GFAP Protein Expression Research Articles

Tirzepatide shows neuroprotective effects via regulating brain glucose metabolism in APP/PS1 mice

Tirzepatide (LY3298176), a GLP-1 and GIP receptor agonist, is fatty-acid-modified and 39-amino acid linear peptide, which ameliorates learning and memory impairment in diabetic rats. However, the specific molecular mechanism remains unknown. In the present study, we investigated the role of tirzepatide in the neuroprotective effects in Alzheimer's disease (AD) model mice. Tirzepatide was administrated intraperitoneal (i.p.) APP/PS1 mice for 8 weeks with at 10 nmol/kg once-weekly, it significantly decreased the levels of GLP-1R, and GFAP protein expression and amyloid plaques in the cortex, it also lowered neuronal apoptosis induced by amyloid-β (Aβ), but did not affect the anxiety and cognitive function in APP/PS1 mice. Moreover, tirzepatide reduced the blood glucose levels and increased the mRNA expression of GLP-1R, SACF1, ATF4, Glu2A, and Glu2B in the hypothalamus of APP/PS1 mice. Tirzepatide increased the mRNA expression of glucose transporter 1, hexokinase, glucose-6-phosphate dehydrogenase, and phosphofructokinase in the cortex. Lastly, tirzepatide improved the energetic metabolism by regulated reactive oxygen species production and mitochondrial membrane potential caused by Aβ, thereby decreasing mitochondrial function and ATP levels in astrocytes through GLP-1R. These results provide valuable insights into the mechanism of brain glucose metabolism and mitochondrial function of tirzepatide, presenting potential strategies for AD treatment.

Heparin-Binding Epidermal-like Growth Factor (HB-EGF) Reduces Cell Death in an Organoid Model of Retinal Damage

In zebrafish and various mammalian species, HB-EGF has been shown to promote Müller glia proliferation and activation of repair mechanisms that have not been fully investigated in human retina. In the current study, 70- to 90-day-old human retinal organoids were treated with 20 μM 4-hydroxytamoxifen (4-OHT), and CRX, REC, NRL, PAX6, VIM, GFAP, and VSX2 gene and protein expression were assessed at various times points after treatment. Organoids with or without 4-OHT-induced damage were then cultured with HB-EGF for 7 days. We showed that 20 μM 4-OHT caused a reduction in the number of recoverin-positive cells; an increase in the number of TUNEL-positive cells; and downregulation of the photoreceptor gene markers CRX, NRL, and REC. Culture of organoids with HB-EGF for 7 days after 4-OHT-induced damage caused a marked reduction in the number of TUNEL-positive cells and small increases in the number of Ki67-positive cells and PAX6 and NOTCH1 gene expression. The current results suggest that treatment of human ESC-derived retinal organoids with 4-OHT may be used as a model of retinal degeneration in vitro. Furthermore, HB-EGF treatment of human retinal organoids increases proliferating Müller cells, but only after 4-OHT induced damage, and may be an indication of Muller reactivity in response to photoreceptor damage. Further studies will aim to identify factors that may induce Müller cell-mediated regeneration of the human retina, aiding in the development of therapies for retinal degeneration.

MiR-503-5p alleviates peripheral neuropathy-induced neuropathic pain in T2DM mice by regulating SEPT9 to inhibit astrocyte activation

