AT-MSCs Antifibrotic Activity is Improved by Eugenol through Modulation of TGF-ß/Smad Signaling Pathway in Rats.

For hepatic failure, stem cell transplantation has been chosen as an alternative therapy, especially for mesenchymal stem cells (MSCs). The aim of this study was to investigate the effect of eugenol (EUG) on the in vivo antifibrotic activity of adipose tissue-derived MSCs (AT-MSCs) and the underlying mechanism. After characterization of MSCs, rats were divided into five groups, Group 1 (normal control), Group 2 (CCl4), Group 3 (CCl4 + AT-MSCs), Group 4 (CCl4 + EUG) and Group 5 (CCl4 + AT-MSCs + EUG). Biochemical and histopathological investigations were performed. Furthermore, expression of type 1 collagen, α-SMA, TGF-ß1, Smad3 and P-Smad3 was estimated. Compared to the single treatment with AT-MSCs, the combination treatment of the fibrotic rats with AT-MSCs and EUG significantly improved the plasma fibrinogen concentration, IL-10 level and proliferating cell nuclear antigen expression, and also significantly decreased the serum levels of liver enzymes, IL-6, IL-1ß, TNF-α, type III collagen, hyaluronic acid, hydroxyproline and the TGF-ß growth factor. Furthermore, the combination treatment significantly decreased the hepatic expression of fibrotic markers genes (Type 1 collagen and α-SMA) and proteins (α-SMA, TGF-ß1 and phospho-Smad3) more than the treatment with AT-MSCs alone. We demonstrated that the combination treatment with EUG and AT-MSCs strongly inhibited the advancement of CCl4-induced hepatic fibrosis, compared with AT-MSCs alone, through TGF-ß/Smad pathway inhibition. This approach is completely novel, so more investigations are necessary to improve our perception of the underlying molecular mechanisms accountable for the effects of EUG on the antifibrotic potential of AT-MSCs.

Preconditioning of Adipose-Derived Mesenchymal Stem-Like Cells with Eugenol Potentiates Their Migration and Proliferation In Vitro and Therapeutic Abilities in Rat Hepatic Fibrosis.

Mesenchymal stem cells (MSCs) have considerable therapeutic abilities in various disorders, including hepatic fibrosis. They may be affected with different culture conditions. This study investigated, on molecular basics, the effect of pretreatment with eugenol on the characteristics of adipose tissue-derived MSCs (ASCs) in vitro and the implication of eugenol preconditioning on the in vivo therapeutic abilities of ASCs against CCl4-induced hepatic fibrosis in rats. The effect of eugenol on ASCs was assessed using viability, scratch migration and sphere formation assays. Expressions of genes and proteins were estimated by immunofluorescence or qRT-PCR. For the in vivo investigations, rats were divided into four groups: the normal control group, fibrotic (CCl4) group, CCl4+ASCs group and CCl4 + eugenol-preconditioned ASCs (CCl4+E-ASCs) group. Eugenol affected the viability of ASCs in a concentration- and time-dependent manner. Eugenol improved their self-renewal, proliferation and migration abilities and significantly increased their expression of c-Met, reduced expression 1 (Rex1), octamer-binding transcription factor 4 (Oct4) and nanog genes. Furthermore, E-ASCs showed more of a homing ability than ASCs and improved the serum levels of ALT, AST, albumin, total bilirubin and hyaluronic acid more efficient than ASCs in treating CCl4-induced hepatic fibrosis, which was confirmed with histopathology. More interestingly, compared to the CCl4+ASCs group, CCl4+E-ASCs group showed a lower expression of inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), cluster of differentiation 163 (CD163) and tumor necrosis factor-α (TNF-α) genes and higher expression of matrix metalloproteinase (MMP)-9 and MMP-13 genes. This study, for the first time, revealed that eugenol significantly improved the self-renewal, migration and proliferation characteristics of ASCs, in vitro. In addition, we demonstrated that eugenol-preconditioning significantly enhanced the therapeutic abilities of the injected ASCs against CCl4-induced hepatic fibrosis.

