1. Effect of G-CSF and TPO on HIBD in neonatal rats
Xue-Mei LIU ; Ai-Min LI ; Yi FENG
Asian Pacific Journal of Tropical Medicine 2015;8(2):132-136
Objective: To observe effect of granulocyte colony-stimulating factor (G-CSF) and restructure human thrombopoietin on hypoxic-ischemic brain damage (HIBD) in new born rats. Methods: A total of 60 neonatal SD rats were selected and divided into 4 groups, with 15 in each group. Group A served as control group. Rats of Groups B-D were prepared for HIBD model by ligation of left common carotid artery combined with hypoxia method. Rats of Group A were only completed with free left common carotid artery without ligation and hypoxia operation. After HIBD model preparation, Group B was administrated with subcutaneous injection of normal saline for placebo treatment; Group C was administrated with cervical subcutaneous injection of 0.5 μg/10 g granulocyte colony stimulating factor (G-CSF) for 5 d (Once a day); Group D was administrated with intraperitoneal injection of 15 U/10 g recombinant human thromobopoietin (rhTPO) for treatment. After modeling for 7, 14 and 21 d, 5 rats were sacrificed in each group, respectively. Brain quality damage (%) conditions of experimental animals in each group were compared in different time points, and cerebral histopathological changes of each group were observed. Expression of nestin in rats of each group was detected by immunohistochemical method. Results: After modeling for 7, 14 and 21 d, brain quality damages (%) of Groups B, C and D were significant higher than that of in Group A (. P<0.05), while brain quality damage (%) degree of Group B was the highest in different time points, followed by Groups D and C, respectively. It was significant different compared among groups (. P<0.05). The histopathological observation showed that degrees of brain damages in Groups C and D were significant lower than that of in Group B. After modeling for 7, 14 and 21 d, nestin positive cell populations in Groups B, C, and D were significant higher than Group A (. P<0.05). Nestin cell populations of Group C in different time points were significant higher than Groups B and D (. P<0.05). There was no significant difference in nestin positive cell populations in the above time points between Groups B and D (. P>0.05). Conclusions: Both G-CSF and TPO can protect the nervous system of HIBD neonatal rats. G-CSF can promote the proliferation and differentiation of neural precursor cells to decrease the degeneration and necrosis of nerve cell. TPO can obviously ameliorate morphology index of HIBD rats. Through regulating ratio of TIMP-1 and MMP-9, TPO can maintain the integrity of blood brain barrier to relieve the occurrence of brain damage.
2.Validation of the Thyrotoxicosis-associated Insomnia Model Induced by Thyroxine through Sympathetic Stimulation: Face, Construct and Predictive Perspectives
Zhifu AI ; Hongwei HE ; Tingting WANG ; Liling CHEN ; Chunhua HUANG ; Changlian CHEN ; Pengfei XU ; Genhua ZHU ; Ming YANG ; Yonggui SONG ; Dan SU
Experimental Neurobiology 2021;30(6):387-400
Insomnia has become a common central nervous system disease. At present, the pathogenesis of insomnia is not clear. Animal models can help us understand the pathogenesis of the disease and can be used in transformational medicine. Therefore, it is very necessary to establish an appropriate model of insomnia. Clinical data show that insomnia patients with high levels of thyroxine and often accompanied by cardiovascular problems, a common mechanism underlying all of these physiological disruptions is the sympathetic nervous system. Combined with the characteristics of chronic onset of clinical insomnia, an insomnia model induced by long-term intraperitoneal injection of thyroid hormone has been created in our laboratory. In this paper, the insomnia-like state of the model was evaluated based on three validity criteria. Face validity has been demonstrated in metabolism, the Morris water maze, electrocardiogram (ECG) and electroencephalogram (EEG). Structure validity has been proved by the results of targeted metabolomics. After treatment with diazepam, a commonly used clinical anti-insomnia drug, the above physiological and pathological disorders were reversed. The results of comprehensive analysis show that the established thyrotoxicosis-associated insomnia model meets the validity requirement to establish an appropriate animal model of insomnia. The model presented in this article might help to study pathogenetic mechanisms of clinical insomnia, as well as to test promising methods of insomnia treatment.
