Identification and functional analysis of an iron-binding protein, ferritin heavy chain subunit, from the swallowtail butterfly, Papilio xuthus.

Ferritin, which is ubiquitous among all living organisms, plays a crucial role in maintaining iron homeostasis, immune response, and detoxification. In the present research, we identified an iron-binding protein, ferritin heavy chain subunit, from Papilio xuthus and named PxFerHCH. The complete complementary DNA of PxFerHCH was 1,252 bp encoding a sequence of 211 amino acids, which includes an iron-responsive element. Phylogenetic analysis showed that PxFerHCH is clustered with Manduca sexta and Galleria mellonella ferritin heavy chain subunits. Expression levels of PxFerHCH in various tissues were analyzed by reverse transcription quantitative polymerase chain reaction, and the results exhibited that PxFerHCH was expressed in all tissues with the highest expression in the fat body. The relative expression level of PxFerHCH in response to bacterial (Escherichia coli and Staphylococcus aureus) challenges sharply increased by about 12 hr postinfection (hpi) and then decreased at 24 hpi. In addition, the iron-binding capacity and antioxidation activity of recombinant PxFerHCH protein were also investigated. These results reveal that PxFerHCH might play an important role in defense against bacterial infection.

Protective effects of selenium against zearalenone-induced apoptosis in chicken spleen lymphocyte via an endoplasmic reticulum stress signaling pathway.

Selenium (Se), an antioxidant agent, provides significant protection from reactive oxygen species (ROS)-induced cell damage in vivo and in vitro. However, it is unclear whether Se can protect against zearalenone (ZEN)-induced apoptosis in chicken spleen lymphocyte. In this study, we investigated the underlying mechanism of the apoptosis induced by ZEN in chicken spleen lymphocyte and further evaluated the protective mechanism of Se on ZEN-induced apoptosis. The results show that ZEN induced an increase in ROS generation and lipid peroxidation, and a decrease in levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione (GSH). The results of apoptosis morphologically from acridine orange/ethidium bromide (AO/EB) fluorescent staining and flow cytometry analysis show apparent apoptosis in the ZEN-treated group, and was confirmed by the upregulation of caspase-3, -12 and downregulation of Bcl-2. Meanwhile, ZEN activated the endoplasmic reticulum (ER) stress by upregulating ER stress-related molecular sensors (GRP78, ATF6, ATF4, IRE). However, co-treatment with Se effectively blocked ROS generation, improved antioxdative capacity, and reversed apoptosis and ER stress-related genes and protein expression. Taken together, these data suggest that oxidative stress and ER stress play a vital role in ZEN-induced apoptosis, and Se had a significant preventive effect on ZEN-induced apoptosis in chicken spleen lymphocyte via ameliorating the ER stress signaling pathway.

GRGM-13 comprising 13 plant and animal products, inhibited oxidative stress induced apoptosis in retinal ganglion cells by inhibiting P2RX7/p38 MAPK signaling pathway.

OBJECTIVE: To determine the effectiveness of GRGM-13 on oxidative stress induced apoptosis of retinal ganglion cells (RGCs) and revealed its possible mechanism. MATERIALS AND METHODS: Caspase-3 activity, MDA level, and glutathione peroxidase level were detected by Caspase-3 assay kit, Lipid Peroxidation MDA Assay Kit, and Total Glutathione Peroxidase Assay Kit, respectively. Protein levels of Bax, Bcl-2, p-p38 and p38 were observed by Western Blot. Reactive oxygen species assay kit was used to determine intracellular ROS level. Apoptotic cells were measured by flow cytometry. RESULTS: GRGM-13 inhibited apoptosis of RGCs and ROS level in rat retinal tissue and RGC-5 cells, and the decrease degree strengthened with the increase of GRGM-13 concentration. In addition, ROS upregulated p-p38 expression, while GRGM-13 reversed this effect. We also found that p38 inhibitor SB202190 did not change L-glutamate (Glu) or H2O2-induced ROS level, while SB202190 inhibited apoptosis of RGC-5 cells. Finally, we observed that P2 × 7R agonist BzATP reversed the inhibition effect of GRGM-13 on RGC-5 cell apoptosis, ROS level and p-p38 expression, while si-P2 × 7R inhibited oxidative stress-induced phosphorylation of p38. CONCLUSION: GRGM-13 could inhibit oxidative stress-induced RGCs apoptosis via inhibiting P2RX7/p38 MAPK pathway, which revealed the possible mechanism of GRGM-13 on stress-induced RGCs apoptosis and provided new Chinese medicine for the treatment of glaucoma.

