Intraperitoneal Injection Of PBS Research Articles

Human Bone Marrow-Derived Mesenchymal Stromal Cells Reduce the Severity of Experimental Necrotizing Enterocolitis in a Concentration-Dependent Manner.

Necrotizing enterocolitis (NEC) is a devastating gut disease in preterm neonates. In NEC animal models, mesenchymal stromal cells (MSCs) administration has reduced the incidence and severity of NEC. We developed and characterized a novel mouse model of NEC to evaluate the effect of human bone marrow-derived MSCs (hBM-MSCs) in tissue regeneration and epithelial gut repair. NEC was induced in C57BL/6 mouse pups at postnatal days (PND) 3-6 by (A) gavage feeding term infant formula, (B) hypoxia/hypothermia, and (C) lipopolysaccharide. Intraperitoneal injections of PBS or two hBM-MSCs doses (0.5 × 106 or 1 × 106) were given on PND2. At PND 6, we harvested intestine samples from all groups. The NEC group showed an incidence of NEC of 50% compared with controls (p < 0.001). Severity of bowel damage was reduced by hBM-MSCs compared to the PBS-treated NEC group in a concentration-dependent manner, with hBM-MSCs (1 × 106) inducing a NEC incidence reduction of up to 0% (p < 0.001). We showed that hBM-MSCs enhanced intestinal cell survival, preserving intestinal barrier integrity and decreasing mucosal inflammation and apoptosis. In conclusion, we established a novel NEC animal model and demonstrated that hBM-MSCs administration reduced the NEC incidence and severity in a concentration-dependent manner, enhancing intestinal barrier integrity.

Probucol enhances the therapeutic efficiency of mesenchymal stem cells in the treatment of erectile dysfunction in diabetic rats by prolonging their survival time via Nrf2 pathway

BackgroundIntracavernous injection of mesenchymal stem cells (MSCs) is a promising method for diabetic mellitus-induced erectile dysfunction (DMED), but short survival time of MSCs in cavernous is a fatal defect for therapy. This study investigated therapeutic efficiency and potential mechanism of probucol combined with MSCs.MethodsIn vivo study, a total of forty-eight 10-week-old male Sprague-Dawley (SD) rats were used. Twelve rats received intraperitoneal injection of PBS as the sham group; the rest received intraperitoneal injection of 60 mg/kg streptozotocin to establish DM models. DM rats were randomly divided into three groups: received intracavernosal (IC) injection of either PBS (DM group), MSCs (M group), or administrated probucol after intracavernosal injection of MSCs (P + M group). Erectile function was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure measurement. After euthanasia, penile tissue was investigated for histologic examination and Western blotting. In in vitro experiment, H2O2 was used to create oxidative stress environment to detect changes in cell viability. CCK8 was used to measure cell viability of MSCs treated with or without probucol. Intracellular ROS changes were detected by flow cytometry. Autophagy and apoptosis were detected by Western blotting and confocal microscopy.ResultsRecovery of erectile function was observed in the P + M group. The combination therapy decreased fibrosis and increased endothelial function compared with MSC therapy alone. Western blotting results confirmed the increased expression of Nrf2 and HO-1 in cavernous body. H2O2 induced high oxidative stress and reduced cell viability in vitro, which was gradually reversed with increased concentration of probucol. H2O2 reduced Nrf2 expression, which was reversed by probucol’s intervention. Furthermore, the expression of Bax, Caspase3, and Cleaved-Caspase3 decreased, and the expression of Bcl-2 increased in a dose-dependent manner because of probucol’s intervention. In addition, Beclin1 and LC3II both increased in a dose-dependent manner. Meanwhile, the expression of P62 decreased. In the study of autophagy flux, we found probucol did not block it.ConclusionProbucol enhanced therapeutic efficiency of MSCs in DMED by prolonging their survival time, which mediated through improving the transplanted microenvironment of MSCs, increasing self-antioxidant ability of MSCs, strengthening protective autophagy, and inhibiting apoptosis of MSCs via Nrf2 pathway.Graphical abstractSchematic model showing combined probucol and MSCs to improve DMED. Probucol increases self-antioxidant ability of MSCs, strengthening protective autophagy and inhibiting apoptosis via Nrf2/HO-1 and Nrf2/autophagy pathways.

