Synergic Effect of Combined Therapy of Hyperbaric Oxygen and Adipose-Derived Mesenchymal Stem Cells on Improving Locomotor Recovery After Acute Traumatic Spinal Cord Injury in Rat Mainly Through Downregulating Inflammatory and Cell-Stress Signalings.

This study tested whether combined hyperbaric oxygen (HBO) and allogenic adipose-derived mesenchymal stem cells (ADMSCs) would be superior to either one for improving the locomotor recovery in rat after acute traumatic spinal cord injury (TSCI) in rat. Adult-male Sprague-Dawley rats were equally categorized into group 1 (sham-operated control), group 2 (TSCI), group 3 (TSCI + HBO for 1.5 h/day for 14 consecutive days after TSCI), group 4 (TSCI + ADMSCs/1.2 × 106 cells by intravenous injection at 3 h and days 1/2 after TSCI), and group 5 (TSCI + HBO + ADMSCs), euthanized, and spinal cord tissue was harvested by day 49 after TSCI. The protein expressions of oxidative-stress (NOX-1/NOX-2), inflammatory-signaling (TLR-4/MyD88/IL-1ß/TNF-α/substance-p), cell-stress signaling (PI3K/p-AKT/p-mTOR), and the voltage-gated sodium channel (Nav1.3/1.8/1.9) biomarkers were highest in group 2, lowest in group 1, and significantly lower in group 5 than in groups 3/4 (all P <0.0001), but they did not differ between groups 3 and 4. The spinal cord damaged area, the cellular levels of inflammatory/DNA-damaged biomarkers (CD68+/GFAP+/γ-H2AX+ cells), mitogen-activated protein kinase family biomarkers (p-P38/p-JNK/p-ERK1/2), and cellular expressions of voltage-gated sodium channel (Nav.1.3, Nav.1.8, and Nav.1.9 in NF200+ cells) as well as the pain-facilitated cellular expressions (p-P38+/peripherin+ cells, p-JNK+/peripherin+ cells, p-ERK/NF200+ cells) exhibited an identical pattern of inflammation, whereas the locomotor recovery displayed an opposite pattern of inflammation among the groups (all P < 0.0001). Combined HBO-ADMSCs therapy offered additional benefits for preserving the neurological architecture and facilitated the locomotor recovery against acute TSCI.

Melatonin and hyperbaric oxygen therapies suppress colorectal carcinogenesis through pleiotropic effects and multifaceted mechanisms.

Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal carcinogenesis is frequently induced by hypoxia to trigger the reprogramming of cellular metabolism and gain of malignant phenotypes. Previously, hyperbaric oxygen (HBO) therapy and melatonin have been reported to alter the hypoxic microenvironment, resulting in inhibiting cancer cell survival. Accordingly, this study tested the hypothesis whether HBO and melatonin effectively inhibited CRC carcinogenesis. In vitro results indicated that melatonin therapy significantly suppressed the malignant phenotypes, including colony formation, growth, invasion, migration and cancer stemness with dose-dependent manners in CRC cell lines through multifaceted mechanisms. Similar to in vitro study, in vivo findings further demonstrated the melatonin, HBO and combined treatments effectively promoted apoptosis (cleaved-caspase 3/ cleaved-PARP) and arrested tumor proliferation, followed by inhibiting colorectal tumorigenesis in CRC xenograft tumor model. Moreover, melatonin, HBO and combined treatments modulated multifaceted mechanisms, including decreasing HIF-1α expression, alleviating AKT activation, repressing glycolytic metabolism (HK-2/PFK1/PKM2/LDH), restraining cancer stemness pathway (TGF-ß/p-Smad3/Oct4/Nanog), reducing inflammation (p-NFκB/ COX-2), diminishing immune escape (PD-L1), and reversing expression of epithelial mesenchymal transition (E-cadherin/N-cadherin/MMP9). In conclusion, melatonin and HBO therapies suppressed colorectal carcinogenesis through the pleiotropic effects and multifaceted mechanisms, suggesting melatonin and HBO treatments could be novel therapeutic strategies for CRC treatment.

Umbilical cord-derived MSC and hyperbaric oxygen therapy effectively protected the brain in rat after acute intracerebral haemorrhage.