Protective effects of a composition of Chinese herbs-Gurigumu-13 on retinal ganglion cell apoptosis in DBA/2J glaucoma mouse model.

AIM: To explore the concrete mechanism of a Mongolian compound medicine-Gurigumu-13 (GRGM) for glaucoma treatment. METHODS: DBA/2J mice, as glaucoma models, were intragastric administrated with GRGM to study the effect of GRGM on retinal ganglion cells (RGCs). The loss of RGCs was evaluated with the number of RGCs and axons. The expression of the target protein of RGCs or mouse retinas was determined by Western blot. The relative content of malondialdehyde (MDA) was examined by ELISA assay. RESULTS: GRGM distinctly improved retina damage via increasing the number of neurons, RGCs and axons in a concentration dependent manner. Meanwhile, GRGM obviously decreased the high level of MDA and the expression of oxidative stress-related proteins in retinas of DBA/2J mice, but promoted the expression of antioxidant proteins. Additionally, GRGM also significantly inhibited the protein expression of Bip and Chop, which were markers of endoplasmic reticulum stress-induced apoptosis. CONCLUSION: GRGM have obvious protective effects on RGCs in DBA/2J mice, and increase the number of RGCs and axons via inhibiting oxidative stress and endoplasmic reticulum stress.

The MiR-135b-BMAL1-YY1 loop disturbs pancreatic clockwork to promote tumourigenesis and chemoresistance.

Circadian disruption has been implicated in tumour development, but the underlying mechanism remains unclear. Here, we show that the molecular clockwork within malignant human pancreatic epithelium is disrupted and that this disruption is mediated by miR-135b-induced BMAL1 repression. miR-135b directly targets the BMAL1 3'-UTR and thereby disturbs the pancreatic oscillator, and the downregulation of miR-135b is essential for the realignment of the cellular clock. Asynchrony between miR-135b and BMAL1 expression impairs the local circadian gating control of tumour suppression and significantly promotes tumourigenesis and resistance to gemcitabine in pancreatic cancer (PC) cells, as demonstrated by bioinformatics analyses of public PC data sets and in vitro and in vivo functional studies. Moreover, we found that YY1 transcriptionally activated miR-135b and formed a 'miR-135b-BMAL1-YY1' loop, which holds significant predictive and prognostic value for patients with PC. Thus, our work has identified a novel signalling loop that mediates pancreatic clock disruption as an important mechanism of PC progression and chemoresistance.

5-fluorouracil attenuates dextran sodium sulfate-induced acute colitis in mice.

5­Fluorouracil (5­FU) has been predominantly used in the clinic for cancer chemotherapy. Previous studies have demonstrated that 5­FU has an anti­inflammatory function. In the current study, the potential therapeutic role of 5­FU in dextran sodium sulfate (DSS)­induced acute mouse colitis was investigated. Effects on the severity of colitis were studied via histochemical and immunohistochemical staining, cytokine levels were determined by reverse transcriptoin­quantitative polymerase chain reaction and the effect of 5­FU on NF­κB was examined by western blotting. Administration of 5­FU ameliorated the severity of acute DSS­induced colitis. The disease activity score was significantly lower in the 5­FU + DSS­treated mice compared with the DSS­treated group (P<0.01). Tumor necrosis factor­α, interleukin­1ß and interferon γ mRNA expression levels were significantly downregulated in the colon tissue of DSS mice treated with 5­FU compared with the untreated DSS mice (P<0.05). In addition, the number of CD4+ T cells in the colonic lamina propria and myeloperoxidase activity were significantly decreased in the 5­FU + DSS­treated mice (P<0.05). Furthermore, 5­FU treatment significantly reduced p­NF­κB­p56 protein expression levels in the colon tissue of DSS­treated mice (P<0.05). The present results demonstrated that 5­FU minimizes the abnormal immune cytokine response and relieves the pathophysiological disorders associated with experimental acute colitis. Thus, the modulating inflammatory response role of 5­FU may be partially associated with inhibiting NF­κB activation and 5­FU may be a novel therapeutic strategy for the treatment of inflammatory bowel disease.

[The effect of rhubarb ethanol-extract on hyperlipidemia and liver fatty in rabbits].

