Cadmium disrupts spermatogenic cell cycle via piRNA-DQ717867/p53 pathway.

Cadmium (Cd) is a harmful environmental pollutant that disrupts public health, including respiratory, digestive, and reproductive systems. In this study, male rats were exposed to CdCl2 at a dose of 3 mg/kg by oral for 28 days to investigate the impact on spermatogenesis. Testis tissue samples were collected after sacrifice, and piRNA expression levels were measured using piRNA microarray and qPCR. PiRNAs, specialized molecules involved in spermatogenesis, were examined. CdCl2 exposure led to disrupted piRNA expression, particularly in piRNA-DQ759395 in rats. This piRNA was found to have a binding site with p53, and a similar piRNA-DQ717867 was discovered in mice. In GC-2spd cells, CdCl2 exposure increased piRNA-DQ717867 expression, which resulted in cell cycle arrest and abnormal expression of cell cycle-related proteins. The activation of p53-related pathways and disruptions in cell cycle regulation were also observed. Antagomir-717867 transfections and PFT-a pretreatment in GC-2spd cells supported the involvement of piRNA-DQ717867 in regulating cell cycle-related proteins. This study suggests that Cd exposure induces abnormal expression of piRNA-DQ759395 in rat testis and that piRNA-DQ717867 may regulate p53, causing cell cycle abnormalities in GC-2spd cells. These findings help understand the mechanisms of male reproductive toxicity caused by Cd exposure and emphasize the role of piRNAs in cell cycle regulation and male reproductive health.

Associations of IFT20 and GM130 protein expressions with clinicopathological features and survival of patients with lung adenocarcinoma.

BACKGROUND: Lung cancer is the leading cause of malignancy-related mortality and lung adenocarcinoma accounts for about 40% of lung malignancies. The aim of this study was to investigate the associations of intraflagellar transport protein 20 (IFT20) and Golgi matrix protein 130 (GM130) expression with clinicopathological features and survival in patients with lung adenocarcinoma. METHODS: The expressions of IFT20 and GM130 protein in cancerous and matched adjacent lung tissues of 235 patients with lung adenocarcinoma were assessed by tissue microarray and immunohistochemistry, which were indicated by the mean optical density (IOD/area), the rate of positive staining cells and staining intensity score. The correlation between IFT20 and GM130 protein was assessed by Spearman's rank correlation. Associations of IFT20 and GM130 protein expression with clinicopathological features of patients were analyzed by multivariate logistic regression models. The survival analysis of patients was performed by Cox proportional hazard regression models. RESULTS: With adjustment for multiple potential confounders, each one-point increase in IFT20 protein staining intensity score was significantly associated with 32% and 29% reduced risk for TNM stage in II ~ IV and lymphatic metastasis of patients, respectively (P < 0.05). And each one-point increase in GM130 protein staining intensity score was associated with a significant reduction in the risk of poor differentiation and tumors size > 7 cm by 29% and 38% for lung adenocarcinoma patients, respectively (P < 0.05). In stratified Cox model analysis, enhanced IFT20 staining intensity score was significantly decreased the risk of death by 16% for patients without distant metastasis. And elevated the IOD/area of GM130 expression significantly decreased the death risk of lung adenocarcinoma patients with tumor size > 7 cm or distant metastasis by 54% and 65%, respectively (P < 0.05). CONCLUSION: IFT20 and GM130 protein expressions were negatively associated with tumor differentiated types, size, TNM stage and lymphatic metastasis of lung adenocarcinoma. Both IFT20 and GM130 proteins have some protective effects on the survival of lung adenocarcinoma patients with specific clinicopathological features.

A pH-targeted and NIR-responsive NaCl-nanocarrier for photothermal therapy and ion-interference therapy.

