Effects Of Cirbp On Lead-induced Cytotoxicity And Molecular Mechanisms

Background:Lead is a common environmental and industrial contaminant. Lead has toxin effects on human health, especially children. Children are sensitive to lead toxicity due to the characteristics of the metabolism and development. Lead exposure can cause irreversible damage to children’s nervous system, which interferes with mental development, impairs cognitive and cognitive function. In China, the situation of lead contamination is severe in children. Lead poisoning incidents in community have occurred frequently, with a negative impact on society. Meanwhile, a large number of sporadic lead poisoning cases also happened. Previous surveys demonstrated that there are still 10% to 15% of urban children and 20% of rural children’s blood lead concentrations are higher than 100 μg/L. Thus, lead exposure to children is not only a medical but also a social problem. It’s urgent priority to further explore the mechanism of lead neurotoxicity and appropriate protective measures.The molecular mechanisms of lead neurotoxicity are still unclear. Cold-inducible RNA-binding protein(Cirbp, CIRP, also known as A18 hnRNP) is the first mild cold-shock protein identified in mammals. It is first isolated and identified from mouse testis in 1997. Cirbp belongs to the glycine-rich RNA-binding protein family(glycine-rich RNA-binding protein family, GRP), containing a consensus sequence of the amino terminus of the RNA-binding domains(consensus Sequence RNA binding domain, the CS-RBD) and a carboxy-terminal glycine-rich region. Cirbp protein may be closely linked to the nervous system development, embryonic development, circadian rhythm, and many other functions. The expression of Cirbp is significantly increased under mild cold temperature, UV, hypoxia, and osmotic pressure changes.In recent years, researches have shown that the protein level of antioxidant thioredoxin(TRX) is reduced during lead exposure. By specific binding with the 3’-UTR of TRX’s mRNA, Cirbp can induce the translation of TRX protein and suppress apoptosis induced by TNF-α at mild cold temperatures, accompanied by significantly increased expression of p-ERK. Collectively, these results suggest that Cirbp can play an anti-apoptotic effect through the activation of the ERK signaling pathway. Therefore, the purpose of our experiment is to establish a model of the cytotoxicity induced by lead, to observe the functions of Cirbp during lead exposure, and eventually explore the detailed molecular regulatory mechanisms underlying the activities of Cirbp. Aim:The purpose of the present study was, by an in vitro lead cytotoxicity model, to investigate the possible relationship between lead exposure and the expression of Cirbp protein, and to further explore the possible pathway and its regulatory mechanisms, which may eventually provide a new way to prevent/treatment of lead toxicity. Methods:1. PC12 cells were exposed to different concentrations of lead acetate(0, 2.5μmol/L, 5μmol/L, and 10μmol/L). The MTT assay was used to measure the proliferation of PC12 cells; DCFH-DA probe method, Rh123, and MDA methods were carried out to detect mitochondrial ROS generation, membrane potential, and lipid peroxidation; TUNEL and Western blot were performed to detect the apoptosis of PC12 cells.2. Cirbp antisense oligonucleotide sequence was designed and synthesized. Then we built a p LVX-Cirbp-mCMV-ZsGreen-purolentiviral expression Vector and infected HEK-293 T cells. The virus titer was determined in 293 T cells by the expression level of the green fluorescent protein. We used the recombinant lentivirus to infect PC12 cells and the expression level of Cirbp was detected by RT-PCR assay.3. Both cells with over-expressed Cirbp and control Vectors were exposed to lead(5μmol/L). MTT assay was used to detect the proliferation of cells after up-regulation of Cirbp. Western blot method was used to detect the protein expression of Cirbp, antioxidant TRX, and apoptosis-related protein Bax and Bcl-2; DCFH-DA probe method and TBARS methods assay were used to evaluate mitochondrial ROS generation and lipid peroxidation.4. After pretreatment with ERK inhibitor PD98059for30 min, PC12 cells were further treated with lead(5μmol/L, 24h). Western blot was performed to detect the protein expression of p-ERK, ERK, Cirbp, TRX, BaxandBcl-2; DCFH-DA probe and MDA assay were employed to evaluate mitochondrial ROS generation and lipid peroxidation. Results:1. Lead exposure induced mitochondrial damage and oxidative stress in PC12 cells. After lead exposure for 24 h, MTT results showed that lead inhibited the proliferation of PC12 cells in a dose- and time- dependent manner; DCFH-DA and MDA test results indicated significantly increased intracellular ROS and MDA formation; Rh123 results showed that mitochondrial membrane function was damaged; the TUNEL and Bax/Bcl-2 results showed an induction of apoptosis.2. The protein level of Cirbp decreased after lead exposure, and it may be related to the cytotoxicity of lead. Western blot results indicated that the expression of Cirbp protein reduced during lead exposure. Lentiviral Vector infecting was carried out to obtain PC12 cells with overexpressed Cirbp. Western blot and RT-PCR results showed that mRNA and protein level were both increased significantly.3. Cirbp protected cells from lead toxicity. MTT results indicated that, compared with the control Vector group cells, the expression of Cirbp enhanced cell proliferation. DCFH-DA, MDA, and Bax/Bcl-2results showed that overexpression of Cirbp decreased ROS generation, lipid peroxidation, and the ratio of Bax/Bcl-2 significantly. These results indicated that Cirbp has protective effect on cytotoxicity induced by lead exposure.4. Cirbp positively regulated the expression of TRX protein. The protein level of TRX decreased under lead exposure in a dose-dependent manner, but overexpression of Cirbp reversed TRX expression reduced by lead exposure. Then we established overexpressed TRX by lentiviral transfection. Western blot, DCFH-DA, and MDA results showed that the overexpression of TRX decreased ROS generation, lipid peroxidation, and the ratio of Bax/Bcl-2 significantly. Meanwhile, overexpression of TRX had no significant effect on Cirbp expression. These results indicated that Cirbp exert its protective effect on cytotoxicity induced by lead exposure through inducing the expression of TRX.5. Overexpression of TRX was related to the ERK signaling pathway. In PC12 cells, lead significantly enhanced phosphorylation of ERK1/2 levels dose-dependently. Overexpressed Cirbp or TRX inhibited phosphorylation of ERK1/2. Pretreatment of ERK inhibitor(PD98059) reduced lead-induced ROS generation, lipid peroxidation, and the ratio of Bax/Bcl-2. These results demonstrated that overexpression of TRX may be related to the level of p-ERK1/2 in response to lead exposure. ConclusionIn the present study, we established an in vitro cytotoxicity model induced by lead, and cells with over-expressed Cirbp and over-expressed TRX by lentiviral Vector. Our results indicated that Cirbp regulated the expression of TRX protein during lead exposure. Meanwhile, the inhibited expression of TRX was related to ERK pathway after lead exposure. The results may provide new theoretical basis for the prevention and / or treatment of lead toxicity.