This study tested the hypothesis that combined therapy with human umbilical cord-derived mesenchymal stem cells (HUCDMSCs) and hyperbaric oxygen (HBO) was superior to either one on preserving neurological function and reducing brain haemorrhagic volume (BHV) in rat after acute intracerebral haemorrhage (ICH) induced by intracranial injection of collagenase. Adult male SD rats (n = 30) were equally divided into group 1 (sham-operated control), group 2 (ICH), group 3 (ICH +HUCDMSCs/1.2 × 106 cells/intravenous injection at 3h and days 1 and 2 after ICH), group 4 (ICH +HBO/at 3 hours and days 1 and 2 after ICH) and group 5 (ICH +HUCDMSCs-HBO), and killed by day 28 after ICH. By day 1, the neurological function was significantly impaired in groups 2-5 than in group 1 (P < .001), but it did not differ among groups 2 to 5. By days 7, 14 and 28, the integrity of neurological function was highest in group 1, lowest in group 2 and significantly progressively improved from groups 3 to 5 (all P < .001). By day 28, the BHV was lowest in group 1, highest in group 2 and significantly lower in group 5 than in groups 3/4 (all P < .0001). The protein expressions of inflammation (HMGB1/TLR-2/TLR-4/MyD88/TRAF6/p-NF-κB/IFN-γ/IL-1ß/TNF-α), oxidative stress/autophagy (NOX-1/NOX-2/oxidized protein/ratio of LC3B-II/LC3B-I) and apoptosis (cleaved-capspase3/PARP), and cellular expressions of inflammation (CD14+, F4/80+) in brain tissues exhibited an identical pattern, whereas cellular levels of angiogenesis (CD31+/vWF+/small-vessel number) and number of neurons (NeuN+) exhibited an opposite pattern of BHV among the groups (all P < .0001). These results indicate that combined HUCDMSC-HBO therapy offered better outcomes after rat ICH.

Circulatory Rejuvenated EPCs Derived from PAOD Patients Treated by CD34+ Cells and Hyperbaric Oxygen Therapy Salvaged the Nude Mouse Limb against Critical Ischemia.

This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice (n = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood prior to CD34+ cell and HBO treatment (EPCPr-T) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood after CD34+ cell and HBO treatment (EPCAf-T) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 (p < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all p < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all p < 0.0010). The number of small vessels and endothelial cell surface markers (CD31+/vWF+) in the CLI area displayed an identical pattern of INBF (all p < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all p < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.

Protective effect of combined therapy with hyperbaric oxygen and autologous adipose-derived mesenchymal stem cells on renal function in rodent after acute ischemia-reperfusion injury.

BACKGROUND: This study tested the hypothesis that combined hyperbaric oxygen (HBO) and autologous adipose-derived mesenchymal stem cell (ADMSC) therapy was superior to either alone at protecting renal function in rodents after acute ischemia-reperfusion (IR) injury. METHODS AND RESULTS: Adult-male SD rats (n = 40) were equally categorized: group 1 (sham-operated control); group 2 (IR + 50 µg medium intra-renal artery administration); group 3 [IR + HBO (at 1.5 h and days 1 and 2 after IR)]; group 4 [IR + ADMSC (2.0×106 cells/5.0×105/per each renal artery and 1.0×106 by intravenous injection at 1.5 h after IR]; and group 5 (IR + HBO-ADMSC). By 72 hr after IR, the circulating levels of BUN/creatinine and ratio of urine protein/creatinine were significantly highest in group 2, lowest in group 1, significantly increased in group 5 than in groups 3 and 4, but not different between latter two groups, whereas the circulating levels of EPCs and soluble-angiogenesis biomarkers (SDF-1α/HIF-1α) exhibited an opposite pattern to BUN/creatinine among the five groups (all P<0.001). The kidney injury score, ROS (fluorescent intensity of H2DCFDA dye in kidney), inflammation (F4/80+, CD14+ cells) and glomerular-tubular injury score (WT-1/KIM-1) displayed an identical pattern whereas the integrity of podocyte components exhibited an opposite pattern to BUN/creatinine among the five groups (all P<0.0001). The protein expressions of inflammatory (MMP-9/TNF-α/NF-κB/ICAM-1), oxidative-stress (NOX-1/NOx-2/oxidized protein) and apoptotic (mitochondrial-Bax/cleaved-caspase3/PARP) markers showed an identical pattern to BUN/creatinine (all P<0.001). CONCLUSION: Combined ADMSC-HBO therapy was superior to either one alone at protecting the kidney from acute IR injury.