Anti-nerve growth factor antibody reduces airway hyperresponsiveness in a mouse model of asthma by down-regulating the level of autophagy in lungs

To study the effects of anti-nerve growth factor (NGF) antibody on the level of autophagy in lung tissue antigen presenting cells in a mouse model of asthma. The 24 BALB/c mice aged 6 weeks were divided into control group (PBS sensitization, PBS challenge) , asthma group [ovalbumin (OVA) sensitization, OVA challenge] and anti-NGF group (OVA sensitization, OVA challenge) using the random number table, 8 mice in each group. Sensitization was carried out by intraperitoneal injection method while challenge by aerosol inhalation. The anti-NGF group received intraperitoneal injection of anti-NGF antibody for intervention 30 min before the atomization, while the control group and asthma group received intraperitoneal injection of PBS 30 min before the atomization. The lung tissue and serum were harvested 24 h after the last sensitization. Immunoelectron microscopy was used to observe the level of autophagy in mouse airway dendritic cells. Western blot assay was used to detect the autophagy marker molecule MHP1 LC3-II level in lung tissue. ELISA was used to detect the serum levels of IL-4, INF-γ and NGF. Immunohistochemical technique was used to detect the expression levels of costimulatory molecules CD₈₀, CD₈₆ and CD₄₀, and the major histocompatibility complex class II molecule (MHC-II) on dendritic cells of lung tissue. The level of autophagy in lung tissue was lower in anti-NGF group (0.28 ± 0.07) than in asthma group (0.46 ± 0.10) but higher than in control group (0.17 ± 0.08) (F = 15.571, P < 0.05) ; and Pairwise comparison showed that all the intergroup differences were statistically significant (all P values<0.05) . The serum NGF concentration was lower in anti-NGF group [(0.65 ± 0.04) µg/L] than in asthma group [(0.75 ± 0.10) µg/L] but still higher than the control group [(0.52 ± 0.05) µg/L] (F = 61.972, P < 0.05); and Pairwise comparison showed that all the intergroup differences were statistically significant (all P values<0.05) . Both the costimulatory molecules CD₈₀, CD₈₆ and CD₄₀ and the MHC-II on antigen presenting cells of lung tissue were significantly lower in anti-NGF group than in asthma group. Anti-NGF group had lower serum IL-4 but higher IFN-γ level than asthma group. Anti-NGF antibody attenuates Th2-dominant airway inflammation in this mouse model of asthma by down-regulating the level of autophagy in lung tissue to inhibit the antigen-presenting cell functions.

Abstract 3782: Chloroquine induces defective autophagic flux and enhances pancreatic cancer cell death induced by pegylated-arginine deiminase

Abstract We have previously shown that a majority of human pancreatic adenocarcinomas are deficient in argininosuccinate synthetase (ASS), a key enzyme in arginine synthesis and therefore sensitive to arginine deprivation by arginine deiminase (ADI). ASS deficient cell lines undergo cell death when deprived of arginine, but also undergo autophagy. The role of autophagy and its contribution to cell death is controversial. We hypothesized that autophagy is protective in the setting of arginine deprivation and that inhibition of autophagy by hydroxychloroquine (ChQ) would increase cell death. Methods: The human pancreatic cancer cell line MIA-PaCa2 was treated in vitro and in vivo with pegylated arginine deiminase (PEG-ADI) alone or in the presence of ChQ. Cell death was measured by propidium iodide-FACS and apoptosis was evaluated by Annexin V-PI flow cytometry. Caspase 3 cleavage was evaluated by western blotting and ELISA as was expression of LC3. In vivo experiments were conducted by creation of subcutaneous xenografts in athymic mice followed by treatment with intraperitoneal injections of PBS, PEG-ADI, ChQ and combination of PEG-ADI and ChQ. At sacrifice, tumors were removed for analysis. Tumor lysates were analyzed by western blot for caspase 3 and p62. Immunohistochemistry for activated caspase 3 and DNA fragmentation (TUNEL) were also performed. Results: In vitro treatment of MIA-PaCa2 with PEG-ADI induced caspase dependent cell death as well as autophagy at 72 hours. ChQ at low doses inhibited autophagy induced by PEG-ADI, but increased cell death. In vivo, addition of ChQ to a sub-therapeutic dose of PEG-ADI increased tumor suppression decreased autophagy and increased caspase 3 activation compared to either treatment alone. Conclusion: Arginine deprivation induces autophagy and cell death in cell lines deficient in ASS. Autophagy appears to play a protective role, and its inactivation by hydroxychloroquine leads to enhanced tumor suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2011-3782

Resveratrol prevents the development of abdominal aortic aneurysm through attenuation of inflammation, oxidative stress, and neovascularization