Transport ions into cells through nanocarrier to achieve ion-interference therapy provides new inspiration for cancer treatment. In this work, a pH-targeted and NIR-responsive NaCl-nanocarrier is prepared using surfactant Vitamin E-O(EG2-Glu) and modified with polydopamine (PDA) and pH-sensitive zwitterionic chitosan (ZWC). The NaCl-nanocarrier is decorated with NH4HCO3 and IR-780 to introduce near-infrared (NIR)-responsive performance and imaging. Once the NaCl-nanocarrier is exposed to NIR laser, the temperature rises rapidly because of the excellent photothermal conversion ability of PDA, then NH4HCO3 is decomposed into NH3 and CO2, which burst the nanocarrier, resulting in Cl- and Na+ "bomb-like" release. This pH-targeted nanocarrier accumulates more at tumor site and when irradiating the site with NIR light, the temperature rises and excessive Cl- and Na+ are released to destroy the ion homeostasis and inhibit tumor growth effectively. Through this strategy, the unique combination of ion interference therapy and photothermal therapy is achieved.

[Impact of Cadmium on the expressions of piRNAs in the rat testis].

OBJECTIVE: To study the impact of cadmium (Cd) on the expressions of PIWI-interacting RNAs (piRNA) in the rat testis and its possible action mechanism. METHODS: Twelve 6-week-old SD rats were randomly divided into a Cd-exposure and a control group, the former gavaged with CdCl2 at 3 mg/kg/d and the latter with normal saline, all for 28 successive days. Then the testicular tissues were collected from the rats, sperm concentration and motility were obtained by computer-assisted sperm analysis (CASA), and piRNA sequencing was performed using the gene chip, followed by bioinformatics analysis of differentially expressed piRNAs. RESULTS: Compared with the controls, the rats in the Cd-exposure group showed significantly decreased sperm concentration and motility (P < 0.05). The expressions of 272 piRNAs were up-regulated and 402 down-regulated after 28 days of Cd exposure, and 4 of the up-regulated piRNAs were consistent with the results of gene chip verification. Bioinformatics analysis showed that the 4 up-regulated piRNA target genes were involved in 50 biological processes, such as negative regulation of apoptosis, positive regulation of gene expression and positive regulation of GTPase activity, and mainly concentrated in 13 signaling pathways including transcription dysregulation, calcium and mitogen-activated protein kinase signaling pathways in cancer. Among them, PIRNA-DQ765261 had a binding site with Bcl-2. CONCLUSION: Cadmium can induce changes in the expressions piRNAs in the rat testicular tissue, and some piRNAs may be involved in the autophagy and apoptosis of sperm. Bcl-2 may be the target of PIRNA-DQ765261.

Reproductive toxicity of cadmium in pubertal male rats induced by cell apoptosis.

Cadmium (Cd) is a heavy metal that is widely present in modern industrial production. It is a known, highly toxic environmental endocrine disruptor. Long-term exposure to Cd can cause varying degrees of damage to the liver, kidney, and reproductive system of organisms, especially the male reproductive system. This study aimed to explore the mechanism of Cd toxicity in the male reproductive system during puberty. Eighteen healthy 6-week-old male Sprague-Dawley rats were randomly divided into three groups (control group, low-dose group, and high-dose group) according to their body weight, with six in each group. Cd (0, 1, and 3 mg/kg/day) was given by gavage for 28 consecutive days. The results showed that Cd exposure to each dose group caused a decrease in the testicular organ coefficient and sperm count, compared with the control group. Cd exposure resulted in significant changes in testicular morphology in the 3 mg/kg/day Cd group. In the 1 and 3 mg/kg/day Cd groups, serum testosterone decreased and apoptosis of testicular cells increased significantly (p < 0.05). In addition, compared with the control group, the activity of glutathione peroxidase and superoxide dismutase in each Cd exposure dose group decreased, but the content of malondialdehyde in the high-dose, 3 mg/kg/day Cd treatment group significantly increased (p < 0.05). Although Cd exposure caused an increase in the messenger RNA (mRNA) levels of Bcl-2, Caspase-3 and Caspase-9 in the testicular tissues (p < 0.05), Bcl-2 expression was unchanged (p > 0.05). The expression level of Akt mRNA in testicular tissue of rats in the high-dose 3 mg/kg/day Cd group was increased (p < 0.05). Our data suggest that Cd affected testosterone levels, and apoptosis was observed in spermatids.

