Animal Ethics Committee Research Articles

Aerobic exercise training prevents skeletal myopathy by myomiRs regulation in heart failure

Abstract Introduction Heart failure (HF) is the endpoint of systemic arterial hypertension. Exercise intolerance is a common symptom, partly due, to changes in the skeletal muscle mass (SM) and fiber type profile. Otherwise, aerobic exercise training (ET) has been used as an important non-pharmacological therapy in HF. MyomiRs are a muscle-specific class of miRNAs, which regulate genes that inhibiting the expression of proteins in pathological and physiological conditions controlling phenotypic changes in the SM, however little is known about these changes in ET-induced HF Purpose To elucidate the molecular mechanisms of ET involved in the metabolic alterations of SM in HF rats of hypertensive etiology. Methods The study was approved by the animal ethics committee (USP-No. 2020/01). 20 male rats, spontaneously hypertensive (SHR), and 10 Wistar Kyoto rats (WKY), SHR controls, nine-months-old, were divided into three groups: sedentary WKY (WKY-S), sedentary SHR (SHR-S), and trained (SHR-T). The ET consisted of swimming sessions with 60 minutes, 1x/day, 5x/week, for 10 weeks, with 5% of body overload. After ET protocol, blood pressure (BP), cardiac morphology and function (Echocardiography), exercise tolerance test, maximal oxygen uptake (VO2 peak), mitochondrial oxygen consumption (Oroboros), immunohistochemistry of the SM, expression of miRNAs (RT-qPCR) were evaluated. Statistical analyzes were performed by one-way ANOVA followed by the Tukey test. The results were expressed as mean ± standard error. Results ET reduced blood pressure levels and cardiac dysfunction in SHR-T compared to SHR-S. The SHR-S group covered smaller distance in the exercise tolerance test (255±22 meters) compared to the WKY-S (419±19 meters, p<0.0001), however ET reestablished the exercise tolerance (SHR-T: 365±20 meters; SHR-S: p<0.001 and WKY-S: p>0.05). The HF induced changes in type I and II fibers composition (I: 73±0.6% and II: 24±0.9%), VO2 peak (50±1.5 mL kg–1 min–1), mitochondrial oxygen consumption (State 3: 3.0±0.2 nmol O2 min–1 mg protein–1) and myomiRs expression (miRNA-208b: 65±4%, -499: 73±5%, -1: 153±10%) in the soleus muscle of SHR-S compared to WKY-S (I: 94±0.6%, II: 6±0.6%, p<0.001; VO2 peak: 59±2.3 mL kg–1 min–1, p<0.01; State 3: 4.0±0.2 nmol O2 min–1 mg protein–1, p<0.05; miRNAs: p<0.01). ET minimized changes in metabolic profile by counteract the muscle fiber type switching, and the oxygen consumption impairment, and myomiRs expression dysregulation (I: 90±0.5%, II: 9±0.6%, SHR-S p<0.01; VO2 peak: 79±2.4 mL kg–1 min–1, SHR-S: p<0.0001; State 3: 5.45±0.32 nmol O2 min–1 mg protein–1, SHR-S: p<0.0001; miRNA-208b: 91±5%, -499: 106±8%, -1: 100±9%; SHR-S: p<0.01). Conclusions ET reestablished structural and metabolic changes in SM, resulting from the progression of HF, through the regulation of myomiRs, improving exercise tolerance. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): The Coordination for the Improvement of Higher Education Personnel (CAPES): Academic Excellence Program (Proex).

Electroacupuncture in promoting neural repair after spinal cord injury: Inhibiting the Notch signaling pathway and regulating downstream proteins expression.

