Palmitoylethanolamide Ameliorates Carbon Tetrachloride-Induced Liver Fibrosis in Rats

Background: Liver fibrosis is a complex inflammatory and fibrogenic process, and the progression of fibrosis leads to cirrhosis. The only therapeutic approaches are the removal of injurious stimuli and liver transplantation. Therefore, the development of anti-fibrotic therapies is desired. Palmitoylethanolamide (PEA) is an endogenous fatty acid amide belonging to the N-acylethanolamines family and contained in foods such as egg yolks and peanuts. PEA has therapeutic anti-inflammatory, analgesic, and neuroprotective effects. However, the effects and roles of PEA in liver fibrosis remain unknown. Here we investigated the therapeutic effects of PEA in rats with liver fibrosis. Methods: We conducted in vitro experiments to investigate the effects of PEA on the activation of hepatic stellate cells (HSCs, LX-2). Liver fibrosis was induced by an intraperitoneal injection of 1.5 mL/kg of 50% carbon tetrachloride twice a week for 4 weeks. Beginning at 3 weeks, PEA (20 mg/kg) was intraperitoneally injected thrice a week for 2 weeks. Then rats were sacrificed and we performed histological and quantitative reverse-transcription polymerase chain reaction analyses. Results: The expression of α-smooth muscle actin (SMA) induced by transforming growth factor (TGF)-β1 in HSCs was significantly downregulated by PEA. PEA treatment inhibited the TGF-β1-induced phosphorylation of SMAD2 in a dose-dependent manner, and upregulated the expression of SMAD7. The reporter gene assay demonstrated that PEA downregulated the transcriptional activity of the SMAD complex upregulated by TGF-β1. Administration of PEA significantly reduced the fibrotic area, deposition of type I collagen, and activation of HSCs and Kupffer cells in rats with liver fibrosis. Conclusion: Activation of HSCs was significantly decreased by PEA through suppression of the TGF-β1/SMAD signaling pathway. Administration of PEA produced significant improvement in a rat model of liver fibrosis, possibly by inhibiting the activation of HSCs and Kupffer cells. PEA may be a potential new treatment for liver fibrosis

Effects of human amnion-derived mesenchymal stromal cell transplantation in rats with radiation proctitis

Background aims. Mesenchymal stromal cells (MSCs) have been reported to be a promising cell source in cell therapy, and large amounts of MSCs can easily be isolated from human amnion. Therapeutic irradiation for intra-pelvic cancer often causes radiation proctitis; however, there is currently no effective treatment. We therefore investigated the effect of transplantation of human amnion derived MSCs (AMSCs) in rats with radiation proctitis. Methods. Amnion was obtained at cesarean delivery, and AMSCs were isolated and expanded. Sprague-Dawley rats were gamma-irradiated (5 Gy/d) at the rectum for 5 days. On day 5, AMSCs (1 x 10(6) cells) were intravenously transplanted. Rats were killed on day 8. Histological analyses were performed, and messenger RNA expression of inflammatory mediators was measured. In vitro, after gamma-irradiation of rat intestinal epithelial cells (IEC-6), the cells were cultured with AMSC-conditioned medium (CM). The effect of AMSC-CM was evaluated by measurement of caspase-3/7 activity, p53 transcription activity and quantitative reverse-transcription polymerase chain reaction for p53-target genes. Results. Histological examination demonstrated that epithelial injury and infiltration of inflammatory cells in the rectum were significantly suppressed by transplantation of AMSCs. In vitro, the cell injury in IEC-6 cells induced by gamma-irradiation was inhibited by AMSC-CM, which also inhibited the upregulation of p53 transcription activity, caspase-3/7 activity and p21 expression. Conclusions. Transplantation of AMSCs improved radiation proctitis, possibly through inhibition of cell injury and inflammatory reactions. AMSC transplantation should be considered as a new treatment for radiation proctitis

Therapeutic effects of human amnion-derived mesenchymal stem cell transplantation and conditioned medium enema in rats with trinitrobenzene sulfonic acid-induced colitis

