Human platelets inhibit liver fibrosis in severe combined immunodeficiency mice

AIM:To investigate the role of human platelets in liver fibrosis.METHODS:Severe combined immunodeficiency(SCID)mice were administered CCl4and either phosphate-buffered saline(PBS group)or human platelet transfusions(hPLT group).Concentrations of hepatocyte growth factor(HGF),matrix metallopeptidases(MMP)-9,and transforming growth factor-β(TGF-β)in the liver tissue were compared between the PBS and the hPLT groups by enzyme-linked immunosorbent assay(ELISA)and Western blotting.The effects of a human platelet transfusion on liver fibrosis included the fibrotic area,hydroxyproline content,and-smooth muscle actin(α-SMA)expression,which were evaluated by picrosirius red staining,ELISA,and immunohistochemical staining using an anti-mouse-SMA antibody,respectively.Phosphorylations of mesenchymal-epithelial transition factor(Met)and SMAD3,downstream signals of HGF and TGF-β,were compared between the two groups by Western blotting and were quantified using densitometry.Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling.Furthermore,the accumulation of human platelets in the liver 2 h after platelet transfusion was compared between normal and fibrotic livers by immunohistochemical staining using an anti-human CD41 antibody.RESULTS:The fibrotic area and hydroxyproline content in the liver were both significantly lower in the hPLT group when compared to the PBS group(fibrotic area,1.7%±0.6%vs 2.5%±0.6%,P=0.03;hydroxyproline content,121±26 ng/g liver vs 156±47 ng/g liver,P=0.04).There was less α-smooth muscle actin staining in the hPLT group than in the PBS group(0.5%±0.1%vs 0.8%±0.3%,P=0.02).Hepatic expression levels of mouse HGF and MMP-9were significantly higher in the hPLT group than in the PBS group(HGF,109±13 ng/g liver vs 88±22 ng/g liver,P=0.03;MMP-9,113%±7%/GAPDH vs 92%±11%/GAPDH,P=0.04).In contrast,the concentration of mouse TGF-β in the liver tissue was significantly lower in the hPLT group than in the PBS group(22±5ng/g liver vs 39±6 ng/g liver,P=0.02).Phosphorylation of Met was more prevalent in the hPLT group than in the PBS group(37%±4%/GAPDH vs 20%±8%/GAPDH,P=0.03).Phosphorylation of SMAD3was weaker in the hPLT group than in the PBS group(60%±12%/GAPDH vs 84%±12%/GAPDH,P=0.1),although this difference was not significant.Furthermore,a lower rate of hepatocyte apoptosis was observed in the hPLT group than in the PBS group(5.9%±1.7%vs 2.9%±2.1%,P=0.02).Significant human platelet accumulation was observed in the fibrotic liver tissues,whereas few platelets accumulated in the normal liver.CONCLUSION:Human platelets inhibit liver fibrosis in SCID mice.Increased concentration of HGF in the liver suppresses hepatic stellate cell activation,induces MMPs,and inhibits hepatocyte apoptosis.

Anticancer effect of linalool via cancer-specific hydroxyl radical generation in human colon cancer

AIM To investigate the anticancer mechanisms of the monoterpenoid alcohol linalool in human colon cancer cells.METHODS The cytotoxic effect of linalool on the human colon cancer cell lines and a human fibroblast cell line was examined using the WST-8 assay. The apoptosisinducing effect of linalool was measured using the terminal deoxynucleotidyl transferase d UTP nickend labeling assay and flow cytometry with Annexin V. Oxidative stress was investigated by staining for diphenyl-1-pyrenylphosphine, which is a cellular lipid peroxidation marker, and electron spin resonance spectroscopy. Sixteen SCID mice xenografted with human cancer cells were randomized into 3 groups for in vivo analysis: control and low-dose and high-dose linalool groups. The control group was administered tap water orally every 3 d. The linalool treatment groups were administered 100 or 200 μg/kg linalool solution orally for the same period. All mice were sacrificed under anesthesia 21 d after tumor inoculation, and tumors and organs were collected for immunohistochemistry using an anti-4-hydroxynonenal antibody. Tumor weights were measured and compared between groups. RESULTS Linalool induced apoptosis of cancer cells in vitro, following the cancer-specific induction of oxidative stress, which was measured based on spontaneous hydroxyl radical production and delayed lipid peroxidation. Mice in the high-dose linalool group exhibited a 55% reduction in mean xenograft tumor weight compared with mice in the control group(P < 0.05). In addition, tumor-specific lipid peroxidation was observed in the in vivo model.CONCLUSION Linalool exhibited an anticancer effect via cancerspecific oxidative stress, and this agent has potential for application in colon cancer therapy.

