Establishment of insomnia model of chronic unpredictable stress in rats.

It is well known that stressful situation is one of the important factors causing insomnia, however, the underlying mechanism is still elusive. Therefore, the establishment of a suitable animal model of stress insomnia will be of great help to solve this problem. In this study, by combining with chronic unpredictable stress (multitude of stressors) and sleep deprivation, we attempted to establish a rat model of stress insomnia. It was observed that rats with stress insomnia showed significant weight loss, and less sleep quality in pentobarbital sodium induced sleep test and electroencephalogram detection. Moreover, rats with stress insomnia showed greater depression and anxiety detected by forced swimming, sucrose preference test and open field. Since oxidative stress has been reported to be involved in insomnia, we further evaluated the production of oxidative stress and found that the levels of lipid peroxidation product malondialdehyde (MDA) in liver, serum total bilirubin and urine biopyrrin were all significantly increased in rats with stress insomnia. In addition, we also found that the memory of these rats with stress insomnia was also obviously reduced in water maze. Taken together, these results demonstrate that the emotional behaviors, memory, oxidative and metabolism of the rats were all significantly changed after modeling, indicating a rat model of stress insomnia was successful establishment, and this animal model will provide basis to further explore the underlying mechanism of chronic stress in insomnia.

Establishment of a chronic insomnia rat model of sleep fragmentation using unstable platforms surrounded by water.

Chronic fragmented sleep is a very common type of insomnia that affects the daily lives of numerous people around the world. However, its pathogenesis is not very clear and a corresponding rat model has not been reported for this purpose at present. The present study aimed to establish a rat model of chronic insomnia with sleep fragmentation using self-made multiple strings of unstable platforms surrounded by shallow water. During the establishment of the models, changes in body weight and differences in food and water intake in the daytime and at night were acquired. The rat models were assessed using several tests, including the Morris water maze test, pentobarbital sodium-induced sleep, infrared monitoring and electroencephalogram/electromyography during sleep. The expression levels of certain inflammatory factors and orexin A were detected in the serum and brain tissues using ELISAs, immunohistochemistry and immunofluorescence. The expression levels of orexin 1 receptor (orexin 1r) were also detected in the brain. Polysomnography indicated that the model rats were successfully prepared with reduced non-rapid eye movement (non-REM) sleep in the daytime, which was increased at night, and considerably lower REM duration during the day and night. The number of instances of sleep arousals were also increased in the day and at night, and the average duration of each sleep bout was decreased in the daytime. The body weights of the model rats increased at a normal rate. However, the reduction of body weight in the daytime and increased in body weight at night were significantly less than those of the control rats. The daytime food and water consumption of the model rats increased significantly compared with that of the control rats, but was similar to that of the control group at night. The Morris water maze test indicated that the model rats were slow to learn to escape the platforms and performed a lower number of target crossings. The pentobarbital-induced sleep experiment confirmed that the model rats exhibited a longer sleep latency and shorter sleep time. The serum IL-1ß, IL-6, TNF-α and orexin A levels of the model rats were significantly increased, whereas their serum IL-10 levels were significantly decreased compared with those of the control rats. The expression levels of IL-1ß, IL-6, orexin A and orexin 1r in the brain tissues of the model rats were also significantly increased. In conclusion, these data indicate that learning and memory function, sleep time, arousal times, diurnal and nocturnal body weight changes, food and water intake, and expression levels of the specific inflammatory factors orexin A and orexin 1r were altered in the model rats. This suggests the chronic insomnia rat model with sleep fragmentation was successfully established using multiple strings of unstable platforms surrounded by water.

Kidney Mesenchymal stem cells alleviate cisplatin-induced kidney injury and apoptosis in rats.

OBJECTIVE: This experiment was designed to demonstrate Mesenchymal stem cells (MSCs) derived from kidney can alleviate cisplatin-induced kidney injury and renal cell apoptosis through paracrine pathway. METHODS: Firstly, MSCs were isolated from kidney of young rats, and their surface-specific markers were identified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunofluorescence staining. Self-renewal ability of Kidney Mesenchymal Stem Cells (KMSCs) was observed by cell counting and 5-Bromo-2'-deoxyuridine (BrdU) fluorescence staining. KMSCs at logarithmic growth stage were traced and injected into rat through tail vein. RESULTS: The results showed that KMSCs homed in the kidney tissues, decreased the secretion of inflammatory factors (CRP, TNFα, IL-1ß, IL-6), and alleviated renal function. Hematoxylin and Eosin (H＆E), Masson and Periodic Acid-silver Methenamine (PASM) staining showed that KMSCs could alleviate pathological damage in rats. Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) assay showed that KMSCs could reduce the apoptosis of rat kidney cells induced by cisplatin. Finally, Immunohistochemistry (IHC) results showed that cisplatin could induce higher expression of the pro-apoptotic protein Bax and lower expression of anti-apoptotic Bcl-2 in kidney tissues. However, KMSCs could reverse the pro-apoptotic effect of cisplatin on kidney cells and improve the survival rate of rats. CONCLUSIONS: In conclusion, KMSCs were successfully isolated from kidney tissues, and KMSCs have therapeutic effects on rat kidney injury induced by cisplatin.

