[Protective effects and mechanism of N-acetylcysteine on cardiac injury induced by ischemia/reperfusion in diabetic rats].

AIM: To study the protective effects and mechanism of N-acetylcysteine on cardiac injury induced by ischemia/reperfusion in diabetic rats. METHODS: Diabetes mellitus was induced by intraperitoneal injection of streptozotocin(STZ). The animals were randomly reassigned into sham-operated group, I/R group and I/R+treated with NAC group. The content of GSH and GSSG, and the activity of Caspase-3 were measured. The apoptosis index (AI) by TUNEL staining was calculated. In addition, the apoptosis of Cardiomyocyte was also confirmed by DNA Ladder. RESULTS: Treatment with NAC decreased the activity of Caspase-3, the content of GSSG, the values of AI but increased the content of GSH in both non-diabetic and diabetic rats (P<0.05), but there were still significant difference about the values of above parameters between diabetic rats and non-diabetic rats (P<0.05). CONCLUSION: NAC can attenuated cardiomyocyte apoptosis by decreasing the activity of Caspase-3 and increasing the content of GSH, which has protective effect on ischemic/reperfused myocardium injury in both non-diabetic and diabetic rats, but the cardioprotective effect is less effective in diabetic rats than that in non-diabetic rats.

The immunosuppressive effect of gossypol in mice is mediated by inhibition of lymphocyte proliferation and by induction of cell apoptosis.

AIM: To investigate the immunosuppressive effect of gossypol in mice both in vitro and in vivo. METHODS: The in vitro effect of gossypol on the proliferation of lymphocytes isolated from lymph nodes of BALB/c mice was determined by CFSE staining and by an MTS assay. Lymphocyte activation and lymphoblastic transformation were evaluated with immunostaining. Cell apoptosis was detected by Annexin-V and Hoechst 33342 staining. The in vivo immunosuppressive effect of gossypol on the DTH reaction was evaluated using a mouse DTH model induced by 2,4-dinitro-1-fluorobenzene (DNFB). The thickness of the ears was measured, and the histological changes of the mouse auricles were observed after hematoxylin-eosin staining. The proliferation capacity of lymphocytes from DTH mice was also assayed. RESULTS: In vitro, gossypol could significantly inhibit the proliferation of mouse lymphocytes stimulated with phorbol ester plus ionomycin in a dose-dependent manner. Although the expression of the early activation antigen CD69 was not affected, the lymphoblastic transformation of both T and B lymphocyte subsets was significantly suppressed by gossypol. Moreover, gossypol could induce apoptosis of lymphocytes, and the effect was time- and dose-dependent. In vivo, the DTH reaction in mice was markedly alleviated by gossypol injected intraperitoneally. Lymphocytes from drug-treated DTH mice had a reduced proliferation capacity as compared with lymphocytes from untreated DTH mice. Gossypol treatment also markedly reduced the number of infiltrated lymphocytes in the auricles of DTH mice. CONCLUSION: Gossypol exhibited immunosuppressive effects in mice, probably by inhibition of lymphocyte proliferation and by induction of cell apoptosis.

[Protective effect of N-acetylcysteine on liver and lung in mice after ischemia-reperfusion injury].

AIM: To investigate protective effect of N-acetylcysteine (NAC) on liver and lung in mice after hepatic ischemia/reperfusion injury. METHODS: BALB/s mice were used in a model of partial hepatic ischemia/reperfusion (I/R) injury. They are divided randomly to sham-operated control group (SH), hepatic I/R group or NAC pretreated in hepatic I/R group (I/R-NAC). The level of TNF-alpha in portal vein and plasma ALT were measured at 1 hour and 3 hour, respectively after reperfusion. Lung tissue wet-to-dry (W/D) weight ratio compared. RESULTS: Lung tissue W/D ratio showed significant difference between two groups; The expressions of TLR2/4 mRNA in liver and lung increased obviously after hepatic I/R injury. Histological evaluation showed several changes in lung tissue in I/R group. The level of TNF-alpha and ALT declined significantly in the group pretreated by NAC. CONCLUSION: N-acetylcysteine can inhibit the activation of TLR2/4 and reduce TNF-alpha secretion resulted from I/R injury it might abate liver and lung injury following partial hepatic ischemia-reperfusion in mice.

[Effect of anisomycin on allogeneic skin transplantation in mice].

AIM: To investigate the effect of anisomycin on the reactivity of lymphocytes and the rejection in the skin transplantation in mice. METHODS: The reactivity of T cells in unilateral or bilateral mixed lymphocyte reaction was detected by MTT assay. The skins from C57BL/6 mice were transplanted to BALB/c mice and the survival period of the grafts was assessed. The recipients were divided into three groups (physiological saline group, cyclosporin A group and anisomycin group). They were injected with physiological saline (0.1 mL), cyclosporin A (20 mg/kg) and anisomycin (12.5 mg/kg) separately once a day for 3 days before the operation and 7 days after the operation. RESULTS: With the optimal dose of 10.0 microg/L, anisomycin inhibited the reactivity of lymphocyte cells in both unilateral and bilateral mixed lymphocyte reactions. The survival period of the grafts from anisomycin group was longer than that from cyclosporin A group and physiological saline group. CONCLUSION: Anisomycin can inhibit the reaction of lymphocytes and prolong the survival period of the grafts in skin transplantation effectively. It could be used as a new way for transplantation rejection treatment.