New drug delivery system for liver sinusoidal endothelial cells for ischemia-reperfusion injury

AIM: To investigate the cytoprotective effects in hepatic ischemia-reperfusion injury, we developed a new formulation of hyaluronic acid(HA) and sphingosine 1-phophate.METHODS: We divided Sprague-Dawley rats into 4 groups: control, HA, sphingosine 1-phosphate(S1P), and HA-S1 P. After the administration of each agent, we subjected the rat livers to total ischemia followed by reperfusion. After reperfusion, we performed the following investigations: alanine aminotransferase(ALT), histological findings, Td T-mediated d UTP-biotin nick end labeling(TUNEL) staining, and transmission electron microscopy(TEM). We also investigated the expressionof proteins associated with apoptosis, hepatoprotection, and S1 P accumulation. RESULTS: S1 P accumulated in the HA-S1 P group livers more than S1 P group livers. Serum ALT levels, TUNEL-positive hepatocytes, and expression of cleaved caspase-3 expression, were significantly decreased in the HA-S1 P group. TEM revealed that the liver sinusoidal endothelial cell(LSEC) lining was preserved in the HA-S1 P group. Moreover, the HA-S1 P group showed a greater increase in the HO-1 protein levels compared to the S1 P group.CONCLUSION: Our results suggest that HA-S1 P exhibits cytoprotective effects in the liver through the inhibition of LSEC apoptosis. HA-S1 P is an effective agent for hepatic ischemia/reperfusion injury.

Development of the nervous system in mouse liver

BACKGROUND The role of the hepatic nervous system in liver development remains unclear.We previously created functional human micro-hepatic tissue in mice by co-culturing human hepatic endodermal cells with endothelial and mesenchymal cells.However,they lacked Glisson’s sheath[the portal tract(PT)].The PT consists of branches of the hepatic artery(HA),portal vein,and intrahepatic bile duct(IHBD),collectively called the portal triad,together with autonomic nerves.AIM To evaluate the development of the mouse hepatic nervous network in the PT using immunohistochemistry.METHODS Liver samples from C57BL/6J mice were harvested at different developmental time periods,from embryonic day(E)10.5 to postnatal day(P)56.Thin sections of the surface cut through the hepatic hilus were examined using protein gene product 9.5(PGP9.5)and cytokeratin 19(CK19)antibodies,markers of nerve fibers(NFs),and biliary epithelial cells(BECs),respectively.The numbers of NFs and IHBDs were separately counted in a PT around the hepatic hilus(center)and the peripheral area(periphery)of the liver,comparing the average values between the center and the periphery at each developmental stage.NF-IHBD and NF-HA contacts in a PT were counted,and their relationship was quantified.SRYrelated high mobility group-box gene 9(SOX9),another BEC marker;hepatocyte nuclear factor 4α(HNF4α),a marker of hepatocytes;and Jagged-1,a Notch ligand,were also immunostained to observe the PT development.RESULTS HNF4αwas expressed in the nucleus,and Jagged-1 was diffusely positive in the primitive liver at E10.5;however,the PGP9.5 and CK19 were negative in the fetal liver.SOX9-positive cells were scattered in the periportal area in the liver at E12.5.The Jagged-1 was mainly expressed in the periportal tissue,and the number of SOX9-positive cells increased at E16.5.SOX9-positive cells constructed the ductal plate and primitive IHBDs mainly at the center,and SOX-9-positive IHBDs partly acquired CK19 positivity at the same period.PGP9.5-positive bodies were first found at E16.5 and HAs were first found at P0 in the periportal tissue of the center.Therefore,primitive PT structures were first constructed at P0 in the center.Along with remodeling of the periportal tissue,the number of CK19-positive IHBDs and PGP9.5-positive NFs gradually increased,and PTs were also formed in the periphery until P5.The numbers of NFs and IHBDs were significantly higher in the center than in the periphery from E16.5 to P5.The numbers of NFs and IHBDs reached the adult level at P28,with decreased differences between the center and periphery.NFs associated more frequently with HAs than IHBDs in PTs at the early phase after birth,after which the number of NF-IHBD contacts gradually increased.CONCLUSION Mouse hepatic NFs first emerge at the center just before birth and extend toward the periphery.The interaction between NFs and IHBDs or HAs plays important roles in the morphogenesis of PT structure.

Platelet therapy:A novel strategy for liver regeneration,anti-fibrosis,and anti-apoptosis

Platelets contain bio-physiological substances, including insulin-like growth factor-1, vascular endothelial growth factor, platelet-derived growth factor,hepatocyte growth factor, serotonin, transforming growth factor-β, adenosine diphosphate, adenosine tri-phosphate, and epidermal growth factor. Platelets have conventionally been considered to exacerbate the inflammatory response and liver injury. Recently,platelets were discovered to have a positive impact on the liver. In this review, we present experimenta and clinical evidence indicating that platelets accelerate liver regeneration and have anti-fibrosis and antiapoptosis activity, and we detail the mechanisms of action. Platelets accelerate liver regeneration by three different mechanisms:(1) a direct effect on hepatocytes,(2) a cooperative effect with liver sinusoidal endothelial cells, and(3) a collaborative effect with Kupffer cells. Platelets exert anti-fibrotic activity by deactivating hepatic stellate cells via the adenosinecyclic adenosine 5'-monophosphate signaling pathway.Platelets prevent hepatocyte apoptosis by activating the Akt pathway and up-regulating Bcl-x L, which sup-presses caspase-3 activation. Platelet therapy with thrombopoietin, thrombopoietin receptor agonists, and platelet transfusion has the advantages of convenience and cost-efficiency over other treatments. We propose that in the future, platelet therapy will play a promising role in the treatment of the various liver disorders that currently challenge the surgical field, such as liver failure after a massive hepatectomy, hepatectomy of a cirrhotic liver, and small grafts in liver transplantation.