AIM: To observe the effect of rhubarb ethanol-extract on hyperlipidemia and liver fatty in rabbits. METHODS: Thirty healthy male white rabbits were divided randomly into five groups, six rabbits in each group. The rabbits in control group were fed with common forage. The rabbits in model group were fed with high lipid forage. The rabbits in three different rhubarb groups were fed with high lipid forage and treated with different level rhubarb ethanol-extract (REE). In the process of experiment, periodically measured serology index of the rabbits and observed common physiology index. The rabbits were killed at the end of tenth week, liver fatty degeneration degree and liver coefficient were measured and compared. RESULTS: REE could decrease serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C), and increase serum high density lipoprotein cholesterol (HDL-C), and reduce liver fatty de generation and protect liver cell function. And the dose-effect relation was showed among different dose REE groups. CONCLUSION: REE can significantly reduce blood lipid, prevent and treat hyperlipidemia and liver fatty.

[Effects of extracts from different parts of Folium perillae (L.) Britt. on Hemorrheological parameters in rats].

In order to provide experimental data for the development and application of drug in clinic, we determined the effects of extracts from different parts of folium perillae (L. ) Britt. on hemorheological parameters, extracted from leaves (folium perillae), seeds (fructus perillae) and peduncles (caulis perillae). The results showed that all extracts from different parts of folium perillae (L. ) Britt. can significantly reduce the whole blood viscosity at low shear rate (10 s(-1)), erythrocyte aggregation index, erythrocyte electrophoresis index (P<0.05), and the whole blood reductive viscosity at low shear rate (10 s(-1)) (P<0.01). Extracts from folium perillae and caulis perillae can significantly decrease erythrocyte deformation index (P<0.05), whereas extracts from fructus perillae can not. Extracts from fructus perillae and caulis perillae can significantly decrease plasma viscosity at low shear rate(10 s(-1)), but extracts of folium perillae can not. Aspirin can only decrease the whole blood reductive viscosity at low shear rate and plasma viscosity (P<0.05). All extracts from different parts of folium perillae (L. ) Britt. had no significant effects on hematocrit, erythrocyte rigidity index, fibrinogen concentration , the whole blood viscosity and the whole blood reductive viscosity at middle and high shear rate (60 s(-1),120 s(-1)).

Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation.

Dendritic cells (DCs) are the most potent antigen-presenting cells that play crucial roles in the regulation of immune response. Triptolide, an active component purified from the medicinal plant Tripterygium wilfordii Hook F., has been demonstrated to act as a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. However, little is known about the effects of triptolide on DCs. The present study shows that triptolide does not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10ng/ml, as demonstrated by phosphatidylserine exposure, mitochondria potential decrease, and nuclear DNA condensation. Triptolide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that the anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.

[The effects of two TCM prescriptions, Tonifying Kidney and Strengthening Vital Energy, on NF-kappaB activing in aged mouse T cells].

AIM: The aim of the present study was to explore the immunologic mechanism of delaying senescence by Strengthening Vital Energy(SVE), Tonifying Kidney (TK) and combined TK and SVE. METHODS: Mice of 20 months were used as senescence model. The effects of the prescriptions on anti-CD3 antibody-induced NF-kappaB activity and the expression of NF-kappaB in T cells from aged mice were analyzed by electrophoretic mobility shift assay (EMSA) and Western blot, respectively. RESULTS: NF-kappaB activity in anti-CD3 antibody-induced T cells from the aged mice was lower than that from young ones. The three prescriptions raised the activity of NF-kappaB in the T cells from the aged mice to certain extent. Combined TK and SVE had no influence on p65 and p50 subunits expressions. CONCLUSION: The increased NF-kappaB activity may be one of mechanisms underlying the improvement of immune response in aged mice by Chinese medicines.

[The expression of mRNA and SDS-PAGE of L1210 cell strains and its cloned cells].

OBJECTIVE: To examine the expression difference of mRNA of L1210 cell strains and its cloned cells and discuss the methods for quality control of cell strains. METHODS: We used SDS-PAGE to observe the difference of protein and performed in situ hybridization to examine the expression of mRNA with the use of 6 cDNA probes that were marked by biotin. RESULTS: The number of protein bands of L1210 from Beijing Cancer Institute was 32. The number of protein bands of the two cloned cells L3E11 and L3F9 was 31. The 6 cDNA probes (p16, c-fos, c-jun, c-myc, p21, and p53 mRNA) were found to be existing in Beijing Cancer Institute L1210 and two different cloned cell strains. Expression of c-myc, c-fos, p53 mRNA could distinguish L3E11 and L3F9 cloned cells.