Chlorogenic Acid Inhibits Progressive Pulmonary Fibrosis in a Diabetic Rat Model.

Background: Chlorogenic acid (CGA) is known to have antifibrotic and hypoglycemic effects and may play a role in preventing diabetes-induced pulmonary fibrosis. This study aimed to determine the effect and optimum dose of CGA on diabetes-induced pulmonary fibrosis. Methods: Thirty Wistar rats (two-month-old, 150-200 grams) were randomly divided into six groups, namely control, six weeks diabetes mellitus (DM1), eight weeks DM (DM2), and three DM2 groups (CGA1, CGA2, and CGA3) who received CGA doses of 12.5, 25, and 50 mg/Kg BW, respectively. After six weeks, CGA was administered intraperitoneally for 14 consecutive days. Lung tissues were taken for TGF-ß1, CTGF, SMAD7, Collagen-1, and α-SMA mRNA expression analysis and paraffin embedding. Data were analyzed using one-way ANOVA and the Kruskal-Wallis test. P<0.05 was considered statistically significant. Results: TGF-ß1 expression in the CGA1 group (1.01±0.10) was lower than the DM1 (1.33±0.25, P=0.05) and DM2 (1.33±0.20, P=0.021) groups. α-SMA expression in the CGA1 group (median 0.60, IQR: 0.34-0.64) was lower than the DM1 (median 0.44, IQR: 0.42-0.80) and DM2 (median 0.76, IQR: 0.66-1.10) groups. Collagen-1 expression in the CGA1 group (0.75±0.13) was lower than the DM1 (P=0.24) and DM2 (P=0.26) groups, but not statistically significant. CTGF expression in CGA groups was lower than the DM groups (P=0.088), but not statistically significant. There was an increase in SMAD7 expression in CGA groups (P=0.286). Histological analysis showed fibrosis improvement in the CGA1 group compared to the DM groups. Conclusion: CGA (12.5 mg/Kg BW) inhibited the expression of profibrotic factors and increased antifibrotic factors in DM-induced rats.

Targeting hepatic oxidative stress rescues bone loss in liver fibrosis.

OBJECTIVE: Chronic liver diseases often involve metabolic damage to the skeletal system. The underlying mechanism of bone loss in chronic liver diseases remains unclear, and appropriate therapeutic options, except for orthotopic liver transplantation, have proved insufficient for these patients. This study aimed to investigate the efficacy and mechanism of transplantation of immature hepatocyte-like cells converted from stem cells from human exfoliated deciduous teeth (SHED-Heps) in bone loss of chronic liver fibrosis. METHODS: Mice that were chronically treated with CCl4 received SHED-Heps, and trabecular bone density, reactive oxygen species (ROS), and osteoclast activity were subsequently analyzed in vivo and in vitro. The effects of stanniocalcin 1 (STC1) knockdown in SHED-Heps were also evaluated in chronically CCl4 treated mice. RESULTS: SHED-Hep transplantation (SHED-HepTx) improved trabecular bone loss and liver fibrosis in chronic CCl4-treated mice. SHED-HepTx reduced hepatic ROS production and interleukin 17 (Il-17) expression under chronic CCl4 damage. SHED-HepTx reduced the expression of both Il-17 and tumor necrosis factor receptor superfamily 11A (Tnfrsf11a) and ameliorated the imbalance of osteoclast and osteoblast activities in the bone marrow of CCl4-treated mice. Functional knockdown of STC1 in SHED-Heps attenuated the benefit of SHED-HepTx including anti-bone loss effect by suppressing osteoclast differentiation through TNFSF11-TNFRSF11A signaling and enhancing osteoblast differentiation in the bone marrow, as well as anti-fibrotic and anti-ROS effects in the CCl4-injured livers. CONCLUSIONS: These findings suggest that targeting hepatic ROS provides a novel approach to treat bone loss resulting from chronic liver diseases.

Accelerated Senescence and Apoptosis in the Rat Liver during the Progression of Diabetic Complications.

