The Role And Mechanism Of Mitochondrial Calcium Uptake 1(MICU1) In The Mouse Model Of Allergic Rhinitis

Background:Allergic rhinitis（AR）is a common allergic inflammatory disease of nasal mucosa in rhinology,which is mediated by immunoglobulin E（Ig E）and driven by T helper type 2（Th2）cells after the body contacts allergens and inhales them.It has a high incidence rate and is also called allergic rhinitis.Its clinical symptoms are mainly manifested as sneezing,runny nose,nasal itching,nasal obstruction and olfactory dysfunction.Apoptosis of nasal mucosal epithelial cells can induce inflammatory response,leading to Ig E production and promote the occurrence of AR,but the specific mechanism is not clear.The occurrence and development of AR is accompanied by a large number of apoptosis of nasal mucosal epithelium,blood vessels and glands.The overload of mitochondrial calcium ions(Ca²⁺)is the main cause of apoptosis.Mitochondrial calcium uptake 1（MICU1）,as a mitochondrial monolayer transmembrane protein,can affect the expression of apoptosis related proteins by regulating the concentration of Ca²⁺,thereby promoting apoptosis.When the expression of MICU1 is abnormal,it will cause mitochondrial Ca²⁺overload,leading to an increase in mitochondrial reactive oxygen species production,increased expression of apoptosis related proteins,and the initiation of apoptosis program,and ultimately leading to apoptosis.However,the relationship between MICU1 and AR is unclear.Objective:This study aims to elucidate the role and related mechanisms of MICU1 in the mouse model of allergic rhinitis through in vitro and in vivo experiments,providing new molecular targets for the treatment of allergic rhinitis in clinical practice.Methods:1.BALB/c strain mice were used to establish AR mouse models.The model group was sensitized by repeated intraperitoneal injection of ovalbumin（OVA）and repeated intranasal drip of OVA provocation.The control group was replaced by the same dose of normal saline.Purification and cultivation of mouse nasal mucosal epithelial cells were carried out simultaneously using the digestion adhesion method and conditioned medium.Behavioral scores were used to evaluate the animal models.Enzyme linked immunosorbent assay（ELISA）was used to detect the expression of ovalbumin-specific Ig E antibodies（OVA-s Ig E）and related cytokine;Quantitative Real-time polymerase chain reaction（q PCR）was used to detect the m RNA expression of specific transcription factors;Flow cytometry（FCM）was used to detect the proportion of Th2 cells and Treg cells,as well as apoptosis;Immunohistochemical staining（IHC）,Western Blot（WB）,and q PCR were used to detect the expression of MICU1;Observation of mitochondrial changes in nasal mucosal epithelial cells and in vitro cells using transmission electron microscopy;The concentration of Ca²⁺in tissues and cells was detected by calcium content colorimetry.TUNEL staining was used to detect the apoptosis of tissue.WB was used to detect the expression of apoptosis related proteins and MICU1.2.The mouse nasal epithelial cells were purified and cultivated by using the digestion adhesion method and conditional culture medium,and the cell type and purity were determined by immunofluorescence staining.MICU1 was overexpressed in the mouse nasal mucosa epithelial cells,FCM were used to detect the apoptosis;CCK-8 method and plate cloning were used to detect cell proliferation activity;The concentration of Ca²⁺in cells was detected by calcium content colorimetry;Apoptosis related proteins were detected by WB,q PCR,and immunofluorescence;Mitochondrial changes were observed under transmission electron microscopy.3.The mouse model of AR was constructed,and treated with MICU1 specific antibody.Behavioral scores were used to evaluate the therapeutic effect;ELISA was used to detect the expression of OVA-s Ig E and related cytokine;QPCR was used to detect the m RNA expression of specific transcription factors;FCM was used to detect the ratio of Th2 cells and Treg cells;WB was used to detect the expression of MICU1 and apoptosis related proteins;The concentration of Ca²⁺in the tissue was detected by calcium content colorimetry;Mitochondrial changes were observed under transmission electron microscopy;TUNEL staining was used to detect the apoptosis of tissue.Results:1.The role of MICU1 in AR mice:Compared with the control group,the behavioral score of the model group increased,and ELISA showed a significant increase in OVA-s Ig E levels;The expression levels of IL-4,IL-5,and IL-13 were significantly upregulated,while the expression of IL-10 was significantly downregulated.QPCR showed a significant upregulation of GATA3 expression and a significant downregulation of Foxp3 expression in the model group mice.FCM showed that the proportion of Th2 cells was significantly upregulated and the proportion of Treg cells was significantly downregulated in the model group mice;OVA treatment promoted apoptosis of mouse nasal mucosal epithelial cells.QPCR,WB,and IHC showed a significant upregulation