Studies On The Effect And Action Mechanism Of LW And Active Fraction Combination On Learning And Memory Impairment Induced By Abnormal Immune Function

There is a two-way adjustment mechanism between the nervous system and the immune system,and one system change affects the function of another system.The nervous system can regulate the function of the immune system through biological active substances such as neurotransmitters and neuropeptides.The immune system can regulate the nervous system function through immunologically active substances such as cytokines,resulting in changes in neuropsychology and behavior.Among them,the influence of peripheral immune system on central learning and memory function is one of the research hotspots of life sciences in recent years.In order to study the effect of immune dysfunction on central learning and memory function,three representative congenital immunodeficiency mice and an acquired immune dysfunction,namely lipopolysaccharide(LPS)induced immune function abnormalities were selected in this study.Meanwhile,the improvement effect and possible mechanism of the traditional Chinese medicine Liuwei Dihuang Decoction(LW)and its active fraction combination LW-AFC were studied.The results included the following two parts:Part 1:The impact of abnormal immune function on learning and memory ability1.Research on learning and memory ability of congenital immunodeficient miceIn congenital immunodeficient mice,Nude mice are congenitally athymic,unable to differentiate T cells normally,resulting in T lymphocyte defects;SCID mice show severe joint immunodeficiency symptoms,normal NK cells,macrophages and granulocytes with T and B lymphocyte dysfunction;NOD-SCID mice lack T,B lymphocytes and NK cells.Three congenital immunodeficient mice,2 months old male Nude mouse(BALB/c),SCID and NOD-SCID,were selected,and BALB/c mice were used as controls.Morris water maze test results showed that compared with BALB/c mice,the escape latency,the number of crossing cycles and the target quadrant residence time of SCID mice did not change significantly during the test period;the escape latency of NOD-SCID mice decreased significantly during the test period(P<0.05),there was no significant change in the number of crossing cycles and the target quadrant residence time.The results of the shuttle box experiment showed that compared with BALB/c mice,the number of active avoidance in Nude mice was significantly lower during the test period(P<0.01).There was no significant change in the number of active avoidance in SCID and NOD-SCID mice during the test period,suggesting T lymphocytes Cell defect Nude mice actively avoid ability decreased significantly.The above experimental results show that congenital T lymphocyte defect can reduce the ability of actively avoid learning and memory.2.Effect of LPS-induced immune dysfunction on the ability of learning and memory in miceLPS is a complex of lipids and polysaccharides in the cell wall of Gram-negative bacteria.After intraperitoneal injection of LPS,it triggers an inflammatory reaction,killing and clearing pathogens,and also causing damage to host tissue cells,causing immune damage.In this study,we used a single intraperitoneal injection of LPS to induce acute immune dysfunction and long-term multiple intraperitoneal injection of LPS-induced chronic immune dysfunction in mice to study the effects of acquired immune dysfunction on central learning and memory function.2.1 Effects of single intraperitoneal injection of LPS on spatial learning and memory in miceMale C57 mice of 2-3 months old were selected and the Morris water maze test was performed after the environment was adapted.The experiment was performed once a day for 6 days,the 1-5 days were the training period,and the sixth day was the test period.LPS(250 μg/kg)was administered intraperitoneally(250 Rg/kg)before the first day of study,after the first day of study,before the third day of study,and on the sixth day(test period)to obtain,consolidate and extract information from learning and memory.The results showed that LPS was injected before the first day of learning.Compared with the control group,there were no significant changes in the indexes of the model group during the learning and test periods,suggesting that LPS may have no significant effect on learning and memory acquisition in mice.Immediately injection of LPS after the first of study,compared with the control group,the escape latency of the model group was significantly increased on the 2nd day(P<0.05),and there was no significant change in the follow-up period and the test period,suggesting that LPS may affect the memory consolidation and extraction of mice.LPS was injected before the 3rd