SOMAN诱发惊厥大鼠丘脑内c－fos的表达

用ＡＢＣ免疫组化技术研究了Ｓｏｍａｎ诱发大鼠产生强直阵挛性惊厥后的ｃ－ｆｏｓ原癌基因在丘脑中的表达。结果显示：在低剂量Ｓｏｍａｎ中毒但未出现惊厥的动物和戊巴比妥抗惊厥的动物丘脑内都未见ｃ－ｆｏｓ表达的增加。而致惊厥的动物则从惊厥１ｈ至３ｈ，丘脑内ｃ－ｆｏｓ表达均有显著增加，表达呈恒定的核特异定位分布。出现群集ＦＯＳ样免疫阳性神经元的核团是：丘脑中线核群的丘脑室旁核、菱形核、粘合核；板内核群的中央旁核、中央内侧核和中央外侧核；丘脑前核诸亚核以及丘脑网状核。上述结果表明，丘脑非特异性核团的协同活动以及丘脑前核可能参与惊厥活动的整合。

SOMAN诱发惊厥后c-fos在大鼠杏仁核簇─氧化氮神经元中的表达

运用FOS免疫组化结合NADPH－d组化双重标记技术，研究了soman诱发惊厥大鼠杏仁核内c－fos高表达神经元与NADPH－d阳性神经元之间的关系。结果显示：在soman诱发惊厥后的1．5h至48h，杏仁核簇内c－fos呈现持续过度表达、FOS免疫阳性神经元的分布具有显著的亚核定位特征。NADPH－d活性在惊厥后明显增强。有10％的FOS阳性神经元同时也呈现NADPH－d阳性。而几乎所有的NADPH－d阳性神经元均呈FOS染色阳性。鉴于FOS表达的诱导和NADPH－d的激活具有共同的上游事件，即NMDA介导的钙内流，可以认为FOS引发的目的基因表达及NO的神经毒作用可能存在于杏仁核的神经元损伤机制中。

Soman诱发惊厥后脑内信号物质及细胞程序死亡相关基因表达变化的定位研究

1．用Beckman－6300型高效氨基酸分析仪分析显示，谷氨酸和天门冬氨酸的水平在惊厥相关的大脑皮质、海马、嗅球、小脑等明显下降。2用ABC免疫组化法对Soman惊厥大鼠脑内Fos的表达变化显示：Soman诱发惊厥可引起大鼠脑内c－fos的广泛表达。惊厥和c－fos表达相伴出现。C-fos。S表达具有时相持异性和恒定的解剖学图布。3．研究了Soman惊厥后c－fos表达与NOS神经元在单个神经元的关系。结果：杏仁核片几乎所有的NOS神经元均表达。－fos双标记神经元约是C—fos表达神经元的1／10。在下丘脑视上核和室分核内短时表达c-fos，介几乎所有的NOS神经元也呈现FOS阳性。4．用ABC免疫络化法研究，Bcl－2、Bax、P53三个与细胞程序死亡密切相关的基因。结果在短时C-fos表达的脑区，惊厥2天后Bcl－2、Bax、P的表达增加；发生c－fos延时延迟表达的脑区，Bax的表达明显强于Bcl－2。Bax与Bcl—2的相互作用和比例决定细胞的生存。Bax－Bax同源二聚体占优势时，细胞发生程序死亡。因此，本文结果强烈支持：由Soman惊厥诱发的脑损伤是一种程序性细胞死亡，c－fos，P53，bcl－2、bax可能涉入其基因路径。详细的分子机制尚待进一步研究。

分子旋光度的密度泛函计算及Soman绝对构型确定

手性化合物绝对构型的确定一直是有机化学特别是手性药物合成研究过程中经常遇到的问题之一．利用密度泛函DFT／B3LYP量子化学计算方法,通过计算两种已知旋光度分子的旋光度,确认计算条件和验证计算结果的可靠性,并将此方法应用于Soman旋光度的计算．结合文献报道的Soman分子实验旋光度测定结果,首次采用理论计算方法确定了梭曼(Soman)分子四个异构体的绝对构型,分别为P(R),C(S)-Soman→P(+)C(+),P(R),C(R)-Soman→P(+)C(-), P(S),C(S)-Soman→P(-)C(+),P(S),C(R)-Soman→P(-)C(-)．

