Sujun Zheng

Vitamin D in liver diseases: From mechanisms to clinical trials

Traditionally regarded as a typical vitamin regulating calcium and phosphorus homeostasis, vitamin D is now discovered as a highly versatile molecule with emerging roles in immunity, cancer, infectious diseases, fibrosis, fatty liver diseases, and alcoholic liver diseases. A large body of clinical evidence has demonstrated the prevalence and risks of vitamin D deficiency in various chronic diseases. Biologically active vitamin D, 1,25-dihydroxylvitamin D3, is synthesized in two distinct systems. In addition to the classic two-step hydroxylation in the liver and kidneys, 1,25-dihydroxylvitamin D3 can also be produced locally by immune cells in response to infection. The bioactive vitamin D generated in these two pools apparently functions differently: while the former facilitates calcium adsorption and homeostasis, the latter confers immune regulation. The immune regulatory functions of vitamin D are demonstrated by induction of antimicrobial peptides, suppression of innate immune response, induction of Th2 cytokines, and stimulation of T-regulatory T cells. Vitamin D deficiency or insufficiency is overwhelmingly associated with viral hepatitis, cirrhosis, and fatty liver diseases. Recent clinical trials have shown that vitamin D supplements significantly enhance the efficacy of interferon plus ribavirin therapy through sustained virological response. A recent study showed that 25-dihydroxyvitamin D rather than 1,25-dihydroxyvitamin D could directly suppress hepatitis C virus assembly. Moreover, clinical evidence has shown that vitamin D deficiency is associated with alcoholic and non-alcoholic fatty liver diseases. In this review, we highlight some recent advances in vitamin D researches and clinical trails.

Vitamin D Signaling through Induction of Paneth Cell Defensins Maintains Gut Microbiota and Improves Metabolic Disorders and Hepatic Steatosis in Animal Models

Metabolic syndrome (MetS), characterized as obesity, insulin resistance, and non-alcoholic fatty liver diseases (NAFLD), is associated with vitamin D insufficiency/deficiency in epidemiological studies, while the underlying mechanism is poorly addressed. On the other hand, disorder of gut microbiota, namely dysbiosis, is known to cause MetS and NAFLD. It is also known that systemic inflammation blocks insulin signaling pathways, leading to insulin resistance and glucose intolerance, which are the driving force for hepatic steatosis. Vitamin D receptor (VDR) is highly expressed in the ileum of the small intestine, which prompted us to test a hypothesis that vitamin D signaling may determine the enterotype of gut microbiota through regulating the intestinal interface. Here, we demonstrate that high-fat-diet feeding (HFD) is necessary but not sufficient, while additional vitamin D deficiency (VDD) as a second hit is needed, to induce robust insulin resistance and fatty liver. Under the two hits (HFD+VDD), the Paneth cell-specific alpha-defensins including α-defensin 5 (DEFA5), MMP7 which activates the pro-defensins, as well as tight junction genes, and MUC2 are all suppressed in the ileum, resulting in mucosal collapse, increased gut permeability, dysbiosis, endotoxemia, systemic inflammation which underlie insulin resistance and hepatic steatosis. Moreover, under the vitamin D deficient high fat feeding (HFD+VDD), Helicobacter hepaticus, a known murine hepatic-pathogen, is substantially amplified in the ileum, while Akkermansia muciniphila, a beneficial symbiotic, is diminished. Likewise, the VD receptor (VDR) knockout mice exhibit similar phenotypes, showing down regulation of alpha-defensins and MMP7 in the ileum, increased Helicobacter hepaticus and suppressed Akkermansia muciniphila. Remarkably, oral administration of DEFA5 restored eubiosys, showing suppression of Helicobacter hepaticus and increase of Akkermansia muciniphila in association with resolving metabolic disorders and fatty liver in the HFD+VDD mice. An in vitro analysis showed that DEFA5 peptide could directly suppress Helicobacter hepaticus. Thus, the results of this study reveal critical roles of a vitamin D/VDR axis in optimal expression of defensins and tight junction genes in support of intestinal integrity and eubiosis to suppress NAFLD and metabolic disorders.