A fluorescent probe for bioimaging of Hexosaminidases activity and exploration of drug-induced kidney injury in living cell.

Hexosaminidases (Hexs) as an exoglycosidase participates in the catalytic hydrolysis of non-reducing end of glycoconjugates in the biological system. The fluctuation of Hexs level could cause many hereditary neurodegenerative diseases such as Tay-Sachs and Sandhoff. The Hexs activity is significantly up-regulated in colorectal cancer and kidney injury tissue so that it is particularly important to construct a fluorescent probe with significant signal change to understand its physiological role. In this work, DyOH was selected as fluorophore scaffolds to synthesize probe Hex-1 for detection of Hexs with good water solubility, high specificity, large stokes shift and quick response. Hex-1 can sensitively detect Hexs with the low detection limit (0.025 mU mL-1) in vitro by "naked eye" due to superior spectral properties of DyOH. Furthermore, Hex-1 was not only employed for imaging Hexs in living cells with low toxicity, but also successfully applied to evaluate the fluctuation of Hexs activity during drug induced kindey injury in living HK-2 cells. These results indicated that Hex-1 could be used as a potential image tool to further explore the pathogenesis of kidney disease and cancer.

Gelatin nanoparticles transport DNA probes for detection and imaging of telomerase and microRNA in living cells.

Telomerase and microRNA (miRNA) are biomarkers closely related to tumors. Simultaneous detection of both markers can improve accuracy and reliability of early diagnosis. Based on the mechanism of fluorescence resonance energy transfer (FRET), two fluorescent DNA probes were designed for telomerase and miRNA-21. The probes were wrapped by gelatin through electrostatic interaction to form nanoparticles. After that, we synthesized molecularly imprinted coating of transferrin on the surface of gelatin nanoparticles, which can avoid the immune stress response and macrophage phagocytosis to help gelatin nanoparticles enter into the cells smoothly through endocytosis. Following with the degradation of gelatin in the cells, DNA probes were released to react with telomerase and miRNA-21 and lead to the change of the fluorescence signal. Thereby the simultaneous imaging of telomerase and miRNA-21 were successfully achieved in HeLa cells and HepG2 cells. The proposed strategy shows the simultaneous imaging for different biological markers with DNA probes by preventing them from being hydrolyzed with nucleases before the determination and achieves reliable method for early diagnosis of cancer.

Carbon black nanoparticles induce HDAC6-mediated inflammatory responses in 16HBE cells.