B serum proteome profiles revealed dysregulated proteins and mechanisms associated with insomnia patients: A preliminary study.

Background: Insomnia is a clinical problem of significant public health importance; however, the underlying pathogenesis of this disorder is not comprehensively understood. Methods: To identify potential treatment targets and unfold one of the gaps that were involved in insomnia pathological mechanisms, we employed a tandem mass tag-based (TMT) quantitative proteomics technology to detect differentially expressed proteins (DEPs) in serum from patients with insomnia and controls. DEPs were further analyzed by bioinformatics platforms. In addition, parallel reaction monitoring (PRM) was used to verify the TMT results. Results: Patients with insomnia had poorer sleep quality compared with healthy controls. A total of 106 DEPs were identified among patients with insomnia and controls. They were mainly enriched in immune and inflammation-related biological functions and signaling pathways. Using the protein-protein interaction network, we screened the 10 most connected proteins as key DEPs. We predicted that four key DEPs were subject to targeted regulation by natural compounds of herbs. Eight key DEPs were validated using PRM in an additional 15 patients with insomnia and 15 controls, and the results also supported the experimental findings. Conclusion: We identified aberrantly expressed proteins in insomnia that may be involved in the immune-inflammatory response. The 10 key DEPs screened may be potential targets for insomnia, especially FN1, EGF, HP, and IGF1. The results of this study will broaden our understanding of the pathological mechanisms of insomnia and provide more possibilities for pharmacotherapy.

Proteomics Reveals Molecular Changes in Insomnia Patients with More Dreams.

Background: Insomnia is a sleep disorder and the cause of many healthy problems. However, there are few studies on patients with insomnia and dreaminess at present. Therefore, this study is aimed at exploring the pathological molecular mechanisms and potential diagnostic and therapeutic targets related to insomnia patients with more dreams. Methods: Sleep characteristics of 36 primary insomnia patients with more dreams and 36 well sleeping participants were assessed using polysomnography (PSG) and Pittsburgh Sleep Quality Index (PSQI). Serum samples from 9 insomnia patients and 9 controls were randomly selected for proteomic detection. Differentially expressed proteins (DEPs) between the two groups were identified; enrichment analysis and PPI network were performed. The top 10 most connected proteins in the PPI network were subjected to targeted drug prediction and screened key proteins. Proteins with targeted drugs were recognized as key proteins and subjected to ELISA detection. Results: Insomnia patients had a distinct REM behavior disorder signature compared with controls. Proteomic sequencing identified 76 DEPs. Enrichment analysis found that DEPs were significantly enriched in the complement and coagulation cascades. Metabolic responses were also activated in insomnia patients. Among the hub proteins screened in the PPI network, APOA1, APOB, F2, and SPARC may be targeted by many herbal medicines and considered as key proteins. ELISA assays validated their differential expression between insomnia and controls. Conclusion: In this study, we identified the potential key proteins of insomnia patients with more dreams. The pathological process may associate with inflammation and metabolic response. These results provide molecular targets for diagnostic and therapeutic targets. The results of our analysis suggest that the expression changes of key proteins have a good predictive diagnostic role for the occurrence of insomnia with more dreams in patients.

Pre-clinical study of human umbilical cord mesenchymal stem cell transplantation for the treatment of traumatic brain injury: safety evaluation from immunogenic and oncogenic perspectives.

