments, CIRP deficiency suppressed hepatocyte proliferation, and CIRP overexpression accelerated hepatocyte proliferation. This observation indicated that CIRP may be a potential therapy target to promote liver regeneration after hepatectomy, and that simultaneous administration of C23 counteracted the effects of CIRP released as DAMPs.
Previous study showed that eCIRP has an ability to bind with IL-6 receptor, activating p-STAT3 to promote macrophage endotoxin tolerance(Zhou, et al., 2020). Therefore, we speculated whether eCIRP can promote hepatocyte proliferation by binding to the IL-6R on the hepatocyte. However, administration of different concentrations of rCIRP to HepG2 cell did not increase the cell vitality. Meanwhile, protein expression of p-STAT3, cyclinD1 and PCNA remained on the baseline, indicating that eCIRP had no direct effect on hepatocyte proliferation. Our previous study has shown that eCIRP treatment can lead to persistent ER stress in pancreatic acinar cell(Liu, et al., 2023). In this study, up-regulated BIP, p-IRE1α, XBP1s and PDI were observed after rCIRP stimulation in HepG2 cells, which were reversed by TAK242. Therefore, eCIRP may cause ER stress in hepatocyte partially through TLR4 receptor.
There were some limitations in our research. Firstly, we just adopted the model of partial hepatectomy to prove the role of CIRP, but whether CIRP can promote liver regeneration in other models such as APAP or CCL4-induced acute liver injury remained unknown. Secondly, though CIRP has been identified as a positive regulator in liver regeneration, partial hepatectomy should be conducted in mice with over-expressed CIRP to verify the effect of CIRP overexpression on liver regeneration in vivo. Thirdly, the mechanism and signaling pathway via which CIRP exerted its effects on STAT3 phosphorylation were still vague. Meanwhile, whether other signal pathways of liver regeneration were involved in the regulation of CIRP keep unsolved. All of these mentioned above require further investigation.