Long-term inhalation of carbon black nanoparticles (CBNPs) leads to pulmonary inflammatory diseases. Histone deacetylase 6 (HDAC6) has been identified as an important regulator in the development of inflammatory disorders. However, the direct involvement of HDAC6 in CBNPs-induced pulmonary inflammatory responses remains unclear. To explore whether HDAC6 participates in CBNPs-induced pulmonary inflammation, human bronchial epithelial cell line (16HBE cells) was transfected with HDAC6 small interference RNA (siRNA) and then exposed to CBNPs at concentrations of 0, 25, and 50 µg/ml for 24 h. Intracellular HDAC6 and intraflagellar transport protein 88 (IFT88) mRNA and protein were determined by real-time polymerase chain reaction and Western blot, respectively. The secretions of inflammatory cytokines including interleukin (IL)-8, tumor necrosis factor (TNF)-α, IL-6, and IL-1ß were measured by enzyme-linked immunosorbent assay. CBNPs induced a significant increase in the expressions of IL-8 and IL-6, accompanied by a high level of intracellular HDAC6 mRNA when compared with a blank control group (p < 0.05). However, there were no significant changes in the levels of TNF-α secretion, intracellular HDAC6 and IFT88 protein induced by CBNPs (p > 0.05). The HDAC6 mRNA expression was significantly suppressed in HDAC6 siRNA-transfected cells (p < 0.05). The secretions of IL-8, TNF-α, and IL-6 were significantly less in HDAC6 siRNA-transfected cells than that in normal 16HBE cells with exposure to 25 or 50 µg/ml of CBNPs, but intracellular IFT88 mRNA expression was markedly increased in HDAC6 siRNA-transfected cells when compared with normal 16HBE cells exposed to 50 µg/ml of CBNPs (all p < 0.05). Downregulation of the HDAC6 gene inhibits CBNPs-induced inflammatory responses in bronchial epithelial cells, partially through regulating IFT88 expression. It is suggested that CBNPs may trigger inflammatory responses in bronchial epithelial cells by an HDAC6/IFT88-dependent pathway.

The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat.

Di (2-ethylhexyl) phthalate (DEHP) is a known environmental endocrine disruptor that impairs development of testis and spermatogenesis. This study aims to explore the effects of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat. 24 6-week-old male Sprague-Dawley rats were randomly divided into 4 groups (Control, low-dose, middle-dose and high-dose group) and were treated with increasing concentration of DEHP (0, 250, 500, 1000 mg/kg/day) respectively for 28 consecutive days by intragastric administration. Our results showed that DEHP exposure induced obvious morphological changes of testis, decreased organ coefficient of testis and sperm count, and increased testicular cell apoptosis in the 500 and 1000 mg/kg/day DEHP groups (p < .05). The serum testosterone decreased in a dose-dependent manner after treatment with DEHP. Furthermore, the exposure of DEHP elevated the levels of oxidative stress accompanied by upregulated expression of p53 and reduced expression of STAT3. In addition, compared with the control group, the expression of PI3K, p-Akt and p-mTOR proteins significantly decreased, whereas the downstream autophagy-related proteins phosphorylated ULK1, Beclin-1, Atg7, LC3-II obviously increased in the 250 mg/kg/day DEHP group (p < .05). The expression of p62 was reduced in DEHP-treated groups. Our data indicated that autophagy could be activated to protect testes from DEHP-induced reproductive damage by inhibiting PI3K-Akt-mTOR signaling pathway in the 250 mg/kg/day DEHP group. STAT3/p53-mediated mitochondrial apoptosis pathway might play a major role to cause testis injury and reproductive dysfunction in the 500 and 1000 mg/kg/day DEHP groups.

Influence of silica particles on mucociliary structure and MUC5B expression in airways of C57BL/6 mice.

Purpose: Impaired mucociliary clearance is an initial characteristic of recurrent cough, respiratory infection and chronic respiratory diseases. It has been demonstrated that prolonged inhalation of respirable silica particles results in a variety of pulmonary diseases, but whether the mucociliary system is involved in this process is unclear. This study aims to evaluate the effects of silica particles on mucociliary structure and MUC5B production in respiratory tract.Materials and Methods: C57BL/6 mice were administered with 2.5 mg silica particles through a single intratracheal instillation. The changes of mucociliary structure and MUC5B expression in trachea was evaluated by HE and AB-PAS staining, transmission electron microscopy and immunohistochemistry on days 1, 7, 28 and 84 post-exposure.Results: The mucociliary structure of airway epithelium was obviously impaired by silica particles, showing disordered, shortened or partially lost cilia on the surface, increased mucus in mucous layer and submucosal glands from day 7 to day 84. A variety of ultrastructural abnormalities were discovered in silica-exposed airway cilia, including absence of central pair microtubules, disorganized microtubules and clusters of axoneme on day 1 and 7. The numbers of ciliary axonemes and basal bodies in ciliated epithelial cells were significantly decreased, whereas the proportion of abnormal axonemes was gradually increased with exposure to silica particles (P < 0.05). In addition, silica particles significantly decreased MUC5B expression on the surface of airway epithelium on day 28 and 84, but obviously increased its production in submucosal glands from day 1 to day 84 (P < 0.01).Conclusions: Silica particles could lead to ultrastructural defects in airway cilia, mucus hypersecretion and altered MUC5B expression in trachea, indicating that impaired mucociliary structure and altered MUC5B production might participate in the development of silica-related respiratory diseases.