Diabetic peripheral neuropathy (DPN) is a common complication of type 2 diabetes mellitus (T2DM) that causes peripheral and autonomic nervous system dysfunction. Dysregulation of miRNAs plays a crucial role in DPN development. However, the role of miR-503-5p in DPN remains unknown. Herein, T2DM mice (db/db) were used as a DPN model in vivo, and astrocytes isolated from db/db mice were induced with high glucose levels as a DPN model in vitro. MiR-503-5p expression was analyzed using qRT-PCR. GFAP, MCP-1, and SEPT9 protein levels were analyzed using western blotting and immunofluorescence. Luciferase assays were performed to investigate the interaction between miR-503-5p and SEPT9. We found that miR-503-5p expression decreased in the spinal cord of DPN model mice and astrocytes treated with high glucose (HG). The db/db mice displayed higher body weight and blood glucose, lower mechanical withdrawal threshold and thermal withdrawal latency, and higher GFAP and MCP-1 protein levels than db/m mice. However, tail vein injection of agomiR-503-5p remarkably reversed these parameters, whereas antigomiR-503-5p enhanced them. HG markedly facilitated GFAP and MCP-1 protein expression in astrocytes, whereas miR-503-5p mimic or inhibitor transfection markedly blocked or elevated GFAP and MCP-1 protein expression, respectively, in astrocytes with HG. SEPT9 was a target of miR-503-5p. In addition, SEPT9 protein levels were found to be elevated in db/db mice and astrocytes treated with HG. Treatment with agomiR-503-5p and miR-503-5p mimic was able to reduce SEPT9 protein levels, whereas treatment with antigomiR-503-5p and miR-503-5p inhibitor led to inhibition of the protein. Furthermore, SEPT9 overexpression suppressed the depressing effect of miR-503-5p overexpression in astrocytes subjected to HG doses. In conclusion, miR-503-5p was found to alleviate peripheral neuropathy-induced neuropathic pain in T2DM mice by regulating SEPT9 expression.

Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis.

Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone). Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment. Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.

Hydralazine alleviates noise-induced hearing loss by scavenging acrolein

Noise-induced hearing loss (NIHL) is the most common cause of hearing loss. This study investigates the therapeutic efficacy of the acrolein scavenger hydralazine for NIHL in rats. NIHL was induced by exposure to a continuous pure tone of 10 kHz. Auditory function was evaluated by auditory brainstem response (ABR) testing and scanning electron microscopy. The expression of acrolein and glial cell markers GFAP and OX42 was assessed by immunofluorescence staining. The protein and mRNA expression of GFAP, OX42, interleukin-18 (IL-18), IL-1β, and fractalkine (FTK) was measured by qRT-PCR and western blotting. A rat model of NIHL was successfully developed, as evidenced by increased ABR thresholds. The results showed that noise exposure increased the expression of acrolein, GFAP, OX42, FTK, IL-1β, and IL-18 in the rat cochlear nucleus. Furthermore, hydralazine alleviates NIHL by reversing the effects of acrolein. These results demonstrate that acrolein is involved in glial cell activation and NIHL, it’s also a therapeutic potential target for NIHL.

Effect of electroacupuncture on proliferation and differentiation of endogenous neural stem cells and Jagged1/Notch1 pathway in hippocampus of APP/PS1 model mice

To investigate the effect of electroacupuncture(EA) stimulation on proliferation and diffe-rentiation of endogenous neural stem cells as well as Jagged1/Notch1 pathway in AD model mice, so as to explore its mechanism underlying amelioration of AD. A total of 40 6-week-old male APP/PS1 transgenic AD mice were randomly divided into EA group (n=20) and AD model group ( n=20), and other 20 normal C57BL/6J mice of the same age were used as the normal control group. The mice in the EA group received EA (10 Hz, 2 mA) at "Baihui"(GV20), "Fengfu"(GV16) and bilateral "Shenshu" (BL23) for 20 min, once daily, 6 days a week for 16 weeks. The mice's learning-memory ability was detected by Morris water maze tests. The Aβ senile plaques in the hippocampal CA1 region were detected by Congo red staining, the immunofluorescence double label of BrdU, neuronal nuclear antigen (NeuN) and astrocyte specific protein GFAP in dentate gyrus of hippocampus was performed for detecting the proliferation and differentiation of the endogenous neural stem cells. The expression levels of Nestin (neuron specific protein) and GFAP were detected by Western blot, and those of Jagged1 and Notch1 mRNAs and proteins in the hippocampus were detected by real-time fluorescence quantifative PCR and Western blot. Compared with the normal control group, the escape latencies at 2nd, 3rd and 4th day, and Aβ senile plaques were significantly increased (P<0.05, P<0.01), whereas the platform crossing times and time spent in the target quadrant, the expression levels of Jagged1 mRNA and Nestin protein were remarkably down-regulated (P<0.05) in the model group. Following EA intervention, the escape latencies at the 3rd and 4th day, Aβ senile plaques, immunofluorescence density of BrdU/GFAP, and GFAP protein expression were pronouncedly decreased (P<0.05, P<0.01), while the platform crossing times, platform quadrant residence time, immunofluorescence density of BrdU/NeuN, expression levels of Jagged1 and Notch1 mRNAs and proteins and Nestin protein evidently increased (P<0.05, P<0.01), suggesting an enhancement of proliferation and diffe-rentiation of endogenous neural stem cells into neurons and a suppression of the proliferation and differentiation towards astrocytes in the hippocampus. EA at GV20, GV16 and BL23 can improve the learning-memory ability, promote the proliferation and differentiation of endogenous neural stem cells towards neurons and inhibit the proliferation and differentiation of endogenous neural stem cells towards astrocytes in the hippocampus, which may be achieved by regulating Jagged1/Notch1 pathway.

