ABSTRACT
Objective To investigate the effect of resveratrol (Res) on paraquat (PQ)-induced acute lung injury (ALI) and mortality in mice and the mechanism of nuclear factor-κB (NF-κB) inflammatory pathway. Methods Sixty-eight healthy male ICR mice with grade SPF were enrolled, among them 20 mice were used for mortality observation (n = 10), and other 48 were used for determination of related parameters (n = 6). The mice were randomly divided into four group s: normal saline (NS) control group, Res control group, PQ group and PQ + Res group. The mice in the latter two groups were subdivided into 6, 24, 72 hours subgroups. The PQ poisoning model of mice was reproduced by one injection of 30 mg/kg PQ intraperitoneally. The mice in PQ + Res group were given 60 mg/kg Res intraperitoneally on the contralateral side after PQ injection. The mice were sacrificed at 6, 24, 72 hours after PQ poisoning, and lung tissue was harvested. The serum levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6 and IL-1β) were determined by enzyme linked immunosorbent assay (ELISA). The pathological changes in lung tissue were observed with electron microscopy. Apoptosis cells in the lung were identified by terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) for the estimation of apoptosis rate. The protein expression of NF-κB p65 was determined by Western Blot. Results Compared with PQ group, the death number of mice at 48, 72, 96 hours in PQ + Res group was slightly decreased (0 vs. 2, 2 vs. 5, 4 vs. 6) but without statistically significant difference (all P > 0.05). Under electron microscope, the lung injury in PQ group was severer than that in NS control group, and Res was found to be able to alleviate the lung injury. Compared with NS control group [(2.45±0.61)%], the apoptosis rate at 6 hours in PQ group was significantly increased [(8.42±1.48)%], and peaked at 72 hours [(21.23±3.47)%]. Res could decrease the apoptosis rate after PQ poisoning [6 hours: (5.56±1.31)% vs. (8.42±1.48)%, 24 hours: (11.14±2.07)% vs. (16.88±2.96)%, 72 hours: (13.28±2.32)% vs. (21.23±3.47)%, all P < 0.05]. The serum levels of TNF-α, IL-6, and IL-1β, and NF-κB p65 in lung tissue were all markedly increased after PQ poisoning, and they were significantly decreased after Res intervention as compared with those of PQ group [TNF-α (ng/L): 2.62±0.29 vs. 4.06±0.74 at 6 hours, 3.98±0.41 vs. 6.79±0.80 at 24 hours, 5.06±0.75 vs. 11.00±0.75 at 72 hours; IL-6 (ng/L): 14.19±1.54 vs. 16.55±1.24 at 6 hours, 13.21±1.37 vs. 19.73±0.85 at 24 hours, 13.72±0.56 vs. 22.45±0.72 at 72 hours; IL-1β (ng/L): 8.54±1.64 vs. 12.59±0.66 at 6 hours, 10.15±0.29 vs. 16.24±1.03 at 24 hours, 16.14±0.70 vs. 19.55±0.56 at 72 hours; 6-hour NF-κB p65: (1.34±0.07) folds vs. (1.86±0.11) folds when the expression in NS control group was represented as 1, all P < 0.05]. Conclusions Res cannot lower the mortality in mice with PQ poisoning, but it seems to be able to attenuate PQ-induced ALI and cell apoptosis. The mechanism responsible for the latter maybe the inhibitive effect of Res on NF-κB p65 translocation and cytokines production.
ABSTRACT
Objective To establish antigen capture ELISA methed to detect Kaposi's sarcoma-as-sociated herpesvirus(KSHV)antigen,and to evaluate its feasibility for clinical application.Methods The BALB/c mice and New Zealand white rabbits were injected with purified recombinant KSHV gpK8.1 proteins to prepare the monoclonal antibody(McAb)and polyclonal antibody(PcAb)anti-gpK8.1,respectively.A new antigen capture ELISA method was established for KSHV antigen detection.The detection reproducibili-ty as well as the sensitivity and specificity of this new assay were determined by the optimization test,which antibody pairs were analyzed to choose the best coating antibody and detecting antibody.The 3 KSHV posi-tive patients sera and 257 patients sera from sexually transmitted disease,cancers or gynecological diseases were detected with this assay to evaluate its value for clinical application.Results When the McAb as coat-ing antibody at concentration of 5 μg/ml and PcAb as detecting antibody at concentration of 1.6μg/ml were selected,the highest P/N value could be obtained.The sensitive analysis of this test could detect recombi-nant KSHV gpK8.1 antigen of 31.28 ng/ml.Meanwhile,it is highly specific to detect KSHV antigen with-out cross reaction to Epstein-Barr vims(EBV),herpes simplex virus(HSV)-1 or HSV-2.All of three KSHV-positive sera and 4 sera from 257 clinical samples were positive with this new assay.which indicated that it could be used for capturing KSHV antigen.Conclusion A sensitive and specific McAb-based anti-gen capture ELISA method to detect KSHV antigen were established successfully.It is of great potential val-ue to develop reagent for KSHV clinical serologic dingnosis.