| Objective:Bronchial asthma,one of the common chronic respiratory diseases,affects more than 300 million people all over the world.It also imposes a huge economic burden on the world healthcare system.Studies have demonstrated that acupuncture could reduce asthma airway inflammation by modulating T-lymphocyte immunity,and this effect may be associated with miR-223/NLRP3 related pyroptosis pathway.In this study,we observed the effects of acupuncture on the airway inflammation in OVA-sensitized asthmatic C57BL/6J mice by acupuncture of Dazhi,Feishu and Zusanli,and the role of miR-223/NLRP3 related pyroptosis pathway and T lymphocyte immunity in asthmatic inflammation.Method:1.C57BL/6J mouse model of bronchial asthma was established by OVA-AAI combined sensitization,and the mice were treated with acupuncture at the Dazhui,Feishu and Zusanli on alternate days for Acu group;2.The histopathological changes of lung tissues were detected by HE staining,etc.Combining with ELISA result of IgE levels in serum;we aim to investigate the effect of acupuncture on airway inflammation in asthma mice.3.The inflammatory factors in serum or bronchoalveolar lavage fluid(BALF)were measured by ELISA to evaluate effect of acupuncture on T cell subsets,by the level of Tlymphocyte-associated factor release,such as TGF-β,IL-4,IL-17A,IFN-γ in mice;4.The differences in the expression levels of miR-223/NLRP3 related pyroptosis pathway were verified by PCR,WB,ELISA,immunohistochemistry,fluorescence staining and LDH level in serum.Results:1.The histological pathological test of mouse lung showed that compared with the Con group,the lung tissue of the Mod group and Sham group showed airway inflammation and remodeling changes such as disorders in bronchial epithelial cells,inflammatory cell aggregation and infiltration of hyaline secretion in the bronchial lumen,wall thickening,significantly increased deposition of collagen fibers,and obvious thickening of myofibers in the tracheal wall.After acupuncture treatment,the pathological manifestations of lung tissue were significantly alleviated(P<0.05).The result of ELISA in serum of Ig-E in Mod group were significantly higher than Con group(P<0.05),the results showed that asthma airway model was established successfully,and acupuncture could reduce asthma airway inflammation.2.Compared with Con group,the pro-inflammatory factor TGF-β,IL-4 and IL-17A in serum or BALF of Mod group were increased,and the anti-inflammatory factor IFN-y was decreased(P<0.05),and the levels of T lymphocyt-related factors were significantly improved after acupuncture treatment(P<0.05).The results indicated that acupuncture could regulate the release of T lymphocyte related factors.3.Compared with Con group,PCR results of Mod group and Sham group showed no significant changes in miR-223 gene in lung tissue(P>0.05),while NLRP3 pathway related genes,such as NLRP3,ASC and IL-1β,were significantly increased(P<0.05).NLRP3,Cleaved GSDMD,Caspase-1/p20 levels were significantly increased by WB detection(P<0.05).NLRP3 pathway related cytokines IL-1β and IL-18 in ELISA results were significantly increased(P<0.05).After acupuncture treatment,the expression of miR-223 gene was significantly increased in Acu group(P<0.05),and the NLRP3 pathway related factors were significantly decreased in PCR,WB and ELISA results(P<0.05).The results of NLRP3-mediated ASC protein immunohistochemistry and GSDMD protein fluorescence were consistent with those of WB results.LDH detection in Serum showed that the LDH concentration after acupuncture was significantly decreased compared with Mod group(P<0.05).It is suggested that acupuncture can activate miR-223 and inhibit NLRP3-related pyroptosis pathway.Conclusion:Acupuncture can effectively reduce airway inflammation by regulating T lymphocyte immunity in asthmatic mice,which may be related to the miR-223/NLRP3 related pyroptosis pathway.Objective:The reticular gene analysis of sputum and peripheral blood of asthmatic patients showed that miR-223 was a negative factor in regulating asthma