Mice hepatic organoids for modeling nonalcoholic fatty liver disease and drug response.

Nonalcoholic fatty liver disease (NAFLD) is a serious disease. There are no specific drugs for it, in part because of the lack of effective models to aid drug development. However, it has been shown that three-dimensional organoid culture systems can reproduce the organ structure and maintain the gene expression profile of the original tissue. Therefore, we aimed to construct NAFLD models from liver organoids for pharmacological and mechanism studies. We successfully observed morphological changes in normal liver tissue in mouse liver organoids with positive albumin (ALB) expression and potential for differentiation toward hepatocyte-like cells. The mRNA expression of the hepatocyte markers ALB and hepatocyte nuclear factor 4 alpha increased after liver organoid differentiation. We observed free fatty acid (FFA)-induced lipid accumulation in organoids with significant increases in alanine aminotransferase, aspartate aminotransferase, total bilirubin, and triglyceride levels. Moreover, FFA-induced inflammatory cytokines (interleukin-6, tumor necrosis factor-α, and nitric oxide) and fibrosis indicators (collagen type I alpha 1 and laminin α1) were also increased. In addition, RNA sequencing results showed that the expression of key genes (NOD-like receptor family apoptosis inhibitory protein, interferon regulatory factor (IRF)3 and IRF7) involved in NAFLD metabolic abnormalities and insulin resistance in the NOD-like receptor signaling pathway was altered after FFA induction of the liver organoids. Finally, we found that JC2-11 and lanifibranor limited the FFA-induced increase in oil-red lipid droplets, liver damage, inflammation, and liver fibrosis. In conclusion, tissue structure, gene expression, and the response of mouse liver organoids to drugs can partially mimic in vivo liver tissue. Liver organoids can successfully construct NAFLD models for drug discovery research.

Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma.

Ferroptosis is a newly characterized form of iron-dependent non-apoptotic cell death, which is closely associated with cancer progression. However, the functions and mechanisms in regulation of escaping from ferroptosis during hepatocellular carcinoma (HCC) progression remain unknown. In this study, we reported that the RNA binding motif single stranded interacting protein 1 (RBMS1) participated in HCC development，and functioned as a regulator of ferroptosis. Clinically, the downregulation of RBMS1 occurred in HCC tissues, and low RBMS1 expression was associated with worse HCC patients survival. Mechanistically, RBMS1 overexpression inhibited HCC cell growth by attenuating the expression of glutathione peroxidase 4 (GPX4)and further facilitated ferroptosis in vitro and in vivo. More importantly, a novel circIDE (hsa_circ_0000251) was identified to elevate RBMS1 expression via sponging miR-19b-3p in HCC cells. Collectively, our findings established circIDE/miR-19b-3p/RBMS1 axis as a regulator of ferroptosis, which could be a promising therapeutic target and prognostic factor.

The anti-alcoholism drug disulfiram effectively ameliorates ulcerative colitis through suppressing oxidative stresses-associated pyroptotic cell death and cellular inflammation in colonic cells.

BACKGROUND: Oxidative stress, cell pyroptosis and inflammation are considered as important pathogenic factors for ulcerative colitis (UC) development, and the traditional anti-alcoholism drug disulfiram (DSF) has recently been reported to exert its regulating effects on all the above cellular functions, which makes DSF as ideal therapeutic agent for UC treatment, but this issue has not been fully studied. METHODS: Dextran sulfate sodium (DSS)-induced animal models in C57BL/6J mice and lipopolysaccharide (LPS)-induced cellular models in colonic cell lines (HT-29 and Caco-2) for UC were respectively established. Cytokine secretion was determined by ELISA. Cell viability and proliferation were evaluated by MTT assay and EdU assay. Real-Time qPCR, Western Blot, immunofluorescent staining assay and immunohistochemistry (IHC) were employed to evaluate gene expressions. The correlations of the genes in the clinical tissues were analyzed by using the Pearson Correlation analysis. RESULTS: DSF restrained oxidative stress, pyroptotic cell death and cellular inflammation in UC models in vitro and in vivo, and elimination of Reactive Oxygen Species (ROS) by N-acetyl-l-cysteine (NAC) rescued cell viability in LPS-treated colonic cells (HT-29 and Caco-2). Further experiments suggested that a glycogen synthase kinase-3ß (GSK-3ß)/Nrf2/NLRP3 signaling cascade played critical role in this process. Mechanistically, DSF downregulated GSK-3ß and NLRP3, whereas upregulated Nrf2 in LPS-treated colonic cells. Also, the regulating effects of DSF on Nrf2 and NLRP3 were abrogated by upregulating GSK-3ß. Moreover, upregulation of GSK-3ß abolished the protective effects of DSF on LPS-treated colonic cells. CONCLUSIONS: Taken together, data of this study indicated that DSF restrained oxidative damages-related pyroptotic cell death and inflammation via regulating the GSK-3ß/Nrf2/NLRP3 pathway, leading to the suppression of LPS-induced UC development.

A pH-sensitive and sustained-release oral drug delivery system: the synthesis, characterization, adsorption and release of the xanthan gum-graft-poly(acrylic acid)/GO-DCFP composite hydrogel.