3.Full spectrum analysis of chemical constituents of Sargassum fusiforme and its in vitro anti-neuroinflammatory activity
Hong PENG ; Pinzhe HUANG ; Yonggui SONG ; Huanhua XU ; Mingyue ZHOU ; Genhua ZHU ; Ming YANG ; Zhifu AI ; Dan SU
China Pharmacy 2022;33(7):800-807
OBJECTIVE To study the composition of chemical constituents of Sargassum fusiforme and its in vitro anti- neuroinflammatory activity ,and to provide reference for its development and utilization and the study of pharmacodynamic substances. METHODS UHPLC-QTOF-MS/MS analysis method and GC-MS/MS method were used to analyze the chemical constituents of S. fusiforme . The lipopolysaccharide (1 μg/mL)was adopted to establish the inflammatory model of neuromicroglia BV2. Using paroxetine (5 μg/mL)as positive control ,CCK-8 assay was used to detect the effects of the extracts of S. fusiforme (20,40,60,80,100 μg/mL)on the activity and morphology of neuromicroglia BV 2. The effects of the extracts of S. fusiforme (40,60,80 μg/mL)on the contents of tumor necrosis factor α(TNF-α)and interleukin- 6(IL-6)in cell supernatant were detected by ELISA. RESULTS A total of 103 non-volatile constituents were identified by UHPLC-QTOF-MS/MS ,and 60 volatile constituents were obtained by GC-MS/MS. The extracts of S. fusiforme (40,60,80 μ g/mL) could significantly reduce the abnormally increased activation of neuromicroglia BV 2 and the contents of TNF-α and IL-6 due to lipopolysaccharide (P<0.05 or P<0.01). CONCLUSIONS The study establish the full spectrum of chemical constituents of S. fusiforme ,and it is confirmed that fusiforme has certain in vitro anti-neuroinflammatory activity.
4.Effects of raw and wine-processed Schisandra chinensis on neuro-endocrine-immune network in insomnia mice and its mechanism
Ruiying WANG ; Dan SU ; Huizhen LI ; Yali LIU ; Genhua ZHU ; Ming YANG ; Zhifu AI ; Tao LUO ; Bing XUE ; Yonggui SONG
China Pharmacy 2023;34(5):525-530
OBJECTIVE To investigate the effect of raw and wine-processed Schisandra chinensis on neuro-immune-endocrine network in insomnia mice and its mechanism. METHODS Fifty mice were randomly divided into blank group, model group, diazepam group, raw S. chinensis group and wine-processed S. chinensis group, with 10 mice in each group. Except for blank group, the mice in the other groups were intraperitoneally injected with thyroxine solution to establish mice model of insomnia; at the end of each day’s modeling, the corresponding doses of diazepam,raw and wine-processed S. chinensis were given by gavage. The blank group and model group were given constant volume of normal saline. The general state of the mice was observed and recorded, and the total activity distance and upright times of the mice were detected; the EEG and EMG signals of mice were recorded, and the time ratio of sleep wake time (wake), non-rapid eye movement (NREM) and rapid eye movement (REM) was analyzed; the contents of neurotransmitters [γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE), cortisol (CORT)] in brain suprachiasmatic nucleus (SCN) were detected; and the expressions of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were detected; the mRNA expressions of clock gene Bmal1, circadian clock gene Clock and cycle gene Per2 were all detected. RESULTS Compared with the blank group, the mental state of the model group mice was relatively depressed, the amount of food and water increased, the body mass decreased, the hair was rough and shiny, and the circadian rhythm was irregular; the total activity distance and upright times decreased significantly; the time ratio of wake increased significantly, while the time ratios of REM and NREM decreased significantly; the content of 5- HT in brain SCN decreased significantly, while the content of NE, DA and CORT increased significantly; the fluorescence intensity of IL-1β and TNF-α was significantly increased; the relative expression level of Bmal1 and Clock mRNA was significantly increased, while the relative expression level of Per2 mRNA was significantly decreased (P<0.05 or P<0.01). Compared with the model group, the general state of mice in diazepam group, raw S. chinensis group and wine-processed S. chinensis group was improved obviously, and most of the above index levels were significantly reversed (P<0.05 or P<0.01). CONCLUSIONS Raw and wine-processed S. chinensis have a certain therapeutic effect on insomnia mice, the mechanism of which may be related to the regulation of neuro-endocrine-immune system related biological indicators in insomnia mice.
