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Project #1:


Bile Acid and Sphingosine-1-phosphate Receptor-mediated Signaling in Cholestasis

Supported by NIH/NIDDK 1R01DK104893 from 2015-2020

Cholangiocytes, which form the bile duct system in the liver, are the major target cells in a number of human cholestatic liver diseases.  Although there has been a significant improvement in the understanding of the pathophysiology of disease progression over the past two decades, the underlying cellular/molecular mechanisms remain largely unknown. Cholangiocytes are continuously exposed to high concentrations of bile salts at their apical membranes.  Bile acids taken up by an apical sodium bile acid transporter (ASBT) on cholangiocytes are unidirectionally transported across the cell and secreted via specific transporters (MDR3 and Ostα/Ostβ) on the basolateral membrane. ASBT has been reported to be regulated by changes in bile acid concentration and inflammatory cytokines. Bile acids taken up by cholangiocytes have been reported to activate a number of intracellular signaling pathways including: PKC, PI3K, MAP Kinase, and ERK 1/2 allowing for normal physiological homeostasis.  Loss of ASBT allows only conjugated bile acids (CBAs) to activate plasma membrane receptors as hydrophilic bile acids cannot easily enter cells by simple diffusion.  We have recently reported that CBAs activate the AKT and ERK1/2 signaling pathways via the G protein-coupled receptor (GPCR) sphingosine-1-phosphate receptor 2 (S1PR2) in hepatocytes and cholangiocytes. The levels of CBAs in serum and liver are significantly elevated in chronic cholestasis, which is correlated with bile duct obstruction. Our preliminary data indicates that: 1) S1PR2 is the predominant S1P receptor expressed in cholangiocytes; 2) taurocholate (TCA)-induced cell proliferation and migration are inhibited by a specific shRNA and an antagonist of S1PR2 in cholangiocytes; 3) bile duct ligation (BDL) induces the up-regulation of S1PR2 gene expression and down-regulation of ASBT expression in mouse primary cholangiocytes; 4) BDL-induced cholangiocyte proliferation and liver fibrosis are significantly reduced in S1PR2-/- mice; 5) both S1PR2 and SphK2 are up-regulated in the liver of mdr2-/- mice (a PSC mouse model). In addition, it has been reported that TCA concentration was dramatically elevated in the liver and serum after BDL in mice.  Based on these studies and our preliminary results, we HYPOTHESIZE that CBA-mediated activation of the S1PR2/SphK2 signaling cascades plays a critical role in promoting chronic cholangiopathy in cholestatic liver diseases.  Three specific aims are proposed to test our central hypothesis. 1) To define the role of S1PR2 and SphK2 in CBA-mediated cholestatic liver injury using the BDL mouse model; 2) To identify the molecular/cellular mechanisms by which CBA-mediated S1PR2/SphK2 activation promotes cholestatic liver injury; 3) To test the therapeutic strategy for cholestatic liver injury by targeting S1PR2/SphK2 using chemical inhibitors and genetic tools in mdr2-/- mice, a cholestasis model of PSC. Completion of these specific aims will not only identify the potential cellular/molecular mechanisms involved in the initiation and progression of cholestatic liver diseases, but will also establish a novel theory in bile acid and S1P biology.

Project #2:


