Cellular mechanism underlying leptin-induced anion secretion of rat epididymal epithelial cells.

BACKGROUND: A large number of studies have shown that leptin plays an important role in the regulation of fertility via the hypothalamus-pituitary-gonad axis. However, its peripheral function in epididymis was still elusive. OBJECTIVE: The purpose of this study was to determine the pro-secretion effect of leptin on the rat epididymal epithelium. MATERIALS AND METHODS: In the present study, real-time quantitative polymerase chain reaction, western blot, and immunohistochemical analysis were employed to detect the expression pattern of leptin receptors in rat epididymis. The pro-secretion effect of leptin on epididymal epithelial cells was measured by short-circuit current, and the prostaglandin E2 and cyclic adenosine monophosphate level was evaluated by enzyme-linked immunosorbent assay. RESULTS: We verified that the leptin receptor was located on the epididymal epithelium, with a relatively high expression level in corpus and cauda epididymis. Ussing chamber experiments showed that leptin stimulated a significant rise of the short-circuit current in rat epididymal epithelial cells, which could be abolished by the specific leptin receptor antagonist peptide Allo-aca, or by removing the ambient Cl- and HCO3 -. Furthermore, the leptin-stimulated short-circuit current response could be abrogated by blocking the apical cystic fibrosis transmembrane regulator or the basolateral Na+-K+-2Cl- cotransporter. Our pharmacological experiments manifested that interfering with the prostaglandin H synthase-2-prostaglandin E2-EP2/EP4-adenylate cyclase pathways could significantly blunt the cystic fibrosis transmembrane regulator-mediated anion secretion induced by leptin. The enzyme-linked immunosorbent assay demonstrated that leptin could induce a substantial increase in prostaglandin E2 release and cyclic adenosine monophosphate synthesis of primary cultured rat cauda epididymal epithelial cells. Our data also suggested that JAK2, ERK, and PI3K-dependent phosphorylation may be involved in the activation of prostaglandin H synthase-2 and the subsequent prostaglandin E2 production. CONCLUSIONS: The present study demonstrated the pro-secretion function of leptin in rat epididymal epithelium via the activation of cystic fibrosis transmembrane regulator and Na+-K+-2Cl- cotransporter, which was dependent on the paracrine/autocrine prostaglandin E2 stimulated EP2/EP4-adenylate cyclase pathways, and thus contributed to the formation of an appropriate microenvironment essential for sperm maturation.

Mannan Oligosaccharide Could Attenuate Ochratoxin A-Induced Immunosuppression with Long-Time Exposure Instead of Immunostimulation with Short-Time Exposure.

Our previous study showed that ochratoxin A (OTA), one of the most common mycotoxins in feed, could induce immunosuppression with long-time exposure but immunostimulation with short-time exposure. However, limited studies for the control of OTA-induced two-way immune toxicity were carried out. This study explored the effects of mannan oligosaccharide (MOS), a glucomannoprotein complex with immunoregulatory capability derived from the yeast cell wall, on OTA-induced immune toxicity and its underlying mechanisms. Surprisingly, the results showed that MOS significantly attenuated immunosuppression induced by long-time OTA treatment but did not provide protection against immunostimulation induced by short-time OTA treatment on porcine alveolar macrophages (PAMs), as demonstrated by the expressions of inflammatory cytokines and the capability of migration and phagocytosis. Further, MOS increased the OTA-inhibited autophagy level and the JNK phosphorylation level on PAMs with long-time OTA treatment. In addition, the inhibition of autophagy by 3-MA or the inhibition of JNK phosphorylation by SP600125 could partly block the protective effects of MOS on OTA-induced immunosuppression. Importantly, the inhibition of JNK phosphorylation down-regulated the MOS-promoted autophagy level. In conclusion, MOS could attenuate OTA-induced immunosuppression with short-time exposure on PAMs through activating JNK-mediated autophagy but had no significant effects on OTA-induced immunostimulation with short-time exposure. Our study provides new insights into the application of MOS as an immunoregulator against mycotoxin-induced immune toxicity.

Low-level contamination of deoxynivalenol: A threat from environmental toxins to porcine epidemic diarrhea virus infection.