Effect of Fetal Bovine Serum or Basic Fibroblast Growth Factor on Cell Survival and the Proliferation of Neural Stem Cells: The Influence of Homocysteine Treatment.

In vitro cell culture is a routinely used method which is also applied for in vitro modeling of various neurological diseases. On the other hand, media used for cell culture are often not strictly standardized between laboratories, which hinders the comparison of the obtained results. Here, we compared the effects of homocysteine (Hcy), a molecule involved in neurodegeneration, on immature cells of the nervous system cultivated in basal medium or media supplemented by either fetal bovine serum or basic fibroblast growth factor. The number of cells in basal media supplemented with basic fibroblast growth factor (bFGF) was 2.5 times higher in comparison to the number of cells in basal media supplemented with fetal bovine serum (FBS). We also found that the neuron-specific β-3-tubulin protein expression dose dependently decreased with increasing Hcy exposure. Interestingly, bFGF exerts a protective effect on β-3-tubulin protein expression at a concentration of 1000 µM Hcy compared to FBS-treated neural stem cells on Day 7. Supplementation with bFGF increased SOX2 protein expression two-fold compared to FBS supplementation. GFAP protein expression increased five-fold on Day 3 in FBS-treated neural stem cells, whereas on Day 7, bFGF increased GFAP expression two-fold compared to FBS-treated neural stem cells. Here, we have clearly shown that the selection of culturing media significantly influences various cellular parameters, which, in turn, can lead to different conclusions in experiments based on in vitro models of pathological conditions.

Multisensory Stimulation Reverses Memory Impairment in Adrβ3KO Male Mice.

Norepinephrine plays an important role in modulating memory through its beta-adrenergic receptors (Adrβ: β1, β2 and β3). Here, we hypothesized that multisensory stimulation would reverse memory impairment caused by the inactivation of Adrβ3 (Adrβ3KO) with consequent inhibition of sustained glial-mediated inflammation. To test this, 21- and 86-day-old Adrβ3KO mice were exposed to an 8-week multisensory stimulation (MS) protocol that comprised gustatory and olfactory stimuli of positive and negative valence; intellectual challenges to reach food; the use of hidden objects; and the presentation of food in ways that prompted foraging, which was followed by analysis of GFAP, Iba-1 and EAAT2 protein expression in the hippocampus (HC) and amygdala (AMY). The MS protocol reduced GFAP and Iba-1 expression in the HC of young mice but not in older mice. While this protocol restored memory impairment when applied to Adrβ3KO animals immediately after weaning, it had no effect when applied to adult animals. In fact, we observed that aging worsened the memory of Adrβ3KO mice. In the AMY of Adrβ3KO older mice, we observed an increase in GFAP and EAAT2 expression when compared to wild-type (WT) mice that MS was unable to reduce. These results suggest that a richer and more diverse environment helps to correct memory impairment when applied immediately after weaning in Adrβ3KO animals and indicates that the control of neuroinflammation mediates this response.

Comparison of motor function recovery and brain changes in intracerebral hemorrhagic and ischemic rats with similar brain damage.

