| Perioperative neurocognitive disorders(PND)refers to the destruction or change of cognitive function that occurs before and after surgery.It is a hot and difficult point of research.The main causes are age,underlying diseases,surgery,and inflammation.Ageing and surgery are currently considered to be the main risk factors for PND.So far,although a lot of manpower and material resources have been invested in cognitive research,there is currently no effective evidence-based treatment.Microglia are the most important immune effector cells.A large number of studies have confirmed that microglia are closely related to the occurrence of neurodegenerative diseases,neurodevelopmental disorders,mental diseases,and chronic neuropathic pain.Chronic inflammation is the pathological basis of most neurodegenerative diseases,and microglia are the central link of inflammation.TNF-?,IL-1?,and IL-6 are the main pro-inflammatory cytokines synthesized and released after activation of microglia.Inhibiting the inflammatory response and oxidative stress induced by the activation of microglia can reduce the degree of nerve damage,so it may become a new direction for alleviating and treating neuroinflamm-ation-related diseases.TLR4,as a "portal" protein,initiates the body's inflammatory chain reaction,which is mainly expressed by microglia in the brain and is one of the key signal transduction receptors for its immune function.Microglia regulate neuronal activity through TLR4.TLR4 activated can promotes the massive secretion of pro-inflammatory factors TNF-?,IL-1?,and IL-6,and exerts a powerful inflammatory effect.Esketamine is an optical isomer of ketamine,which interacts with a variety of receptors.Studies have shown that ketamine has antidepressant and antiinflammatory effects.At present,the research of esketamine is mainly focused on antidepressant,and the effects of different doses and duration of esketamine on learning and memory have not been clarified.On this basis,this study puts forward the following hypothesis that the use of subanesthetic dose of esketamine in the postoperative stage of elderly mice can inhibit the proliferation of microglia and inhibit the TLR4/NF-?B signaling pathway,thereby reducing the production of inflammatory factors,thus improve the postoperative neurocognitive function of elderly mice.We hope this study can provide experimental basis and new ideas for finding and determining the treatment target of PND.Part one The influence of esketamine on postoperative cognitive function and dose selectionObjective: To explore the optimal subanesthetic dose of esketamine to improve postoperative cognitive dysfunction in mice.Methods: Using 18-month-old C57BL/6 mice,they were randomly divided into two groups,namely the control group(C group)and the operation group(S group).The laparotomy exploratory operation model was established.Both groups were given different concentrations of esketamine(0,5,10,20mg/kg)by injected into the abdominal cavity one week,the open field test(OFT)was performed to detect the spontaneous activity of the animals,and the Morris water maze was subjected to a behavioral test.Result:1.In the open field experiment,compared with the control group,the mice in the operation group decreased their distance,moving speed,and the number of crossings(P<0.01),and the number of rearing(P<0.05).In the water maze experiment,the escape latency of the mice in the operation group was longer than that in the control group(P<0.01);in probe trial,the time spent in target quadrant and the number of crossing platforms in operation group were less than those in the control group(P<0.001).2.After administering different doses of esketamine,the open field experiment was doing again.It showed that there was no statistical difference between the control groups in the distance,moving speed,frequency of crossing,time spent in the central grid,and the number of rearing;in the operation groups,distance and moving speed in the SK0 group was decreased compared with the control groups(P<0.01)and SK2group(P<0.05).Compared with the control groups,the frequency of crossing was significantly reduced in SK0 and SK3 groups,and the number of rearing in SK0 group was significantly less than the control group(P<0.05).The difference is Statistical significance.The water maze experiment showed that after the administration of different doses of esketamine,the escape latency of the control groups and the SK1 and SK2 groups decreased with the increase of training days.In the operation groups,the SK0 and SK3 groups had no significant improve in escape latency with the increase of training days.In the probe trial,the time spent in the target quadrant of the SK0 group and the SK3 group was shorter than that of the control group,and the target quadrant stay time of the SK0 group was also shorter than that of the SK2 group(P<0.05).The platform crossover number in the SK0 group was less compared with the control group and SK2 group(P<0.05).Conclusion : The exploratory laparotomy can cause postoperative cognitive dysfunction in aged mice;a certain subanesthetic dose of esketamine has neurocognitive protection,and esketamine 10mg/kg is selected as the drug treatment dose.Part two Subanesthetic esketamine in mediating microglia through TLR4/NF-?B pathway in postoperative neurocognitive disordersObjective: To study the effect of subanesthetic dose of esketamine through TLR4/NF-?B signaling pathway to regulate microglia on central inflammation on postoperative neurocognition.Methods: 18-month-old C57BL/6 mice were used to establish a laparotomy exploratory surgery model,lipopolysaccharide(LPS)was used to stimulate microglial cell proliferation,and a subanesthetic dose of