Stem cell therapy is a promising strategy for the treatment of traumatic brain injury (TBI). However, animal experiments are needed to evaluate safety; in particular, to examine the immunogenicity and tumorigenicity of human umbilical cord mesenchymal stem cells (huMSCs) before clinical application. In this study, huMSCs were harvested from human amniotic membrane and umbilical cord vascular tissue. A rat model of TBI was established using the controlled cortical impact method. Starting from the third day after injury, the rats were injected with 10 µL of 5 × 106/mL huMSCs by cerebral stereotaxis or with 500 µL of 1 × 106/mL huMSCs via the tail vein for 3 successive days. huMSC transplantation decreased the serum levels of proinflammatory cytokines in rats with TBI and increased the serum levels of anti-inflammatory cytokines, thereby exhibiting good immunoregulatory function. The transplanted huMSCs were distributed in the liver, lung and brain injury sites. No abnormal proliferation or tumorigenesis was found in these organs up to 12 months after transplantation. The transplanted huMSCs negligibly proliferated in vivo, and apoptosis was gradually observed at later stages. These findings suggest that huMSC transplantation for the treatment of traumatic brain injury displays good safety. In addition, huMSCs exhibit good immunoregulatory function, which can help prevent and reduce secondary brain injury caused by the rapid release of inflammatory factors after TBI. This study was approved by the Ethics Committee of Wuhan General Hospital of PLA (approval No. 20160054) on November 1, 2016.

Proteomic Profiling Reveals the Molecular Changes of Insomnia Patients.

BACKGROUND: Insomnia is an economic burden and public health problem. This study is aimed at exploring potential biological pathways and protein networks for insomnia characterized by wakefulness after sleep. METHOD: Proteomics analysis was performed in the insomnia group with wakefulness and the control group. The differentially expressed proteins (DEPs) were enriched; then, hub proteins were identified by protein-protein interaction (PPI) network and verified by parallel reaction monitoring (PRM). RESULTS: Compared with the control group, the sleep time and efficiency of insomnia patients were decreased, and awakening time and numbers after sleep onset were significantly increased (P < 0.001). The results of proteomic sequencing found 68 DEPs in serum under 1.2-fold changed standard. These DEPs were significantly enriched in humoral immune response, complement and coagulation cascades, and cholesterol metabolism. Through the PPI network, we identified 10 proteins with the highest connectivity as hub proteins. Among them, the differential expression of 9 proteins was verified by PRM. CONCLUSION: We identified the hub proteins and molecular mechanisms of insomnia patients characterized by wakefulness after sleep. It provided potential molecular targets for the clinical diagnosis and treatment of these patients and indicated that the immune and metabolic systems may be closely related to insomnia characterized by wakefulness after sleep.

A Longitudinal, Prospective Study to Evaluate the Effects of Treatment on the Inhibitory Control Function After Transsphenoidal Surgery for Pituitary Adenomas.

OBJECTIVES: Injured cognitive abilities have been reported in patients with pituitary adenoma. However, to date, few researchers have directly investigated the electrophysiological study of inhibitory control function of pituitary patients both pre- and postsurgery. Thus, this study aimed to identify the factors affecting the inhibitory control function of pituitary patients. METHODS: Thirty presurgery pituitary patients were recruited and 26 patients of them completed the postsurgery follow-up. Thirty healthy people were recruited for control group. Visual Go/Nogo tasks were carried out by the patients and controls to assess the inhibitory control function before surgery and 6 months after the surgery, respectively. The function of inhibitory control was analyzed with the components of N2 and P3. RESULTS: Across 3 groups, Nogo stimuli evoked larger frontal-central N2nogo and P3nogo than Go stimuli did. Furthermore, N2d of presurgery patients (-1.14 µV) and postsurgery patients(-0.61 µV) were significantly decreased compared with that of control group (-3.09 µV), F(2, 83) = 13.92, P < .01, whereas no difference was detected between pre- and postsurgery groups. There was no remarkable difference in the amplitude of P3d among the 3 groups, F(2, 83) = 0.19, P > .05. With regard to the amplitude of P3 for Go condition, The P3 amplitude of healthy group (4.38 µV) was larger than both pre- and postsurgery (1.00 µV and 3.01 µV). With regard to the amplitude of P3 for Nogo condition, The P3 amplitude of healthy group (5.25 µV) was larger than both pre- and postsurgery groups (2.35 µV and 4.18 µV). CONCLUSIONS: These results indicated that presurgery patients showed the dysfunction of inhibition, due to the nerve tissue damage or brain structure alteration caused by the presurgery physical pressure from tumor and abnormal hormone levels. Postsurgery patients showed a tendency toward recovery, but there was no obvious improvement in the inhibitory control function after successful treatments.

Response Activation and Inhibition in Patients With Prolactinomas: An Electrophysiological Study.