of MICU1 expression in the nasal mucosa tissue of model group mice.Transmission electron microscopy observed swelling,vacuolization,and cleavage of mitochondria in the nasal mucosal epithelial cells and in vitro cells of the model group mice.The Ca²⁺concentration showed that the Ca²⁺concentration in the nasal mucosa tissue and cells of the model group mice increased.TUNEL staining showed a significant increase in the number of positive apoptotic cells in the nasal mucosa tissue of the model group mice.WB showed that the expression level of MICU1 protein was significantly upregulated,the expression of pro apoptotic proteins Bim and Bid was upregulated,and the expression of anti apoptotic proteins Bcl-2 and MCL-1 was downregulated in the nasal mucosal epithelial cells of the model group mice.2.MICU1 regulates the apoptosis mechanism of nasal mucosa epithelial cells in mice with AR:Compared with the Vector group,overexpression of MICU1 can promote apoptosis of mouse nasal mucosal epithelial cells and inhibit cell proliferation,and clone formation.Overexpression of MICU1 upregulates Ca²⁺concentration in mouse nasal mucosal epithelial cells.Transmission electron microscopy observed swelling,vacuolization,and cleavage of the body crest of the mouse nasal mucosal epithelial cells after MICU1 transfection.QPCR,WB,and immunofluorescence showed that compared with the Vector group,overexpression of MICU1 resulted in a decrease in the expression of anti apoptotic proteins Bcl-2 and MCL-1,while the expression of pro apoptotic proteins Bim and Bid was significantly upregulated.3.MICU1 antibody has a protective effect on AR mice:The behavioral scores showed that the model group scored over 5 points,while the treatment group scored significantly lower at 2.4points.ELISA showed that compared with the control group,the level of OVA-s Ig E in the model group was significantly increased;The use of MICU1 antibody therapy can significantly reduce the levels of OVA-s Ig E.The expression levels of IL-4,IL-5,and IL-13 in the model group mice were significantly upregulated,while the expression of IL-10 was significantly downregulated;After treatment with MICU1antibody,the expression levels of IL-4,IL-5,and IL-13 were significantly reduced,while the expression of IL-10 was significantly upregulated.QPCR showed a significant upregulation of GATA3 expression and a significant downregulation of Foxp3 expression in the model group mice;After treatment with MICU1 antibody,the expression of GATA3 was significantly reduced and the expression of Foxp3 was upregulated.FCM showed that the proportion of Th2 cells was significantly upregulated and the proportion of Treg cells was significantly downregulated in the model group mice;After treatment with MICU1 antibody,the proportion of Th2 cells was significantly reduced and the proportion of Treg cells was upregulated.WB showed that the expression of MICU1 was significantly reduced in the treatment group;The expression of pro apoptotic proteins Bim and Bid in the nasal mucosa of model group mice was significantly upregulated,while the expression of anti apoptotic proteins Bcl-2 and MCL-1 was downregulated;After MICU1 antibody treatment,the expression of pro apoptotic proteins Bim and Bid in the treatment group was effectively reduced,while the expression of anti apoptotic proteins Bcl-2 and MCL-1 was upregulated.The concentration of Ca²⁺in the nasal mucosa tissue of mice in the treatment group decreased.Transmission electron microscopy observed that the mitochondria in the nasal mucosal epithelial cells of the model group mice showed swelling,vacuolization,and somatic ridge rupture changes.The mitochondrial structure in the nasal mucosal epithelial cells of the treatment group mice was relatively normal,and the proportion of vacuolization decreased.The phenomenon of somatic ridge rupture was also improved.TUNEL staining showed that the number of positive apoptotic cells in the nasal mucosa tissue of the model group mice was significantly increased;MICU1 antibody treatment can effectively reduce the number of apoptotic cells in the nasal mucosa tissue of mice in the treatment group.Conclusions:During the occurrence and development of allergic rhinitis in mice,the abnormally high expression level of MICU1 causes Ca²⁺overload and changes in mitochondrial structure,which leads to increased expression of apoptosis related proteins,and ultimately leads to apoptosis of nasal mucosa epithelial cells.MICU1antibody can alleviate the symptoms of allergic rhinitis mice while inhibiting apoptosis of nasal mucosa epithelial cells.MICU1 antibody has a protective mechanism in the course of allergic rhinitis mice,protecting the mitochondria of nasal mucosa epithelial cells from damage and maintaining the normal survival of cells.This study aims to elucidate the role and related mechanisms of MICU1 in the mouse model of allergic rhinitis through in vitro and in vivo experiments,providing new molecular targets for the treatment of allergic rhinitis in clinical practice.