hUMSCs Transplantation Regulates AMPK/NR4A1 Signaling Axis to Inhibit Ovarian Fibrosis in POI Rats

The mechanism of human Umbilical Cord Mesenchymal Stem Cells (hUMSCs) transplantation to improve ovarian function in the rats with Premature Ovarian Insufficiency (POI) is still unclear. The aim of this study is to investigate the signal axis mechanism that is involved in the ovarian function recovery of POI rats following hUMSCs transplantation. The rat model with POI was established by intraperitoneal injection of cisplatin. The hUMSCs were transplanted by caudal vein injection into POI rats. Hematoxylin–eosin (H&E) staining was performed to examine the morphology of rat ovarian tissue. Masson staining, Sirus red staining and immunofluorescence were used to observe the fibrosis extent of ovarian tissue. The levels of serum sex hormones and the expression of fibrosis related markers in ovarian tissues were measured by enzyme-linked immunosorbent assay (ELISA). The expression of NR4A1, Phospho-NR4A1 and AMP-activated protein kinase (AMPK) signaling in rat ovarian tissues was measured by immunohistochemistry and immunofluorescence. The role of AMPK/NR4A1 signaling axis in the regulation of ovarian function recovery in POI rats following hUMSCs transplantation was further investigated by adenovirus and siRNA intervention in isolated stromal cells. The results showed that the hUMSCs transplantation significantly inhibited ovarian tissue fibrosis and restored the ovarian function in POI rats. The level of NR4A1 and AMPK expression in ovarian tissue of POI rats after hUMSCs transplantation was significantly increased compared with the control group. In the cultured ovarian stromal cells, the similar results were obtained on the expression of NR4A1 and its regulation on fibrosis related molecular markers in Cisplatin (CDDP) damaged stromal cells following hUMSCs supernatant treatment. Both hUMSCs supernatant treatment and the addition of AMPK inhibitors increased NR4A1 expression in stromal cells. And after NR4A1 molecular intervention, fibrosis-related indicators in stromal cells changed. The data suggests that the AMPK/NR4A1 signaling axis is involved in the ovarian function changes in POI rats following hUMSCs transplantation. The data from this study indicate that the inhibition of tissue fibrosis and recovery of ovarian function is regulated by AMPK/NR4A1 signaling axis in POI rats following hUMSCs transplantation.


Introduction
Premature ovarian insufficiency (POI) is a subclass of ovarian dysfunction in which the cause is within the ovary. The mechanism to cause the ovarian dysfunction is not completely understood. As reported, one of the reasons to cause ovarian damage is due to the side effects of chemotherapy treatment in cancer patients [1][2][3]. The clinical manifestations of POI disease are secondary or primary amenorrhea for at least 4 months, the serum estradiol (E 2 ) level decreases, and the gonadotropin level increases [4,5]. The basic pathological changes of ovarian tissues in POI patients include fibrosis and follicular development disorders [6,7]. In clinical, the hormone replacement therapy (HRT) is the primary treatment for POI patients. However, HRT therapy might increase the risk of cancer recurrence [8,9]. In recent years, stem cell therapy has attracted a great attention as a promising method to treat POI patients. In the animal study, the results have shown that mesenchymal stem cell transplantation can significantly improve the ovarian function in POI rats [10][11][12]. In our previous published studies, the data also have shown that mesenchymal stem cell transplantation can improve ovarian function in POI rats by reducing the ovarian fibrosis [13]. However, its exact mechanism is still unclear. In this study, we investigate the mechanism on how hUMSCs transplantation could inhibit tissue fibrosis and induce ovarian function recovery in POI rats.
In addition to the in vivo animal study, the in vitro study on the isolated ovarian stromal cells also is performed to investigate its mechanism to promote the recovery of ovarian function in POI rats following hUMSCs supernatant treatment. The stromal cells, as a component of ovarian structure, play an important role in follicular development. It can differentiate into follicular theca cells or myofibroblasts during the preantral follicular stage. Theca cells maintain the structural integrity of the follicles, while myofibroblasts secrete the extracellular matrix, including Collagen Type I (Collagen I), Collagen Type III (Collagen III), which influence follicular development and atresia. Currently, the studies on stromal cells are mostly focused on polycystic ovary disease. There is lack of research on its role in the development of POI disease. In this study, we further investigated the effect of hUMSCs transplantation on the differentiation of stromal cells into myofibroblasts, and explored the mechanism of its inhibition of tissue fibrosis in POI rats.
The orphan nuclear receptor 4A1 (NR4A1, also known as NUR77, TR3, NGFI-B or NAK-1) is a member of the NR4A subfamily of nuclear receptors and plays an important role in maintaining cell homeostasis and diseases [14][15][16]. It is mainly used as a transcription factor to promote or inhibit the expression of downstream genes, thereby participating in various cellular activities. Studies have shown that NR4A1 can be used as an effective target to inhibit fibrosis in the fibrosis process of scleroderma and endometriosis [17,18]. However, its role in the ovarian tissue fibrosis of POI disease has not been clearly reported. Also, the other signaling related to NR4A1 regulation such as AMP-activated protein kinase (AMPK) is investigated to determine the pathway that is involved in the ovarian function recovery following hUMSCs transplantation. The goal of the study is to elucidate the mechanism mediated in the ovarian tissue recovery following hUMSCs transplantation and provide new pharmaceutical approach to treat the POI disease in clinical.

