| Objective: To observe the effect of chlorpyrifos(CPF)on the expression of autophagy-related proteins in hippocampal neurons of rats,and then investigate the role of autophagy in central nervous injury of acute CPF poisoning.Methods: An animal model of CPF poisoning was prepared with the dose of 81.5mg/kg(1/2LD50).60 SD rats were divided into two groups of thirty each.30 SD rats in the first group were used for the preparation of pathological tissue samples,and the other 30 SD rats were applied to detect the levels of autophagy related proteins.The 30 SD rats in the two groups were then randomly divided into 6 groups(control,1/2d,2d,7d,14 d and 21d)of five each according to their weight.The SD rats in the experimental groups were given a one-time gavage,while the SD rats in the control group were given olive oil gavage.The general conditions and poisoning symptoms of poisoned SD rats were observed and their body weight were measured once a day.The hippocampal tissue samples and severed head blood of SD rats were collected according to the observation time point,while the Ach E plasma test was used to evaluate the preparation of the poisoned model.The pathological change of hippocampal tissue was observed according to thionin staining,while the formation of autophagosomes and expression of LC3 protein were also observed by immunofluorescence staining.Besides,the autophagy-related proteins expression levels in hippocampus,such as Beclin1,P62/SQSTM1 and LC3,were detected by Western blot.On the other hand,an additional 15 SD rats were randomly divided into 3 groups(control,CPF and CPF+3-MA)of five each.The SD rats in the two experimental groups were poisoned by CPF(81.5mg/kg)with a one-time gavage,and the control group was given a gavage of olive oil.Half an hour after exposure,the CPF+3-MA group was given 3-MA solution with the dose of 50ug/100 g by intraperitoneal injection,while the control group and the CPF group were given the same dose of saline by intraperitoneal injection.Then the general conditions of the SD rats were observed and recorded.The hippocampal tissue samples were taken after poisoned two days.Whereafter,the expression levels of autophagy-related proteins(Beclin1,P62/SQSTM1 and LC3)and apoptosis-related proteins(Bax and Bcl2)were detected by Western blot.Results:1 The preparation of SD rat model of acute CPF poisoning.All the experimental SD rats were survived during the preparation of the poisoned model.The rats in the poisoning group gradually showed obvious symptoms of acute chlorpyrifos poisoning after gavage.The poisoning symptoms were gradually worsened within poisoning 24 hours,and then gradually alleviated within 24 to 48 hours.After 48 hours,the poisoning symptoms were disappeared basically.Compared with the control group,the body weight of SD rats in the poisoning groups decreased obviously after poisoning,and the lowest body weight was appeared at the 3rd day.After that,the body weight curve of the poisoning group increased slowly until the 12 th day which was comparable to the control group.And the statistical analysis showed that significant differences in the body weight values were observed from 2nd day to 11 th day(P < 0.05).1.2 The determination of acetylcholinesterase(Ach E)in plasma.Compared with the control group(2.53±0.35 U/ml),the Ach E activity in plasma of CPF group was decreased significantly after poisoning 12h(0.45±0.26 U/ml,P < 0.01),and it remained lower level until 48 h(0.44±0.29 U/ml,P < 0.01).Although the Ach E activity level in CPF group was recovered somewhat at 7th day(1.61±0.40 U/ml,P < 0.01),it was still obviously lower than that in the control group.Then the Ach E activity level in plasma of 14 th and 21 th days(2.63±0.62 U/ml and 2.70±0.43 U/ml,P > 0.05)were returned to that of the control group.2 Effect of CPF on expression of autophagy-related proteins in hippocampal neurons2.1 Thionine stainingThe hippocampal neurons in the control group showed complete morphology and orderly arrangement,and it did not change significantly in the 1/2d group.The number of neurons in the 2d group decreased slightly,and the arrangement of these neurons was unconsolidated a bit.But the cell morphology was still recognizable in this group.It also can be seen that the number of neurons in the 7d group decreased significantly,while the cells were blurred and disordered.The morphology of neurons in the 14 d and 21 d groups was more completely and orderly,and the