Background: Screening and preliminary identification of DNMT1 high expression related LncRNA, which is involved in various inter-related signaling pathways, has revealed a theoretical basis for various types of disease mechanisms. Methods: LncRNA and mRNA differential expression profiles were identified on a microarray. The 10 LncRNAs with high levels of variation were identified by qRT-PCR. KEGG and GO analyses were used to dissect out differentially expressed mRNAs. Results and Conclusions: Six signal pathways were selected based on the KEGG results of the LncRNA-mRNAs expression network analysis.We found a total of 6987 differentially expressed LncRNAs and 7421 differentially expressed mRNAs were obtained from the microarrays in experimental and control groups ( P <0.05; Fold Change >2.0x). GO analysis KEGG pathway analysis showed high expression of DNMT1 in esophageal epithelial cells. Nine pathways involved mRNA up-regulation including natural killer cell mediated cytotoxicity and many other prominent biochemical pathways. Forty six pathways were associated with down regulated mRNAs and ribosomes involving multiple biological pathways. Co-expression network analysis showed that the 8 mRNA and 16 LncRNA were linked to the P53 signaling pathway. Interactions occurred between 22 LncRNA and 11 mRNA in the ErbB signaling pathway, between 19 LncRNA and 8 mRNA in epithelial cell signal transduction of Helicobacter pylori infection. Interactions were present in 19 LncRNAs and 5 mRNAs in the sphingolipid signaling pathway along with 21 with LncRNA and 12 with mRNAs in the PI3K-Akt signaling pathway. Cytotoxicity interactions occurred with 22 LncRNAs and 9 mRNAs in natural killer cells.

The mechanisms by which cigarette smoke (CS) exposure has a detrimental effect on the male reproductive system is still not fully understood. We aimed to elucidate the role of cigarette smoke-induced injury by the Fas/FasL pathway by using a Sprague-Dawley rat model of cigarette smoking exposure. Here, 200 rats were randomaly divided into five groups with different smoking exposure durations. Forty animals per group were further divided into four groups: a control group, and groups exposed to cigarette smoke at doses of 10, 20 or 30 cigarettes/day. The testes were harvested and the effects of CS exposure on the testis were characterized on the basis of morphological changes, oxidative stress, and a significant elevation in the expression of FAS/FASL pathway related genes, such as FAS, FASL, FADD, caspase 8 and caspase 3. Oxidative stress was reflected by significant time-dependent changes in SOD and GSH-Px activity, and MDA content. Taken together, our data suggest that CS exposure induces testis injury, which is related to the increased oxidative stress and activation of the FAS/FASL apoptotic pathway in the testes.

OBJECTIVE:To screen differentially expressed miRNAs in the testis of male rats exposed to cigarette smoke (CS) and identify the early molecular markers of CS-induced apoptosis of testicular cells.METHODS:We randomly divided 200 SPF male SD rats into blank control and low-dose (10 non-filter cigarettes/d), medium-dose (20 non-filter cigarettes/d) and high-dose (30 non-filter cigarettes/d) CS exposure groups. After 2, 4, 6, 8 and 12 weeks of CS exposure, we observed the histopathological changes of the testis by HE staining, detected the apoptosis of the testicular cells by TUNEL, and determined the expressions of caspase-3 and caspase-9 in the testis tissue by immunohistochemistry, RT-PCR and Western blot. Based on the laboratory results, we selected 4 testicular samples from the 12-week high-dose group and another 4 from the control for miRNA microarray-based screening, bioinformatics analysis, and verification of differentially expressed miRNAs in all the animals by RT-PCR.RESULTS:Compared with the controls, the CS-exposed rats showed dose- and time-dependent increase in the atrophy of the testis and significantly increased number of apoptotic testis cells from the 6th week of exposure (P < 0.05), with dramatically up-regulated expressions of caspase-3 (P < 0.01) and caspase-9 protein and mRNA (P < 0.05) in the testis tissue. Microarray-based screening and RT-PCR revealed 5 differentially expressed miRNAs in the testis of the CS-exposed rats, of which miR-138-5p, miR-181d-5p, miR-19a-3p and miR-3588 were down-regulated, and miR-155-5p up-regulated, and the target genes of the differentially expressed miRNAs positively regulated the apoptosis of the testicular cells.CONCLUSIONS:The differentially expressed miRNAs miR-155-5p, miR-138-5p, miR-181d-5p, miR-19a-3p and miR-3588 regulate CS-induced apoptosis of testicular cells, and may become biomarkers for early diagnosis and prognosis of CS-induced spermatogenesis obstruction.《.

