| A growing number of studies have demonstrated that inflammatory damage is the pathogenesis of central nervous system diseases such as depression,Alzheimer's disease and the like.As a classical inflammatory stimulator,lipopolysaccharide?LPS?binds to the receptor and initiates a series of phosphorylation processes,which promotes transcription factor-?B?NF-?B?into the nucleus and releases a large number of inflammatory factors,such as tumor necrosis factor??TNF-??,interleukin 6?IL-6?,interleukin-1??IL-1??,interleukin 10?IL-10?and so on.A recent study showed that the offspring exposed in utero to infection/inflammation had a significantly higher risk of developing autism.Hypothetically,prenatal maternal inflammation may increase cerebral susceptibility to LPS injury in rat offspring.However,the underlying mechanisms remain unclear.It is well established that COX2 plays an important role in inflammation-involved diseases.Among all the PGs produced by the oxidation of arachidonic acid by COX,prostaglandin D2?PGD2?is the most abundant prostaglandin in human brain tissue.PGD2 exerts a variety of physiological and pathophysiological effects through the prostaglandin D1 receptor?DP1?and the prostaglandin D2 receptor?DP2?.This study mainly observed the effect of prenatal maternal inflammation on brain injury induced by LPS in rat offspring,and preliminarily explored its mechanism from COX2-PGD2-DPs pathway.Part ? Effect of prenatal maternal inflammation on LPS-induced brain injury in offspring ratsObjective:To observe the effects of prenatal inflammation on LPS-induced brain injury in offspring rats and the changes of COX2-PGD2-DPs pathway in rat brain tissue.Through COX2intervention,the correlation between COX2-PGD2-DPs pathway and the influence of prenatal inflammation on susceptibility of offspring rats to LPS brain injury was preliminarily confirmed.Method:1.The construction of an inflammatory model during pregnancyIn this experiment,paired rats were checked daily for vaginal plugs,indicating pregnancy.Pregnant females?n=30?were housed singly and LPS diluted in saline was injected intraperitoneally?i.p.?at the dose of300?g/kg into pregnant rats?n=21?on gestational day?GD?11,14 and18,respectively.The control group?n=9?consisted of pregnant rats injected with normal saline on the same days.2.Establishment of LPS-induced brain injury model in offspring rats70 male offspring rats treated with LPS or NS during pregnancy were randomly selected on the 60th day for the experimental modeling.5?l LPS?12?g/?l?or equal volume artificial cerebrospinal fluid?CSF?were injected into the lateral ventricles of both sides.3.The experimental groupSeven experimental groups were studied,including normal control?NS+CSF group?,prenatal infection?LPS+CSF group?,postnatal infection?NS+LPS group?,prenatal and postnatal infection?LPS+LPS group?,prenatal infection+MXC?LPS+CSF+MXC group?,postnatal infection+MXC?NS+LPS+MXC group?and prenatal and postnatal infection+MXC?LPS+LPS+MXC group?.On the second day after modeling,3mg/kg meloxicam?MXC?was administered intragastrically for one week,and the other groups were given an equal volume of 0.5%CMC.4.Main observation indicators and methodsThe water maze test,open field test and spontaneous activity test were used to detect the behavioral changes of the offspring rats.HE staining was employed to measure the morphologic change of hippocampal and cortical neurons.The contents of PGD2,IL-6,TNF-?and IL-1?in rat hippocampus and cortex were determined by ELISA.The biochemical method was adopted to detect SOD activity and MDA content in rat cortex and hippocampus.The expression of APP,A?,COX2,DP1and DP2 were measured by Western blot.Results:1.Compared with NS+CSF group,LPS+CSF rats have longer latency of finding platform,fewer times of shuttling platform,fewer exploring behaviors and autonomic movement activities;neurons in hippocampus and cortex both showed little karyopyknosis;the contents of PGD2 and TNF-?in cortex increased significantly;the contents of IL-6 and IL-1?in hippocampus increased significantly;SOD activity in cortex and hippocampus decreased significantly and MDA content in cortex increased significantly;the protein expressions of A?,COX2 and DP2 in cortex increased significantly,while the protein expression of DP1 decreased significantly;the protein expressions of APP,A?and COX2 in hippocampus increased significantly;however,there was no significant difference in the contents of IL-1?,IL-6 and the protein expression of APP in cortex;there was no significant difference in the levels TNF-?,PGD2 as well as the protein expressions of DP1 and DP2in hippocampus.2.Compared with the NS+CSF group,NS+LPS and LPS+LPS rats have longer latency of finding platform,fewer times of shuttling platform,fewer exploring behaviors and autonomic movement activities;neurons in hippocampus and cortex both showed obviously karyopyknosis;the contents of PGD2,IL-6 