Cell therapy with mesenchymal stem cells (MSCs) is expected to provide a new strategy for the treatment of inflammatory bowel disease (IBD). Large amounts of MSCs can be obtained from human amnion. Therefore, we investigated the effect of transplantation of human amnion-derived MSCs (hAMSCs) or enema of conditioned medium (CM) from hAMSCs into rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. In the first experiment, 10-week-old male Sprague-Dawley rats were intravenously injected with hAMSCs (1 × 10^6 cells) 3 h after rectal administration of TNBS (45 mg/kg). In the second experiment, rats with TNBS-induced colitis received CM by enema into the colon for 3 days. Colitis was investigated by endoscopy, histology, immunohistochemistry, and by measuring mRNA expression of inflammatory mediators. Administration of hAMSCs or CM enema significantly improved the endoscopic score. In addition, these two interventions resulted in significantly decreased infiltration of neutrophils and monocytes/macrophages and decreased expression levels of TNF-α, CXCL1, and CCL2. In conclusion, transplantation of hAMSCs and CM enema provided significant improvement in rats with TNBS-induced colitis. CM from hAMSCs and hAMSCs may be new strategies for the treatment of IBD

Oral administration of conditioned medium obtained from mesenchymal stem cell culture prevents subsequent stricture formation after esophageal submucosal dissection in pigs

Background and Aims: Endoscopic submucosal dissection (ESD) for esophageal cancer often causes postoperative stricture when more than three fourths of the circumference of the esophagus is dissected. Mesenchymal stem cells are a valuable cell source in regenerative medicine, and conditioned medium (CM) obtained from mesenchymal stem cells reportedly inhibits inflammation. In this study we evaluated whether CM could prevent esophageal stricture after ESD. Methods: We resected a semi-circumference of pig esophagus by ESD. We prepared CM gel by mixing with 5% carboxymethyl cellulose and endoscopically applied it onto the wound bed immediately after ESD and on days 8 and 15 (weekly CM group) or administered it orally from days 1 to 4 (daily CM group). We also injected triamcinolone acetonide into the remaining submucosa immediately after ESD (steroid group). We killed the pigs on day 8 or day 22 to measure the stricture rate and to perform histologic analysis. Results: Stricture rate in weekly and daily CM groups and steroid groups were significantly lower than in the control group on day 22. Moreover, CM significantly attenuated the number of activated myofibroblasts and fiber thickness on day 22. CM also significantly decreased the infiltration of neutrophils and macrophages compared with the control group on day 8. Conclusions: CM gel prevents esophageal stricture formation by suppressing myofibroblast activation and fibrosis after the infiltration of neutrophils and macrophages. Oral administration of CM gel is a promising treatment for the prevention of post-ESD stricture

Extracellular Vesicles from Amnion-Derived Mesenchymal Stem Cells Ameliorate Hepatic Inflammation and Fibrosis in Rats

Background. There are no approved drug treatments for liver fibrosis and nonalcoholic steatohepatitis (NASH), an advanced stage of fibrosis which has rapidly become a major cause of cirrhosis. Therefore, development of anti-inflammatory and antifibrotic therapies is desired. Mesenchymal stem cell- (MSC-) based therapy, which has been extensively investigated in regenerative medicine for various organs, can reportedly achieve therapeutic effect in NASH via paracrine action. Extracellular vesicles (EVs) encompass a variety of vesicles released by cells that fulfill functions similar to those of MSCs. We herein investigated the therapeutic effects of EVs from amnion-derived MSCs (AMSCs) in rats with NASH and liver fibrosis. Methods. NASH was induced by a 4-week high-fat diet (HFD), and liver fibrosis was induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) twice a week for six weeks. AMSC-EVs were intravenously injected at weeks 3 and 4 in rats with NASH (15 μg/kg) and at week 3 in rats with liver fibrosis (20 μg/kg). The extent of inflammation and fibrosis was evaluated with quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The effect of AMSC-EVs on inflammatory and fibrogenic response was investigated in vitro. Results. AMSC-EVs significantly decreased the number of Kupffer cells (KCs) in the liver of rats with NASH and the mRNA expression levels of inflammatory cytokines such as tumor necrosis factor- (Tnf-) α, interleukin- (Il-) 1β and Il-6, and transforming growth factor- (Tgf-) β. Furthermore, AMSC-EVs significantly decreased fiber accumulation, KC number, and hepatic stellate cell (HSC) activation in rats with liver fibrosis. In vitro, AMSC-EVs significantly inhibited KC and HSC activation and suppressed the lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway. Conclusions. AMSC-EVs ameliorated inflammation and fibrogenesis in a rat model of NASH and liver fibrosis, potentially by attenuating HSC and KC activation. AMSC-EV administration should be considered as a new therapeutic strategy for chronic liver disease