近年来,伴随全球吸烟人数剧增,对吸烟所致雄性生殖毒性损伤关注也日益增多.研究表明,长期大量吸烟可致男性睾丸损伤,表现为精液质量下降等,然而其具体机制不详.为探讨线粒体凋亡通路在香烟烟雾暴露所致大鼠睾丸凋亡损伤中的调控作用,选用SPF级SD雄鼠并随机分为高、中、低香烟烟雾暴露组(30支/d、20支/d和10支/d)和对照组,采用静式染毒法处理各组,分别于2、4、6、8和12周后麻醉处死动物,计算睾丸脏器系数,检测睾丸组织病理学改变及细胞凋亡情况.同时检测细胞凋亡激活因子1(cell apoptosis activation factor 1,Apaf-1)、胱天蛋白酶9(caspase-9)及Bcl-2家族中Bim、Bcl-w和Bak在mRNA和蛋白质水平的表达.结果显示,随香烟烟雾暴露剂量增加,暴露时间延长,暴露组大鼠的体重逐渐下降,睾丸组织呈现出明显的病理学改变,睾丸脏器系数逐渐减少.染毒第8周,中、高剂量组及第12周各剂量组睾丸脏器系数较对照组显著减少(P＜0.05);睾丸阳性凋亡细胞数也随染毒时间延长而逐渐增多,从第6周起呈现统计学差异(P＜0.05);香烟烟雾暴露12周后,睾丸Apaf-1和胱天蛋白酶9表达均较空白组显著上调(P＜0.05);促凋亡基因Bak的mRNA转录水平在染毒初期逐渐下调,从第8周开始,其mRNA转录水平逐渐上调;Bak的蛋白质水平上调,在染毒12周时,Bak的mRNA和蛋白质水平均出现显著性上调(P＜0.05);抗凋亡基因Bcl-w的mRNA转录表达在染毒第6周开始出现显著性下调(P＜0.05),Bcl-w在染毒12周时出现显著性下调(P＜0.05),BH3 only基因Bim表达虽有上调趋势,但未出现统计学差异(P＞0.05).以上结果提示,长期大量吸烟可激活线粒体凋亡通路,促使睾丸组织的不可逆性凋亡损伤.这为进一步探讨吸烟所致男性生殖损伤的分子机制提供了科学依据.

Long-term cigarette smoking (CS) can cause testicular toxicity, which interferes with normal spermatogenesis and leads to male infertility. One possible mechanism for this is the activation of the apoptosis signaling pathway, which leads to the irreversible apoptosis of testicular cells. However, the exact mechanism for this is not completely understood. Cell viability, cell apoptosis, and lactate dehydrogenase release assays were performed to elucidate the function of micro RNA (miRNA) in the pathogenesis of male testicular cell injury induced by CS. The results suggested that testicular cell injury was associated with CS both in vitro and in vivo. CS extract (CSE)-treated Leydig and Sertoli cells showed noticeable apoptosis. Based on the results of Agilent miRNA microarray and bioinformatics analyses, miRNA-138-5p was used in subsequent experiments. Quantitative polymerase chain reaction and Western blot assays showed a negative correlation between miR-138-5p and Caspase-3 expression. Transfection of miR-138-5p mimic significantly inhibited apoptosis and downregulated the expression of Caspase-3 in TM3 and TM4 cells. Furthermore, a dual-luciferase reporter assay demonstrated that miR-138-5p directly targeted Caspase-3 to regulate the apoptosis of testicular cells mediated by CSE. In addition, overexpression of miR-138-5p markedly downregulated the expression of p53 and Bak, which played critical roles in the Bcl-2 pathway. These results demonstrate that miRNA-138-5p inhibits CS-induced apoptosis in testicular cells by targeting Caspase-3 through the Bcl-2 signaling pathway.

An understanding of the causative mechanisms of the harmful effects of cigarette smoke on the male reproductive system remains incomplete. Here, we investigated three different inhaled cigarette smoke doses over five different exposure durations to identify how the testis is affected. The effects of cigarette smoke exposure on testicular germ cells were characterized by morphological changes and a significant elevation in the number of apoptotic cells. Caspase 3 activation increased dramatically after cigarette smoke exposure, accompanied by significant time-dependent expression of the pro-apoptotic proteins Bak (B cell lymphoma/leukemia 2 [Bcl-2] homologous antagonist killer), Bcl2l11 (a BH3 domain-only protein related to Bcl-2), Apaf1 (Apoptotic protease-activating factor-1), and Caspase 9. Conversely, the abundance of anti-apoptotic Bcl2l2 decreased. Taken together, our findings suggest that extensive inhalation of cigarette smoke damages testicular germ cells through the induction of the mitochondrial apoptotic pathway through the Bcl-2 protein family.

The effects of differences in smoke concentration and exposure duration in Sprague Dawley rats to determine variation in type and severity of the testis apoptosis were evaluated. The daily dosages were 10, 20 and 30 non-filter cigarettes for a period of 2, 4, 6, 8 and 12weeks. Mainstream smoke exposure suppressed body weight gain in all regimens. A dose-related increase in plasma nicotine concentration was observed in smoke-exposed groups for 4, 6, 8 and 12week regimens. Histopathological examination of the exposed groups showed disturbances in the stages of spermatogenesis, tubules atrophying and these appeared to be dose-related. Cytoplasmic caspase-3 immunostaining was detected both in Sertoli cells and germ cells in smoke-exposure groups. An increase in TUNEL-positive cells of testicular cells was observed after 6weeks of cigarette exposure. The results indicate that cigarette exposure concentration and duration have interaction effect to induce apoptosis in the rat testes.