LncRNA H19 in Cholestatic Liver Diseases

Supported by NIH/NIDDK 1R01DK115377 from 2017-2022

Cholangiopathies, such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), are characterized by damage and dysfunction of bile duct epithelial cells (cholangiocytes). Recently, long noncoding RNAs (lncRNAs) have been identified as a novel class of master regulators of gene expression and are linked to many fundamental biological processes and various human diseases including various liver diseases. However, little is known regarding the role of lncRNAs in the regulation of cholangiocyte function and pathogenesis of hepatobiliary diseases.  The overall goal of the current application is to identify the roles and mechanisms of lncRNAs in biliary dysfunction under cholestatic conditions and to create a fundamental base for developing novel therapeutic strategies for cholangiopathies. The expression of lncRNAs is tissue-, cell type- and differentiation stage-specific. LncRNA H19 is the first identified imprinted lncRNA and is highly conserved across lineages. It has been reported that H19 is the most strongly differentially expressed lncRNA during liver development and has been linked to hepatic metastases from a range of human carcinomas and cholestatic liver injury. However, the regulatory role of H19 in cholangiocyte pathophysiology remains unknown and is the focus of the current application.  Our most recent studies discovered that H19 is highly expressed in cholangiocytes, but not in hepatocytes under physiological conditions and hepatic H19 expression levels are correlated with upregulation of S1PR2 and cholestatic liver injury in the multi-drug resistance 2 knockout (Mdr2-/-) mouse, a well-established mouse model of PSC and PSC patient liver. Our preliminary data further showed that 1) BDL significantly up-regulated H19 and down-regulated the apical sodium bile acid transporter (ASBT) and sodium/taurocholate co-transporting polypeptide (NTCP); 2) BDL-induced cholestatic liver injury was markedly reduced in H19ΔExon1/+ mouse; 3) Knocking down H19 not only significantly reduced taurocholate (TCA)-induced expression of fibrotic genes and S1PR2 in cholangiocytes, but also markedly upregulated hepatic small heterodimer partner (SHP) expression and reduced cholestatic injury in Mdr2-/- mice; 4) Hepatic H19 level was also significantly upregulated in carbon tetrachloride (CCl4)-induced cholestatic liver injury mouse model. Based on these observations, we HYPOTHESIZE that lncRNA H19 plays an important role in the regulation of hepatobiliary epithelial function by disruption of hepatic bile acid homeostasis.  Two specific aims are proposed to test this hypothesis. 1) To define the role of lncRNA H19 in the regulation of bile acid-mediated cholangiocyte growth and remodeling during cholestatic liver injury; 2) To identify the mechanisms by which bile acids upregulate lncRNA H19 in cholestatic conditions. Completion of the proposed studies will make a significant conceptual advance by linking the lncRNA H19-mediated regulation of biliary epithelial function with cholestatic biliary injury in patients with cholangiopathies and will provide a translational mechanism of how bile acids and lncRNA H19 mediate hepatobiliary fibrosis.

Project #3:


Sphingosine-1 phosphate signaling in alcoholic liver disease

Supported by NIH/NIAAA, 1 R21 AA026629 from 2019-2021


Alcoholic liver disease (ALD) remains the most common chronic liver diseases worldwide. During last several decades, extensive studies have shown that ALD progression involves multiple events such as hepatic lipid accumulation, intestinal barrier dysfunction, and activation of an inflammatory response. Currently, no FDA approved therapy is available for any stage of ALD. Therefore, the unmet need to identify novel targets for the development of effective therapeutics against ALD is urgent.  It has been shown that alcohol alters bile acid metabolism and disrupts intestinal barrier function, which results in leaky gut and bacterial translocation and activation of systemic and hepatic inflammation. Bile acids are important signaling molecules involved in regulating lipid metabolism. Our lab first discovered a link between conjugated bile acids and sphingosine-1 phosphate (S1P) signaling in regulating hepatic lipid and glucose metabolism. The conjugated bile acids activate S1P receptor 2 (S1PR2), which further activate sphingosine kinase 2 (SphK2). S1P is a membrane-derived lipid mediator, which is synthesized from sphingosine by either SphK1 or SphK2.  It has been identified that S1P can regulate various fundamental cellular responses either as an intracellular signaling molecule or a ligand for five cell membrane G-protein coupled receptors (GPCRs), S1PR1-5. SphK1 is primarily located in the cytoplasm of mammalian cells, whereas SphK2 is located in the nucleus and mitochondria. SphK2-generated nuclear S1P is a powerful natural inhibitor of histone deacetylases (HDAC1/2). Increased histone acetylation is often associated with an increase in the transcriptional activity of genes involved in cell proliferation, migration and angiogenesis. We recently reported that both S1PR2 and SphK2 knock out mice (S1PR2-/- and SphK2-/-) are highly susceptible to high fat diet-induced fatty liver and hepatic lipotoxicity. Activation of SphK2 in response to ER stress ameliorates hepatic steatosis. The preliminary results further showed that SphK2-/-mice developed more severe fatty liver and hepatic injury as indicated by increased hepatic lipid accumulation and inflammation after 10-day’s feeding with 5% ethanol liquid diet (Lieber-DeCarli) followed by a binge via 31.5% ethanol gavage. In addition, alcohol-feeding significantly increased intestinal permeability and bacterial translocation in SphK2-/- mice. Furthermore, both LPS and thapsigargin (an ER stress inducer) inhibited intestinal organoid growth. Based on these key findings, we HYPOTHESIZE that disruption of SphK2/S1P-mediated signaling pathways plays a critical role in alcohol-induced liver injury. Two specific aims are proposed to test our central hypothesis:1) To investigate the role of SphK2 in alcohol-induced liver injury. 2) To identify the potential mechanisms by which lack of SphK2 promotes hepatic injury in response to chronic alcohol consumption. Results from these studies will provide novel information for the development of effective therapeutics not only for ALD but also for other related metabolic diseases. Therefore, the subject matter of this proposal is timely, important, and has direct clinical application.

