Browsing by Author "Dong, Charlie X."

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    Elucidating the Role of Biliary Senescence and Mast Cell-Mediated Therapy in Non-Alcoholic Fatty Liver Disease
    (2023-05) Kundu, Debjyoti; Francis, Heather; Dong, Charlie X.; Alpini, Gianfranco; Linnemann, Amelia; Ekser, Burcin
    Non-alcoholic fatty liver disease, or NAFLD, is characterized by excess fat deposition in the liver. Cellular senescence is a critical hallmark of NAFLD. Cholangiocytes in the liver plays a significant role in the progression of fatty liver by contributing to senescence. p16 is the main senescent protein expressed by cholangiocytes in primary sclerosing cholangitis (PSC). Thus, we aimed to downregulate p16 by vivo-morpholino and evaluate the disease phenotypes and signaling mechanisms in a murine model of NAFLD. We found that downregulation of p16 reduced i) steatosis), ii) inflammation, iii) fibrosis, and cholangiocyte proliferation in HFD mice compared to the HFD-fed, control vivo-morpholino injected mice. Moreover, the downregulation of p16 reduced insulin-like growth factor-1 (IGF-1) in cholangiocytes, previously identified by our laboratory as a principal SASP factor secreted from cholangiocytes during NAFLD. By ingenuity pathway analysis, we found that p16 might regulates IGF-1 expression via the E2F1/FOXO1axis. Further analyses indicate that p16 downregulation reduces E2F1 mRNA transcription, inhibiting FOXO1 and subsequent IGF-1 expression in cholangiocytes. The presence of mast cells in the liver has been implicated in multiple cholangiopathies. Our lab demonstrated that mast cell stabilization by cromolyn sodium treatment reduced histamine secretion, fibrosis, and biliary proliferation in Mdr2-/- mice, a model of PSC. Thus, we aimed to determine mast cell stabilization as a therapeutic approach to managing NAFLD and its more advanced form, NASH. We found that cromolyn sodium ameliorated i) serum histamine levels, ii) intrahepatic mast cells, iii) inflammation, iv) fibrosis, v) steatosis, and cholangiocyte proliferation in methionine choline deficient diet-fed mice compared to the saline controls. Overall, we report that amelioration of senescence is a critical factor in improving the disease phenotypes in NAFLD. Biliary senescence plays a crucial role in modulating the disease progression in NAFLD, and mast cell stabilization can be used as a therapeutic approach to reduce pathological hallmarks of fatty liver.
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    Melatonin-MT1 Signaling Axis Ameliorates the Phenotypes of Primary Sclerosing Cholangitis
    (2023-03) Ceci, Ludovica; Alpini, Gianfranco; Francis, Heather; Ekser, Burcin; Dong, Charlie X.; Maiers, Jessica L.
    Background: Primary Sclerosing Cholangitis (PSC) is characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N-acetyltransferase (AANAT) in cholangiocytes. We found that: (i) the MT1 receptor is primarily expressed in cholangiocytes; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases ductular reaction (DR) and liver fibrosis in cholestatic rats by downregulation of MT1 and clock genes. Melatonin administration to male Mdr2-/- mice (PSC model) reduces angiogenesis and portal inflammation via decreased miR-200b. Downregulation of maspin triggers angiogenesis during tumorigenesis by interaction with glutathione S-transferase (GST). We aimed to evaluate the effects of long-term melatonin treatment and MT1 signaling on PSC phenotypes in Mdr2-/- mice. Methods: Male FVB/NJ and Mdr2-/- mice had access ad libitum to drinking water with/without melatonin for 3 months. Immortilized-SV40-cholangiocytes isolated from human liver samples (control and PSC) were treated with melatonin (10-3 mol/L) for 24 hr. Male C3H-Hej (WT for MT1-/-), FVB/NJ (WT for Mdr2-/-), MT1-/-, Mdr2-/- mice and MT1-/-/Mdr2-/- mice were euthanized at 12 wk. We analyzed liver damage, PSC phenotypes, angiogenesis and AANAT, melatonin receptors and clock genes by immunohistochemistry, immunofluorescence, ELISA and western blots in liver samples and isolated cholangiocytes. Melatonin signaling was evaluated in human control and PSC samples. Results: Long-term melatonin treatment and inhibition of MT1 receptor ameliorates cholestatic liver phenotypes in Mdr2-/-mice by decreasing the immunoreactivity of melatonin enzymes and clock genes. GST activity and maspin expression decreased in Mdr2-/- mice and human PSC samples compared to controls; the phenotypes were reversed by melatonin. Conclusion: Chronic melatonin treatment improves liver histology and restores biliary circadian rhythm by interaction with MT1. Suppression of MT1 ameliorates biliary/liver phenotypes through changes in clock genes and melatonin enzymes. Restoration of the circadian rhythm by modulation of melatonin/MT1 signaling may be key for PSC management.
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    β-Cell Autophagy in the Pathogenesis of Type 1 Diabetes
    (2021-12) Muralidharan, Charanya; Linnemann, Amelia K.; Dong, Charlie X.; Sims, Emily K.; Kaplan, Mark H.
    Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors. One of the factors implicated in disease pathogenesis is early life viral infection. A typical immune response to viral infection includes production of type 1 interferons (IFN), such as IFN-α, which can induce stress in the pancreatic β-cells. Reactive oxygen species (ROS) accumulation occurs after exposure to other inflammatory cytokines, causing oxidative stress that may be linked to T1D pathogenesis. Therefore, we hypothesized that IFN-α may also elicit β-cell ROS accumulation. Our in vivo and in vitro experiments with human islets showed rapid and heterogenous ROS accumulation with IFN-α. Although T1D is characterized by autoimmune destruction of β-cells, some cells survive this persistent attack. We hypothesized that survival/ death of β-cells could be attributed to the ability to effectively mitigate ROS accumulation. One mechanism to mitigate ROS is autophagy, which degrades and recycles cellular components to promote cellular homeostasis. We observed an impairment in autophagy in β-cells of donors with T1D as well as in islets of diabetic non-obese diabetic (NOD) mouse model of autoimmune diabetes. Autophagic flux was also impaired in diabetic NOD mouse islets, further confirming impairment of autophagy. Interestingly, we observed an induction of autophagy after acute treatment with IFN-α both in vitro and in vivo, suggesting compensatory upregulation of autophagy to restore homeostasis. Similarly, we observed an increase in autophagosomes and telolysosomes in β-cells of normoglycemic autoantibody positive organ donors compared to nondiabetic organ donors. Together, these data implicate a defect in the final degradation step of autophagy involving lysosomes. Therefore, we analyzed the activity and expression of lysosomal cysteine protease Cathepsin H (CTSH, a T1D susceptibility locus), and found both to be increased in islets of pre-diabetic NOD mice. Together, these data support compensatory hyperactivation of lysosomal enzymes prior to overt diabetes, potentially to rid the cell of ROS and degradation-resistant oxidized proteins and lipids. We also observed that C57Bl/6J mice lacking a key autophagy enzyme, ATG7, in their β-cells, spontaneously developed hyperglycemia. Collectively, these data highlight the importance of -phagic degradation process in the pathogenesis of T1D.