We investigated the effects of pulsed electromagnetic fields (PEMFs) of 20 Hz/2 mT on the osteogenic and adipogenic differentiation of bone marrow stem cells (BMSCs). Sprague Dawley rat BMSCs were isolated and cultured in vitro. The BMSCs of the third passage were obtained and stimulated by PEMFs of 20 Hz/2 mT. The alkaline phosphatase (ALP) activity was measured according to the ALP assay kit manufacturer instructions, the BMSC osteogenic and adipogenic indicators were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and oil red O staining was used to observe the adipose-induced adipogenic differentiation of BMSCs. PEMFs of 20 Hz/2 mT significantly promoted the activity of ALP in the BMSCs (P < 0.01) and mRNA expression of osteogenic proteins (osteocalcin and osteopontin). The PEMFs inhibited the expression of adipogenic transcription factors such as adipokines and adipocyte-binding protein-2, and the adipogenic differentiation of BMSCs. PEMFs of 20 Hz/2 mT can promote osteogenic differentiation and inhibit adipogenic differentiation in BMSCs.

Objective Investigate the changes and effects of cyclooxygenase-2 in the development of neuropathic pain induced by chronic nonbacterial prostatitis in rats. Methods The rats were assigned randomly into the 0-, 2-, 4-, 6-, 8-, 12-Week Group, and the 12-Week Control Group. A chronic nonbacterial prostatitis model was established by the immunization method. The expressions of COX2 in the prostate gland and the posterior horn of the lumbar segment of spinal cord were detected by the immunohistochemical SABC method. Results Compared with those in the 0-Week Group, there were statistically significant differences of the COX2 expressions in the prostate gland and the posterior horn of spinal cord in the 2-, 4-, 6-, 8-, and 12-Week Groups, respectively. Compared with those in the 2-,4-,6-Week Group, there were statistically differences of the COX 2 expressions in the prostate gland in the 8- and 12-Week Groups; Compared with those in the 2-Week Group, there were statistically differences of the COX 2 expressions in the posterior horn of spinal cord in the6-, 8- and 12-Week Groups. The COX 2 expressions in the prostate gland and the posterior horn of spinal cord were positively correlated. Conclusion These data indicated that COX 2 might be involved in development of neuropathic pain induced by chronic nonbacterial prostatitis.

To evaluate the expression and differential significance of c-Jun, p73, Casp-9 and N-ras in thymic epithelial tumors (TETs) with the aim to provide useful information for tumor biology and prospective therapy.

OBJECTIVE:To study the correlation between amplification of chromosome 1 and histological typing and clinical staging of thymic epithelial tumors according to the WHO classification. METHODS:Amplification of chromosome 1 was detected by interphase fluorescence in-situ hybridization (FISH) in 60 cases of thymic epithelial tumors, including type A thymoma (2 cases), type AB (19 cases), B1 (4 cases), B2 (14 cases), B3 (11 cases), metaplastic thymoma (2 cases), and thymic carcinoma (8 cases) and 11 samples of normal thymus. RESULTS:Gain on chromosome 1 was found in 19 cases (31.7%) of thymic epithelial tumors, and none was detected in normal thymic tissues (P < 0.05). The positive rates of gain on chromosome 1 were statistically different among various histological subtypes of thymic epithelial tumors (P < 0.05), in which the highest rate of detection was in thymic carcinoma (6/8), the second, type B3 (6/11), followed by type A (1/2), type AB (4/19), type B2 (2/14) and type B1 (0). The positive rate of gain on chromosome 1 in type B3 had no statistical difference from thymic carcinoma (P > 0.05), but significantly higher than that in other types of thymoma (P < 0.05). In addition, the polysomy rate of chromosome 1 was significantly different among the thymic epithelial tumors at different clinical stages (P = 0.023), and that at stages III and IV was statistically higher than that in stages I and II (P = 0.003) but there was no significant difference between stage I and stage II tumors (P = 0.750). CONCLUSIONS:Gain on chromosome 1 is more common in thymic carcinoma and type B3 thymoma than that in other subtypes of thymic epithelial tumors. Thymoma of type B3 may have different genetic features from other subtypes. Detection of gain on chromosome 1 by FISH is helpful in the differential diagnosis and prediction of prognosis in patients with thymic epithelium tumors.

To study the effect of manganese with different valences to apoptosis, human neuroblastoma cell line (SH-SY5Y) has been chosen to study the induction of Mn2+ and Mn3+ on apoptosis and the content of dopamine in vitro. The results showed that both Mn2+ and Mn3+ induced a time-dependent decrease in SH-SY5Y cell viability, as determined by MMT assay. In the meantime, apoptosis was inducted by flow cytometer analysis (FCAS) after culture of Mn2+ or Mn3+ (0.5-2 mmol/L) with SH-SY5Y cell for 24-72 h. The ultrastructural changes of apoptosis such as nuclear chromosome condensation, was seen by transmission electron microscope. In addition, DA content was decreased by measured with HPLC. The major path of cell death induced by Mn2+ and Mn3+ may be due to apoptosis. Both Mn2+ and Mn3+ could induce the reduction of DA content. All these results suggest that Mn3+ appeared to be more cytotoxic than Mn2+.