Effect of agonist on gamma-aminobutyric acid B receptor subunit expression in the hippocampus of epileptic rats

BACKGROUND： An amino acid imbalance has been considered to be responsible for epilepsy pathogenesis. Gamma-aminobutyric acid-B receptor （GABABR） inhibits voltage-sensitive calcium ion channels and GABA or glutamic acid （Glu） neurotransmitter release, which promotes or inhibits onset and development of epilepsy. OBJECTIVE： To explore the effect of baclofen on GBRla and GBR2 mRNA expression in the hippocampus of epileptic rats following kainic acid （KA） induction, and to study the adaptability of GABABR subunits. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment based on molecular biology was performed at the Laboratory Research Center of Second Hospital Affiliated to Soochow University from November 2005 to March 2006. MATERIALS： KA was provided by Sigma, USA. In situ hybridization detection kit of GBRla and GBR2 was provided by Wuhan Boster Biological Technology, China. GABABR agonist （baclofen） was provided by Sigma, USA. METHODS： Forty-four epileptic rats were randomly allocated to epileptic （n = 28） and drug intervention （n = 16） groups. The epileptic group was further divided into post-epileptic subgroups at different time points： 6, 12 hours, 1, 3, 7, 15, and 30 days （n = 4）. The drug intervention group was further divided into intervention controls subgroups at various time points： 6 hours, 1 day, and 3 days （n = 4）. Four additional rats were considered the normal control group and not modeled, but were injected with saline in the hippocampal CA3 region. MAIN OUTCOME MEASURES： GBRla and GBR mRNA expression was detected in the right hippocampal CA1, CA3, and dentate gyrus （DG） areas of the control, epileptic, and interference groups at various time intervals according to in situ hybridization results. RESULTS： （1） During the early stage of epilepsy （6 and 12 hours）, GBRla and GBR2 mRNA expression was decreased, and expression was less than the control group at one day after KA induction （P 〈 0.05）. mRNA expression was increased in the DG, but was greater than the control group at day 3 （P 〈 0.05）. Expression in the hippocampal CA1 and CA3 regions remained low （P 〈 0.05）, but gradually recovered to control levels. （2） The time points when subunit expression was decreased were prolonged following baclofen intervention, and expression was significantly greater than the epileptic group （P 〈 0.05）. CONCLUSION： Both mRNA expressions of GABABR subunits were up-regulated following decreased expression in the epileptic group, suggesting that the temporal lobe exhibited endogenous antiepileptic mechanisms during the early stages of epilepsy onset. Baclofen promoted mRNA expression of GBRla and GBR2.

Effect of macrophage polarization regulated by miR-29b,B7H3 on CD4^(+)T cell differentiation in asthma

Objective:To explore the mechanism that miR-29b and B7H3 regulate the polarization of macrophages and thus affect the differentiation of CD4^(+)T.Methods:1.PBMC was extracted from peripheral blood mononuclear cells of children with asthma and normal children in the affiliated Children's Hospital of Soochow University,and RNA was extracted and reverse transcribed.The expression of miR-29b and B7H3mRNA was determined by real-time quantitative polymerase chain reaction(Q-PCR).The family history of asthma and history of allergic diseases were collected.2.THP-1 cells were induced into macrophages,miR-29b interference,miR-29b overexpression and normal control were induced by LV526,LV527 and NC virus infection.After 24 hours of culture,the cells were collected to detect the expression of STAT3 and B7H3 genes and proteins.3.It was verified that STAT3 was the target gene of miR-29b:after inoculating THP-1 cells and culturing with PMA with final concentration of 50ng/ml for 6 hours,the macrophages without PMA were cultured for 24 hours,then the macrophages infected by LV528,LV529 and NC virus were induced to form miR-29b interference,miR29b overexpression and normal control group.Luciferase analysis was performed at 48 hours to verify that STAT3 was the target gene of miR-29b.STAT3-3'UTR luciferase reporter gene plasmids were constructed and divided into three groups:"miR-29b+STAT3-3'UTR","miR-29b+STAT3-mut-3'UTR"and"miR-29b+luciferase empty load".4.Macrophages with different treatments were co-cultured with initial T cells for 3 days.The relative expressions of T-bet,GATA3 and ROR-γt were detected by Q-PCR.Result:1.The incidence of allergic disease in the acute attack group(68%)was higher than that in the other two groups(34.8%,33.3%),and the family history of asthma in the normal group(0%)was much lower than that in the other two groups(52%,60.9%).The difference was statistically significant(P<0.05).2.The expression of B7H3 in PBMC in acute attack group was higher than that in non-acute attack group and normal group.The expression of miR-29b in PBMC in normal group was significantly higher than that in non-acute attack group and acute attack group(P<0.0001).The expression of miR-29b in non-acute attack group was significantly higher than that in acute attack group(P=0.007).3.After silencing the expression of miR-29b,IL-4Rα,IL-4,IL-5,IL-13 and CD206 of macrophages increased significantly,while IFN-γdecreased,suggesting that miR-29b can promote the polarization of macrophages to M2.4.The overexpression of miR-29b,STAT3 and B7H3 gene and protein level in macrophages decreased,while the increase of miR-29b,STAT3 and B7H3 gene and protein expression was inhibited.5.There was a significant positive correlation between the expression of STAT3 and B7H3mRNA in macrophages(r=0.9737,P<0.0001).6.STAT3 is the target gene of miR-29b.7.Co-culture of macrophages with CD4^(+)T cells can promote the differentiation of primary T cells,namely Th 0 cells,into Th2,and the promoting effect of macrophages with downregulation of miR-29b is more obvious.Conclusion:The expression of miR-29b in PBMC of children with asthma is lower than that of normal children,while the expression of B7H3 is higher than that of normal children.It is speculated that miR-29b has a protective effect on children with asthma,while B7H3 aggravates the inflammatory response.Down-regulation of miR-29b,in macrophages can promote macrophages to M2 polarization,increase the expression of B7H3 and STAT3 in macrophages,make Th0 cells differentiate into Th2 cells,and aggravate the inflammatory response in patients with asthma.