Anti-fibrosis Effect On Endometrium Of All-trans Retinoic Acid In Intrauterine Adhesions Model And Its Mechanism

Uterine is female internal genital organ,as well as the vital organs of fetus growth.In the childbearing age women,Endometrium experience cyclical hyperplasia,stripped and repair under the action of estrogen and progesterone.Some scholars also called the endometrial cycle repair as without scar repair.When exogenous endometrial trauma happened,sometimes different degrees of intrauterine adhesions appeared.The difficulty in treatment is how to prevent postoperative adhesion formation.Transforming growth factor beta 1(TGF-beta 1)is recognized as the strongest fibrosis factor.Previous study detected high expression of TGF-beta 1 in patients with intrauterine adhesions.Some scholars found that matrix metalloproteinases 9(MMP9)insulin-like growth factor-I(IGF-I)also promote the fibroblast activation to secrete extracellular matrix.All trans retinoic acid(ATRA)has a wide range of biological activities.In recent years,a growing number of studies have found that retinoic acid in vivo and in vitro also has the anti-fibrosis effect.Currently,there is no report about retinoic acid in the field of intrauterine adhesions.Objective:In endometrial stromal cells(ESCs)fibrosis models,the antifibrosis effect of all-trans retinoic acid on endometriumwas investigated in vitro.We observed the effect of all trans retinoic acid on human endometrial stromal cell into myofibroblast.To observe the preventive effect of all-trans retinoic acid on intrauterine adhesions and explore its mechanism,further explore its role in endometrial fibrosis.This study aimed to offer us new strategy for clinical intervention and treatment caused by intrauterine adhesions and provide experimental evidence for the selection of treatment time.Methods:Chapter Ⅰ:In endometrial stromal cells(ESCs)fibrosis models,cells were treated with all-trans retinoic acid.Real-time quantitative RT-PCR was used to observe COL1A1,alpha SMA mRNA level.WB was used to detect COL1A1,alpha SMA protein expression.Chapter Ⅱ:Intrauterine adhesion animal model was established and experimental group with all trans retinoic acid(low dose group:1 mg/Kg/d;medium dose group:2.5 mg/Kg/d,high dose group:5 mg/Kg/d).Using HE staining to observe endometrial histological morphology changes and glands count.Using Masson staining to observe endometrial stromal collagen deposition.Using immune histohemical method to detect TGF-beta 1 expression in endometrium and its quanity.Using HE staining to observe heart,liver and kidney tissue structure.The TGF-beta 1,smad4,COL1A1 and COL1A2 mRNA expression were detected by real-time quantitative RT-PCR.The TGF-beta 1,smad4,COL Ⅰ protein expression were detected by Western blot.Chapter Ⅲ:Intrauterine adhesion animal model was established and all trans retinoic acid was injected in different stage.HE staining,Masson staining,IHC,RT-PCR and WB were the same with Chapter Ⅱ.Results:Chapter Ⅰ:Persist TGF-beta 1 could stimulat ESCs to myofibroblasts.The COL1A1,alpha SMA mRNA expression were significantly lowered in the experimental group treated by ATRA.The COL1A1,alpha SMA protein expression were significantly lowered in the experimental group treated by ATRA.Chapter Ⅱ:In preventive study,HE staining showed that all trans retinoic acid treatment could increase the count of glands.Masson staining showed that all trans retinoic acid could reduce the collagen deposition.Immunohistochemical result showed that TGF-beta 1 protein expression was reduced in all trans retinoic acid group significantly.In each group,the morphology in heart,liver and renal cells were normal,no necrosis,no edema,no hemorrhage.The Real-Time qRT-PCR showed that compared with intrauterine adhesions group,TGF-beta 1,smad4,COL1A1 and COL1A2mRNA expression in all trans retinoic acid group was significantly decreased with statistical difference.Western blot results showed that compared with intrauterine adhesions group,TGF-beta 1,smad4,COLI protein expression in all trans retinoic acid group were significantly decreased with statistical difference,which in corresponding with mRNA level changes.Chapter Ⅲ:HE staining showed:In early treatment group,endometrial gland counts increased with statistical difference compared to intrauterine adhesions model group.Masson staining showed:In early treatment group,endometrial fibrosis area was reduced with statistical difference.Real-Time qRT-PCR results showed that:Compared with intrauterine adhesions model group,TGF-beta 1,smad4,COL1 A1 and COL1 A2mRNA expression in all-trans retinoic acid early treatment group was significantly reduced.Western blot results show that TGF-beta 1.smad4,COLI protein expression reduced significantly in all-trans retinoic acid early treatment group compared with intrauterine adhesions model group.All-trans retinoic acid failed to improve the endometrial fibrosis in the late treatment.Conclusion:All trans retinoic acid could prevent endometrial stromal cells into myofibroblasts in vitro and reduce the expression of collagen COL1 A1 to exert its preventive role in endometrial fibrosis.All trans retinoic acid could exert protective effects on endometrial fibrosis in intrauterine adhesions by decreasing smad4 and its downstream Collagen I mRNA and protein expression in TGF-beta 1/smad signaling pathways to delay the endometrial fibrosis and promote the endometrium regeneration.All-trans retinoic acid could treat intrauterine adhesions in the early treatment group by inhibiting TGF-beta 1/smad signaling pathways.All-trans retinoic acid failed to treat intrauterine adhesions in the late treatment.All trans retinoic acid showed on toxicity to cardiovascular,hepatic and renal tissue pathologically.