Xiaodong Han

The toxic effects of microcystin-LR on the reproductive system of male rats in vivo and in vitro.

The aim of this study was to investigate whether microcystin-LR, one of the most common cyanobacterial toxins has toxic effects on reproductive system in vivo or Leydig cells in vitro. Male rats were treated with MC-LR (i.p.) at a dose of 0, 5, 10 or15 microg/(kgday) for 28 days. Leydig cells were cultured with a culture medium including 0, 0.5, 5, 50 or 500 nM MC-LR. In vivo study, we observed exposure to 5 microg/(kgday) of MC-LR decreased the sperm motility, increasing the sperm abnormality rate, 15 microg/(kgday) of MC-LR led to the decrease of testis weight and sperm concentration, decreased the levels of serum testosterone, FSH and LH. The histological findings showed that the seminiferous tubules atrophied and obstructed. In vitro study evaluated MC-LR-induced toxicity and oxidative stress in Leydig cells. It was observed 50 and 500 nM MC-LR significantly decreased the cell viability, increasing the apoptotic DNA fragmentation, and increasing the ratio of necrotic cells. The Leydig cells exposed to MC-LR decreased testosterone production. 500 nM MC-LR increased ROS production, 50 or 500 nM MC-LR enhanced the lipid peroxidation. All Leydig cells exposed to MC-LR showed decreased SOD activity. The results of this study showed that the oxidative stress of MC-LR might lead to cytotoxicity, which may play an important role in cell apoptosis. Then could reduce the production of testosterone in Leydig cells and result in reproductive toxicity.

Nonylphenol induces apoptosis in rat testicular Sertoli cells via endoplasmic reticulum stress

Nonylphenol (NP) is a widely distributed environment contaminant and has been documented to disrupt testicular development and decrease male fertility. Amongst possible targets of this compound are testicular Sertoli cells, which play a crucial role in supporting and nourishing sperm cells. In the present study, we found that NP treatment could cause dramatic morphological changes as well as decreased cell viability of Sertoli cells, while the following Annexin V-PI staining demonstrated that NP treatment led to increased proportion of cell apoptosis, which was evidenced again by the detection of condensation and marginal changes of chromatins using Hoechst staining and transmission microscopy observation. In addition, increased intracellular Ca(2+) levels and changes of endoplasmic reticulum (ER) ultrastructure were also observed in NP-treated groups, indicating the action of NP on ER. The subsequent data showed that the expressions of ER-stress signaling targeted genes GRP78 and gadd153 were elevated, suggesting the activation of ER-stress signal pathway. Furthermore, the detection of ER-stress related proteins by western blotting revealed that the expression of gadd153 was upregulated by NP, whereas the expressions of GRP78 and ERp57 were both first upregulated and then inhibited. Taken together, it is suggested that NP can induce ER stress in Sertoli cells, which may plays an important role in the induction of apoptosis.

Decline of sperm quality and testicular function in male mice during chronic low-dose exposure to microcystin-LR.

The effects of chronic low-dose exposure to microcystins were preliminarily studied on sperm quality and testicular function in male mice. Microcystin-LR (MC-LR) was orally administered to male mice at 0, 1, 3.2, and 10 μg/L for 3 and 6 months. Our preliminary study found in three-month group, sperm quality declined at 3.2 and 10 μg/L doses, testosterone dropped at 10 μg/L, levels of LH and FSH increased, and Leydig cells exhibited apoptosis. Similar, but more pronounced, effects were observed in groups treated with MC-LR for 6 months. Compared to control (0 μg/L), the rate of sperm abnormality was higher and testosterone levels were lower following administration of 3.2 and 10 μg/L MC-LR and structural damage to the testis was observed with 10 μg/L dose. Thus, chronic low-dose treatment with MC-LR results in substantial toxicity to male reproduction, causing declines in sperm quality, decreased levels of serum testosterone, and injury to the testis.

The toxic effects of microcystin-LR on rat spermatogonia in vitro.