In this study, we compared the mechanisms of brain recovery in intracerebral hemorrhage and ischemia, focusing on synapses, glial cells, and dopamine expression, which are considered fundamental for neural recovery after stroke. Male Wistar rats were divided into intracerebral hemorrhage, ischemia, and sham surgery (SHAM) groups. The intracerebral hemorrhage group was injected with a collagenase solution, the ischemia group was injected with an endothelin-1 solution, and the SHAM group was injected with physiological saline. The motor function of these rats was evaluated using a rotarod test on days 7, 14, 21, and 28 post-surgery. On postoperative day 29, lesion volume was analyzed using Nissl staining. In addition, the protein expression levels of NeuN, GFAP, tyrosine hydroxylase, and PSD95 were analyzed in the striatum and motor cortex. There was no significant difference between the ischemia and intracerebral hemorrhage groups in terms of lesion volume in the striatum; however, the motor recovery of the intracerebral hemorrhage group occurred more rapidly than that of the ischemia group, and the intracerebral hemorrhage group exhibited higher GFAP protein expression in the motor cortex. The rapid motor recovery in intracerebral hemorrhage rats relative to that in ischemia rats may be associated with changes in astrocytes in brain regions remote from the injury site.

AD−1 Small Molecule Improves Learning and Memory Function in Scopolamine-Induced Amnesic Mice Model through Regulation of CREB/BDNF and NF-κB/MAPK Signaling Pathway

Cognitive decline and memory impairment induced by oxidative brain damage are the critical pathological hallmarks of Alzheimer's disease (AD). Based on the potential neuroprotective effects of AD-1 small molecule, we here explored the possible underlying mechanisms of the protective effect of AD-1 small molecule against scopolamine-induced oxidative stress, neuroinflammation, and neuronal apoptosis. According to our findings, scopolamine administration resulted in increased AChE activity, MDA levels, and decreased antioxidant enzymes, as well as the downregulation of the antioxidant response proteins of Nrf2 and HO-1 expression; however, treatment with AD-1 small molecule mitigated the generation of oxidant factors while restoring the antioxidant enzymes status, in addition to improving antioxidant protein levels. Similarly, AD-1 small molecule significantly increased the protein expression of neuroprotective markers such as BDNF and CREB and promoted memory processes in scopolamine-induced mice. Western blot analysis showed that AD-1 small molecule reduced activated microglia and astrocytes via the attenuation of iba-1 and GFAP protein expression. We also found that scopolamine enhanced the phosphorylation of NF-κB/MAPK signaling and, conversely, that AD-1 small molecule significantly inhibited the phosphorylation of NF-κB/MAPK signaling in the brain regions of hippocampus and cortex. We further found that scopolamine promoted neuronal loss by inducing Bax and caspase-3 and reducing the levels of the antiapoptotic protein Bcl-2. In contrast, AD-1 small molecule significantly decreased the levels of apoptotic markers and increased neuronal survival. Furthermore, AD-1 small molecule ameliorated scopolamine-induced impairments in spatial learning behavior and memory formation. These findings revealed that AD-1 small molecule attenuated scopolamine-induced cognitive and memory dysfunction by ameliorating AChE activity, oxidative brain damage, neuroinflammation, and neuronal apoptosis.

Hypoxia Induces Early Neurogenesis in Human Fetal Neural Stem Cells by Activating the WNT Pathway.

Fetal neural stem cells (FNSCs) present in the human fetal brain differentiate into cells of neuronal and glial lineages. The developing fetus is exposed to lower oxygen concentrations than adults, and this physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the subventricular zone of aborted fetal brains (n = 5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression using immunocytochemistry and flow cytometry, respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48h, and hypoxia exposure (n = 5) was validated. Whole transcriptome analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4 × 44K human array slides) highlighted that genes associated with neurogenesis were enriched upon exposure to hypoxia. The pathway analysis of these enriched genes (using Metacore) showed the involvement of the WNT signaling pathway. Microarray analyses were validated using neuronal and glial lineage commitment markers, namely, NEUROG1, NEUROG2, ASCL1, DCX, GFAP, OLIG2, and NKX2.2, using qPCR (n = 9). DCX, ASCL1, NGN1, and GFAP protein expression was analyzed by Western blotting (n = 3). This demonstrated upregulation of the neuronal commitment markers upon hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n = 9) and increased protein expression of CTNNB1 (β-catenin) and ID2 by Western blot (n = 3) indicated its involvement in mediating neuronal differentiation upon exposure to hypoxia.

Neural Differentiation Potential of Mesenchymal Stem Cells Enhanced by Biocompatible Chitosan-Gold Nanocomposites.