inflammation.The previous study showed that the expression level of miR-223 gene was increased after acupuncture.In order to further understand whether acupuncture plays a therapeutic role in airway inflammation of asthmatic mice by activating miR-223 gene and inhibiting the NLRP3-mediated cytoscorposis pathway.In this study,miR-223 agomir was used to statically overexpress miR-223 gene in NLRP3 knock out mice(NLRP3-/-mice)and ordinary mice,and the effects of acupuncture on miR-223/NLRP3 mediated pyroptosis pathway and T lymphocyte immunity in airway inflammation of asthmatic mice were observed.Method:1.NLRP3-/-mice were constructed by CRISPR/cas9 technique;12 NLRP3-/-mice and 18 C57BL/6J mice were randomly divided into:Model group(Mod group),miR-223 overexpression group(miR-223 group),miR-223 overexpression+NLRP3 knock out group(miR-223-/-group),acupuncture+ miR-223 overexpression group(miR-223+Acu group),acupuncture+miR-223 overexpression+NLRP3 knock out group(miR-223-/-+Acu group).OVA-AAI was used to construct bronchial ordinary mice and NLRP3-/-mouse models of asthma inflammation.After nasal injection of miR-223 agomir to stabilize the high expression of miR-223,interday acupuncture intervention was performed.2.The histopathological changes of lung tissues were detected by HE staining,etc.Combining with ELISA result of IgE levels in serum;we aim to investigate the effect of acupuncture on airway inflammation in asthma mice after overexpression of miR-223;3.The inflammatory factors in serum or bronchoalveolar lavage fluid(BALF)were measured by ELISA to evaluate effect of acupuncture on T cell subsets,by the level of Tlymphocyte-associated factor release,such as TGF-β,IL-4,IL-17A,IFN-γ in overexpression of miR-223 of mice;4.The differences in the expression levels of miR-223/NLRP3 related pyroptosis pathway were verified by PCR,WB,ELISA,immunohistochemistry,fluorescence staining and LDH level in serum in each group after overexpression of miR-223 in mice.Results:1.Histopathological examination of mouse lung showed that compared with the Mod group,the lung histopathological manifestations were reduced in the miR-223 overexpression group,the bronchial structure was still intact,a small amount of inflammatory cell infiltration and mucus secretion was seen in the lumen along the bronchial wall,the arrangement of mucosal epithelial cells was slightly disturbed,collagen fiber deposition was reduced,and there was no significant thickening of the outer muscle fibers in the wall.After overexpression of miR-223,the improvement of lung pathology was more obvious in ordinary mice and NLRP3-/-mice after acupuncture treatment.ELISA results showed that the IgE level in serum of miR-223 group was significantly decreased compared with that in the Mod group(P<0.05).After overexpression of miR-223,the IgE level in serum was more significantly decreased in the normal mice and NLRP3-/-mice treated with acupuncture(P<0.05).The results indicated that overexpression of miR-223 could reduce airway inflammation in asthmatic mice,and the therapeutic effect of acupuncture might be enhanced after overexpression of miR-223.2.The levels of pro-inflammatory cytokines TGF-β,IL-4 and IL-17A in serum or BALF of miR-223 group were decreased(P<0.05),and the levels of anti-inflammatory cytokines IFN-y were increased compared with those of Mod group(P<0.05),and NLRP3 pathway related cytokines IL-1β and IL-18 were also significantly decreased(P<0.05).After overexpression of miR-223,the change trend of T lymphocyt-related factors was similar between the normal mice and NLRP3-/-mice treated with acupuncture,showing a lower release level of T lymphocyt-related factors(P<0.05).The results indicated that acupuncture can regulate the release of T lymphocyt-related factors by regulating miR-223,so as to reduce airway inflammation of asthma.And this effect may be related to cytokines released by NLRP3 pathway.3.Compared with the Mod group,PCR results showed that after overexpression of miR223,the expression level of miR-223 in lung tissues of each group