Spinal cord injury (SCI) is one of the serious central nervous system injuries and the incidence of SCI continues to increase. Previous studies have indicated that electroacupuncture (EA) is beneficial for promoting recovery after SCI. In the present study, we attempted to evaluate how EA can promote the neural repair in SCI model rats by observing changes in the Notch signaling pathway. Experimental rats were randomly divided into four groups. Each group had its own intervention period: 1 day, 7 days, 14 days, and 28 days, and five randomized subgroups: blank control (B) group, blank electroacupuncture (BE) group, sham operation (S) group, model control (M) group and EA group. Animals in the EA group and the BE group were treated with EA at Dazhui (GV14) and Mingmen (GV4) acupoints for 20 min. After the intervention period, the Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurological function. We found that BBB score increased in EA-treated groups. Hematoxylin and eosin staining was used to observe pathological changes in the injured spinal cord and the results showed that EA therapy could promote the repair of injured spinal cord tissue. Immunohistochemistry and Western blot methods were used to detect the expression of proteins Delta1, Presenilin1, Hes1, and Hes5 in the injured spinal cord. The results showed that the expression levels of Delta1, Presenilin1, Hes1, and Hes5 increased significantly after SCI and decreased after EA treatment. Our study suggested that the possible mechanism by which EA could benefit the recovery after SCI in rats may include inhibiting the Notch signaling pathway and regulating the downstream proteins expression. In addition, our study can provide reference for selecting acupoints and treatment cycle in the treatment of SCI.

Histone Demethylase KDM5C Deficiency Inhibits Bone Formation and Fracture Healing in Aged Mice by Epigenetically Activating Erdr1

Background: Fractures are one of the most common clinical injuries, especially for elders with osteoporosis. KDM5C specifically demethylate di- and tri-methylated lysine 4 on histone. It is frequently mutated in X-linked intellectual disability patients exhibiting physical and behavioral abnormalities. However, it is unclear whether KDM5C plays a role in bone formation and fracture repair. Methods: We used KDM5C conventional knockout mice model to investigate its role on bone formation. Western blotting, immunofluorescence staining, RNAseq, and ChIP assay were applied to demonstrate the molecular mechanism. Open femur fracture model was used to investigate the effects of KDM5C and Erdr1 on fracture healing. Findings: We found that the expression level of KDM5C was constantly increased during the osteogenesis of MSCs. Silencing endogenous KDM5C inhibited osteogenic differentiation of MSCs in vitro and KDM5C knockout mice displayed decreased bone mass and abnormal bone development. Further, RNA-seq analysis showed that PI3K-Akt signaling pathway and Erdr1 were most significantly influenced by KDM5C. KDM5C deficiency resulted in increased Erdr1 expression by histone modification. While knockdown of Erdr1 could promote osteogenesis and angiogenesis. Finally, using an experimental mouse femur fracture model, we found that KDM5C deficiency would impair fracture healing, which could be rescued by silencing Erdr1. More importantly, we found that KDM5C was decreased in bone samples of osteoporotic fracture. Interpretation: Taken together, these results showed that KDM5C was a novel regulator of osteogenesis and angiogenesis. KDM5C or Erdr1 might be potential therapeutic targets for the treatment of fracture and other bone diseases. Funding: The work was supported by grants from the National Natural Science Foundation of China (NSFC No. 81871778). Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: All surgical interventions, treatments and postoperative animal care procedures were strictly performed in accordance with the guidelines for Animal Welfare and Ethics Committee of Guangzhou University of Chinese medicine. Human bone tissues were obtained in the Second Affiliated Hospital of Guangzhou Medical University. All animal experiments were performed in accordance with protocols that were approved and authorized by the Animal Welfare and Ethics Committee of Guangzhou University of Chinese medicine.

Effects of two formulations containing Phyllanthus emblica and Tinospora cordifolia with and without Ocimum sanctum in immunocompromised mice

