Fecal microbiota transplantation alleviates experimental colitis through the Toll-like receptor 4 signaling pathway

BACKGROUND Fecal microbiota transplantation(FMT)has shown promising therapeutic effects on mice with experimental colitis and patients with ulcerative colitis(UC).FMT modulates the Toll-like receptor 4(TLR4)signaling pathway to treat some other diseases.However,it remains unknown whether this modulation is also involved in the treatment of UC.AIM To clarify the necessity of TLR4 signaling pathway in FMT on dextran sodium sulphate(DSS)-induced mice and explain the mechanism of FMT on UC,through association analysis of gut microbiota with colon transcriptome in mice.METHODS A mouse colitis model was constructed with wild-type(WT)and TLR4-knockout(KO)mice.Fecal microbiota was transplanted by gavage.Colon inflammation severity was measured by disease activity index(DAI)scoring and hematoxylin and eosin staining.Gut microbiota structure was analyzed through 16S ribosomal RNA sequencing.Gene expression in the mouse colon was obtained by transcriptome sequencing.RESULTS The KO(DSS+Water)and KO(DSS+FMT)groups displayed indistinguishable body weight loss,colon length,DAI score,and histology score,which showed that FMT could not inhibit the disease in KO mice.In mice treated with FMT,the relative abundance of Akkermansia decreased,and Lactobacillus became dominant.In particular,compared with those in WT mice,the scores of DAI and colon histology were clearly decreased in the KO-DSS group.Microbiota structure showed a significant difference between KO and WT mice.Akkermansia were the dominant genus in healthy KO mice.The ineffectiveness of FMT in KO mice was related to the decreased abundance of Akkermansia.Gene Ontology enrichment analysis showed that differentially expressed genes between each group were mainly involved in cytoplasmic translation and cellular response to DNA damage stimulus.The top nine genes correlating with Akkermansia included Aqp4,Clca4a,Dpm3,Fau,Mcrip1,Meis3,Nupr1 L,Pank3,and Rps13(|R|>0.9,P<0.01).CONCLUSION FMT may ameliorate DSS-induced colitis by regulating the TLR4 signaling pathway.TLR4 modulates the composition of gut microbiota and the expression of related genes to ameliorate colitis and maintain the stability of the intestinal environment.Akkermansia bear great therapeutic potential for colitis.

化学合成小干扰RNA对大鼠脑内AQP4蛋白沉默效果验证与筛选

目的针对大鼠脑内AQP4蛋白使用化学合成法制备数条小干扰RNA链(siRNA),对其中两条链进行沉默效果验证,并筛选出效果显著的小干扰RNA链。方法根据大鼠的AQP4mRNA序列选择2个不同的靶点,采用化学合成的方法分别合成2条siRNA,并同时合成阴性对照siRNA。雄性成年Wistar大鼠18只,随机分为注射阴性对照液组、注射725链组和注射1006链组,并分别经侧脑室注射相应药物。6 h后将其全部处死,利用免疫组织化学法和原位杂交法分别检测脑内AQP4蛋白和mRNA的表达水平。结果给药6 h后,注射725链siRNA组大鼠脑内AQP4在蛋白质和基因两个水平均显著低于注射阴性对照液组和注射1006链组(P<0.05)。结论脑室注射725链siRNA可以在6 h内有效沉默大鼠脑内AQP4的表达。

Early hyperbaric oxygen therapy inhibits aquaporin 4 and adrenocorticotropic hormone expression in the pituitary gland of rabbits with blast-induced craniocerebral injury

In the present study, rabbits were treated with hyperbaric oxygen for 1 hour after detonator-blastinduced craniocerebral injury. Immunohistochemistry showed significantly reduced aquaporin 4 expression and adrenocorticotropic hormone expression in the pituitary gland of rabbits with craniocerebral injury. Aquaporin 4 expression was positively correlated with adrenocorticotropic hormone expression. These findings indicate that early hyperbaric oxygen therapy may suppress adrenocorticotropic hormone secretion by inhibiting aquaporin 4 expression.

Aquaporin 4 expression and ultrastructure of the blood-brain barrier following cerebral contusion injury

This study aimed to investigate aquaporin 4 expression and the ultrastructure of the blood-brain barrier at 2-72 hours following cerebral contusion injury, and correlate these changes to the formation of brain edema. Results revealed that at 2 hours after cerebral contusion and laceration injury, aquaporin 4 expression significantly increased, brain water content and blood-brain barrier permeability increased, and the number of pinocytotic vesicles in cerebral microvascular endothelia cells increased. In addition, the mitochondrial accumulation was observed. As contusion and laceration injury became aggravated, aquaporin 4 expression continued to increase, brain water content and blood-brain barrier permeability gradually increased, brain capillary endothelial cells and astrocytes swelled, and capillary basement membrane injury gradually increased. The above changes were most apparent at 12 hours after injury, after which they gradually attenuated. Aquaporin 4 expression positively correlated with brain water content and the blood-brain barrier index. Our experimental findings indicate that increasing aquaporin 4 expression and blood-brain barrier permeability after cerebral contusion and laceration injury in humans is involved in the formation of brain edema.

Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

Objective To investigate the role of extracellular signal-regulated kinase1/2（ERK1/2） pathway in the regulation of aquaporin 4（AQP4） expression in cultured astrocytes after scratch-injury. Methods The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase（LDH） leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2（p-ERK1/2） expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 μmol/L U0126（ERK1/2 inhibitor） was incubated in the medium at 30 min before the scratch-injury in some groups. Results Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. Conclusion These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Attenuation of opioid addiction in mice lacking aquaporin 4

OBJECTIVE To examine the effects of aquaporin 4(AQP4) on opioid addiction and underlie the mechanism behind it. METHODS(1) In the heroin-induced self-administration(SA) experiment,we explored the role of AQP4 on heroin-induced psychological addiction. After the mice were trained to learn heroin-induced SA under a fixedratio1(FR1) reinforcement program for 7 d,we randomly switched the heroin doses to 0.00625,0.0125,0.025,0.05 or 0.1 mg·kg^(-1)per infusion to counterbalance assignment design. In the end,all mice underwent extinction training and reinstatement testing.(2) In oral sucrose self-administration,5% sucrose solution was used for the mice and the procedures were similar to heroin SA.(3) In morphine-induced hyperactivity test,mice were habituated in the test apparatus for 30 min and then were given saline(10 mL·kg^(-1),sc) or morphine(10 or 20 mg·kg^(-1),sc) to record the locomotion for 1.5 h.(4) For the in vivo microdialysis experiment,mice were surgically implanted with intracranial guide cannula into nucleus accumbens(AP +1.4 mm,ML ±0.9 mm,DV-3.8 mm from bregma). After 5 d of recovery from surgery,the mice were challenged by saline(10 mL·kg^(-1),sc)or morphine(10 mg·kg^(-1),sc),and then samples were collected every 20 min. RESULTS We found that AQP4 deletion had no effects on sucrose-seeking and sucrose-taking,but it significantly attenuated heroin-taking and heroin-seeking behaviors in heroin self-administration. Besides these,AQP4 deletion had no effects on basal level of locomotion,but dramatically decreased morphine-induced hyperactivity.Furthermore,the in vivo microdialysis studies showed that AQP4 deficiency inhibited morphine(10mg · kg^(-1),sc)-induced elevation of extracellular dopamine levels in nucleus accumbens in mice.CONCLUSION Our present findings demonstrate that AQP4 was potentially involved in the properties of opioid rewarding by inhibiting dopamine release in nucleus accumbens(NAc).

Serum anti-aquaporin 4 antibody in neuromyelitis patients is not correlated with the length of injured spinal cord segments

Clinical information and serum samples of 20 neuromyelitis patients and 30 patients with multiple sclerosis were collected in this study. The expression of anti-aquaporin 4 antibody in the serum of all patients was detected with an indirect immunofluorescence assay, using human embryonic kidney 293 cell line that stably express human-derived aquaporin 4 as a substrate. The characteristics of head and spinal magnetic resonance imaging were also observed in patients who had neuromyelitis and were positive for anti-aquaporin 4 antibody. Results showed that the expression of anti-aquaporin 4 antibody was significantly different between multiple sclerosis patients and neuromyelitis patients. There were 13 out of 20 neuromyelitis patients （including high-risk syndrome） that were positive for anti-aquaporin 4 antibody. The magnetic resonance imaging examinations of the head and spinal cord found that among the 13 positive patients, nine cases showed normal cerebral hemisphere and optic nerve, two cases had optic nerve changes, and one case had an atypical lesion in the brain. All 30 multiple sclerosis patients were negative for this antibody. The experimental findings indicate that patients with neuromyelitis optica had more than three lesioned segments in the spinal cord by magnetic resonance imaging, and the segment length of the injured spinal cord was not associated with the titer of aquaporin 4 antibody in neuromyelitis patients.