Effect of soman,sarin and VX on the nicotinic acetylcholine receptor

The highly specific ligand of the N-acetylcholine receptor（N-AChR）,alpha-bungarotoxin,was used to determine the effect of soman,sarin and VX onN-AChR of the diaphragm and extensor digitorum Iongus muscle of mice andrats.The effects of the three anti-cholinesterase agents on N-AChR weredifferent.Sarin did not act directly on N-AChR and cause a change in the numberof N-AChR.VX decreased the binding sites of the receptor by binding with N-AChRdirectly.The LD50was 0.054mg/kg in mousse.Soman increased the binding sites,e.g.1～1.5 LD50soman increased the number of N-AChR of mouse diaphragm by 25%.The peak increaseof N-AChR was reached 0.5 h after poisoning and could last 96h.The receptornumber was still 22% higher than that of the control on the fourth day aftersoman poisoning in rats.Soman mainly increased the number of extrasynapticN-AChR,leading to the enhancement of sensitivity of cholinergic effectors toacetylcholine（ACh）,which is similar to the hypersensitiveness resulting fromdenervation.These findings are of significance in probing the receptor mecha-nisms and treatment of soman poisoning.

Effect of soman on the neuro-muscle junctional and extrajunctional nicotinic acetylcholine receptors

Autoradiography of nicotinic acetytcholine receptors(N-ACHR)with the application ofhistochemical staining location of cholinesterase was used to observe the effect of soman onjunctional and extrajunctional N-AChR.Testing with the diaphragms and extensor digitorum longusmuscles of mice and rats,we found that soman mainly increased the number of extrajunctionalN-AChR.It did not alter the number of junctional N-AChR significantly,nor did it have any pro-nouneed effects on the gtycoprotein property and isoelectfic point(pI)of junctional andextrajunctional N-AChR.The change of extrajunctional N-AChR number caused by somanis similar to the phenomenon of increased extrajunctional N-AChR number and sensitivity resultingfrom denervation,but the mechanism of action is different from the latter.The increase ofN-AChR number is one of the important characteristics of soman poisoning which make it differ-ent from other nerve agents.To maintain the metabofic balance of N-AChR may be an importantnew approach to the treatment of soman poisoning.

Effects of Soman on Lymphocyte Proliferation in Mice

Experiments to investisate the effects of soman on lymphocyte proliferation werecarried out on balb/c mice,which were injected subcutaneously with soman in the doseof 154μg/kg (0.8 LD50),96μg/kg (0.5 LD50) and 38.4μg/kg (0.2 LD50) respectively.Itwas found that from the 1st to the 7th day after poisoning with 154μg/kg soman,B-lymphocyte proliferation was severely inhibited （P【0.05）,and it returned to the controllevel on the 10th day.Con A-stimulated T-cell proliferation showed a diphasic change,increasing at first and then markedly decreasing thereafter,after soman poisoning.The re-sults imply that the mitogenic response of both B- and T- cells and the primary increaseof T- cell response are closely related to the dosage of soman but not with the changes ofwhole blood cholinesterase activity.

Effects of hypoxia, soman and their combination on PC12 cells

To investigate the effects of hypoxia, soman and their combined ones on PC12 cells. Methods: After the PC12 cells were exposed to an atmosphere containing different concentrations of oxygen and cultured in a medium containing different concentrations of soman, the amount of lactic dehydrogenase (LDH) released by the cells and their survival rate were determined to observe the dose-dependent and time-dependent cytotoxic effects. Student’s t test and two-way ANOVA were employed to determine the statistical differences and interaction between hypoxia and soman exposure. Results: 1) Both hypoxia and soman exposures exerted dose-dependent cytotoxic effects on PC12 cells and the interaction between the two injurious factors was significant; 2)The combined effects of the two factors were equal to the sum of those exerted by each one separately; and the combined application of the two factors resulted in a more severe cytotoxicity than that caused by either agent used singly; 3) The amount of LDH released from PC12 cells could serve as a more sensitive indicator of cytotoxicity than the survival rate of the cells. Conclusion: This study demonstrates the cytotoxic effects of the combined exposure to hypoxia and soman acted in a summative manner, which suggests that the two factors might induce intracellular release of LDH in PC12 cells through different mechanisms.