was increased(P<0.05),and NLRP3 path-related genes such as ASC and IL-1β were significantly decreased(P<0.05).After overexpression of miR-223,the expression level of miR-223 gene was higher in all groups treated by acupuncture,and the expression level of NLRP3 pathway genes was decreased more significantly.It is suggested that acupuncture can inhibit NLRP3 pathway by activating miR-223 gene(P<0.05).Compared with the Mod group,the NLRP3 pathway protein level in the miR-223 group was decreased by WB results(P<0.05),After the miR223 overexpression group,the NLRP3 pathway protein expression in the normal mice and NLRP3-/-mice after acupuncture treatment was more significantly decreased by WB results(P<0.05).The results of NLRP3-mediated ASC protein immunohistochemistry and GSDMD protein fluorescence were consistent with those of WB.LDH content in Serum also found that the LDH level in serum of miR-223 group was decreased compared with that of Mod group,and the decrease was more obvious in the groups after acupuncture treatment.The results indicated that acupuncture could inhibit NLRP3-related pyroptosis pathway,regulate T lymphocyte subsets,and reduce airway inflammation of asthma by activating miR-223 gene expression.Conclusion:Acupuncture can activate miR-223,inhibit the NLRP3-related pyroptosis pathway,regulate T lymphocyte immunity and reduce airway inflammation in asthmatic mice.Objective:Activation of NLRP3 inflammasome pathway is considered to be one;of the important causes of the inflammatory response in bronchial asthma.In the last two sections,acupuncture has been found to play a protective role in the inflammatory model of asthmatic mice by activating miR-223 gene expression and inhibiting the NLRP3-related pyroptosis pathway.However,in airway inflammation of asthmatic mice,whether acupuncture first activates miR-223 and then inhibits NIRP3-mediated pyroptosis pathway,or first inhibits NLRP3-mediated pyroptosis pathway and activates miR-223 gene,thus reducing inflammation remains to be discussed.In order to further understand whether acupuncture inhibits the NLRP3 inflammatory pyroptosis pathway by activating the miR-223 pathway,NLRP3-/-and NLRP3 agonists were used in this section to investigate the effects of acupuncture on the miR-223/NLRP3 pathway and T lymphocyte immunity in asthmatic inflammatory NLRP3-/-mice.In addition,293T cells were used to perform dual luciferase assay in vitro to verify the targeted regulation of miR-223 and NLRP3 gene.Method:1.18 C57BL/6J mice and NLRP3-/-mice were randomly divided into blank group(Con group),model group(Mod group),Nigerian agonist group(ModNi group),NLRP3 knock out group(Mod-/-group),acupuncture knockout group(Acu-/-group)and acupuncture group(Acu group).Except Con group,OVA-AAI combined sensitization was used to construct asthmatic inflammatory mouse model,interday acupuncture intervention was performed.2.The histopathological changes of lung tissues were detected by HE staining,etc.Combining with ELISA result of IgE levels in serum;we aim to investigate the effect of NLRP3 gene on airway inflammation in asthmatic mice;3.The inflammatory factors in serum or bronchoalveolar lavage fluid(BALF)were measured by ELISA to explore the effect of NLRP3 gene when acupuncture regulated T cell subsets,by the level of T-lymphocyte-associated factor release,such as TGF-β,IL-4,IL-17A、IFN-y in mice;4.The differences in the expression levels of miR-223/NLRP3 related pyroptosis pathway were verified by PCR,WB,ELISA,immunohistochemistry,fluorescence staining and LDH level in serum in each group,we aim to investigate the effect of NLRP3 gene on the expression of miR-223/NlRP3-mediated pyroptosis pathway in each group of mice;5.NLRP3-miR-223-3p-WT(wild type)plasmid and NLRP3-miR-223-3p-Mut(mutant type)plasmid were constructed,and hsa-miR-223-3p mimic or mimic NC were transfected into the above plasmids,and double luciferase detection was performed after transfection for 24h.Results:1.The histological pathological examination of mouse lung showed that compared with the Mod