Effects of Avastin on Expression of AQP4 in Müller Cells under Hypoxia

The aim of this study was to investigate the effects of Avastin on aquaporin4(AQP4) expression in human retinal Müller cells in vitro under hypoxia,so as to explore the mechanism of Avastin treating retinal edema.The human Müller cells were cultured using the enzymatic digestion method.Müller cells were identified under the transmission electron microscopy and by using immunofluorescence staining.By using semi-quantitative reverse transcription polymerase chain reaction(RT-PCR),the expression of AQP4 mRNA and VEGF mRNA in Müller cells cultured with 500 μmol/L CoCl 2 for 0,3,6,12 and 24 h,and with 0,100,300,500 and 700 μmol/L CoCl 2 for 24 h was detected.The expression of AQP4 mRNA in Müller cells cultured with 50 ng/mL exogenous vascular endothelial growth factor(VEGF) for 0,0.5,1,2 and 4 h,and with 0,25,50 and 75 ng/mL VEGF for 24 h was detected.Amplified cDNA products of AQP4 mRNA in Müller cells cultured with 500 μmol/L CoCl 2 and 200 μg/mL Avastin for 24 h were detected.The results showed that more than 95% cells displayed positive immunofluorescence reaction.Characteristic 8-10 nm intracellular filaments could be seen in the cytoplasm under the transmission electron microscopy.In the CoCl 2 experimental groups,the expression of AQP4 mRNA and VEGF mRNA in Müller cells was increased as compared with the control group.Alteration of AQP4 mRNA and VEGF mRNA levels showed a significantly positive correlation(r 2 =0.822,P<0.05).The expression of AQP4 mRNA in Müller cells was increased by VEGF.The expression of AQP4 mRNA was significantly decreased by Avastin as compared with the control group.It is suggested that Avastin can decrease the expression of AQP4 mRNA in human Müller cells under chemical hypoxic conditions partially via VEGF path,which may be one of the mechanisms of Avastin treating retinal edema.

Lights at night:does photobiomodulation improve sleep?

Sleep is a critical part of our daily routine.It impacts every organ and system of our body,from the brain to the heart and from cellular metabolism to immune function.A consistent daily schedule of quality of sleep makes a world of difference to our health and well-being.Despite its importance,so many individuals have trouble sleeping well.Poor quality sleep has such a detrimental impact on many aspects of our lives;it affects our thinking,learning,memory,and movements.Further,and most poignantly,poor quality sleep over time increases the risk of developing a serious medical condition,including neurodegenerative disease.In this review,we focus on a potentially new non-pharmacological treatment that improves the quality of sleep.This treatment,called photobiomodulation,involves the application of very specific wavelengths of light to body tissues.In animal models,these wavelengths,when applied at night,have been reported to stimulate the removal of fluid and toxic waste-products from the brain;that is,they improve the brain’s inbuilt house-keeping function.We suggest that transcranial nocturnal photobiomodulation,by improving brain function at night,will help improve the health and well-being of many individuals,by enhancing the quality of their sleep.

Inhibiting phosphatase and actin regulator 1 expression is neuroprotective in the context of traumatic brain injury

Studies have found that the phosphatase actin regulatory factor 1 expression can be related to stroke,but it remains unclear whether changes in phosphatase actin regulatory factor 1 expression also play a role in traumatic brain injury.In this study we found that,in a mouse model of traumatic brain injury induced by controlled cortical impact,phosphatase actin regulatory factor 1 expression is increased in endothelial cells,neurons,astrocytes,and microglia.When we overexpressed phosphatase actin regulatory factor 1 by injection an adeno-associated virus vector into the contused area in the traumatic brain injury mice,the water content of the brain tissue increased.However,when phosphatase actin regulatory factor 1 was knocked down,the water content decreased.We also found that inhibiting phosphatase actin regulatory factor 1 expression regulated the nuclear factor kappa B signaling pathway,decreased blood-brain barrier permeability,reduced aquaporin 4 and intercellular adhesion molecule 1 expression,inhibited neuroinflammation,and neuronal apoptosis,thereby improving neurological function.The findings from this study indicate that phosphatase actin regulatory factor 1 may be a potential therapeutic target for traumatic brain injury.

Hyperbaric oxygen therapy improves local microenvironment after spinal cord injury

Clinical studies have shown that hyperbaric oxygen therapy improves motor function in patients with spinal cord injury. In the present study, we explored the mechanisms associated with the recovery of neurological function after hyperbaric oxygen therapy in a rat model of spinal cord injury. We established an acute spinal cord injury model using a modification of the free-falling object method, and treated the animals with oxygen at 0.2 MPa for 45 minutes, 4 hours after injury. The treatment was administered four times per day, for 3 days. Compared with model rats that did not receive the treatment, rats exposed to hyperbaric oxygen had fewer apoptotic cells in spinal cord tissue, lower expression levels of aquaporin 4/9 mRNA and protein, and more NF-200 positive nerve fibers. Furthermore, they had smaller spinal cord cavities, rapid recovery of somatosensory and motor evoked potentials, and notably better recovery of hindlimb motor function than model rats. Our findings indicate that hyperbaric oxygen therapy reduces apoptosis, downregulates aquaporin 4/9 mRNA and protein expression in injured spinal cord tissue, improves the local microenvironment for nerve regeneration, and protects and repairs the spinal cord after injury.

