Preliminary Study Of Acetate In Ameliorating Postoperative Neurocognitive Disorder In Aged Mice

BackgroundPostoperative neurocognitive disorder is usually manifested as disturbances in consciousness,orientation,memory,cognition,and sleep after surgery with anesthesia.The incidence of postoperative neurocognitive disorder is close to 10%-54%in the postoperative weeks.The postoperative process has long-term effects on the human body,including incomplete postoperative function,increased mortality,prolonged hospital stay,long-term cognitive decline,and dementia.Although the specific pathophysiological mechanisms are not completely clear at present,more and more evidence indicate that neuroinflammation plays a key role in the occurrence and development of postoperative neurocognitive disorder.Therefore,in-depth study of the mechanisms of postoperative neurocognitive disorder is of great significance for its potential intervention and therapeutic targets.Acetate is a short-chain fatty acids?SCFAs?,which is mainly derived from the intestinal microorganisms'fermentation of dietary fiber.It is an important metabolite of the human body.Acetate can be used as a source of cellular energy and plays a key role in energy supply,fatty acid synthesis,and lipid metabolism;meanwhile,acetate has been reported to regulate immune and inflammatory responses.However,its role in postoperative neurocognitive disorder has not been explored.This study aims to explore the effects of acetate in postoperative neurocognitive disorder and the possible mechanisms,to provide potential intervention and treatment for it.Methods1.Model establishment of postoperative neurocognitive disorder and detection of pro-inflammatory cytokines'levels in hippocampus.?1?Model construction of postoperative neurocognitive disorder in aged mice:Male C57BL/6 aged mice?12 months?were randomly divided into normal group and surgery group.Exploratory laparotomy with isoflurane anesthesia was used to construct the model of postoperative neurocognitive disorder.?2?Behavioral tests:The training phase of Morris water maze?MWM?was performed for 5 consecutive days before surgery,so that all mice learned to find the hidden platform,which eliminating the known cognitive deficits before surgery;in order to exclude the effects of surgery on the motor function of aged mice,open field test?OFT?was used to detect the spontaneous activity of the mice on postoperative day 3?POD 3?;the testing phase of MWM was performed to observe the altered neurocognitive function in aged mice on POD 3 and POD 7.?3?Expression of pro-inflammatory cytokines in hippocampus:Mice hippocampus tissues were obtained on day 1 and day 7 after surgery,and the expression of inflammatory factors were detected by ELISA,including TNF-?,IL-6 and IL-1?.2.The role of exogenous acetate in postoperative neurocognitive disorder and neuroinflammation?1?The effects of acetate on postoperative neurocognitive function in aged mice:the aged mice were randomly divided into 4 groups:normal group,acetate group,surgery group and acetate intervention group.Before the model construction,all mice were trained for 5 consecutive days in MWM.Mice in surgery and acetate intervention groups were undergone laparotomy with isoflurane;mice in the acetate and acetate intervention groups were given drinking water containing acetate at a concentration of 200 m M and fed for 7 consecutive days before surgery.Open field test was performed on POD 3,and MWM tests were performed on POD 3 and POD 7.?2?The function of acetate on peripheral and hippocampal inflammation:After construction of animal model for 6 hours,peripheral blood of mice was acquired and the levels of inflammatory factors?TNF-?,IL-6,IL-1??in serum were determined;meanwhile,the expression of pro-inflammatory cytokines?TNF-?,IL-6 and IL-1??in hippocampus were also measured at 6 hours post-surgery,on POD 1 and POD 7,respectively.?3?The influence of acetate on inflammatory signaling pathways in hippocampus:Western Blot was used to detect the expression of inflammatory signaling pathways including NF-?B and p38 MAPK in hippocampus at 6 hours post-surgery,on POD 1 and POD 7,respectively.?4?The effects of acetate on oxidative stress in hippocampus:Hippocampus tissues of aged mice were obtained on POD 1 and POD 7.Western Blot was applied to measure the levels of oxidative stress molecules,including NADPH oxidase 2?Nox-2?and inducible nitric oxide synthase?iNOS?;meanwhile,the expression of 8-hydroxydeoxyguanosine?8-OH-d G?in hippocampus was detected by immunofluorescence.?5?The function of exogenous acetate on microglia activation:On POD 1 and POD 7,hippocampus tissues of aged mice were obtained.The expression of microglia activation's marker?IBA-1?in CA1 region of hippocampus was measured through immunofluorescence.3.The effects of exogenous acetate on inflammation in BV2 microglia?1?Different concentrations of acetate exerted function in LPS induced inflammation at different time points:BV2 cells were divided into the following groups:control group,LPS group,and acetate intervention group with different concentrations?10 m M,20 m M,and 50 m M?.Acetate was added 30 minutes before or after LPS stimulation.After LPS?100 ng/ml?stimulation for 6 hours,the cell supernatant was collected to detect the expression of pro-inflammatory factors,including TNF-?and IL-6.?2?The effects of different concentrations of acetate on the cell viability of BV2microglia:BV2 cells were divided into control group and LPS group.Different concentrations of acetate?0 m M,10 m M,20 m M,and 50 m M?were added in each well.After LPS?100 ng/ml?stimulation for 6 hours,cell viability was measured through the MTT method.?3?The influence of acetate on inflammatory signaling pathways in BV2 microglia:BV2 cells were divided into the following 4 groups:control group,acetate group,LPS group,and acetate intervention group.After LPS?100 ng/ml?stimulation for 30 