SeMet alleviates AFB1-induced oxidative stress and apoptosis in rabbit kidney by regulating Nrf2//Keap1/NQO1 and PI3K/AKT signaling pathways.

The purpose of this study was to explore the protective effect of SeMet on renal injury induced by AFB1 in rabbits and its molecular mechanism. Forty rabbits of 35 days old were randomly divided into control group, AFB1 group (0.3 mg AFB1/kg b.w), 0.2 mg/kg Se + AFB1 group (0.3 mg AFB1/kg b.w + 0.2 mg SeMet/kg feed) and 0.4 mg/kg Se + AFB1 group (0.3 mg AFB1/kg b.w + 0.4 mg SeMet/kg feed). The SeMet treatment group was fed different doses of SeMet diets every day for 21 days. On the 17-21 day, the AFB1 treatment group, the 0.2 mg/kg Se + AFB1 group and the 0.4 mg/kg Se + AFB1 group were administered 0.3 mg AFB1 /kg b.w by gavage (dissolved in 0.5 ml olive oil) respectively. The results showed that AFB1 poisoning resulted in the changes of renal structure, the increase of renal coefficient and serum biochemical indexes, the ascent of ROS and MDA levels, the descent of antioxidant enzyme activity, and the significant down-regulation of Nrf2, HO-1 and NQO1. Besides, AFB1 poisoning increased the number of renal apoptotic cells, rised the levels of PTEN, Bax, Caspase-3 and Caspase-9, and decreased the levels of PI3K, AKT, p-AKT and Bcl-2. In summary, SeMet was added to alleviate the oxidative stress injury and apoptosis of kidney induced by AFB1, and the effect of 0.2 mg/kg Se + AFB1 is better than 0.4 mg/kg Se + AFB1.

Selenomethionine attenuates ochratoxin A-induced small intestinal injury in rabbits by activating the Nrf2 pathway and inhibiting NF-κB activation.

The aim of this study was to investigate whether selenomethionine (SeMet) could attenuate intestinal injury in rabbits induced by ochratoxin A (OTA). Sixty 35-day-old IRA rabbits with similar weights were randomly assigned to the control group, OTA group (0.2 mg OTA/kg b.w), OTA+ 0.2 mg/kg Se (0.2 mg OTA/kg b.w + 0.2 mg SeMet/kg feed), OTA+ 0.4 mg/kg Se (0.2 mg OTA/kg b.w + 0.4 mg SeMet/kg feed) and OTA+ 0.6 mg/kg Se (0.2 mg OTA/kg b.w + 0.6 mg SeMet/kg feed). The rabbits were examined after oral administration of different doses of SeMet for 21 days and were intragastrically administered OTA for 7 consecutive days. The results showed that pretreatment with different doses of SeMet protected against the changes in serum biochemical indicators and the decline in production performance caused by OTA exposure. In addition, the activities of SOD, GSH-PX and T-AOC were significantly increased, and the levels of MDA and ROS were decreased after SeMet pretreatment; thus, oxidative damage in rabbit jejunum tissue due to OTA exposure was inhibited. SeMet stimulates Nrf2 and inhibits the NF-κB signalling pathway; the anti-inflammatory response and antioxidative stress in rabbits were improved, and the intestinal barrier damage caused by OTA exposure was improved. In summary, SeMet alleviates OTA-induced intestinal toxicity in rabbits by activating the Nrf2 pathway and inhibiting NF-κB activation. Moreover, 0.4 mg/kg SeMet induced the most significant improvement.

SeMet attenuates AFB1-induced intestinal injury in rabbits by activating the Nrf2 pathway.

