Transcriptome Analysis and Epigenetics Regulation in the Hippocampus and the Prefrontal Cortex of VPA-Induced Rat Model.

Autism spectrum disorders (ASD) are a highly heterogeneous group of neurodevelopmental disorders caused by complex interaction between various genes and environmental factors. As the hippocampus and prefrontal cortex are involved in social recognition, they are the regions of the brain implicated in autism. The effects of prenatal exposure to valproic acid (VPA) can induce an ASD phenotype in both humans and rats; this tool is commonly used to model the complexity of ASD symptoms in the laboratory. However, researchers rarely undertake epigenetic regulation of the brain regions using this model. The present study has addressed this gap by examining gene expression abnormalities in the hippocampus and prefrontal cortex in the VPA rat model of ASD. mRNA and miRNA sequencing was performed on samples from the hippocampus and prefrontal cortex of the VPA model of autism. According to the analysis, 3000 mRNAs in the hippocampus and 2187 mRNAs in the prefrontal cortex showed a significant difference in expression between the VPA and saline groups. In addition, there were 115 DE miRNAs in the hippocampus and 14 DE miRNAs in the prefrontal cortex. Further, the predicted and validated target mRNA of DE miRNA enriched pathways involved neurotransmitter uptake, long-term synaptic depression, and AMPA receptor complex (anti-GluA2-b) in the hippocampus; as well as the neuroactive ligand-receptor interaction and regulation of postsynaptic membrane potential in the prefrontal cortex. This revealed the negative regulation network of miRNAs-mRNAs in the hippocampus and prefrontal cortex, while filtering out key genes (miR-10a-5p and Grm3). Finally, the significant variable miR-10a-5p and its negative regulated genes (Grm3) were verified in both brain regions by QPCR. Importantly, the fact that miR-10a-5p downregulated Grm3 in both the hippocampus and the prefrontal cortex may play a potentially significant role in the occurrence and development of autism. This study suggests that the VPA model has the potential to reproduce ASD-related hippocampus and prefrontal cortex abnormalities, at the epigenetic and transcriptional levels. Furthermore, the network of miRNAs-mRNAs was confirmed; this negative regulatory relationship may play a key role in determining the occurrence and development of autism. The study of this topic help better understand the pathogenesis of ASD.

Released dsDNA-triggered inflammasomes serve as intestinal radioprotective targets.

Objectives: Intestinal mucositis is the major side effect during abdominal or pelvic radiotherapy, but the underlying immunogen remains to be further characterised and few radioprotective agents are available. This study investigated the role of dsDNA-triggered inflammasomes in intestinal mucositis during radiotherapy. Methods: Pro-inflammatory cytokines were detected by ELISA. Radiation-induced intestinal injury in mice was analyzed by means of survival curves, body weight, HE staining of intestines, and intestinal barrier integrity. Western blot, immunofluorescence staining, co-immunoprecipitation assay and flow cytometry were used to investigate the regulatory role of dsDNA on inflammasomes. Results: Here, we show that a high level of IL-1ß and IL-18 is associated with diarrhoea in colorectal cancer (CRC) patients during radiotherapy, which accounts for intestinal radiotoxicity. Subsequently, we found that the dose-dependently released dsDNA from the intestinal epithelial cells (IECs) serves as the potential immunogenic molecule for radiation-induced intestinal mucositis. Our results further indicate that the released dsDNA transfers into the macrophages in an HMGB1/RAGE-dependent manner and then triggers absent in melanoma 2 (AIM2) inflammasome activation and the IL-1ß and IL-18 secretion. Finally, we show that the FDA-approved disulfiram (DSF), a newly identified inflammasome inhibitor, could mitigate intestinal radiotoxicity by controlling inflammasome. Conclusion: These findings indicate that the extracellular self-dsDNA released from the irradiated IECs is a potential immunogen to stimulate immune cells and trigger the subsequent intestinal mucositis, while blunting the dsDNA-triggered inflammasome in macrophages may represent an exciting therapeutic strategy for side effects control during abdominal radiotherapy.

Peripheral CD4 +CD8 + double positive T cells: A potential marker to evaluate renal impairment susceptibility during systemic lupus erythematosus.

