| Background Neuroinflammation plays a crucial role in the pathophysiologic mechanisms of neuro-degeneration and leads to cognitive impairment.The mainly symptoms of neurodegenera-tion and cognitive impairment include the impairment of learning and memory ability,which seriously influence the quality of patients’ daily life and pose enormous burdens on families and society.The pathological characteristics of neuroinflammation include the activation of microglia in the central nervous system(CNS),which leads to inflammation induced by the release of pro-inflammatory factors and chemokines,and the oxidative stress caused by the increase release of reactive oxygen species(Reactive oxygen species,ROS).In addition,the accumulation of A beta amyloid and cell apoptosis also contribute to the development of neuroinflammation,and eventually lead to neuronal damage.Microglia,as the main immunocyte in the CNS,play an important role in the process of innate immunity and protect the brain from damages.However,the neurotoxic effect mediated by the excessive activation of microglia is an important pathological mechanism for neurodegenerative diseases.Lipopolysaccharide(LPS),the main pathogenic compon-ent of gram-negative bacteria endotoxin,is a powerful activator of microglia.Studies have shown that LPS can adhere to the toll-like receptors(TLR)4 on the surface of microglia,and lead to the activation of microglia by up-regulating the nuclear transcription factor(NF-κB),mitogen activated protein kinase(MAPK)pathway and the expression of interleukin-1 beta(IL-1β)related kinases,and eventually lead to the release of pro-inflammatory cytokines,neurotoxic molecules,proteases,and ROS.Oxidative stress,which mainly induced by the release of ROS and the destruction of redox defense system,is another important pathological mechanism of neurodegenerative diseases and cognitive impairment mediated by neuroinflammation.Oxidative stress is closely associated with the activation of microglia.Oxidative burst is an early biochemical event of the activation of microglia.The Nicotinamide adenine dinucleotide phosphate(NADPH)oxidase will be activated by the activation of microglia,and lead to the release of oxygen radicals and hydrogen peroxide.The process of apoptosis is a spontaneous programmed cell death so as to maintain the stability of the internal environment,and is essential for homeostasis in multicellular life forms.Studies have shown that neuronal apoptosis induced by neuroinflammation in CNS is considered to be another potential mechanism for cognitive impairment.The cell apoptosis caused by neuroinflammatory injury,which leads to synaptic dysfunction,plays an important role in animal models of cognitive impairment.Methane,as the simplest alkane chain organic,plays an important role in “greenhouse effect”.Due to the physical and chemical properties of gas,methane could freely cross cell membranes and enter into organelles by the manner of simple diffusion.In addition,methane could freely cross biological barriers,such as the blood-brain barrier(BBB).Studies have showed that methane plays protective roles in myocardial injury caused by ischemia/reperfusion(IR),spinal cord injury,pancreatitis,hepatitis,acute lung injury,and even diabetic retinopathy via the anti-inflammatory,antioxidative and anti-apoptotic pathways.However,whether methane also has a protective effect on neuroinflammation and cognitive impairment has not been reported.This study established an animal model of neuroinflammatory injury,and explored the potential mechanisms of LPS-induced neuroinflammatory injury and cognitive impairment from the ethology and molecular biology aspects.Furthermore,the anti-inflammatory,antioxidative and anti-apoptotic functions of methane on neuroinflammatory injury in mice were further investigated and verified in vitro experiments.Methods 1.The research of methane on the improvement of cognitive impairment induced by neuroinflammatory injury in mice Male C57BL/6 mice(20-25 g)at 8-12 weeks were randomly divided into control,methane-rich-saline(MS),LPS and LPS+MS groups.After 5 days Morris water maze(MWM)navigation training,single dose injection of LPS(5 mg/kg)was applied by intraperitoneal(i.p.)to establish a neuroinflammatory injury model of mice.MS(20 m L/kg,MS and LPS+MS groups)or normal saline(20 m L/kg,control and LPS groups)was