Experimental study on therapy of ultraviolet blood irradiation and oxygenation in acute soman intoxication in rabbits

Objective:To study the therapy effect of ultraviolet blood irradiation and oxygenation （UBIO） on blood AChe activity and lung injury due to acute soman intoxication in rabbits. Methods:Forty rabbits were randomly divided into 4 groups: normal control group, intoxication group, routine therapy group and UBIO therapy group. Blood AChe activity and artery blood gas were analyzed 2 h after intoxication. ACP and AKP activities in BALF were determined respectively. Results:Blood AChe activity in intoxication group was lower than that in normal control group （P<0.05）. BALF ACP and AKP activities in intoxication group were higher than that in normal control group. Blood AChe activities in UBIO therapy group increased and were higher than that in intoxication and routine therapy groups. Compared with intoxication group, BALF ACP and AKP activities were decreased （P<0.05） in UBIO therapy group, while artery blood pH, PaO2 and SaO2 increased （P<0.05）. Conclusion: UBIO therapy can elevate blood AChe activity and alleviate lung injury induced by soman intoxication. So it may be a new way to treat acute soman intoxication.

The influence of ultraviolet blood irradiation and oxygenation on oxygen free radicals metabolism in rabbits with soman intoxication

Objective: To investigate the effect of ultraviolet blood irradiation and oxygenation (UBIO) on the metabolism of oxygen free radicals in rabbits with acute soman intoxication. Methods: One hundred rabbits were randomly divided into 5 groups: normal control group, intoxication group, routine therapy group. UBIO therapy group and combined therapy group. After 14 d, the concentration of malondiadehyde(MDA) and activity of superoxide dismutase(SOD), glutathionperoxidase(GSH-Px), catalase (CAT) and total antioxidative capacity (T-AOC) in serum were determined respectively. Results: Compared with the normal control group, the concentration of MDA and activity of CAT in the intoxication group were significantly higher (P < 0. 05). but SOD. GSH-Px activity and T-AOC were significantly lower (P<0. 05). After UBIO or combined therapy, serum MDA level was significantly lower in comparison with intoxication group (P<0. 05). but the activity of SOD. GSH-Px, CAT and T-AOC were higher than intoxication group(P<0. 05). Conclusion: There is an obvious oxygen free radical injury in rabbits with a-cute soman intoxication. UBIO can improve the antioxidation ability of rabbits and may be applied to treat acute soman intoxication as adjunctive therapy.

Protooncogene c-fos overexpression in NADPH-d positive neurons of the amygdala in soman induced seizures in the rat

Fos immunohistochemistry and NADPH-d histochemistry and double labeling of the 2 methods were used to study therelationship between c-Fos expressed neurons and NADPH-d positive neurons of the amygdala in soman-induced seizures in the rat.It was found that protooncogene c-fos was overexpressed in the amygdala from 1.5 h to 2 d after soman-induced seizures occurred.c-fos overexpression was in the persisting and delayed pattem. The distribution of Fos immunoreactively positive neurons was sub-nuclear specific. About 10% of the Fos-positive neurons were NADPH-d positive and almost all the NADPH-d positive neuronswere also stained with Fos immunohistochemistry. Our findings together with those reported in our previous paper in which c-fosexpression and NOS activation were found to share the common upstream of the intracellular signal transducing cascade mediatedthrough NMDA receptor depended Ca2+ influx suggest that c-fos expression and nitric oxide neurotoxicity mighy exist in the amygdala lesions after soman induced seizures occurred.

TRPV4 Regulates Soman-Induced Status Epilepticus and Secondary Brain Injury via NMDA Receptor and NLRP3 Inflammasome

