Hydroxymethylation and Epigenetic Drugs: New Insights into the Diagnosis and Treatment in Epigenetics of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal and heterogeneous malignancy with multiple genetic alternations and complex signaling pathways. The complexity and multifactorial nature of HCC pose a tremendous challenge regarding its diagnosis and treatment. Emerging evidence has indicated an important regulatory role of epigenetic modifications in HCC initiation and progression. Epigenetic modifications are stably heritable gene expression traits caused by changing the accessibility of chromatin structure and genetic activity without alteration in the DNA sequence and have been gradually recognized as a hallmark of cancer. In addition, accumulating data suggest a potential value of altered hydroxymethylation in epigenetic modifications and therapeutics targeting the epigenetically mediated regulation. As such, probing the epigenetic field in the era of precision oncology is a valid avenue for promoting the accuracy of early diagnosis and improving the oncological prognosis of HCC patients. This review focuses on the diagnostic performance and clinical utility of 5-hydroxymethylated cytosine, the primary intermediate product of the demethylation process, for early HCC diagnosis and discusses the promising applications of epigenetic-based therapeutic regimens for HCC.

Mesoporous Nanoparticles for Diagnosis and Treatment of Liver Cancer in the Era of Precise Medicine.

Primary liver cancer is the seventh-most-common cancer worldwide and the fourth-leading cause of cancer mortality. In the current era of precision medicine, the diagnosis and management of liver cancer are full of challenges and prospects. Mesoporous nanoparticles are often designed as specific carriers of drugs and imaging agents because of their special morphology and physical and chemical properties. In recent years, the design of the elemental composition and morphology of mesoporous nanoparticles have greatly improved their drug-loading efficiency, biocompatibility and biodegradability. Especially in the field of primary liver cancer, mesoporous nanoparticles have been modified as highly tumor-specific imaging contrast agents and targeting therapeutic medicine. Various generations of complexes and structures have been determined for the complicated clinical management requirements. In this review, we summarize these advanced mesoporous designs in the different diagnostic and therapeutic fields of liver cancer and discuss the relevant advantages and disadvantages of transforming applications. By comparing the material properties, drug-delivery characteristics and application methods of different kinds of mesoporous materials in liver cancer, we try to help determine the most suitable drug carriers and information media for future clinical trials. We hope to improve the fabrication of biomedical mesoporous nanoparticles and provide direct evidence for specific cancer management.

[Antimotion sickness effects of ginsenosides combined with dexamethasone in rats].

OBJECTIVE: To investigate the antimotion sickness effects of ginsenosides combined with dexamethasone in rats. METHODS: Fifty SD rats were randomly divided into 5 groups: normal saline, scopolamine-treated, ginsenosides-treated, dexamethasone-treated and ginsenosides plus dexamethasone-treated groups. There were 10 rats in each group. The rats in each group were fed with corresponding ingredients respectively, and then the rats were exposed to abnormal acceleration for one hour. The motion sickness index, the level of kaolin consumption and the course and time of spontaneous activity were observed. RESULTS: The motion sickness index and the level of kaolin consumption of acceleration-exposed rats in ginsenosides plus dexamethasone-treated group were significantly lower than those in normal saline group. And the course and time of spontaneous activity of acceleration-exposed rats in ginsenosides plus dexamethasone-treated group were significantly higher than those in normal saline group. The level of body weight increment of acceleration-exposed rats in ginsenosides plus dexamethasone-treated group was significantly higher than that in dexamethasone-treated group. CONCLUSION: Ginsenosides combined with dexamethasone can significantly increase tolerance to acceleration of rats, and the drug combination can decrease side effects of methylprednisolone, such as body weight loss.

The effect of psychological stress on iron absorption in rats.