Project #4:


Mechanisms of Berberine for the Treatment of Non-Alcoholic Fatty Liver Disease

Supported by VA Merit Review Award  1I01BX003676, 2017-2021


Nonalcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease, especially in US veterans. NAFLD is critically linked to inflammation, insulin resistance, dyslipidemia, and metabolic syndrome. Recent advances in NAFLD and non-alcoholic steatohepatitis (NASH) studies indicate that gut microbiota exerts a significant role in the disease progression by affecting host metabolic balance and immune response. The “metabolic endotoxemia” and elevated circulating levels of lipopolysaccharide (LPS) and free long-chain fatty acids (FFA) are implicated in the stimulation of systemic inflammation and insulin resistance, both of which are positively correlated with the development and progression of NAFLD. Although the mechanism by which LPS/FFA induce hepatic lipotoxicity is still not fully understood, LPS/FFA-induced expression of inflammatory cytokines such as TNF-a and activation of the endoplasmic reticulum (ER) stress signaling pathway, known as the unfolded protein response (UPR), are major contributors. Furthermore, micro-RNAs (miR), small noncoding RNAs (~22 nucleotides), have recently been found to be potent regulators of lipid and cholesterol metabolism. Alterations in miR expression have also been linked to LPS/FFA-induced inflammatory response, hepatic lipotoxicity and insulin resistance as well as disease progression of NAFLD in the clinic. Berberine (BBR), an isoquinoline alkaloid isolated from many medicinal herbs, has been used to treat various infectious disorders for more than 3,000 years in Asia. Numerous studies have shown that BBR has various pharmacological activities including anti-inflammatory, hypoglycemic and lipid-lowering effects. Importantly, BBR represents a novel cholesterol-lowering drug through a unique mechanism distinct from the current statin therapy. These studies strongly indicate that BBR is a promising natural therapeutic agent for NAFLD/NASH. However, the molecular mechanisms underlying BBR’s anti-inflammatory and lipid-lowering properties remain to be fully identified. We have demonstrated that BBR inhibits ER stress-mediated TNF-a and IL-6 expression through regulating the RNA-binding protein (RBP) HuR in macrophages. Recent advances in miR research have identified specific panels of miRs as major regulators of inflammation, lipid metabolism and metabolic disorders, such as miR-155, miR-125a-5p, miR-33, miR-34a, and miR-122. In addition, it has been reported that BBR exerted protective effects against high fat diet (HFD)-induced NAFLD/NASH via modulating gut microbiota. Our preliminary studies also indicate that 1) BBR significantly inhibited HFD-induced hepatic lipid accumulation and systemic inflammation in animal models; 2) BBR significantly changed gut microbiota composition and bile acid metabolism; 3) BBR inhibited FFA-induced ER stress and lipid accumulation in hepatocytes; 4) BBR inhibited FFA-induced miR-34a expression in hepatocytes and LPS-induced miR-125a-5p expression in macrophages; 4) The expression of miR-34a was significantly down-regulated in the chop-/- primary hepatocytes. Based on these observations, we hypothesize that BBR inhibits HFD-induced hepatic lipotoxicity by modulating gut microbiota and inhibiting ER stress and inflammatory response.  Three specific aims are proposed to test this hypothesis. Aim 1: To determine the mechanism by which BBR inhibits LPS/FFA-induced ER stress and inflammatory response in hepatocytes and macrophages.  Aim 2: To identify the mechanism by which BBR inhibits HFD-/FFA-induced hepatic lipotoxicity. Aim 3: To further examine the therapeutic effect of BBR on HFD-induced hepatic lipotoxicity in the in vivo NAFLD/NASH model.

Huiping Zhou, Ph.D., AGAF, FAASLD


Department of Microbiology and Immunology

Medical College of Virginia

Virginia Commonwealth University 

Research Career Scientist

McGuire VA Medical Center

Richmond, VA




Kontos Medical Sciences Building

Room 534, 511, 515

1217 E Marshall Street

Richmond, VA 23298​

Tel:804-827-1556 or 804-828-2332

McGuire VA Medical Center


1201 Broad Rock Blvd

Richmond, VA 23249-0001



Molecular Medicine Research Building

Room 5-044

1220 E Broad Street, Richmond VA 23298

Tel: 804-828-6817

McGuire VA Medical Center


1201 Broad Rock Blvd

Richmond, VA 23249-0001

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