Study On The Mechanism Of Acupuncture At Yingxiang Point To Regulate Nasal Mucosal Immunity In Mice With Allergic Rhinitis Based On The Linkage Changes Of "Calcitonin Gene-related Peptide And DC-T Cells"

Objective1.To study the effects of Calcitonin gene related peptide（CGRP）on the differentiation and antigen presentation of bone marrow-derived dendritic cells（DC）and the effects of CGRP-intervened DC on the differentiation of naive CD4+T cells;2.To study the effect of intranasal injection of exogenous allogeneic DC on nasal mucosal immunity in mice with allergic rhinitis（AR）after CGRP intervention of DC;3.To study the effect of acupuncture at Yingxiang point on nasal mucosal immunity of AR mice;4.To provide evidence for acupuncture at Yingxiang point to regulate nasal mucosal immunity of AR mice based on the linkage change of"CGRP and DC-T cells".Method1.Effects of CGRP on DC surface molecules and cytokines:BALB/c male mice aged 4-6weeks were selected for isolation and culture of bone marrow derived DCs in vitro.The DCs on the seventh day of culture were collected,the morphology of the DCs was observed by light microscope,the DCs were identified by transmission electron microscopy,and the purity of the cultured DCs was detected by flow cytometry.Different concentrations of CGRP(10^-6M,10^-8M,10^-10M)were used to intervene DCs,and lipopolysaccharide（LPS）group and blank control group were set separately.After 48 hours of intervention,the DC phenotypes CD11c,MHCII,CD80 and CD86 in each group were analyzed by flow cytometry;DC Toll-like receptors in each group were detected by real-time fluorescence quantitative PCR;the cytokines IL-10 and IL-12 in the supernatant of each group were detected by ELISA.2.Effects of CGRP-intervened DCs on naive CD4+T cell differentiation:The spleen of BALB/c male mice aged 4-6 weeks was taken out and the naive CD4+T cells were prepared by magnetic bead sorting.DCs in each group were co-cultured with CD4+T cells at a ratio of 1:5,and Ovalbumin（OVA）was added for 72 hours.The expression of CD69 on CD4+T cells was detected by flow cytometry;the expression of T-bet and GATA3 m RNA in cells was detected by real-time fluorescence quantitative PCR;the levels of CD4+T cell cytokines IFN-γand IL-4 were detected by ELISA.3.Study on the immunity of mouse nasal mucosa after DC isolated and cultured in vitro injected into nasal mucosa:（1）The 6-week-old BALB/c male mice were selected,and the AR mouse model was established by intraperitoneal injection of OVA combined with intranasal instillation.（2）BALB/c male mice were randomly divided into two groups,blank control group and AR model group.Bone marrow-derived immature DCs were fluorescently labeled with PKH26 and injected into the nasal mucosa of two groups of mice,respectively.After 48 hours,the mouse nasal mucosa was taken for frozen section,which were labeled with CD11c and CD80 fluorescence respectively,and observed by fluorescence microscope;another nasal mucosa was taken,and the samples were prepared by conventional transmission electron microscope,and the morphology of mouse nasal mucosa was observed by transmission electron microscope.（3）BALB/c male mice were randomly divided into blank control group,AR model group and capsaicin nasal drop group.The AR model of capsaicin blocking neuropeptide secretion was established,and the expression of CGRP in the nasal mucosa of mice was observed by immunohistochemistry.（4）Balb/c male mice,established the AR model of capsaicin blocking neuropeptide secretion,and were randomly divided into group A and group B.In group A,DCs intervened by CGRP 10^-6M were injected into nasal mucosa,while in group B,DCs not intervened by CGRP were injected into nasal mucosa.48 hours after injection,the behavioral scores of mice were performed;the nasal mucosa of mice was observed by HE staining;the levels of IFN-γand IL-4 in serum and nasal mucosa of mice were detected by ELISA.4.Effect of acupuncture at Yingxiang point on nasal mucosal immunity in AR mice:Balb/c male mice were randomly divided into four groups:blank control group,AR model group,sham acupuncture group,and acupuncture group.After the model was successfully established,the acupuncture group selected the Ying Xiang point and acupuncture was performed once a day,five times a week for a fortnight.During this period,the acupuncture group,the sham acupuncture group,and the AR model group were sensitized by nasal instillation of 4%ovalbumin,and the blank control group was intranasally instilled with normal saline,once every other day for two consecutive weeks.24 hours after the last intervention,the behavioral scores of the mice were performed;the nasal mucosa of the mice was observed by HE staining;the expression of CGRP in mouse nasal mucosa was detected by immunohistochemistry;the levels of CD11c,CD80,CD86,and MHCII on the DC surface of mouse nasal mucosa were measured by flow cytometry;the levels of serum IFN-γand IL-4 and the expressions of T-bet and GATA-3 were detected by ELISA.Result1.