## 4 Discussion
Human CDR1 as is located mainly in the cytoplasm and has more than 70 binding sites to miR-7 [14; 15; 37]. MiR-7 is involved in the regulation of genes in the brain [38; 39; 40]. The binding of miR-7 to CDR1 as has been found in cell lines, and thus CDR1 as is thought to exert a sponge effect on miR-7 by reducing the number of freely available miR-7 molecules [15]. MiR-7 binding sites are only partially complementary to CDR1 as, ensuring that CDR1 as is not cleaved by AGO2 bound to the miR-7:CDR1 as complex. CDR1 as also has a binding site for miR-671. In contrast with the binding site for miR-7, this binding site is almost completely complementary to miR-671, and thus miR-671 may use this binding site to mediate the slicing of CDR1 as, potentially releasing its miR-7 carrier [41]. In our study, we found a significant decrease in CDR1 as expression levels and an increase in miR-671 expression levels in the Pb-exposed group of mice under long-term Pb exposure conditions with increasing duration of toxicity, which we hypothesize may be caused by Pb exposure promoting miR-671 expression to induce cleavage of CDR1 as by AGO2. The downregulation of CDR1 as due to Pb exposure implies a possible upregulation of miR-7 as it dissociates from CDR1 as. Accumulating evidence suggests that Pb exposure can induce NF-kB hyperactivation, which promotes overexpression of APP and BACE1 and aberrant tau protein hyperphosphorylation, thereby accelerating learning and memory deficits in human and animal models [42; 43]. Our previous 3-month Pb exposure study in mice revealed a dose-dependent decrease in the expression of total NF-kB. In this study, total NF-kB protein expression levels showed no difference between mice in each group. Interestingly, previous in vitro studies have proposed that CDR1 as may act as an upstream regulator of NF-kB, facilitating its nuclear translocation and subsequent activation of downstream gene expression, including AD-related genes [44]. However, a lack of corresponding in vivo experimental evidence has hindered the validation of this hypothesis. This study showed that chronic Pbexposure promoted the migration of NF-xB proteins in the cytoplasm to the nucleus, which is consistent with the results of our previous study in which mice were Pb-exposed for 3 months. These results suggested a potential interaction between CDR1 as and NF-xB in the context of Pb exposure, which warrants further investigation. Focusing solely on the total levels of NF-xB may not provide a complete picture of its regulatory roles in the cell, as the transcriptional activity of NF-xB is primarily determined by its nuclear localization. Although our study revealed the effects of lead exposure on the expression levels of CDR1 as and miR-671 in the hippocampal region of mice and preliminarily investigated the molecular mechanisms that may be involved in these changes, there are still some limitations of the present study that need to be addressed in future studies. First, the cerebral cortex, a crucial brain area in AD research, was not analyzed. Future research should broaden the sample scope to encompass the cerebral cortex, thereby providing a more comprehensive evaluation of Pb's effects on various brain regions. In addition, direct measurements of miR-7 levels were not feasible in this study due to experimental and temporal constraints, which limits our understanding of the dynamic interactions between miR-7, CDR1 as, and miR-671. In future studies, we will endeavor to overcome these limitations by directly measuring miR-7 levels and the transcript and protein levels of its target genes (e.g., RelA, NLRP3/Nalp3, etc.) to explore more deeply the regulatory mechanisms of these genes in response to lead exposure. Second, while a hypothesis has been posed suggesting that CDR1 as might release miR-7 via an AGO2-mediated cleavage process, current conditions and time limitations precluded direct validation through RNA immunoprecipitation (RIP) or Ago2-KO cell experiments.