Nerve agents are used in civil wars and terrorist attacks,posing a threat to public safety.Acute exposure to nerve agents such as soman(GD)causes serious brain damage,leading to death due to intense seizures induced by acetylcholinesterase inhibition and neuronal injury resulting from increased excitatory amino-acid levels and neuroinflammation.However,data on the anticonvulsant and neuroprotective efficacies of currently-used countermeasures are limited.Here,we evaluated the potential effects of transient receptor vanilloid 4(TRPV4)in the treatment of soman-induced status epilepticus(SE)and secondary brain injury.We demonstrated that TRPV4 expression was markedly up-regulated in rat hippocampus after soman-induced seizures.Administration of the TRPV4 antagonist GSK2193874 prior to soman exposure significantly decreased the mortality rate in rats and reduced SE intensity.TRPV4-knockout mice also showed lower incidence of seizures and higher survival rates than wild-type mice following soman exposure.Further in vivo and in vitro experiments demonstrated that blocking TRPV4 prevented NMDA receptor-mediated glutamate excitotoxicity.The protein levels of the NLRP3 inflammasome complex and its downstream cytokines IL-1βand IL-18 increased in soman-exposed rat hippocampus.However,TRPV4 inhibition or deletion markedly reversed the activation of the NLRP3 inflammasome pathway.In conclusion,our study suggests that the blockade of TRPV4 protects against soman exposure and reduces brain injury following SE by decreasing NMDA receptor-mediated excitotoxicity and NLRP3-mediated neuroinflammation.To our knowledge,this is the first study regarding the“dual-switch”function of TRPV4 in the treatment of soman intoxication.

Nitric oxide and soman poisoning

目的：探查索曼中毒机制是否有ＮＯ参与．方法：预先侧脑室注射Ａｒｇ、ＮＡＭＥ，观察索曼中毒小鼠惊厥潜伏期、死亡率及脑中ＮＯＳ活性变化．结果：Ａｒｇ预处理时，潜伏期从５２ｍｉｎ（对照组）缩短到４３ｍｉｎ（Ａｒｇ１６０ｎｍｏｌ），死亡率由５０％（对照组）增加至８１％（Ａｒｇ１６０ｎｍｏｌ）．ＮＡＭＥ预处理时，潜伏期从４０ｍｉｎ延长到１４５ｍｉｎ（ＮＡＭＥ２２０μｍｏｌ），死亡率由８７％（对照组）减少至５０％（ＮＡＭＥ２２０μｍｏｌ）．索曼的小鼠毒性被Ａｒｇ增强、被ＮＡＭＥ减弱，均有剂量依赖性．索曼中毒可使小鼠大脑、小脑及海马的ＮＯＳ活性分别增加４％，１５％及１１％．

Y-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitro-phenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcatt= 198 min-1 and the rate enhancement kcatkuncat = 122 419. When 0.16 mg · mL-1 EP6 was preincubated in vitro with 0.132 mmol·L-1 (220 μg·kg-1 =1.1×LD95) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP6 could prolong the latent time of spasm and death when mice were passively immunized with EP6 intravenously 15 min before 1×LD95 of soman challenge. These results demonstrate that EP6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.

补硒与锌对梭曼中毒大鼠乙酰胆碱酯酶活力和抗氧化力的影响

目的 探讨抗氧化剂硒 (Se)和锌 (Zn)预防梭曼中毒的可能性。方法 雄性大鼠 4 0只 ,按体重随机分为正常对照组、染毒组、硒治疗组、锌治疗组。正常对照组和染毒组每日 5mL·kg- 1的生理盐水(NS)ig ;硒治疗组每日 1mg·kg- 1Na2 Se0 3的NS液ig和锌治疗组每日 4mg·kg- 1Zn(C6 H11O7) 2 的NS液ig ,共ig 9d。d 10除正常对照组外其余各组大鼠均sc 0 .9LD50 梭曼 ,2h后断头处死并取样 ,测定大鼠全血乙酰胆碱酯酶 (AChE)活性及血清、大脑、肝脏的超氧化物歧化酶 (SOD)活性、总抗氧化力 (T AOC)和一氧化氮合酶 (NOS)活性。结果 梭曼中毒大鼠后 ,染毒组大鼠全血AChE活性被明显抑制 ,血清、大脑、肝脏的SOD活性、T AOC显著下降 ,NOS含量明显升高 ,而硒和锌预防组大鼠对梭曼引起的AChE、SOD、NOS活性和T AOC变化明显减轻。

丝网印刷胆碱酯酶电极测定神经性毒剂沙林、梭曼

将丝网印刷电化学传感器制作技术与戊二醛蒸汽熏蒸的酶固定化方法相结合制作了一次性使用胆碱酯酶电极（ＡＣhＥ），并用于神经性毒剂沙林和梭曼的测定。以氯化硫代乙酰胆碱 （ＡＴＣh）为底物，该酶电极在pＨ７．２、温度３７℃时有最大响应。酶催化反应的动力学常数为：Ｋm＝３．５ｍｍｏｌ／Ｌ，Ｉｍａｘ＝２．５ｕＡ。沙林、梭曼抑制酶电极响 应，百分抑制率为毒剂浓度的对数呈线性关系。当抑制时间为２ｍｉｎ时，测定沙林。