Background: Chronic hyperglycaemia of diabetes causes long-term damage and impaired function of multiple organs. However, the pathological changes in the liver following long-term diabetes remain unclear. This study aimed to determine the pathological complications of long-term diabetes in the rat liver. Methods: Intraperitoneal injection of streptozotocin (STZ) was used to induce diabetes in rats at a single dose (60 mg/kg body weight [BW]). Rats were euthanised at 1 month (DM1 group), 2 months (DM2 group) and 4 months (DM4 group) following diabetes induction with six rats in each group. Immunohistochemistry was performed against SOD1, CD68, p53 and p16 antibodies. Messenger RNA (mRNA) expressions of SOD1, SOD2, GPx, CD68, p53, p21 and caspase-3 genes were measured by reverse transcription-polymerase chain reaction. Results: Hepatic p53 mRNA expression was significantly higher in DM1, DM2 and DM4 groups compared to the control group. The p21 and caspase-3 mRNA expressions were significantly upregulated in the DM2 and DM4 groups. The p16-positive cells were obviously increased, particularly in the DM4 group. Bivariate correlation analysis showed mRNA expressions of p21 and caspase-3 genes were positively correlated with the p53 gene. Conclusion: Diabetic rats exhibited increased apoptosis and senescence in the liver following a longer period of hyperglycaemia.

Biliary atresia-specific deciduous pulp stem cells feature biliary deficiency.

BACKGROUND: Biliary atresia (BA) is a severe hepatobiliary disease in infants that ultimately results in hepatic failure; however, its pathological mechanism is poorly elucidated. Current surgical options, including Kasai hepatoportoenterostomy and orthotopic liver organ transplantations, are palliative; thus, innovation in BA therapy is urgent. METHODS: To examine whether BA-specific post-natal stem cells are feasible for autologous cell source for BA treatment, we isolated from human exfoliated deciduous teeth, namely BA-SHED, using a standard colony-forming unit fibroblast (CFU-F) method and compared characteristics as mesenchymal stem cells (MSCs) to healthy donor-derived control SHED, Cont-SHED. BA-SHED and Cont-SHED were intrasplenically transplanted into chronic carbon tetrachloride (CCl4)-induced liver fibrosis model mice, followed by the analysis of bile drainage function and donor integration in vivo. Immunohistochemical assay was examined for the regeneration of intrahepatic bile ducts in the recipient's liver using anti-human specific keratin 19 (KRT19) antibody. RESULTS: BA-SHED formed CFU-F, expressed MSC surface markers, and exhibited in vitro mesenchymal multipotency similar to Cont-SHED. BA-SHED showed less in vitro hepatogenic potency than Cont-SHED. Cont-SHED represented in vivo bile drainage function and KRT19-positive biliary regeneration in chronic carbon tetrachloride-induced liver fibrosis model mice. BA-SHED failed to show in vivo biliary potency and bile drainage function compared to Cont-SHED. CONCLUSION: These findings indicate that BA-SHED are not feasible source for BA treatment, because BA-SHED may epigenetically modify the underlying prenatal and perinatal BA environments. In conclusion, these findings suggest that BA-SHED-based studies may provide a platform for understanding the underlying molecular mechanisms of BA development and innovative novel modalities in BA research and treatment.

Ethanolic Extract of Centella asiatica Treatment in the Early Stage of Hyperglycemia Condition Inhibits Glomerular Injury and Vascular Remodeling in Diabetic Rat Model.

BACKGROUND: Diabetes mellitus (DM) is marked by oxidative stress, inflammation, and vascular dysfunction that caused diabetic nephropathy that resulted in end-stage renal disease (ESRD). Vascular dysfunction is characterized by an imbalance in vasoconstrictor and vasodilator agents which underlies the mechanism of vascular injury in DM. Additionally, diminished podocytes correlate with the severity of kidney injury. Podocyturia often precedes proteinuria in several kidney diseases, including diabetic kidney disease. Centella asiatica (CeA) is known as an anti-inflammatory and antioxidant and has neuroprotective effects. This research aimed to investigate the potential effect of CeA to inhibit glomerular injury and vascular remodeling in DM. METHODS: The DM rat model was induced through intraperitoneal injection of streptozotocin 60 mg/kg body weight (BW), and then rats were divided into 1-month DM (DM1, n = 5), 2-month DM (DM2, n = 5), early DM concurrent with CeA treatment for 2 months (DMC2, n = 5), and 1-month DM treated with CeA for 1-month (DM1C1, n = 5). The CeA (400 mg/kg BW) was given daily via oral gavage. The control group (Control, n = 5) was maintained for 2 months. Finally, rats were euthanized and kidneys were harvested to assess vascular remodeling using Sirius Red staining and the mRNA expression of superoxide dismutase, podocytes marker, ACE2, eNOS, and ppET-1 using RT-PCR. RESULTS: The DM groups demonstrated significant elevation of glucose level, glomerulosclerosis, and proteinuria. A significant reduction of SOD1 and SOD3 promotes the downregulation of nephrin and upregulation of TRPC6 mRNA expressions in rat glomerular kidney. Besides, this condition enhanced ppET-1 and inhibited eNOS and ACE2 mRNA expressions that lead to the development of vascular remodeling marked by an increase of wall thickness, and lumen wall area ratio (LWAR). Treatment of CeA, especially the DMC2 group, attenuated glomerular injury and showed the reversal of induced conditions. CONCLUSIONS: Centella asiatica treatment at the early stage of diabetes mellitus ameliorates glomerulosclerosis and vascular injury via increasing antioxidant enzymes.