BACKGROUND: Psychological stress (PS) is recognized as an important pathogenic factor which leads to metabolism disorder in many diseases. Previous studies have shown that systemic iron homeostasis in mammalians was changed under specific stress conditions. METHODS: In present study, we used communication box to create psychological stress model and investigated the iron apparent absorption, iron accumulation in the apical poles of villous enterocytes and protein expressions of ferroportin 1 (FPN1), ferritin, divalent metal transporter 1 (DMT1). RESULTS: Our study showed that iron apparent absorption decreased and iron significantly accumulated in the apical poles of villous enterocytes in 3 d and 7 d PS groups. The expression of intestinal FPN1 in 3 d and 7 d PS groups was lower than that of control, while the change of intestinal ferritin was opposite. However, the expression of DMT1 did not change. CONCLUSION: These results demonstrate that PS can decrease iron absorption in rats, which might be related to regulation expression of iron transporters.

Psychological stress induces hypoferremia through the IL-6-hepcidin axis in rats.

Anemia is a widespread public health problem. The psychological stress decreases serum iron level and inhibits erythropoiesis. However, the molecular mechanisms involved, leading to iron mal-regulation are not well known. We used a communication box paradigm to induce psychological stress and found that serum iron level decreased after 3d while liver iron storage increased after 7d. Moreover, psychological stress up-regulated expressions of interleukin-6 (IL-6) and hepcidin, while down-regulating ferroportin expression after 3d. These changes were blocked by the injection of IL-6 monoclonal antibody. In conclusion, the IL-6-hepcidin axis is up-regulated by psychological stress in rats, resulting in hypoferremia and increase of hepatic iron storage.

Hsp70 may protect cardiomyocytes from stress-induced injury by inhibiting Fas-mediated apoptosis.

Expression of Hsp70 is an endogenous mechanism by which living cells adapt to stress and the protection of Hsp70 may interfere with the apoptotic machinery in a variety of ways. Here, we observed the change of Hsp70 expression in rat myocardium under stress and explored the protective effect of Hsp70 on the Fas-mediated pathway to cardiomyocyte apoptosis. The results showed that restraint stress led to cardiac dysfunction and structural damage of the myocardium, as well as activation of the Fas pathway. A similar increase in the Fas expression level, caspase-8/3 activity, and the apoptotic rate of the cardiomyocyte also were found, which indicated that Fas-mediated apoptosis of cardiomyocytes might be one of the mechanisms of cardiomyocyte injury induced by stress. Changes in Hsp70 levels and distribution occurred during the stress process, which correlated with the severity of myocardium injury. Heat preconditioning induced the upregulation of Hsp70 synthesis, which in turn may have mitigated subsequent restraint stress-induced damage, including electrocardiography (ECG) abnormality, myocardium damage, and cell death. Moreover, Hsp70 overexpression induced by heat preconditioning had no effect on Fas expression in the cardiomyocyte, but could inhibit activation of caspase-8/3 induced by the Fas signaling pathway and, as a result, prevent cell apoptosis. These results suggest that Hsp70 is capable of protecting the cardiomyocyte from stress-induced injury by inhibiting Fas-mediated apoptosis, and Hsp70 could be considered a target in future drugs to prevent cardiovascular injury caused by stress.

miR-212 mediates counter-regulation on CRH expression and HPA axis activity in male mice.

The mechanisms of hypothalamic-pituitary-adrenal (HPA) axis regulation have been studied persistently but still are not elucidated. Considering the emerging roles of microRNA in stress response, we conducted a microRNA microarray in mice hypothalamus to identify the potential role of microRNAs in regulating the HPA axis. In total, 41 microRNAs changed during heat stress in which we found that miR-212 contains a binding sequence with corticotropin-releasing hormone (Crh) 3'UTR according to a sequence analysis. We observed that miR-212 expression in the hypothalamus was escalated by repeated heat and restraint stress. By overexpression or inhibition of miR-212 and the dual-luciferase reporter assay, we proved that miR-212 could bind with Crh 3'UTR to regulate its expression in mice hypothalamus primary cells and in the hippocampus neuron cell line HT-22. In addition, we injected miR-212 agomir or antagomir in mice hypothalamus to overexpress or inhibit miR-212, which leads to alterations of CRH expression and HPA axis activity in vivo Furthermore, miR-212 and CRH were both transcribed by the cAMP response element-binding protein (CREB). Overexpression and inhibition of miR-212 affect CREB-dependent CRH expression. Taken together, our results suggest an inhibitory role of miR-212 on the HPA axis, which acts in a counter-regulatory manner.

