Role Of Nrf2/HO-1 Pathway In Protection Against Lead-induced Neurotoxicity

Lead pollution is a global public health issue due to its persistence and ubiquitous existence in the environment, as well as serious health threat to various organisms. Over the past few decades, adverse effects of lead have received considerable attention due to the nervous system is the most sensitive to lead exposure. However, the toxicological mechanisms of lead are still unclear. Previous studies suggested that oxidative stress may mediate lead-induced neurotoxicity reactions. The present study focused on molecular mechanisms of Nrf2/HO-1 in the protection against neurotoxicity of lead, to provide the reasonable scientific evidence for making a new strategy to protect against nervous system damage.Part One: Role of Nrf2 in protection against lead-induced toxicityObjective: Role of Nrf2 in protection against lead-induced toxicity was investiaged.Methods:SH-SY5Y cells were treated with PbAc at the indicated concentrations of PbAc. Then cell viability were measured by CCK-8; ROS levels were done by DCHF; protein levels of HO-1, y-GCS and nuclear Nrf2 were detected by Western Blot; mRNA levels of Hmoxl, Gstal, Gclc, Gclm and Nqol genes were measured by q-RT-PCR. After knocking down Nrf2 in the PbAc-treated cells, the mRNA and protein levels of the genes mentioned above were detected. Cell viability were measured after co-treatment of PbAc and either NAC or DPI. Cells with Nrf2-overexpression were exposed to PbAc, cell viability, ROS level, apoptosis rate and protein expression of Bax, Bc12, CytoC and pro-Caspase 3 were measured, respectively.Results:PbAc-treated SH-SY5Y cells exhibited its neurotoxicity through oxidant-based processes, however, it could be inhibited by NAC or DPI. As a defense response, PbAc induced activation of Nrf2, and then promoted a rapid increase in Nrf2 nuclear accumulation, as well as Nrf2-ARE binding activities in a ROS-dependent manner. Results relating to Nrf2-regulated gene expression showed that PbAc could induce the transcriptional levels of Hmox1, Gstal, Gclc, Gclm and Nqol. Additionally, upregulated expression of HO-1 and y-GCS proteins were also found. In SH-SY5Y cells with silencing Nrf2 expression, the mRNA and protien expression of PbAc-induced genes were inhibited. Furthermore, in SH-SY5Y cells with Nrf2 overexpression, PbAc decreased ROS generation and inhibited cell apoptosis, but increased cell viability.Conclusion:Nrf2-ARE system exhibited a protective role in Pb-induced neurotoxicity.Part Two:Role of HO-1 in protection against lead-induced toxicityObjective:Role of HO-1 in protection against lead-induced toxicity was investigated.Methods:mRNA and protein expression of HO-1 were measured in PbAc-treated primary rat hippocampal neurons and PbAc-treated SH-SY5Y cells. In SH-SY5Y cells treated with PbAc, protein expression of certain genes involved in the P38, ERK1/2, JNK, STAT3 and PI3K/AKT signaling pathways were measured. Then the inhibitors targeting these five signaling pathways were used to treat the cells, thereafter the protein levels of HO-1 were detected. In PbAc-treated SH-SY5Y cells with HO-1 overexpression or knockdown, cell viability, ROS level, apoptosis rate and protein expression of Bax, Bc12 and Bcl-xl were measured, respectively.Results: PbAc induced an increase in intracellular ROS, and decreased cell viability in SH-SY5Y cells. PbAc induced the mRNA transcription and protein expression of HO-1 in primary rat hippocampal neurons and SH-SY5Y cells. Then we found that HO-1 could be induced by PbAc via the P38, ERK1/2 and PI3K/AKT signaling pathways in a ROS-dependent manner, and via JNK in a ROS-independent manner. Further investigation revealed that overexpression of HO-1 was able to decrease the ROS generation and restrain PbAc-induced cell apoptosis in SH-SY5Y cells. Moreover, knockdown of HO-1 aggravated PbAc-induced ROS generation and cell apoptosis.Conclusion:HO-1 could efficiently inhibit PbAc-induced oxidative stress and apoptosis in nerve system.Part Three: t-BHQ protects against lead-induced neurotoxicity through the Nrf2/HO-1 pathway.Objective: Mechanisms underlying protective effects of Nrf2/HO-1 against lead-induced neurotoxicity were investigatedMethods:Developmental SD rats were coexposed to PbAc and t-BHQ. After rats were sacrificed, the MDA levels, SOD activities and GSH contents in the hippocampus and frontal cortex were detected. Then, neuron death was analyzed by Nissl staining. In addition, nuclear Nrf2 protein expression and Nrf2-ARE binding activity were detected. SH-SY5Y cells were pretreated with t-BHQ, CHX or Act.D, and then treated with PbAc, the mRNA and protein expression of Nrf2 and HO-1 were measured by q-RT-PCR, Western Blot or immunofluorescence, respectively. After cells with knocking down Nrf2 or HO-1 were treated with PbAc, cell viability, ROS level, Caspase 3/7 activity and protein expression of Bax and Bc12 were measured, respectively.Results: t-BHQ attenuated oxidative stress in the hippocampus and frontal cortex of developmental rats with a decrease in the MDA levels, and an increase in the SOD activities and GSH contents. In addition, neuronal apoptosis was inhibited in the t-BHQ-treated Rats. Furthermore, t-BHQ suppressed ROS production and Caspase 3/7 activity, and increased intracellular GSH contents in PbAc-treated SH-SY5Y cells. Results from in vivo and in vitro studies showed that t-BHQ enhanced the nuclear translocation of Nrf2 and binding to ARE areas, but did not induce the Nrf2 transcription, subsequently, expression of HO-1, NQO1 and GCLC proteins were upregulated. However, knockdown of Nrf2 or HO-1 adversely affected the protective effects of t-BHQ against lead toxicity.Conclusion:t-BHQ could protect against lead neurotoxicity through the Nrf2/HO-1 pathway.