ABSTRACT
BACKGROUND@#Ballooned hepatocytes (BH) are a key histological hallmark of nonalcoholic steatohepatitis (NASH), yet their consequences for liver-specific functions are unknown. @*METHODS@#In our previous study, an experimental model of human induced-BHs (iBH) has been successfully developed based on cell sheet technology. This study aimed to determine the functions of iBHs in the primary human hepatocyte/ normal human dermal fibroblast (PHH/NHDF) co-culture cell sheets. Normal hepatocytes in the PHH/3T3-J2 co-culture cell sheets were set as a control, since 3T3-J2 murine embryonic fibroblasts have exhibited previously long term maintenance of PHH functions. @*RESULTS@#It was found that, albumin secretion was not affected in iBHs, but urea synthesis as well as cytochrome P450 enzyme (CYP) activities including CYP1A2 and CYP3A4, were significantly reduced in iBHs. Besides, loss of bile canaliculi was observed in iBHs. These findings are consistent with clinical studies of human NASH. In addition, PHH/ NHDF cell sheets demonstrated two fold higher TGF-b1 secretion compared with PHH/3T3-J2 cell sheets. Furthermore, treatment with a TGF-b inhibitor and a semi-synthetic bile acid analogue (obeticholic acid, phase 3 trial of NASH therapy) ameliorated the histological appearance of established iBHs. @*CONCLUSION@#In summary, this study demonstrates the priority of iBHs in recapitulating not only histology but also clinically relevant hepatic dysfunctions in human NASH and suggests TGF-b and bile acid related signal pathway may play important roles in the formation of iBHs.
ABSTRACT
BACKGROUND@#Ballooned hepatocytes (BH) are a key histological hallmark of nonalcoholic steatohepatitis (NASH), yet their consequences for liver-specific functions are unknown. @*METHODS@#In our previous study, an experimental model of human induced-BHs (iBH) has been successfully developed based on cell sheet technology. This study aimed to determine the functions of iBHs in the primary human hepatocyte/ normal human dermal fibroblast (PHH/NHDF) co-culture cell sheets. Normal hepatocytes in the PHH/3T3-J2 co-culture cell sheets were set as a control, since 3T3-J2 murine embryonic fibroblasts have exhibited previously long term maintenance of PHH functions. @*RESULTS@#It was found that, albumin secretion was not affected in iBHs, but urea synthesis as well as cytochrome P450 enzyme (CYP) activities including CYP1A2 and CYP3A4, were significantly reduced in iBHs. Besides, loss of bile canaliculi was observed in iBHs. These findings are consistent with clinical studies of human NASH. In addition, PHH/ NHDF cell sheets demonstrated two fold higher TGF-b1 secretion compared with PHH/3T3-J2 cell sheets. Furthermore, treatment with a TGF-b inhibitor and a semi-synthetic bile acid analogue (obeticholic acid, phase 3 trial of NASH therapy) ameliorated the histological appearance of established iBHs. @*CONCLUSION@#In summary, this study demonstrates the priority of iBHs in recapitulating not only histology but also clinically relevant hepatic dysfunctions in human NASH and suggests TGF-b and bile acid related signal pathway may play important roles in the formation of iBHs.
ABSTRACT
The promotion of fatty acid metabolism, to which PPARα contributes, has been suggested that it would be participate to maintain the proximal tubular cell function in kidney. The loading on the proximal tubular cell of fatty acids could arise the inflammation and cell death in obesity. One of the “Kampo” medicines, Boiogito (BO) is used for the remedy of overweight women exhibiting chronic fatigues as well as edema in the lower extremities or knees. BO would exhibit the prevention of the proximal tubular cell damage and improvement of kidney function by reducing the portion of fatty acids. In this study, BO was orally administered high fatty acid combined with bovine serum albumin for mice to evaluate the mRNA expression of PPARα quantified by PCR. The increase of PPARα mRNA expression was observed BO administration, followed by reduce the volume of fatty acids in kidney.KEY WORDS: Boiogito, Fatty acid metabolism, PPARα, Proximal Tubular CellINTRODUCTIONObesity is a risk factor for incidence of albuminuria and chronic kidney disease 1, 2, and an accumulating visceral fat would be involved in the regulation of primary stage of nephropathy 3, microalbuminuria. Fatty acids are major contributor to these kidney disorders caused by obesity 4. The binding fatty acids with albumin represents in blood generally, taking up by proximal tubular cells after glomerular filtration from albumin. A peroxisome proliferator - activated receptor (PPARα) has been suggested that it would regulate the fatty acid metabolism. Because the glomerular filtration rate and renal blood flow would increase in overweight patients 5, a large quantity of free fatty acids should be loaded into proximal tubular cells. Therefore, the investigation concerning to PPARα stimulator can be regarded as the fatty acid metabolism - regulation. One of the “Kampo” medicines, Boiogito (BO) is used for the remedy of the inflammation and cell death in obesity, is composed of eight crude drugs: Aluminum Silicate Hydrate with Silicon Dioxide, Astragalus Root, Atractylodes Rhizome, Ginger, Glycyrrhiza, Jujube, Sinomenium Stem and Rhizome. In this study, to clarify the therapeutic mechanisms of BO, we focused on the up - regulating for fatty acid metabolism through the PPARα activation.METHODSKampo formulaeBO was prepared according to the prescription for a one-day dose 6: 3.0 g Aluminum Silicate Hydrate with Silicon Dioxide, 5.0 g Astragalus Root, 3.0 g Atractylodes Rhizome, 1.0 g Ginger, 2.0 g Glycyrrhiza, 4.0 g Jujube, 4.0 g Sinomeniumstem and Rhizome.
ABSTRACT
We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintaed intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.