| Airway allergic diseases mainly include allergic rhinitis,allergic cough and bronchial asthma,is a specific IgE and Th2 cytokine-mediated upper and lower respiratory tract type I hypersensitivity reaction,and there is no effective treatment.Peroxisome proliferators-active receptors γ is one of the super-family members of the nuclear hormone receptors on which the ligand depends,and PPAR can also play an antioxidant stress role in addition to regulating fat and sugar metabolism and fat cell differentiation.Oxidative stress also played an important role in airway allergic inflammation.Mast cells are derived from bone marrow hematologic progenitor cells,migrate to outer tissues to differentiate mature and participate in immune response.Mast cell degranulation is considered to be the "total valve" of type I hypersensitive reactions.Objective:To study the therapeutic effect of PPARy agonists in mouse allergic rhinitis model,and the inhibitory effect of PPARγ in the function of bone marrow-derived mast cells(BMMCs)and their differentiation and maturation.And to Study the effect of PPARγactivation on mast cell degranulation in allergic reaction and its mechanism.Methods:1.The therapeutic effect of PPARγ agonists on allergic rhinitisSPF grade female C57BL/6J mice aged from 8 to 10 weeks were randomly divided into 4 groups with six mice in each group.The times of sneezing and nose scratching were recorded.Inflammatory nasal cell infiltration was detected by HE staining in coronal nasal section.Serum specific IgE,IL-5 and IFNy in nasal lavage fluid were detected by ELISA.2.PPARγ Agonists inhibit mast cell maturation and induce apoptosisBMMC were collected from the femur and tibia of C57BL/6J female mice(8-10 weeks old).To evaluate the maturation and differentiation of BMMC,CD117 and FCεRIα were detected by flow cytometry.MRNA expression detection of-6 and PPARγby qRT-PCR level.The effects of different concentrations of PIO on BMMC cell proliferation were measured by Alamar Blue method.β-Hexosaminidase assay to detect BMMC degranulation inhibition.Annexin V/PI apoptosis detection kit was used to detect mast cell apoptosis.3.The mechanism of PPARγ’s inhibitory effect on mast cell degranulationConstruction of PPARy overexpression cell line in RBL-2H3 cells(RBL-2H3 PPARy OE).CCK-8 kit was used to detect the effect of different concentrations of PIO on the survival rate of RBL-2H3 cells.Degranulation test was used to detect the βhexosaminidase release rate of RBL-2H3 cells and RBL-2H3 PPARy OE cell line under the intervention of PIO.MRNA expression Mast cell cytokines IL-4,IL-5,IL-13 and TNF α were detected by qRT-PCR.Western blotting was used to detect the key protein phosphorylation level shift in FcεRI signaling pathway of RBL-2H3 cells treated with PIO.We also compared the phosphorylation levels of key proteins in this signaling pathway in RBL-2H3 WT cells and RBL-2H3 PPARy OE cells.Result:1.The Therapeutic effect of PPARy agonists on allergic rhinitis.1)The times of sneezing and scratching the nose in PIO group were 12.50±1.78 and 42.17±5.59 respectively,and those in OVA group were 52.00±6.53 and 72.83±5.07 respectively.There was significant difference between the two groups(P<0.0001).The times of sneezing and scratching the nose in the antagonistic group were 64.00±6.03 and 62.67±5.14 respectively,which were significantly different from those in the PIO group(P<0.0001).2)Nasal HE staining showed that the epithelial continuity of nasal mucosa in OVA group was disrupted,and a large number of inflammatory cells infiltrated under the mucosa.Compared with OVA group,the nasal mucosa epithelium of PIO group was intact,and the infiltration of inflammatory cells was significantly reduced.The pathological manifestations of the antagonistic group were similar to those in OVA group.The number of eosinophils in nasal submucosa of OVA group was 80.67±5.64.After PIO treatment,the number of eosinophils was 38.83±4.47.There was significant difference between the two groups(P<0.001).The number of eosinophils in nasal mucosa in antagonistic group was 62.50±3.68,which is significantly higher than that in PIO group(P<0.001).3)The serum specific IgE concentration in PIO group was 61.81±2.45ng/ml and that in OVA group was 28.73±1.63ng/ml.There was significant difference between the two groups,P=0.023.The concentration of IgE in the antagonistic group was 56.36±3.49ng/ml,which was significantly different from that in the PIO group(P<0.05).4)The concentration of IL-5 in OVA group was 40.13±2.45ng/ml and that in PIO group was 