5.Compatibility Advantage of Scutellariae Radix-Coptidis Rhizoma for Anti-neuroinflammation and Its Potential Targets for Regulating TLR4/MyD88/NF-κB Signaling Pathway
Hongjie ZHANG ; Dan SU ; Genhua ZHU ; Yonggui SONG ; Bugao ZHOU ; Shanshan LI ; Changhua ZHANG ; Zhifu AI
Chinese Journal of Experimental Traditional Medical Formulae 2022;28(22):58-67
ObjectiveTo explore the compatibility advantage of Scutellariae Radix-Coptidis Rhizoma in the prevention and treatment of neuroinflammation, and to elucidate the action characteristics and mechanism of the compatibility advantage based on Toll like receptor (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappaB (NF-κB) pathway. MethodRepresentative mouse microglia cells (BV2) in vitro were selected and divided into 8 groups: control group, model group, Scutellariae Radix-Coptidis Rhizoma group, Piracetam group, Scutellariae Radix group and Coptidis Rhizoma group. The BV2 cell inflammatory model was established by lipopolysaccharide (LPS), and the cell activity was detected by cell counting kit-8 (CCK-8). Cell morphology was observed under bright field. The production and release of pro-inflammatory factors in BV2 cells were determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay, and the mRNA expressions of TLR4, MyD88 and NF-κB were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). The nuclear translocation of NF-κB p65 was detected by immunofluorescence, and TLR4 signal transduction inhibitor (CLI-095) and NF-κB inhibitor (PDTC) were used to confirm the anti-neuroinflammation targets of Scutellariae Radix-Coptidis Rhizoma. ResultCompared with the conditions in the control group, most cells in LPS-induced model group were activated, and the contents of IL-6, TNF-α and IL-1β in culture medium and cells and the mRNA expressions of TLR4, MyD88, NF-κB p50 and NF-κB p65 were increased (P<0.01), with obvious nuclear entry of NF-κB p65. Compared with the conditions in the model group, BV2 cell morphology was mostly recovered after pretreatment in Scutellariae Radix-Coptidis Rhizoma and Piracetam groups, and the levels of IL-6, TNF-α and IL-1β and the mRNA expressions of TLR4, MyD88, NF-κB p50 and NF-κB p65 were decreased (P<0.05, P<0.01), with NF-κB p65 mostly observed in cytoplasm. Compared with the conditions in the model group, cell morphology was slightly recovered in Scutellariae Radix group and Coptidis Rhizoma group, and the levels of pro-inflammatory factors and mRNA expressions of TLR4, MyD88, NF-κB p50 and NF-κB p65 were reduced. In terms of inhibitory effect on pro-inflammatory factors, Scutellariae Radix group and Coptidis Rhizoma group were lower than Scutellariae Radix-Coptidis Rhizoma group (P<0.05). Compared with the model group, the "Scutellariae Radix-Coptidis Rhizoma+CLI-095" group and "Scutellariae Radix-Coptidis Rhizoma+PDTC" group had lowered mRNA expressions of TLR4, MyD88, NF-κB p50 and NF-κB p65 (P<0.05, P<0.01), and the transfer of NF-κB p65 into nucleus was obviously inhibited. ConclusionThe anti-neuroinflammation effect of Scutellariae Radix-Coptidis Rhizoma was significantly better than Scutellariae Radix or Coptidis Rhizom alone, and the anti-neuroinflammation advantage was closely related to the inhibition of activation of TLR4/MyD88/NF-κB signaling pathway in microglial cells. It was confirmed that TLR4, MyD88 and NF-κB were potential targets for Scutellariae Radix-Coptidis Rhizoma to exert the compatibility advantage.