Zinc Ameliorates Tripterygium Glycosides-Induced Reproductive Impairment in Male Rats by Regulating Zinc Homeostasis and Expression of Oxidative Stress-Related Genes.

Tripterygium glycosides (TG) can seriously damage male reproductive function, and the reproductive system is difficult to restore after stopping the administration of TG in male rats. Zinc (Zn) is one of the most important trace elements in the human body and plays an important role in maintaining male fertility. The aim of this study was to investigate whether zinc supplementation could improve the testicular reproductive damage induced by TG toxicity in rats and to investigate its mechanism of action. The results showed that zinc sulfate (ZnSO4) could improve testicular tissue structure and semen parameters, promote testosterone synthesis, increase zinc-containing enzyme activity, increase zinc concentration in serum and testicular tissues, and maintain zinc homeostasis in male rats induced by TG toxicity. Zinc supplementation activated relevant signalling molecules in the KEAP1-NRF2/ARE pathway and alleviated TG-induced oxidative stress. Therefore, this study concluded that zinc supplementation could improve reproductive damage by regulating zinc homeostasis and the expression of genes related to oxidative stress.

Pt single atoms meet metal-organic frameworks to enhance electrocatalytic hydrogen evolution activity.

The electrochemical hydrogen evolution reaction (HER) effectively produces clean, renewable, and sustainable hydrogen; however, the development of efficient electrocatalysts is required to reduce the high energy barrier of the HER. Herein, we report two excellent single-atom (SA)/metal-organic framework (MOF) composite electrocatalysts (PtSA-MIL100(Fe) and PtSA-MIL101(Cr)) for HER. The obtained PtSA-MIL100(Fe) and PtSA-MIL101(Cr) electrocatalysts exhibit overpotentials of 60 and 61 mV at 10 mA cm-2, respectively, which are close to that of commercial Pt/C (38 mV); they exhibit overpotentials of 310 and 288 mV at 200 mA cm-2, respectively, which are comparable to that of commercial Pt/C (270 mV). Theoretical simulations reveal that Pt SAs modulate the electronic structures of the MOFs, leading to the optimization of the binding strength for H* and significant enhancement of the HER activity. This study describes a novel strategy for preparing desirable HER electrocatalysts based on the synergy between SAs and MIL-series MOFs. Using MIL-series MOFs to support SAs could be valuable for future catalyst design.

Ochratoxin A causes mitochondrial dysfunction, apoptotic and autophagic cell death and also induces mitochondrial biogenesis in human gastric epithelium cells.

Ochratoxin A (OTA) is a common natural contaminant found in human and animal food worldwide. Our previous work has shown that OTA can cause oxidative DNA damage, G2 arrest and malignant transformation of human gastric epithelium (GES-1) cells. Mitochondria are considered to be target for the action of many cytotoxic agents. However, the role of mitochondria in the cytotoxicity of OTA remains unknown. The aim of this study is to explore the putative role of mitochondria on OTA cytotoxicity by analyzing mitochondrial changes in GES-1 cells. The results showed that OTA treatment (5, 10, 20 µM) for different times caused increases in the production of reactive oxygen species, and induced mitochondrial damage, shown by loss of mitochondrial membrane potential (ΔΨM), and decrease in cellular ATP concentration. Subsequently, the mitochondrial apoptotic pathway was activated, presented by increase of apoptotic rate and activation of apoptotic proteins. Autophagic cell death was also triggered, demonstrated by the conversion of light chain 3B (LC3B)-I to LC3B-II and elevated levels of green fluorescent protein-LC3 (GFP-LC3) puncta. Moreover, Parkin-dependent mitophagy was also activated presented by the colocalization of MitoTracker with LysoTracker or GFP-LC3 puncta. The inhibition of autophagy and mitophagy by inhibitors or siRNA attenuated the toxic effect of OTA on cell growth. Interestingly, OTA treatment also enhanced mitochondrial biogenesis confirmed by activation of AMPK/PGC-1α/TFAM pathway and promoted cell survival. Collectively, the effects of OTA on mitochondria of GES-1 cells are complex. OTA could cause mitochondrial function disturbance, apoptotic and autophagic cell death and also induce mitochondrial biogenesis.