BackgroundCurrent pandemic has led us to explore the role of traditional system of medicine to look for formulations that enhance immunity. ObjectiveThe obejctive of this experimental study was to evaluate the immunomodulatory effects of two formulations, Tinospora cordifolia (Tc) and Phyllanthus emblica (Pe) with and without coating of Ocimum sanctum (Os). Materials and methodsAfter obtaining Institutional Animal Ethics Committee approval, present experimental study was conducted to evaluate the immunomodulatory effects of plant drug formulations against infection induced in mice subjected to major surgical stress. Hemisplenectomy was selected to induce major stress and the procedure for hemisplenectomy was standardized. A model of secondary fungal infection after hemisplenectomy was established followed by the treatment of mice with plant drugs and controls. They were subjected to hemisplenectomy or sham operation and 105 C. albicans were injected intravenously. The therapy continued for next 14 days. Kidneys were isolated to estimate fungal load. Fungal load of the kidneys was estimated on post-operative Day 15. ResultsThe test formulations Tc + Pe and Tc + Pe + Os showed significant reduction in the fungal burden of kidneys as compared to hemisplenectomized control group. However, Tc alone exerted better degree of protection as compared to Tc + Pe and Tc + Pe + Os. ConclusionThe formulations, Tc + Pe and Tc + Pe + Os that were developed on the basis of theoretical concepts were not found to be superior to Tc. Though the individual ingredients have been shown to possess immunnostimulant activities, in combination, Pe and Os blunted the effects of Tc. The basis for this drug interaction needs further exploration. Thus, the current experimental study validates immunomodulatory role of Tc. However, the addition of Pe with bhavana of Os does not lead to any augmentation of immunomodulatory activity of Tc. This study also underlines the need to generate data on Ayurveda formulations to understand the rationality of the multi-ingredient Ayurvedic formulations.

Biomimetic chitosan scaffolds with long-term controlled release of nerve growth factor repairs 20-mm-long sciatic nerve defects in rats.

Although autogenous nerve transplantation is the gold standard for treating peripheral nerve defects of considerable length, it still has some shortcomings, such as insufficient donors and secondary injury. Composite chitosan scaffolds loaded with controlled release of nerve growth factor can promote neuronal survival and axonal regeneration after short-segment sciatic nerve defects. However, the effects on extended nerve defects remain poorly understood. In this study, we used chitosan scaffolds loaded with nerve growth factor for 8 weeks to repair long-segment (20 mm) sciatic nerve defects in adult rats. The results showed that treatment markedly promoted the recovery of motor and sensory functions. The regenerated sciatic nerve not only reconnected with neurons but neural circuits with the central nervous system were also reconstructed. In addition, the regenerated sciatic nerve reconnected the motor endplate with the target muscle. Therefore, this novel biomimetic scaffold can promote the regeneration of extended sciatic nerve defects and reconstruct functional circuits. This provides a promising method for the clinical treatment of extended peripheral nerve injury. This study was approved by the Animal Ethics Committee of Capital Medical University, China (approval No. AEEI-2017-033) on March 21, 2017.

Nerve bundle formation during the promotion of peripheral nerve regeneration: collagen VI-neural cell adhesion molecule 1 interaction.

The formation of nerve bundles, which is partially regulated by neural cell adhesion molecule 1 (NCAM1), is important for neural network organization during peripheral nerve regeneration. However, little is known about how the extracellular matrix (ECM) microenvironment affects this process. Here, we seeded dorsal root ganglion tissue blocks on different ECM substrates of peripheral nerve ECM-derived matrix-gel, Matrigel, laminin 521, collagen I, and collagen IV, and observed well-aligned axon bundles growing in the peripheral nerve ECM-derived environment. We confirmed that NCAM1 is necessary but not sufficient to trigger this phenomenon. A protein interaction assay identified collagen VI as an extracellular partner of NCAM1 in the regulation of axonal fasciculation. Collagen VI interacted with NCAM1 by directly binding to the FNIII domain, thereby increasing the stability of NCAM1 at the axolemma. Our in vivo experiments on a rat sciatic nerve defect model also demonstrated orderly nerve bundle regeneration with improved projection accuracy and functional recovery after treatment with 10 mg/mL Matrigel and 20 μg/mL collagen VI. These findings suggest that the collagen VI-NCAM1 pathway plays a regulatory role in nerve bundle formation. This study was approved by the Animal Ethics Committee of Guangzhou Medical University (approval No. GY2019048) on April 30, 2019.

Baicalin attenuates blood-spinal cord barrier disruption and apoptosis through PI3K/Akt signaling pathway after spinal cord injury.

