Dasatinib Plus Quercetin on Uterine Age-Related Dysfunction and Fibrosis in Mice

Female reproductive function is negatively impacted by age. Animal and human studies show that fibrosis of the uterus contributes to gestational outcomes. Collagen deposition in the myometrial and endometrial layers is the main change related to uterine aging. Senolytic therapies are a potential option for reducing diseases and health complications related to aging. We investigated effects of aging and the senolytic drug combination of dasatinib plus quercetin (D+Q) on uterine fibrosis. A total of 40 mice, 20 young females (03-months) and 20 old females (18-months), were analyzed. Young (Y) and old (O) animals were divided into groups of 10 mice, with one treatment (T) group (YT and OT) and another control (C) group (YC and OC). Comparative analysis of Pi3k/Akt1/mTor and p53 gene expression among the 4 groups was performed to test effects of age and treatment on collagen deposition in uterine tissue. Uterine levels of microRNAs (miR34a, miR34b, miR34c, miR146a, miR449a, miR21a, miR126a, and miR181b) were evaluated. Aging promoted downregulation of genes of the Pi3k/Akt1/mTor signaling pathway (p=0.005, p=0.031, and p=0.028, respectively) as well as a reduction in expression of miR34c (p=0.029), miR126a (p=0.009), and miR181b (p=0.007). D+Q treatment increased p53 gene expression (p=0.041) and decreased levels of miR34a (p=0.016). Our results demonstrate a role for the Pi3k/Akt1/mTor signaling pathway in uterine aging and suggest for the first time a possible anti-fibrotic effect in the uterus of D+Q senolytic therapy.


43
The reproductive profile of women has been changing over the last few decades. Older 44 maternal age by the first gestation and an increased number of pregnancies after 40 years of age 45 are phenomena observed worldwide, impacting directly on gestational results [1]. Studies in 46 humans and animals suggest a strong relationship between pregnancy loss and maternal age [2, 47 3]. In addition to advanced age, other gynecological conditions are related to fertility, such as 48 polycystic ovary syndrome, leiomyomas, and endometriosis [4]. It is known that ovarian 49 dysfunction is the major factor responsible for these poor reproductive outcomes, but other 50 reproductive organs are involved in this complex process [2]. 51 An increase in uterine volume with aging is common in some species of rodents, mostly 52 due to endometrial cystic hyperplasia, as opposed to what occurs in menopausal women, in 53 whom uterine atrophy is usually evident [3,5]. The most obvious histological change in the aged 54 uterus is the collagen deposition (fibrosis) in the muscle layers and stroma [3]. Mechanisms 55 involved in this uterine fibrosis remain unclear [6]. Collagen deposition in tissues occurs as a 56 result of chronic inflammatory processes involving several pathways: inflammatory interleukins, 57 growth factors, caspases, oxidative stress products, and accumulation of senescent cells [6]. 58 These chronic inflammatory pathways are also involved in undesired obstetric outcomes such as 59 loss of pregnancies and preterm delivery [7]. 60 The phosphoinositide 3-kinase (Pi3k)/ protein kinase B (Akt)/ mammalian target of 61 rapamycin (mTor) pathway is an intracellular signaling mechanism that regulates several cellular 62 functions, including cell growth, proliferation, differentiation, transformation, and survival, 63 among others. Etiological processes underlying many gynecological conditions have not yet been completely identified. The Pi3k/Akt1/mTor and p53 signaling pathways appear to be involved in 65 the pathophysiological mechanisms of gynecopathies including polycystic ovarian syndrome, 66 premature ovarian failure, leiomyoma, endometriosis, and gynecological cancers [8][9][10][11][12][13][14][15]. This 67 signaling pathway has also been implicated in fibrosis in different tissues, such as the kidney, 68 lung, and liver [16][17][18][19][20].

77
MicroRNAs are non-coding RNAs that act as transcriptional silencers and are involved in 78 different cellular functions through post-transcriptional regulation of gene expression. Several 79 microRNAs have been associated with the fibrosis process in different tissues (lung, liver, 80 kidney, heart, skin) involving different mechanisms. Some of the most studied microRNAs in the It is important to understand more completely the physiological mechanisms of uterine 96 aging, as well as to discover therapies that delay this process. This could contribute to 97 improvement in gestational outcomes. The aim of our study was to test the impact of aging on 98 uterine fibrosis and the potential anti-fibrotic structural and molecular effects of the senolytic 99 D+Q combination on uterine aging.      System Fast RT-PCR system (Applied Biosystems™). For each assay, 40 PCR cycles were run 154 (95 C for 3s and 62 C for 30s), and a dissociation curve was performed at the end of the reaction 155 to verify the amplification of a single PCR product. Each assay included a negative control using 156 RNase-free water.

Genes
Primers sequences (forward and reverse) Product Size Reference NCBI Mammalian target of rapamycin (mTOR) Data were normalized using beta-2 microglobulin (β2m) as a housekeeping gene. To

301
In our experiment, we showed the effect of aging on downregulation of the 302 Pi3k/Akt1/mtTor signaling pathway in uterine tissue, but interestingly D+Q treatment did not 303 promote an inhibitory effect on this pathway by either reducing Pi3k/Akt1/mTor gene expression or increasing Pten mRNA. As reviewed above, the effect of single or combined use of these 305 drugs may be dose-, duration of treatment-, as well as tissue-dependent. In our study, the specific 306 D+Q protocol used may explain the absence of a uterine anti-fibrotic effect due to the short 307 duration of the intervention.

308
Importantly, our study indicated that D+Q treatment significantly increased the 309 expression of p53 mRNA, the tumor suppressor gene that is related to a higher incidence of 310 cancer in elderly people, which is not only due to a high frequency of mutant forms but its

566
Values are shown as mean ± standard error of the mean. Two-way ANOVA was performed and 567 p values for age, treatment, and their interaction are presented (p<0.05).