| Part I Effects of ACR intracerebroventricular injection on striatum and cerebellum of ratsObjectives:To investigate the neurotoxicity of ACR by intracerebroventricular injection with pathological change, and to study the effects of ACR on MAPKs, Nrf2, and NF-?B signaling pathways in rat striatum and cerebellum.Methods:Healthy SD male rats, weigh 200?220 g, were randomly divided into 5 groups,7 rats per group, including the solvent control group (artificial cerebrospinal fluid, ACSF),6-OHDA positive control group (0.5 mg/kg), and three ACR treatment groups (0.5,2.5, and 12.5 mg/kg). After intracerebroventricular injection, the general behavior changes were observed. All animals were killed, and the striatum and cerebellum tissues were collected and preserved in -80? after ACR treatment for 24 h. The pathological alterations of striatum and cerebellum neurons, especially dopaminergic neurons, were investigated by Nissl staining, immunohistochemistry, and Western blot technology. The number of apoptotic neurons was determined by TUNEL method. MDA levels were assayed with a spectrophotometer, and GSH contents were detected with a microplate reader. TNF-a and IL-6 levels were determined by ELISA method. The expression of MAPKs, Nrf2, and NF-?B signaling pathways key proteins were measured by immunofluoresecence and Western blot analysis.Results:(1) The general behavior changes:The rats in 12.5 mg/kg ACR group exhibited a slightly gait disorder. (2) Nissl staining:The striatal neurons and cerebellar Purkinje cells in 2.5 and 12.5 mg/kg ACR and positive control groups performed the decreased Nissl body number, cellular swelling, and irregular arrangement. (3) Tyrosine hydroxylase (TH) expression:Compared with that in the solvent control group, the number of TH-positive cells in striatum and cerebellum in 2.5 and 12.5 mg/kg ACR and positive control groups significantly decreased (P <0.05). TH protein expression in striatum of all ACR and positive control groups and in cerebellum of 12.5 mg/kg ACR and positive control groups significantly decreased (P<0.01). (4) Apoptosis in neurons:Apoptosis in striatum and cerebellum of 12.5 mg/kg ACR and positive control groups significantly increased compared with that in the solvent control group (P<0.05). (5) Oxidative stress and cytokines:Compared with that in the solvent control group, MDA levels in striatum of 2.5 mg/kg ACR group and in cerebellum of 12.5 mg/kg ACR group significantly increased (P<0.05). GSH contents in striatum and cerebellum of all ACR and positive control groups significantly dereased (P<0.01). The striatal TNF-a levels in 12.5 mg/kg ACR and positive control groups and IL-6 levels in 2.5 mg/kg ACR group significantly increased (P<0.05). The cerebellar TNF-a levels in 12.5 mg/kg ACR group and IL-6 levels in 12.5 mg/kg ACR and positive control groups were significantly elevated (P <0.05). (6) MAPKs signaling pathway:In cerebellum, p-ERK1/2 protein expression in 12.5 mg/kg ACR group, p-JNK1/2 expression in positive control group, and p-p38 expression in 2.5 and 12.5 mg/kg ACR and positive control groups significantly increased compared with that in the solvent control group (P<0.05). In striatum, p-ERK1/2 expression in positive control group, p-JNK.1/2 expression in 12.5 mg/kg ACR group, and p-p38 expression in 2.5 mg/kg ACR group were also significantly enhanced compared with that in the solvent control group (P<0.05). (7) Nrf2 transcriptional factor:Compared with the solvent control, immunofluoresecence assay showed that 12.5 mg/kg ACR and 6-OHDA improved Nrf2 expression in striatum (P <0.05), and 2.5 and 12.5 mg/kg ACR enhanced Nrf2 expression in cerebellum (P <0.01). (8) NF-?B transcriptional factor:Compared with the solvent control, immunofluoresecence results showed that NF-?B expression in striatum and cerebellum significantly increased in 12.5 mg/kg ACR and 6-OHDA groups (P <0.05).Conclusion:The intracerebroventricular injection model of ACR neurotoxicity in rats was established. Intracerebroventricular injection with 0.5 mg/kg ACR had no influence on striatal and cerebellar neurons. Treatment with 2.5 and 12.5 mg/kg ventricle ACR could lead to neuron pathologic change in striatum and cerebellum, and the decline of dopaminergic neurons. Exposure to 12.5 mg/kg ACR even caused