Baicalin is a natural active ingredient isolated from Scutellariae Radix that can cross the blood-brain barrier and exhibits neuroprotective effects on multiple central nervous system diseases. However, the mechanism behind the neuroprotective effects remains unclear. In this study, rat models of spinal cord injury were established using a modified Allen's impact method and then treated with intraperitoneal injection of Baicalin. The results revealed that Baicalin greatly increased the Basso, Beattie, Bresnahan Locomotor Rating Scale score, reduced blood-spinal cord barrier permeability, decreased the expression of Bax, Caspase-3, and nuclear factor κB, increased the expression of Bcl-2, and reduced neuronal apoptosis and pathological spinal cord injury. SH-SY5Y cell models of excitotoxicity were established by application of 10 mM glutamate for 12 hours and then treated with 40 µM Baicalin for 48 hours to investigate the mechanism of action of Baicalin. The results showed that Baicalin reversed tight junction protein expression tendencies (occludin and ZO-1) and apoptosis-related protein expression (Bax, Bcl-2, Caspase-3, and nuclear factor-κB), and also led to up-regulation of PI3K and Akt phosphorylation. These effects on Bax, Bcl-2, and Caspase-3 were blocked by pretreatment with the PI3K inhibitor LY294002. These findings suggest that Baicalin can inhibit blood-spinal cord barrier permeability after spinal cord injury and reduce neuronal apoptosis, possibly by activating the PI3K/Akt signaling pathway. This study was approved by Animal Ethics Committee of Xi'an Jiaotong University on March 6, 2014.

Pharmacological blockade of neurokinin1 receptor restricts morphine-induced tolerance and hyperalgesia in the rat

The most notable adverse side effects of chronic morphine administration include tolerance and hyperalgesia. This study investigated the involvement of dorsal root ganglion (DRG) protein kinase Cɛ (PKCɛ) expression during chronic morphine administration and also considered the relationship between DRG PKCɛ expression and the substance P- neurokinin1 receptor (SP- NK1R) activity. Thirty-six animals were divided into six groups (n=6) in this study. In the morphine and sham groups, rats received 10µg intrathecal (i.t.) morphine or saline for eight consecutive days, respectively. Behavioral tests were performed on days 1 and 8 before and after the first injectionsand then 48h after the last injection (day 10). In thetreatment groups, rats received NK1R antagonist (L-732,138, 25µg) daily, either alone or 10min before a morphine injection, Sham groups received DMSO alone or 10min before a morphine injection. Animals were sacrificed on days 8 and 10, and DRG PKCɛ and SP expression were analyzed by western blot and immunohistochemistry techniques, respectively. Behavioral tests indicated that tolerance developed following eight days of chronic morphine injection. Hyperalgesia was induced 48h after the last morphine injection. Expression of SP and PKCɛ in DRG significantly increased in rats that developed morphine tolerance on day 8 and hyperalgesia on day 10, respectively. NK1R antagonist (L-732,138) not only blocked the development of hyperalgesia and the increase of PKCɛ expression but also alleviated morphine tolerance. Our results provide evidence that DRG PKCɛ and SP-NK1R most likely participated in the generation of morphine tolerance and hyperalgesia. Pharmacological inhibition of SP-NK1R activity in the spinal cord suggests a role for NK1R and in restricting some side effects of chronic morphine. All experiments were performed by the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised1996) and were approved by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.MSP.REC.1396.130).

Early mature bone formation using a bioactive membrane in dogs

The purpose of study was to compare the amounts of mature bone formation between a new bioactive collagen membrane (BCM) and a conventional collagen membrane (CCM) when used in a canine model of dental guided bone and tissue regeneration (GBR). GBR surgery was performed in 36 skeletally mature (9-14kg / 20-30lb) Beagle dogs using a split mouth design. The implant site was prepared with a titanium implant in the tooth socket and remaining void filled with a granulated bone substitute. The collagen membranes were trimmed and placed over the implant, and the gingiva closed around the treatment site. Animals were sacrificed (n=6 each group) at 4, 8, and 12 weeks after implant placement. Bone formation and tissue reactions were measured by micro-CT and histomorphometry. The study was approved by the institutional Animal Ethics committee. Bone volume in sites treated with both collagen membranes was significantly greater than controls at 12 weeks. However, the BCM displayed comparatively more bone formation at the early time point of 4 weeks than the CCM. Further, histological assessment of sites treated with the bioactive membrane revealed that mature cortical bone formation occurred as early as 8 weeks, which was likely due to the minimal inflammatory reaction observed and retention of the bioactive barrier structure. The new bioactive collagen membrane induces early mature bone formation in a canine model of dental GBR.