The venom of spider Haplopelma hainanum suppresses proliferation and induces apoptosis in hepatic cancer cells by caspase activation in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Spiders and spider venoms have been used in traditional Chinese medicine to treat various ailments for more than 1000 years. For instance, several large spiders have been utilized by the Li People, who mainly live in Hainan Island of China, in their own unique traditional Chinese medicine therapy. Recent studies have indicated that spider venoms may be an important source of bioactive compounds for anti-tumor treatments. However, the specific mechanisms underlying these activities are not yet completely understood. AIM OF THE STUDY: The present study investigated how the venom of the spider Haplopelma hainanum regulate proliferation and apoptosis in HepG2 cells via the underlying molecular mechanisms. MATERIALS AND METHODS: We treated HepG2 cells with various concentrations of the spider venom (0, 10, 50, 100 and 200 µg/mL) for 48 h, and then analyzed anti-proliferation activity, apoptosis-inducing effects, mitochondrial membrane potential (Δψm) and changes in the pro-apoptotic pathway. The anti-proliferation activity was detected by the MTT assay and Western blotting. Flow cytometry was used to analyze both apoptosis and mitochondrial membrane potential. The key pro-apoptotic molecules in the caspase-3 and -9 dependent mitochondrial pathway, including Bcl2 family, were assessed through realtime PCR, Western blotting and enzymatic test. RESULTS: Obvious morphological changes induced by the spider venom included decreased cell numbers, shorter cell length and reduced cell adhesion. MTT and Western blotting demonstrated that the spider venom potently suppressed cell proliferation in a dose- and time-dependent manner with IC50 of 126.00 µg/mL for 48 h. In addition, the spider venom caused a reduction in the mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm under the participation of Bax. Finally, cytochrome c activated caspase-3 and caspase-9, and induced the apoptosis in the HepG2 cells. CONCLUSION: The results indicated that the venom of H. hainanum exhibited potent inhibition effects in HepG2 cells through suppressing proliferation, reducing the mitochondrial membrane potential, activating caspase-3 and caspase-9, and inducing the apoptosis through a mitochondrial-dependent pathway.

Sterigmatocystin-induced checkpoint adaptation depends on Chk1 in immortalized human gastric epithelial cells in vitro.

Sterigmatocystin (ST) is a common contaminant detected in food and animal feed that has been recognized as a possible human carcinogen. Our previous studies demonstrate that ST causes DNA damage and subsequently triggers cell cycle arrest in G2 and apoptosis in immortalized human gastric epithelial cells (GES-1). Recently, studies have shown that in certain contexts, cells with DNA damage may escape checkpoint arrest and enter mitosis without repairing the damage. The term for this process is "checkpoint adaptation," and it increases the risk of unstable genome propagation, which may contribute to carcinogenesis. Thus, we aimed to investigate whether checkpoint adaptation occurs in GES-1 cells treated with ST and explored the underlying molecular mechanisms that contribute to this phenotype. In this study, we found that ST treatment for 24 h in GES-1 cells led to an initial G2 arrest; however, a fraction of GES-1 cells became large and rounded, and the number of p-H3-positive cells increased sharply after ST treatment for 48 h. Moreover, collection of the large and rounded cells by mechanical shake-off revealed that the majority of these large cells were found in the mitotic phase of the cell cycle. Importantly, we found that these rounded cells entered mitosis despite damaged DNA and that a small subset of this cell population survived and continued to propagate. These results suggest that ST induces an initial G2 arrest that is subsequently followed by G2 phase checkpoint adaptation, which may potentially promote genomic instability and result in tumorigenesis. Furthermore, we showed that activation of Chk1 contributes to the G2 arrest in GES-1 cells that are treated with ST for 24 h and that prolonged treatment of cells with ST for 48 h led to a decrease in the total protein and phosphorylation levels of Chk1 in mitotic cells, indicating that checkpoint adaptation may be driven by inactivation of Chk1. Knockdown studies confirmed that cells entered mitosis following inactivation of Chk1. Taken together, we show that ST treatment for 24 h activates Chk1 and induces a G2 arrest in GES-1 cells. However, prolonged ST treatment for 48 h led to Chk1 inactivation in GES-1 cells, which promotes checkpoint adaptation and entry of cells into mitosis despite damaged DNA. Importantly, checkpoint adaptation in GES-1 cells treated with ST may potentially promote genomic instability and drive tumorigenesis.