Lupus nephritis (LN) has a high incidence in systemic lupus erythematosus (SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4 +CD8 + double positive T (DPT) lymphocytes and LN. The study included patients with SLE without renal impairment (SLE-NRI), LN, nephritic syndrome (NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group ( t=4.012, P<0.001), NS group ( t=3.240, P=0.001), and nephritis group ( t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times (95% confidence interval, 2.115-12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.

Pharmaceutical targeting of succinate dehydrogenase in fibroblasts controls bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix in the lung with fibroblast-to-myofibroblast transition, leading to chronically compromising lung function and death. However, very little is known about the metabolic alterations of fibroblasts in IPF, and there is still a lack of pharmaceutical agents to target the metabolic dysregulation. Here we show a glycolysis upregulation and fatty acid oxidation (FAO) downregulation in fibroblasts from fibrotic lung, and perturbation of glycolysis and FAO affects fibroblasts transdifferentiation. In addition, there is a significant accumulation of succinate both in fibrotic lung tissues and myofibroblasts, where succinate dehydrogenase (SDH) operates in reverse by reducing fumarate to succinate. Then succinate contributes to glycolysis upregulation and FAO downregulation by stabilizing HIF-1α, which promotes the development of lung fibrosis. In addition, we identify a near-infrared small molecule dye, IR-780, as a targeting agent which stimulates mild inhibition of succinate dehydrogenase subunit A (SDHA) in fibroblasts, and which inhibits TGF-ß1 induced SDH and succinate elevation, then to prevent fibrosis formation and respiratory dysfunction. Further, enhanced cell retention of IR-780 is shown to promote severe inhibition of SDHA in myofibroblasts, which may contribute to excessive ROS generation and selectively induces myofibroblasts to apoptosis, and then therapeutically improves established lung fibrosis in vivo. These findings indicate that targeting metabolic dysregulation has significant implications for therapies aimed at lung fibrosis and succinate dehydrogenase is an exciting new therapeutic target to treat IPF.

Acceleration of ageing via disturbing mTOR-regulated proteostasis by a new ageing-associated gene PC4.

Research on ageing-associated genes is important for investigating ageing and anti-ageing strategies. Here, we firstly reported that the human positive cofactor 4 (PC4), a multifunctional and highly conserved nucleoprotein, is accumulated and activated during ageing and causes global accelerated ageing process by disrupting proteostasis. Mechanistically, PC4 interacts with Sin3-HDAC complex and inhibits its deacetylated activity, leads to hyper-acetylation of the histones at the promoters of mTOR-related genes and causes mTOR signalling activation. Accordingly, mTOR activation causes excessive protein synthesis, resulting in impaired proteostasis and accelerated senescence. These results reveal a new biological function of PC4 in vivo, recognizes PC4 as a new ageing-associated gene and provides a genetically engineered mouse model to simulate natural ageing. More importantly, our findings also indicate that PC4 is involved in histone acetylation and serves as a potential target to improve proteostasis and delay ageing.

Artesunate induces ER-derived-ROS-mediated cell death by disrupting labile iron pool and iron redistribution in hepatocellular carcinoma cells.

Aberrant iron homeostasis is a typical characteristic of Hepatocellular carcinoma (HCC), and perturbation of iron metabolism is an effective strategy for HCC therapy. However, there are few safe and effective targeting agents available in clinical practices. The artemisinin and its derivatives have shown potential anti-cancer activity by disturbing cellular iron homeostasis, but the specific mechanism is still unclear. In this study, we demonstrate that Artesunate (ART), a water-soluble anti-malaria agent in clinical use, can regulate the labile iron pool (LIP) and effectively induce ROS-dependent cell death in multiple HCC cells. Mechanistically, ART increases the LIP by promoting lysosomal degradation of iron-storage protein ferritin through acidizing lysosomes. Then the accumulation of labile iron in the endoplasmic reticulum (ER) promotes excessive reactive oxygen species (ROS) production and severe ER disruption, which leads to cell death. Our results provide a new understanding of how ART modulates iron metabolism in HCC cells at the subcellular level, demonstrate the significance of endoplasmic reticulum as iron-vulnerability of HCC cells. More importantly, our findings suggest ART is a safe and potential anti-HCC agent via disturbing iron homeostasis.

