The effect and mechanism of resveratrol on autophagy regulation in nasopharyngeal carcinoma CNE-2 cells / 天津医药

Objective To investigate the effect of resveratrol on autophagic flux of nasopharyngeal carcinoma CNE-2 cells, and to explore the underlying mechanism. Methods Nasopharyngeal carcinoma CNE-2 cells were divided into control group and resveratrol group. Cells in control group were normally cultured at 37℃and received no further treatment. Resveratrol group was added 40 μmol/L resveratrol 2 h before cells were culture at 37 ℃. Western blot analysis was performed to detect protein expressions of LC3B, p62, Beclin-1, phospho-mTOR (p-mTOR) and phospho-S6 (p-S6). The autophagic flux was detected under the confocal laser scanning microscopy through different color spots, after cells were transfected with adenovirus encoding GFP-mRFP-LC3. Results (1) The protein expression of LC3B was significantly increased and the protein expression of p62 was significantly decreased in resveratrol group compared with those of control group (P<0.05). There was no significant difference in Beclin-1 expression between two groups. (2) Compared to control group, expressions of p-mTOR and p-S6 were significantly decreased in resveratrol group (P<0.05). (3) Compared to control group, the red mRFP puncta were significantly increased, and the yellow GFP puncta were significantly decreased in resveratrol group (P<0.05). Conclusion Resveratrol promotes the autophagic flux of nasopharyngeal carcinoma CNE-2 cells, and the effects are possibly dependent on the activation of mTOR pathway-related proteins.

Effects and mechanisms of periostin overexpression on invasion and migration of the nasopharyngeal carcinoma 6-10B cell line / 解剖学报

Objective To explore the effects and mechanisms of periostin overexpression on migration and invasion of nasopharyngeal carcinoma ( NPC) cell line.Methods The recombinant plasmids [ pCMV-neo ( +)-periostin ] and control plasmids [pCMV-neo (+)] were transfected into 6-10B cells using lipofectamine 2000TM reagent.The expression of periostin was detected with PCR and Western blotting .Transwell chamber invasion assay was employed to assay the migration and invasion of 6-10B cells before and after transfection .A gelatin zymogram was used to detect the activity of MMP-2 and MMP-9 in cultivated supernatant of 6-10B cells before and after transfection .The expression of integrin-αvβ5 was detected by immunohistochemistry ( IHC) in 6-10Bperiostin cells, 6-10Bvector cells and 6-10B cells as well as normal nasopharyngeal mucosa ( NNM) and NPC and at the same time periostin also was detected by immumohistochemistry in NNM and NPC, and densitometry analysis using image-pro plus 6.0 software, and the correlation between periostin and integrin-αvβ5 on NPC was assayed with statistics .Results Over expression of periostin promoted cell migration and invasion.The expression levels of integrin-αvβ5 in primary NPC and 6-10Bperiostin cells were significantly higher than those in NNM and 6-10Bvector, 6-10B cells.The expression in NPC of integrin-αvβ5 showed positively correlated with the expression of periostin (r=0.682, P<0.01).Conclusion Periostin plays an important role in regulation of cell migration and invasion probably by combining with integrin-αvβ5 to improve the activities of MMPs .

Effect of PFT-α on apoptosis of spermatogenic cells caused by enorchia / 中南大学学报(医学版)