High nuclear expression of Twist1 in the skeletal extramedullary disease of myeloma patients predicts inferior survival.

The aim of the study was to investigate the expression of epithelial to mesenchymal transition (EMT)-inducing transcription factors, including Twist1 and ZEB1, in skeletal extramedullary disease (EMD) of multiple myeloma (MM) patients and to clarify the effects on clinical outcomes. The expression of Twist1 and ZEB1 in the bone marrow (BM) and the masses of skeletal EMD from 70 MM cases with skeletal EMD and 30 MM patients without skeletal EMD were determined by immunohistochemistry. The results demonstrated that the percentage of high nuclear staining for Twist1 was 24.3% (17/70) in skeletal EMD, which was significantly higher than in the BM of these patients as well as those without skeletal EMD (P=0.030 and P=0.011). The microvessel density (MVD, P=0.004) was significantly higher in patients with high nuclear expression of Twist1 (Twist1-high) than in those with low expression. Patients with Twist1-high experienced a lower rate of progression-free survival (PFS, 11.8% vs. 35.0%, P=0.000) and overall survival (OS, 52.5% vs. 83.7%, P=0.001) compared to those with low expression. Multivariate analysis showed that Twist1-high was independently associated with inferior PFS (HR=2.161; 95%CI: 1.116-4.183; P=0.022) and OS (HR=3.111; 95%CI: 1.114-8.685; P=0.030). We concluded that Twist1-high is associated with a poor prognosis and may be correlated with angiogenesis in the skeletal EMD of MM patients.

Malignant transformation of human gastric epithelium cells via reactive oxygen species production and Wnt/ß-catenin pathway activation following 40-week exposure to ochratoxin A.

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogenic to humans. We previously demonstrated that OTA treatment induced oxidative damage in human gastric epithelium cells (GES-1) in vitro. In this study, we found that long-term OTA treatment could result in increased proliferation, migration, and invasion abilities of GES-1 cells and induce anchorage-independent growth of cells in soft agar. Inoculation of OTA-treated GES-1 cells resulted in the formation of tumor xenografts in Balb/c nude mice in vivo, confirming that long-term OTA treatment can induce the malignant transformation of GES-1 cells. In addition, we found that long-term OTA treatment induced oxidative stress and activated the Wnt/ß-catenin pathway, including the nuclear transition of ß-catenin and the upregulation of the downstream molecules of the pathway. Finally, pretreatment with the antioxidant N-acetyl-L-cysteine (NAC) inhibited ROS formation and activation of the Wnt pathway in OTA-transformed GES-1 cells, which decreased the tumor formation abilities of these cells after inoculation in nude mice. These findings suggest that long-term OTA exposure induces the malignant transformation of GES-1 cells via intracellular ROS production and activation of the Wnt/ß-catenin signaling pathway.

[Effect of high-fat diet and food restriction on energy metabolism in obesity-prone and obesity-resistant rats].