number of neurons in these groups increased compared with that in the 7d group.2.2 Western blot assayBeclin1: The Beclin1 protein expression level increased first and then decreased with poisoning time,and the highest expression level was appeared in 2d group.Compared with the control group,the statistical analysis showed that significant differences in the levels of Bclin1 were observed among the groups of 2d and 7d(P < 0.05),and there is no statistically significant difference in terms of 0.5d,14 d and 21 d groups(P > 0.05).LC3: the LC3 Ⅱprotein expression amount increased gradually with infecting time,and the peak value appeared in 2d group.Then,the value decreased gradually until 14 d group,and reach the level of control group.But it increased again in the 21 d group.Compared with the control group,the LC3 Ⅱprotein expression amount increased in the 1/2d,2d,7d and 21 d groups(P < 0.05),and there is no statistically significant difference in terms of the 14 d group(P > 0.05).P62/SQSTM: Compared with the control group,the amount of P62/SQSTM protein expression increased significantly in 1/2d group(P < 0.05),and then the amount decreased obviously in 2d group(P < 0.05).After that,the amount increased to the level of the control group in the 7d and 14 d groups(P > 0.05).The amount of P62/SQSTM protein expression in 21 d was higher than that of the control group(P < 0.05).2.3 Immunofluorescence stainingThe LC3 in control group express uniform and at a lower level,it was not found an obvious LC3 granule in the cytoplasm.The fluorescence intensiⅡ ty in 1/2d group enhanced obviously,compared with the control group.It is indicating that the overall expression level of LC3 was reinforced.Meanwhile,a small amount of LC3 Ⅱparticle aggregation appeared in the cytoplasm.Compared with 2d group,the LC3 expression level in 7d group was reduced slightly,and a relatively small amount of LC3 Ⅱparticle aggregation also appeared in the cytoplasm.The LC3 expression level in 14 d group was reduced significantly,and the level was comparable to that of the control group.It is also found that there was not an obvious LC3 Ⅱgranule in the cytoplasm of 14 d group.Besides,the LC3 expression level in 21 d group was enhanced again,and a slight LC3 granule was appeared in the cytoplasm.Ⅱ 3 The role of autophagy in hippocampal neuron damage caused by acute CPF poisoning 3.1 Western blot assay was used to detect the effect of 3-MA on autophagy-related protein expressionLC3Ⅱ: Compared with the control group,the LC3 expression level in ⅡCPF group increased significantly(P < 0.01).Compared with the CPF group,the LC3 Ⅱexpression level in CPF+3-MA group decreased obviously(P < 0.01),but it has not statistically significant difference compared with the control group(P > 0.05).Bclin1: Compared with the control group,the Bclin1 expression level in CPF group increased significantly(P < 0.05).Meanwhile,the Bclin1 expression level in CPF+3-MA group decreased clearly(P < 0.05),compared with the CPF group.But no statistically significant difference was found compared with the control group(P > 0.05).P62/SQSTM: The P62/SQSTM expression level in CPF group decreased obviously(P < 0.05),compared with the control group.Moreover,compared with the CPF group,the P62/SQSTM expression level in CPF+3-MA group increased visibly(P < 0.05).It is also found that there was no statistically significant difference compared with the control group(P > 0.05).3.2 Western blot assay was used to detect the effect of 3-MA on apoptosis-related protein expressionBax: Compared with the control group,the Bax expression level in CPF group enhanced obviously(P < 0.05).While the Bax expression level in CPF+3-MA group enhanced further,compared with CPF group(P < 0.05).Bcl2: Compared with the control group,the Bcl2 expression level in CPF group reduced clearly(P < 0.05).Meanwhile,the Bcl2 expression level in CPF+3-MA group reduced further,compared with CPF group(P < 0.05).Conclusions:1.The autophagy of hippocampal neuron was activated in the early stage of acute CPF poisoning.Besides,the blocked autophagy degradation and the autophagy dysfunction may be involved in the injury of hippocampal neurons induced by CPF.2.To appropriate active the autophagy level,and then improve the autophagy flux as well as maintain the autophagy homeostasis might be beneficial to alleviate hippocampal neuron injury caused by acute CPF poisoning. |
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