Bee Venom Accelerates Wound Healing in Diabetic Mice by Suppressing Activating Transcription Factor-3 (ATF-3) and Inducible Nitric Oxide Synthase (iNOS)-Mediated Oxidative Stress and Recruiting Bone Marrow-Derived Endothelial Progenitor Cells.

Multiple mechanisms contribute to impaired diabetic wound healing including impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Bee venom (BV) has been used as an anti-inflammatory agent for the treatment of several diseases. Nevertheless, the effect of BV on the healing of diabetic wounds has not been studied. Therefore, in this study, we investigated the impact of BV on diabetic wound closure in a type I diabetic mouse model. Three experimental groups were used: group 1, non-diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice treated with BV. We found that the diabetic mice exhibited delayed wound closure characterized by a significant decrease in collagen production and prolonged elevation of inflammatory cytokines levels in wounded tissue compared to control non-diabetic mice. Additionally, wounded tissue in diabetic mice revealed aberrantly up-regulated expression of ATF-3 and iNOS followed by a marked elevation in free radical levels. Impaired diabetic wound healing was also characterized by a significant elevation in caspase-3, -8, and -9 activity and a marked reduction in the expression of TGF-ß and VEGF, which led to decreased neovascularization and angiogenesis of the injured tissue by impairing EPC mobilization. Interestingly, BV treatment significantly enhanced wound closure in diabetic mice by increasing collagen production and restoring the levels of inflammatory cytokines, free radical, TGF-ß, and VEGF. Most importantly, BV-treated diabetic mice exhibited mobilized long-lived EPCs by inhibiting caspase activity in the wounded tissue. Our findings reveal the molecular mechanisms underlying improved diabetic wound healing and closure following BV treatment. J. Cell. Physiol. 231: 2159-2171, 2016. © 2016 Wiley Periodicals, Inc.

The Impact of Royal Jelly against Hepatic Ischemia/Reperfusion-Induced Hepatocyte Damage in Rats: The Role of Cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF-α Signaling Pathways.

OBJECTIVE: The present study was conducted to elucidate the underlying molecular mechanism as well as the potential hepatoprotective effects of royal jelly (RJ) against hepatic ischemia/ reperfusion (IR) injury. METHODS: Rats were assigned into four groups; sham (received vehicle), IR (30 minutes ischemia and 45 minutes reperfusion), sham pretreated with RJ (200 mg/kg P.O.), and IR pretreated with RJ (200 mg/kg P.O.). The experiment lasted for 28 days. RESULTS: Hepatic IR significantly induced hepatic dysfunctions, as manifested by elevation of serum transaminases, ALP and LDH levels. Moreover, hepatic IR caused a significant up-regulation of P38-MAPK, NF-κB-p65, TNF-α and MDA levels along with marked down-regulation of Nrf-2, HO-1, COX-4, cytoglobin, IκBa, IL-10, GSH, GST and SOD levels. Additionally, marked histopathological changes were observed after hepatic IR injury. On the contrary, pretreatment with RJ significantly improved hepatic functions along with the alleviation of histopathological changes. Moreover, RJ restored oxidant/antioxidant balance as well as hepatic expressions of Nrf- 2, HO-1, COX-4, and cytoglobin. Simultaneously, RJ significantly mitigated the inflammatory response by down-regulation of P38-MAPK, NF-κB-p65, TNF-α expression. CONCLUSION: The present results revealed that RJ has successfully protected the liver against hepatic IR injury through modulation of cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF- α signaling pathways.

The Possible Protective Role of Barley Seeds on the Spleen after Administration of Glucocorticoids in Adult Albino Rats: A Histological and Immunohistochemical Study.

