| Deltamethrin (DM), one of the type II pyrethroids, is employed widely as composition of insecticide, and has drawn widely attention of more and more toxicologists for its latent mammal and human neural toxicity . In recent decades, pyrethroids are thought to be a kind of interfering substance to the web of nerve-endocrine-immune system. Nerve system, endocrine system and immune system interacts each other, many endocrine and immune factors play important roles in the function of nerve system, such as interleukin 1β(IL-1β), which involved in many physical and pathological process (e.g. sleeping, fever, ischemia and hypoxia). The pre-protein of IL-1βis converted to mature form after the dissection by interleukin converting enzyme (ICE) and then is secreted. Nature antagonist of IL-1β(interleukin 1 receptor antagonist, IL-1ra) and the inhibitor of ICE can block the function of IL-1β. The mechanism of neural exciting toxicity induced by DM is similar to ischemia and hypoxia, but the relationship between IL-1βand DM is still unknown. We measured the expression of IL-1βafter DM intoxication, the effect of exogenous IL-1β, IL-1ra and inhibitor of ICE co-operated with DM on the activities of ATPase and the releasing of NO and aminoglutaminic acid (GLU) to explore the role of IL-1βin the nervous lesion induced by DM. Result as follow:Section 1 Expression of IL-1βinduced by DM1. Expression of IL-1βinduced by DM(1). IL-1βmRNA transcription by RT-PCR Aim: To measured the relative expression of IL-1β/ GAPDH. Methods: 15 male SD rats were separated randomly into 5 groups: 1 control group and 4 DM treated groups, 1 h, 6 h, 24 h, 48 h after intraperitoneal injection of 12.5 mg?kg-1 DM, total RNA was extracted from cortex, hippocampus and hypothalamus, the RNA was reverse transcribed to cDNA , IL-1βgene and GAPDH gene were replicated by PCR., and then we measured the relative expression of IL-1β/ GAPDH.Results: The expression of IL-1βmRNA increased in 1 h in cortex, and in hippocampus it showed a transient up-regulation at 24 h, while in hypothalamus it reached maximum at 6 h. Conclusions: DM up-regulated IL-1βmRNA in cortex and hypothalamus rapidly, and IL-1βmRNA expression increased 18 h later in hippocampus.(2). IL-1βprotein content by immunohistochemistryAim: To compared the content of IL-1βin different time after DM injection. Methods: 25 male SD rats were separated randomly into 5 groups: 1 control group and 4 DM treated groups, 1 h, 6 h, 24 h, 48 h after intraperitoneal injection of 12.5 mg?kg-1 DM, we used immunohistochemistry to detect the OD of IL-1βpositive cells in cortex, CA1,CA2,CA3 and DG regions of hippocampus and hypothalamus.Results: In cortex, IL-1βof 1 h and 6 h groups were higher than the control group; in hippocampus, IL-1βwas up-regulated in 1 h, and kept higher than the control group till 48 h, while in the CA2 and CA3 regions of hippocampus, IL-1βwas elevated at 48 h, and in DG IL-1βexpression was up-regulated in 1 h; in hypothalamus IL-1βshowed higher after 24 h compared with the control group.Conclusions: The mRNA and protein expression of IL-1βwere up-regulated simultaneously in cortex and hippocampus; and in hypothalamus, molecular of IL-1βprotein augmented 18 h after its gene expression.2. The changing of the activities of ATPase after DM administration Aim: To detect the activities of ATPase in different times after DM administration. Methods: 25 male SD rats were separated randomly into 5 groups: 1 control group and 4 DM treated groups, 1 h, 6 h, 24 h, 48 h after intraperitoneal injection of 12.5 mg·kg-1 DM, we measured the activities of ATPase in cortex, hippocampus and hypothalamus. Results: The activities of Na+-K+-ATPase in hippocampus decreased in 6 h group and 48 h group, and showed a transient