Cholangiogenic potential of human deciduous pulp stem cell-converted hepatocyte-like cells.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) have been reported to show the in vivo and in vitro hepatic differentiation, SHED-Heps; however, the cholangiogenic potency of SHED-Heps remains unclear. Here, we hypothesized that SHED-Heps contribute to the regeneration of intrahepatic bile duct system in chronic fibrotic liver. METHODS: SHED were induced into SHED-Heps under cytokine stimulation. SHED-Heps were intrasplenically transplanted into chronically CCl4-treated liver fibrosis model mice, followed by the analysis of donor integration and hepatobiliary metabolism in vivo. Immunohistochemical assay was examined for the regeneration of intrahepatic bile duct system in the recipient liver. Furthermore, SHED-Heps were induced under the stimulation of tumor necrosis factor alpha (TNFA). RESULTS: The intrasplenic transplantation of SHED-Heps into CCl4-treated mice showed that donor SHED-Heps behaved as human hepatocyte paraffin 1- and human albumin-expressing hepatocyte-like cells in situ and ameliorated CCl4-induced liver fibrosis. Of interest, the integrated SHED-Heps not only expressed biliary canaliculi ATP-binding cassette transporters including ABCB1, ABCB11, and ABCC2, but also recruited human keratin 19- (KRT19-) and KRT17-positive cells, which are considered donor-derived cholangiocytes, regenerating the intrahepatic bile duct system in the recipient liver. Furthermore, the stimulation of TNFA induced SHED-Heps into KRT7- and SRY-box 9-positive cells. CONCLUSIONS: Collectively, our findings demonstrate that infused SHED-Heps showed cholangiogenic ability under the stimulation of TNFA in CCl4-damaged livers, resulting in the regeneration of biliary canaliculi and interlobular bile ducts in chronic fibrotic liver. Thus, the present findings suggest that SHED-Heps may be a novel source for the treatment of cholangiopathy.

Therapeutic potential of spheroids of stem cells from human exfoliated deciduous teeth for chronic liver fibrosis and hemophilia A.

PURPOSE: Mesenchymal stem cell (MSC)-based cell therapies have emerged as a promising treatment option for various diseases. Due to the superior survival and higher differentiation efficiency, three-dimensional spheroid culture systems have been an important topic of MSC research. Stem cells from human exfoliated deciduous teeth (SHED) have been considered an ideal source of MSCs for regenerative medicine. Thus, in the present study, we introduce our newly developed method for fabricating SHED-based micro-hepatic tissues, and demonstrate the therapeutic effects of SHED-based micro-hepatic tissues in mouse disease models. METHODS: SHED-converted hepatocyte-like cells (SHED-HLCs) were used for fabricating spherical micro-hepatic tissues. The SHED-HLC-based spheroids were then transplanted both into the liver of mice with CCl4-induced chronic liver fibrosis and the kidney of factor VIII (F8)-knock-out mice. At 4 weeks after transplantation, the therapeutic efficacy was investigated. RESULTS: Intrahepatic transplantation of SHED-HLC-spheroids improved the liver dysfunction in association with anti-fibrosis effects in CCl4-treated mice. Transplanted SHED-converted cells were successfully engrafted in the recipient liver. Meanwhile, renal capsular transplantation of the SHED-HLC-spheroids significantly extended the bleeding time in F8-knock-out mice. CONCLUSIONS: These findings suggest that SHED-HLC-based micro-hepatic tissues might be a promising source for treating pediatric refractory diseases, including chronic liver fibrosis and hemophilia A.

Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson's disease.