The Role of CTGF in Inflammatory Responses Induced by Silica Particles in Human Bronchial Epithelial Cells.

BACKGROUND: Prolonged exposure to crystalline silica leads to persistent pulmonary inflammation and progressive fibrosis. Connective tissue growth factor (CTGF) has emerged as a potent proinflammatory and profibrotic regulator to participate in a variety of chronic inflammatory diseases. However, the role of CTGF in silica-induced pulmonary inflammation remains poorly understood. METHODS: To explore the effect of CTGF on inflammatory responses caused by silica particles, human bronchial epithelial cells (16HBE) were transfected with CTGF siRNA and exposed to silica particles at concentrations of 0, 12.5, 25, 50, 100 µg/ml for 48 h. Intracellular CTGF mRNA and protein expressions were determined by RT-PCR and Western blotting, respectively. The levels of inflammatory cytokines including IL-8, TNF-α, IL-6, IL-1ß, IL-17A and TGF-ß1 were measured by ELISA kits. RESULTS: Silica particles induce significantly elevated intracellular CTGF mRNA expression in 16HBE cells in a dose-dependent manner when compared with blank control group (P < 0.05). The secretions of IL-8, TNF-α, IL-6 and IL-17A were also significantly increased by silica particles (P < 0.05). After exposure to 25 or 50 µg/ml silica particles, the expression of intracellular CTGF mRNA was significantly inhibited in 16HBE cells when transfected with CTGF siRNA (P < 0.05). The secreted levels of IL-8, TNF-α, IL-6 and IL-17A induced by silica particles were also significantly lower from CTGF siRNA-transfected cells than that from normal 16HBE cells (P < 0.05). CONCLUSION: Inhibition of CTGF gene attenuates silica-induced inflammatory responses in bronchial epithelial cells, suggesting that CTGF could be a pivotal regulator in the development of silica-induced inflammation.

Di-(2-ethylhexyl) phthalate induces apoptosis of GC-2spd cells via TR4/Bcl-2 pathway.

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used environmental endocrine disruptor. Many studies have reported that DEHP exposure causes reproductive toxicity and cells apoptosis. However, the mechanism by which DEHP exposure causes male reproductive toxicity remains unknown. This study investigated the role of the testicular orphan nuclear receptor4 (TR4)/Bcl-2 pathway in apoptosis induced by DEHP, which resulted in reproductive damage. To elucidate the mechanism underpinning the male reproductive toxicity of DEHP, we sought to investigate apoptotic effects, expression levels of TR4/Bcl-2 pathway in GC-2spd cells, including TR4, Bcl-2 and caspase-3. GC-2spd cells were exposed to various concentrations of DEHP (0, 50, 100, or 200µM). The results indicated that, with the increase of the concentrations of DEHP, the survival rate of cell decreased gradually. DEHP exposure at over 100µM significantly induced apoptotic cell death. DEHP decreased SOD and GSH-Px activity in 200µM group. Compared to the control group, the mRNA levels of caspase-3 increased significantly, however, Bcl-2 mRNA decreased (P<0.05). In addition, there was a significant reduction in TR4, Bcl-2 and procaspase-3 protein levels. Taken together, these results lead us to speculate that in vitro exposure to DEHP might induce apoptosis in GC-2spd cells through the TR4/Bcl-2 pathway.