In this study, graphene oxide (GO) was successfully prepared using the improved Hummers method, and the prepared GO powder was dissolved in distilled water and subjected to ultrasonic stripping. Diclofenac potassium (DCFP) was selected as a model drug to systematically evaluate the adsorption mechanism of DCFP by GO. Different reaction models were constructed to fit the adsorption kinetics and adsorption isotherms of DCFP on GO, in order to further explore the underlying adsorption mechanism. The results demonstrated that the pseudo-second-order kinetic model and Freundlich model could better delineate the adsorption process of DCFP by GO. Both π-π stacking and hydrophobic interaction were mainly involved in the adsorption process, and there were electrostatic interaction and hydrogen bonding at the same time. Then, the xanthan gum-graft-poly(acrylic acid)/GO (XG-g-PAA/GO) composite hydrogel was synthesized by in situ polymerization as a slow-release drug carrier. For this reason, a XG-g-PAA/GO-DCFP composite hydrogel was synthesized, and its in vitro drug release and pharmacokinetic data were assessed. The results showed that the synthesized XG-g-PAA/GO composite hydrogel had a certain mechanical strength and uniform color, indicating that GO is evenly distributed in this composite hydrogel. Moreover, the results of a swelling ratio test demonstrated that the swelling ratios of the XG-g-PAA/GO composite hydrogel were significantly increased with increasing pH values, implying that this material is sensitive to pH. The in vitro drug release experiment showed that the cumulative release of DCFP after 96 h was significantly higher in artificial intestinal fluid than in artificial gastric fluid. These findings indicate that the XG-g-PAA/GO-DCFP composite hydrogel exhibits pH sensitivity under physiological conditions. Besides, the results of in vivo pharmacokinetic analysis revealed that the t 1/2 of DCFP group was 2.03 ± 0.35 h, while that of the XG-g-PAA/GO-DCFP composite hydrogel group was 10.71 ± 2.04 h, indicating that the synthesized hydrogel could effectively prolong the drug action time. Furthermore, the AUC(0-t) of the DCFP group was 53.99 ± 3.18 mg L-1 h-1, while that of the XG-g-PAA/GO-DCFP composite hydrogel group was 116.79 ± 14.72 mg L-1 h-1, suggesting that the bioavailability of DCFP is greatly enhanced by this composite hydrogel. In conclusion, this study highlights that the XG-g-PAA/GO-DCFP composite hydrogel can be applied as a sustained-release drug carrier.

Mechanism of Roux-en-Y gastric bypass treatment for type 2 diabetes in rats.

OBJECTIVES: Roux-en-Y gastric bypass (RYGB) is a novel therapy for diabetes. We aimed to explore the therapeutic mechanism of RYGB. METHODS: After RYGB, animal models were established, and gene expression profile of islets was assessed. Additionally, gastrointestinal hormones were measured using enzyme-linked immunosorbent assays. Ca(2+) was studied using confocal microscopy and patch-clamp technique. The morphology of islets and beta cells was observed using optical microscopy and electron microscopy. RESULTS: RYGB was an effective treatment in diabetic rats. Expression profiling data showed that RYGB produced a new metabolic environment and that gene expression changed to adapt to the new environment. The differential expression of genes associated with hormones, Ca(2+) and cellular proliferation was closely related to RYGB and diabetes metabolism. Furthermore, the data verified that RYGB led to changes in hormone level and enhanced Ca(2+) concentration changes and Ca(2+) channel activity. Morphological data showed that RYGB induced the proliferation of islets and improved the function of beta cells. CONCLUSIONS: RYGB promoted a new metabolic environment while triggering changes to adapt to the new environment. These changes promoted the cellular proliferation of islets and improved the function of beta cells. The quantity of beta cells increased, and their quality improved, ultimately leading to insulin secretion enhancement.

Abnormal mitochondrial function impairs calcium influx in diabetic mouse pancreatic beta cells.

BACKGROUND: Abnormal insulin secretion of pancreatic beta cells is now regarded as the more primary defect than the insulin function in the etiology of type 2 diabetes. Previous studies found impaired mitochondrial function and impaired Ca(2+) influx in beta cells in diabetic patients and animal models, suggesting a role for these processes in proper insulin secretion. The aim of this study was to investigate the detailed relationship of mitochondrial function, Ca(2+) influx, and defective insulin secretion. METHODS: We investigated mitochondrial function and morphology in pancreatic beta cell of diabetic KK-Ay mice and C57BL/6J mice. Two types of Ca(2+) channel activities, L-type and store-operated Ca(2+) (SOC), were evaluated using whole-cell patch-clamp recording. The glucose induced Ca(2+) influx was measured by a non-invasive micro-test technique (NMT). RESULTS: Mitochondria in KK-Ay mice pancreatic beta cells were swollen with disordered cristae, and mitochondrial function decreased compared with C57BL/6J mice. Ca(2+) channel activity was increased and glucose induced Ca(2+) influx was impaired, but could be recovered by genipin. CONCLUSION: Defective mitochondrial function in diabetic mice pancreatic beta cells is a key cause of abnormal insulin secretion by altering Ca(2+) influx, but not via Ca(2+) channel activity.