Mycotoxins are toxic metabolites produced by fungal species that commonly present in the global environment, especially in cereals and animal forages. The changing global environment may further increase the exposure to these toxins, posing a serious threat to humans and animals. Recently, coronavirus has become one of the most important pathogens threatening human and animal health. It is not clear whether environmental toxins, such as mycotoxins, will affect coronavirus infection. Given that pigs are among the animals most affected by coronavirus and highly homologous to humans, weaned piglets and IPEC-J2 cells were respectively chosen as in vivo and in vitro model to explore the impacts of deoxynivalenol (DON), the most abundant trichothecene mycotoxin in feed, on porcine epidemic diarrhea virus (PEDV) infection and the mechanisms involved. In vivo, twenty-seven piglets infected naturally with PEDV were randomly divided into three groups, receiving the basal diet containing 0, 750 and 1500 µg/kg DON, respectively. Significant increases in the diarrhea rates, gut barrier injury and PEDV proliferation of piglets' small intestine were observed in experimental groups compared with the control. Additionally, the autophagosome-like vesicles and the autophagy-related proteins expression were also increased in experimental groups. In vitro, we observed that 0.1, 0.5 and 1.0 µM DON significantly promoted the entry and replication of PEDV in IPEC-J2 cells, along with the induction of a complete autophagy. CRISPR-Cas9-mediated knockout of LC3B indicated a vital role of autophagy in the promotion. Pretreatment with p38 signaling inhibitor could significantly block the induction of autophagy, indicating that DON could promote the PEDV infection by triggering p38-mediated autophagy. Our findings suggest that mycotoxin could influence the prevalence of coronavirus and provide new ideas for the prevention and control of coronavirus.

Long-Time Instead of Short-Time Exposure in Vitro and Administration in Vivo of Ochratoxin A Is Consistent in Immunosuppression.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium, contaminating in a wide variety of foods and feeds. Mycotoxins, including OTA, could cause immunosuppression in almost all previous studies in vivo. However, the vast majority of results in vitro showed that mycotoxins caused immunostimulation. Why the results of studies in vitro are contrary to studies in vivo is unknown. Our study aims to explore the underlying reason and mechanism of the paradoxical effect. In this study, porcine alveolar macrophage cell line 3D4/21 was chosen as an in vitro model and treated with 1.0 µg/mL OTA for different times. Some indexes, such as expression of inflammatory cytokines, migration, phagocytosis, macrophage polarization, autophagy-related proteins, and Akt1 phosphorylation, were detected. The results showed that pro-inflammatory cytokine expression, migration, and phagocytosis were increased, with macrophage polarization to the M1 phenotype at 24 h of OTA exposure. Surprisedly, anti-inflammatory cytokine expression was increased, cell phagocytosis and migration were decreased, and macrophage polarization was switched from M1 to M2 at 72 h of OTA exposure. Furthermore, we found that long-time exposure of OTA also suppressed autophagy, and the autophagy activator blocked the OTA-induced immunosuppression. Phosphorylation of Akt1 plays a positive role in autophagy inhibition. In conclusion, long-time instead of short-time exposure of OTA in vitro induced immunosuppression. The immunosuppression mechanism of OTA in vitro involved inhibition of autophagy through upregulating p-Akt1. Our results provide new insight into research on the mechanism of mycotoxin-induced immunosuppression in vitro.

Aflatoxin B1 Induces Immunotoxicity through the DNA Methyltransferase-Mediated JAK2/STAT3 Pathway in 3D4/21 Cells.