Sevoflurane causes neuronal apoptosis and adaptability changes of neonatal rats

Neonatal exposure to sevoflurane can induce neurodegeneration and learning deficits in developing brain. We hypothesised that with the increase in the concentration and duration of sevoflurane, neurodegeneration of neonatal rats aggravates and causes behaviour changes as the rats grow.

Vitamin D deficiency promotes nonalcoholic steatohepatitis through impaired enterohepatic circulation in animal model

Vitamin D deficiency (VDD) or insufficiency is recognized for its association with nonalcoholic steatohepatitis (NASH), whereas the underlying mechanism remains unknown. Using animal models, we found that vitamin D deficiency promoted the high-fat diet (HFD)-initiated simple steatosis into typical NASH, characterized by elevated hepatic inflammation and fat degeneration. The NASH derived from VDD + HFD was related to poor retention of bile acids in the liver and biliary tree, in line with downregulation of the ileal apical sodium-dependent bile acid cotransporter (iASBT). The impediment of hepatic bile acids by the VDD + HFD mice was related to increased expression of hepatic SREBP-1c and fatty acid synthase, suggesting that VDD may upregulate endogenous fatty acid synthesis into NASH through impaired enterohepatic circulation. Administration of 1,25(OH)2VD3 (calcitriol) corrected the NASH phenotypes in line with restoration of iASBT, promotion of bile filling in the biliary tree, suppression of hepatic lipogenesis, and inflammation. Moreover, administration of a bile acid-sequestering agent suppressed ileal fibroblast growth factor 15 expression, leading to increased iASBT expression to restore bile filling in the liver and biliary tree, which ameliorates steatosis and inflammation in the liver. These results suggest a novel mechanism for NASH development, by which VDD downregulates iASBT expression, resulting in a poor bile acid pool and elevation of hepatic lipogenesis and inflammation. In conclusion, vitamin D and bile acid sequestration may be explored as new strategies to treat or prevent NASH.

Inhibition of glycogen synthase kinase 3β ameliorates D-GalN/LPS-induced liver injury by reducing endoplasmic reticulum stress-triggered apoptosis.

Background Glycogen synthase kinase 3β(GSK3β) is a ubiquitous serine-threonine protein kinase that participates in numerous cellular processes and disease pathophysiology. We aimed to determine therapeutic potential of GSK3β inhibition and its mechanism in a well-characterized model of lipopolysaccharide (LPS)-induced model of acute liver failure (ALF). Methodology In a murine ALF model induced by D-GalN(700 mg/kg)/LPS(10 µg/kg), we analyzed GSK3β mechanisms using a specific chemical inhibitor, SB216763, and detected the role of endoplasmic reticulum stress (ERS). Mice were administered SB216763 at 2 h before or after D-GalN/LPS injection, respectively, and then sacrificed 6 h after D-GalN/LPS treatment to evaluate its prophylactic and therapeutic function. The lethality rate, liver damage, ERS, cytokine expression, MAP kinase, hepatocyte apoptosis and expression of TLR 4 were evaluated, respectively. Whether the inhibition of GSK3β activation protected hepatocyte from ERS-induced apoptosis was investigated in vitro. Principal Findings GSK3β became quickly activated (dephosphorylated) upon D-GalN/LPS exposure. Administration of SB216763 not only ameliorated liver injury, as evidenced by reduced transaminase levels, and well-preserved liver architecture, but also decreased lethality. Moreover, GSK3β inhibition resulted in down-regulation of pro-apoptotic proteins C/EBP–homologous protein(CHOP) and caspase-12, which are related to ERS. To further demonstrate the role of ERS, we found that GSK3β inhibition protected hepatocyte from ERS-induced cell death. GSK3β inhibition down-regulated the MAPK pathways, reduced expression of inflammatory cytokines and decreased expression of TLR4. Conclusions Our findings demonstrate the key function of GSK3β signaling in the pathophysiology of ALF, especially in regulating the ERS, and provide a rationale for targeting GSK3β as a potential therapeutic strategy to ameliorate ALF.