Ethanol extracts from Portulaca oleracea L. attenuated ischemia/reperfusion induced rat neural injury through inhibition of HMGB1 induced inflammation.

It is well demonstrated that the high mobility group box 1 (HMGB1) mediated inflammation has been implicated as one of the important causes for brain damage induced by cerebral ischemia/reperfusion (I/R). In the present study, we assessed the neuro-protective and anti-inflammation effects of the ethanol extracts from Portulaca oleracea L. (EEPO) against cerebral I/R injury in the rat transient middle cerebral artery occlusion (tMCAO) model. Rats were administrated with their respective treatment for 7 days before the MCA occlusion. After that, rats were intraperitoneal injection with chloral hydrate and sacrificed by decapitation, then the serum and brain tissue were collected. The neurological deficit score, infarct size and brain edema were tested. The levels of serum cytokine as TNF-α, IL-1ß, INF-γ, IL-6, and HMGB1 and LDH were detected. The protein level of tissue or nucleus HMGB1, IκB and p-p65 were tested, too. The results showed that pretreatment with EEPO significantly decreased the neurological deficit score, infarct size and brain edema. Moreover, EEPO decreased rat serum cytokine level and rat right cortices p-p65 and IκB protein level. In conclusion all these results suggested that pretreatment with EEFPO provided significant protection against cerebral I/R injury in rats might by virtue of its anti-inflammation property through inhibition of increase of neuleus HMGB1.

[Effects of flavones extracted from Portulaca oleracea on ability of hypoxia tolerance in mice and its mechanism].

OBJECTIVE: To identify anti-hypoxia ingredients extracted from Portulaca oleracea and to find out the possible mechanism of its anti-hypoxia actions. METHODS: Seventy mice were randomly divided into seven groups which were untreated (normal saline), ginsenosides-treated, polysaccharide-treated, acidic components-treated, basic components-treated, alkaloids-treated and flavones-treated groups, and the ingredients of polysaccharide, acidic components, basic components, alkaloids and flavones were extracted from Portulaca olerace. The mice in each group were fed with corresponding ingredients for one week respectively. Then the survival time of mice in hypoxic conditions was observed. Another 90 mice were divided into 3 groups: untreated (normal saline), ginsenosides-treated and flavones-treated groups. The mice in each of these 3 groups were divided into 3 subgroups according to 12-, 24- and 36-hour exposure to hypoxia (10% oxygen and 90% nitrogen), respectively. After exposure to hypoxia, the red blood cell count (RBC), hemoglobin (Hb) concentration and hematocrit (HCT) in mice were determined. The plasma erythropoietin (EPO) levels of mice were detected by enzyme-linked immunosorbent assay (ELISA) and the relative values of EPO mRNA in renal tissue and pallium of mice were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The survival time of mice in hypoxic conditions in flavones-treated group was significantly longer than that in the untreated group. The RBC, Hb concentration, HCT, plasma EPO level and the relative values of EPO mRNA in renal tissue and pallium of mice were significantly higher in the flavones-treated group than those in the untreated group. CONCLUSION: The anti-hypoxia ingredients extracted from Portulaca oleracea are flavones and the anti-hypoxia effects may be obtained by improving the expression level of EPO and accelerating the generations of erythrocyte and Hb.

Ethanol extract from portulaca oleracea L. attenuated acetaminophen-induced mice liver injury.

Acetaminophen-induced liver injury represents the most frequent cause of drug-induced liver failure in the world. Portulaca oleracea L., a widely distributed weed, has been used as a folk medicine in many countries. Previously, we reported that the ethanol extracts of Portulaca oleracea L. (PO) exhibited significant anti-hypoxic activity. In the present study, we investigated the role of PO on acetaminophen (APAP) induced hepatotoxicity. The results demonstrated that PO was an effective anti-oxidative agent, which could, to some extent, reverse APAP-induced hepatotoxicity by regulating the reactive oxygen species (ROS) in the liver of mice. At the same time, PO treatment significantly decreased mice serum levels of IL-6 and TNFα and their mRNA expression in liver tissue IL-α and TNFα play an important role during APAP-induced liver injury. Furthermore, PO inhibited APAP and TNFα-induced activation of JNK, whose activation play an important effect during APAP induced liver injury. These findings suggested that administration of PO may be an effective strategy to prevent or treat liver injury induced by APAP.