esketamine was administered.They were divided into Control group,Model group,Model +esketamine(Model+K2)group,Model+esketamine+LPS(Model+K2+LPS)group,Model+LPS(Model+LPS)group,observing the changes in learning and memory abilities of old mice through behavioral tests;qPCR detect the levels of inflammatory factors TNF-?,IL-1? and IL-6,Western Blot detection Iba-1,TLR4 and p-NF-?Bp65 protein expression levels;immunofluorescence detection Iba-1 and p-NF-?Bp65 protein expression levels.Result:1.The results of the open field test showed that compared with the Control group,the distance and moving speed of the Model group and Model+LPS group were significantly reduced(P <0.01),the distance and moving speed of the Model+K2group were significantly increased compared with the Model+LPS group(P<0.01).The number of crossings in the Model+LPS group was significantly lower than that in the Control group(P<0.01),and the number of crossings in the Model+K2 group and Model+K2+LPS group were higher than that in the Model+LPS group(P<0.05).The time spent in center of the mice in the Model+LPS group was shorter than that in the Control group(P<0.05).Compared with the number of rearing,the mice in the Model group(P<0.05)and Model+LPS group(P<0.01)were significantly less than those in the Control group,and the Model+LPS group was significantly less than the Model+K2 group(P<0.05).2.The results of the MWM showed that as the training days increased,the escape latency of the mice in each group was gradually shortened except for the Model group and the Model+LPS group.The escape latency of mice in Model group and Model+LPS group was increased compared with Control group and Model+K2 group(P<0.01).Compared with Model+LPS group,the escape latency of Model+K2+LPS group was shorter.The probe trial showed that compared with the Control group(P<0.01)and Model+K2 group(P<0.05),the target quadrant stay time and the number of crossing platforms were significantly reduced in the Model group and Model+LPS group.3.The levels of inflammatory factors TNF-?,IL-1? and IL-6 were detected by qPCR.The results showed that compared with the Control group,the levels of TNF-?and IL-1? m RNA in the Model group and the Model+LPS group were significantly increased(P<0.05).Compared with the Control group,the IL-6m RNA levels in the Model+LPS group were also significantly increased(P <0.05),the IL-6 level of Model+K2 group was lower than that of Model+LPS group(P<0.05)4.The Western Blot results showed that the expression of Iba-1,TLR4 and p-NF-?B p65 proteins in Model+LPS group were significantly higher than that in Control group(P<0.01);compared with Model+LPS group,the expression level of Iba-1,TLR4 and p-NF-?Bp65 protein decreased in Model+K2 group.Compared with the Model+LPS group,the expression levels of Iba-1 and TLR4 protein in the Model+K2+LPS group decreased,and the difference was statistically significant.5.Immunofluorescence results showed that compared with the Control group and the Model+K2 group,the Iba-1 and p-NF-?Bp65 protein positive regions in the Model+LPS group increased,and the difference was statistically significant.Conclusion:1.Surgery and LPS stimulation promote the activation of microglia in the central nervous system of aged mice,up-regulate the TLR4/NF-?B signaling pathway,promote neuro-inflammatory response,and cause neurocognitive dysfunction.2.Esketamine inhibits the TLR4/NF-?B signaling pathway of microglia in the central nervous system of aged mice,reduces the level of inflammatory factors,improves neuroinflammatory response,and relieves postoperative neurocognitive dysfunction.Part three Cell experiments confirm the mechanism of esketamine regulating the inflammation of microgliaObjective: In vitro cell experiments confirmed that esketamine reduces inflammation by inhibiting the TLR4/NF-?B signaling pathway in BV2 cells.Methods: The BV2 microglia cell line was used,and the cultured cells were divided into: Control group,LPS group,LPS+K group,LPS+TAK-242 group,LPS+TAK-242+K group.Control group was pretreated with PBS for BV2 cells;LPS group was treated with 1?g/m L LPS injury;LPS+K2 group was treated with 1?g/m L LPS injury and then treated with 5?g/m L esketamine;LPS+TAK-242 group was treated with 1?g/m L LPS injury and then 100nmol/L TAK-242 solution;LPS+TAK-242+K group was treated with 1?g/m L LPS injury,and then treated with100nmol/LTAK-242 solution and 5?g/m L esketamine.24 h later,MTT test to detect cell viability;qPCR to detect the levels of inflammatory factors TNF-?,IL-1? and IL-6;immunofluorescence detection of TLR4 and p-NF-?Bp65 protein expression levels.Results:1.MTT results showed that cell viability decreased in the LPS group compared with the other groups,and the difference was statistically significant(P<0.01).2.The qPCR results showed that compared with the Control group,the levels of TNF-?,IL-1? and IL-6 m RNA in the LPS group were significantly increased(P<0.05).After the intervention of esketamine or TAK-242,inflammation was reduced(P<0.01).After the intervention of esketamine combined with TAK-242,the inflammatory factors IL-1? and IL-6 m RNA were further reduced(P<0.05),and the difference was statistically significant.3.Immunofluorescence results showed that TLR4 and p-NF-?Bp65 protein expression increased after LPS treatment,and TLR4 and p-NF-?Bp65 protein expression decreased after esketamine and TAK-242 treatment.Conclusion: LPS increases the expression of pro-inflammatory factors by activating the TLR4/NF-?B signaling pathway in BV2 cells;esketamine protects the inflammatory response induced by LPS in BV2 cells,and its mechanism may be inhibiting the TLR4/NF-?B signaling pathway. |
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