Effects of the mitochondrial calcium uniporter on cerebral edema in a rat model of cerebral ischemia reperfusion injury

The present study investigated the effects of the mitochondrial calcium uniporter inhibitor ruthenium red and the agonist spermine on cerebral edema in rats with cerebral ischemia reperfusion injury. Left middle cerebral artery occlusion （MCAO） was induced in rats using the suture method. Following 24 hours of ischemic reperfusion, neurological function scores of rats with MCAO, and rats pretreated with ruthenium red and spermine were significantly lower, however, water content of brain tissue, aquaporin 4 expression and immunoglobulin G （IgG） exudation were significantly higher than those of sham-operated rats. Compared with MCAO rats and spermine-treated rats, neurological function scores were considerably higher, and brain tissue water content, aquaporin 4 expression and IgG exudation decreased in ruthenium red-treated rats. These findings suggest that preventive application of the mitochondrial calcium uniporter inhibitor ruthenium red can significantly decrease aquaporin 4 and IgG expression, influence the permeability of the blood brain barrier, and thereby decrease the extent of cerebral edema.

Aggravated inflammation and increased expression of cysteinyl leuko-triene receptors in the brain after focal cerebral ischemia in AQP4-deficient mice

Objective Aquaporin-4 （AQP4）, the main water channel protein in the brain, plays a critical role in water homeostasis and brain edema. Here, we investigated its role in the inflammatory responses after focal cerebral ischemia. Methods In AQP4-knockout （KO） and wild-type mice, focal cerebral ischemia was induced by 30 rain of middle cerebral arterial occlusion （MCAO）. Ischemic neuronal injury and cellular inflammatory responses, as well as the expression and localization of cysteinyl leukotriene CysLT2 and CysLT~ receptors, were determined at 24 and 72 h after MCAO. Results AQP4-KO mice showed more neuronal loss, more severe microglial activation and neutrophil infiltration, but less astrocyte proliferation in the brain after MCAO than wild-type mice. In addition, the protein levels of both CysLT1 and CysLT2 receptors were up-regulated in the ischemic brain, and the up-regulation was more pronounced in AQP4-KO mice. The CysLT1 and CysLT2 receptors were primarily localized in neurons, microglia and neutrophils; those localized in microglia and neutrophils were enhanced in AQP4-KO mice. Conclusion AQP4 may play an inhibitory role in postischemic inflammation.

Baicalin Reduces Early Brain Injury after Subarachnoid Hemorrhage in Rats

Objective: To evaluate the effect of baicalin on subarachnoid hemorrhage(SAH) in rats and explore the potential mechanisms. Methods: Sprague-Dawley rats underwent experimental SAH and received treatment with baicalin at 10 or 50 mg/kg after 2 and 12 h of SAH. Neurological scores, brain water content, Evans-blue extravasation, and levels of glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), myeloperoxidase(MPO), and malondialdehyde(MDA) were measured 24 h after SAH. Expression of nuclear factor erythroid-related factor 2(Nrf2), NAD(P)H: quinone oxidoreductase 1(NQO1), matrix metalloproteinase-9(MMP-9), aquaporin 4(AQP4), occludin, and zonulaoccludens-1(ZO-1) were detected in the brain by Western blot. Heme oxygenase-1(HO-1) was detected by quantitative polymerase chain reaction, and tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were assessed by enzyme-linked immunosorbent assay. Results: Baicalin attenuated EBI 24 h after SAH in rats(P<0.05). Baicalin elevated neurological scores, GSH-Px, SOD, and increased the expression of Nrf2, NQO1, HO-1, occludin, and ZO-1 in SAH rats(P<0.05 or P<0.01). Baicalin reduced MPO, MDA, and the expression of MMP-9, AQP4, TNF-α, and IL-1β(P<0.05 or P<0.01). Conclusion: Baicalin reduced SAH-induced EBI, partially via activation of the Nrf2/HO-1 pathway and inhibition of MMP-9 and AQP4.

Current and emerging therapies for neuromyelitis optica

Neuromyelitis optica(NMO)is an autoimmune demyelinating disease that mainly affects the optic nerve and spinal cord,potentially resulting in blindness and paralysis.Once thought to be a clinical variant of multiple sclerosis,NMO is currently considered as a different disease with its own features due to the identification of a specific autoantibody against aquaporin 4.Given the high risk of disability,treatment should be launched once the diagnosis is established.Evidence from clinical practice showed that traditional immunosuppressive agents affecting the function of T and B cells could attenuate disease exacerbation.Recently,with better understanding pathogenesis of NMO,increasing bodies of novel therapies and therapeutic targets have been discovered.In this review,the authors discuss the current strategies of treating NMO in details and briefly introduce the potential therapies in future.