and IL-1?increased significantly in hippocampus and cortex;SOD activity decreased significantly and MDA content increased significantly;the protein expressions of APP,A?,COX2 and DP2 were significantly increased,whereas the expression of DP1was significantly decreased.There was no significant difference in the level of TNF-?in hippocampus between NS+CSF group and NS+LPS group.3.Compared with NS+LPS group,LPS+LPS rats have longer latency of finding platform,fewer times of shuttling platform,fewer horizontal movement and autonomic movement activities;neurons in hippocampus and cortex both showed obviously karyopyknosis;the contents of IL-6and IL-1?increased significantly in cortex;the contents of PGD2andIL-6increased significantly in hippocampus;SOD activity in cortex and hippocampus decreased significantly and MDA content in hippocampus increased significantly;the protein expressions of APP,A?,COX2 and DP2 were significantly increased,whereas the expression of DP1 was significantly decreased in cortex and hippocampus;however,there was no significant difference in the contents of PGD2?TNF-?and MDA in cortex;there was no significant difference in the contents of IL-1?and TNF-?in hippocampus.4.Compared with LPS+LPS group,LPS+LPS+MXC rats have shorter latency of finding platform,more times of shuttle platform,more exploring behaviors and more autonomic movement activities;the karyopyknosis of hippocampus and cortex neurons decreased;the contents of IL-6,TNF-?and IL-1?in cortex decreased obviously;the contents of PGD2 and IL-6 in hippocampus decreased obviously;SOD activity in cortex and hippocampus increased significantly and MDA content in cortex decreased significantly;the protein expressions of A?,COX2 and DP2 in cortex decreased significantly;the protein expressions of APP,A?,COX2 and DP2 in hippocampus decreased significantly,while the protein expression of DP1 increased significantly;however,there was no significant difference in the levels of PGD2,DP1and the protein expression of APP in cortex;there was no significant difference in the levels of MDA,TNF-?and IL-1?in hippocampus.Conclusion:1.Prenatal inflammation can cause behavioral and pathological abnormalities in offspring rats.2.Prenatal inflammation can cause increased expression of COX2 in cortex of offspring rats,increased expression of DP2,and decreased expression of DP1.3.Prenatal inflammation can increase the susceptibility of offspring rats to LPS-induced brain injury in adulthood.The mechanism may be related to the imbalance of COX2-PGD2-DPs,which leads to oxidative stress and inflammatory reaction,and increases the protein expressions of A?and APP.4.Meloxicam has obvious protective effect on brain injury of LPS-induced in offspring from prenatal inflammation exposure.its mechanism may be related to inhibiting COX2 activity,reestablishing COX2-PGD2-DPs balance,and further reducing oxidative stress and inflammatory response.Part ? Effect of prenatal maternal inflammation on LPS-induced primary neuron injury in offspring ratsObjective:To observe the susceptibility of primary neurons of pregnant rats stimulated by inflammation to LPS-induced neuronal damage,and to explore its mechanism from the COX2-PGD2-DPs pathway.Method:1.Primary neuron culture in SD rats and establishment of LPS-induced neuronal injury model?1?Paired rats were checked daily for vaginal plugs,indicating pregnancy.Pregnant females were housed singly and LPS diluted in saline was injected intraperitoneally?i.p.?at the dose of 300?g/kg into pregnant rats on gestational day?GD?11,14 and 18,respectively.The control group consisted of pregnant rats injected with normal saline on the same days.?2?Primary culture neurons from brain of newborn rat within 24h was cultured.On the 7th day,the purity of neuron was identified by immunocytochemistry of neuron specific enolase.To determine the optimal concentration and time point of LPS for primary culture neurons injury.MTT assay was used to determine the cell survival rate,and the kit was used to determine the leakage rate of LDH.2.The effect of COX2 intervention on LPS-induced primary neuron injury in offspring with prenatal inflammation?1?Normal?non-pregnant inflammatory treatment?primary cultured neurons and LPS-induced damage model of normal primary neurons were used.MTT assay was used to determine the cell survival rate,and the kit was used to determine the leakage rate of LDH to find out the optimal concentration of MXC.?2?Experimental grouping and observation indicatorsCells were divided into 7 groups:NS,LPS,NS+LPS,LPS+LPS,LPS+MXC,NS+LPS+MXC and LPS+LPS+MXC,n=6 in each group.Among them,NS group was normal control;LPS group was the primary neurons of offspring from prenatal inflammation exposure;NS+LPS group was the LPS-treated primary neurons of offspring from normal pregnant rat;LPS+LPS group was the LPS-treated primary neurons of offspring from prenatal inflammation exposure.Main