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Nerve agent not only inhibit acetylcholinesterase ( AChE) at an early stage, but also induce prolonged and progressive neuroinflammation and delayed neurodegeneration.Recently, the US National Institute of Health ( NIH) has sponsored some major programs of toxic mechanisms and treatment of nerve agents, which aims at the development of quick and effective treatment to acute intoxication and delayed effect.The experimentally effective new antidotes mainly include AChE-targeting drugs, broad-spectrum reactivators and scavengers, antiinflamatory and nerve protection drugs.
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Objective To compare the changes in energy metabolism in 2-chloroethyl ethryl sulfide(CEES)-poisoned bronchial epithelial cell 16HBE cultured in media at different glucose concentrations .Methods Bronchial epithelial cell 16HBE was cultured in high (4.5 mg/ml) or low (1.1 mg/ml) glucose medium and exposed to a sulfur mustard simulant CEES of 0.2, 0.5, 1.0 mmol/L.Cell growth and cytotoxicity were tested using MTS .ATP, ADP and AMP were detected by HPLC and the value of ATP/ADP, total adenine nucleotides ( TAN) and energy charge ( EC) was subsequently calculat-ed.Mitochondrial oxidative phosphorylation-related proteins, COX-10 and ISCU, were detected using Western blotting . Rhodamine 123 was applied to detect the mitochondrial membrane potential using flow cytometry .Results Low glucose accelerated the growth and energy metabolism of 16HBE cells in regular culture , and the contens of ADP , TAN, COX-10 and ISCU in low glucose group were significantly higher than those in high glucose group .CEES exposure (≥0.5 mmol/L) significantly affected cell viability in both high and low glucose groups , with significant difference between the two groups exposed to 1.0 mmol/L CEES.In high glucose group, 24 h after 0.5 or 1.0 mmol/L CEES exposure, the contents of ATP, ADP and TAN were significantly increased , while ATP/ADP and EC decreased .In low glucose group , ADP, AMP and TAN significantly decreased, while ATP/ADP and EC increased 24 h after 1.0 mmol/L CEES exposure.The mi-tochondrial membrane potential (MMP) also changed differently after 0.5 mmol/L CEES exposure.MMP in high glucose group marginally increased at 3 h, and significantly increased at 8-12 h (P<0.05), and returned to normal at 24 h. MMP in low glucose group showed a transient decrease at 5 h (P<0.01), and back to normal at 8 h.The protein levels of COX-10 and ISCU were significantly increased in high glucose group 24 h after 0.5-1.0 mmol/L CEES exposure , but sig-nificantly decreased in low one 24 h after 1.0 mmol/L CEES exposure .Conclusion When 16HBE is cultured at a high or low glucose concentration , the cell growth, stress responses and energy metabolism including MMP , COX-10, ISCU and ATP production are in different status before or after CEES exposure .High glucose could protect against CEES exposure .
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Objective To explore the effect of 2-chloroethyl ethyl sulfide(CEES) poisoning on keratinocyte migration and the regulatory role of microRNA(miR)-34a.Methods MTS was used to detect the viability of cells exposed to CEES in order to select an appropriate dose of CEES exposure in this in vitro model.The protein level of keratin 5 and keratin 10 was detected to assess cell differentiation status .Scratch assay was applied to evaluate cell migration ,and miR-34a silencing in keratinocytes was achieved by transfecting chemically synthesized miR-34a specific miRNA inhibitor.t-ERK1/2 and p-ERK1/2 levels closely related to cell migration were detected using Western blotting .Results An in vitro CEES exposure model of keratinocytes was established at the optimal concentration of 0.5 mmol/L CEES in the viability test , and this dose was chosen to evaluate cell migration changes .The migration of cells was significantly inhibited 24 h after CEES exposure , accompanied by no changes in morphology and keratin 5/10 levels.Silencing of miR-34a significantly increased the migration of cells exposed to CEES , which could be blocked by adding 5 μmol/L U0126 , an ERK1/2 phosphorylation selective inhibitor.Conclusion Silencing of miR-34a can significantly increase keratinocyte migration and partially reverse the inhibition of CEES-caused migration , which could be mediated by ERK 1/2 pathway activation .
