| Uteroglobin(UG) is a anti-inflammatory protein secreted from Clara cells, and its biological effect may depend on mediation of uteroglobin binding protein (UGBP), however the function of gene structure of UGBP are poorly understood. At present, research on UGBP is far behind to its natural ligand UG, which will limit the step to investigate the function of UG and its potential clinical application. Therefore, it is great significant for research on UGBP. Our previous results show that UG active fragment, Antiflammin-1 (referred to as AF-1, corresponding to the 39-47 bits of UG(MQMKKVLDS)could bind with UGBP expressed mainly in the membrane and AF-1 could affect mice lung fibroblasts (NIH3T3) in the level of ERK phosphorylation via mediation of UGBP, which suggested that the bioactivity function of AF-1 might be stimulated by UGBP as its receptors. However, there is no study to investigate the effect of AF-1 on lung fibroblasts proliferation in vitro.For that, our study observed the effect of AF-1 on proliferation of lung fibroblast, and explored the role of UGBP in mediating anti-proliferation effects of AF-1 on lung fibroblasts. It provide a new way for revealing the receptor mechanism about inhibiting lung fibrosis of AF-1. Methods:①BrdU-ELISA was used to detect cell proliferation of NIH3T3;②The change of cell cycle was measured by flow cytometry;③RT-PCR and flow cytometry were used to detect mRNA expression of cyclinD1 and p27 respectively in each group;④Anti-UGBP antibody was used as a drug tool to explore biological effect of UGBP.Results:1.Adding 0.5～15ng/ml TGF-β1 to stimulate NIH3T3 for 12 hours, the results showed that TGF-(31 could promote the proliferation of NIH3T3 in a dose-dependant manner in vitro, and the biggest effect was observed at concentration of lOng/ml (P<0.01).2. There is no significant effect of proliferation on NIH3T3 by adding AF-1 from 4×10-7 to 2.5×10-4 mol/L (P(?)0.05). AF-1(4×10-7～2.5×10-4 mol/L) pretreatment could inhibit the proliferation of NIH3T3 induced by TGF-β1(10ng/ml) in a dose-dependant manner in vitro, and the inhibitory effect could be blocked by anti-UGBP antibody(P<0.01).3. Results of flow cytometry showed that TGF-β1(0.5～15ng/ml) was added to stimulate NIH3T3 for 12 hours, the proliferation index (PrI) got to the top (P<0.01); AF-1(10-5mol/L) pretreatment could inhibit increased proliferation index (PrI) of NIH3T3 induced by TGF-β1 and the inhibitory effect could be blocked by anti-UGBP antibody (P<0.01).4. Comparing the differences of G1, S and G2 phase among each group, we found that the percentage of cells can be found decreased more in TGF-β1+AF-1 group than TGF-β1 group in S phase(P<0.01), but no significant difference in G2 phase(P>0.05).5. mRNA expression of cyclinDl and p27 was detected by RT-PCR in NIH3T3. TGF-β1 could promote mRNA expression of cyclinDl (P<0.01); AF-1 pretreatment could inhibit increased cyclinD1 mRNA expression induced by TGF-β1 and the inhibitory effect could be blocked by anti-UGBP antibody (P<0.01). However, in terms of p27, the results were absolutely opposite. TGF-β1 could reduce the expression of p27 mRNA level (P<0.01), but AF-1 could inhibit this effect(P<0.01), and the inhibitory effect of AF-1 could also be blocked by anti-UGBP (P <0 .01).6. TGF-β1 could promote cyclinDl protein level detected by flow cytometry (P<0.01); AF-1 pretreatment could inhibit increased cyclinD1 expression induced by TGF-P 1 and the inhibitory effect could be blocked by anti-UGBP antibody (P<0.01). However, in terms of p27, the results were absolutely opposite. TGF-β1 could reduce the expression of p27(P <0.01), but AF-1 could inhibit this effect of TGF-β1 (P<0.01), and the inhibitory effect of AF-1 could also be blocked by anti-UGBP antibody (P<0.01).Conclusion:1.The inhibitory of AF-1 affected both proliferation and cell circle of NIH3T3 induced by TGF-β1, and the effect depended on mediation of UGBP.2. AF-1 could slow down transformation from G1 to S phase in cell circle, but AF-1 has almost no effect on another limit point which is from S to G2 phase.3. The inhibitory effect of AF-1 on NIH3T3 cell cycle enhanced by TGF-β1 is connected with expression change of cyclinDl and p27, and depended on mediation of UGBP.
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