OBJECTIVE@#To determine the molecular mechanism of germ cell apoptosis via investigating the effect of PFT-α on the expression of p53 and bcl-2/bax during experimental cryptorchid cell apoptosis.@*METHODS@#Male Sprague-Dawley rats were assigned into 4 groups: a sham-operated group, a cryptorchid group, a cryptorchid+p53 inhibitor (p53 inhibitor-alpha, PFT-α) group, and a cryptorchid+dissolvent of PFT-α [dimethyl sulphoxide (DMSO)] group. Unilateral cryptorchidism was surgically induced in the rats of the cryptorchid group, PFT-α group, and cryptorchid+dissolvent of PFT-α group. The rats in the PFT-α group and cryptorchid+dissolvent of PFT-α group were intra-peritoneally injected PFT-α and dissolvent of PFT-α, respectively, once a day. The rats were killed on the 7th day after the surgery. The morphology of spermatogenic epithelium at the side of surgery in the rats was observed under light microscope. The apoptosis of spermatogenic cells in the unilateral cryptorchidism was evaluated by TUNEL and flow cytometry (FCM). The protein expression levels of p53, bcl-2, and Bax were detected by Western blot and immunohistochemical assay in turn.@*RESULTS@#Compared with the cryptorchid groups and the cryptorchid+dissolvent of PFT-α group, the seminiferous epithelium of the cryptorchid+p53 inhibitor group appeared orderly, with thicker cell layers and lower apoptosis index, weak protein expression level of p53/Bax and strong protein expression level of bcl-2.@*CONCLUSION@#PFT-α inhibits the germ cell apoptosis caused by the experimental cryptorchidism via increasing the expression of bcl-2 and decreasing the expression of p53 and bax.

Effects of SNP and L-NAME on spermatogenesis in rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To observe the effects of nitric oxide(NO) on the DNA ploidy of germ cells and to evaluate the role of NO in modulating spermatogenesis by using SNP, a donor of NO and N-nitro-l-arginine-mythel-ester(L-NAME), an inhibitor of nitric oxide synthese(NOS) in rats physically in vivo.</p><p><b>METHODS</b>Forty adult male, Sprague-Dawley rats (60-70 days) were divided into four groups, and injected ultraperitoneally with one of the following agents (once a day, for 12 days): SNP, L-NAME and SNP + L-NAME with normal saline. Two hours after the last injection the rats were sacrificed. The sera were collected and stored at -70 degrees C for subsequent hormone assay. The concentration of serum testosterone was measured by radioimmunoassay. Serum NOx- (nitrite/nitrate) concentration was measured by Greiss method. DNA of spermatogenic cells was detected by flow cytometry(FCM), and the percentage of 1c, 2c and 4c germ cells calculated.</p><p><b>RESULTS</b>In the SNP treatment group, the serum concentration of NOx- was higher, testosterone concentration was lower and the number of 1c cells was smaller compared with the control group. However, in rats treated with L-NAME, the concentration of NOx- was significantly lower, testosterone concentration was higher and the number of 1c cells was larger compared with the control group(P < 0.01). No changes were observed in the SNP + L-NAME group.</p><p><b>CONCLUSION</b>Enhancing ectogenous NO will suppress spermatogenesis while inhibiting NO productive pathway will promote it.</p>

EFFECT OF SODIUM NITROPRUSSIDE AND N-NITRO-L-ARGININE-MYTHEL-ESTER ON APOPTOSIS OF SPERMATOGENIC CELLS IN RAT TESTIS / 解剖学报

Objective To evaluate the effect of sodium nitroprusside(SNP)and N-nitro-l-arginine-mythel-ester (L-NAME) on apoptosis of spermatogenic cells in rats. Methods Fourty adult male Sprague-Dawley rats (60-70days) were divided into four groups.Each group was injected intraperitoneally with one of the following agents, once a day, for 12 days: 1. SNP; 2.L-NAME;3.SNL+L-NAME;4.Normal saline NS group.Two hours after the last time injection the rats were sacrificed.TUNEL staining and flow cytometry analysis were used to detect the apoptosis of spermatogenic cells. Results Sub-monoploid and apoptosis index (AI) in SNP group was significantly higher than that of NS group and sub-monoploid and apoptosis index (AI) in L-NAME group were significantly lower than that of NS group by FCM and TUNEL (P0.5) was found.Conclusion SNP can accelerate the apoptosis of spermatogenic cells and L-NAME can inhibite the apoptosis of spermatogenic cells,The effect of SNP and L-NAME on apoptosis of spermatogenic cells probably occurs through the action of nitric oxide.