| Objective:To investigate the effect and mechanism of sphingosine-1-phosphate receptor 2(S1P2)antagonist JTE-013 on ovalbumin(OVA)-induced asthma inflammation in mice.Methods:Forty healthy BALB/C mice were divided into four groups:normal group,model group and JTE-013 group.The normal group(CON group)received intraperitoneal injection of normal saline,200 ul/mouse,on days 0,7,and 14;from day 21,10 ml of normal saline was nebulized for 30 min;and from day 19,normal saline,200 ul/mouse,was injected intraperitoneally for 7 consecutive days.The model group was intraperitoneally injected with OVA mixed with 200 ul/mouse on days 0,7,and 14;from day 21,10 ml of OVA solution was taken and placed in the nebulizer for challenge for 30 min;from day 19,normal saline was intraperitoneally injected for 7 consecutive days,of which intraperitoneal injection treatment was performed 30 min before challenge on days 21-25.The JTE-013 treatment group was the same as the model group,and 200 ul/mouse of OVA was intraperitoneally injected on days 0,7,and 14;from day 21,10 ml of OVA solution was placed in the nebulizer for challenge for 30 min;from day 19,JTE-013(2 mg/kg),200 ul/mouse of JTE-013(2 mg/kg)was intraperitoneally injected for 7 consecutive days;of which 21 days to 25 days were treated with intraperitoneal injection 30 min before challenge.In the RAC1 inhibitor group,200 ul/mouse of OVA was intraperitoneally injected on days 0,7,and 14;from day 21,10 ml of OVA solution was placed in a nebulizer for challenge for 30 min for a total of 5 days;from day 19,200 ul/mouse of JTE-013(2 mg/kg)was intraperitoneally injected for 7 consecutive days;on days 21-25,JTE-013 was intraperitoneally injected 30 min before challenge.The RAC1 inhibitor group was given 3 mg/kg by tail vein injection on days 0-21.Twenty-four hours after treatment,blood was collected from the eyeballs,and the mice were sacrificed by subsequent cervical dislocation,and lung tissue and bronchoalveolar lavage fluid(BALF)were obtained.HE staining and AB-PAS staining were used to observe the pathological changes of lung tissues;Raynaud’s-Apodemus staining was used to stain the cell smears to detect the total cell number,eosinophil number,neutrophil number,and lymphocyte number in BALF;enzyme-linked immunosorbent assay was used to detect the concentrations of IL-1,IL-4,and IL-5 in BALF and IgE concentration in serum;immunoblotting was used to detect the contents of RAC1,JNK,and autophagy proteins Beclinl,LC-3B,P-P62,and P62 in the lung tissues of the four groups of mice;and immunohistochemical staining was used to detect the expression of Beclinl,LC-3B,and P62 in the lung tissue structures.Results:1.HE staining and AB-PAS staining:In the model group,there was significant inflammatory cell infiltration around the airways and significant goblet cell proliferation in the airway epithelium;after administration of JTE-013,JTE-013 significantly inhibited the production of airway inflammation and the content of goblet cells.2.Swiss-Giemsa staining:The total cell number,eosinophil,neutrophil,and lymphocyte contents in BALF of mice in the model group were significantly increased,with statistically significant differences(P<0.01);after JTE-013 treatment,the above cell contents were significantly decreased,with statistically significant differences compared with the model group(P<0.01).3.Enzyme-linked immunosorbent assay:The contents of IL-1,IL-4 and IL-5 in BALF of OVA-induced asthmatic mice were significantly higher,while after treatment with JTE-013,the contents of cytokines in BALF of mice were decreased compared with the model group,and the differences were statistically significant(P<0.01);the contents of IgE in serum were significantly higher in the model group than in the normal group,and the contents of IgE were decreased after treatment with JTE-013,and the differences were statistically significant(P<0.01).4.Immunohistochemical staining was used to detect the positive expression of autophagy-related proteins in lung tissues:there were no obvious inflammatory cells around the intact tracheal wall in the normal group,Beclin-1 and LC-3B had less protein expression in the airway epithelium,and P62 had relatively more positive expression;inflammatory cells were significantly increased in the model group,Beclin-1 and LC-3B positive expression in the airway epithelium was significantly increased,while P62 protein expression content was decreased;after JTE-013 treatment,Beclin-1 and LC-3B positive expression were significantly attenuated in lung tissues,while P62 protein content was significantly increased,and inflammatory cells around the airways were significantly reduced.5.Western blot was used to detect the expression of related proteins in lung tissues.The contents of RAC1 and JNK and the content of autophagy-related protein Beclin-1 in lung tissues were significantly increased,the conversion of LC-3B Ⅰ to LC3B Ⅱ was enhanced,the accumulation of P62 was decreased,and the phosphorylation expression of P62 was significantly enhanced(P<0.01);JTE-013 significantly decreased the contents of RAC1 and JNK and the content of autophagy-related protein Beclin-1 in lung tissues,and attenuated the conversion of LC-3B Ⅰ to LC3B Ⅱ,increased the accumulation of P62,and decreased the phosphorylation expression of P62 with extremely significant differences compared with the model group(P<0.01).After administration of RAC1 inhibitor,the contents of RAC1,JNK and the content of autophagy-related protein Beclin-1 were significantly increased,the conversion of LC-3B I to LC3B II was enhanced,the accumulation of P62 was decreased,and the activity of P62 was significantly enhanced(P<0.01);the expression of RAC1,JNK and autophagy-related proteins did not change significantly compared with the model group.Conclusion:JTE-013,an inhibitor of sphingosine 1-phosphate receptor 2,inhibits the production and development of asthma inflammation by inhibiting RAC 1/JNK pathway to regulate autophagy. |