As the most toxic mycotoxin of all of the fungal toxins, aflatoxin B1 (AFB1) has carcinogenesis, heptotoxicity, and immunotoxicity. DNA methylation plays a critical role in gene expression regulation of the pathological process. However, the relationship between DNA methylation and AFB1-induced immunotoxicity was not yet reported. Therefore, the objectives of this study were to verify AFB1-induced immunotoxicity and investigate the potential role of the DNA methyltransferase (DNMT) family in AFB1-induced immunotoxicity and the pathway mechanism in 3D4/21 cells. The results showed that AFB1 could induce cytotoxicity, apoptosis, pro-inflammatory cytokine expression, DNA damage, and oxidative stress and decrease phagocytotic capacity. Meanwhile, the levels of DNMT1 and DNMT3a were significantly increased in 0.04 and 0.08 µg/mL AFB1 compared to the control. Inhibition of DNMT1 and DNMT3a by 5-Aza-2dc could reverse changes of the above parameters. Further, the JAK2/STAT3 pathway was significantly activated in 0.04 µg/mL AFB1. Inhibition of p-JAK2 and p-STAT3 by AG490 could alleviate AFB1-induced immunotoxicity. Moreover, inhibition of DNMT1 and DNMT3a by 5-Aza-2dc could suppress the phosphorylation of JAK2 and STAT3. Taken together, AFB1-induced immunotoxicity is related to the JAK2/STAT3 pathway mediated by DNMTs in 3D4/21 cells.

Mannan Oligosaccharide Protects against the Aflatoxin-B1-Promoted Influenza Replication and Tissue Damages in a Toll-Like-Receptor-4-Dependent Manner.

Our previous study reported that aflatoxin B1 (AFB1) promoted influenza replication. Mannan oligosaccharide (MOS), derived from the cell walls of yeast, is a potent immunomodulator. Here, we investigated the role of MOS in AFB1-promoted influenza replication and further explored the underlying mechanisms. In vitro and in vivo, the exposure to AFB1 alone resulted in significantly decreased weight gain and increased viral replication as well as lung and spleen damages. Increased influenza replication coupled with increases in toll-like receptor 4 (TLR4), phosphorylated nuclear factor κB, and tumor necrosis factor α (TNF-α) levels. However, MOS given in conjunction with exposure to AFB1 significantly reversed these above changes. A further study indicated that MOS activity was abolished by TLR4 knockout or TLR4 overexpression. Surprisingly, TNF-α played no role in the MOS-mediated protective effects. Collectively, our data suggest that MOS alleviates the AFB1-promoted influenza replication, inflammation, and tissue damages in a TLR4-dependent manner.

Aflatoxin B1 Promotes Influenza Replication and Increases Virus Related Lung Damage via Activation of TLR4 Signaling.

Aflatoxin B1 (AFB1), which alters immune responses to mammals, is one of the most common mycotoxins in feeds and food. Swine influenza virus (SIV) is a major pathogen of both animals and humans. However, there have been few studies about the relationship between AFB1 exposure and SIV replication. Here, for the first time, we investigated the involvement of AFB1 in SIV replication in vitro and in vivo and explored the underlying mechanism using multiple cell lines and mouse models. In vitro studies demonstrated that low concentrations of AFB1 (0.01-0.25 µg/ml) markedly promoted SIV replication as revealed by increased viral titers and matrix protein (M) mRNA and nucleoprotein (NP) levels in MDCK cells, A549 cells and PAMs. In vivo studies showed that 10-40 µg/kg of AFB1 exacerbated SIV infection in mice as illustrated by significantly higher lung virus titers, viral M mRNA levels, NP levels, lung indexes and more severe lung damage. Further study showed that AFB1 upregulated TLR4, but not other TLRs, in SIV-infected PAMs. Moreover, AFB1 activated TLR4 signaling as demonstrated by the increases of phosphorylated NFκB p65 and TNF-α release in PAMs and mice. In contrast, TLR4 knockdown or the use of BAY 11-7082, a specific inhibitor of NFκB, blocked the AFB1-promoted SIV replication and inflammatory responses in PAMs. Furthermore, a TLR4-specific antagonist, TAK242, and TLR4 knockout both attenuated the AFB1-promoted SIV replication, inflammation and lung damage in mice. We therefore conclude that AFB1 exposure aggravates SIV replication, inflammation and lung damage by activating TLR4-NFκB signaling.