Restoration of intrahepatic regulatory T cells through MMP-9/13-dependent activation of TGF-β is critical for immune homeostasis following acute liver injury

During the acute liver injury, immune responses are provoked into eliciting inflammation in the acute phase. In the healing phase, the inflammation is terminated for wound healing and restoration of immune homeostasis. In this study, we sought to address how regulatory T cells (Tregs) are involved in the progression of liver injury and repair. In the acute phase, intrahepatic Tregs (CD4(+)FoxP3(+)Helios(+)) diminished promptly through apoptosis, which was followed by inflammation and tissue injury. In the healing phase, a new subset of Tregs (CD4(+)Foxp3(+)Helios(-)) was generated in correlation with the matrix metalloproteinase (MMP) cascade and transforming growth factor-beta (TGF-β) activation that were manifested mainly by hepatic stellate cells. Moreover, the induction of induced Tregs and wound healing were both impaired in mice lacking TGF-β signaling or MMPs. The depletion of induced Tregs also impeded wound healing for tissue repair. Together, this study demonstrates the mechanism that the loss of nTregs through apoptosis in the acute phase may facilitate inflammation, while regenerated Tregs through MMP9/13-dependent activation of TGF-β in the healing phase are critical to terminate inflammation and allow for wound healing.

Spontaneous liver fibrosis induced by long term dietary vitamin D deficiency in adult mice is related to chronic inflammation and enhanced apoptosis.

Epidemiological studies have revealed an association between vitamin D deficiency and various chronic liver diseases. However, it is not known whether lack of vitamin D can induce spontaneous liver fibrosis in an animal model. To study this, mice were fed either a control diet or a vitamin D deficient diet (VDD diet). For the positive control, liver fibrosis was induced with carbon tetrachloride. Here we show, for the first time, that liver fibrosis spontaneously developed in mice fed the VDD diet. Long-term administration of a VDD diet resulted in necro-inflammation and liver fibrosis. Inflammatory mediators including tumor necrosis factor-α, interleulin-1, interleukin-6, Toll-like-receptor 4, and monocyte chemotactic protein-1 were up-regulated in the livers of the mice fed the VDD diet. Conversely, the expression of Th2/M2 markers such as IL-10, IL-13, arginase 1, and heme oxygenase-1 were down-regulated in the livers of mice fed the VDD diet. Transforming growth factor-β1 and matrix metalloproteinase 13, which are important for fibrosis, were induced in the livers of mice fed the VDD diet. Moreover, the VDD diet triggered apoptosis in the parenchymal cells, in agreement with the increased levels of Fas and FasL, and decreased Bcl2 and Bclx. Thus, long-term vitamin D deficiency can provoke chronic inflammation that can induce liver apoptosis, which consequently activates hepatic stellate cells to initiate liver fibrosis.

Serum sphingomyelin has potential to reflect hepatic injury in chronic hepatitis B virus infection

OBJECTIVE To explore the relation between serum sphingolipids and hepatic injury in chronic HBV infection. METHODS A cohort of participants including 48 healthy persons, 103 chronic HBV-infected patients containing chronic hepatitis B (CHB) and HBV-related cirrhosis were included. High performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was performed to detect serum sphingolipids. The serological indicators were detected and quantified. The valid liver biopsy specimens were acquired from twenty five CHB. RESULTS Twenty four serum sphingolipids were detected. There were eighteen sphingolipids showing significant differences between the healthy control and chronic HBV infection groups. In patients with chronic HBV infection, fourteen sphingolipids differed significantly between CHB and HBV-related cirrhosis. Among sphingolipids with a significant difference in both HBV infection vs healthy control and CHB vs cirrhosis, seven sphingolipids were independently related to the presence of cirrhosis. SM(d18:1/24:0), a sphingomyelin (SM) compound, was found to have a negative correlation with model for end-stage liver disease (MELD) score. Additionally, SM(d18:1/24:0) was demonstrated to have a correlation with inflammation grades by liver biopsy in CHB patients. CONCLUSIONS Serum sphingolipids have close relation with hepatic injury in chronic HBV infection, especially that SM(d18:1/24:0) might be a potential serum biomarker.