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Objective To investigate the effects of anticholinergic antidote and rhodosin on the brain injury induced by soman intoxication combined with hypobaric hypoxia in rats. Methods A total of 72 Wistar rats were divided into 4 groups: hypoxia control (HC), hypoxia plus soman (HS), hypoxia plus soman plus anticholinergic antidote (HSAA), and hypoxia plus soman plus anticholinergic antidote plus rhodosin (HSAAR). The animals after soman intoxication (72 ?g/kg) were placed in a hypobaric (62 kPa) apparatus for hypoxic exposure for 48 h. Rats were sacrificed for brain tissue detachment at the time points of 12, 24, and 48 h. Evans blue (EB) content and PLA 2 activity were detected biochemically. CaM concentration was determined by radioimmuno assay. Results Compared with the rats in HC, soman induced significant increases of brain EB, PLA 2, and CaM at 12, 24, and 48 h in HS. Elevated EB, PLA 2, and CaM induced by hypoxia and soman intoxication in rats in group HSAA were obviously attenuated by anticholinergic antidote. More significant decreases of brain EB, PLA 2, and CaM were found in rats in group HSAA. Conclusion Both anticholinergic antidote and anticholinergic antidote plus rhodosin have the preventive effect on rat brain damage induced by soman intoxication combined with hypoxia.
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Objective To investigate the effect of soman on the Janus kinases (JAKs) expression in cell line PC 12 . Methods The PC 12 cell was used in these experiments and treated with soman at a concentration of 20 ?mol/L . RT PCR and Western blotting were employed to detect the mRNA and protein expressions of JAK1, JAK2 and JAK3 at the time points of 0, 6, 12 and 24 h. The products were sequenced by Sanger's double strand DNA sequence determination. Results The expression levels of JAK1, JAK2 and JAK3 mRNAs and proteins increased at 2 h, reached the highest at 12 h and decreased at 24 h, but they were still higher than those of the control. It was shown that the sequences of amplification products by RT PCR were the same to corresponding ones in GenBank. Conclusion Soman intoxication enhances the expression of Janus kinases in PC 12 cells. JAKs genes may play an important role in brain injury due to soman intoxication.
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Objective To illustrate the features of soman induced signal transducers and activators of transcription (STATs) gene and protein expressions in cell line PC 12 . Methods The expression levels of STAT1, 3 and 5 mRNAs and protein in PC 12 cells were detected by semi quantitative RT PCR and Western blotting. PC 12 cells at 5～8 passages were randomly divided into 5 groups: control, intoxication groups for 2, 6, 12 and 24 h respectively. The products were sequenced by Sanger's double strand DNA sequence determination. Results The expression levels of STAT1, 3 and 5 mRNAs and proteins increased in PC 12 cell at 2 h and reached the highest at 12 h, then decreased at 24 h, but they were still higher than those of the control. The sequences of amplification products by RT PCR were the same to those in GenBank. Conclusion Soman intoxication can enhance the expression of STATs in PC 12 cells. STAT genes may possibly play an important role in brain injury.
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Objective To explore the changes of interleukin-4,8 and TPK in PC_(12) cells exposed to hypoxia and soman intoxication.Methods PC_(12) cells were randomized into 4 groups: control,hypoxia only,soman only,hypoxia plus soman.The activity of IL-4,IL-8 was measured by ELISA and the activity of tyrosine protein kinase in cells were detected by RIA.Results The activity of IL-4,IL-8 in PC_(12) cell supernatant and TPK in PC_(12) cell cytoplasm,nucleus were increased in 2 h and reached the highest at 12 h,decreased at 24 h in soman group,correspondingly the activity of IL-4,IL-8 in PC_(12) cell supernatant and TPK in PC_(12) cell cytoplasm,nucleus were increased at 2 h and reached the highest at 6 h,decreased at 12 h in soman plus hypoxia group,all higher than that of control.Conclusion TPK,cytokine and its correlated signal pathway lead to an important modulatory role in the brain damage after combined soman and hypoxia injury.
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Objective To observe the changes of the concentration of Ca2+, contents of cAMP, CaM and activity of Ca2+/CaM-PK II in pheochromocytoma PC12 cells after combined soman and hypoxia injury. Methods The changes of ［Ca2+］, and activity of CaM, cAMP and Ca2+/CaM-PK II in PC12 cells were studied after combined soman and hypoxia injury with radioimmunoassay. Results The changes of ［Ca2+］, the contents of CaM, cAMP were significantly higher in hypoxic and soman intoxicated group than in soman intoxicated group and control group under hypoxia; but the activity of Ca2+/CaM-PK Ⅱ were significantly decreased. Conclusion ［Ca2+］, CaM, cAMP and Ca2+/CaM-PK Ⅱ exert important role in the damage of PC12 after combined soman and hypoxia injury.
