Effect Of 1,25-Dihydroxyvitamin D3 On NLRP3 Inflammasome In The Mice Model Of Asthma

Objective:By establishing the mice model of asthma,we aim to explore the effect of 1,25-dihydroxyvitamin D3 on NLRP3 inflammasome in asthma-related airway inflammation.Methods:Twenty-four Specific pathogen-free（SPF）female Balb/c mice with an average weight of（20±2）g were divided into three groups according to the complete randomization method（n=8 for each group）.Three groups include control group,asthma group,and intervention group.ovalbumin（OVA）sensitizing solution（50μg OVA emulsified with 4 mg AL（OH）₃）was injected intraperitoneally three times on days 1,8 and 15 in asthma group and intervention group.Then mice were atomized with 1%OVA solution by ultrasonic atomizer on days 22-28.For control,mice were given the same amount of normal saline instead of OVA intraperitoneal injection and atomization inhalation,and the rest operation steps were the same as those in the asthma model group.1,25（OH）₂D₃ was administrated into mice in the intervention group by intraperitoneal injections 30 min before each aerosol inhalation.In the same time,the same amount of normal saline instead of 1,25（OH）₂D₃ was injected intraperitoneally in control group and asthma group.12 hours after the last challenge,the bronchoalveolar lavage fluid（BALF）samples were collected and the lungs were harvested.Histopathological examination of the lung specimens was carried out using the hematoxylin-eosin（HE）staining and Periodic Acid-Solferino（PAS）staining.The expression of NLRP3 and Caspase-1 were observed by immunohistochemistry and were detected by Western blot.The levels of IL-1βand IL-18 in BALF supernatant were determined by ELISA.The correlation between NLRP3 and Caspase-1 was analyzed,and the correlation between the two and the levels of IL-18 and IL-1βwas analyzed.Results:1.At the sensitization stage,dietary activities,hair color and body weight of mice in each group had no significant changes.At the challenge stage,the mice in asthma group experienced accelerating breathing,moving restlessly,panting and other symptoms.The above symptoms can be relieved 1h after atomization.Increasing exposure to aerosols consisting of 1%OVA over time,the mice were poor spirited and had rough,shaggy fur.The manifestations of mice in intervention group were similar,just to a lesser degree.The asthma-related symptoms can be relieved30min after atomization.In control group,the activity of mice increased within 3minutes at the beginning of atomization,but soon returned to normal.After repeated atomization,the hair,diet and activity were not significantly affected.2.In asthma group,HE staining showed narrowing of the lumen,disturbing the structure of the smooth muscle of the airways and inflammatory cell infiltration especially eosnophils.PAS staining showed increased mucus secretion and goblet cell proliferation.The structure of the airways in intervention group were relatively intact.The degree of the infiltration of inflammatory cells were less than the asthma group.The pathological changes of mucus secretion and goblet cell hyperplasia were mild.The structure of the airways in control group were smooth and intact.No pathological changes such as mucus secretion and goblet cell hyperplasia were observed.3.Expresstion of NLRP3 and Caspase-1 were observed in the cytosol of the epithelial cells by immunohistochemistry.Compared with control group as well as intervention group,the positive expression area of NLRP3 and Caspase-1 in asthma group was increased.Intervention group showed increased expression of NLRP3 and Caspase-1 than that in control group.Western blot showed the expression of NLRP3and Caspase-1 in asthma group significantly increased（P<0.01）.Compare with asthma group,the expression of NLRP3 and Caspase-1 decreased after 1,25（OH）₂D₃intervention,with statistical difference.（P<0.05）.4.Levels of IL-18 and IL-1β:The levels of IL-18（131.25±7.26ng/L）and IL-1β（114.25±4.45ng/L）of BALF in asthma group were significantly higher than those in control group（92.85±7.27ng/L,65.43±4.33ng/L）,with statistical difference.（P<0.05）.And IL-18（116.02±5.66ng/L）,IL-1β（84.29±4.38ng/L）levels in intervention group were higher than those in control group,with statistical difference.（P<0.05）.5.The Person correlation test showed that the expression of NLRP3 and Caspase-1 has a positive correlation（r=0.9088,P<0.001）.The level of the expression of NLRP3 was positively correlated to the levels of IL-18（r=0.8585,P<0.001）and IL-1β（r=0.9428,P<0.001）in alveolar lavage fluid.The expression level of Caspase-1 was positively correlated to the levels of IL-18（r=0.8422 P<0.001）and IL-1β（r=0.8964P<0.001）.Conclusions:1.NLRP3 inflammasome was activated in the mice model of asthma,increasing the release of mature IL-1βand IL-18,which is associated with airway inflammation and airway structure disturbance.2.1,25（OH）2D3 reduced airway inflammation and relieved the asthma-related symptoms by inhibiting the NLRP3/Caspase-1/IL-1βpathway.