day and the test period,Compared with the control group,there were no significant changes in the indexes of the model group during the learning and test periods,suggesting that LPS has no effect on the memory extraction of mice.The above results suggest that a single intraperitoneal injection of LPS(250μg/kg)on day 1 can significantly reduce the ability of mice to consolidate and extract information in learning and memory,which might be related to the immune irnflammatory response caused by intraperitoneal injection of LPS.To determine whether the above-mentioned impairment of learning and memoryability is related to the immune inflammatory response caused by LPS,the change of swimming speed,body weight,body temperature,pain response,and cytokines in plasma and hippocampus were observed.The results showed that the autonomic activity,body weight and body temperature of the mice were significantly decreased(P<0.001),and there was no significant change in swimming speed and pain response;IL-6 levels in plasma and hippocampus were significantly increased(P<0.001).These results suggest that the ability to consolidate and extract information in spatial learning and memory caused by a single intraperitoneal injection of LPS(250μg/kg)after day 1 is associated with an inflammatory response induced by LPS.2.2 Effects of long-term intraperitoneal injection of LPS on spatial learning and memory in miceTwo-month-old male C57 mice were injected intraperitoneally with LPS(250μg/kg)for a long time(once a day for two months).Morris water maze test was performed after environmental adaptation.The results showed that compared with the control group,the escape latency of the model group was significantly increased during the learning period(P<0.01);the escape latency was significantly increased during the test period(P<0.001),and the number of crossing cycles and target quadrant residence time were significantly lower(P<0.001).These results suggest that long-term intraperitoneal injection of LPS(250μg/kg)can damage the spatial learning and memory ability of mice.Part 2:Effect and mechanism of LW and its active fractjion combination on learning and memory impairment induced by LPSLiuwei Dihuang Recipe(LW)is a classic representative of traditional Chinese medicine "Ziyin Bushen".LW-AFC is a component Chinese medicine consisting of active ingredient groups isolated from Liuwei Dihuang Decoction.Studies have shown that LW can improve the immune function of immunocompromised animals;LW-AFC can significantly improve the spatial learning and memory ability of Rapid aging model mice SAMP8 and APP/PS1 transgenic AD model mice.However,it is unclear whether LW and its active fraction combination LW-AFC can improve the learning and memory impairment induced by LPS.1.Effect of LW and its active fraction combination on spatiial learning and memory ability of normal miceThree-month-old male C57 mice were selected and given LW(1.17g/kg)and LW-AFC(0.4g/kg,0.8g/kg,1.6g/kg,once/day for 4 weeks).Morris water maze experiments were performed to observe the effects of different administration time on spatial learning and memory ability of normal mice.Compared with the control group,there were no significant changes in the indexes of the learning period and the test period escape latency after LW and LW-AFC were administered for 1 week,2 weeks and 4 weeks.The above results suggest that the spatial learning and memory ability of mice is not affected by intragastric administration of LW and its active fraction combination.2.Effects of LW and its active fraction combination on LPS-induced spatiallearning and memory impairment2.1 Effects of LW and its active fraction combination on learning and memoryimpairment induced by single intraperitoneal injection of LPSThree-month-old male C57 mice were selected and intragastrically administered LW(1.17g/kg),LW-AFC(0.4g/kg,0.8g/kg,1.6g/kg)and the positive drug TLR4 inhibitor TAK-242(3 mg/g,ip)Morris water maze test was performed two weeks later,and LPS(250 μg/kg)was intraperitoneally injected after the first day to observe the effect on learning and memory impairment in mice.The results showed that compared with the control group,the escape latency of the model group was significantly prolonged on the 2nd day after intraperitoneal injection of LPS(P<0.05),and the TAK-242 group had significant improvement(P<0.05),LW and LW-AFC group there was no significant change in the escape latency,and there was no significant effect on the escape latency,the number of piercing cycles and the target quadrant residence time during the test period.The above results suggest that LW and LW-AFC can not improve the spatial learning and memory impairment caused by single intraperitoneal