neuronal apoptosis in striatum and cerebellum. Moreover, ACR could induce oxidative stress and inflammation, and activate MAPKs pathway, and increase Nrf2 and NF-?B expression in striatum and cerebellum.Part II The molecular mechanism of ACR-induced cytotoxicity in PC 12 cellsObjectives:To establish ACR poisoning model in vitro through investigating cell viability, oxidative stress and inflammation in PC12 cells, and to study the activation of MAPKs, Nrf2, and NF-?B signaling pathways and the cross-talk among them through special inhibitors application and siRNA transfection technology.Methods:Cell treatment:Cells were treated with 0.6,1.25,2.5, and 5 mmol/L ACR for 12 or 24 h. Inhibitors application or cell transfection:Cells were pretreated with 10?mol/L U0126,20?mol/L SP600125,10?mol/L SB202190, and 5?mol/L BAY 11-7082 for 2 h, respectively, or transfected with 20 nmol/L Nrf2 siRNA for 4 h before exposed with 2.5 mmol/L ACR for 24 h. Cell viability in PC 12 cells were determined by MTT assay. Cell morphological changes were observed under an optical microscopy. ROS generation was determined using DCFH-DA fluorescence probe. MDA levels were measured with a spectrophotometer. GSH contents and GSH-Px activity were determined with a microplate reader. TNF-? and IL-6 levels were assayed by both ELISA and RT-PCR methods. Nrf2 and NF-?B nuclear transcription was observed by immunofluoresence. The nuclear and cytosolic Nrf2, keapl, HO-1, NQO-1, the nuclear and cytosolic NF-?B, I?B?, TNF-?, and COX-2 protein expression were quantified by Western blot analysis. HO-1 and NQO-1 mRNA levels were determined by RT-PCR.Results:(1) Cell viability:Compared with controls, cells treated with ACR at doses of 2.5 and 5 mmol/L showed the decreased cell number, neurite shrink, reduced adherence, and ball float. ACR treatment significantly decreased cell viability in a dose and time-dependent manner (P<0.01), compared with controls.(2) Oxidative stress and inflammation:ACR treatment at doses of 2.5 and 5 mmol/L significantly increased ROS and MDA levels compared with controls (P<0.05). GSH contents significantly decreased in 1.25,2.5, and 5 mmol/L ACR treatment groups, compared with that in controls(P<0.01). ACR improved TNF-? and IL-6 expression in a dose- and time- dependent manner. TNF-? contents and mRNA expression significantly increased after 2.5 and 5 mmol/L ACR treatment for 24 h (P<0.05). IL-6 contents and mRNA expression were enhanced by 5 mmol/L ACR (P<0.01).(3) MAPKs signaling pathway:Compared with controls, different concentrations of ACR significantly decreased p-ERK1/2 protein expression (P<0.05). ACR treatment at doses of 2.5 and 5 mmol/L promoted p-JNK1/2 and p-p38 protein levels (P<0.05). It indicated that ACR could activate MAPKs pathways in a dose-dependent manner. Compared with 2.5 mmol/L ACR alone treatment, MEK inhibitor U0126, JNK inhibitor SP600125, and p38 inhibitor SB202190 respective pretreatment for 2 h effectively reduced Nrf2 and NF-?B protein expression, and HO-1, NQO-1, and TNF-? mRNA levels (P<0.05). Results showed that the inhibition of MAPKs pathways suppressed the activation of Nrf2 and NF-?B pathways induced by ACR.(4) Nrf2 signaling pathway:Compared with controls, ACR promoted Nrf2 nuclear transcription, exhibiting the significant enhancement of nuclear Nrf2 protein expression (P<0.01). ACR treatment at doses of 5 mmol/L significantly increased keapl protein expression (P<0.01). ACR treatment at doses of 0.6,1.25, and 2.5 mmol/L significantly increased Nrf2 downsteam HO-1 protein expression (P<0.05). ACR treatment at doses of 1.25,2.5, and 5 mmol/L significantly increased NQO-1 protein level (P<0.05). It showed that ACR induced the activation of Nrf2 signaling pathway in a dose-dependent manner. Compared with siRNA control, Nrf2 siRNA transfection effectively silenced Nrf2 gene expression and significantly reduced NF-?B protein expression (P<0.01), which revealed that Nrf2 gene silence suppressed NF-?B factor activation induced by ACR.