group,the lung tissue of ModNi group showed more serious manifestations of bronchial epithelial cell disorder,inflammatory cell aggregation,and hyaline secretion infiltration,with thickened or even obstructed tube walls and collagen fiber deposition diffused around the bronchi,while the NLRP3-/-group mice showed significantly less manifestations of each pathology.The improvement of lung pathology was more obvious in ordinary mice and NLRP3-/-mice after acupuncture treatment.ELISA results showed that compared with the Mod group,IgE level in serum in the ModNi group was significantly increased(P<0.05),Ig-E level in serum in the NLRP3-/-group was significantly decreased(P<0.05).and there was no significant difference in IgE level between the NLRP3-/-and normol mice after acupuncture treatment(P>0.05).These results indicated that NLRP3 related pyroptosis aggravated airway inflammation in asthmatic mice,and acupuncture could reduce airway inflammation by inhibiting NLRP3 pathway.2.Compared with the Mod group,the pro-inflammatory factor TGF-β,IL-4 and IL-17A in serum or BALF in ModNi group were increased,and the anti-inflammatory factor IFN-γwas decreased(P<0.05),while the T-lymphocyt-related factors in NLRP3-/-group showed a trend of decreasing pro-inflammatory factor level and increasing anti-inflammatory factor level.NLRP3-/-and normal mice treated with acupuncture showed more significant improvement in the release level of T lymphocyte proinflammatory cytokines(P<0.05),indicating that the enhancement or attenuation of NLRP3 pathway can affect the airway inflammation level of asthma mice.Acupuncture can reduce airway inflammation of asthma by regulating the release of T lymphocyte related factors,and this effect is related to the inhibition of NLRP3 pathway.3.The PCR results showed that compared with the Mod group,the level of miR-223 gene in the ModNi group had no significant changes(P>0.05),while the NLRP3 inflammasome pathway related genes,such as NLRP3,ASC and IL-1β,were significantly increased(P<0.05).There was no significant increase in miR-223 gene(P>0.05),while NLRP3 pathway related expression was significantly decreased(P<0.05).Compared with Mod group,NLRP3-/-mice and normal mice after acupuncture treatment had significantly increased miR-223 gene,and the expression of NLRP3 pathway genes was significantly decreased,suggesting that acupuncture could inhibit the NLRP3 inflammasome pathway by activating miR-223,thereby affecting T lymphocyte immunity and reducing airway inflammation in mice.WB detection showed that compared with Mod group,NLRP3 pathway protein level in ModNi group was significantly increased(P<0.05),while NLRP3-/group was significantly decreased(P<0.05).Acupuncture treatment of normal and NLRP3-/mice showed a more significant decrease in NLRP3 pathway protein level(P<0.05).The results of NLRP3-mediated ASC protein immunohistochemistry and GSDMD protein fluorescence were consistent with the results of WB in each group.Serum LDH content detection also found that compared with the Mod group,the serum LDH level in the ModNi group was increased,and the serum LDH level in the NLRP3-/-group was decreased(P<0.05),and the decrease was more obvious in the groups after acupuncture treatment.It is suggested that acupuncture can inhibit NLRP3-mediated pyroptosis pathway,regulate T lymphocyte subsets,and reduce airway inflammation of asthma by activating miR-223 gene expression.4.After transfection with hsa-miR-223-3p mimic,the dual luciferase activity of WT group was significantly decreased(P<0.05),indicating that hsa-miR-223-3p could negatively regulate the expression of NLRP3 gene.This effect is eliminated when NLRP3 is mutated,that is,hsa-miR-223-3p can bind to NLRP3 and inhibit NLRP3 gene expression.Conclusion:miR-223-3p can bind to NLRP3 and inhibit the expression of NLRP3 gene.Acupuncture can activate miR-223/NLRP3-related pyroptosis pathway to regulate T lymphocyte immunity and effectively reduce airway inflammation in asthmatic mice. |
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