Near-infrared oxidative phosphorylation inhibitor integrates acute myeloid leukemia-targeted imaging and therapy.

Acute myeloid leukemia (AML) is a deadly hematological malignancy with frequent disease relapse. The biggest challenge for AML therapy is the lack of methods to target and kill the heterogeneous leukemia cells, which lead to disease relapse. Here, we describe a near-infrared (NIR) fluorescent dye, IR-26, which preferentially accumulates in the mitochondria of AML cells, depending on the hyperactive glycolysis of malignant cell, and simultaneously impairs oxidative phosphorylation (OXPHOS) to exert targeted therapeutic effects for AML cells. In particular, IR-26 also exhibits potential for real-time monitoring of AML cells with an in vivo flow cytometry (IVFC) system. Therefore, IR-26 represents a novel all-in-one agent for the integration of AML targeting, detection, and therapy, which may help to monitor disease progression and treatment responses, prevent unnecessary delays in administering upfront therapy, and improve therapeutic efficiency to the residual AML cells, which are responsible for disease relapse.

Mitigation of radiation-induced pulmonary fibrosis by small-molecule dye IR-780.

Radiation-induced pulmonary fibrosis (RIPF) is a common complication during thoracic radiotherapy, but there are few effective treatments. Here, we identify IR-780, a mitochondria-targeted near-infrared (NIR) dye, can selectively accumulate in the irradiated lung tissues. Besides, IR-780 significantly alleviates radiation-induced acute lung injury and fibrosis. Furthermore, our results show that IR-780 prevents the differentiation of fibroblasts and the release of pro-fibrotic factors from alveolar macrophages induced by radiation. Besides, IR-780 downregulates the expression of glycolysis-associated genes, and 2-Deoxy-d-glucose (2-DG) also prevents the development of fibrosis in vitro, suggesting radioprotective effects of IR-780 on RIPF might be related to glycolysis regulation. Finally, IR-780 induces tumour cell apoptosis and enhances radiosensitivity in representative H460 and A549 cell lines. These findings indicate that IR-780 is a potential therapeutic small-molecule dye during thoracic radiotherapy.

Improvement of obesity-associated disorders by a small-molecule drug targeting mitochondria of adipose tissue macrophages.

Pro-inflammatory activation of adipose tissue macrophages (ATMs) is causally linked to obesity and obesity-associated disorders. A number of studies have demonstrated the crucial role of mitochondrial metabolism in macrophage activation. However, there is a lack of pharmaceutical agents to target the mitochondrial metabolism of ATMs for the treatment of obesity-related diseases. Here, we characterize a near-infrared fluorophore (IR-61) that preferentially accumulates in the mitochondria of ATMs and has a therapeutic effect on diet-induced obesity as well as obesity-associated insulin resistance and fatty liver. IR-61 inhibits the classical activation of ATMs by increasing mitochondrial complex levels and oxidative phosphorylation via the ROS/Akt/Acly pathway. Taken together, our findings indicate that specific enhancement of ATMs oxidative phosphorylation improves chronic inflammation and obesity-related disorders. IR-61 might be an anti-inflammatory agent useful for the treatment of obesity-related diseases by targeting the mitochondria of ATMs.

PC4 serves as a negative regulator of skin wound healing in mice.

BACKGROUND: Human positive cofactor 4 (PC4) was initially characterized as a multifunctional transcriptional cofactor, but its role in skin wound healing is still unclear. The purpose of this study was to explore the role of PC4 in skin wound healing through PC4 knock-in mouse model. METHODS: A PC4 knock-in mouse model (PC4+/+) with a dorsal full-thickness wound was used to investigate the biological functions of PC4 in skin wound healing. Quantitative PCR, Western blot analysis and immunohistochemistry were performed to evaluate the expression of PC4; Sirius red staining and immunofluorescence were performed to explore the change of collagen deposition and angiogenesis. Proliferation and apoptosis were detected using Ki67 staining and TUNEL assay. Primary dermal fibroblasts were isolated from mouse skin to perform cell scratch experiments, cck-8 assay and colony formation assay. RESULTS: The PC4+/+ mice were fertile and did not display overt abnormalities but showed an obvious delay in cutaneous healing of dorsal skin. Histological staining showed insufficient re-epithelialization, decreased angiogenesis and collagen deposition, increased apoptosis and decreased cell proliferation in PC4+/+ skin. Our data also showed decreased migration rate and proliferation ability in cultured primary fibroblasts from PC4+/+ mice in vitro. CONCLUSIONS: This study suggests that PC4 might serve as a negative regulator of skin wound healing in mice.