BACKGROUND: Glucocorticoids (GCs) are the main treatment strategy in many autoimmune disease and inflammatory diseases; however, they have immunosuppressive effect on many organs. The barley seeds contain many antioxidant compounds, which may improve the antioxidant status and related physiological functions. Our aim in this work is to evaluate the possible protective role of barley seeds on some immune cells in the spleen against immunosuppressive effect of GCs in adult albino rats. MATERIALS AND METHODS: Forty-five adult albino rats were equally divided into 3 groups. Group I: normal vehicle control (n = 15), Group II: steroid-treated animals (n = 15), and Group III: steroid/barley-treated group (n = 15). Specimens from spleen were processed for light and electron microscopy. RESULTS: In steroid-treated group, the histological changes in white and red pulp were in the form of loss of architecture and wide empty spaces among the cells. Most of the cells showed degenerative change, dilatation of blood sinusoids, and deposition of fibrinoid material among the cells of the RP. However, multiple lysosomal bodies were observed in both dendritic and macrophage cells. These changes are improved in steroid/barley-treated group in the form of increasing the number and size of the lymphatic follicles. Most of the splenic cells regained normal structure. Dendritic cell marker CD86 and macrophage marker CD68 expression are increased. CONCLUSION: Barley protects the spleen tissues from steroid-induced structural changes; this could be mediated through its antioxidant effects, so barely is recommended as a healthy diet in patients consuming steroids.

Periostin expression and characters of human adipose tissue-derived mesenchymal stromal cells were aberrantly affected by in vitro cultivation.

BACKGROUND: Human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) have been under focus in regenerative medicine since their discovery as a suitable source of MSCs. AD-MSCs are heterogeneous cells and exhibit variations in population doubling time, morphology and proliferative capacity. This study investigated if human AD-MSCs are developing, during in vitro long-term cultivation, in an unwanted or aberrant way. METHODS: This study monitored AD-MSCs during their in vitro culture till the tenth passage investigating proliferation kinetics, DNA index and surface markers expression. Also, periostin gene expression was examined. RESULTS: The proliferation capacity and colony forming unit were decreased after passage 6 and the population doubling time was increased. Flow cytometric analysis revealed that newly cultivated population strongly expressed MSCs markers, furthermore, reduction of CD105 expression appeared in passage 5 onwards, the later was associated with significant increase in expression of CD34 (a hematopoietic cell marker). Also, reduction of CD73 and CD90 expression was observed from passage 8. Furthermore, during the first six passages, periostin expression was significantly unchanged, with significant upregulation in late passages. CONCLUSIONS: Long-term cultivation of human AD-MSCs changed their characters in an aberrant way and the first four passages might be the most appropriate passages for therapy. More investigation and understanding of these variations are needed to help in standardizing the expansion of MSCs-based therapies.

Blocking type I interferon signaling rescues lymphocytes from oxidative stress, exhaustion, and apoptosis in a streptozotocin-induced mouse model of type I diabetes.

Elevated levels of type I interferon (IFN) during type 1 diabetes mellitus (T1D) are associated with a defective immune response. In the present study, we investigated whether blocking type I IFN signaling during streptozotocin- (STZ-) induced T1D in mice improves lymphocyte proliferation and escape from continuous apoptosis. Three groups of mice were examined: diabetic mice, type I IFN signaling-incompetent diabetic mice, and control nondiabetic mice. We first found that diabetes induction was accompanied by an elevation in the plasma levels of reactive oxygen species (ROS), hydroperoxide, malondialdehyde (MDN), and the proinflammatory cytokines IL-1 α, IL-1 ß, IL-6, and CXCL10. Blocking type 1 IFN signaling in diabetic mice significantly decreased the levels of oxidative stress and proinflammatory cytokines. In addition, lymphocytes from diabetic mice exhibited a marked reduction in their proliferative capacity, increased apoptosis, upregulation of the exhaustion marker PD-1, and aberrant phosphorylation of STAT1, STAT2, AKT and I κ B- α. Interestingly, following the blocking of type I IFN signaling in diabetic mice, the lymphocytes exhibited restored proliferative capacity, decreased apoptosis, normal expression of PD-1, and normal phosphorylation of STAT1, STAT2, AKT and I κ B- α. Our data suggest that elevated levels of type I IFN during T1D trigger lymphocyte exhaustion and a defective lymphocyte-medicated immune response.