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reression of M 2 receptor mRNA of rat myocardium intoxicated by soman under high altitude hypoxia. Methods The expressions of M 2 receptor mRNA under hypoxia, soman intoxication and soman intoxication under hypoxia were detected by semi quantitative RT PCR, respectively. Results The expression of M 2 receptor mRNA increased in the high altitude hypoxia group. Both simple soman intoxication and combined soman intoxication and hypoxia decreased the expression of M 2 receptor rapidly. But under hypoxia, the expression increased significantly at 12 h and 24 h. Conclusion M 2 receptor was sensitive to nerve agents. Compared to simple soman intoxication group, the expression of M 2 receptor increased in combined soman intoxication and hypoxia group. This may be one of the major factors leading to aggravation of the injury of heart function by nerve agents in high altitude area.
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Objective To explore the effects of oxidative stress and pulmonary vascular permeability on lung injury in rats treated with sodium cyanide(NaCN) and /or hypobaric hypoxia.Methods A total of 72 male SD rats were randomly divided into 2 groups: the NaCN intoxicated group in 308m altitude and the hypobaric hypoxia combined NaCN intoxicated group in 4 000m high altitude.The animals for the experiment of hypoxia were processed in an artificial hypobaric chamber to simulate the designated high altitude hypoxia(4 000m,61kPa).NaCN was injected subcutaneously to the rats in the both groups at the dosage of 3.6mg/kg.Broncho-alveolar lavage fluid(BALF) and lung tissues were prepared at the time points of 0,0.5,1.0,2.0,4.0 and 6.0h.The activity of SOD,GSH-PX,ACE,LDH,AKP and the content of MDA,GSH,Evan's blue(EB) were detected by spectrophotometric method.Results Combined acute hypobaric hypoxia and NaCN intoxication produced a significant increasing effect on EB content in the rats.The activity of ACE,LDH and AKP in BALF,and the contents of MDA in BALF and lung tissue reached the highest value at 0.5h preparation at the combined hypobaric hypoxia and NaCN intoxicated group,but they were still higher than that of control group(NaCN intoxicated only);correspondingly the activity of SOD,GSH-PX,content of GSH in lung tissue and BALF reached the lowest value at 0.5h preparation at the combined hypobaric hypoxia and NaCN intoxicated group,but they were still lower than that of control NaCN intoxicated group.Conclusion The results suggest that under the condition of hypoxia,NaCN intoxication may produce severe harmful effects on the lungs,increase the pulmonary vascular permeability,and cause severe interference on to the oxidative stress level.
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Objective To investigate the complex effect of soman and hypoxia on PC12 cell line as shown by the expression changes in IL-6/GP130, JAK1 and STAT1. Methods A cell model of intoxication by combining effect of hypoxia and soman was reproduced, and it was divided into four groups: control, soman intoxication, hypoxia combined with soman intoxication, and Genistein inhibition groups. PC12 cells were cultured in RPMI 1640 and were treated with NGF (50ng/ml) for seven days. The differentiated PC12 cells were then exposed to hypoxia in an incubator containing 5% CO2, 95% N2 and (or) incubated with soman (20?mol/L) for 2, 6, 12 or 24 hours. The expression levels of IL-6/GP130, JAK1, STAT1 mRNA and protein were assessed by RT-PCR and Western blot in PC12 cells. The products were sequenced by Sanger's double strand DNA sequence determination. Results In soman intoxicated group, the expression levels of IL-6/GP130, JAK1, STAT1 mRNA and protein were elevated in PC12 cell, reaching the peak level at 12 hours, and then lowered at 24 hours, but remaining higher than that of control group. In combined soman intoxicated and hypoxia group, the expression levels of IL-6/GP130, JAK1, STAT1 mRNA and protein reached the peak value at 6 hours, being higher than that of control group, soman intoxicated group and Genistein inhibition group. It was shown that the sequences of the products as amplified by RT-PCR were the same as that found in the GenBank. Conclusion Soman intoxication or (and) hypoxia up-regulate the expression of IL-6/GP130. Both hypoxic condition and soman treatment can up-regulate the expression of JAK1/STAT1 mRNA and protein in PC12 cells. JAK-STAT pathway may play a role in the mechanisms of brain injury resulted from hypoxia and soman poisoning.