Prenatal Immune Challenge In Rats Worsens Susceptibility To Cerebral Ischemic Injury In Adolescent Offspring Via COX-2-PGD2-DPs Pathway Activation

In recent years,the scientific community has generally accepted the theory that stress response during pregnancy and embryonic development has a significant impact on the occurrence and development of adult diseases in offspring.Many studies on the pathogenesis of diseases have begun to pay attention to the relationship between maternal and offspring.Stroke is one of the three leading diseases that cause human death,which seriously affects human life expectancy and quality of life.Although a large number of literatures have found that ischemic brain injury is the result of a combination of many factors,including free radical damage,excitatory amino acid toxicity,calcium overload,apoptosis,oxidative stress,inflammatory response,etc.,but its earliest occurrence time and mechanism is not yet clear.Accordingly,further studies and discussions on how prenatal immune would cause and exacerbate stroke-induced brain injury could contribute to the discovery of potential new drug targets and help the development of novel therapies.Numerous studies have shown that the inflammatory response plays an important role in the stroke-induced brain injury.COX-2,which is an inducible cyclooxygenase produced by the body under pathological conditions,oxidizes free fatty acids such as arachidonic acid（AA）and polyenoic fatty acids in the form of combination.COX-2 also participates in a variety of inflammatory responses and induces apoptosis by modulating inflammation.This study mainly observed whether prenatal inflammation exposure could activate COX-2-PGD2-DPs pathway to alter susceptibility of offspring to stroke-induced brain injury in vivo and in vitro.In addition,this study explored the mechanism which COX-2-PGD2-DPs pathway involved in prenatal inflammation exposure on later stroke susceptibility and nerve cell injury in offspring rats.Part I Changes of cerebral cortical and hippocampal COX-2-PGD2-DPs pathway in prenatal inflammation-exposured offspring rat subjected to focal cerebral ischemia reperfusionAim:To observe the changes of cerebral cortical and hippocampal COX-2-PGD2-DPs pathway in prenatal LPS-exposured offspring rat subjected to focal ischemia reperfusion,and preliminarily identify that prenatal LPS-exposure can magnify the susceptibility of offspring rat to focal ischemia reperfusion-induced brain injury.Method:1.Experimental treatment and grouping:30 pregnant Sprague-Dawley（SD）rats were used.After 1 week of acclimation,pregnant rats were randomly divided into the normal group（n=10）and prenatal LPS-exposure rats（n=20）.All pregnant rats were injected intraperitoneally（i.p.）saline or LPS（300μg/kg）on gestational days（GD）11,14 and 18,respectively.Pups from the prenatal LPS-exposure and control groups were fed on postnatal day（PD）60.At PD 60,the male offspring of rats（220–250 g,n=48）were randomly divided into four groups:（i）the N+Sham group,given 0.5mL,i.p.,saline during pregnancy and animals received sham operation;（ii）the LPS+Sham group,given 300μg/kg,i.p.,LPS during pregnancy and received sham operation;（iii）the N+MCAO group,given 0.5mL,i.p.,saline during pregnancy and animals received MCAO;（iv）the LPS+MCAO group,given300μg/kg,i.p.,LPS during pregnancy and animals received MCAO,n=12for each groups.2.Observation index:neurologic dysfunction score and TTC staining in rats.HE staining was employed to measure the morphologic change of neurons.The SOD activity and MDA content were detected according to biochemical enzymology.The levels of IL-1β,IL-6,TNF-αand PGD2 in rat hippocampus and cortex were determined by ELISA.Western blotting was used to determine the expression of COX-2,DP_1/2,caspase-3,cleaved caspase-3,caspase-9 and Bcl-2 in rat hippocampus and cortex.Results:（1）There was no significant difference in neurological deficit scores between the LPS+Sham group and the N+Sham group（P>0.05）.In the N+MCAO group and the LPS+MCAO group,the neurological deficit scores of rats were significantly increased compared with the N+Sham group and the LPS+Sham group,respectively（P<0.01）.Compared with the N+MCAO group,the neurological deficit scores of rats were significantly increased in the LPS+MCAO group（P<0.01）.