Glutamine Deficiency Promotes PCV2 Infection through Induction of Autophagy via Activation of ROS-Mediated JAK2/STAT3 Signaling Pathway.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds. We previously reported that glutamine (Gln) deficiency promoted PCV2 infection in vitro. Here, we established a Gln deficiency model in vivo and further investigated the detailed molecular mechanisms. In vivo and in vitro, Gln deficiency promoted PCV2 infection, which was evident through increased viral yields and PCV2 Cap protein synthesis. It also induced autophagy, as demonstrated by the increases in LC3-II conversion, SQSTM1 degradation, and GFP-LC3 dot accumulation. Autophagy inhibition abolished the effects of Gln deficiency on PCV2 infection. Inhibition of ROS generation alleviated the Gln deficiency-activated JAK2/STAT3 signaling pathway, thereby inhibiting autophagy induction. In vitro, the inhibition of STAT3 by an inhibitor or RNA interference blocked autophagy, thus reversing the effects of Gln deficiency on PCV2 infection. These results indicate that Gln deficiency activates autophagy by upregulating ROS-medicated JAK2/STAT3 signaling and thereby promoting PCV2 infection.

Activation of AMPK-dependent SIRT-1 by astragalus polysaccharide protects against ochratoxin A-induced immune stress in vitro and in vivo.

Recent studies have highlighted the immune stress caused by ochratoxin A (OTA), but little attention was paid to its alleviation. In the present study, the protective effects of astragalus polysaccharide (APS) against OTA-induced immune stress in vitro and in vivo and its mechanism/(s) involved were investigated. The in vitro results showed that APS (20 µg/ml) induced a significant decrease in cytotoxicity, apoptosis and pro-inflammatory cytokine expressions elevated by OTA (1.5 µg/ml) in porcine alveolar macrophages (PAMs). In vivo, APS (200 mg/kg b.w.) significantly alleviated OTA-induced (75 µg/kg b.w.) spleen damages and decreased the expressions of OTA-promoted apoptosis-related protein and pro-inflammatory cytokine. Further study indicated that APS caused significant enhancement of AMPK/SIRT-1 and inhibition of NFκB in PAMs and mice. The down-regulation of SIRT-1 by EX527 in vivo or EX527 and SIRT-1 knockdown in vitro abolished the inhibitory effects of APS on OTA-induced cytotoxicity, apoptosis, spleen damages and pro-inflammatory cytokine expressions. Taken together, these findings indicate that APS could attenuate the immune stress induced by OTA in vitro and in vivo via activation of the AMPK/SIRT-1 signaling pathway.

A rare case of isolated castrate resistant bilateral testicular metastases in advanced prostate cancer.

Testicular metastasis is rare with the prostate being the most common site of primary cancer. We report a case of a 72-year-old man with castration-resistant prostate cancer (CRPC) and known metastases to bone and lymph nodes, who developed bilateral painful swollen testes 3 years after the initial diagnosis of prostate cancer. He had first presented with lower urinary tract symptoms (LUTS) with suspicious findings on digital rectal examination of the prostate, and an elevated serum prostate specific antigen (PSA) level of 129 ng/mL. Transrectal prostate biopsy revealed Gleason 4 + 5 adenocarcinoma. Radiological staging showed locally advanced prostate cancer with extensive metastases to bone and pelvic and retroperitoneal lymph nodes. He was given hormonal therapy for over 2 years until progression to CRPC. Six months later he developed painful bilateral testicular swellings, and serum markers for testicular germ cell cancer were normal. Bilateral orchiectomy was performed, showing metastatic prostate cancer (Gleason 4 + 5) on histology. One month postoperatively his PSA level dropped to 0.1 ng/mL from a presurgery level of 6.24 ng/mL.

A retrospective discussion of the prognostic value of combining prothrombin time(PT) and fibrinogen(Fbg) in patients with Hepatocellular carcinoma.

