RESUMO
Mild therapeutic hypothermia has been shown to mitigate cerebral ischemia, reduce cerebral edema, and improve the prognosis of patients with cerebral ischemia. Adipose-derived stem cell-based therapy can decrease neuronal death and infiltration of inflammatory cells, exerting a neuroprotective effect. We hypothesized that the combination of mild therapeutic hypothermia and adipose-derived stem cells would be neuroprotective for treatment of stroke. A rat model of transient middle cerebral artery occlusion was established using the nylon monofilament method. Mild therapeutic hypothermia (33°C) was induced after 2 hours of ischemia. Adipose-derived stem cells were administered through the femoral vein during reperfusion. The severity of neurological dysfunction was measured by a modified Neurological Severity Score Scaling System. The area of the infarct lesion was determined by 2,3,5-triphenyltetrazolium chloride staining. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The regeneration of microvessels and changes in the glial scar were detected by immunofluorescence staining. The inflammatory responses after ischemic brain injury were evaluated by in situ staining using markers of inflammatory cells. The expression of inflammatory cytokines was measured by reverse transcription-polymerase chain reaction. Compared with mild therapeutic hypothermia or adipose-derived stem cell treatment alone, their combination substantially improved neurological deficits and decreased infarct size. They synergistically reduced the number of TUNEL-positive cells and glial fibrillary acidic protein expression, increased vascular endothelial growth factor levels, effectively reduced inflammatory cell infiltration and down-regulated the mRNA expression of the proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α and interleukin-6. Our findings indicate that combined treatment is a better approach for treating stroke compared with mild therapeutic hypothermia or adipose-derived stem cells alone.
RESUMO
To investigate the effect of a new reactive oxygen metabolites (ROM) scavenger as immune adjuvant in NK cell-mediated killing effect on K562 cell, IL-2 and PHA were used to activate monocyte to produce ROM, and different concentrations of tiopronin as ROM scavenger was used in the cultivated systems with different ratio of monocytes plus NK cells and K562 cells, while histamine dihydrochloride (DHT) with different concentrations was used as positive control. The reuslts indicated that after IL-2 and PHA were supplemented in the cultivated systems mixing with NK cells and K562 cells as the E/T ratio was 10/1, the ROM production increased from 33.17 +/- 25.02 U/ml to 223.59 +/- 59.41 U/ml (P < 0.05) while K562 cell inhibition rate (KIR) increased from 65.56% to 85.89% (P < 0.05). When the monocytes as the E/MO ratios of 10/2, 10/5 and 10/10 were supplemented respectively, ROM production increased correspondingly (ROM production was 389.79 +/- 43.83 U/ml, 456.74 +/- 42.77 U/ml, 601.42 +/- 21.92 U/ml, respectively), and KIR was on the other round (KIR was 82.36%, 81.36%, 48.09% respectively). Tiopronin, DHT were used in the K562 + NK + MO + IL-2/PHA cultivated systems as the E/MO ratio was 10/2, the ROM production also decreased from 389.79 +/- 43.83 U/ml to -1.20 +/- 60.70 U/ml, 50.21 +/- 22.4 U/ml (P < 0.05), respectively, however KIR increased from 82.53% to 96.09% and 94.64% either (P < 0.05). Higher concentrations of tiopronin and DHT were used, ROM production decreased accordingly. There showed a reverse correlation between ROM production and KIR (r = -0.518). When E/MO ratio was 10/5 or 10/10, tiopronin at any testing concentration and DHT at the higher testing concentration could reduce the ROM production (P < 0.05), but did not improve KIR significantly (P > 0.05). Tiopronin was as good as DHT in ameliorating KIR (P > 0.05) and better than DHT in scavenging ROM (P < 0.05). It is concluded that (1) Monocytes are the major resources of ROM, and the ROM derived from monocytes can disable NK cells in killing neoplasm cells (K562 cells); (2) A new ROM scavenger, tiopronin, can scavenge ROM effectively, and reverse the ROM induced inhibition of NK cell-mediated killing of K562 cell in a certain extent. And tiopronin is better than DHT in scavenging ROM, and as good as DHT in up-regulating KIR. The new ROM scavenger tiopronin with less side effect may take the place of DHT as adjuvant during the adoptive immuno-therapy in leukemia.