OBJECTIVE: To explore the effect of high-fat diet and food restriction on energy metabolism in obesity-prone (OP) and obesity-resistant (OR) rats. METHODS: Sixty male Sprague-Dawley (SD) rats were divided into OP, OR and control groups according to their body weight gain after fed with high-fat diet for 3 wk. OP and OR groups were fed with high-fat diet in the following 12 wk to promote the development of obesity. Then one-half of the rats of each group began to food restriction and were allowed access to 50% of their individual baseline mean daily food intake each day, while the other half were maintained on ad libitum food for 2 wk. Basal metabolic rate (BMR), resting metabolic rate (RMR) of each group were measured by indirect calorimetry during the high-fat diet feeding and food restriction conditions. After the rats were sacrificed, body fat content was measured. RESULTS: OR rats had significantly higher BMR and RMR than the other two groups during high-fat diet feeding condition. There was no significant difference between OP and control group. Food restriction led to a reduction in BMR and RMR in all groups. OR rats showed a significantly greater reduction. OP group showed a significant decrease in body fat weight and fat content during the food restriction period, while there was no significant differences in OR rats. CONCLUSION: There are significant differences between OP and OR rats in BMR and RMR either in high-fat diet feeding condition or food restricted state. OR rat has the ability to sense and respond to energy imbalance more accurately than OP rat.

Effect of bone morphogenetic protein 7 on differentiation of adipose derived mesenchymal stem cells into brown adipocytes in rats.

OBJECTIVE: To evaluate the effect of bone morphogenetic protein(BMP7)on the differentiation of adipose derived mesenchymal stem cells(AD-MSCs)isolated from different adipose tissues into brown adipocytes in rats. METHODS: Primary AD-MSCs were isolated from rate interscapular brown adipose tissue(iBAT),inguinal subcutaneous white adipose tissue(sWAT),and epididymal white adipose tissue(eWAT),respectively,and then cultivated in vitro. Differentiation of AD-MSCs into brown adipocytes was induced by BMP7. The characteristics of brown adipocytes were detected by immunofluorescence staining and oil red staining of cells. The expression levels of brown adipocyte-related genes were detected by polymerase chain reaction. RESULTS: AD-MSCs from iBAT and sWAT were differentiated into cluster multilocular cells,which were stained red by oil red "O"staining and showed uncoupling protein 1-positive by immunofluorescent staining method. AD-MSCs from eWAT had a small number of scattered multilocular cells and showed uncoupling protein 1-negative. The results of reverse transcription-polymerase chain reaction showed that the uncoupling protein 1 gene was highly expressed in the iBAT group and sWAT group but was negative in the eWAT group. CONCLUSION: AD-MSCs isolated from different adipose tissues in rats have different gene expression profiles and differentiation potentials.

[Anti-tumor effect of cisplatin combined with DC vaccine on tumor-bearing mice].

OBJECTIVE: To explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice. METHODS: B16 melanoma cells were treated with cisplatin at the final concentration of 20 µg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-ß were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin (100 µg/mouse i.p., for sequential 3 days) and intratumoral injection of DC cells (3×10(6)/mouse, twice with a 7-day interval) in the tumor-bearing mouse models was evaluated. Expression of MHC II, ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination. Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation. The distribution of T-reg and CD8(+) T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8(+) T/T-reg was determined. The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay. RESULTS: Cisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC II, ICAM-1 and CD86 on DC cells were (47.5 ± 8.8)%, (35.5 ± 8.3)% and (36.2 ± 9.2)%, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was (2.1 ± 0.6) g, that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g, showing a significant inhibition of tumor growth (P < 0.01). Furthermore, the CD8(+) T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine. When the effector-to-target ratio was 20:1, 10:1 and 5:1, the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)%, (22.0 ± 6.0)% and (14.0 ± 4.0)%, respectively, significantly higher than (8.2 ± 3.6)%, (6.7 ± 1.8)% and (3.6 ± 1.9)%, respectively, in the control group (all P < 0.01). CONCLUSION: Cisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.