[Differentially expressed genes associated with cold acclimation].

To investigate the upregulated genes associated with cold acclimation, a cold acclimation model was established based on Balb/C mouse. mRNA of muscle and liver were isolated, and the upregulated genes of these tissues were studied by representational differential analysis (RDA). The upregulated genes then were sequenced and searched by Blast software in GenBank database. The results showed that some genes were upregulated and possibly associated with cold acclimation. Three of these genes, transferrin, fibrinogen B-beta-chains and a new gene fragment (Genbank ID: AF454762), were confirmed to be upregulated by RNA slot-blot analysis. The finding of these genes might contribute to further understanding of the molecular mechanisms of cold acclimation.

Ethanol extract of Portulaca Oleracea L. reduced the carbon tetrachloride induced liver injury in mice involving enhancement of NF-κB activity.

Acute hepatic injury causes high morbidity and mortality world-wide. Management of severe acute hepatic failure continues to be one of the most challenging problems in clinical medicine. In present study, carbon tetrachloride (CCl4) was used to induce acute liver damage in mice and the protective effects of ethanol extract of Portulaca Oleracea L. (PO) were examined. The aminotransferase activities were biochemical estimated and the liver damage was tested by morphological histological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The role of PO on the activity of NF-κB was determined by luciferase reporter gene assay and immunohistochemistry. The level of p-p65 was tested by western blot. Our results showed that PO administration on mice would decrease the serum aminotransferase level and reduced the liver histological damage. We also found that nuclear translocation of p65 was enhanced in liver tissues of mice treated with PO compared with control animals. In addition, in cultured hepatic cells, PO increased the NF-κB luciferase reporter gene activity and upregulated the level of phosphorylation of p65, but had no effects on mice liver SOD activity and MDA level. Collectively, PO attenuated CCl4 induced mice liver damage by enhancement of NF-κB activity.

Psychological stress induced hippocampus zinc dyshomeostasis and depression-like behavior in rats.

There are strong evidences showed that psychological stress (PS) could result in depression. Recently, many attentions were paid to the roles of corticosterone (CORT) and zinc dyshomeostasis in the development of depression. In this study, we investigated the zinc level in rat hippocampus after exposure to PS and accompanied behavior change. Male SD rats were randomly divided into the control and PS groups. Each group had two subgroups: 7-d group and 14-d group. A communication box was used to produce the PS model in rats. Compared to control group, the PS-treated group showed decreased total zinc levels and increased free zinc levels observed by TSQ staining in hippocampus. Meanwhile, there were significant decreases in mRNA expressions of zinc transporters including ZnT1, ZnT3 and ZIP1 and metallothionein (MT) contents in hippocampus. Moreover, the increased immobility time in forced swim test (FST), lower movement time and total movement distance and longer immobile time in spontaneous activity test were demonstrated in rats after PS exposure. These results suggested that the depression-like behavior in PS-treated rats might be correlated with zinc dyshomeostasis including decreased zinc contents and increased free zinc in hippocampus which was related to changes in zinc transporters and MT expressions.

Anti-hypoxic activity of the ethanol extract from Portulaca oleracea in mice.