（1）DC observation under light microscope:the first three days of culture,the cells were small and round,without burr-like protrusions;from the fourth to seventh day of culture,the cells began to form colonies,and burr-like protrusions could be seen on the surface of the cells.Observation under the electron microscope of DC:DC cells were irregular in shape,with slender dendritic protrusions extending from the cell surface;the nuclei were irregular and slanted to one side,and the intracellular organelles were abundant,with more mitochondria,rough endoplasmic reticulum and smooth endoplasmic reticulum,and vesicles were visible.The purity of the cultured DC reached 98.3%,which met the experimental needs.（2）Compared with the blank control group,the expressions of DC phenotypes CD11c,MHCII,CD80 and CD86 in the LPS group were all enhanced,with statistically significant differences.Compared with the blank control group,the expressions of DC phenotypes CD11c and MHCII in the CGRP10^-6M and CGRP10^-8M groups were all attenuated,and the expressions of CD80 and CD86 were enhanced,with statistically significant differences.（3）Compared with blank control group,the expressions of TLR2 m RNA,TLR3 m RNA and TLR4 m RNA in LPS group were decreased,and the differences were statistically significant.Compared with blank control group,TLR3 m RNA and TLR4 m RNA expression were decreased in CGRP10^-6M group;TLR2 m RNA expression was decreased and TLR3 m RNA expression was increased in CGRP 10^-8M group and CGRP10^-10M group,and the differences were statistically significant.（4）Compared with the blank control group,the levels of IL-12 in LPS group,CGRP10^-6M group,CGRP10^-8M group,and CGRP10^-10M group all increased,with statistically significant differences;the level of IL-10 in CGRP10^-10M group decreased,with a statistically significant difference.2.Compared with blank control group,the expression of CD69 in LPS group and CGRP10^-6M group was enhanced,with statistically significant differences.Compared with the blank control group,the expression of T-bet and GATA3 m RNA in the LPS group,CGRP10^-6M group and CGRP10^-10M group were decreased,with statistically significant differences.Compared with blank control group,the levels of IL-4 in LPS group,CGRP10^-6M group,CGRP10^-8M group and CGRP10^-10M group were decreased,while the levels of IFN-γwere increased,with statistically significant differences.3.（1）Establishment of AR mouse model:Compared with the blank control group,the behavioral scores of the mice in the AR model group increased,and the concentration of OVA-s Ig E in serum increased.HE staining showed that nasal mucosa cilia were disordered and absent,lamina propria glands and blood vessels were dilated,and inflammatory cells were infiltrated,which indicated that the model was successfully established.（2）The cell membrane of DC was uniformly marked in red by PKH26.The results of immunofluorescence showed that after the injection of immature DC into the nasal mucosa,the dendritic cells in the AR model group were mainly distributed in the epithelial layer and the lamina propria,and the dendritic cells in the blank control group were mainly distributed in the epithelial layer.The results of electron microscopy showed that the cilia of nasal mucosa in AR model group were exfoliated and sparse,mitochondria in epithelial cells were significantly increased,and some mitochondria were swollen,body crista were broken,decreased,disordered,and even body crista disappeared.In the blank control group,the cilia were dense,scattered and twisted locally,and the mitochondria were slightly edematous.