Impairment of executive function has been reported in patients with prolactinomas. However, few studies have investigated the electrophysiological mechanisms of response activation and response inhibition in these patients. In this study, we employ an event-related potentials (ERPs) technique to quantitatively assess response activation and inhibition before and after the surgical treatment of prolactinomas. A 64-electrode electroencephalogram (EEG) skullcap was used to record the brain activity in 20 pre-operative patients, 20 follow-up post-operative patients, and 20 healthy controls (HCs) while performing the visual Go/Nogo task. As expected, we identified P300 across all study populations that could reflect response activation and inhibition. Across the three groups, the Nogo stimuli evoked larger frontal-central P300 than the Go stimuli did. In contrast, the Go trials elicited larger parietal P300 than the Nogo trials did. The peak latency of P300 was significantly delayed in both the pre-operative and the post-operative groups compared to the HCs. The amplitude of P300 in both the Go and the Nogo conditions was significantly decreased in the pre-operative patients compared with that of the HCs. At 6 months post-operatively, the prolactinoma patients showed an increase in amplitude of P300 during both the Go and the Nogo tasks. These findings indicate that the prolactinoma patients suffer from deficits in response activation and inhibition, which could be improved by surgical treatment.

Establishment of a rat model with ageing insomnia induced by D-galactosef and para-chlorophenylalanine.

The current study aimed to establish a rat model of ageing insomnia induced by D-galactose and/or para-chlorophenylalanine. Following establishment of the model, body weights were measured, and Morris water maze and pentobarbital-induced sleep tests were performed. The serum levels of inflammatory mediators and the neural levels of neurotransmitters were detected. The results demonstrated that the body weights of PCPA+D-gal-induced ageing insomnia rats decreased significantly. Ageing insomnia rats exhibited longer latencies to the platform in the Morris water maze tests and fewer target crossings. The sleep latency of the model rats was longer and sleep time was shorter by contrast. The relative expression of hippocampal IL-6, TNF-α, NF-κB and mGluR2 mRNA of the PCPA+D-gal-induced ageing insomnia group was higher, while the relative expression of 5-HT1AR and GABAARa1 mRNA were lower. The serum levels of IL-1ß, IL-6, TNF-α and brain level of glutamate increased in the PCPA+D-gal-induced ageing insomnia group, while the levels of 5-HT and GABA decreased. In conclusion, memory function, sleep time, expression of inflammatory factors and neurotransmitters are altered in ageing insomnia rats induced by D-galactose and para-chlorophenylalanine, indicating the successful establishment of a murine model of ageing insomnia.

Tumor inhibition effects and mechanisms of Angelica sinensis and Sophorae flavescentis ait decoction combined with cisplatin in xenograft mice.

BACKGROUND: To investigate tumor inhibition effects and mechanisms of Angelica sinensis and Sophorae flavescentis ait decoction (ASSF) combined with diamine-dichloroplatinum (DDP). MATERIALS AND METHODS: Bodyweight, tumor inhibition rate and q value were calculated for single ASSF or ASSF combined with DDP on H22 carcinoma xenograft KM mice. Biochemical methods for serum LDH, AST, ALT, and AKP, ELISA method for serum HIF-1α, pathological assessemnt of thymus, immunohistochemistry detection of tumor tissue caspase3 and mutant p53 protein, and qRT-PCR detection of bax/ bcl-2 mRNA were applied. RESULTS: Compared with DDP control group, the bodyweight increased in ASSF-DDP group (p<0.01). Tumor inhibition rates for DDP, ASSF, ASSF-DDP were 62.7%. 43.7% and 71.0% respectively, with a q value of 0.90. Compared with other groups, thymus of DDP control group had obvious pathological injury (p<0.01), serum LDH, AST, ALT, AKP increased significantly in DDP control group (p<0.01), while serum HIF-1α was increased in the model control group. Compared with this latter, the expression of mutant p53 protein and bcl-2 mRNA were decreased in all treatment groups (p<0.01), but there were no statistical difference between DDP control p and ASSF-DDP groups. The expression of caspase3 protein and bax mRNA was increased in all treatment groups, with statistical differences between the DDP and ASSF-DDP groups (p<0.01). CONCLUSIONS: ASSF can inhibit bodyweight decrease caused by DDP, can inhibit tumor growth synergistically with DDP mainly through increasing serum HIF-1α and pro-apoptotic molecules such as caspase 3 and bax, rather than through decreasing anti-apoptotic mutant p53 and bcl-2. ASSF can reduce DDP toxicity due to decreasing the release of LDH, AST, ALT, AKP into blood and enhancing thymus protection.