administered by i.p.0.5 hours after the LPS injection at 12 hours intervals for continuous 7 days and a total of 14 times.The spontaneous activity of mice was evaluated by the open field test(OFT)at 24 hours and the 7th day.MWM spatial probe tests were performance to evaluate the spatial memory ability of mice at the 7th day 2 hours after the OFT.2.The research of methane alleviates neuroinflammatory injury of mice via the anti-inflammatory function Experiment 1: Male C57BL/6 mice(20-25 g)at 8-12 weeks were randomly divided into control and LPS groups.single dose injection of LPS(5 mg/kg)was applied by i.p.to establish a neuroinflammatory injury model of mice.The hippocampus and prefrontal cortex were harvested(8 hours in control group;2,4,6,8 hours respectively in LPS groups)to detect the expression of pro-inflammatory factors by Enzyme-linked immunosorbent assay(ELISA).Experiment 2: Male C57BL/6 mice(20-25 g)at 8-12 weeks were randomly divided into control,MS,LPS and LPS+MS groups.Single dose injection of LPS(5 mg/kg)was applied by i.p.to establish a neuroinflammatory injury model of mice.MS(20 m L/kg,MS and LPS+MS groups)or normal saline(20 m L/kg,Control and LPS groups)was administered by i.p.0.5 hour after the LPS injection at 12 hours intervals for 2 times.The hippocampus and prefrontal cortex were harvested 4 hours after LPS injection to detect the expression of pro-inflammatory factors by ELISA.The brains were harvested(24 hours and 7 days after LPS injection)to detect the expression of(Ionized calcium binding adaptor molecule-1,IBa-1)by immunofluorescent staining.The hippocampus was harvested(24 hours and 7 days after LPS injection)to detect the expression and phosphorylation of P-65-NF-κB and P-38-MAPK by western-blot.3.The research of methane alleviates neuroinflammatory injury of mice via the antioxidative function Experiment 1: Male C57BL/6 mice(20-25 g)at 8-12 weeks were randomly divided into control,MS,LPS and LPS+MS groups.Single dose i.p.injection of LPS(5 mg/kg)was used to establish a neuroinflammatory injury model of mice.MS(20 ml/kg,MS and LPS+MS groups)or normal saline(20 ml/kg,Control and LPS groups)was administered by i.p.0.5 hour after the LPS injection at 12 hours intervals for 2 times.The hippocampus and prefrontal cortex were harvested at 24 hours after LPS injection to detect the expression Malonaldehyde(MDA)and the activity of Superoxide dismutase(SOD).Experiment 2: The paraffin embedded brain section samples from the second part of the study were collected to detect the expression of oxidative stress marker 8-Hydroxy-2 deoxyguanosine(8-OHd G)in hippocampus by immunofluorescent staining.Furthermore,the protein-denatured hippocampus samples from the second part of the study were collected to detect the expression of inducible Nitric oxide synthase(i NOS)and NOX-2 by western-blot.4.The research of methane alleviates neuroinflammatory injury of mice via the anti-apoptotic function The paraffin embedded brain section samples from the second part of the study were collected to detect cell apoptosis in hippocampus by hippocampus by Td T-mediated d UTP Nick-End Labeling(TUNEL).Furthermore,the protein-denatured hippocampus samples from the second part of the study were collected to detect the expression of Bax and Bcl-2 by western-blot.5.The verification research of the anti-inflammatory,antioxidative and anti-apoptotic functions of methane in vitro experiments Experiment 1: The BV-2 cells were plated and divided into control and LPS groups.The medium in the four groups were stimulated with the same amount of serum-free DMEM or LPS(100 ng/m L)respectively.After that,each group was divided into 4 subgroups,and the MS was added with the concentration of 0,5,10,and 20 μL/m L respectively.The cell viability of BV-2 cells in each group was detected by MTT assay.Experiment 2: The BV-2 cells were plated and divided into control,MS,LPS and LPS+MS groups.0.5 hour after the pre-treatment of normal saline or MS(20 μL/m L),the BV-2 cells in each group was stimulated with serum-free DMEM or LPS(100 ng/m L)respectively.The supernatant samples were collected 6 hours after LPS stimulation to detect the expression of proinflammatory factors by ELISA.Experiment 3: The BV-2 cells were plated and divided into control,MS,LPS and LPS+MS groups.0.5 hour after the pre-treatment of normal saline or