injection of LPS.2.2 Effects of LW and its active fraction combination on learning and memoryimpairment induced by long-term intraperitoneal injection of LPSThree-month-old male C57 mice were selected and intraperitoneally injected with LPS(250 p,g/kg,once a day)for 2 months.LW(1.17g/kg)and LW-AFC(1.6g/kg)were administered by intragastric administration.A positive drug(TAK-242,3 mg/kg)was administered intraperitoneally,followed by Morris water maze test.The results showed that compared with the control group,the escape latency of the model group was significantly prolonged(P<0.001),the number of crossing cycles was significantly decreased(P<0.05),and the target quadrant residence time was significantly shortened(P<0.01).Compared with the model group,LW and LW-AFC significantly shortened the escape latency of the test period(P<0.001),increased the number of crossing cycles(P<0.05),and prolonged the target quadrant residence time(P<0.05).The TLR4 inhibitor TAK-242 acts equally well.The above results suggest that LW and LW-AFC can improve spatial learning and memory impairment caused by long-term intraperitoneal injection of LPS.3.Mechanism of LW and its active fraction combination to improve learning and memory impairment induced by LPSIntraperitoneal injection of LPS can cause an immune inflammatory reaction,and the production of small molecule inflammatory substances into the brain causes the activation of microglia,which leads to neuroinflammation in the brain and causes learning and memory damage.According to the results of the first part of the study,the time of learning and memory impairment(14 hr)after intraperitoneal injection and the time of recovery of learning and memory impairment(day 5)were taken to measure the biochemical parameters of mouse plasma and whole brain.Immunofluorescence staining was used to observe the activation of microglia in the brain.The results showed that compared with the control group,the integral optical density(IOD)of the whole brain Ibal+cells increased significantly(P<0.01);the positive drug TLR4 inhibitor TAK-242 showed obvious lowering effect(P<0.001);LW and LW-AFC significantly reduced the IOD of whole brain Ibal+ cells(P<0.001).When the learning and memory impairment was restored,there was no significant change on Ibal+cell IOD in the model group compared with the control group.Compared with the model group,TAK-242,LW and LW-AFC had no Significant impact on Ibal+ cell IOD in the mouse brain.The results suggest that microglia in the brain of mice is activated by intraperitoneal injection of LPS,and LW and LW-AFC can inhibit the activation of microglia.Detection of pro-inflammatory factors(IL-6,TNF-a,IL-1β,RANTES,MCP-1,GM-CSF,IFN-y)and anti-inflammatory factors(IL-4,IL-10)and the content of vascular endothelial growth factor(VEGF)in plasma and hippocampus by using Luminex technology.The results showed that the level of pro-inflammatory factors IL-6 and RANTES in the plasma and hippocampus of the model group was significantly higher(P<0.001)and the level of anti-inflammatory factor IL-10 in plasma was significantly lower than that of the control group(P<0.001),TAK-242 significantly decreased the level of pro-inflammatory factors(P<0.05);compared with the model group,the levels of pro-in:flammatory factors IL-6 and MCP-1 in the hippocampus of LW-AFC group were significantly decreased(P<0.05).When the learning and memory impairment was restored,there was no significant change in the levels of inflammatory related cytokines in the plasma and hippocampus of the model group compared with the control group.These results suggest that the levels of pro-inflammatory factors and anti-inflammatory factors are decreased in plasma and hippocampus of mice after intraperitoneal injection of LPS;LW-AFC can inhibit the release of pro-inflammatory cytokines.The above discussion demonstrates that LW-AFC can reduce the levels of pro-inflammatory cytokines and inhibit the activation of microglia,thereby improving learning and memory impairment induced by LPS.Based on the above findings,this study can draw the following conclusions:1.Congenital defects of T cells can cause damage to the ability of mice to active avoid response.2.Abnormal immune function caused by intraperitoneal injection of LPS can cause damage to the central learning and memory function.3.Administration of LW and LW-AFC did not affect the ability of learning and memory of normal mice.4.LW and LW-AFC can improve the spatial learning and memory impairment caused by abnormal immune function caused by intraperitoneal injection of LPS,which might be owing to the activation of microglia in the brain and reducing the elevated inflammation Cytokine levels in plasma and hippocampus.