Chitosan (Chi) is a natural polymer that has been demonstrated to have potential as a promoter of neural regeneration. In this study, Chi was prepared with various amounts (25, 50, and 100 ppm) of gold (Au) nanoparticles for use in in vitro and in vivo assessments. Each as-prepared material was first characterized by UV-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Dynamic Light Scattering (DLS). Through the in vitro experiments, Chi combined with 50 ppm of Au nanoparticles demonstrated better biocompatibility. The platelet activation, monocyte conversion, and intracellular ROS generation was remarkably decreased by Chi–Au 50 pm treatment. Furthermore, Chi–Au 50 ppm could facilitate colony formation and strengthen matrix metalloproteinase (MMP) activation in mesenchymal stem cells (MSCs). The lower expression of CD44 in Chi–Au 50 ppm treatment demonstrated that the nanocomposites could enhance the MSCs undergoing differentiation. Chi–Au 50 ppm was discovered to significantly induce the expression of GFAP, β-Tubulin, and nestin protein in MSCs for neural differentiation, which was verified by real-time PCR analysis and immunostaining assays. Additionally, a rat model involving subcutaneous implantation was used to evaluate the superior anti-inflammatory and endothelialization abilities of a Chi–Au 50 ppm treatment. Capsule formation and collagen deposition were decreased. The CD86 expression (M1 macrophage polarization) and leukocyte filtration (CD45) were remarkably reduced as well. In summary, a Chi polymer combined with 50 ppm of Au nanoparticles was proven to enhance the neural differentiation of MSCs and showed potential as a biosafe nanomaterial for neural tissue engineering.

Exosomes derived from bone-marrow mesenchymal stem cells alleviate cognitive decline in AD-like mice by improving BDNF-related neuropathology

BackgroundAlzheimer's disease (AD) is a neurodegenerative disease characterized by a progressive decline in cognitive ability. Exosomes derived from bone-marrow mesenchymal stem cells (BMSC-exos) are extracellular vesicles that can execute the function of bone-marrow mesenchymal stem cells (BMSCs). Given the versatile therapeutic potential of BMSC and BMSC-exos, especially their neuroprotective effect, the aim of this study was to investigate the potential effect of BMSC-exos on AD-like behavioral dysfunction in mice and explore the possible molecular mechanism.MethodsBMSC-exos were extracted from the supernatant of cultured mouse BMSCs, which were isolated from the femur and tibia of adult C57BL/6 mice, purified and sorted via flow cytometry, and cultured in vitro. BMSC-exos were identified via transmission electron microscopy, and typical marker proteins of exosomes were also detected via Western blot. A sporadic AD mouse model was established by intracerebroventricular injection of streptozotocin (STZ). Six weeks later, BMSC-exos were administered via lateral ventricle injection or caudal vein injection lasting five consecutive days, and the control mice were intracerebroventricularly administered an equal volume of solvent. Behavioral performance was observed via the open field test (OFT), elevated plus maze test (EPM), novel object recognition test (NOR), Y maze test (Y-maze), and tail suspension test (TST). The mRNA and protein expression levels of IL-1β, IL-6, and TNF-α in the hippocampus were measured via quantitative polymerase chain reaction (qPCR) and Western blot, respectively. Moreover, the protein expression of Aβ1-42, BACE, IL-1β, IL-6, TNF-α, GFAP, p-Tau (Ser396), Tau5, synaptotagmin-1 (Syt-1), synapsin-1, and brain-derived neurotrophic factor (BDNF) in the hippocampus was detected using Western blot, and the expression of GFAP, IBA1, Aβ1−42 and DCX in the hippocampus was measured via immunofluorescence staining.ResultsLateral ventricle administration, but not caudal vein injection of BMSC-exos improved AD-like behaviors in the STZ-injected mouse model, as indicated by the increased number of rearing, increased frequency to the central area, and increased duration and distance traveled in the central area in the OFT, and improved preference index of the novel object in the NOR. Moreover, the hyperactivation of microglia and astrocytes in the hippocampus of the model mice was inhibited after treatment with BMSC-exos via lateral ventricle administration, accompanied by the reduced expression of IL-1β, IL-6, TNF-α, Aβ1-42, and p-Tau and upregulated protein expression of synapse-related proteins and BDNF. Furthermore, the results of the Pearson test showed that the preference index of the novel object in the NOR was positively correlated with the hippocampal expression of BDNF, but negatively correlated with the expression of GFAP, IBA1, and IL-1β. Apart from a positive correlation between the hippocampal expression of BDNF and Syt-1, BDNF abundance was found to be negatively correlated with markers of glial activation and the expression of the inflammatory cytokines, Aβ1-42, and p-Tau, which are characteristic neuropathological features of AD.ConclusionsLateral ventricle administration, but not caudal vein injection of BMSC-exos, can improve AD-like behavioral performance in STZ-injected mice, the mechanism of which might be involved in the regulation of glial activation and its associated neuroinflammation and BDNF-related neuropathological changes in the hippocampus.