[Premature delivery induced by LPS in syngenetically impregnated BALB/c and NOD/SCID mice].

AIM: To extend understanding of the mechanism of lipopolysaccharide (LPS)-induced preterm delivery in syngenetically impregnated BALB/c and NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. METHODS: Strategies of LPS stimulation were pursued with or without previous Toll-like receptor 4 (TLR4) blocking. The incidence of LPS-induced preterm delivery and fetal death were compared between the BALB/c and NOD/SCID groups. Guided by the time when all expected preterm deliveries have occurred in the first experiment (i.e., day 16 of gestation), the LPS-stimulated mice, with or without previous TLR4 blocking, were killed at the beginning of preterm labor and pooled placentas were collected in each mouse in the second experiment. The expression of cell surface TLR4, CD80, and intracellular TNF-alpha in placenta CD45(+) cell population was determined by flow cytometry(FCM). RESULTS: It displayed that preterm delivery could be induced by LPS in BALB/c mice, while the NOD/SCID mice seemed to be resistant to LPS induction. Upon LPS stimulation, TLR4 expression was not changed either in BALB/c or in NOD/SCID mice, but the CD45(+) CD80(+) cell percentage was elevated in both groups. However, the CD45(+) TNF-alpha(+) cell percentage was increased merely in BALB/c mice after stimulation, while no such trend was observed in NOD/SCID mice. In BALB/c mice, the effect of LPS on CD80 and TNF-alpha expression could be abrogated by previous TLR4 blocking, which subsequently prevented LPS-induced preterm delivery. CONCLUSION: Although LPS do not alter TLR4 expression, it interacts with this receptor, triggers the mobilization of CD45(+) CD80(+) cells, results in elevated production of inflammatory cytokines, and finally results in preterm delivery. The diversity of sensitivity to LPS induction observed in BALB/c and NOD/SCID mice implies that the lack of functional T and NK cells in the NOD/SCID may be the reason why the NOD/SCID appeared to be resistant to LPS-induced premature labor.

[The effect of p38 on the cycloheximide-induced HL-60 cell death through mitochondria pathway].

OBJECTIVE: To study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway. METHODS: Inhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points. RESULTS: The sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01). CONCLUSION: CHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

[Examination expression of CD3 and CD69 in nasal polyps].

OBJECTIVE: To investigate the infiltration of T lymphocytes with CD3 expression as surface marker and the activation of T lymphocytes with CD69 expression as activation marker. METHODS: Nasal polyp tissue samples and peripheral blood were obtained from 21 patients. The normal inferior turbinate mucosa and peripheral blood were obtained as comparison. Flow cytometry was adopted to detect the expression of CD3 and CD69 of T lymphocytes. RESULTS: Nasal polyp tissue consisted of abundant T lymphocytes. Activation marker CD69 was expressed in T lymphocytes of nasal polyps (36.96 +/- 2.50)% and peripheral blood (4.66 +/- 0.18)% from the same patient. The expression rates of CD69 after a short-term stimulation (5 h) in response to PDB were (59.88 +/- 2.59)% and (92.76 +/- 0.55)% respectively. While T lymphocytes was rarely detected in normal inferior turbinate and the expression of CD69 was low in peripheral blood from normal human but almost all T lymphocytes were activated after stimulation. CONCLUSIONS: There were generous of T lymphocytes infiltrating in nasal polyps. The expression of CD69 in T lymphocytes was abnormally high, which indicated that T lymphocytes infiltrating in nasal polyps were in activated state immunologically.

[Application of vital dye CFDA-SE to study lymphocytic proliferation].

AIM: To explore the application value of vital dye CFDA-SE to study of lymphocytic proliferation. METHODS: CFDA-SE staining, fluorescence antibody labeling, flow cytometry and related software were used to detect the fluorescence intensity and analysis the proliferation kinetics of lymphocytes and their subsets after stimulation with polyclonal stimulators. RESULTS: Lymphocytes divided after stimulation of PDB+ionomycin or ConA for 48 h, manifesting the serial halving of fluorescence intensity. CsA inhibited the proliferative effect of ConA on lymphocytes and no CFSE fluorescence halving were seen. Proliferation of CD4(+) T cells and CD8(+) T cells were asynchronous after ConA stimulation for 48 h, which became more obvious at the time of 72 h. Proliferation-related indexs got ten from ModFit(TM) software showed that the proliferative effect of ConA on CD8(+) T cells was stronger than that on CD4(+) T cells. CONCLUSION: CFDA-SE staining combined with fluorescent antibody labeling and cytometry were powerful tools for analysis of lymphocytic proliferation.