Histochemistry of microinfarcts in the mouse brain after injection of fluorescent microspheres into the common carotid artery.

The mouse model of multiple cerebral infarctions, established by injecting fluorescent microspheres into the common carotid artery, is a recent development in animal models of cerebral ischemia. To investigate its effectiveness, mouse models of cerebral infarction were created by injecting fluorescent microspheres, 45-53 µm in diameter, into the common carotid artery. Six hours after modeling, fluorescent microspheres were observed directly through a fluorescence stereomicroscope, both on the brain surface and in brain sections. Changes in blood vessels, neurons and glial cells associated with microinfarcts were examined using fluorescence histochemistry and immunohistochemistry. The microspheres were distributed mainly in the cerebral cortex, striatum and hippocampus ipsilateral to the side of injection. Microinfarcts were found in the brain regions where the fluorescent microspheres were present. Here the lodged microspheres induced vascular and neuronal injury and the activation of astroglia and microglia. These histopathological changes indicate that this animal model of multiple cerebral infarctions effectively simulates the changes of various cell types observed in multifocal microinfarcts. This model is an effective, additional tool to study the pathogenesis of ischemic stroke and could be used to evaluate therapeutic interventions. This study was approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (approval No. D2021-03-16-1) on March 16, 2021.

Identification of key genes involved in axon regeneration and Wallerian degeneration by weighted gene co-expression network analysis.

Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration. Therefore, investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system. In this study, we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury. We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments, respectively. Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration, while the differentially expressed genes in distal modules promoted neurodegeneration. Next, we constructed hub gene networks for selected modules and identified a key hub gene, Kif22, which was up-regulated in both nerve segments. In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway. Collectively, our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments, and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration. All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University, China (approval No. S20210322-008) on March 22, 2021.

The mechanism behind activation of the Nod-like receptor family protein 3 inflammasome in Parkinson's disease.

Previous studies have shown that the ATP-P2X4 receptor signaling pathway mediates the activation of the Nod-like receptor family protein 3 (NLRP3) inflammasome. The NLRP3 inflammasome may promote renal interstitial inflammation in diabetic nephropathy. As inflammation also plays an important role in the pathogenesis of Parkinson's disease, we hypothesized that the ATP-P2X4 receptor signaling pathway may activate the NLRP3 inflammasome in Parkinson's disease. A male rat model of Parkinson's disease was induced by stereotactic injection of 6-hydroxydopamine into the pars compacta of the substantia nigra. The P2X4 receptor and the NLRP3 inflammasome (interleukin-1β and interleukin-18) were activated. Intracerebroventricular injection of the selective P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) or knockdown of P2X4 receptor expression by siRNA inhibited the activation of the NLRP3 inflammasome and alleviated dopaminergic neurodegeneration and neuroinflammation. Our results suggest that the ATP-P2X4 receptor signaling pathway mediates NLRP3 inflammasome activation, dopaminergic neurodegeneration, and dopamine levels. These findings reveal a novel role of the ATP-P2X4 axis in the molecular mechanisms underlying Parkinson's disease, thus providing a new target for treatment. This study was approved by the Animal Ethics Committee of Qingdao University, China, on March 5, 2015 (approval No. QYFYWZLL 26119).

Long non-coding RNA MEG3 regulates autophagy after cerebral ischemia/reperfusion injury

Severe cerebral ischemia/reperfusion injury has been shown to induce high-level autophagy and neuronal death. Therefore, it is extremely important to search for a target that inhibits autophagy activation. Long non-coding RNA MEG3 participates in autophagy. However, it remains unclear whether it can be targeted to regulate cerebral ischemia/reperfusion injury. Our results revealed that in oxygen and glucose deprivation/reoxygenation-treated HT22 cells, MEG3 expression was obviously upregulated, and autophagy was increased, while knockdown of MEG3 expression greatly reduced autophagy. Furthermore, MEG3 bound miR-181c-5p and inhibited its expression, while miR-181c-5p bound to autophagy-related gene ATG7 and inhibited its expression. Further experiments revealed that mir-181c-5p overexpression reversed the effect of MEG3 on autophagy and ATG7 expression in HT22 cells subjected to oxygen and glucose deprivation/reoxygenation. In vivo experiments revealed that MEG3 knockdown suppressed autophagy, infarct volume and behavioral deficits in cerebral ischemia/reperfusion mice. These findings suggest that MEG3 knockdown inhibited autophagy and alleviated cerebral ischemia/reperfusion injury through the miR-181c-5p/ATG7 signaling pathway. Therefore, MEG3 can be considered as an intervention target for the treatment of cerebral ischemia/reperfusion injury. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University, China (approval No. XF20190538) on January 4, 2019.