Microcystin-leucine arginine (MC-LR), a cyclic heptapeptide produced by several bloom-forming cyanobacteria, has strong reproductive toxicity. We examined whether MC-LR could enter spermatogonia and investigated the toxic effects of MC-LR on spermatogonia in vitro. Multispecific organic anion-transporting polypeptides (Oatps), which transported MCs, were screened as well. Spermatogonia were exposed to 0, 0.5, 5, 50, and 500 nmol/L (nM) MC-LR for 6 h. Cell viability and total antioxidant capacity significantly decreased, meanwhile, the ratio of apoptotic cells, reactive oxidative species (ROS) production, mitochondrial membrane potential (MMP), and intracellular free Ca²⁺ increased after exposure to 5 nM and higher concentrations of MC-LR. MC-LR can immigrate into spermatogonia. At least 5 Oatps (Oatp1a5, -3a1, -6b1, -6c1, and -6d1) were detected at the mRNA level in spermatogonia, and the expression of these Oatps was affected by MC-LR, especially Oatp3a1. This study demonstrated that MC-LR can be transported into spermatogonia and leads to cytotoxicity.

Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/beta-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/beta-catenin signaling were determined, suggested down-regulation of Wnt/beta-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3alpha can inhibit the epithelial differentiation of MSCs. A loss of beta-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated beta-catenin expression and subsequently decreased beta-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/beta-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

Protective effect of l-theanine on carbon tetrachloride-induced acute liver injury in mice

We studied effects of L-theanine, a unique amino acid in tea, on carbon tetrachloride (CCl(4))-induced liver injury in mice. The mice were pre-treated orally with L-theanine (50, 100 or 200 mg/kg) once daily for seven days before CCl(4) (10 ml/kg of 0.2% CCl(4) solution in olive oil) injection. L-theanine dose-dependently suppressed the increase of serum activity of ALT and AST and bilirubin level as well as liver histopathological changes induced by CCl(4) in mice. L-theanine significantly prevented CCl(4)-induced production of lipid peroxidation and decrease of hepatic GSH content and antioxidant enzymes activities. Our further studies demonstrated that L-theanine inhibited metabolic activation of CCl(4) through down-regulating cytochrome P450 2E1 (CYP2E1). As a consequence, L-theanine inhibited oxidative stress-mediated inflammatory response which included the increase of TNF-α and IL-1β in sera, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in livers. CCl(4)-induced activation of apoptotic related proteins including caspase-3 and PARP in mouse livers was also prevented by L-theanine treatment. In summary, L-theanine protects mice against CCl(4)-induced acute liver injury through inhibiting metabolic activation of CCl(4) and preventing CCl(4)-induced reduction of anti-oxidant capacity in mouse livers to relieve inflammatory response and hepatocyte apoptosis.

Administration of a herbal immunoregulation mixture enhances some immune parameters in carp (Cyprinus carpio)

The herbal immunoregulation mixture (HIRM) were extracts of several traditional Chinese medicines (TCMs): Astragalus membranaceus (from the root and stem), Polygonum multiflorum Thunb. (from the root), Isatis tinctoria L. (from the root), Glycyrrhiza glabra (from the stem). Immune parameters, which included macrophage phagocytic activity, macrophage reactive oxygen species (ROS), activity of serum lysozyme, nitric oxide synthetase (NOS) and superoxide dismutase (SOD), levels of total serum protein, globulin, albumin, triglyceride and cholesterol, were determined in carp that had been fed diets containing HIRM at 0.5% or 1% for 30xa0days. The results showed that, compared with those in the control group, the diets with 0.5% and 1% HIRM resulted in significant increase in macrophage phagocytic activity, macrophage ROS and the levels of total protein, globulin, albumin and NOS activity in serum (Pxa0<xa00.05), and no significant difference was found in SOD, lysozyme activities and triglyceride level (Pxa0>xa00.05). On the other hand, 0.5% HIRM led to evident enhancement of NOS activity and cholesterol level compared to 1% HIRM. These results indicated that HIRM might elevate the function of immunity in carp (Cyprinus carpio).