The aim of this study was to investigate the role of selenomethionine (SeMet) in alleviating AFB1 induced intestinal injury by inhibiting intestinal oxidative stress. Forty 35-day-old rabbits were divided randomly into 4 groups (control group, AFB1 group, 0.2 mg/kg Se + AFB1 group, 0.4 mg/kg Se + AFB1 group). From the first day of the experiment, the two treatment groups were fed 0.2 mg/kg SeMet or 0.4 mg/kg SeMet daily for 21 days. On the 17th day, all rabbits in the model group and the two treatment groups were given intragastric AFB1 daily for 5 days. The ADG, ADFI and FCR of the rabbits were examined. Rabbit jejunum tissue was collected for hematoxylin- eosin staining (HE), PCNA detection, immunofluorescence and WB. Intestinal tissue IL-1ß, IL-6 and TNF-α were examined by enzyme-linked immunosorbent assay (ELISA). The results showed that the production performance was decreased, the levels of ROS and MDA were increased in intestinal tissues, the activity of antioxidant enzymes was decreased and the expression levels of Nrf2 and HO-1 were decreased in AFB1-exposed rabbits. In addition, AFB1 induces an inflammatory response in the jejunum and promotes the expression of TNF-α, IL-6 and IL-1ß. SeMet pretreatment significantly improved the performance of the rabbits, alleviated intestinal oxidative stress and the inflammatory response. Therefore, we confirmed that SeMet protects against AFB1 induced oxidative damage and improves productivity in rabbits by activating the Nrf2/HO-1 signaling pathway.

Comparative Proteome Analysis Indicates The Divergence between The Head and Tail Regeneration in Planarian.

OBJECTIVE: Even a small fragment from the body of planarian can regenerate an entire animal, implying that the different fragments from this flatworm eventually reach the same solution. In this study, our aim was to reveal the differences and similarities in mechanisms between different regenerating fragments from this worm. MATERIALS AND METHODS: In this experimental study, we profiled the dynamic proteome of regenerating head and tail to reveal the differences and similarities between different regenerating fragments using 2-DE combined with MALDITOF/ TOF MS. RESULTS: Proteomic profiles of head and tail regeneration identified a total of 516 differential expressed proteins (DEPs) and showed a great difference in quantity and fold changes of proteome profiles between the two scenarios. Briefly, out of the 516 DEPs, 314 were identified to be specific for anterior regeneration, while 165 were specific for posterior regeneration. Bioinformatics analysis showed a wide discrepancy in biological activities between two regenerative processes; especially, differentiation and development and signal transduction in head regeneration were much more complex than that in tail regeneration. Protein functional analysis combined with protein-protein interaction (PPI) analysis showed a significant contribution of both Wnt and BMP signaling pathways to head regeneration not but tail regeneration. Additionally, several novel proteins showed completely opposite expression between head and tail regeneration. CONCLUSION: Proteomic profiles of head and tail regeneration identified a total of 516 differential expressed proteins (DEPs) and showed a great difference in quantity and fold changes of proteome profiles between the two scenarios. Briefly, out of the 516 DEPs, 314 were identified to be specific for anterior regeneration, while 165 were specific for posterior regeneration. Bioinformatics analysis showed a wide discrepancy in biological activities between two regenerative processes; especially, differentiation and development and signal transduction in head regeneration were much more complex than that in tail regeneration. Protein functional analysis combined with protein-protein interaction (PPI) analysis showed a significant contribution of both Wnt and BMP signaling pathways to head regeneration not but tail regeneration. Additionally, several novel proteins showed completely opposite expression between head and tail regeneration.

The Upstream Pathway of mTOR-Mediated Autophagy in Liver Diseases.

Autophagy, originally found in liver experiments, is a cellular process that degrades damaged organelle or protein aggregation. This process frees cells from various stress states is a cell survival mechanism under stress stimulation. It is now known that dysregulation of autophagy can cause many liver diseases. Therefore, how to properly regulate autophagy is the key to the treatment of liver injury. mechanistic target of rapamycin (mTOR)is the core hub regulating autophagy, which is subject to different upstream signaling pathways to regulate autophagy. This review summarizes three upstream pathways of mTOR: the phosphoinositide 3-kinase (PI3K)/protein kinase (AKT) signaling pathway, the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and the rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-extracellular activated protein kinase kinase (MEK)/ extracellular-signal-regulated kinase (ERK) signaling pathway, specifically explored their role in liver fibrosis, hepatitis B, non-alcoholic fatty liver, liver cancer, hepatic ischemia reperfusion and other liver diseases through the regulation of mTOR-mediated autophagy. Moreover, we also analyzed the crosstalk between these three pathways, aiming to find new targets for the treatment of human liver disease based on autophagy.

