To investigate the molecular mechanism of leptin regulating insulin secretion through determining the regulation of insulin secretion and the insulin receptor substrate (IRS)-2-associated phosphoinositide 3-kinase (PI3K) activity by leptin in the isolated rat pancreatic islets, pancreatic islets were isolated from male SD rats by the collagenase method. The purified islets were incubated with leptin 2 nmol/L for 1 h in the presence of 5.6 mmol/L or 11.1 mmol/L glucose. Insulin release was measured using radioimmunoassay. IRS-2-associated activity of PI3K was determined by immunoprecipitate assay and Western blot. The results showed that in the presence of 5.6 mmol/L glucose, leptin had no significant effect on both insulin secretion and IRS-2-associated PI3K activity, but in the presence of 11.1 mmol/L glucose, insulin release was significantly inhibited after the islets were exposed to leptin for 1 h (P<0.01). PI3K inhibitor wortmannin blocked the inhibitory regulation of leptin on insulin release (P<0.05). Western Blot assay revealed that 2 nmol/L leptin could significantly increase the IRS-2-associated activity of PI3K by 51.5% (P<0.05) in the presence of 11.1 mmol/L glucose. It was concluded that Leptin could significantly inhibit insulin secretion in the presence of 11.1 mmol/L glucose by stimulating IRS-2-associated activity of PI3K, which might be the molecular mechanism of leptin regulating insulin secretion.

To explore the anti-apoptotic role of electroacupuncture (EA) and its molecular mechanisms after cerebral ischemia/reperfusion (IR) of rats, by using animal model of middle cerebral artery occlusion (MCA), the changes of the cleavage of PARP were observed by Western blot and the mRNA of heat shock protein (Hsp) 70 and Hsp90ß detected by competitive RT-PCR after cerebral IR and EA treatment. The results were as follows: (1) The cleavage of PARP was increased in ischemic hippocampus, and EA treatment could attenuate the level of the cleavage remarkably: (2) The mRNA expression of Hsp70 was increased in the ischemic cortex and hippocampus and was futher increased after EA treatment: (3) The mRNA expression of Hsp90ß was decreased in ischemic cortex and hippocampus and the decrease was relatively slight after EA treatment. The above results demonstrated EA treatment could protect neurons from apoptosis after cerebral IR. One of the molecular mechanisms was the promotion of the inducible expression of Hsp70 and the improvement of the inhibition of the expression of Hsp90.

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-κB were labled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-κB in PMA group was more than that in PMA+PDTC group. It is suggested that detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.

To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in preventing graft-versus-host disease (GVHD), the CD34⁺ cells transfected with FasL or not, used as stimulus cells, were mixed with allo-antigen specific T lymphocytes in presence or absence of IFN-γ and IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry (FCM). The affects of these two cytokines on CD34⁺ cells in the graft were also compared. The ratio of apoptosis of T cells was 12.1±1.5% when CD34⁺ cells transfected with FasL was used as stimulus cells, much higher than that of CD34⁺ cells non-transfected (3.2±1.1%,P<0.01). And in presence of IFN-γ or IL-2, the ratio reached 20.1±2.3%, 17.6±1.3% respectively (P<0.01). However, IFN-γ up-regulated Fas expression of CD34⁺ cells and increased the sensibility of CD34⁺ cells to soluble FasL(sFasL); IL-2 showed no such affect. It is possible to induce apoptosis of the allo-antigen specific T cells of grafts activated by allo-antigen by exogenous Fas ligand expressed on recipient cells and this might provide a new approach for preventing GVHD and IL-2 may be more suitable for clinical application.