Cholestasis liver injury is a pathophysiological process induced by bile secretion and excretion disorders. A variety of factors, such as drugs, oxidative stress, inflammatory injury and immune disorders, are considered as the causes of cholestasis . Currently, adults with different phenotypes of cholestasis have increasingly been evaluated for variants in these genes to identify specific cholestasis-related genes [14,15]. Excessive accumulation of bile components, including bile acid, cholesterol and bilirubin in hepatic and systemic circulation is considered as the major driver of liver injury. Long-term continuous cholestasis could develop into liver fibrosis and even cirrhosis .
Although UDCA could slow disease progression in some cholestasis patients, UDCA unsensitivity and intolerance remain outstanding issues . The limited treatment effect of UDCA cholestasis indicates that novel therapeutic approaches are required.
Currently, MSC transplantation is increasingly applicated in researches due to its therapeutic potential in a variety of liver diseases. Investigators suggested the effect of MSCs in promoting liver tissue repair and survival rates in acute liver failure, hepatectomy, hepatitis B virus-related acute-on-chronic liver failure, ischemic-type biliary lesions, liver fibrosis, liver transplantation and related graft-vs-host disease, et, al [17–24]. Although rash and fever (37–38°C) that resolved without additional treatment were observed in several patients , no MSC transplantation-related safety issues were detected in either short- or long-term follow-up [26,27]. All these previous studies indicate that MSC transplantation is an ideal candidate for cholestasis treatment.
In this study, we demonstrated that MenSCs could attenuate the development of DDC-induced liver injury. Injected MenSCs could accumulate in mouse livers and significantly improve emaciation, jaundice and mortality. Intrahepatic bile duct dilation, cholestasis and concomitant fibrosis are the main pathological changes in DDC mice and could be reduced by MenSC therapy.
However, previous studies indicated that although transplanted MSCs were recruited to injured liver sites, few cells differentiated into HLCs . Most researchers believe that MSCs exhibit treatment effects mainly through paracrine activities . Thus, we mainly focused on identifying the target of MenSC treatment in DDC mouse models.
According to TEM images, MenSCs could repair TJ structural injury in the liver caused by DDC feeding. The blood–bile barrier (BBlB) is primarily composed of TJs , represents a physical barrier formed by liver epithelial cells and hepatocytes, which separates bile from blood sinusoids . Loss of BBIB is believed to be the main cause of cholestasis liver injury . To determine the mechanism of MenSC treatment in DDC-induced cholestasis liver injury, TMT-based quantitative proteomic analysis was used to select target pathways and molecules. After the GSEA, GO and KEGG analysis, TJ pathway is selected as one of the regulators involved in liver damage and treatment. Combined with western blot verification, we found that MenSCs could restore the expression of claudins (including Claudin-1, Claudin-3, Claudin-5 and Claudin-7) and Occludin, the two major families of tetraspanins at TJs in DDC mouse livers. Despite the proteomics results, considering that cholestasis could be partly attributed to bile transport function disorder [32,33] we detected expression changes in OATP2, BSEP and NTCP1 to evaluate bile transport functions in different groups. We found that MenSC could also restore bile transporter levels inhibited by DDC feeding.
Liver fibrosis is believed to be the most important pathological change caused by cholestasis. Our results showed that MenSC transplantation could reduce liver fibrosis and downregulate TGF-β1 and α-SMA expressions in DDC mice. We hypothesize that MenSCs may downregulate fibrosis-related pathways by improving cholestasis, thereby inhibiting the progression of liver fibrosis.
Furthermore, we demonstrated that β-Catenin is a key target of MenSC treatment. MenSCs promote the repair of TJs and bile transport function damage by upregulating the expression of liver β-Catenin, which is inhibited by DDC, thereby inhibiting the progression of liver fibrosis. We found that hepatic β-Catenin knockdown did not cause significant liver damage in normal mice but inhibited the therapeutic effect of MenSC transplantation in DDC-induced hepatic cholestasis and fibrosis. Furthermore, β-Catenin knockdown could inhibit MenSCs’ regulation of TJ and bile transport function related proteins and pathways in DDC mice.
Thompson et al. reported that the upregulation of β-Catenin in mice could enhance the resolution of intrahepatic cholestasis after chronic DDC administration for 150 d . According to Tao et al., mice with β-catenin-deficient hepatocytes demonstrated increased liver injury following the DDC diet . These studies are consistent with our results.
However, the research by Saggi et al. suggested that β-catenin might play an opposite role relative to that in our study in the DDC-induced liver injury model . According to Saggi et al., in the mice fed a DDC diet for 2 weeks, inhibiting β-catenin could result in decreased liver injury. Conflicting results were also noticed by Utiey et al. . In their study, β-catenin upregulation did not improve liver injury based on the assessment of AST and ALT levels, which is consistent with the results of Thompson et al. After two weeks of DDC feeding, excessive β-catenin expression and activation failed to improve serum biochemical markers of hepatic injury . However, researchers observed that β-catenin was over-activated and the liver function tests improved at 150 d. We hypothesized that β-catenin may play different regulatory roles at different stages of DDC-induced liver injury. This hypothesis needs further explorations, and the efficiency of MSC treatment at different stages of injury on the efficacy requires further investigation.
In addition, although we hypothesized that MenSCs transplantation could inhibit liver fibrosis induced by cholestasis, an in-depth study of the mechanism was not conducted. To date, a series of studies have indicated that β-catenin could be a protective factor against cholestasis-induced fibrosis.
Interestingly, we noticed that β-catenin aggravates hepetic fibrosis in carbon tetrachloride (CCl4)-induced models. Li et al. reported that inhibiting the Wnt/β-catenin signaling pathway could attenuate CCl4-induced hepatic fibrosis in rats . According to Rao et al., β-catenin promotes hepatic fibrosis by activating hepatic stellate cells in CCl4-induced mouse models . The different regulatory effects of β-catenin in different liver fibrosis models attracted our attention. The role of β-catenin in hepatic fibrosis induced by a variety of pathological reasons is worthy of further exploration.