minutes,acetate with the concentration of 20 m M was added in the well.After 6 hours of LPS stimulation,the cells were collected to detect the expression of NF-?B and p38 MAPK.?4?The effects of acetate on oxidative stress in BV2 microglia:BV2 cells were cultured and divided into 4 groups mentioned above.After LPS?100 ng/ml?stimulation for 30 minutes,acetate with the concentration of 20 m M was added in the well.After 6hours of LPS stimulation,the cells were obtained to measure the expression of Nox-2and iNOS.4.Exploring the preliminary mechanism of acetate regulation of inflammatory response?1?BV2 cells were cultured in vitro,the small interfering RNA was used to knock down the expression of G protein-coupled receptors including GPR41 and GPR43 on the membrane of BV2 cells.After the interference for 48 hours,cells were stimulated with LPS?100 ng/ml?,followed by acetate?20 m M?.After 6 hours of LPS stimulation,the cell supernatant was collected to measure TNF-?and IL-6 levels.At the same time,after interference with si-GPR41 and si-GPR43,the cells were collected to determine the m RNA levels of GPR41 and GPR43.?2?BV2 cells were cultured in vitro,and si-G_q?was applied to knock down the G_q? subunit.Then cells were stimulated with LPS?100 ng/ml?and acetate?20 m M?.After 6hours of LPS treatment,the supernatant was obtained to detect TNF-?and IL-6expression;meanwhile,after interference of G_q?subunit,the m RNA expression of G_q?in BV2 cells was detected.In addition,the inhibitor of G_i/o?subunit?pertussis toxin,PTX,50 ng/ml?was added in the cell well,followed by LPS?100 ng/ml?and acetate?20m M?treatment.The cell supernatant was obtained after 6 hours of LPS treatment to detect TNF-?and IL-6 expression.Results1.After the construction of PND model,aged mice showed changes in neurocognitive function accompanied by inflammatory response in hippocampus?1?Aged mice both in normal and surgery groups could find the hidden platform in the training phase of MWM for 5 consecutive days before surgery.There was no significant difference in latency and average speed between the two groups during the 5days.On POD 3,the open field test showed that there was no significant difference in the total distance and pause time between the two groups.In the testing phase of MWM,the numbers of crossing times in the surgery group were significantly lower than that in the normal group;while the percentage of time spent and distance traveled in target quadrant had no statistic difference compared with the normal group.Compared with the normal group,the numbers of crossing times,and the percentage of time spent as well as distance traveled in target quadrant were significantly decreased on POD 7.?2?Surgery stimulation could induce high expression of pro-inflammatory factors including TNF-?,IL-6,and IL-1?in hippocampus on POD 1.On POD 7,IL-1?expression in hippocampus were still up-regulated in surgery group,while the levels of TNF-?and IL-6 had no statistic difference compared with normal group.2.Acetate partially improved the cognitive function of PND aged mice and reduced the inflammatory response as well as oxidative stress in hippocampus?1?Aged mice in 4 groups could find the hidden platform during 5 consecutive days' training in MWM before surgery.There was no significant difference in the latency and average speed between the four groups.On POD 3,open field test indicated that there was no significant difference in total distance and pause time.Exogenous acetate supplementation could improve the neurocognitive function of aged mice after surgery,which were manifested as the increase of crossing times,and percentage of time spent as well as distance traveled in target quadrant both on POD 3 and POD 7.?2?Acetate supplementation could decrease the levels of peripheral and central inflammatory factors?TNF-?,IL-6 and IL-1??in the acute phase after surgery.It may inhibit the expression of signaling pathways?NF-?B/p38 MAPK?and suppress oxidative stress factors'levels?Nox-2,iNOS,8-OH-d G?in hippocampus.In addition,acetate significantly suppressed the expression of IBA-1,the marker of microglia activation in hippocampus.3.Acetate inhibited the inflammatory response and oxidative stress induced by LPS in BV2 microgliaAcetate supplementation in vitro effectively reduced the levels of TNF-?and IL-6 in BV2 microglia induced by LPS;meanwhile,acetate could inhibit the expression of inflammatory signaling pathways?NF-?B/p38 MAPK?and oxidative stress factors?Nox-2,iNOS?in BV2 microglia.4.Acetate exerted anti-inflammatory function by initiating GPR43-Gq?signaling?1?Using small interfering RNA to knock down gene expression of short-chain fatty acid receptors including GPR41 and GPR43 in BV2 microglia.When GPR41 was knocked down,acetate effectively reduced LPS-induced expression of TNF-?and IL-6.When the short chain fatty acid receptor GPR43 was knocked down,acetate could not exert the anti-inflammatory effects mentioned above.?2?Besides,using si-G_q?to knock down the expression of G_q?subunit in BV2 cells,acetate could not significantly decrease the expression of TNF-?and IL-6 after LPS stimulation.After the use of G_i/o?subunit inhibitor?PTX?,acetate supplement still effectively inhibited LPS-induced inflammation in BV2 microglia.ConclusionSupplementation of acetate,a short-chain fatty acid,may effectively inhibit hippocampal microglia activation,reduce inflammatory response and oxidative stress in hippocampus of aged mice,thereby improving postoperative neurocognitive function;acetate treatment in vitro could significantly suppress LPS-induced TNF-?and IL-6levels in BV2 microglia,inhibit the expression of inflammatory signaling pathways?NF-?B and p38 MAPK?and decrease the levels of oxidative stress factors?Nox-2 and iNOS?.Acetate may regulate microglial activity mainly through initiating GPR43-G_q?signaling,and then suppressing the activation of NF-?B as well as p38 MAPK signaling pathways.