Taurine supplementation improves hippocampal metabolism in immature rats with intrauterine growth restriction (IUGR) through protecting neurons and reducing gliosis.

Taurine as an essential amino acid in the brain could play an important role in protecting the fetal brain of intrauterine growth restriction (IUGR). The hippocampus with IUGR showed neural metabolic disorder and structure changed that affected memory and learning ability. This study was aimed to identify the effect of taurine supplementation on the metabolism alterations and cellular composition changes of the hippocampus in IUGR immature rats. Metabolite concentrations were determined by magnetic resonance spectroscopy (MRS) in the hippocampus of juvenile rats with IUGR following taurine supplementation with antenatal or postnatal supply. The composition of neural cells in the hippocampus was observed by immunohistochemical staining (IHC) and western blotting (WB). Antenatal taurine supplementation increased the ratios of N-acetylaspartate (NAA) /creatine (Cr) and glutamate (Glu) /Cr of the hippocampus in the IUGR immature rats, but reduced the ratios of choline (Cho) /Cr and myoinositol (mI) /Cr. At the same time, the protein expression of NeuN in the IUGR rats was increased through intrauterine taurine supplementation, and the GFAP expression was reduced. Especially the effect of antenatal taurine was better than postpartum. Furthermore, there existed a positive correlation between the NAA/Cr ratio and the NeuN protein expression (R = 0.496 p < 0.001 IHC; R = 0.568 p < 0.001 WB), the same results existed in the relationship between the mI/Cr ratio and the GFAP protein expression (R = 0.338 p = 0.019 IHC; R = 0.440 p = 0.002 WB). Prenatal taurine supplementation can better improve hippocampal neuronal metabolism by increasing NAA / Cr ratio related to the number of neurons and reducing Cho / Cr ratio related to the number of glial cells.

The analgesic activities of total alkaloids of the ethnic medicine Cynanchum komarovii Al. Iljinski

Ethnopharmacological relevanceCynanchum komarovii Al. Iljinski is a ethnomedicinal herb and this ethno-medicine is used mainly to treat arthritis, toothache, reducing phlegm, relieving cough. Total alkaloids of Cynanchum komarovii Al. Iljinski (TACKI) is the main active compound of Cynanchum komarovii Al. Iljinski. Previous investigations have revealed that TACKI can significantly inhibit rat foot swelling caused by carrageenan; it has a significant inhibitory effect on granulation tissue proliferation. Pharmacology study showed that Cynanchum komarovii Al. Iljinski has analgesia, anti-inflammatory, antibacterial, anti-tumor, relieving cough and relieving asthma. However, there is no any investigation on the mechanism of analgesia and anti-inflammation. Aim of the studyTo clarify the analgesic effect and material basis of Cynanchum komarovii Al. Iljinski, determine the analgesic effect of TACKI, and provide experimental data support for its traditional application in the treatment of various pains. Materials and methodsTACKI were prepared by the traditional acid extraction and alkaline precipitation method, and TACKI was analyzed through classic animal models of acute antinociceptive animal models and chronic antinociceptive. Evaluation of analgesic effects, and preliminary discussion of the mechanism of its analgesic effects were performed in this work. ResultsAcute toxicity experiments showed that the LD50 of TACKI mice was 2960.88 mg/kg, and symptoms of poisoning appeared. Patholog of liver and kidney studies have shown that TACKI reduces eosinophils and increases basophils in kidney glomeruli. In the study of analgesic effects, TACKI had analgesic activity through the PWL, formalin test, and acetic acid writhing test. In the chronic inflammatory antinociceptive study, the latency of the withdrawal reflex in the TACKI group was prolonged, and the mechanical withdrawal reflex threshold was significantly increased. The protein expression of NMDA, GFAP and Iba-1 in rat brain tissue can be reduced significantly byTACKI. Meanwhile, the content of TNF-α and IL-6 in rat brain tissue is reduced. ConclusionTACKI has a significant analgesic activities. It may be related to inhibiting the activation of astrocytes and reducing the content of inflammatory mediators.