observation indicators and methodsThe contents of PGD2,IL-6,TNF-?and IL-1?in the primary culture neurons were determined by ELISA.The biochemical method was adopted to detect SOD activity and MDA content in the primary culture neurons.Analysis of neuronal apoptosis by flow cytometry.The expressions of APP,A?,COX2,DP1 and DP2 were measured by Western blot.3.The effect of DPs intervention on LPS-induced primary neuron injury in offspring with prenatal inflammation?1?Normal?non-pregnant inflammatory treatment?primary cultured neurons and LPS-induced damage model of normal primary neurons were used.MTT assay was used to determine the cell survival rate,and the kit was used to determine the leakage rate of LDH to find out the optimal concentration of DP1 agonist?BW245C?,DP1 antagonist?BWA868C?,DP2 agonist?DK-PGD2?and DP2 antagonist?AZD1981?,respectively.?2?Experimental grouping and observation indicatorsCells were divided into seven groups:NS,LPS,NS+LPS,LPS+LPS,LPS+drug,NS+LPS+drug and LPS+LPS+drug groups.These drugs including DP1 agonist?BW245C?,DP1 antagonist?BWA868C?,DP2agonist?DK-PGD2?,and DP2 antagonist?AZD1981?were used.Main observation indicators and methodsMTT assay was used to determine the cell survival rate,and the kit was used to determine the leakage rate of LDH.Analysis of neuronal apoptosis by flow cytometry.Result:1.When 1?g/ml LPS acts on neurons for 24 hours,the cell survival rate decreases obviously,while LDH leakage rate increases obviously.2.The effect of COX2 intervention on LPS-induced primary neuron injury in offspring with prenatal inflammation?1?Compared with the NS+LPS group,1×10-8M MXC could significantly increase the cell survival rate and reduce the leakage rate of LDH of LPS-induced neurons,then 1×10-8M was chosen as the optimal concentration of MXC.?2?Compared with NS group,MTT value of primary neurons in LPS group was significantly decreased;LDH leakage rate and apoptosis increased significantly;the contents of PGD2,TNF-?and IL-1?increased significantly;SOD activity decreased significantly and MDA content increased significantly;the protein expressions of APP,A?,COX2 and DP2 increased significantly;however,there was no significant difference the protein expression of DP1 and the content of IL-6.Compared with NS group,the MTT value of neurons in NS+LPS group and LPS+LPS group decreased significantly;LDH leakage rate and apoptosis increased significantly;the contents of PGD2,IL-6,TNF-?and IL-1?increased significantly;SOD activity decreased significantly and MDA content increased significantly;the protein expressions of APP,A?,COX2,DP1 and DP2 increased significantly.Compared with NS+LPS group,the MTT value of primary neurons in LPS+LPS group decreased significantly;LDH leakage rate and apoptosis increased significantly;SOD activity decreased significantly;the protein expressions of APP,COX2,DP1 and DP2 increased significantly;however,there was no significant difference in the contents of PGD2,IL-6,TNF-?,IL-1?,MDA and the protein expression of A?.Compared with LPS+LPS group,the MTT value of primary neurons in LPS+LPS+MXC group increased significantly;LDH leakage rate and apoptosis decreased significantly;SOD activity increased significantly;the protein expressions of APP,A?,COX2 and DP2 decreased significantly;however,there was no significant difference in the contents of PGD2,IL-6,TNF-?,IL-1?,MDA and the protein expression of DP1.3.The effect of DPs intervention on LPS-induced primary neuron injury in offspring with prenatal inflammation?1?Compared with the NS+LPS group,1×10-5M DP1 agonist?BW245C?or 1×10-5M DP2 antagonist?AZD1981?can significantly increased the cell viability and decreased LDH leakage rate of LPS-induced neurons,1×10-5M was selectedastheoptimal administration concentration.Compared with the NS+LPS group,1×10-5M DP1 antagonist?BWA868C?or 1×10-5M DP2 agonist?DK-PGD2?can significantly decreased the cell viability and increased LDH leakage rate of LPS-induced neurons,therefore,1×10-5M was selected as the optimal administration concentration.?2?Compared with LPS+LPS treatment,after DP1 agonist?BW245C?or DP2 antagonist?AZD1981?intervention,neuron survival rate increased significantly,LDH leakage rate and apoptosis decreased significantly.However,after administration of DP1 antagonist?BWA868C?or DP2 agonist?DK-PGD2?,neuron survival rate decreased significantly,LDH leakage rate and apoptosis increased significantly.Conclusion:1.The cell viability was decreased,LDH leakage rate while cell apoptosis and the expression of COX2 and its downstream products were significantly increased in primary neurons of offspring with prenatal inflammation.2.Prenatal inflammation increases susceptibility of offspring neurons to LPS injury.3.The mechanism by which prenatal inflammation increases susceptibility of offspring neurons to LPS injury may involve imbalance of COX2-PGD2-DPs pathway. |
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