（3）Compared with the blank control group,the expression of CGRP in the nasal mucosa of mice in the AR group was enhanced,with statistically significant differences.Compared with the AR model group,the expression of CGRP in the nasal mucosa of mice in the capsaicin nasal drop group was attenuated,and there was a statistically significant difference.Compared with the blank control group,the expression of CGRP in nasal mucosa of mice in the capsaicin nasal infusion group had no significant change,and there was no statistically significant difference.This suggests that the AR model of capsaicin blocking neuropeptide secretion was successfully established.（4）There was no statistical difference in the behavioral scores of AR mice between the DC nasal mucosa injection group with CGRP10^-6M intervention and the DC nasal mucosa injection group without CGRP intervention.The results of HE staining showed that the infiltration of inflammatory cells in the nasal mucosa of mice in the DC group with CGRP10^-6M intervention was more than that in the DC group without CGRP intervention.Compared with the DC group without CGRP intervention,the levels of IFN-γin serum and nasal mucosa of mice in the DC group with CGRP10^-6M intervention were decreased,and the levels of IL-4 in serum and nasal mucosa were increased,with statistically significant differences.4.（1）Compared with the blank control group,the behavioral scores of mice in the AR model group and the sham acupuncture group were increased,and the difference was statistically significant.Compared with the AR model group,the behavioral scores of mice in the acupuncture group were decreased,and the difference was statistically significant.（2）The results of HE staining showed that the nasal mucosal epithelial cells in the blank control group were arranged neatly,and no obvious inflammatory cell infiltration was observed.In the AR model group and the sham acupuncture group,nasal mucosa cilia were disordered and absent,and inflammatory cell infiltration was observed.In the acupuncture group,the nasal mucosal epithelial cells were basically arranged neatly,and no obvious inflammatory cell infiltration was observed.（3）The results of CGRP immunohistochemistry showed that compared with the blank control group,the expression of CGRP in the nasal mucosa of the AR model group and the sham acupuncture group was enhanced,with statistically significant differences.Compared with AR model group,CGRP expression in nasal mucosa of acupuncture group was decreased,and the difference was statistically significant.（4）Flow cytometry test results of nasal mucosa DC phenotype:Compared with the blank control group,the expression of CD11c+/CD80+and CD11c+/CD86+on the surface of nasal mucosa DC in the acupuncture group was increased,with a statistically significant difference.Compared with the AR model group,the expression of CD11c+/MHCII+,CD11c+/CD80+,and CD11c+/CD86+on the DC surface of the nasal mucosa in the acupuncture group increased significantly,with a statistically significant difference.Compared with the blank control group,the serum levels of OVA-s Ig E,IL-4,and GATA-3 in the AR model group increased,while IFN-γand T-bet levels decreased,with a statistically significant difference.Compared with the AR model group,the serum levels of OVA-s Ig E,IL-4,and GATA-3 in the acupuncture group decreased,while IFN-γand T-bet levels increased,with a statistically significant difference.Conclusion1.CGRP could reduce the expression of CD11c and MHCII,and enhance the expression of CD80 and CD86 in bone marrow derived DC isolated and cultured in vitro,weaken the expression of TLR2 m RNA and TLR4 m RNA in DC,and increase the level of cytokine IL-12 in DC.CGRP-intervened DC can promote the activation of naive CD4+T cells.2.Immature DCs from bone marrow were mainly distributed in the nasal epithelium and lamina propria of AR mice and the nasal epithelium of normal mice after injection into the nasal mucosa of mice.3.The AR model of blocking the secretion of neuropeptides by capsaicin was successfully established.After the injection of CGRP-intervened DC into the nasal mucosa of AR mice,the Th2-type immune response of AR mice was aggravated.4.Acupuncture at Yingxiang point can reduce the expression of CGRP in mouse nasal mucosa,promote the expression of CD11c+/MHCII+,CD11c+/CD80+,CD11c+/CD86+molecules on the surface of DC cells,reduce the levels of OVA-s Ig E,IL-4,and GATA-3 in mouse serum,and increase serum IFN-γand T-bet levels,thereby improving allergic inflammation.5.Acupuncture at Yingxiang acupoint may regulate the balance of Th1/Th2 cells by reducing CGRP,but this process is not through DC.