recovery in 24 h group; the activities of Na+-K+-ATPase of hypothalamus decreased in 1 h group.Conclusions: Na+-K+-ATPase in hippocampus and hypothalamus tended to be damaged by DM but cortex seemed be tolerant to DM. In this section, IL-1βinduced by DM showed different peak point times in different cerebral regions, and when we analyzed relationship between IL-1βand the lesion of ATPase we concluded that IL-1βmay play a protective role in cortex and hypothalamus, while may aggravate the hippocampus lesion.Section 2 Effect of exogenous IL-1βon the neural toxicity of DMAs the IL-1βshowed different roles involved in different CNS regions in section 1, we wanted to know whether exogenous IL-1βand the nature antagonist of IL-1β, IL-1ra, could intensify or block the DM toxicity in vitro. Cultured neural cells were applied. 1. Prepared compt. cultured neural cellsAim: To establish the model of compt. cultured neural cells for the next experiment in vitro. Methods: Male adult rats were decapitated, cortex, hippocampus and hypothalamus were digested by 0.125% trypsogen at 37℃for 30min, blew the dish per 5～6min, then added 1:1 volume of serum to stop digest, centrifuged at 1500rpm for 10min, cells were suspended with Hank's buffer and cultured in CO2 incubator, after 1 h and 6 h, the unicells were dyed with trypan blue to detect the cell survival rates.Results: The cell survival rate in 1 h was >90%, and at 6 h, the cell survival rate was >70%. Conclusion: Cultured neural cells model was established successfully.2. Choose the dose of DM administration and its action time Aim: Observing the effect of different dose of DM on the activities of Na+-K+-ATPase to choose the dose and the action time of DM.Methods: Cortex, hippocampus and hypothalamus were separated from cerebrum and dissected to unicells, the neural cells were treated with 2×10-6mol/L and 2×10-5mol/L DM, after 1 h and 6 h the cells were harvest for activities of Na+-K+-ATPase.Results: The activities of Na+-K+-ATPase of all the three brain region cells'were damaged by different dose DM, even the 6 h control group showed lower activity of Na+-K+-ATPase than the 1 h control group. Conclusions: Dose 2×10-6mol/L and 1 h time point were chosen to the next experiments in vitro.3. Effect of exogenous IL-1βand IL-1ra on the activities of ATPase Aim: To observe the effect of exogenous IL-1βand IL-1ra on the activities of Na+-K+-ATPase Methods: Cells of three encephalic regions were separated into 5 groups: control group, 2×10-6mol/L DM group, 100U/ml IL-1βgroup, DM+IL-1βgroup and 0.1μg/ml IL-1ra+DM group, 1 h after DM treated, cells were collected for activities of Na+-K+-ATPase.Results: DM group, IL-1βgroup and DM+IL-1βgroup manifested decreasing of the activities of ATPase of cortex cells, IL-1ra+DM group showed higher activities of ATPase compared with the DM group of cortex and hypothalamus cells.Conclusion: 100U/ml IL-1βand DM had no synergistic effect on ATPase damage, but IL-1ra showed protective effect on the activities of ATPase of cortex and hypothalamus cells.4. Effect of exogenous IL-1βand IL-1ra on the releasing of NOAim: To observe the effect of exogenous IL-1βand IL-1ra on the releasing of NO. Methods: Cells of three encephalic regions were separated into 5 groups: control group, 2×10-6mol/L DM group, 100U/ml IL-1βgroup, DM+IL-1βgroup and 0.1μg/ml IL-1ra+DM group, 1 h after DM treating, supernatant were collected for the NO content.Results: The NO release of DM group in cortex and hippocampus neural cells were higher than the control group; and IL-1βgroup of cortex was higher than the control group also, but the NO release of hypothalamus was lower than the control group; DM+IL-1βgroups of cortex, hippocampus and hypothalamus manifested a drastic up-regulation compared