Wilson's disease (WD) is an inherited metabolic disease arising from ATPase copper transporting beta gene (ATP7B) mutation. Orthotoropic liver transplantation is the only radical treatment of fulminant WD, although appropriate donors are lacking at the onset of emergency. Given the hepatogenic capacity and tissue-integration/reconstruction ability in the liver of stem cells from human exfoliated deciduous teeth (SHED), SHED have been proposed as a source for curing liver diseases. We hypothesized the therapeutic potential of SHED and SHED-converted hepatocyte-like- cells (SHED-Heps) for fulminant WD. SHED and SHED-Heps were transplanted into WD model Atp7b-mutated Long-Evans Cinnamon (LEC) rats received copper overloading to induce a lethal fulminant liver failure. Due to the superior copper tolerance via ATP7B, SHED-Hep transplantation gave more prolonged life-span of fulminant LEC rats than SHED transplantation. The integrated ATP7B-expressing SHED-Heps showed more therapeutic effects on to restoring the hepatic dysfunction and tissue damages in the recipient liver than the integrated naïve SHED without ATP7B expression. Moreover, SHED-Heps could reduce copper-induced oxidative stress via ATP7B- independent stanniocalcin 1 secretion in the fulminant LEC rats, suggesting a possible role for paracrine effect of the integrated SHED-Heps. Taken together, SHED-Heps offer a potential of functional restoring, bridging, and preventive approaches for treating fulminant WD.

In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver fibrosis in mice.

INTRODUCTION: Liver transplantation is a gold standard treatment for intractable liver diseases. Because of the shortage of donor organs, alternative therapies have been required. Due to their potential to differentiate into a variety of mature cells, stem cells are considered feasible cell sources for liver regeneration. Stem cells from human exfoliated deciduous teeth (SHED) exhibit hepatogenic capability in vitro. In this study, we investigated their in vivo capabilities of homing and hepatocyte differentiation and therapeutic efficacy for liver disorders in carbon tetrachloride (CCl4)-induced liver fibrosis model mice. METHODS: We transplanted SHED into CCl4-induced liver fibrosis model mice through the spleen, and analyzed the in vivo homing and therapeutic effects by optical, biochemical, histological, immunological and molecular biological assays. We then sorted human leukocyte antigen-ABC (HLA-ABC)-positive cells from primary CCl4-damaged recipient livers, and analyzed their fusogenicity and hepatic characteristics by flow cytometric, genomic DNA, hepatocyte-specific gene assays. Furthermore, we examined the treatment effects of HLA-positive cells to a hepatic dysfunction by a secondary transplantation into CCl4-treated mice. RESULTS: Transplanted SHED homed to recipient livers, and expressed HLA-ABC, human hepatocyte specific antigen hepatocyte paraffin 1 and human albumin. SHED transplantation markedly recovered liver dysfunction and led to anti-fibrotic and anti-inflammatory effects in the recipient livers. SHED-derived HLA-ABC-positive cells that were sorted from the primary recipient liver tissues with CCl4 damage did not fuse with the host mouse liver cells. Sorted HLA-positive cells not only expressed human hepatocyte-specific genes including albumin, cytochrome P450 1A1, fumarylacetoacetase, tyrosine aminotransferase, uridine 5'-diphospho-glucuronosyltransferase, transferrin and transthyretin, but also secreted human albumin, urea and blood urea nitrogen. Furthermore, SHED-derived HLA-ABC-positive cells were secondary transplanted into CCl4-treated mice. The donor cells homed into secondary recipient livers, and expressed hepatocyte paraffin 1 and human albumin, as well as HLA-ABC. The secondary transplantation recovered a liver dysfunction in secondary recipients. CONCLUSIONS: This study indicates that transplanted SHED improve hepatic dysfunction and directly transform into hepatocytes without cell fusion in CCl4-treated mice, suggesting that SHED may provide a feasible cell source for liver regeneration.

Therapeutic potential of mesenchymal stem cell transplantation in a nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: The aim of this study was to evaluate the efficacy of mesenchymal stem cells (MSCs) in a nitrofen-induced congenital diaphragmatic hernia (CDH) rat model. METHODS: Pregnant rats were exposed to nitrofen on embryonic day 9.5 (E9.5). MSCs were isolated from the enhanced green fluorescent protein (eGFP) transgenic rat lungs. The MSCs were transplanted into the nitrofen-induced E12.5 rats via the uterine vein, and the E21 lung explants were harvested. The study animals were divided into three: the control group, the nitrofen-induced left CDH (CDH group), and the MSC-treated nitrofen-induced left CDH (MSC-treated CDH group). The specimens were morphologically analyzed using HE and immunohistochemical staining with proliferating cell nuclear antigen (PCNA), surfactant protein-C (SP-C), and α-smooth muscle actin. RESULTS: The alveolar and medial walls of the pulmonary arteries were significantly thinner in the MSC-treated CDH group than in the CDH group. The alveolar air space areas were larger, while PCNA and the SP-C positive cells were significantly higher in the MSC-treated CDH group, than in the CDH group. MSC engraftment was identified on immunohistochemical staining of the GFP in the MSC-treated CDH group. CONCLUSIONS: MSC transplantation potentially promotes alveolar and pulmonary artery development, thereby reducing the severity of pulmonary hypoplasia.