Objective We sought to examine the effect of resveratrol (3,4′,5-trihydroxy-trans-stilbene), a plant-derived polyphenolic compound, on the development of abdominal aortic aneurysm (AAA). Methods AAA was induced in mice by periaortic application of CaCl 2. NaCl (0.9%)-applied mice were used as a sham group. Mice were treated with intraperitoneal injection of PBS (Sham/CON, AAA/CON, n = 30 for each) or resveratrol (100 mg/kg/day) (AAA/RSVT, n = 30). Six weeks after the operation, aortic tissue was excised for further examinations. Results Aortic diameter was enlarged in AAA/CON compared with Sham/CON. Resveratrol treatment reduced the aneurysm size and inflammatory cell infiltration in the aortic wall compared with AAA/CON. Elastica Van Gieson staining showed destruction of the wavy morphology of the elastic lamellae in AAA/CON, while it was preserved in AAA/RSVT. The increased mRNA expression of monocyte chemotactic protein-1, tumor necrosis factor-α, intercellular adhesion molecule-1, CD68, vascular endothelial growth factor-A, p47, glutathione peroxidase (GPX)1 and GPX3 were attenuated by resveratrol treatment (all p < 0.05). Administration of resveratrol decreased protein expression of phospho-p65 in AAA. The increased 8-hydroxy-2′-deoxyguanosine-positive cell count and 4-hydroxy-2-nonenal-positive cell count in AAA were also reduced by resveratrol treatment. Zymographic activity of matrix metalloproteinase (MMP)-9 and MMP-2 was lower in AAA/RSVT compared with AAA/CON (both p < 0.05). Compared with AAA/CON, Mac-2 + macrophages and CD31 + vessels in the aortic wall were decreased in AAA/RSVT (both p < 0.05). Conclusion Treatment with resveratrol in mice prevented the development of CaCl 2-induced AAA, in association with reduced inflammation, oxidative stress, neoangiogenesis, and extracellular matrix disruption. These findings suggest therapeutic potential of resveratrol for AAA.

Alphastatin inhibits tumor angiogenesis in nude mice bearing human gastric cancer xenografts

To explore the effect and mechanism of alphastatin on tumor angiogenesis in nude mice bearing human gastric cancer xenografts. Nude mice were injected subcutaneously with matrigel matrix into matrigel plug. The effect of alphastatin on neovasculature inside Matrigel plug was observed. Human gastric cancer cells (BGC823) were injected subcutaneously in nude mice. After intraperitoneal injection of PBS or alphastatin,tumor volume, weight and microvascular density were analyzed. Sphingosine kinase(SPK) activity assay was used to evaluate the effect of alphastatin on tumor endothelial cells. In vivo, alphastatin was sufficiently potent to suppress nude mice neovascularization. Daily administration of alphastatin produced significant tumor growth suppression [tumor volume:(612.65+/-23.45) microm(3),(1145.96+/-29.89) microm(3) vs(1771+/-31.05) microm(3) (P<0.05), tumor weight: (0.31+/-0.03) g, (0.12+/-0.02) g vs (0.67+/-0.02) g (P<0.05)]. Immunohistochemical studies of tumor tissues revealed decreased microvessel density in alphastatin-treated animals as compared with controls. Alphastatin significantly inhibited tumor endothelial cells SPK activity. Alphastatin shows potent antiangiogenic properties in nude mice,which might be closely associated with down-regulation of tumor endothelial cells SPK activity.

A Focal Fibrotic Reaction of the Lung

We sought to examine the effect of resveratrol (3,4′,5-trihydroxy-trans-stilbene), a plant-derived polyphenolic compound, on the development of abdominal aortic aneurysm (AAA).AAA was induced in mice by periaortic application of CaCl2. NaCl (0.9%)-applied mice were used as a sham group. Mice were treated with intraperitoneal injection of PBS (Sham/CON, AAA/CON, n = 30 for each) or resveratrol (100 mg/kg/day) (AAA/RSVT, n = 30). Six weeks after the operation, aortic tissue was excised for further examinations.Aortic diameter was enlarged in AAA/CON compared with Sham/CON. Resveratrol treatment reduced the aneurysm size and inflammatory cell infiltration in the aortic wall compared with AAA/CON. Elastica Van Gieson staining showed destruction of the wavy morphology of the elastic lamellae in AAA/CON, while it was preserved in AAA/RSVT. The increased mRNA expression of monocyte chemotactic protein-1, tumor necrosis factor-α, intercellular adhesion molecule-1, CD68, vascular endothelial growth factor-A, p47, glutathione peroxidase (GPX)1 and GPX3 were attenuated by resveratrol treatment (all p < 0.05). Administration of resveratrol decreased protein expression of phospho-p65 in AAA. The increased 8-hydroxy-2′-deoxyguanosine-positive cell count and 4-hydroxy-2-nonenal-positive cell count in AAA were also reduced by resveratrol treatment. Zymographic activity of matrix metalloproteinase (MMP)-9 and MMP-2 was lower in AAA/RSVT compared with AAA/CON (both p < 0.05). Compared with AAA/CON, Mac-2+ macrophages and CD31+ vessels in the aortic wall were decreased in AAA/RSVT (both p < 0.05).Treatment with resveratrol in mice prevented the development of CaCl2-induced AAA, in association with reduced inflammation, oxidative stress, neoangiogenesis, and extracellular matrix disruption. These findings suggest therapeutic potential of resveratrol for AAA.