（2）No obvious infarction was observed in the N+Sham group and the LPS+Sham group.However,in the N+MCAO group and the LPS+MCAO group,the infarct volume was significantly increased compared with the N+Sham group and the LPS+Sham group,respectively（P<0.001）.Compared with the N+MCAO group,the infarct volume was significantly increased in the LPS+MCAO group（P<0.001）.（3）In the N+Sham group and the LPS+Sham group,nerve cells were arranged tightly in order,and most cells appeared round or oval with large cell bodies.There were significant differences in the mortality of nerve cells between the N+Sham group and the LPS+Sham group（P<0.05&P<0.01）.In the N+MCAO group and the LPS+MCAO group,rat hippocampal CA1subfield and cortical nerve cells presented obvious nuclear pyknosis,irregular arrangement and a reduction in the number of neurons.Compared with the N+Sham group and the LPS+Sham group,the mortality of nerve cells increased significantly in the N+MCAO group and the LPS+MCAO group,respectively（P<0.001）.Compared with the N+MCAO group,the mortality of nerve cells increased significantly in the LPS+MCAO group（P<0.001）.（4）The activity of SOD in the LPS+Sham group was significantly lower than that of the N+Sham group,and the levels of TNF-α,IL-6,IL-1β,PGD2 and the MDA contents increased significantly in the hippocampus and cortex（P<0.001&P<0.05）.Compared with the N+Sham group and the LPS+Sham group,the activity of SOD was significantly decreased in the N+MCAO group and the LPS+MCAO group,respectively,and the levels of TNF-α,IL-6,IL-1β,PGD2 and the MDA contents increased significantly in the hippocampus and cortex（P<0.001&P<0.01&P<0.05）.Compared with the N+MCAO group,the activity of SOD was decreased significantly in the LPS+MCAO group,and the levels of TNF-α,IL-6,IL-1β,PGD2 and the contents of MDA increased significantly in the hippocampus and cortex（P<0.001&P<0.01&P<0.05）.（5）Compared with the N+Sham group,the expression levels of COX-2,DP₂,caspase-3,cleaved caspase-3 and caspase-9 were significantly increased in the LPS+Sham group,whereas the expression levelsofDP₁andBcl-2weresignificantlydecreased（P<0.05&P<0.01&P<0.001）.The expression levels of COX-2,DP₂,caspase-3,cleaved caspase-3 and caspase-9 in N+MCAO group and the LPS+MCAO group were significantly increased compared with the N+Sham group and LPS+Sham group,respectively,whereas the expression levelsofDP₁andBcl-2weresignificantlydecreased（P<0.05&P<0.01&P<0.001）.The expression levels of COX-2,DP₂,caspase-3,cleaved caspase-3 and caspase-9 in the LPS+MCAO group were significantly increased compared with the N+MCAO group,whereas the expression levels of DP₁ and Bcl-2 were significantly decreased（P<0.05&P<0.01&P<0.001）.Conclusion:1.Maternal LPS exposure during pregnancy can magnify brain damage of offspring rats induced by MCAO/R.2.The imbalance of cerebral cortical and hippocampal COX-2-PGD2-DPs pathway in prenatal inflammation-exposured offspring rat subjected to focal cerebral ischemia reperfusion enhanced oxidative stress and inflammatory response,which further promoted neuronal apoptosis.Part II Effect of COX-2 intervention on OGD/R-treated primary neuron in prenatal inflammation-exposured offspring ratsAim:To observe that prenatal LPS-exposured can magnify the susceptibility to OGD/R-induced injury in prenatal LPS-exposured primary neuron and the COX-2-PGD2-DPs signaling pathway after intervention with COX-2 inhibitor in prenatal LPS-exposured primary neuron after OGD/R.Method:1.Establishment of an OGD/R-treated prenatal LPS-exposured primary neuron model:primary neurons were prepared from the prenatal LPS-exposured offspring rats at postnatal day（PD）0.The purity of primary cultured neurons was identified by Immunofluorescence staining on day 7after primary culture.Primary neurons were treated by oxygen-glucose deprivation and reoxygenation（OGD/R）to determine the suitable time point of OGD/R.2.Effect of COX-2 inhibitor on the prenatal LPS-exposured primary neuron after OGD/R.（1）Different concentrations of celecoxib acted in OGD/R-treated primary neurons.CCK-8 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 celecoxib.