The effects of phorbol-12,13-dibuterate (PDBu) on total sodium current (I_Na-total), tetrodotoxin-resistant sodium current (I_Na-TTXr), 4-AP-sensitive potassium current (I_A) and TEA-sensitive potassium current (I_K) in trigeminal ganglion (TG) neurons were investigated. Whole-cell patch clamp techniques were used to record ion currents in cultured TG neurons of rats. Results revealed that 0.5 μmol/L PDBu reduced the amplitude of I_Na-total by (38.3±4.5)% (n=6, P<0.05), but neither the G-V curve (control: V_0.5=−17.1±4.3 mV, k=7.4±1.3; PDBu: V_0.5=−15.9±5.9 mV, k=5.9±1.4; n=6, P>0.05) nor the inactivation rate constant (control: 3.6±0.9 ms; PDBu: 3.6±0.8 ms; n=6, P>0.05) was altered. 0.5 μmol/L PDBu could significantly increase the amplitude of I_Na-TTXr by (37.2±3.2)% (n=9, P<0.05) without affecting the G-V curve (control: V_0.5=−14.7±6.0 mV, k=6.9± 1.4; PDBu: V_0.5=−11.1±5.3 mV, k=8.1±1.5; n=5, P>0.05) or the inactivation rate constant (control: 4.6±0.6 ms; PDBu: 4.2±0.5 ms; n=5, P>0.05). 0.5 μmol/L PDBu inhibited I_K by (15.6±5.0) % (n=16, P<0.05), and V_0.5 was significantly altered from − 4.7±1.4 mV to −7.9 ±1.8 mV (n=16, P<0.05). I_A was not significantly affected by PDBu, 0.5 μmol/L PDBu decreased I_A by only (0.3±3.2)% (n=5, P>0.05). It was concluded that PDBu inhibited I_Na-total but enhanced I_Na-TTXr, and inhibited I_K without affecting I_A. These data suggested that the activation of PKC pathway could exert the actions.

In order to investigate the effects of verapamil on the proliferation of meningiomas cells in vitro and in vivo, the cultured meningiomas cells were cultured with verapamil at different concentrations for 24 h and the inhibitory effects of verapamil on cell proliferation were observed by MTT method. The meningiomas model was established by implanting the newly removed tumor fragments into the nude mice subcutaneously. The nude mice with tumors were divided into two groups: verapamil-treated group and control group. Tumor volumes were measured and after 12 weeks the tumors were taken out and examined histologically. The expression of proliferating cell nuclear antigen (PCNA) in the tumors was detected by using immunohistochemistry. It was found that verapamil could inhibit the growth of cultured meningiomas cells in a concentration-dependant manner. The inhibitory effect could be observed in the concentration of 1 μmol/L verapamil and the most obvious effects appeared in the concentration of 100 μmol/L. Tumor volume in the verapamiltreated group was obviously smaller than that in the control group (211.40±5.50 vs 163.94±3.62, P<0.01) and the expression of PCNA was also lower (1.52±0.24 vs 2.86±0.53, P<0.05). Tumor inhibition rate was about 22.45%. It was suggested that verapamil could inhibit the proliferation and growth of meningiomas cells in vitro and in vivo.

Our previous study showed that transmembrane TNF-α (TM-TNF-α) had broader tumoricidal spectrum than secretory TNF-α (s-TNF-α). This study examined the difference between the two kinds of TNF-α in inducing cells and the relationship between the apoptosis induced by TM-TNF-α and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-α on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-staining were used for determining the phase of apoptotic cells. Our results showed that TM-TNF-α could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-α predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-α mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G₁ phase. It is concluded that the cytotoxic effects of the two TNF differ substantially. Since TM-TNF-α works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.

To construct a pUCP18/lasR^antisense plasmid carrying the reversed gene and analyze its effect on the virulence of Pseudomonas aeruginosus, LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and reversely recombined with plasmid pUCP18. The recombinant pUCP18/lasR^antisense was verified by enzyme digestion, PCR and sequencing. The biological effects of pUCP18/lasR^antisense were examined by using RT-PCR, NAD method and the assay of pyocyanin. Our results showed that the expected full length lasR fragment (721 bp) was extended from Pseudomonas aeruginosus gene with PCR. And it is consistent with LasR gene of Pseudomonas aeruginosa in GenBank (No. NC_002516). The recombinant plasmid was successfully constructed and transferred into Pseudomonas aeruginosus. The antisense nucleic acid of LasR gene could reduce the virulence of Pseudomonas aeruginosus and might serve as a new target site for treatment purpose.

To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic β-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.