Myeloid-specific blockade of Notch signaling ameliorates nonalcoholic fatty liver disease in mice.

Rationale: Macrophages play a central role in the development and progression of nonalcoholic fatty liver disease (NAFLD). Studies have shown that Notch signaling mediated by transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBP-J), is implicated in macrophage activation and plasticity. Naturally, we asked whether Notch signaling in macrophages plays a role in NAFLD, whether regulating Notch signaling in macrophages could serve as a therapeutic strategy to treat NAFLD. Methods: Immunofluorescence staining was used to detect the changes of macrophage Notch signaling in the livers of human patients with NAFLD and choline deficient amino acid-defined (CDAA) diet-fed mice. Lyz2-Cre RBP-Jflox or wild-type C57BL/6 male mice were fed with CDAA or high fat diet (HFD) to induce experimental steatohepatitis or steatosis, respectively. Liver histology examinations were performed using hematoxylin-eosin (H&E), Oil Red O staining, Sirius red staining and immunohistochemistry staining for F4/80, Col1α1 and αSMA. The expression of inflammatory factors, fibrosis or lipid metabolism associated genes were evaluated by quantitative reverse transcription (qRT)-PCR, Western blot or enzyme-linked immunosorbent assay (ELISA). The mRNA expression of liver samples was profiled by using RNA-seq. A hairpin-type decoy oligodeoxynucleotides (ODNs) for transcription factor RBP-J was loaded into bEnd.3-derived exosomes by electroporating. Mice with experimental NAFLD were treated with exosomes loading RBP-J decoy ODNs via tail vein injection. In vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. Results: The results showed that Notch signaling was activated in hepatic macrophages in human with NAFLD or in CDAA-fed mice. Myeloid-specific RBP-J deficiency decreased the expression of inflammatory factors interleukin-1 beta (IL1ß) and tumor necrosis factor alpha (TNFα), attenuated experimental steatohepatitis in mice. Furthermore, we found that Notch blockade attenuated lipid accumulation in hepatocytes by inhibiting the expression of IL1ß and TNFα in macrophages in vitro. Meanwhile, we observed that tail vein-injected exosomes were mainly taken up by hepatic macrophages in mice with steatohepatitis. RBP-J decoy ODNs delivered by exosomes could efficiently inhibit Notch signaling in hepatic macrophages in vivo and ameliorate steatohepatitis or steatosis in CDAA or HFD mice, respectively. Conclusions: Combined, macrophage RBP-J promotes the progression of NAFLD at least partially through regulating the expression of pro-inflammatory cytokines IL1ß and TNFα. Infusion of exosomes loaded with RBP-J decoy ODNs might be a promising therapy to treat NAFLD.

Cellular senescence primes liver fibrosis regression through Notch-EZH2.

Cellular senescence plays a pivotal role in wound healing. At the initiation of liver fibrosis regression, accumulated senescent cells were detected and genes of senescence were upregulated. Flow cytometry combined with single-cell RNA sequencing analyses revealed that most of senescent cells were liver nonparenchymal cells. Removing senescent cells by dasatinib and quercetin (DQ), alleviated hepatic cellular senescence, impeded fibrosis regression, and disrupted liver sinusoids. Clearance of senescent cells not only decreased senescent macrophages but also shrank the proportion of anti-inflammatory M2 macrophages through apoptotic pathway. Subsequently, macrophages were depleted by clodronate, which diminished hepatic senescent cells and impaired fibrosis regression. Mechanistically, the change of the epigenetic regulator enhancer of zeste homolog2 (EZH2) accompanied with the emergence of hepatic senescent cells while liver fibrosis regressed. Blocking EZH2 signaling by EPZ6438 reduced hepatic senescent cells and macrophages, decelerating liver fibrosis regression. Moreover, the promoter region of EZH2 was transcriptionally suppressed by Notch-Hes1 (hairy and enhancer of split 1) signaling. Disruption of Notch in macrophages using Lyz2 (lysozyme 2) Cre-RBP-J (recombination signal binding protein Jκ) f/f transgenic mice, enhanced hepatic cellular senescence, and facilitated fibrosis regression by upregulating EZH2 and blocking EZH2 abrogated the above effects caused by Notch deficiency. Ultimately, adopting Notch inhibitor Ly3039478 or exosome-mediated RBP-J decoy oligodeoxynucleotides accelerated liver fibrosis regression by augmenting hepatic cellular senescence.

Age-related liver endothelial zonation triggers steatohepatitis by inactivating pericentral endothelium-derived C-kit.

Aging leads to systemic metabolic disorders, including steatosis. Here we show that liver sinusoidal endothelial cell (LSEC) senescence accelerates liver sinusoid capillarization and promotes steatosis by reprogramming liver endothelial zonation and inactivating pericentral endothelium-derived C-kit, which is a type III receptor tyrosine kinase. Specifically, inhibition of endothelial C-kit triggers cellular senescence, perturbing LSEC homeostasis in male mice. During diet-induced nonalcoholic steatohepatitis (NASH) development, Kit deletion worsens hepatic steatosis and exacerbates NASH-associated fibrosis and inflammation. Mechanistically, C-kit transcriptionally inhibits chemokine (C-X-C motif) receptor (CXCR)4 via CCAAT enhancer-binding protein α (CEBPA). Blocking CXCR4 signaling abolishes LSEC-macrophage-neutrophil cross-talk and leads to the recovery of C-kit-deficient mice with NASH. Of therapeutic relevance, infusing C-kit-expressing LSECs into aged mice or mice with diet-induced NASH counteracts age-associated senescence and steatosis and improves the symptoms of diet-induced NASH by restoring metabolic homeostasis of the pericentral liver endothelium. Our work provides an alternative approach that could be useful for treating aging- and diet-induced NASH.

Effect of comparability of Coptis chinensis and Scutellaria baicalensis on five sub-enzymatic activities of liver microsomes in rats / 中国中药杂志

To study the mechanism of metabolic interaction between Coptis chinensis and Scutellaria baicalensis. Rats were given C. chinensis and S. baicalensis for 7 days to produce hepatic microsomal enzyme. Cocktail probe substrate and liver microsome in vitro temperature incubation method were adopted. Meanwhile, the metabolic elimination percentages of the five probe substrates were detected with HPLC, in order to evaluate the effect of each administration group on the enzymatic activity of rat liver microsome CYP450. Compared with the blank group, C. chinensis obviously inhibited CYP2D6 and CYP1A2, and S. baicalensis remarkably inhibited CYP1A2, CYP2E1 and CYP2C9. The compatibility of C. chinensis and S. baicalensis with the ratio of 1:1 not only inhibited CYP1A2, but also remarkably activated CYP2D6 and CYP3A4. However, their activation effect disappeared under the ratio of 2: 1, and turned into the inhibitory effect on CYP1A2 and CYP2C9. The results showed that C. chinensis and S. baicalensis had an inhibitory effect on CYP450, but their compatibility with certain ratio resulted in double effects of activation and inhibition, which was related to their compatibility ratio. It is speculated that the inhibitory and inducing effects of C. chinensis and S. baicalensis on metabolic enzymes are among causes for their attenuation and synergistic effects.