（2）According to the optimal concentration of celecoxib,the neurons were divided into N+N group,LPS+N group,N+OGD/R group,LPS+OGD/R group,LPS+N+celecoxib group,N+OGD/R+celecoxib group,LPS+OGD/R+celecoxib group;CCK-8 assay was used to determine the cell survival rate,the kit was used to determine the leakage rate of LDH,flow cytometry analysis and TUNEL assay were used to determine the neuronal apoptosis,biochemical enzymology was used to determine the SOD activity and MDA content,ELISA was used to determine the levels of IL-1β,IL-6,TNF-αand PGD2 and Western blotting was used to determine the expression of COX-2,DP_1/2,caspase-3,cleaved caspase-3,caspase-9 and Bcl-2proteins.Results:1.Primary neurons cultured for 7 days aggregated into clumps,strong stereoscopic,faintly visible nucleolus profile and synaptic interwoven into a dense network with coarse;the purity of neurons was over 97%by immunofluorescence staining;compared with the N+N group,the survival rate of primary neuron after OGD/R of different time treatment for was significantly decreased（P<0.001）.The leakage rate of LDH was significantly increased（P<0.001）.For the proper proportion of primary neuron of damage,we selected oxygen-glucose deprivation for 2 h and reoxygenation for 24 h as a condition for OGD/R-treated prenatal LPS-exposured primary neuron model establishment conditions.2.Effects of COX-2 inhibitor on the prenatal LPS-exposured primary neuron after OGD/R.（1）Compared with the N+N group,the survival rate of primary neurons in the LPS+N group and the N+OGD/R group were significantly decreased,and the leakage rate of LDH was increased（P<0.001&P<0.01）;the survival rate of primary neurons in LPS+OGD/R group was significantly decreased compared with the LPS+N group and N+OGD/R group,respectively,whereas the leakage rate of LDH was increased（P<0.001）;after celecoxib treatment,compared with the LPS+N group,the survival rate of primary neurons in the LPS+N+celecoxib group was significantly increased（P<0.01）,and the leakage rate of LDH was decreased（P<0.01）;compared with the N+OGD/R group,the survival rate of primary neurons in the N+OGD/R+celecoxib group was significantly increased（P<0.05）,and the leakage rate of LDH was decreased（P<0.001）;compared with the LPS+OGD/R group,celecoxib significantly increased cell survival rate（P<0.05）and decreased LDH leakage rate（P<0.001）.（2）Compared with the N+N group,the number of apoptotic cells in the LPS+N group and the N+OGD/R group were significantly higher than those in the N+N group（P<0.001&P<0.05）;the number of apoptotic cells in the LPS+OGD/R group were significantly increased compared with the LPS+N group and the N+OGD/R group,respectively（P<0.001&P<0.01）;after celecoxib treatment,compared with the N+OGD/R group,the number of apoptotic cells in the N+OGD/R+celecoxib group were significantly decreased（P<0.05）;compared with the LPS+OGD/R group,celecoxib significantly decreased the number of apoptotic cells（P<0.01）.（3）The activity of SOD in the LPS+N group and the N+OGD/R group were significantly lower than that of the N+N group,and the levels of TNF-α,IL-6,IL-1β,PGD2 and the MDA contents increased significantly（P<0.001&P<0.05）;compared with the LPS+N group and the N+OGD/R group,the activity of SOD was significantly decreased in the LPS+OGD/R group,respectively,and the levels of TNF-α,IL-6,IL-1β,PGD2 and the MDA contents increased significantly（P<0.001&P<0.01&P<0.05）;after celecoxib treatment,compared with the LPS+N group,the activity of SOD was increased significantly in the LPS+N+celecoxib group,and the levels of TNF-α,IL-6,IL-1β,PGD2 and the contents of MDA decreased significantly（P<0.01&P<0.05）;compared with the N+OGD/R group,the activity of SOD was increased significantly in the N+OGD/R+celecoxib group,and the levels of TNF-α,IL-6,IL-1β,PGD2 and the contents of MDA decreased significantly（P<0.001&P<0.05）;compared with the LPS+OGD/R group,theactivityofSODwasincreasedsignificantlyinthe LPS+OGD/R+celecoxib group,and the levels of TNF-α,IL-6,IL-1β,PGD2and the contents of MDA decreased significantly（P<0.001）.