Preventive effect of mesenchymal stem cell culture supernatant on luminal stricture after endoscopic submucosal dissection in the rectum of pigs

Background: Mesenchymal stem cells (MSCs) are valuable in regenerative medicine, and MSC culture supernatant (MSC-CS) reportedly inhibits inflammation and fibrosis. We investigated whether colorectal luminal stricture develops after circumferential endoscopic submucosal dissection (ESD) in the colorectum, and whether the development of luminal stricture could be prevented by using MSC-CS enema. Methods: In the first experiment, we performed circumferential ESD in the rectums or distal colons of pigs (n = 4 in each group). We sacrificed the pigs on Day 22 and measured the degree of luminal stricture. In the second experiment, we performed circumferential ESD in the rectums of pigs and administered an MSC-CS gel or a control gel enema after ESD for 4 days. We sacrificed the pigs on Day 8 (n = 3 in each group) or 22 (n = 3 in each group) to measure the degree of luminal stricture, and performed histological analysis. Results: Severe luminal stricture was observed in the rectum but not in the distal colon. Moreover, fiber accumulation in the submucosa and hypertrophy of the muscularis propria were observed in the rectum but not in the distal colon. The degree of luminal stricture in the rectum was significantly lower in the MSC-CS group than in the control group. Furthermore, MSC-CS attenuated myofibroblast activation and hypertrophy of the muscularis propria on Day 22, and reduced inflammatory cell infiltration on Day 8. Conclusions: Luminal stricture after ESD developed only in the rectum because of the difference in myofibroblast activation and fiber accumulation. In addition, MSC-CS enema prevented luminal stricture after ESD, possibly by inhibiting the inflammatory reaction and fibrosis

Human Amnion-Derived Mesenchymal Stem Cell Transplantation Ameliorates Liver Fibrosis in Rats

Background: Mesenchymal stem cells (MSCs) are a valuable cell source in regenerative medicine. Recently, several studies have shown that MSCs can be easily isolated from human amnion. In this study, we investigated the therapeutic effect of transplantation of human amnion-derived MSCs (hAMSCs) in rats with liver fibrosis. Methods: Liver fibrosis was induced by an intraperitoneal injection of 2 mL/kg of 50% carbon tetrachloride twice a week for 6 weeks. At 3 weeks, hAMSCs (1 × 10^6 cells) were transplanted intravenously. Rats were sacrificed at 7 weeks, and histological analyses and quantitative reverse-transcription polymerase chain reaction were performed. In vitro experiments were conducted to investigate the effect of hAMSCs on the activation of Kupffer cells. Results: Transplantation of hAMSCs significantly reduced the fibrotic area, deposition of type-I collagen, the number of α-smooth muscle actin-positive hepatic stellate cells, and CD68-positive Kupffer cells in the livers. messenger RNA expression of α-smooth muscle actin and tissue inhibitor of metalloproteinase-1 was significantly decreased and the expression of matrix metalloproteinase-9 and hepatocyte growth factor was significantly increased in the liver of hAMSC-treated rats. Transplantation of hAMSCs at 3 weeks plus 5 weeks did not have an additive effect. In vitro experiments demonstrated that Kupffer cell activation induced by lipopolysaccharide was significantly decreased by culturing with conditioned medium obtained from hAMSCs. Conclusions: Transplantation of hAMSCs provided significant improvement in a rat model of liver fibrosis, possibly through the inhibition of Kupffer cell and hepatic stellate cell activation. hAMSCs may be a potential new treatment for liver fibrosis

