Interferon-α mediating the functional damage of CD56dimCD57+natural killer cells in peripheral blood of systemic lupus erythematosuss / 北京大学学报(医学版)

OBJECTIVE@#To investigate the regulatory effect of interferon-α (IFN-α) on the apoptosis and killing function of CD56dimCD57+ natural killer (NK) cells in systemic lupus erythematosus (SLE) patients, and to explore the specific mechanism.@*METHODS@#A total of sixty-four newly treated SLE patients and sixteen healthy controls (HC) enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects. And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction. CD56dimCD57+ NK cells were co-cultured with the K562 cells, and the apoptotic K562 cells were labeled with Annexin-Ⅴ and 7-amino-actinomycin D. Peripheral blood mononuclear cells were treated with 20, 40, and 80 μmol/L hydrogen peroxide (H2O2), and treated without H2O2 as control, the expression level of perforin (PRF) was detected by flow cytometry. The concentration of IFN-α in serum was determined by enzyme linked immunosorbent assay. The expression levels of IFN-α receptors (IFNAR) on the surface of CD56dimCD57+ NK cells were detected by flow cytometry, and were represented by mean fluorescence intensity (MFI). CD56dimCD57+ NK cells were treated with 1 000 U/mL IFN-α for 24, 48 and 72 h, and no IFN-α treatment was used as the control, the apoptosis and the expression levels of mitochondrial reactive oxygen species (mtROS) were measured by flow cytometry and represented by MFI.@*RESULTS@#Compared with HC(n=3), the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients (n=3) were decreased (1.24±0.41 vs. 0.57±0.12, P=0.05). Compared with HC(n=5), the ability of peripheral blood CD56dimCD57+ NK cells in the SLE patients (n=5) to kill K562 cells was significantly decreased (58.61%±10.60% vs. 36.74%±6.27%, P < 0.01). Compared with the control (n=5, 97.51%±1.67%), different concentrations of H2O2 treatment significantly down-regulated the PRF expression levels of CD56dimCD57+ NK cells in a dose-dependent manner, the 20 μmol/L H2O2 PRF was 83.23%±8.48% (n=5, P < 0.05), the 40 μmol/L H2O2 PRF was 79.53%±8.56% (n=5, P < 0.01), the 80 μmol/L H2O2 PRF was 76.67%±7.16% (n=5, P < 0.01). Compared to HC (n=16), the serum IFN-α levels were significantly increased in the SLE patients (n=45) with moderate to high systemic lupus erythematosus disease activity index (SLEDAI≥10) [(55.07±50.36) ng/L vs. (328.2±276.3) ng/L, P < 0.001]. Meanwhile, compared with HC (n=6), IFNAR1 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=6) were increased (MFI: 292.7±91.9 vs. 483.2±160.3, P < 0.05), and compared with HC (n=6), IFNAR2 expression in peripheral blood CD56dimCD57+ NK cells of the SLE patients (n=7) were increased (MFI: 643.5±113.7 vs. 919.0±246.9, P < 0.05). Compared with control (n=6), the stimulation of IFN-α (n=6) significantly promoted the apoptosis of CD56dimCD57+ NK cells (20.48%±7.01% vs. 37.82%±5.84%, P < 0.05). In addition, compared with the control (n=4, MFI: 1 049±174.5), stimulation of CD56dimCD57+ NK cells with IFN-α at different times significantly promoted the production of mtROS in a time-dependent manner, 48 h MFI was 3 437±1 472 (n=4, P < 0.05), 72 h MFI was 6 495±1 089 (n=4, P < 0.000 1), but there was no significant difference at 24 h of stimulation.@*CONCLUSION@#High serum IFN-α level in SLE patients may induce apoptosis by promoting mtROS production and inhibit perforin expression, which can down-regulate CD56dimCD57+ NK killing function.

Myeloid-derived suppressor cells aggravate Sjögren's syndrome by inhibiting the response of Th2 cells / 医学研究生学报

