Effect And Mechanism Of Nicotine On Reducing Hippocampal Neuroinflammation And Neuronal Death Induced By Crystalline Silica Dust In Mice

Objective:The inhalation of crystalline silica dust（c Si O₂）can lead to the development of silicosis as well as mood disorders.Notably,the nicotine present in cigarettes may offer temporary relief against the mood disorders caused by c Si O₂ exposure.The purpose of this study is to explore the impact and underlying mechanisms of silica dust exposure,nicotine consumption,and the combined exposure of silica dust and nicotine on the behavior,neuroinflammation,and neuronal apoptosis in the hippocampus of mice.Methods:（1）To establish a mouse model exposed to nicotine and silica dust.Male C57BL/6mice were randomly divided into the Veh group,Sil group,Nic group,and Nic-Sil group.Nic group,Nic-Sil group drank 200μg/m L nicotine solution;Veh group and Sil group drank 1%saccharin sodium.In the Sil group,and Nic-Sil group,mice were injected with c Si O₂ suspension（50 mg/m L,50μL）every 3 days,5 times in total;Veh group and Nic group were given equal volumes of normal saline.Weigh daily.（2）Successive open field test（OFT）,elevated plus maze test（EPM）,and marble buring test（MBT）were conducted in the 3rd,6th,and 9th week,respectively,with tissue samples being taken in batches.（3）An ELISA kit was employed to identify inflammatory factors present in the peripheral blood.（4）The assessment of the blood-brain barrier’s integrity was conducted by injecting Evans blue into the posterior orbital vein of mice.（5）Differentially expressed genes（DEGs）were identified and confirmed through q PCR.RNA-seq sequencing analysis and DEGs screening were performed on the hippocampus of three mice from each group.Following this,genes related to inflammation and apoptosis were screened and verified through q PCR and immunofluorescence staining.（6）Histological staining and WB were utilized to identify astrocyte activation and neuronal apoptosis.Results:（1）The construction of the mice model for double exposure to nicotine and c Si O₂was successful.The Sil group of mice showed a significant decrease in body weight compared to the Veh group,with the most significant difference observed on the third day of exposure.In contrast,the Nic group did not exhibit any significant changes in body weight.Furthermore,there was no significant difference in body weight between the Nic-Sil group and the group exposed to c Si O₂ alone.（2）The experimental data reveals a marked reduction in the total distance traversed by mice in the Sil group during the OFT,accompanied by an increase in the duration of immobility,with the most substantial difference being observed in the third week.Conversely,the Nic-Sil group exhibited improvement compared to the Sil group.Moreover,the Sil group of EPM exhibited a significant decrease in the number of mice entering the open arm,while the MBT showed no significant variation among the groups.（3）The Sil group exhibited a significant increase in serum IL6 levels.However,after double exposure,nicotine was found to decrease the expression of IL6.（4）The third week of c Si O₂ exposure resulted in an increase in BBB permeability specifically within the hippocampus,accompanied by a reduction in Cldn-5 expression,as well as an elevation in both Albumin and EB levels.（5）In the comparison between Sil and Veh,517 genes were observed to be up-regulated while 263 genes were down-regulated,with Lcn2 being the most significantly up-regulated among them.Similarly,Nic-Sil vs Sil showed 487up-regulated genes and 263 down-regulated genes.Genes that displayed a decrease in down-regulation or remained down-regulated in the Nic-Sil group,as well as those that were down-regulated in the Sil group,were identified as Weaken Genes.Out of 227genes associated with neuroinflammation and apoptosis,Lcn2 exhibited the most significant variation.The q PCR results supported this finding.Immunofluorescence staining results indicated that Lcn2 was mainly secreted by astrocytes.（6）Upon exposure to c Si O₂,the expression of STAT3,IL6/NF-κB were found to be up-regulated.The number of neurons decreased as observed by Nissl and Neu N staining.TUNEL and BAX were also up-regulated,indicating an increase in neuron apoptosis.Furthermore,Lcn2 was significantly increased in activated astrocytes.The"triad"structure was observed to increase by Iba1/GFAP/Neu N staining.However,nicotine was found to inhibit astrocytes activation,thereby reducing neuronal apoptosis and the number of"triad"structure.Conclusion:Exposure to c Si O₂ has been found to trigger neuroinflammation and neuronal apoptosis,leading to the development of mood abnormalities.However,nicotine has been shown to possess neuroprotective properties by alleviating neuroinflammation-induced aberrant behavior in nicotine and c Si O₂ double-exposed mice.Figure[18]table[2]reference[107]...