ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.
Objective To study the effects of adenosine 2A receptor activation on activation, proliferation, and toxicity of T lymphocytes stimulated by phytohemagglutinin (PHA) in vitro. Methods A model of activated T cells was established by stimulating the cells with PHA. Those T cells were treated with different concentrations of adenosine 2A receptors agonist (0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, and 10 μmol/L CGS21680). The expressions of CD69, CD25 and proliferation of T cells were measured by fluorescent antibody stain and flow cytometry. ELISA method was used to detect IL-2 and INF-γ levels. Results All concentrations of CGS21680 significantly inhibited the expressions of CD25 and CD69 on PHA-stimulated T cells surface and proliferation of T cells （Plt;0.05， Plt;0.01）. IL-2 and INF-γ secreted by T cells were significantly suppressed, too （Plt;0.01）. Conclusion Activation of adenosine 2A receptor can effectively inhibit the activation, proliferation, and toxicity of T cells in vitro.
Objective To investigate the mechanism of adenosine-tri phosphate (ATP) activated mammal ian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signal pathway in the physiology and pathology of spinal cord injury (SCI). Methods Ninety-six adult healthy female Sprague-Dawley rats were randomly divided into 4 groups (groups A, B, C and D, n=24). In groups A, B and C, the rats were made the SCI models at T8-10 levels by using a modified Allen’ s stall, and in group D, rats were given laminectomy without SCI. The rats were subjected to the administration of ATP (40 mg/kg) for 7 days in group A, to the administration of physiological sal ine (equal-volume) for 7 days in group B, to the administration of ATP (40 mg/kg) and rapamycin (3 mg/kg) for 7 days in group C, and to the administration of physiological sal ine (equal-volume) for 7 days in group D. Locomotor activity was evaluated using the Basso-Beattie-Bresnahan rating scale at the postoperative 1st, 2nd, 3rd, and 4th weeks. Then, the expressions of spinal cord cell marker [Nestin, neuron-specific enolase (NSE), gl ial fibrillary acidic protein (GFAP)] and the mTOR/STAT3 pathway factors (mTOR, STAT3) were detected at the postoperative 1st, 2nd, 3rd, and 4th weeks by immunohistochemistry analysis, Western blot assay, and real-time fluorescence PCR analysis. Results The BBB scores in group A showed a steady increase in the postoperative 1st-4th weeks and were significantly higher than those in groups B and C (P lt; 0.01), but were lower than that in group D (P lt; 0.01). Real-time fluorescence PCR results showed that the mRNA expressions of mTOR, STAT3, NSE of group A steadily increased, however, the Nestin mRNA expression gradually decreased in the postoperative 1st-4th weeks, which were all significantly higher than those of groups B, C, and D (P lt; 0.01). The mRNA expression of GFAP showed a steady increase in group A and was significantly less than those of groups B and C, but was higher than that of group D (P lt; 0.01). There were significant differences (Plt; 0.01) in all markers between groups B, C, and group D; there were significant differences in mTOR, P-mTOR, STAT3, and P-STAT3 mRNA between groups B and C at 1st-4th weeks (P lt; 0.05). The similar changes were found by Western blot assay. Conclusion ATP can activate the mTOR/STAT3 pathway to induce endogenic NSCs to prol iferate and differentiate into neurons in rats, it enhances the heal ing of SCI.
The retina of SD rats was incubated in four types of the Eagle solution respectively. The results showed the cAMP level of retinas was the lowest in the hGnMg(high glucose with normal magnesium) solution but the cAMP level was significantly increased in the hGhMg(high glucose with high magnesium) and higher than that of normal control group. The cAMP level was the highest in the nGhMg(normal glucose with high magnesium). The results suggested that magnesium might play an important role in maintaining the normal metabolism of glucose of the retinal tissue.
（Chin J Ocul Fundus Dis,1992,8:138-140）
ObjectiveTo systematically review the clinical efficacy and effects on pregnancy outcomes of S-adenosy-L-methionine combined with ursodesoxycholic acid in the treatment of intrahepatic cholestasis of pregnancy.
MethodsDatabases such as PubMed, The Cochrane Library, CNKI, VIP, WanFang Data were searched for the studies about the clinical efficacy and effects on pregnancy outcomes of S-adenosy-L-methionine combined with ursodesoxycholic acid in the treatment of intrahepatic cholestasis of pregnancy up to December 31st, 2013. Two reviewers independently screened literature, extracted data and evaluated methodological quality. Then meta-analysis was conducted using RevMan 5.0.24 software.
ResultsA total of 11 RCTs involving 776 patients were included. The results of meta-analysis showed that, combined medication reduced blood biochemical indexes inlcuding ALT (MD=3.63, 95%CI 0.63 to 6.64, P=0.02), TB (MD=3.70, 95%CI 1.45 to 5.96, P=0.001), and AST (MD=7.61, 95%CI 2.47 to 12.75, P=0.004). Combined therapy significantly decreased the rates of amniotic fluid contamination (OR=0.29, 95%CI 0.19 to 0.45, P=0.000 01), cesarean section (OR=0.53, 95%CI 0.36 to 0.79, P=0.002), postpartum hemorrhage (OR=0.32, 95%CI 0.12 to 0.90, P=0.03), preterm birth (OR=0.36, 95%CI 0.24 to 0.55, P < 0.000 01), fetal distress (OR=0.33, 95%CI 0.19 to 0.58, P=0.000 1) and neonates asphyxia (OR=0.30, 95%CI 0.19 to 0.47, P < 0.000 01). Combined therapy was also beneficial to improving pruritus symptoms (MD=0.20, 95%CI 0.08 to 0.31, P=0.000 08) and benefiting fetus growth (MD=0.45, 95%CI 0.23 to 0.66, P < 0.000 1).
ConclusionThe combination of S-adenosy-L-methionine and ursodesoxycholic acid is superior to ursodesoxycholic acid alone in improving clinical symptoms and pregnant outcomes of patients with intrahepatic cholestasis of pregnancy.