Integrated mRNA-Seq and miRNA-Seq analysis of PLCγ2-overexpressing hepatocarcinoma cells and identification of the associated miRNA-mRNA network.

Our previous study has discovered the positive effect of phospholipase Cγ 2 (PLCγ2) on the growth of hepatocarcinoma cells; however, the underlying mechanism is far from being understood. For this reason, this study attempts to identify the differently expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) in PLCγ2-overexpressing hepatocarcinoma cells. The results showed that totally 596 differently-expressed genes (DEGs) were identified in PLCγ2-expressed cells, including 314 upregulated and 282 downregulated ones; according to gene ontology analysis, these DEGs were involved in different cellular processes. Concurrently, 34 differently-expressed miRNAs (DEMs) were also detected in PLCγ2-expressing hepatocarcinoma cells. Moreover, the integrative analysis of miRNA and mRNA expression profiles identified the potential regulatory network linked to hepatocarcinoma-related biological processes, including metabolic activity, gene expression, cell cycle, cell migration, and so on. To our knowledge, it is the first study on the effect of PLCγ2 on miRNA and mRNA expressions in hepatocarcinoma cells, and the findings provide new insights into the mechanism supporting the growth-promoting effect of PLCγ2 in hepatocarcinoma cells.

PLCγ2 promotes apoptosis while inhibits proliferation in rat hepatocytes through PKCD/JNK MAPK and PKCD/p38 MAPK signalling.

OBJECTIVES: The PLCG2 (PLCγ2) gene is a member of PLC gene family encoding transmembrane signalling enzymes involved in various biological processes including cell proliferation and apoptosis. Our earlier study indicated that PLCγ2 may be involved in the termination of regeneration of the liver which is mainly composed of hepatocytes, but its exact biological function and molecular mechanism in liver regeneration termination remains unclear. This study aims to examine the role of PLCγ2 in the growth of hepatocytes. MATERIALS AND METHODS: A recombinant adenovirus expressing PLCγ2 was used to infect primary rat hepatocytes. PLCγ2 mRNA and protein levels were detected by qRT-PCR and Western blot. The subcellular location of PLCγ2 protein was tested by an immunofluorescence assay. The proliferation of hepatocytes was measured by MTT assay. The cell cycle and apoptosis were analysed by flow cytometry. Caspase-3, -8 and -9 activities were measured by a spectrophotometry method. Phosphorylation levels of PKCD, JNK and p38 in the infected cells were detected by Western blot. The possible mechanism underlying the role of PLCγ2 in hepatocyte growth was also explored by adding a signalling pathway inhibitor. RESULTS: Hepatocyte proliferation was dramatically reduced, while cell apoptosis was remarkably increased. The results demonstrated that PLCγ2 increased the phosphorylation of PKCD, p38 and JNK in rat hepatocytes. After PKCD activity was inhibited by the inhibitor Go 6983, the levels of both p-p38 and p-JNK MAPKs significantly decreased, and PLCγ2-induced cell proliferation inhibition and cell apoptosis were obviously reversed. CONCLUSIONS: This study showed that PLCγ2 regulates hepatocyte growth through PKCD-dependently activating p38 MAPK and JNK MAPK pathways; this result was experimentally based on the further exploration of the effect of PLCγ2 on hepatocyte growth in vivo.

Silencing of phospholipase C gamma 2 promotes proliferation of rat hepatocytes in vitro.