Fluorescence evaluation of EUS-FNA specimens

Purpose: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is the most reliable method for the histological diagnosis of pancreatic tumors. Rapid on-site fluorescence-guided histological diagnosis was evaluated by topically applying an enzymatically activatable probe onto the EUS-FNA samples; the probe fluoresces in the presence of γ-glutamyltranspeptidase (GGT). Procedures: We evaluated GGT expression in pancreatic cancer cell lines in vitro. EUS-FNA was performed in 10 pancreatic tumors. After topical application of the probe, signal intensity was measured using a fluorescence imaging system for 13 min. Results: GGT was expressed in Panc-1, AsPC-1, and AR42J, but not in KP4 cells. In samples from six cases, several regions of the specimens fluoresced and contained adequate tissue for pathological diagnosis. The remaining four non-fluorescent samples contained very small amounts of carcinoma, normal epithelial cells, or no epithelial cells. The signal intensity at 5 min was 25.5 ± 7.7 and 7.7 ± 0.5 in fluorescent and non-fluorescent regions, respectively (p < 0.05). Conclusions: Application of enzymatically activatable probe onto EUS-FNA samples would be feasible for the rapid evaluation of tissues suitable for histological diagnosis

Fluorescent imaging of superficial head and neck squamous cell carcinoma using a γ-glutamyltranspeptidase-activated targeting agent : a pilot study

Background: Detecting superficial head and neck squamous cell carcinoma (HNSCC) by endoscopy is challenging because of limited morphological hallmarks, and iodine cannot be applied to head and neck lesions due to severe mucosal irritation. γ-glutamyltranspeptidase (GGT), a cell surface enzyme, is overexpressed in several cancers, and it has been reported that γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), a fluorescent targeting agent which can be enzymatically activated and becomes fluorescent after cleavage of a GGT-specific sequence, can be activated within a few minutes after application to animal models. We investigated whether early HNSCC can be detected by applying gGlu-HMRG to clinical samples. Methods: gGlu-HMRG was applied to four HNSCC cell lines, and fluorescence was observed by fluorescence microscopy and flow cytometry. Immunohistological examination was performed in three recent cases of endoscopic submucosal dissection (ESD) to investigate GGT expression. Fluorescence imaging with gGlu-HMRG in eight clinical samples resected by ESD or surgery was performed, and fluorescence intensity of tumor and normal mucosa regions of interest (ROI) was prospectively measured. Results: All four gGlu-HMRG-applied cell lines emitted green fluorescence. Immunohistological examination demonstrated that GGT was highly expressed in HNSCC of the recent three ESD cases but barely in the normal mucosa. Fluorescence imaging showed that iodine-voiding lesions became fluorescent within a few minutes after application of gGlu-HMRG in all eight resected tumors. Tumor ROI fluorescence intensity was significantly higher than in the normal mucosa five minutes after gGlu-HMRG application. Conclusions: Fluorescence imaging with gGlu-HMRG would be useful for early detection of HNSCC

Autograft of Demineralized Dentin Matrix Prepared Immediately after Extraction for Horizontal Bone Augmentation of the Anterior Atrophic Maxilla: A First Case of Non-Vital Tooth-Derived Dentin

Onlay bone augmentation is a challenging field, especially in esthetic zones. In implant dentistry, labial bone loss is generally recovered through autologous bone and/or biomaterials. Dental pioneers have been applying a patient-own demineralized dentin matrix (DDM) for bone regeneration. The aim of this study was to histologically and radiologically evaluate horizontal bone augmentation on an anterior atrophic maxilla using an autograft of non-vital tooth-derived DDM for implant. A 56-year-old female patient presented with severe labial bone loss. Autologous DDM was immediately prepared for horizontal ridge augmentation. DDM blocks were fixed with titanium screws for lateral augmentation. DDM granules were grafted on the atrophic bone between the DDM blocks. Twelve months postoperatively, bone biopsy was performed from the implant placement point for histological observation. X-ray computed tomography (CT) was performed before and after the DDM graft surgery. Hematoxylin and eosin sections showed that new bones were directly generated on the DDM residue. CT images showed the original buccal bone lines and root-type and wall-type DDM. The grafted DDMs were clearly found on the original bone. The outlines in the 22nd region indicated an 8.11 mm horizontal width after the implant placements compared to 4.95 mm before the augmentation. Additionally, the width at the upper level increased from 3.38 mm to 5.92 mm. DDM scaffolds contributed to the horizontal bony support required to place the implants. The patient experienced therapeutic success with DDM immediate autograft and implant surgery