Isolation and characterization of lung resident mesenchymal stem cells capable of differentiating into alveolar epithelial type II cells

Controversies and risks continue to be reported about exogenous mesenchymal stem cell‐based therapies. In contrast with employing exogenous stem cells, making use of lung resident mesenchymal stem cells (LR‐MSCs) could be advantageous. Our study sought to isolate the LR‐MSCs and explore their potential to differentiate into alveolar epithelial type II cells (ATII cells). Total lung cells were first precultured, from which the Sca‐1+CD45−CD31− population was purified using fluorescence activated cell sorting (FACS). By these methods, it would seem that the Sca‐1+CD45−CD31− cells were LR‐MSCs. Similar to bone marrow derived mesenchymal stem cells (BM‐MSCs), these cells express Sca‐1, CD29, CD90, CD44 and CD106, but not CD31 or CD45. They share the same gene expression file with the BM‐MSCs and have a similar DNA content during long‐term culturing. Furthermore, they could be serially passaged with all these properties being sustained. Above all, LR‐MSCs could differentiate into ATII cells when co‐cultured with ATII cells in a trans‐well system. These findings demonstrated that the Sca‐1+CD45−CD31− cells appear to be LR‐MSCs that can differentiate into ATII cells. This approach may hold promise for their use in the treatment of lung disease.

Proteomic Analysis of Changes Induced By Nonylphenol in Sprague-Dawley Rat Sertoli Cells

Nonylphenol (NP) is a common environmental contaminant that is known to disrupt the reproductive system. The testicular Sertoli cells play a pivotal role in the regulation of spermatogenesis and are susceptible to NP-induced reproductive lesions. Our goal was to ascertain whether NP could induce apoptosis in Sertoli cells and to explore the preapoptotic changes in Sertoli cells at low NP concentrations, similar to environmental conditions. In order to survey events that occur at the protein level in Sertoli cells after exposure to NP, we used a proteomic approach with two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify proteins with altered expression in rat Sertoli cells treated with 0.01 and 0.1 microM NP for 24 h. We separated 63 protein spots and identified 41 that were differently expressed in the NP-treated groups and the control. Of these 41 spots, we focused on Raf kinase inhibitor protein (RKIP), Annexin A7 (ANXA7), ERp57, and Peroxiredoxin 6 (PRDX6) for further analysis by Western blot. These proteins are involved in the response of Sertoli cells to programmed cell death. These data help to outline mechanisms by which NP might induce apoptotic tendencies in Sertoli cells.

Inhibition of Wnt/β-catenin signaling promotes epithelial differentiation of mesenchymal stem cells and repairs bleomycin-induced lung injury

Idiopathic pulmonary fibrosis is a progressive lung disorder of unknown etiology. Previous studies have shown that aberrant activation of the Wnt/β-catenin signaling cascade occurs in lungs of patients with idiopathic pulmonary fibrosis. Given the important roles of the Wnt/β-catenin signaling pathway in the development of pulmonary fibrosis, we targeted this pathway for the intervention of pulmonary fibrosis with XAV939, a small molecule that specifically inhibits Tankyrase 1/2, eventually leading to the degradation of β-catenin and suppression of the Wnt/β-catenin signaling pathway. Our results demonstrated that XAV939 significantly inhibited the activation of Wnt/β-catenin signaling and attenuated bleomycin-induced lung fibrosis in mice, and thus improved the survival of mice with lung injury. Interestingly, previous investigations have confirmed that endogenous and exogenous mesenchymal stem cells could be recruited to the injured lung, although the exact effects of these cells are debatable. To determine the effect of Wnt/β-catenin signaling in the epithelial differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs), we established a coculture system that contains BM-MSCs and alveolar type II epithelial cells. The in vitro experiments demonstrated that XAV939 could promote the differentiation of BM-MSCs into an epithelium-like phenotype in the coculture system. We also found that XAV939 could inhibit the proliferation and myofibroblast differentiation of NIH/3T3 fibroblasts. This work supports that inhibition of the Wnt/β-catenin signaling pathway may be exploited for the treatment of idiopathic pulmonary fibrosis for which effective treatment strategies are still lacking.