Molecular Structural Evolution of Near-Infrared Cationic Aggregation-Induced Emission Luminogens: Preclinical Antimicrobial Pathogens Activities and Tissues Regeneration

Molecular Structural Evolution of Near-Infrared Cationic Aggregation-Induced Emission Luminogens: Preclinical Antimicrobial Pathogens Activities and Tissues Regeneration

Urinary Metabolites of Type 2 Diabetes Rats Fed With Palm Oil Enriched-High Fat Diet

High fat diet (HFD) is one of the risk factors to obesity and diabetes. Recommended diet regimen for diabetes is difficult to abide especially for HFD as it adds flavour to the taste buds. In this study, palm oil-enriched HFD and control diet (ND) were fed to nicotinamide-induced type 2 diabetes rats, respectively for six weeks. Additionally, metformin, a common drug used to treat diabetes was given to rats under treatment groups. We evaluated the change of urinary metabolites of diabetes rats caused by HFD, and also after metformin treatment. Rats were divided into six-groups with different feeding diets, disease condition and with or without metformin treatment. Rats’ urine were collected at the end of six weeks feeding program and subjected to 1H-NMR and multivariate data analysis to evaluate their metabolite profiles. At the early phase of diabetes, metabolites changes in diabetic rats were associated with the disease itself. Our data showed that continuous consumption of HFD altered various metabolic pathways of diabetic rats and caused detrimental effects to the rats. Metformin treatment combined with ND lessened the physiological impacts caused by diabetes condition. Funding Information: This research project was supported by USM RUI topdown grant (1001/PFARMASI/870034) funded by Universiti Sains Malaysia. Declaration of Interests: None to declare. Ethics Approval Statement: All experiment procedures were approved by the Animal Ethics Committee Universiti Sains Malaysia (USM/Animal Ethics Approval/2016/(717)).

Profile of the RNA in exosomes from astrocytes and microglia using deep sequencing: implications for neurodegeneration mechanisms.

Glial cells play an important role in signal transduction, energy metabolism, extracellular ion homeostasis and neuroprotection of the central nervous system. However, few studies have explained the potential effects of exosomes from glial cells on central nervous system health and disease. In this study, the genes expressed in exosomes from astrocytes and microglia were identified by deep RNA sequencing. Kyoto Encyclopedia of Genes and Genomes analysis indicated that several pathways in these exosomes are responsible for promoting neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. Gene ontology analysis showed that extracellular exosome, mitochondrion and growth factor activity were enriched in exosomes from the unique astrocyte group, while extracellular exosome and mitochondrion were enriched in exosomes from the unique microglia group. Next, combined with the screening of hub genes, the protein-protein interaction network analysis showed that exosomes from astrocytes influence neurodegenerative diseases through metabolic balance and ubiquitin-dependent protein balance, whereas exosomes from microglia influence neurodegenerative diseases through immune inflammation and oxidative stress. Although there were differences in RNA expression between exosomes from astrocytes and microglia, the groups were related by the hub genes, ubiquitin B and heat shock protein family A (Hsp70) member 8. Ubiquitin B appeared to be involved in pleiotropic regulatory functions, including immune regulation, inflammation inhibition, protein catabolism, intracellular protein transport, exosomes and oxidative stress. The results revealed the clinical significance of exosomes from glia in neurodegenerative diseases. This study was approved by the Animal Ethics Committee of Nantong University, China (approval No. S20180102-152) on January 2, 2018.