Objective Changes in the number and function of myeloid-derived suppressor cells (MDSC) were reported in clinical and experimental Sjögren’s syndrome (SS), whereas the underlying mechanisms of MDSCs in SS remain to be elucidated. This study was to observe the changes in the pathologic structure and function of the submandibular gland and salivary flow in SS mice after adoptive transfer or deletion of MDSCs and explore the action mechanisms of MDSCs. Methods Ten 4-week-old non-obese diabetic (NOD) mice (without SS-like symptoms) received adoptive transfer of purified MDSCs at 1×106 per mouse (the MDSC group, n = 5) or injection of PBS (the PBS group, n = 5). Another ten 10-week-old NOD mice were injected intraperitoneally with anti-Gr1 antibodies (the anti-Gr1 group, n = 5) or commensurable Rat IgG2b isotype antibodies (the Rat IgG2b group, n = 5). At 2 weeks after treatment, we determined the salivary flow rate, examined lymphocytic infiltration in the submandibular glands, and counted the MDSCs on Th2 cells in different groups of the mice. Results Compared with the PBS group, the NOD mice of the MDSC group showed significantly reduced Th2 cells in the peripheral blood ([0.67 ± 0.13] % vs [0.16 ± 0.07] %, P < 0.05) and spleen ([0.80 ± 0.13] % vs [0.37 ± 0.04] %, P < 0.05) and salivary flow ([78.70 ± 6.80] vs [33.85 ± 11.25] µL, P < 0.05), but increased numbers of MDSCs in the peripheral blood ([1.54 ± 0.14] vs [5.47 ± 1.54] ×105, P < 0.05) and spleen ([1.09 ± 0.23] vs [4.50 ± 1.04] ×105, P < 0.05). In comparison with the Rat IgG2b group, the animals of the anti-Gr1 group exhibited remarkably decreased Th2 cells in the peripheral blood ([0.55 ±0.09] % vs [0.92 ± 0.10] %, P < 0.05) and spleen ([0.63 ± 0.08] % vs [1.10 ± 0.06] %, P < 0.05) and salivary flow ([56.48 ± 14.18] vs [121.20 ± 10.34] µL, P < 0.05), as well as decreased numbers of MDSCs in the peripheral blood ([1.53 ± 0.12] vs [0.35±0.16] ×105, P < 0.05) and spleen ([2.53 ± 1.10] vs [0.91±0.07] ×105, P < 0.05). The adoptive transfer of MDSCs aggravated while the injection of anti-Gr1 antibodies attenuated lymphocytic infiltration in the submandibular gland of the mice. Conclusion MDSCs participate in the pathogenesis Sjögren’s syndrome by suppressing the response of Th2 cells, which suggests that increasing the response of Th2 cells by inhibiting MDSCs could be a novel target for the treatment of Sjögren’s syndrome.

Pharmacokinetic interaction between aminophylline and metronidazole in rats / 中国药学杂志

OBJECTIVE: To establish an HPLC-UV method for the simultaneous determination of theophylline and metronidazole in rat plasma, so as to explore the pharmacokinetic interaction between theophylline and metronidazole. METHODS: Eighteen male Sprague-Dawley (SD) rats were divided into aminophylline group, metronidazole group, aminophylline and metronidazole group. Blood samples were collected at intervals after each intervenous administration. Then the concentrations of theophylline and metronidazole were determined by HPLC, and the pharmacokinetic parameters were calculated by DAS2.0 software and evaluated by one-way analysis of variance using SPSS19.0 software. Drug-drug interaction was tested by 95% confidence interval. RESULTS: Compared with single mediacation groups, the pharmacokinetic parameters t1/2 MRT, AUC0-t, Vd and CL of the combined group showed no significant difference (P > 0.05). CONCLUSION: Aminophylline and metronidazole do not have pharmacokinetic interaction in rats. Copyright 2013 by the Chinese Pharmaceutical Association.

Effect of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.</p><p><b>METHODS</b>Using whole-cell patch-clamp techniques applied to NG108-15 cells, investigate the effect of opioid receptor agonist on the delayed rectified potassium channels by administration of Guanosine-5'-0'-2-thiociphosphate (GDP beta S), Pertusis Toxin (PTX), Tetroacetic acid nueleoside diphosphate kinase (NDPK) and Adenosine-3' 5' cyclic monophosphate cAMP in the pipette solution.</p><p><b>RESULTS</b>(1) GDP beta S could block the changes induced by both high and low concentration of (D-Pen2.5)-enkephalin (DPDPE) (P < 0.05). (2) PTX could inhibit the excitative regulation on K+ channel by high concentration of DPDPE (P < 0.05). But CTX had no effect on K+ channel caused by DPDPE. (3) UDP could block the excitative effect of K+ channel by high concentration of NDPK, while have no changes on the inhibitory effect caused by low concentration of opioid agonists. (4) cAMP took part in the regulation in high concentration of agonist administration (P < 0.05), while no changes for low concentration of agonists.</p><p><b>CONCLUSIONS</b>Dual changes were observed on delayed rectifier potassium channel by agonist treatment on NG108-15 cells. The excitative effect was Gi/o coupled in high concentration of agonist incubation, related to cAMP. While the inhibitory effect was possibly induced by G protein beta gamma subunit directly.</p>

Dual regulation by delta opioid receptor agonists on the delayed rectified potassium channels in NG108-15 cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the dual effects by the delta opioid receptor agonists DPDPE on the delayed rectified potassium channels in NG108-15 cells.</p><p><b>METHODS</b>A series of outward currents were evoked in NG108-15 cells by depolarizing voltage from -50 mV to +80 mV at holding potential of -90 mV. These currents were delayed rectified potassium currents. Relatively selected delta opioid receptor agonists DPDPE of higher and lower concentrations were used to modulate the delayed rectified K+ current in NG108-15 cells. Opioid receptor antagonist Naloxone (NAL) and relatively selected delta opioid receptor antagonist Naltrindole (NTI) were used in the present experiments for the characterization of the actions of opioid receptors.</p><p><b>RESULTS</b>The relatively higher concentrations of delta opioid receptor agonist DPDPE (> or = 10(-6) mol/L) significantly increased the amplitude of the delayed rectified K+ current. On the contrary, the relatively lower concentrations of DPDPE (< or = 10(-12) mol/L) decreased the amplitude of the delayed rectified K+ current (P < 0.05). Furthermore both the increase and decrease were time-dependent.</p><p><b>CONCLUSIONS</b>delta opioid receptor agonist has dual regulatory effects on the delayed rectified potassium channels in NG108-15 cells.</p>