（4）Compared with the N+N group,the expressions of COX-2,DP_1/2,caspase-3,cleaved caspase-3 and caspase-9 in the LPS+N group and the N+OGD/R group were significantly increased,whereas the expression of Bcl-2 was significantly decreased（P<0.001&P<0.01&P<0.05）;the expression levels of COX-2,DP_1/2,caspase-3,cleaved caspase-3 and caspase-9 in the LPS+OGD/R group were significantly increased compared with the LPS+N group and the N+OGD/R group,respectively,whereas the expressionlevelofBcl-2wassignificantlydecreased（P<0.001&P<0.01&P<0.05）;after celecoxib treatment,compared with the LPS+N group,the expressions of COX-2,DP_1/2,caspase-3,cleaved caspase-3 and caspase-9 in the LPS+N+celecoxib group were significantly decreased（P<0.01）,whereas the expression of Bcl-2 was significantly increased（P<0.01）;compared with the N+OGD/R group,the expressions of COX-2,DP_1/2,caspase-3,cleaved caspase-3 and caspase-9 in the N+OGD/R+celecoxib group were significantly decreased（P<0.05）,whereas the expression of Bcl-2 was significantly increased（P<0.05）;compared with the LPS+OGD/R group,the expressions of COX-2,DP_1/2,caspase-3,cleaved caspase-3 and caspase-9 in the LPS+OGD/R+celecoxib group were significantly decreased（P<0.05）,whereas the expression of Bcl-2 was significantly increased（P<0.01）.Conclusion:1.An OGD/R-treated prenatal LPS-exposured primary neuron model was successfully established after oxygen-glucose deprivation for 2 h and reoxygenation for 24 h applied to primary neuron.2.Maternal LPS exposure increased the susceptibility of primary neurons to OGD/R-treated injury in offspring rats;SOD activity of primary neurons with OGD/R treatment decreased significantly,MDA,TNF-a,IL-6,IL-1βand PGD2 content increased significantly;COX-2,DP_1/2,caspase-3,cleaved caspase-3 and caspase-9 protein expression increased significantly,while Bcl-2 protein expression decreased significantly.3.Celecoxib,a COX-2 inhibitor,can significantly improve the primary neuronal damage induced by OGD/R in prenatal LPS exposured rats.Its mechanism may involve inhibiting COX-2,reducing the production of PGD2,down-regulating the expression of DP_1/2,caspase-3,cleaved caspase-3 and caspase-9,and up-regulating the expression of Bcl-2.Part III Effect of DP_1/2 intervention on OGD/R-treated primary neuron in prenatal inflammation-exposured offspring ratsAim:To observe the effects of DP_1/2 agonist and antagonist on prenatal LPS-exposured primary neuron subjected to OGD/R.Method:1.Different concentrations of DP_1/2 intervention groups were induced with OGD/R followed by DP receptor agonists or antagonists.Intervention groups were treated with agonists of DP₁（BW245C）,DP₂（DK-PGD2）or antagonists of DP₁（BWA868C）,DP₂（AZD1981）,respectively.CCK-8assay 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 DP_1/2 agonist and antagonist.2.According to the optimal concentration of DP_1/2 agonist and antagonist,the neurons were divided into N+N group,LPS+N group,N+OGD/R group,LPS+OGD/R group,LPS+N+intervention group,N+OGD/R+intervention group,LPS+OGD/R+intervention group;CCK-8assay was used to determine the cell survival rate,the kit was used to determine the leakage rate of LDH,flow cytometry analysis was used to determine the neuronal apoptosis.Results:compared with the N+N group,the survival rate of primary neurons in the LPS+N group and the N+OGD/R group were significantly decreased,and the leakage rate of LDH was increased（P<0.001&P<0.01）;the survival rate of primary neurons in LPS+OGD/R group was significantly decreased compared with the LPS+N group and N+OGD/R group,respectively,whereas the leakage rate of LDH was increased（P<0.001）;after DP_1/2 agonist and antagonist treatment,compared with the LPS+N group,N+OGD/R group and LPS+OGD/R group,the neuron viability increased significantly after administration of DP₁ agonists（BW245C）and DP₂ antagonist（AZD1981）,whereas DP₁ antagonist（BWA868C）and DP₂ agonists（DK-PGD2）decreased significantly neuron viability.In particular,the LDH leakage rate decreased significantly after administration of DP₁ agonists（BW245C）and DP₂ antagonist（AZD1981）,whereas DP₁ antagonist（BWA868C）and DP₂ agonists（DK-PGD2）increased significantly LDH leakage rate.Moreover,the number of apoptotic cells decreased efficiently after administration of DP₁ agonists（BW245C）and DP₂ antagonist（AZD1981）,whereas DP₁ antagonist（BWA868C）and DP₂ agonists（DK-PGD2）increased efficiently the number of apoptotic cells.Conclusion:1.Activation of DP₁ can significantly improve the primary neuronal damage of prenatal LPS-exposured rats treated with OGD/R,and inhibition of DP₁ can significantly aggravate the primary neuronal damage of prenatal LPS-exposured rats treated with OGD/R.2.Activation of DP₂ can significantly aggravate the primary neuronal damage of prenatal LPS-exposured rats treated with OGD/R,and inhibition of DP₂ can significantly improve the primary neuronal damage of prenatal LPS-exposured rats treated with OGD/R.