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Introduction Diabetic retinopathy (DR) is one of the major leading causes for vision loss globally. Current study illustrates the role of miR-7a in DR. Material and Methods Retinal pericytes (RPs) and Endothelial cells (ECs) were isolated from mouse model of DR. qRT-PCR was done for expression of miR-7a and target gene mRNA, Western blot for protein expression. Identification of miR-7a target gene was done by TargetScan and Luciferase assay. Cell viability and invasion was done by MTT and Transwell chamber assay. Results The expression of miR-7a was down-regulated whereas level of IRS-2 was unregulated in isolated RPs and ECs. Luciferase assay suggested correlation between miR-7a and IRS-2, over-expression of miR-7a using a mimic resulted in suppression in viability and invasion capacity of RPs and ECs and inhibited the protein levels of PI3K/Akt cascade and IRS-2, and however the inhibitor reversed them respectively. Transfection of siRNA targeting IRS-2 caused alteration in miR-7a mediated changes in ECs suggesting that miR-7a may decrease angiogenesis in DR by inhibiting the levels of IRS-2. Conclusion miR-7a suppresses PI3K/Akt cascade via targeting IRS-2, thus decreasing the viability and invasion capacity of RPs and ECs, suggesting an interesting treatment target for DR.

The number of text pages: 21The number of figures: 7 The number of references: 44 The number of words in the Abstract: 276 The number of words in the Introduction: 625 The number of words in the Discussion: 1231 Abbreviations pyrrolizidine alkaloids, PAs; cytochrome P450, CYP; retrorsine, RTS; gestation day, GD; glutathione, GSH; glyceraldehyde phosphate dehydrogenase, GAPDH; 4-dimethylaminobenzaldehyde, DABA; hematoxylin and eosin, HE; declustering potential, DP; collision energy, CE; collision cell exit potential, CXP; highperformance liquid chromatography, HPLC; multiple-reaction monitoring, MRM; ATP-binding cassette, ABC AbstractPyrrolizidine alkaloids (PAs) are extensively synthesized by plants and are commonly present in herbs and foodstuffs, which exhibit hepatotoxicity requiring metabolic activation by cytochrome P450 (CYP) 3A to form the electrophilic metabolites-pyrrolic esters. PAs also cause embryo toxicity, but the metabolic profiles of PAs in fetus and placenta have been far from clear. In this study, we determined the basal metabolic activation of retrorsine (RTS) in rat maternal liver, placenta and fetal liver in vitro, and examined the fetal toxicity and bioactivation of RTS in vivo. Detection of microsomal RTS metabolites in vitro showed that the basal metabolic activity of fetal liver and placenta to RTS was much weaker than that of maternal liver. In addition, higher rate of pyrrolic ester formation was found in normal male fetal liver, compared with that of female pups. In vivo exposure to RTS caused fetal growth retardation as well as placenta and fetal liver injury. Little difference in serum RTS was observed in dams and fetuses, but the content of pyrrole-protein adduction in fetal liver was much lower than that in mother liver, which was consistent with basal metabolic activity. Unexpectedly, compared with basal metabolism in fetal liver, exposure to RTS during middle and late pregnancy caused an opposite gender difference in RTS metabolism and CYP3A expression in fetal liver. For the first time, our study proved that RTS was capable of permeating placenta barrier and entering fetal circulation, while the intrauterine pyrrolic metabolite was mainly generated by fetal liver, but not transported from mother circulation. Induction of CYP3A by RTS was gender-dependent in fetal liver, which was probably responsible for RTS-induced fetal

Pyrrolizidine alkaloids (PAs) are a type of natural phytotoxin that contaminate food and feed and become an environmental health risk to humans and livestock. PAs exert toxicity that requires metabolic activation by cytochrome P450 (CYP) 3A, and case reports showed that fetuses are quite susceptible to PAs toxicity. The aim of this study was to explore the characteristics of developmental toxicity and fetal hepatotoxicity induced by retrorsine (RTS, a typcial toxic PA) and the underlying mechanism. Pregnant Wistar rats were intragastrically administered with 20 mg/(kg•day) RTS from gestation day (GD) 9 to 20. Results showed that prenatal RTS exposure lowered fetal bodyweights, reduced hepatocyte numbers, and potentiated hepatic apoptosis in fetuses, particularly females. Simutaneously, RTS increased CYP3A expression and pregnane X receptor (PXR) activation in female fetal liver. We further confirmed that RTS was a PXR agonist in LO2 and HepG2 cell lines. Furthermore, agonism or antagonism of androgen receptor (AR) either induced or blocked RTS-mediated PXR activation, respectively. As a PXR agonist, RTS toxicity was exacerbated in female fetus due to the increased CYP3A induction and self-metabolism, while the inhibitory effect of AR on PXR activation reduced the susceptibility of male fetus to RTS. Our findings indicated that PXR may be a potential therapeutic target for PA toxicity.