Tumour-derived IL-10 within tumour microenvironment represses the antitumour immunity of Socs1-silenced and sustained antigen expressing DCs.

It has been shown that silencing of suppressor of cytokine signalling 1 (Socs1) or stably expressing transgenic protein Ags in antigen-presenting dentritic cells (DCs) strongly enhances antigen-specific anti-tumour immunity. However, whether the strong and long-lasting T cell responses induced by the modified DCs could modulate the immunosuppressive tumour microenvironment has not been clarified. In this study, we explored the anti-tumour immunity of DCs modified by Socs1-shRNA lentiviral transduction combined with sustained expression of TRP2 in different tumour models. We showed that transfer Socs1-silenced or tumour antigen TRP2 persistent expressed DCs, or DCs modified by combination of Socs1-silencing and sustaining TRP2 expression prior to inoculation of tumour cells delayed B16 tumour cell growth, prolonged mouse survival and increased the ratio of CD8+ T/Treg as well as the CTL activity in tumours. However, there was no significant effect on tumour growth and mouse survival rate upon tumour established. Further, we showed that tumour cell secreted IL-10 counteracted the immunity of modified DCs in established tumour model, injection of Socs1-shRNA and TRP2 antigen modified significantly inhibited growth of the established B16-IL-10(-/-) tumours. These data indicated that the high level of IL-10 within tumour microenvironment is one of factors that compromise DC vaccine functions.

Review of the genus Plistobunus Pocock, 1903, with description of a new species from Hainan Island, China (Opiliones, Laniatores, Epedanidae).

The genus Plistobunus Pocock, 1903 and its type species Plistobunus rapax Pocock, 1903 are redescribed based on the type material deposited in the British Museum of Natural History (BMNH), London. In addition, a new Plistobunus species from Hainan Island is described and illustrated of Plistobunus columnariussp. n. The new species is diagnosed by having a row of 12 setiferous tubercles on anterior margin of carapace, and the femur of pedipalpus ventrally with 13 setiferous tubercles in male.

[Immunoadjuvant effect of Hsp70L1 in tumor vaccine].

AIM: To explore the immune enhancement of Hsp70L1 in the tumor cell vaccines. METHODS: TRP2(153-243) and Hsp70L1 genes were obtained by RT-PCR from B16 cells in murine melanoma and from spleens of C57BL/6 mice and then were inserted into pcDNA3.1/V5-His eukaryotic expression vectors respectively. The recombinants of pTRP2, pHSP70L1 or pTRP2-Hsp fusion gene were obtained and transfected into B16 cells respectively. TRP2(153-243), HSP70L1 or TRP2-Hsp fusion gene-expressing B16 cells were then induced to necrosis by freezing-thawing or to apoptosis by mitomycin C. C57BL/6 mice were immunized with the necrotic or apoptotic B16 cells twice, then the live B16 tumor cells were transplanted into the immunized mice and the tumor growth was observed in some tumor-bearing mice. IFN-gamma-producing cells in splenocytes were measured by flow cytometry and the CTL activity of spleno-lymphocyte was detected by LDH release assay. RESULTS: After the normal mice were immunized with the necrotic or apoptotic tumor vaccines modified with TRP2(153-243), Hsp70L1 or TRP2-Hsp fusion genes, CTL lysis activity and IFN-gamma production from the splenic lymphocytes were promoted in the groups of Hsp70L1 and TRP2-Hsp modified tumor vaccines (P<0.05 or P<0.01). Additionally, the tumor growth was inhibited obviously in the groups of mice immunized with necrotic tumor vaccines (P<0.05 or P<0.01). However, no marked inhibition of tumor growth was observed in the groups of mice immunized with apoptotic tumor vaccines (P>0.05). CONCLUSION: Hsp70L1 remarkably improves the immunogenicity of B16 tumor vaccines, especially that of necrotic tumor vaccines.