Exploration of the mechanism by which icariin modulates hippocampal neurogenesis in a rat model of depression

Icariin (ICA) has a significant capacity to protect against depression and hippocampal injury, but it cannot effectively cross the blood-brain barrier and accumulate in the brain. Therefore, the mechanism by which ICA protects against hippocampal injury in depression remains unclear. In this study, we performed proteomics analysis of cerebrospinal fluid to investigate the mechanism by which ICA prevents dysfunctional hippocampal neurogenesis in depression. A rat model of depression was established through exposure to chronic unpredictable mild stress for 6 weeks, after which 120 mg/kg ICA was administered subcutaneously every day. The results showed that ICA alleviated depressive symptoms, learning and memory dysfunction, dysfunctional neurogenesis, and neuronal loss in the dentate gyrus of rats with depression. Neural stem cells from rat embryonic hippocampi were cultured in media containing 20% cerebrospinal fluid from each group of rats and then treated with 100 μM corticosterone. The addition of cerebrospinal fluid from rats treated with ICA largely prevented the corticosterone-mediated inhibition of neuronal proliferation and differentiation. Fifty-two differentially expressed proteins regulated by chronic unpredictable mild stress and ICA were identified through proteomics analysis of cerebrospinal fluid. These proteins were mainly involved in the ribosome, PI3K-Akt signaling, and interleukin-17 signaling pathways. Parallel reaction monitoring mass spectrometry showed that Rps4x, Rps12, Rps14, Rps19, Hsp90b1, and Hsp90aa1 were up-regulated by chronic unpredictable mild stress and down-regulated by ICA. In contrast, HtrA1 was down-regulated by chronic unpredictable mild stress and up-regulated by ICA. These findings suggest that ICA can prevent depression and dysfunctional hippocampal neurogenesis through regulating the expression of certain proteins found in the cerebrospinal fluid. The study was approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine of China in March 2017.

Effects Of Acute Exercise And Acupuncture Treatment On Collagen Of Skeletal Muscle Extracellular Matrix

PURPOSE: Skeletal muscle ultrastructure and content changes of different collagen types in the rat skeletal muscle extracellular matrix after acute eccentric exercise was measured to elucidate the effects of acupuncture on exercise-induced skeletal muscle damage and remodeling of the skeletal muscle extracellular matrix. METHODS: Eight-week-old Sprague-Dawley rats (n = 96, body wt 220 ± 6.9 g) were randomly assigned to four groups: sedentary control (C), acupuncture control (CA), exercise (E) and acupunctured exercise (EA). Animals were adapted to the environment for 3 days and made to run on a motor-driven treadmill with −16° downhill tracks at a speed of 16 m/min for 90 min. CA and EA rats underwent intervention after exercise. Tissues were collected from the vastus intermedius muscle of the experimental rats immediately, and at 24 hours, 48 hours, 72 hours, and 120 hours after exercise. A transmission electron microscope was used to observe the skeletal muscle ultrastructure, a scanning electron microscope was used to analyze the morphological changes of collagen fibers, and western blotting were used to detect gene and protein expression of type I, II, and IV collagen. All animal experiments were approved by the Animal Welfare and Ethics Committee of Zhengzhou University. RESULTS: Acute eccentric exercise increased collagen fiber expression, whereas acupuncture inhibited it. Scanning electron microscope results revealed that collagen hyperplasia was increased in the skeletal muscle of E rats but not EA rats 120 hours post exercise. mRNA levels of type I and III collagen of E rats were upregulated to a maximal level 24 hours after exercise compared with immediately killed rats (Type I: 3.865 ± 1.181 to 11.651 ± 3.526, p = 0.28; Type III: 1.032 ± 0.270 to 2.657 ± 0.883, p = 0.17). The mRNA levels of type I and III collagen in EA rats significantly increased 48 hours after exercise compared with immediately killed rats (Type I: 1.024 ± 0.359 to 4.537 ± 1.621, p = 0.56; Type III: 0.680 ± 0.251 to 2.334 ± 0.945, p = 0.32). CONCLUSION: Acute eccentric exercise may stimulate increase collagen levels in rat skeletal muscle. Acupuncture altered collagen expression to facilitate the recovery and regeneration of injured skeletal muscle.