维生素A和维生素E对梭曼染毒大鼠自由基损伤的预防作用

目的 探讨梭曼染毒自由基损伤及维生素A(VA)和维生素E(VE)对该损伤的保护作用。方法 实验设对照组 ,梭曼染毒模型组及VA 和VE 实验组。在梭曼染毒前 ,对照组和梭曼染毒模型组ig处理过的菜子油 (1mL·kg- 1·d- 1) ,VA 及VE 实验组分别按VA2mg·kg- 1和VE2 .5mg·kg- 1的剂量ig ,共 9d。d 10除对照组外 ,其余大鼠均用梭曼腹部sc(6 3μg·kg- 1) ,中毒 2h后宰杀取血和肝脏组织 ,观察大鼠血清和肝组织中相关指标的变化。结果 结果显示 ,染毒模型组AChE严重下降 ,血清和肝组织中MDA含量明显升高 ,血清和肝组织中SOD活性下降 ,全血和肝组织中GSH Px活性呈显著下降趋势。VA 和VE 实验组血清MDA含量下降 ,在血清和肝组织中SOD及全血和肝组织中GSH Px活性均上升。结论 梭曼染毒引起AChE活性严重下降 ,同时伴有严重的自由基损伤 ,染毒前大鼠用VA 和VEig处理 ,能够保护AChE活性 ,同时作为抗氧化剂的VA和VE

化学中毒与急性缺氧的双因素联合效应的实验研究

目的 研究化学中毒与急性缺氧两因素的联合效应。方法 建立常压常氧、常压缺氧和低压缺氧 3种模型 ,以梭曼 (Soman)、氰化钠 (NaCN)和 4 DMAP ( 4 dimethylaminophenol)为代表 ,测定大鼠、小鼠、家猫、PC1 2 细胞、兔血红细胞等在3种模型条件下对毒物药物的行为、生化和生理指标变化。结果 梭曼单独作用可导致动物协调运动下降、脑等组织含水率增加、脑组织AChE活性降低、MR下调、NE和cAMP含量增加。急性缺氧单独作用也可引起动物协调运动及自主活动的降低、脑等组织含水率的增加、外周血和脑组织AChE活性升高、MR受体上调、NE和cAMP含量增加、4 DMAP药效升高。化学中毒与急性缺氧同时作用 ,上述变化更加复杂。结论 化学中毒与急性缺氧两种因素同时作用于机体 ,对机体产生广泛而复杂的损伤作用。此种作用为两因素的联合效应。缺氧引起的机体功能下降对联合效应的增加部分贡献较大。急性缺氧既可使重要的组织成分发生质或量的变化 ,也会影响某些抗毒剂的作用强度。

硒、锌对梭曼诱导大鼠过氧化损伤的保护机制研究

目的 : 研究梭曼 (Soman)对大鼠急性中毒的自由基损伤作用及硒、锌的抗氧化保护作用。方法 : 雄性大鼠 40只 ,按体重随机分为阴性组 (正常对照组 )、阳性组 (损伤对照组 )、硒组 (加硒保护组 )及锌组 (加锌保护组 )。测定指标有血清、大脑、肝脏的维生素 E(VE)、总抗氧化能力 (T- AOC)及一氧化氮合成酶 (NOS)。结果 : 梭曼中毒后 ,T- AOC水平、VE含量明显下降 ,NOS活性显著提高 ;硒、锌组较阳性对照组损伤指标变化幅度较小。结果 : 梭曼中毒有明显的自由基损伤 ,硒、锌对梭曼中毒有显著的抗氧化作用。

模拟4000m高原梭曼中毒犬的血流动力学变化

麻醉犬在模拟4000m高原停留2h后开胸,经静脉输注梭曼(10μg/kg)中毒后,测定左心室压力(LVP)及其变化最大速率(±dp/dtmax)、主动脉压力(AP)、肺动脉压力(PAP)、心输出量(CO)、心脏指数(CI)和心率(HR)。结果表明,模拟高原能加重梭曼中毒对犬血流动力学及心脏功能的抑制作用,与平原组值比较,具有显著性差异(P<0.05)。