Protection of Intestinal Barrier in Uremic Mice by Electroacupuncture via Regulating the Cannabinoid 1 Receptor of the Intestinal Glial Cells.

Intestinal barrier injuries are common in uremia, which aggravates uremia. The goal of this study is to learn moreabout how electroacupuncture regulates gastrointestinal function, as well as to identify the importance of microglia in electroacupuncture regulation and the cannabinoid receptor signaling pathway in controlling the activity of intestinal glial cells. The mice were arbitrarily assigned to four groups: control, CKD, electroacupuncture stimulation, or AM251 (CB1 receptor antagonist). The mice model of uremia was established by adenine gavage. Western blotting revealed the development of tight junction proteins ZO-1, cannabinoid 1 receptor, glial specific GFAP, occludin, S100 β, claudin-1, and JNK. GFAP and CB1R protein expression and co-localization of the intestinal glial cells were observed by double-labeled fluorescence. The expression of cannabinoid 1 receptor CB1R in the intestinal glial cells was increased after electroacupuncture. The expression of tight junction protein, GFAP, S100 β, and CB1R protein was up-regulated after electroacupuncture, and the dysfunction of the intestinal barrier in uremia was corrected. Nevertheless, AM251, a CB1R antagonist, reversed the effect of electroacupuncture. Electroacupuncture can protect the intestinal barrier through the intestinal glial cell CB1R, and the effect is achieved by inhibiting the JNK pathway.

Investigating Markers of the NLRP3 Inflammasome Pathway in Alzheimer's Disease: A Human Post-Mortem Study.

Neuroinflammatory mechanisms with glial cell activation have been implicated in the pathogenic process of Alzheimer’s disease (AD). Activation of the NLRP3 inflammasome is an essential component of the neuroinflammatory response. A role for NLRP3 activation in AD is supported by both in vitro and in vivo preclinical studies with little direct investigation of AD brain tissue. RNA expression of genes of three glial cell markers, HLA-DRA, AIF-1 and GFAP; the components of the NLRP3 inflammasome NLRP3, ASC, and caspase-1; and downstream pre-inflammatory cytokines IL-1 β and IL-18, were investigated in the temporal cortex of AD patients and age- and sex-matched controls. Protein expression of GFAP was also assessed. Increases in both mRNA and protein expression were observed for GFAP in AD. There were no significant changes in other NLRP3 activation markers between groups. Our results indicate the involvement of astrocyte activation in AD, particularly in more severe patients. We found no evidence for the specific involvement of the NLRP3 inflammasome.

Effects of Pulsed Radiofrequency on Nerve Repair and Expressions of GFAP and GDNF in Rats with Neuropathic Pain.