The management of hepatic failure is undoubtedly difficult, and poor results have led to the search for novel therapeutic approaches. Nowadays, anti-apoptotic gene therapy is considered as an ideal approach. It has been proved that phospholipase Cγ2 (PLCγ2) is involved in the apoptosis of immune cells and tumor cells; however, whether this gene is related to hepatocyte death is still unclear. This study examined the role of PLCγ2 by inhibiting its expression in rat hepatocytes with siRNA. We also further analyzed the cellular mechanism by which the expression inhibition of PLCγ2 induces cell death. Silencing PLCγ2 gene by adenovirus vector expressing PLCγ2-targeted siRNA caused the great decline in the number of G1- and G2/M phase cells, the significant increase in the number of S phase cells, and the obvious reduction in apoptosis index. In addition, silencing PLCγ2 gene relieved the rat hepatocyte damage, such as the cell shrinkage and chromatin condensation, nuclear fragmentation. Further analysis of Ad-PLCγ2 siRNA-transfected hepatocytes demonstrated that suppression of PLCγ2 gene expression could cause the caspase dependent cell death by inhibiting the signal pathway MEKK1/MKK4/JNK1/2/c-Jun. In conclusion, these findings suggest that interference with PLCγ2 expression could relieve the inhibitory effect of PLCγ2 on hepaocyte apoptosis, thus, promote proliferation through inactivating PKCδ-mediated JNK1/2 signaling pathway.

Effect of Food Additive Citric Acid on The Growth of Human Esophageal Carcinoma Cell Line EC109.

OBJECTIVE: Today, esophageal cancer (EC) has become one of the most common cancer types in China. Therefore, new drug and therapeutic strategies are urgently needed to improve postoperative survival rate of patients with EC. As a food additive, several lines of evidence have shown that citric acid can be served as glycolysis suppressor to inhibit growth of some tumor cells. However, little is known about the effect of this organic acid on the growth of human esophageal carcinoma cell line, EC109. MATERIALS AND METHODS: In this experimental study, cell proliferation rate was determined using MTT assay. Apoptotic morphological changes were evaluated by fluorescent microscopy using Hoechst 33258 staining. Cell apoptosis rate and mitochondrial membrane potential (MMP) were detected using flow-cytometry. Effect of citric acid on cellular membrane permeability was assessed by measuring lactate dehydrogenase (LDH) activity, using LDH assay kit. RESULTS: Compared to the control group, there was a marked decrease in cells proliferation when the cells were treated with higher citric acid concentrations (800, 1600 µg/ml). Typical apoptotic morphology of EC109 cells was observed upon treatment with citric acid, such as chromatin condensation and appearance of apoptotic body. Cell apoptotic indexes were significantly increased (P<0.01) after treatment with citric acid at the concentration of 400-1600 µg/ml. Extracellular LDH activity and loss of MMP in all of the treated groups were significantly higher than control (P<0.05), in a dose-dependent manner. CONCLUSION: These results suggest that citric acid prevents EC109 cell growth by inhibiting cell proliferation and inducing apoptosis, which perhaps offers some theoretical guidance for its application in EC treatment.

Effects and mechanisms of melatonin on the proliferation and neural differentiation of PC12 cells.

Melatonin, a lipophilic molecule that is mainly synthesized in the pineal gland, performs various neuroprotective functions. However, the detailed role and mechanisms of promoting neuronal differentiation remains limited. This study demonstrated that 10 µM melatonin led to significant increases in the proliferation and neurite outgrowth of PC12 cells. Increased expression of microtubule-associated protein 2 (MAP2, a neuron-specific protein) was also observed. However, luzindole (melatonin receptor antagonist) and PD98059 (MEK inhibitor) attenuated these increases. LY294002 (AKT inhibitor) inhibited melatonin-mediated proliferation in PC12 cells and did not affect melatonin-induced neural differentiation. The expression of p-ERK1/2/ERK1/2 was increased by melatonin treatment for 14 days in PC12 cells, whereas luzindole or PD98059 reduced the melatonin-induced increase. These results suggest that the activation of both the MEK/ERK and PI3K/AKT signaling pathways could potentially contribute to melatonin-mediated proliferation, but that only the MEK/ERK pathway participates in the melatonin-induced neural differentiation of PC12 cells. Altogether, our study demonstrates for the first time that melatonin may exert a positive effect on neural differentiation via melatonin receptor signalling and that the MEK/ERK1/2 signalling may act down stream from the melatonin pathway.