Interleukin-1 as an Injury Signal Mobilizes Retinyl Esters in Hepatic Stellate Cells through Down Regulation of Lecithin Retinol Acyltransferase

Retinoids are mostly stored as retinyl esters in hepatic stellate cells (HSCs) through esterification of retinol and fatty acid, catalyzed by lecithin-retinol acyltransferase (LRAT). This study is designated to address how retinyl esters are mobilized in liver injury for tissue repair and wound healing. Initially, we speculated that acute inflammatory cytokines may act as injury signal to mobilize retinyl esters by down-regulation of LRAT in HSCs. By examining a panel of cytokines we found interleukin-1 (IL-1) can potently down-regulate mRNA and protein levels of LRAT, resulting in mobilization of retinyl esters in primary rat HSCs. To simulate the microenvironment in the space of Disse, HSCs were embedded in three-dimensional extracellular matrix, by which HSCs retaine quiescent phenotypes, indicated by up-regulation of LRAT and accumulation of lipid droplets. Upon IL-1 stimulation, LRAT expression went down together with mobilization of lipid droplets. Secreted factors from Kupffer cells were able to suppress LRAT expression in HSCs, which was neutralized by IL-1 receptor antagonist. To explore the underlying mechanism we noted that the stability of LRAT protein is not significantly regulated by IL-1, indicating the regulation is likely at transcriptional level. Indeed, we found that IL-1 failed to down-regulate recombinant LRAT protein expressed in HSCs by adenovirus, while transcription of endogenous LRAT was promptly decreased. Following liver damage, IL-1 was promptly elevated in a close pace with down-regulation of LRAT transcription, implying their causative relationship. After administration of IL-1, retinyl ester levels in the liver, as measured by LC/MS/MS, decreased in association with down-regulation of LRAT. Likewise, IL-1 receptor knockout mice were protected from injury-induced down-regulation of LRAT. In summary, we identified IL-1 as an injury signal to mobilize retinyl ester in HSCs through down-regulation of LRAT, implying a mechanism governing transition from hepatic injury to wound healing.

Persistence of cirrhosis is maintained by intrahepatic regulatory T cells that inhibit fibrosis resolution by regulating the balance of tissue inhibitors of metalloproteinases and matrix metalloproteinases

Fibrosis is the result of the abnormal accumulation of the extracellular matrix and ineffective clearance of fibroplasia. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunosuppressive lymphocytes that are highly expressed in the fibrotic tissues and peripheral blood of patients with cirrhosis or hepatocellular carcinoma. The role of Tregs in the progression of liver fibrosis is not well understood. Our experiments reveal that abundant of Tregs was scattered around sites of fibroplasia. Conversely, the depletion of Tregs promoted the resolution of liver fibrosis. As a consequence of Tregs depletion, the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was altered; mmp9 and timp1 were reduced, whereas mmp2 and mmp14 were enhanced. The mmp9/timp1, mmp13/timp1, and mmp14/timp2 ratios were significantly increased in association with fibrosis resolution. Kupffer cells (KCs) are the main source of MMP. We observed that when KCs were cocultured with Tregs, the Tregs were able to inhibit MMP expression of KCs even at a low ratio; and anti-transforming growth factor-β (TGF-β) significantly reversed the inhibition of Tregs on MMP. Meanwhile, we also found that after Tregs depletion, TGF-β levels decreased in the mice liver, unlike in fibrosis. Furthermore, double depletion of both KCs and Tregs did not cause fiber resolution in mice. Thus, our results demonstrate that the persistence of liver cirrhosis is maintained by increased Tregs in the sites of fibroplasia and the subsequent regulation of the MMP/TIMP balance and that the suppression of KC-mediated MMP expression contributed to the regulatory process.