Metformin mitigates gastrointestinal radiotoxicity and radiosensitises P53 mutation colorectal tumours via optimising autophagy.

BACKGROUND AND PURPOSE: There is an urgent but unmet need for mitigating radiation-induced intestinal toxicity while radio sensitising tumours for abdominal radiotherapy. We aimed to investigate the effects of metformin on radiation-induced intestinal toxicity and radiosensitivity of colorectal tumours. EXPERIMENTAL APPROACH: Acute and chronic histological injuries of the intestine from mice were used to assess radioprotection and IEC-6 cell line was used to investigate the mechanisms in vitro. The fractionated abdominal radiation model of HCT116 and HT29 tumour grafts was used to determine the effects on colorectal cancer. KEY RESULTS: Metformin alleviated radiation-induced acute and chronic intestinal toxicity by optimising mitophagy which was AMPK-dependent. In addition, our data indicated that metformin increased the radiosensitivity of colorectal tumours with P53 mutation both in vitro and in vivo. CONCLUSION AND IMPLICATIONS: Metformin may be a radiotherapy adjuvant agent for colorectal cancers especially those carrying P53 mutation. Our findings provide a new strategy for further precise clinical trials for metformin on radiotherapy.

Cordycepin prevents radiation ulcer by inhibiting cell senescence via NRF2 and AMPK in rodents.

The pathological mechanisms of radiation ulcer remain unsolved and there is currently no effective medicine. Here, we demonstrate that persistent DNA damage foci and cell senescence are involved in radiation ulcer development. Further more, we identify cordycepin, a natural nucleoside analogue, as a potent drug to block radiation ulcer (skin, intestine, tongue) in rats/mice by preventing cell senescence through the increase of NRF2 nuclear expression (the assay used is mainly on skin). Finally, cordycepin is also revealed to activate AMPK by binding with the α1 and γ1 subunit near the autoinhibitory domain of AMPK, then promotes p62-dependent autophagic degradation of Keap1, to induce NRF2 dissociate from Keap1 and translocate to the nucleus. Taken together, our findings identify cordycepin prevents radiation ulcer by inhibiting cell senescence via NRF2 and AMPK in rodents, and activation of AMPK or NRF2 may thus represent therapeutic targets for preventing cell senescence and radiation ulcer.

Transcriptional positive cofactor 4 promotes breast cancer proliferation and metastasis through c-Myc mediated Warburg effect.

BACKGROUND: The human positive cofactor 4 (PC4) is initially identified as a transcriptional cofactor and has an important role in embryonic development and malignant transformation. However, the clinical significance and the molecular mechanisms of PC4 in breast cancer development and progression are still unknown. METHODS: We investigated PC4 expression in 114 cases of primary breast cancer and matched normal breast tissue specimens, and studied the impact of PC4 expression as well as the molecular mechanisms of this altered expression on breast cancer growth and metastasis both in vitro and in vivo. RESULTS: PC4 was significantly upregulated in breast cancer and high PC4 expression was positively correlated with metastasis and poor prognosis of patients. Gene set enrichment analysis (GSEA) demonstrated that the gene sets of cell proliferation and Epithelial-Mesenchymal Transition (EMT) were positively correlated with elevated PC4 expression. Consistently, loss of PC4 markedly inhibited the growth and metastasis of breast cancer both in vitro and in vivo. Mechanistically, PC4 exerted its oncogenic functions by directly binding to c-Myc promoters and inducing Warburg effect. CONCLUSIONS: Our study reveals for the first time that PC4 promotes breast cancer progression by directly regulating c-Myc transcription to promote Warburg effect, implying a novel therapeutic target for breast cancer.