MS(20 μL/m L),the BV-2 cells in each group was stimulated with the same amount of serum-free DMEM or LPS(100 ng/m L)respectively.The cells samples were collected 1 hour after LPS stimulation to detect the expression and phosphorylation of P-65-NF-κB,P-38-MAPK,and the expression of i NOS,NOX-2,Bax and Bcl-2 by western-blot.Results 1.Methane improved cognitive impairment induced by neuroinflammatory injury Each group mouse could find the hidden platform during the MWM navigation training,and the average escape latency,average swimming distance were gradually shortened as training times go on.The comparison of intra-group showed that the other three groups mice showed no significant statistical difference in the average speed in each day except for the LPS group.The comparison between groups showed that there was no significant statistical difference in the average escape latency,average swimming distance and average swimming speed in each day.The results of OFT showed that methane intervention could increase the distance and active time of mice at 24 hours after LPS-induced neuroinflammatory injury;however,there was no significant statistical difference in the distance and active time in each group mouse at the 7th day.There was no statistically significant difference in swimming distance and swimming speed between groups during MWM space exploration test.Methane intervention could increase the crossing times and the distance traveled in target quadrant%.2.Methane alleviated neuroinflammatory injury via anti-inflammatory function The expression of pro-inflammatory factors reached peak at 4 hours after LPS-induced neuroinflammatory injury.Methane could inhibit the expression of pro-inflammatory factors(IL-6,TNF-α and IL-1β)at 4 hours.In addition,methane could weaken the positive signal intensity of IBa-1 and the cell size/cell body ratio in hippocampus at 24 hours and the 7th day after LPS-induced neuroinflammatory injury so as to inhibit the activation of microglia.Furthermore,methane intervention could reduce the inflammation by inhibiting the phosphorylation of P-38-MAPK and P-65-NF-κB at 24 hours after LPS challenge,and the phosphorylation of P-38-MAPK at the 7th day.3.Methane suppressed LPS induced oxidative stress Methane could reduce the expression of MDA and elevate SOD activity at 24 hours after LPS-induced neuroinflammatory injury in mice.In addition,methane could weaken the positive signal intensity of 8-OHd G in hippocampus,and reduce the expression of i NOS and NOX-2 so as to suppress oxidative stress during the acute phase of LPS-induced neuroinflammatory injury.4.Methane mitigated LPS-induced apoptosis via anti-apoptotic function Methane could reduce the TUNEL-labeled apoptotic cells at 24 hours and the 7th day after LPS-induced neuroinflammatory injury in mice.Furthermore,the cell apoptosis was inhibited via the increase expression of anti-apoptotic protein Bcl-2 and the decrease of Bax/Bcl-2 ratio.5.Methane alleviated inflammation of BV-2 cell induced by LPS via an-inflammatory,antioxidative and anti-apoptotic functions Methane or LPS had no cytotoxic effect on the cell viability of BV-2 cells.In addition,methane could inhibit the expression of pro-inflammatory factors(IL-6,TNF-α and IL-1β)stimulated by LPS.Furthermore,methane could inhibit the phosphorylation of P-38-MAPK and P-65-NF-κB,suppress the proteins expression of i NOS and NOX-2,and increase the expression of anti-apoptotic protein Bcl-2 along with the decrease of Bax/bcl-2 ratio so as to exert anti-inflammatory,antioxidant and anti-apoptotic functions in vitro.Conclusion The cognitive impairment resulted from LPS-induced neuroinflammatory injury in mice could be improved by the treatment of methane.The protective role of methane may be related to the inhibition of P-65-NF-κB and P-38-MAPK phosphorylation,thereby inhibiting the activation of microglia and the release of pro-inflammatory factors.This effect may be also related to the inhibition of i NOS and NOX-2 expression,thereby inhibiting oxidative stress.The inhibition of cell apoptosis via the decrease of Bax/bcl-2 system may be also involved in this beneficial effect of methane.This research provided theoretical evidences for the further clinical exploration of the prevention and treatment of neurodegenerative diseases and cognitive dysfunction caused by neuroinflammation. |