Study on injury effect of food additive citric acid on liver tissue in mice.

To investigate the damaging effect and action mechanism of the food additive citric acid (CA) on mouse liver, 40 healthy male Kunming mice were randomly divided into control group (0.9 % saline), low CA dose (120 mg/kg), middle dose (240 mg/kg) and high dose groups (480 mg/kg). All experimental mice have received peritoneal injection of the corresponding reagent each week for 3 weeks. After 7 days since the third injection, morphological changes were observed by light microscope; activities of T-SOD, glutathione peroxidase (GSH-Px), caspase-3, and contents of hydrogen peroxide (H2O2) and malonyldialdehyde (MDA) in the liver were evaluated using the corresponding assay kits; DNA fragmentation was assayed using agarose gel electrophoresis. Microscopical detection showed a series of hispathological changes in mouse livers treated with CA, such as indiscriminate liver cell cord, blood clot in central veins, and lymphocyte infiltrating. Biochemical examination suggested the gradually but moderately reduced T-SOD activity and elevated H2O2 level with the increase of CA dose (P > 0.05), and the gradually reduced GSH-Px activity and increased MDA content depending on graded doses with a significant difference (P < 0.05) between the high dose group and the control group. According to cell apoptosis assays, caspase-3 activity were significantly higher in all treatment groups than in the control (P < 0.05) in a dose-dependent manner. Contrasting to the control, characteristic DNA laddering was observed when injected with any of the three graded doses. It can be concluded that certain concentrations of CA cause oxidative damage of the liver by means of the decrease of antioxidative enzyme activities, thus resulting in MDA level elevation and DNA fragmentation inducing active caspase-3.

A new method for Schwann-like cell differentiation of adipose derived stem cells.

Peripheral nerve repair can be enhanced by Schwann cell transplantation, but the clinical application of this procedure is limited by donor site morbidity and the inability to quickly generate a sufficient number of cells. Thus, alternative cell systems for the generation of Schwann cells are desired. Schwann-like cell induced from adipose-derived stem cells (ADSCs) may be one of the ideal alternative cell systems for Schwann cell generation. Although co-culture with Schwann cells or chemicals combined with a mixture of glial growth factors are often utilized for Schwann cell-like differentiation of ADSCs, these methods are usually complicated or expensive. In this experiment, the rat sciatic nerve was cut, and then soaked in culture medium for two days. The treated culture medium was used as an induction agent after filtering. The obtained ADSCs were incubated with the above induction culture medium for five days. Then, expression of the typical Schwann cell markers, S-100 and GFAP proteins was determined by immunocytochemical staining and Western blotting. The results showed that almost all of the treated ADSCs displayed a spindle shape like morphology after being incubated with induction culture medium for 24h and expressed S-100 and GFAP proteins after five days. All of these characteristics of differentiated rat ADSCs were similar to genuine Schwann cells. Thus, this new method, which utilized trophic factors secreted from sciatic nerve leachate, was capable of inducing ADSC differentiation into Schwann-like cell.

Transportation stress alters the expression of immunoregulatory cytokines in the porcine thymus.

This study investigated the effects of transportation stress on blood concentrations of the main pro-inflammatory cytokines (interleukins IL-1ß, IL-2 and IL-6; tumour necrosis factor-α) and anti-inflammatory cytokines (IL-4 and IL-10) and the expression of these cytokines and their receptors in the thymus. Pigs were assessed after 1, 2 and 4 h of transportation (n=5 per group), with normal housing conditions as a control (n=4). Serum concentrations of IL-2, IL-6 and IL-10 were highest at 1 h, whereas concentrations of IL-6 and IL-10 were significantly decreased at 4h. Expression of these three cytokines and their receptors was also significantly altered in the thymus during transportation stress. Serum IL-10 concentrations and thymus IL-10 mRNA expression were significantly correlated. The thymus may contribute towards the regulation of cytokines in pigs during transportation.

Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

OBJECTIVE: To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. ANIMALS: 24 pigs (mean weight, 20 +/- 1 kg). PROCEDURES: Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. RESULTS: No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.