Epigenetic knockdown of Notch1 inhibits hepatitis B virus X protein­induced hepatocarcinogenesis of L02/HBx cells.

The present study aimed to investigate the effects of Notch1 on the development of hepatitis B virus X protein (HBx)­induced hepatocarcinogenesis. The L02/HBx cells were transfected with a short hairpin RNA (shRNA) specially targeting Notch1 (Notch1­shRNA). The mRNA and protein expression levels of Notch1 signaling pathway­related molecules (Notch1, Hes1 and NICD) were detected after knockdown of Notch1. The effects of Notch1 knockdown on the proliferation was analyzed by Cell Counting Kit­8 assay, and cell cycle and apoptosis of L02/HBx cells in vitro were investigated by flow cytometry. The in vivo tumor xenograft model was established by subcutaneously injection of mice with Notch1­shRNA or sh­NC transfected cells. The effects of Notch1 knockdown on tumor progression in vivo were then explored by H&E staining and immunohistochemistry. The results showed that knockdown of Notch1 inhibited the activation of the Notch1 signaling pathway. In addition, decreased viability and colony formation ability of L02/HBx cells were detected along with downregulated protein expression levels of Ki­67 and PCNA (proliferating cell nuclear antigen). In addition, knockdown of Notch1 led to L02/HBx cell cycle arrest at G0/G1 phase by decreasing the expression of cyclin D1, CDK4, E2F1 and increasing the expression of p21 and retinoblastoma gene (Rb). Moreover, knockdown of Notch1 promoted the apoptosis of L02/HBx cells by activation of caspase­3 and caspase­9. In vivo experiments demonstrated that knockdown of Notch1 inhibited the tumorigenicity of L02/HBx cells. Our findings revealed that inhibition of the Notch1 signaling pathway may inhibit the development of HBx­induced hepatocellular carcinoma. Notch1 may serve as a promising therapeutic target for HCC.

An NIR-Fluorophore-Based Therapeutic Endoplasmic Reticulum Stress Inducer.

The endoplasmic reticulum (ER) stress signaling or unfolded protein response (UPR) is a common feature of many human diseases, including cancer. Excessive activation of ER stress directly induces cell death, holding a new promising strategy for the therapeutic intervention of cancer. Current ER-stress-inducing agents mainly target UPR components or proteasomes, which exert limited treatment efficacy and undesired side effects due to unselective ER stress and poor tumor-specific distribution. In this study, a unique near-infrared (NIR) fluorophore, IR-34, is synthesized and identified to selectively and efficiently trigger tumoricidal ER stress by targeting the mitochondrial protein NDUFS1. IR-34 is demonstrated to specifically accumulate in living cancer cells for tumor NIR imaging and drastically inhibit tumor growth and recurrence without causing apparent toxicity. Thus, this multifunctional NIR fluorophore may represent a novel theranostic agent for tumor imaging-guided treatment and also strengthens the idea that mitochondria could be a useful target for therapeutic ER stress in cancer cells.

P53 and Murine Double Mimute 2 (MDM2) Expression Changes and Significance in Different Types of Endometrial Lesions.