(5) NF-?B signaling pathway:Compared with controls, ACR at doses of 2.5 and 5 mmol/L promoted NF-?B nuclear transcription, exhibiting the significant increased nuclear NF-?B protein expression (P<0.05). ACR treatment at doses of 0.6,1.25, and 2.5 mmol/L significantly increased I?B? protein expression(P<0.05). ACR treatment at doses of 2.5 and 5 mmol/L significantly increased TNF-a and COX-2 protein levels in NF-?B downsteam (P<0.05). Results showed that ACR activated NF-?B pathway in a dose-dependent manner. Moreover, compared with ACR at 2.5 mmol/L treatment, NF-?B inhibitor BAY 11-7082 pretreatment for 2 h significantly reduced Nrf2 protein expression (P<0.01), which indicated that the inhibition of NF-?B could suppress Nrf2 factor activation induced by ACR.Conclusion:Treatment with 2.5 and 5 mmol/L ACR for 24 h could lead to obvious cytotoxicity in a dose-dependent manner, which performed that the decreased cell viability, oxidative stress, and inflammation. ACR could activate MAPKs pathways, including ERK, JNK, and p38 pathways. ACR induced oxidative stress in PC 12 cells, promoted keapl-Nrf2 dissociation, and increased HO-1, NQO-1 genes expression. Moreover, ACR could lead to cell inflammation, caused I?B?-NF-?B dissociation, and up-regulated TNF-a, COX-2 levels. In the process of ACR-induced cytotoxicity, a cross-talk existed among MAPKs, Nrf2, and NF-?B pathways. MAPKs pathways had a regulatory action on Nrf2 and NF-?B factors, and a dual-direction regulation between Nrf2 and NF-?B factors was also found.Part III The regulatory mechanism of JNK and p38 on ACR-induced apoptosis in PC12 cellsObjectives:To study whether ACR activates the mitochondrion-mediated apoptosis in PC12 cells, which is regulated by JNK and p38 signaling pathways, and to provide theoretical basis for seeking the target of ACR poisoning.Methods:Cells were treated with 0.6,1.25,2.5, and 5 mmol/L ACR for 24 h. In addition, cells were pretreated with 10 ?mol/L U0126,20 ?mol/L SP600125, and 10 ?mol/L SB202190 for 2 h, respectively, before treatment with ACR for 24 h. Cell apoptosis was observed by Hoechst33258 staining. Ultrastructure of cells and mitochondria was also observed under transmission electron microscope (TEM). The rate of cell apoptosis was determined by Flow cytometry. Mitochondrial membrane potential change was assayed by JC-1 staining. Bcl-2, Bax, cytochrome c, caspase-9, and caspase-3 protein expression were analyzed by Western blot method.Results:(1) Cell apoptosis:2.5 and 5 mmol/L ACR treatment promoted chromosome gathering around the cell nucleus and DNA cleavage. Compared with controls,2.5 and 5 mmol/L ACR treatment significantly increased the early apoptosis and late apoptosis (P<0.05). (2) Mitochondrial membrane potential and ultrastructure alterations:Compared with controls,2.5 and 5 mmol/L ACR treatment reduced mitochondrial membrane potential (P<0.01). ACR induced mitochondrial swelling, cristae breakage. (3) Mitochondrial apoptosis pathway-related proteins expression: Following the increased doses of ACR treatment, Bcl-2 expression significantly decreased, Bax levels and mitochondrial cytochrome c release significantly increased compared with controls (P<0.05). (4) Caspase cascade:Compared with controls,2.5 and 5 mmol/L ACR treatment significantly decreased pro-caspase-9 and pro-caspase-3, and increased cleaved caspase-9 and cleaved caspase-3 (P<0.05). (5) The regulation of JNK and p38 pathways on mitochondrion-mediated apoptosis: Compared with ACR treatment, JNK inhibition enhanced Bcl-2 protein expression, and decreased Bax and cytochrome c protein expression, whereas p38 suppression only increased Bcl-2 protein expression (P<0.05). Moreover, JNK pathway blockage induced a significant increase of mitochondrial membrane potential and a decrease of early apoptosis and late apoptosis induced by ACR, whereas p38 pathway inhibition only decreased cell early apoptosis (P<0.05). Results showed that the inhibition of JNK and p38 pathways both suppressed mitochondrion-mediated apoptosis induced by ACR. However, JNK pathway blockage more effectively decreased ACR-induced apoptosis.Conclusion:ACR treatment at doses of 2.5 and 5 mmol/L for 24 h could cause mitochondrial ultrastructure and mitochondrial membrane potential alterations, activate caspase cascade, and eventually induce cell apoptosis. JNK and p38 could regulate mitochondria apoptotic pathway. The inhibition of JNK and p38 pathways could reduce ACR-induced apoptosis, which provided molecular targets for prevention and treatment of ACR poisoning. |
PDF Full Text Request