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To investigate the expressions of the pannexin (Panx) proteins in I-10 Leydig tumor cells and TM3 Leydig cells and their regulatory effect on the Panx channel function in mice.The expressions of the Panx-1 and Panx-2 proteins in the mouse Leydig tumor cells were determined by Western blot. The I-10 Leydig tumor cells were treated with carbenoxolone (CBX) at 100 μmol/L or probenecid (PBN) at 200 μmol/L, the fluorescence resonance energy transfer (FRET) detected by time-lapse fluorescence imaging, and the extracellular adenosine 5'-triphosphate (eATP) level measured with the commercial detection kit. Molecular biological methods were used to interfere with shRNA and overexpress mPanx-1 the Panx-1 gene and regulate the expression and function of the Panx-1 protein.The expressions of Panx-1 (［289.5 ± 55.8］%) and Panx-2 (［264.5 ± 24.6］%) were significantly increased in the I-10 Leydig tumor cells as compared with those in the normal TM3 Leydig cells (both P0.05). FRET was remarkably reduced after treated with CBX (［87.5 ± 17.7］%) and PBN (［89.3 ± 14.3］%) in comparison with that in the control group (both P0.01). At 8, 16 and 24 hours, the eATP level was decreased by (57.3 ± 7.2)%, (56.4 ± 9.6)% and (63.4 ± 6.4)% in the CBX group (P0.01) and (61.7 ± 2.5)%, (35.8 ± 1.6)% and (13.5 ± 8.3)% in the PBN group (P0.01). Molecular biological treatment down-regulated the expression of Panx-1 by (38.3 ± 5.2)% and (31.8 ± 5.1)% in the shRNA1 and shRNA2 groups, respectively (both P0.01), but up-regulated that of Panx-1 by (128.4 ± 7.5)% in the mPanx-1 group (P0.01) as compared with the negative control. FRET was reduced by (72.4 ± 39.4)% in the shRNA group (P0.01) and the eATP level by (14.7 ± 0.1)%, (13.7 ± 0.3)% and (13.1 ± 0.3)% at 8, 16 and 24 hours, respectively (P0.01) while FRET elevated by (122.5 ± 17.1)% in the mPanx-1 group (P0.01) and the eATP level by (886.1 ± 82.1)%, (885.8 ± 83.3)% and (841.5 ± 21.8)% at 8, 16 and 24 hours, respectively (P0.01).The expressions of Panx-1 and Panx-2 are increased in I-10 mouse Leydig tumor cells, and inhibiting the Panx channel with CBX, PBN and shRNA reduces FRET and the eATP level in the I-10 cells.

To observe the inhibitory effect of gefitineb on the proliferation and its inducing effect on the apoptosis of mouse I-10 Leydig testicular cancer cells in vitro.We treated I-10 Leydig testicular cancer cells of mice with gefitineb at 0, 1.25, 2.5, 5, 10, 20, and 40 µmol/L. Then we determined the inhibitory effect of gefitineb on the growth of the cells by MTT, detected their early and late apoptosis by Annexin V-FITC/propidium iodide double staining and Hoechst 33258 nuclear staining, respectively, and observed the expressions of apoptosis-related proteins Bcl-2, Bax and caspase 3/9 by Western blot.Compared with the blank control group, gefitineb significantly inhibited the proliferation of the I-10 cells at 10 and 20 µmol/L (P0.05). The survival rate of the cells was (32.4 ± 2.8)% (P0.01) and their early and late apoptosis rates were (26.7 ± 4.2)% and (59.33 ± 10.2)% in the 40 µmol/L group, significantly different from those in the control (P0.05 and P0.01). In comparison with the blank control group, gefitineb at 10, 20, and 40 µmol/L increased the expression of pro-apoptotic protein Bax by (41.9 ± 7.1), (60.1 ± 9.8), and (69.0 ± 11.3)% (all P0.05), decreased that of apoptosis-inhibitory protein Bcl-2 by (50.3 ± 8.9), (63.9 ± 6.9), and (88.7 ± 13.9)% (all P0.05), and elevated that of the cleft proteins caspase-3 by (69.0 ± 6.9)% (P0.05), (71.5 ± 8.1)% (P0.05), and (110.9 ± 14.2)% (P0.01) and caspase-9 by (51.8 ± 4.9), (54.7 ± 6.7), and (43.8 ± 11.8)% (all P0.05).Gefitineb can increase the cytotoxicity of I-10 Leydig testicular cancer cells of mice and induce their apoptosis via the mitochondria-mediated apoptosis signaling pathway.

To investigate the effect of baicalein on the gap junction intercellular communication (GJIC) in the TM4 Sertoli cells of the mouse testis and its related mechanism.We measured the cytotoxicity of different concentrations of baicalein on the TM4 Sertoli cells in the mouse testis by MTT, detected the fluorescence transfer of the TM4 Sertoli cells by parachute assay, and determined the expression of the protein connexin 43 ( Cx43) in the baicalein-treated cells by Western blot and immunofluorescence assay.Baicalein produced no obvious cytotoxicity on the TM4 Sertoli cells at the concentration below 60 µmol/L but significantly increased their GJIC at 0-20 µmol/L (P0.01). Western blot and immunofluorescence assay showed that 0-20 µmol/L baicalein remarkably elevated the expression of Cx43 in the TM4 cells (P0.01) and on the membrane of the TM4 cells.Baicalein at the concentration of 0-20 µmol/L can significantly enhance GJIC in mouse TM4 Sertoli cells by increasing the expression of the Cx43 protein.

To investigate the effects of total flavonoids of Litsea Coreana (TFLC) on the gap junction (GJ) intercellular communication in TM3 testicular Leydig cells and whether TFLC can reduce the cytotoxicity of oxaliplatin (OHP) in vitro.We detected the effect of TFLC on the dye spread of the in vitro cultured TM3 cells by parachute assay, observed changes in the expression of connexin 43 (Cx43) total protein in the TFLC-treated TM3 cells by Western blot, and determined the effects of TFLC on the expression of Cx43 on the membrane of the TM3 cells by immunofluorescence assay and on the cytotoxicity of OHP by MTT assay.TFLC obviously enhanced the GJ function with the increasing of the TFLC concentration in the TM3 cells. Western blot and immunofluorescence assay confirmed that TFLC significantly enhanced the expression of Cx43 total protein and Cx43 expression on the membrane of the TM3 cells. MTT assay showed that at a high cell density (confluent with GJ formation), 20 microg/ml TFLC enhanced the GJ function of the TM3 cells and reduced the cytotoxicity of OHP (P0.05), while at a low density (preconfluent with no GJ formation), TFLC exhibited no effect on the cytotoxicity of OHP (P0.05).TFLC increases the Cx43 expression and GJ function in normal TM3 Leydig cells, and the enhancement of GJ function reduces the cytotoxicity of OHP.