BACKGROUND Endometrial lesions are common in obstetrics and gynecology, including endometrial polyps, uterine adenomyosis, and malignant endometrial adenocarcinoma. Endometrial lesions seriously affect women's health, fertility, quality of life, and life safety. As a pro-apoptosis gene, p53 is considered to be closely related with human tumors. Murine double mimute 2 (MDM2) is an oncogene that can promote tumor occurrence and development. P53 and MDM2 expression and significance in different types of endometrial lesions have not been fully elucidated. MATERIAL AND METHODS Normal endometrium, endometrial polyps, uterine adenomyosis, and endometrial adenocarcinoma tissue samples were collected. Real-time PCR was used to detect p53 and MDM2 mRNA expression. Immunohistochemical staining and Western blot analysis were applied to test p53 and MDM2 protein expression. Their correlation with clinical staging of endometrial adenocarcinoma was analyzed. RESULTS P53 and MDM2 mRNA and protein expression were significantly elevated in the endometrial polyps group and the endometrial adenocarcinoma group compared with the normal control group (P<0.05). Their levels increased more obviously in endometrial adenocarcinoma compared with endometrial polyps (P<0.05). P53 and MDM2 mRNA and protein expression were slightly enhanced in uterine adenomyosis compared with normal controls, but this difference lacked statistical significance (P>0.05). P53 and MDM2 mRNA and protein level showed a positive correlation. Significantly higher expression of p53 or MDM2 was observed in patients with stage III compared to those in patients with stage II. Higher expression was also observed in patients with stage II than in patients with stage I. CONCLUSIONS P53 and MDM2 mRNA and protein were elevated in endometrial polyps and endometrial adenocarcinoma and their expressions were correlated with clinical staging of endometrial adenocarcinoma. They can promote cancer occurrence and development, and can be treated to assist diagnosis and provide a reference for treatment.

Allyl Isothiocyanate Inhibits the Proliferation of Renal Carcinoma Cell Line GRC-1 by Inducing an Imbalance Between Bcl2 and Bax.

BACKGROUND Because of the insensitivity of renal cell carcinoma (RCC) to both chemotherapy and radiotherapy, surgery remains the primary approach for anticancer treatment. However, patients who do not receive timely diagnoses may not be suitable for surgery, especially in the late phase of tumor development. Thus, the discovery of novel effective treatment is of great importance. Allyl isothiocyanate (AITC) can inhibit the proliferation and induce apoptosis in many cancer cells. In this paper, we report on an in vitro study to determine the effect of AITC on proliferation and apoptosis of RCC line GRC-1. MATERIAL AND METHODS CCK8 assay was used to detect cell proliferation under gradient concentrations of AITC. Flow cytometry was employed to evaluate cell apoptosis. Real-time fluorescent polymerase chain reaction quantified mRNA levels of Bax and Bcl-2 genes. Western blotting was further employed for protein expression assay. RESULTS AITC inhibited GRC-1 cell proliferation and induced cell apoptosis in a dose-dependent manner; it also elevated Bax while suppressing Bcl-2 gene expression at both mRNA and protein levels. In general, increasing concentration of AITC decreased Bcl-2/Bax ratio. CONCLUSIONS The inhibitory effect of AITC on GRC-1 cells is exerted via cell apoptosis, in which the imbalance of Bcl-2/Bax plays a significant role.

The Effects of CYP2C19 genotype on the susceptibility for nephrosis in cardio-cerebral vascular disease treated by anticoagulation.

In recent years, the genetic factor has become one of the important predisposing factors of nephropathy susceptibility. There is a high incidence of nephropathy in CCVd. The CYP2C19 enzyme metabolizes most the drugs, including proton pump inhibitors commonly used medicines to treat CCVd, CYP2C19 genetic polymorphisms is association with multi-pathogenesis factors of nephropathy. The purpose of the study is to reveal the association between CYP2C19 genotype and the susceptibility of nephropathy in the CCVd patients. The study is composed of 623 samples from CCVd treated by anticoagulation. The patients were studied, including CCVd with hyperuricemia, coronary heart disease, diabetes, and other complication. Biochemical tests and CYP2C19 variants measurements were performed by the gene chip method. The association among CYP2C19 variants, complications, and nephropathy was analyzed in the CCVd. There is no correlation between nephropathy and complications in CCVd. In hyperuricemia, coronary heart disease and diabetes groups, the differences of renal function tests were significant between CYP2C19 mutant (P < 0.05). The nephropathy risk of wild genotype is 3.288 times higher than of mutation genotype in hyperuricemic group, 1.928 times higher than mutation genotype in coronary heart disease group, and 5.248 times higher than CYP2C19 mutation genotype in the diabetic group. There was significant correlation between the CYP2C19 wild type and the nephropathy susceptibility in CCVd patients. The CYP2C19 gene plays a potential maker to evaluate nephropathy in CCVd patients. We deduced that identification of CYP2C19 gene type may benefit for reducing and avoiding nephropathy caused by abnormal metabolism function in CCVd patients.