Combined Therapy With Hyperbaric Oxygen and Melatonin Effectively Reduce Brain Infarct Volume and Preserve Neurological Function After Acute Ischemic Infarct in Rat.

This study tested the hypothesis that combined hyperbaric oxygen (HBO) and melatonin (Mel) was superior to either one for protecting the brain functional and parenchymal integrity from acute ischemic stroke (IS) injury. Adult-male Sprague-Dawley rats were divided into groups 1 (sham-operated control), 2 (IS), 3 (IS + HBO), 4 (IS + Mel), and 5 (IS + HBO-Mel). By day 28 after IS, the brain infarct area (BIA) was lowest in group 1, highest in group 2, significantly higher in groups 3 and 4 than in group 5, but not different between groups 3 and 4. The neurological function at day 7, 14, and 28 exhibited an opposite pattern to BIA among the 5 groups. The protein expressions of inflammatory (IL-1ß/IL-6/iNOS/TNF-α/p-NF-κB), apoptotic (cleaved-caspase3/cleaved-PARP/mitochondrial Bax), mitochondrial/DNA-damaged (cytochrome-C/γ-H2AX), oxidative stress (NOX-1/NOX-2), and autophagy (i.e. ratio of CL3B-II/CL3B-I) biomarkers displayed an identical pattern of BIA among 5 groups. Cellular expressions of inflammation (F4/80+/GFAP+) and DNA-damaged biomarker (γ-H2AX+) exhibited an identical pattern, whereas the integrities of myelin sheath/neuron (MPB+/NeuN+), endothelial cell (CD31+/vWF+), and number of small vessels exhibited an opposite pattern of BIA among the 5 groups. Combined HBO-Mel therapy offered an additional benefit in protecting the brain against IS injury.

Hyperbaric oxygen facilitates the effect of endothelial progenitor cell therapy on improving outcome of rat critical limb ischemia.

We tested the hypothesis that hyperbaric oxygen (HBO) (100% oxygen/2.4 atmospheres) facilitated the effect of autologous endothelial progenitor cell (EPC) therapy on restoring the blood flow in rat critical-limb ischemia (CLI). Adult-male-SD rats (n = 8/each group) were categorized into group 1 [sham control (SC)], group 2 (CLI-treated with culture medium), group 3 [CLI-intermittent HBO (3 h/day for 5 consecutive days after CLI), group 4 (CLI-EPC/2.0 × 106 cells), and group 5 (CLI-HBO-EPC). By day 5 after CLI, flow cytometry showed that the circulating EPC (Sca-1/CD31+/C-kit/CD31+/CD34+) levels were highest in group 5 and lowest in group 2 (all P < 0.001). By day 14, laser Doppler demonstrated that the ratio of blood flow (i.e., CLI to normal hind-limb) was highest in group 1, lowest in group 2 and significantly higher in group 5 than in groups 3 and 4 (all P < 0.0001). The protein expressions of endothelial-cell biomarkers (CD31/vWF/eNOS), and numbers of endothelial-cell markers (CD31+/vWF+) and small vessels exhibited a similar pattern to blood-flow ratio among five groups, whereas the angiogenesis parameters in protein (CXCR4/SDF-1α/HIF-1α/VEGF) and cellular (HIF-1α/SDF-1α/CXCR4+) levels were progressively increased from groups 1 to 5 (all P < 0.0001). The protein expression of apoptotic (mitochondrial-Bax/cleaved-capspase-3/PARP), fibrotic (p-Smad3/TGF-ß) and mitochondrial-damaged (cytosolic-cytochrome C) exhibited an opposite pattern, whereas the protein expressions of anti-fibrotic (BMP-2/p-Smad1/5) and mitochondrial integrity (mitochondrial-cytochrome C) exhibited an identical pattern of ratio of blood flow among the five groups (all P < 0.0001). Combined HBO-EPC therapy is superior to either one alone in improving ischemia in rodent CLI.