Aims: The levels of coagulation system tests have been studied in various cancers. In this study, our aim is to evaluate the prognostic value of pretreatment plasma coagulation tests in hepatocellular carcinoma (HCC) patients. Patient and methods: A retrospective study was performed in 539 patients with HCC, and follow-up period was at least 60 months until recurrence or death. The prognostic significance of coagulation system tests (prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen) were determined by univariate and multivariate Cox hazard models. Then, according to the results of the multivariate analyses, we proposed the coagulation-Based Stage, which combined the independent risk factors (prothrombin time and fibrinogen). Results: Coagulation system tests including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fbg) were analyzed. Patients with prolonged PT (≥12.1 sec) levels had significantly poor overall survival (OS) and disease-free survival (DFS), not only in the entire cohort (HR: 1.661, 95%CI: 1.125-2.451, p=0.011 vs. HR: 1.660, 95%CI: 1.125-2.451, p=0.011), but also in the subgroups stratified by pathological stage (stage I-II and stage III-IV). Additionally, high Fbg (≥2.83 g/L) levels experienced significantly decreased OS and DFS (HR: 2.158, 95%CI: 1.427-3.263, p<0.001 vs. HR: 2.161, 95%CI: 1.429-3.267, p<0.001), not only in the entire cohort but also in the subgroups stratified by pathological stage (stage I-II and stage III-IV). All the patients were then stratified (based on combined PT and Fbg) into three groups, The OS for HCC patients were (41.37±17.76), (31.83±19.84) and (18.68±18.41) months, and the DFS for HCC patients were (41.15±17.88), (31.65±19.81) and (18.66±18.39) months. Conclusions: Our findings suggest that the combination of plasma PT and Fbg levels should be evaluated as the valuable predictor of survival in patients with HCC.

Knockdown BMI1 expression inhibits proliferation and invasion in human bladder cancer T24 cells.

B cell-specific moloney murine leukemia virus integration site 1 (BMI1) is a transcriptional repressor of polycomb repressive complex 1, which is involved in the proliferation, senescence, migration, and tumorigenesis of cancer. Experimental researchers have convincingly linked BMI1 to tumorigenesis. However, there is no study about the issue on the role of BMI1 in the proliferation, apoptosis, and migration of bladder cancer. To address this question, we examined the expression of BMI1 in bladder cancer tissues and used siRNA to knockdown BMI1 expression in bladder cancer T24 cells. Then we tested the cell proliferation by CCK8 assay and soft agar colony formation assay, apoptosis by flow cytometry assay, and cell invasiveness by transwell migration assay. Our results revealed that BMI1 promoted proliferation, migration, invasion, and progression in bladder cancer. Over-expression of BMI1 was correlated with tumor clinic-pathological features. BMI1 siRNA effectively inhibited bladder cancer cell proliferation and migration in vitro, and it promoted bladder cancer invasion, maybe by causing epithelial-to-mesenchymal transition. Our findings suggested that BMI1 may represent a novel diagnostic marker and a therapeutic target for bladder cancer, and deserves further investigation.

Investigation on the indication of ipsilateral adrenalectomy in radical nephrectomy: a meta-analysis.

BACKGROUND: With a trend that renal tumors are being detected at an earlier stage, classical radical nephrectomy is being reconsidered. More conservative techniques are being proposed. To clarify the indication for synchronous adrenalectomy in radical nephrectomy for renal cell carcinoma which has been questioned since the 1980s, this study evaluates the role of adrenalectomy and recommends a new indication for adrenalectomy in renal cell carcinoma. METHODS: A systemic search was performed, using PubMed and Google Scholar, of all English language studies published up to March 2012 that compared adrenalectomy with adrenal-sparing surgery, in surgery for renal cell carcinoma. We assessed preoperative imaging for adrenal involvement and the relationship of tumor location with adrenal metastases. Twenty-one studies (20 retrospective and 1 prospective) involving 11 736 patients were included. RESULTS: The mean incidence of ipsilateral adrenal involvement from renal cell carcinoma was 4.5%. Synchronous adrenalectomy did not alter survival (hazard ratio (HR) = 0.89, 95% confidence interval (CI) 0.67 - 1.19, P = 0.43; odds ratio (OR) = 1.10, 95%CI 0.84 - 1.44, P = 0.49). Upper pole tumors were not associated with a higher incidence of ipsilateral adrenal metastases. Pooled preoperative imaging: sensitivity, specificity, positive predictive value and negative predictive value were 92% (95%CI 0.84 - 0.97), 95% (95%CI 0.93 - 0.96), 71.6% and 98.5% respectively. CONCLUSIONS: Adrenal involvement from renal cell carcinoma is rare, even in advanced tumours. Synchronous adrenalectomy does not offer any benefit, even for "high risk" patients. We suggest that only patients with a positive preoperative adrenal finding on preoperative imaging for a solitary adrenal metastasis should undergo adrenalectomy as part of the radical nephrectomy.