Objective To study the effect of pulsed radio frequency (PRF) on nerve repair and the expression of GFAP and GDNF in rats with neuropathic pain. Methods Thirty SPF healthy SD rats were randomly divided into control group (Group C), PSNL group (partial ligation of sciatic nerve) + sham group (Group PS), and PSNL group (partial ligation of sciatic nerve) + PRF group (Group PR), with 10 rats in each group. In group C, the right sciatic nerve was exposed without ligation. In the PS group, the model of neuropathic pain was established by partial ligation of sciatic nerve. The mice in the PR group were treated with PRF after establishing the neuropathic pain model. The general behavior of rats during the treatment was observed. The mechanical and thermal hyperalgesia were measured before operation and 1, 3, 7, and 14 days after operation. The content of inflammatory factors in nerve tissue was detected by ELISA. The pathological condition of nerve tissue was observed by HE. The gene and protein changes of GFAP and GDNF in nerve tissue were determined by QRT PCR and Western blot. Results Rats in the control group were free to move and in good condition. In the PS group, there were different degrees of claudication, weakness of the lower limbs, lateral toe valgus, nerve injury, and other behavioral changes. After the pulsed radiofrequency in the PR group, the above symptoms decreased gradually with the prolongation of the treatment time. The mechanical pain sensitivity and thermal allodynia of the PS group were reduced after the operation. The mechanical pain sensitivity and thermal pain sensitivity of the PR group gradually increased with the prolongation of the treatment time, and the 14 days were basically close to the control group. The levels of TNF-α and IL-6 in ELISA were significantly higher in the PS group than in the control group, and the content in the PR group was gradually reduced, which was close to the control group. HE staining showed that the sciatic nerve fibers disappeared, and the formation of nerve cavities was obvious in the 14-day PS group. The nerve fibers were found in the sciatic tissue of the PR group, and there was no obvious hemorrhagic edema and cell deformation. The expression of GFAP mRNA in the PS group was significantly higher than that in the control group and the PR group (p < 0.05), and the expression of GDNF was opposite (p < 0.05). The results of western blot showed that the expression of GFAP protein in the 14-day PS group was significantly higher than that in the control group. The expression of the PR group decreased compared with the control group, and the expression of GDNF was opposite (p < 0.05). Conclusion Pulsed radiofrequency ablation can promote neurological repair, promote GDNF, and reduce the expression of GFAP in rats with neuropathic pain.

MicroRNA-125b and its downstream Smurf1/KLF2/ATF2 axis as important promoters on neurological function recovery in rats with spinal cord injury.

The purpose of this study is to investigate the role of microRNA‐125b (miR‐125b) and its mechanism in spinal cord injury (SCI) by targeting Smurf1. After loss‐ and gain‐function approaches were conducted in SCI rat models and neural stem cells (NSCs) isolated from foetal rats, the Basso‐Beattie‐Bresnahan (BBB) score was calculated, and related protein expression was determined by Western blot analysis and cell apoptosis by TUNEL staining. NSC viability was detected by CCK‐8, migration abilities by Transwell assay and apoptosis by flow cytometry. The relationship between miR‐125b, Smurf1 and KLF2 was evaluated by dual‐luciferase reporter gene experiments, Co‐IP and in vivo ubiquitin modification assays. Inhibition of miR‐125b and KLF2 and the up‐regulation of Smurf1 and ATF2 were observed in SCI rats. BBB scores were elevated, the expression of Nestin, NeuN, GFAP, NF‐200 and Bcl‐2 protein was enhanced but that of Bax protein was reduced, and cell apoptosis was inhibited in SCI rats after up‐regulating miR‐125b or silencing ATF2. Smurf1 was a target gene of miR‐125b, which promoted KLF2 degradation through its E3 ubiquitin ligase function, and KLF2 repressed the expression of ATF2 in NSCs. The results in vivo were replicated in vitro. miR‐125b overexpression promotes neurological function recovery after SCI.

Very Early Exercise Rehabilitation After Intracerebral Hemorrhage Promotes Inflammation in the Brain

Background Very early exercise has been reported to exacerbate motor dysfunction; however, its mechanism is largely unknown. Objective This study examined the effect of very early exercise on motor recovery and associated brain damage following intracerebral hemorrhage (ICH) in rats. Methods Collagenase solution was injected into the left striatum to induce ICH. Rats were randomly assigned to receive placebo surgery without exercise (SHAM) or ICH without (ICH) or with very early exercise within 24 hours of surgery (ICH+VET). We observed sensorimotor behaviors before surgery, and after surgery preexercise and postexercise. Postexercise brain tissue was collected 27 hours after surgery to investigate the hematoma area, brain edema, and Il1b, Tgfb1, and Igf1 mRNA levels in the striatum and sensorimotor cortex using real-time reverse transcription polymerase chain reaction. NeuN, PSD95, and GFAP protein expression was analyzed by Western blotting. Results We observed significantly increased skillful sensorimotor impairment in the horizontal ladder test and significantly higher Il1b mRNA levels in the striatum of the ICH+VET group compared with the ICH group. NeuN protein expression was significantly reduced in both brain regions of the ICH+VET group compared with the SHAM group. Conclusion Our results suggest that very early exercise may be associated with an exacerbation of motor dysfunction because of increased neuronal death and region-specific changes in inflammatory factors. These results indicate that implementing exercise within 24 hours after ICH should be performed with caution.