Hyperbaric Oxygen Therapy Enhanced Circulating Levels of Endothelial Progenitor Cells and Angiogenesis Biomarkers, Blood Flow, in Ischemic Areas in Patients with Peripheral Arterial Occlusive Disease.

BACKGROUND: This study tested the hypothesis that hyperbaric oxygen (HBO) therapy enhanced the circulating levels of endothelial progenitor cells (EPCs), soluble angiogenesis factors, and blood flow in ischemic areas in patients with peripheral arterial occlusive disease (PAOD). METHODS: In total, 57 consecutive patients with PAOD undergoing the HBO therapy (3 atmospheres (atm) for 2 h each time) were prospectively enrolled into the present study. Venous blood sampling was performed to assess the circulating levels of EPCs and soluble angiogenesis factors prior to and during five sessions of HBO therapy. Additionally, skin perfusion pressure (SPP), an indicator of blood flow in ischemic areas, was measured by moorVMS-PRES. RESULTS: The results demonstrated that the circulating levels of EPCs (cluster of differentiation (CD)34⁺/CD133⁺/CD45dim, CD31⁺/CD133⁺/CD45dim, CD34⁺) and soluble angiogenesis factors-vascular endothelial growth factor/stromal cell-derived factor 1/hepatocyte growth factor/fibroblast growth factor (VEGF/SDF-1α/HGF/FGF) were significantly increased post-HBO therapy as compared to pre-HBO therapy (all p < 0.01). Additionally, Matrigel assay showed that the angiogenesis was significantly increased in post-HBO therapy as compared to prior to therapy (p < 0.001). Furthermore, SPP was significantly increased in the ischemic area (i.e., plantar foot and mean SPP of the ischemic foot) in post-HBO therapy as compared to pre-HBO therapy (all p < 0.01). Importantly, the HBO therapy did appear to result in complications, and all the patients were uneventfully discharged without amputation. CONCLUSIONS: HBO therapy augmented circulating levels of EPCs and angiogenesis factors, and improved the blood flow in the ischemic area.

Combined Therapy With Adipose-Derived Mesenchymal Stem Cells and Ciprofloxacin Against Acute Urogenital Organ Damage in Rat Sepsis Syndrome Induced by Intrapelvic Injection of Cecal Bacteria.

UNLABELLED: We hypothesized that combined treatment with autologous adipose-derived mesenchymal stem cell (ADMSC) and ciprofloxacin is superior to ciprofloxacin only in reducing sepsis-induced urogenital organ damage and mortality in rat sepsis syndrome (SS) caused by intrapelvic injection of cecal bacteria (1.0 × 10(4) cells per milliliter; total, 5.0 ml). Male Sprague-Dawley rats (n = 60) equally divided into group 1 (sham-control), group 2 (SS), group 3 (SS-ADMSC [5.0 × 10(5) intravenously at 0.5, 6, and 18 hours after sepsis induction]), group 4 (SS-ciprofloxacin [3.0 mg/kg, b.i.d.] for 5 days), and group 5 (SS-ADMSC-ciprofloxacin) were sacrificed by day 5. Mortality rate and creatinine level were highest in group 2 and lowest in group 1 and significantly higher in groups 3 and 4 than those in group 5, but there was no difference between groups 3 and 4 (all p < .005). The kidney injury score, inflammatory biomarker expressions at protein (tumor necrosis factor-1α, nuclear factor-κB, matrix metallopeptidase-9, regulated on activation, normal T-cell expressed and secreted, interleukin-1ß) and cellular (CD14+, migratory inhibitor factor positive, CD68+) levels in kidneys and urinary bladder were lowest in group 1 and highest in group 2, higher in group 4 than in groups 3 and 5, and higher in group 3 than in group 5 (all p < .001). Protein expressions of apoptosis (Bax, cleaved caspase 3 and poly[ADP-ribose] polymerase 1, p21 protein [Cdc42/Rac]-activated kinase 2) and oxidative stress (oxidized protein, NADPH oxidase (NOX)-1, NOX-2) in these organs showed an identical pattern compared with that of inflammation in all groups (all p < .001). In conclusion, ADMSC-assisted ciprofloxacin therapy offered an additional benefit by reducing acute urogenital organ damage in rat. SIGNIFICANCE: Autologous adipose-derived mesenchymal stem cell-assisted ciprofloxacin therapy offered an additional benefit by reducing acute urogenital organ damage in rats.