9.03±0.41ng/ml.There was significant difference between the two groups(P<0.0001).The concentration of IL-5 in the antagonistic group was 15.5 6±1.14ng/ml,which was significantly different from that in the PIO group(P<0.001).5)The concentration of IFN-γ in OVA group was 577.59±61.28ng/ml,after PIO treatment the concentration of IFN-γ was 417.97±38.99ng/ml,and there was significant difference between the two groups(P<0.001).2.PPARγ agonists inhibit mast cell maturation and induce apoptosis1)PIO decreased CD117 and FcεRIα expression in a concentration dependent manner,and the expression of surface antigen was significantly reduced at 20μM.P<0.001.2)The survival rate of MCS was negatively correlated with the concentration of PIO treatment.The survival rate of cultured BMMC was significantly decreased to 50.4±7.71%when treated with PIO(20μM),P<0.05.Also,when treated with PIO(20μM),the percentage of AnnexinV+/PI+BMMC cells was 19.2±1.51%,while that in the control group was 14.9±0.87%(P<0.05,n=3).3)Compared with the control group,PIO administration(20μM)decreased the release rate of β-Hexosaminidase by 25.0%,the difference was statistically significant,P<0.05.It shows that PIO can inhibit the degranulation of MCs.4)The expression level of MCP-6 mRNAin BMMC treated with PIO for 4 weeks was significantly down regulated(P<0.05).PIO treatment up-regulated the mRNA expression of PPARy in BMMC three-fold(P<0.05).5)BMMCs of PIO gavage group and control group were cultured in vitro for 3 weeks,and the percentages of CD117/FcεRIα double positive cells were 56.0±4.2%and 78.0±3.5%,respectively(P<0.05,n=3).3.The mechanism of PPARγ’s inhibitory effect on mast cell degranulation.1)Cell proliferation was detected by CCK-8 method.There was no significant difference between PIO treated cells and the control group in 24-hour treatment group(P>0.05).PIO(10μM and 20μM)treatment for 48 hours significantly inhibited cell proliferation(P<0.05),the cell survival rates were 83.3±2.1%and 73.2 ± 2.2%,respectively.After 48 hours of treatment,the apoptosis induced by PIO(20μM)was detected by AnnexinV/PI method.The percentage of AnnexinV+/PI+cells in PIO treatment group was 9.33±0.18%,while that in control group was 7.07±0.63%(P<0.05,n=3).2)In RBL-2H3 cells,when treated with PIO at concentrations of 1,5,10 and 25μM,the release rate of β-hexosaminidase was 15.64±0.18%,16.79±0.91%,15.99±0.55%and 16.06±0.54%,respectively,which were significantly different from the control group(13.3 7±1.21%)(P<0.05,n=3).After DNP-IgE stimulation,the release rate of β-hexosaminidase of RBL-2H3 WT cells and RBL-2H3 PPARγ OE cells were 25.86±0.55%and 17.61±0.75%,respectively.There was significant difference between the two groups(P<0.05,n=3).(P<0.05).3)After DNP-IgE stimulation,the mRNA expression levels of IL-4,IL-5,IL-13 and TNF-α in RBL-2H3 cells of the control group were up-regulated by 32.83±5.21,1.63±0.35,24.6±2.34 and 4.53±2.56 times,while the mRNA expression levels IL-4,IL-5,IL-13 and TNF-α in PIO(10μM)treated RBL2H3 cells were 6.8±1.34,1.12±0.03,8.28±1.45 and 0.15±0.01 times than that of the control group without DNP-IgE stimulation,respectively.There was significant difference between the two groups(P<0.05).4)Compared with the control group,Western blotting showed PIO treatment group has lower key protein phosphorylation levels of mast cell activation signaling pathway mediated by FcεRI.Phosphorylation of Lyn,ERK,PYK,FAK,PLCγ,SHIP1 and GAB2 were lower than control.5)Compared with RBL-2H3 WT cells,the phosphorylation levels of shipl,GAB2,Lyn,ERK,PYK,FAK and PLC γ were lower,which indicates inhibited mast cell activation signaling pathway mediated by FcεRI,and the trend was similar to that of PIO treatment group.6)Breeded PPARγflox/flox mice with Cpa3-Cre mice in the same cage,and got Cpa3-Cre PPARγflox/flox mice,which can specifically knockout PPARγ in MCs。Conclusion:1.PIO can attenuate nasal allergic symptoms and caused by OVA stimulation,and reverse the destruction of epithelial cells,as well as inhibit eosinophil infiltration.2.PPARγ agonist can inhibit the differentiation,maturation and function of mast cells,and have sustained inhibitory effect on BMMC in vitro.3.PPARγ activation can inhibit mast cell degranulation by inhibiting FcεRI mediated mast cell activation signaling pathway.4.PIO can inhibit mast cell degranulation by inhibiting FcεRI mediated mast cell activation in a PPARγ dependent way,and is a new target for the treatment of allergic diseases and the development of new drugs. |
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