with the DM group; IL-1ra showed suppressed effect on the NO release of cortex and hippocampus cells. Conclusions: IL-1βmay be involved in the function of DM neural toxicity in cortex and hippocampus by causing NO releasing.Though the exogenous IL-1βof dose 100U/ml couldn't aggravate the activities losing of ATPase, IL-1ra could protect the ATPase in cortex and hypothalamus, and it could decrease the releasing of NO in cortex and hippocampus, we concluded IL-1βmay involved in DM neural toxicity through NO pathway.Section 3 Effect of the inhibitor of interleukin converting enzyme on the neural toxicity of DMWe had observed the effect of exogenous IL-1βand IL-1ra on the DM neural toxicity, in this section, we wanted to applied inhibitor of interleukin converting enzyme to inhibit the maturation of IL-1βand observed the effect of DM on neural cells. 1. Effect of the ICE inhibitor on the activities of ATPaseAim: To observe the effect of ICE inhibitor on the activities of Na+-K+-ATPase.Methods: Cells of three encephalic regions were separated into 8 groups: control group, 10μmol/LAc-YVAD-CHO group, 15μmol/L Ac-YVAD-CHO group, 2×10-6mol/L DM group, DM1 group(cells were treated with 10μmol/L Ac-YVAD-CHO and 2×10-6DM simultaneously), DM2 group(15min after DM treating 10μmol/L Ac-YVAD-CHO was added into culture),DM3 group(cells were treated with 15μmol/L Ac-YVAD-CHO and 2×10-6DM simultaneously)and DM4 group(15min after DM treating 10μmol/L Ac-YVAD-CHO was added into culture), 1 h after DM treating, cells were collected for activities of Na+-K+-ATPase.Results: The activities of Na+-K+-ATPase of DM1, DM2, DM3 groups of cortex cells were higher than the DM group, while those of DM4 group of hypothalamus cells were higher than the DM group.Conclusions: Ac-YVAD-CHO had protective effect on the activities of ATPase of cortex and hypothalamus.2. Effect of ICE inhibitor on the releasing of NO Aim: To observe the effect of ICE inhibitor on the releasing of NO. Methods: Cells of three encephalic regions were separated into 8 groups: control group, 10μmol/L Ac-YVAD-CHO group, 15μmol/L Ac-YVAD-CHO group, 2×10-6mol/L DM group, DM1 group, DM2 group, DM3 group and DM4 group, 1 h after DM treating, supernatant were collected for the NO content.Results: DM increased the release of NO of cortex and hippocampus cells but reduced NO release of hypothalamus cells compared with the control group; inhibitor of ICE suppressed the release of NO of cortex and hippocampus but induced NO release of hypothalamus. Conclusions: Inhibitor of ICE could block the effect of DM on releasing of NO of cortex and hippocampus cells.3. Effect of ICE inhibitor on the releasing of GLUAim: To observe the effect of ICE inhibitor on the releasing of GLU. Methods: Cells of three encephalic regions were separated into 8 groups: control group, 10μmol/L Ac-YVAD-CHO group, 15μmol/L Ac-YVAD-CHO group, 2×10-6mol/L DM group, DM1 group, DM2 group, DM3 group and DM4 group, 1 h after DM treating, supernatant were collected for the Glu content.Results: DM increased the release of GLU of cortex and hippocampus, while reduced that of hypothalamus; DM1, DM2, DM3 and DM4 groups all showed blocking effect of releasing of GLU induced by DM.Conclusions: IL-1βmay participates in the DM neuronal toxicity by GLU.In this section, we found that the ICE inhibitor can protect the activities of ATPase of cortex and hypothalamus cells, and it can antagonize the effect of DM on the NO and GLU release of cortex and hippocampus.As mentioned above, to cortex and hippocampus, NO and Glu might involved in the function of IL-1βin nerve lesion induced by DM , but to hypothalamus, there might be some other factors playing roles in neural toxicity of DM.
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