Sitagliptin attenuated brain damage and cognitive impairment in mice with chronic cerebral hypo-perfusion through suppressing oxidative stress and inflammatory reaction.

BACKGROUND: Sitagliptin, a new antidiabetic drug that inhibits dipeptidyl peptidase (DPP)-4 enzyme activity, has been reported to possess neuroprotective property. We tested the protective effects of sitagliptin against chronic cerebral hypoperfusion (CHP) in mice after bilateral carotid artery stenosis (BCAS). METHOD: Thirty C57BL/6 mice were divided into three groups: sham control (n = 10), CHP (n = 10) and CHP-sitagliptin (orally 600 mg/kg/day) (n = 10). Working memory was assessed with novel-object recognition test. MRI was performed at day 0 and day 90 after BCAS procedure prior to sacrifice. RESULTS: Immunohistochemical (IHC) staining showed significantly enhanced white matter lesions, microglia activation and astrocytosis of white matter in CHP group than in sham control, but the changes were significantly suppressed after sitagliptin treatment (all P < 0.01). The mRNA expressions of inflammatory [tumour necrosis factor-alpha (TNF-α), monocyte chemoattractant protein (MCP-1) and matrix metalloproteinase (MMP)-2] and apoptotic (Bax) biomarkers showed an identical pattern, whereas the anti-inflammatory (interleukin, IL-10) and antiapoptotic (Bcl-2) biomarkers showed an opposite pattern compared with that of IHC among all groups (all P < 0.01). The protein expressions of oxidative stress (NOX-I, NOX-II, nitrotyrosin, oxidized protein), inflammatory [nuclear factor-kappa B (NF-κB), TNF-α and MMP-2], apoptotic [mitochondrial Bax, cleaved poly(ADP-ribose) polymerase (PARP)] and DNA-damage (γ-H2AX) markers showed an identical pattern, while expression pattern of antiapoptotic marker (Bcl-2) was opposite to that of IHC (all P < 0.01). Glycogen-like peptide-1 receptor protein expression progressively increased from sham control to CHP-sitagliptin (P < 0.01). The short-term working-memory loss and MRI/diffusion tensor imaging (DTI) showed a pattern identical to that of IHC in all groups (all P < 0.01). CONCLUSION: Sitagliptin protected against cognitive impairment and brain damage in a murine CHP model.

Impact of simvastatin and losartan on antiinflammatory effect: in vitro study.

Antiinflammatory properties of losartan are currently unclear. This study tested the hypothesis that losartan itself has an antiinflammatory effect comparable to that of simvastatin. Human umbilical vein endothelial cells (HUVECs) were (1) incubated with culture medium alone, (2) incubated with added C-reactive protein (CRP) (25, 50, 75, and 100 microg/mL) for stimulation, and (3) pretreated with losartan (stepwise increased dose: 100, 300, 500, and 750 micromol/L) and simvastatin (stepwise increased dose: 25, 50, 75, and 100 micromol/L) for 4 hours before adding CRP for stimulation. Surface expression of vascular cell adhesion molecule-1 (VCAM-1) was determined by flow cytometry. Supernatant levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were measured by ELISA. Experimental results showed that the effect of CRP on VCAM-1 expression and supernatant levels of MCP-1 and IL-6 increases stepwise as CRP concentrations increase from 25 to 50 to 75 to 100 microg/mL (all P < 0.001). The effect of CRP on VCAM-1 expression in HUVECs and supernatant levels of MCP-1 and IL-6 were significantly suppressed by 25 micromol/L simvastatin with stepwise increased suppression as simvastatin dose increased to 50, 75, and 100 micromol/L (all P < 0.0001). However, losartan did not significantly suppress CRP's effect on VCAM-1 expression in HUVECs (P > 0.5). Moreover, losartan did not suppress CRP's effect on MCP-1 and IL-6 secretion unless a high dose (> or =500 micromol/L) of losartan was used. Compared with simvastatin, losartan had less effect on suppression of CRP-mediated inflammation.