Effects Of Acute Exercise And Acupuncture Treatment On Regulation MMPs/TIMPs Balance Of Skeletal Muscle

PURPOSE: This study investigated MMPs/TIMPs balance in the rat skeletal muscle extracellular matrix after acute eccentric exercise to elucidate the effects of acupuncture on exercise-induced skeletal muscle damage and remodeling of the skeletal muscle extracellular matrix. METHODS: Eight-week-old Sprague-Dawley rats (n = 96, body wt 220 ± 6.9 g) were randomly assigned to four groups: sedentary control (C), acupuncture control (CA), exercise (E) and acupunctured exercise (EA). Animals were made to run on a motor-driven treadmill with −16° downhill tracks at a speed of 16 m/min for 90 minutes. Tissues were collected from the vastus intermedius muscle immediately, and at 24 hours, 48 hours, 72 hours and 120 hours after exercise. Western blotting was used to detect protein expression of matrix metalloproteinases-2 (MMP2), MMP9, tissue inhibitor of metalloproteinases-1 (TIMP1) and TIMP2. All animal experiments were approved by the Animal Welfare and Ethics Committee of Zhengzhou University. RESULTS: The protein expression of MMP2 (EA: 1.330 ± 0.065, E: 1.341 ± 0.120, C: 1.000 ± 0.060) reached the maximal level 24 hours and MMP9 of EA rats (EA: 3.776 ± 1.253, C: 1.000 ± 0.357, P < 0.05) and E rats (E: 5.828 ± 2.151, C: 1.000 ± 0.357, P = 0.016) reached the maximal level at 48 hours and 24 hours respectively. The protein expression of TIMP1 of EA rats (EA: 2.938 ± 0.437, C: 1.000 ± 0.251, P = 0.032) and E rats (E: 1.984 ± 0.077, C: 1.000 ± 0.251, p = 0.059) reached the maximal level at 72 hours and 48 hours respectively. The protein expression of TIMP2 of EA rats (EA: 1.477 ± 0.199, C: 1.000 ± 0.063, p = 0.067) and E rats (E: 1.371 ± 0.081, C: 1.000 ± 0.063, p = 0.074) reached the maximal level immediately after exercise. CONCLUSION: Acupuncture facilitated the recovery and regeneration of injured skeletal muscle by increasing the expression of MM2 and MMP9. TIMP1 and TIMP2 played different roles in inflammation of injured skeletal muscle.

Observing the Anti-oxidant and Anti-inflammatory Effect of Nigella Sativa Combined With Silybum Marianum Extracts on the Acute Peritonitis Mouse Model

Background and Aim: In addition to free radicals such as Nitric Oxide (NO), inflammation is one of the most important pathophysiological causes of peritonitis. Over thousands of years, Nigella Sativa (NS) and Silybum Marianum (SM) are two plants known for their anti-oxidant and anti-inflammatory properties. However, the effect of its compound is unclear. Thus, in this study, we evaluated the anti-inflammatory effect of NS and SM extracts and their combination on inflammatory diseases like thioglycollate peritoneal. Methods &amp; Materials: Alcoholic extracts of SM and NS were obtained by the soxhlet method. Male Balb/C mice were divided into 5 groups and gavage orally for 14 days with SM, NS, the mixture of extracts of these two, DMSO 30% as the control group, and dexamethasone as the positive control group. The safety profile and acute toxicity in mice were assessed. On day 10, acute peritonitis was induced by thioglycollate 3%. Finally, the total anti-oxidant power and NO concentration were measured by FRAP and Griess method, respectively, in the serum of treated mice. Ethical Considerations: All experimental process was performed following the guidelines according to the Animal Ethics Committee of Arak University of Medical Sciences (IR.ARAKMU.REC.1397.359). Results: Acute toxicity test showed no significant changes in weight and physical appearance of the mice. However, the extract and their mixture decreased NO level significantly (P=0.000) in serum. Also, the mixture significantly increased total anti-oxidant power (P=0.015). Conclusion: Results showed that the SM and NS extract mixture demonstrated anti-inflammatory activity, inhibiting inflammatory mediators such as NO and increasing anti-oxidant power, thus supporting its therapeutic potential in slowing down inflammatory processes in inflammation disorders.