AIM OF THE STUDY: To investigate the effects of the ethanol extract from Portulaca oleracea (EEPO) on hypoxia models mice and to find the possible mechanism of its anti-hypoxic actions so as to elucidate the anti-hypoxia activity and provide scientific basis for the clinical use of Portulaca oleracea. MATERIALS AND METHODS: EEPO was evaluated on anti-hypoxic activity in several hypoxia mice models, including closed normobaric hypoxia and sodium nitrite or potassium cyanide toxicosis. To verify the possible mechanism(s), we detected the activities of pyruvate kinase (PK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and the level of adenosine triphosphate (ATP) in mice cortices. RESULTS: Given orally, the EEPO at doses of 100, 200, 400 mg/kg could dose-dependently enhance the survival time of mice in both of the normobaric and chemical hypoxia models. The activity of the glycolysis enzymes and the level of ATP were higher than those of the control. In the pentobarbital sodium-induced sleeping time test and the open-field test, EEPO neither significantly enhanced the pentobarbital sodium-induced sleeping time nor impaired the motor performance, indicating that the observed anti-hypoxic activity was unlikely due to sedation or motor abnormality. CONCLUSIONS: These results demonstrated that the EEPO possessed notable anti-hypoxic activity, which might be related to promoting the activity of the key enzymes in glycolysis and improving the level of ATP in hypoxic mice.

Foot-shock stress-induced regional iron accumulation and altered iron homeostatic mechanisms in rat brain.

Like in other organs, iron in the brain plays an important role in various biological processes. Previous studies have shown that systemic iron homeostasis in mammalians was changed under specific stress conditions. The present study aimed to investigate effects of stress on brain iron homeostasis in rats using a foot-shock stress model. Young adult male Sprague-Dawley rats were randomly assigned to foot-shock stress group subjected to 30 min of cutaneous foot-shock (0.80 mA, 1 pulse/s, 300 ms duration) daily for 1 week or control group left undisturbed. Then, the rats were sacrificed and iron concentration in serum, liver, and some brain regions were measured using atomic absorption spectrophotometry. Expression of ferritin, Transferrin receptor (TfR), divalent metal transporter 1 (DMT1, with or without iron-responsive element), lactoferrin (Lf), and iron regulatory protein 1 (IRP1) in rat hippocampus were determined using western blot analysis. The results showed that stress induced decreased serum iron concentration, increased liver iron content, and elevated iron contents in specific brain regions including hippocampus, striatum, and frontal cortex. In the hippocampus, stress caused decreased expression of ferritin, increased expression of TfR and IRP1, and no change in expression of DMT1 or Lf. Results of this study demonstrated that foot-shock stress induced region specific iron accumulation and altered iron homeostatic mechanisms in the brain in addition to a changed systemic iron homeostasis characterized by decreased serum iron concentration and increased liver iron content. And, elevated IRP1 expression might be associated with the increased TfR and decreased ferritin expression, leading to subsequent iron accumulation and possible increased vulnerability to oxidative damage in hippocampus.

Protective effect of Portulaca oleracea extracts on hypoxic nerve tissue and its mechanism.

The aim of this study was to investigate whether Portulaca oleracea (PO) extracts have hypoxic neuroprotective effects and if so, by what mechanism. After being orally administrated with the PO extracts or distilled water for seven days, adult male BALB/c mice were adapted to a normobaric low oxygen environment (10% oxygen and 90% nitrogen) for different time and then were sacrificed. The mouse cortices were used for histological analysis by hematoxylin and eosin (H-E staining). The activities of pyruvate kinase (PK), phosphofructokinase (PFK), lactic acid (LD) and the level of lactate dehydroenase (LDH) and ATP were detected, and the mRNA and protein levels of EPO in the cortices were analyzed. PC-12 cells and primarily cultured nerve cells were used for 3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. The degree of LDH in the cell culture medium was tested. The results showed that the PO extracts enhanced the EPO mRNA and protein expression in the mouse cortices. Compared to the control group, the mouse in the group treated with the PO extracts by 1 g/d had significantly higher activities of PF, PFK, LDH and higher levels of ATP in the cortices, especially under the hypoxic environment for 24 hours. Histological analysis indicated that the extracts lessened the inflammation damage of the mouse brain. MTT assay results showed the PO extracts or the herb-containing serum raised the viability of the cells under the tested hypoxic conditions and decreased the degree of LDH in the culture medium in a dose-dependent manner. We thus demonstrated that the PO extracts had protective effects on hypoxic nerve tissue.