Animals
All experimental procedures requiring the use of animals were approved by the Binzhou Medical University Institutional Animal Care and Use Committee. The female rats were purchased from Jinan Pengyue Experimental Animal Breeding Co, Ltd (Shandong, China). All animals at 3 and 6 weeks of age were housed in standard plastic cages and maintained on a 12 h light/dark cycle at room temperature with free access to food and water. The study was conducted in accordance with the National Laboratory Animal Care and Use research committee guidelines.

hUMSCs Cell Culture
As described previously [13], human umbilical cord mesenchymal stem cells (hUMSCs) were isolated from the umbilical cords of healthy pregnant women. The cells marker were identified by flow cytometry and observed the cells morphology using microscopy. Alizarin red staining and oil red O staining were used to detect the differentiation ability to 1 3 osteoblasts and adipocytes. The isolated cells were cultured in DMEM medium for further experiment.

hUMSCs Transplantation in POI Rat Model
The POI rat model was established with CDDP treatment at the dose of 2 mg/kg for 7 days in according to our previous study [13]. The success of POI rat model was confirmed by disturbance of estrous cycle. Two weeks later, the rats were randomly assigned to four experiment groups (n = 20 per group) including Control, POI, POI + hUMSCs and POI + PBS group. Rats in the POI + hUMSCs group were injected with hUMSCs through the tail vein (1 × 10 6 /ml per rat). And rats in the POI + PBS group were injected with the same dose of PBS through the tail vein. One week after hUMSCs treatment, ten rats were randomly selected from each group to examine its fertility. The remaining rats in each group were necropsied for the study.

Ovarian Tissue Staining
During the necropsy, the left ovarian tissue in each rat was taken out and fixed in 4% paraformaldehyde for 24 h. Then half of the collected ovarian tissues were embedded in paraffin and stained with hematoxylin-eosin (H&E) after sectioning to examine morphology of ovarian tissue under microscope. The number of follicles at each stage of development were counted to examine the ovarian function. The ovarian tissue sections also were stained with Masson trichrome and Sirius red to evaluate the degree of fibrosis. Image J software was used for quantitative analysis on the severity of ovarian tissue interstitial fibrosis.

Enzyme-Linked Immunosorbent Assay (ELISA)
The blood was collected at the end of necropsy. After centrifuge, the serum was harvested and stored at -80℃ for analysis. The supernatants of cells from each group were collected and stored at -80℃. The hormone levels of estradiol (E 2 ) (ml002891, Mlbio, China), follicle stimulating hormone (FSH) (ml002872, Mlbio, China), luteinizing hormone (LH) (ml470613, Mlbio, China) and progesterone (P) (ml059114, Mlbio, China) in serum were measured by ELISA kit. The levels of Collagen I (ml003303, Mlbio, China) and Collagen III (YJ718203, Mlbio, China) in the cell supernatants were also measured with ELISA kit.

Immunohistochemical Staining
The expression of NR4A1 and Phospho-NR4A1 in ovarian tissues was measured by immunohistochemical staining. The paraffin sections were incubated with mouse primary antibody polyclonal antibody against rat NR4A1(1:100, Santa Cruz Biotechnology) and rabbit primary antibody polyclonal antibody against rat Phospho-NR4A1 (1:100, Affinity, China) at 4℃ for overnight. The second antibody was incubated with the tissue in accordance with the instruction of immunohistochemistry kit. Diaminobenzidine (DAB) was used as a chromogen for color development. The nucleus was stained with hematoxylin. The staining results were analyzed using German Immune Response Score (IRS) as reported in the literature [19]. And in vitro experiments, the transmembranes of the inserts of the 24-transwell plates were fixed using 4% paraformaldehyde for 15 min. The primary antibody was added to the 24-well plate and incubated with the membrane overnight at 4 ℃. Primary antibodies include NR4A1 (1:100, Santa Cruz Biotechnology) and Phospho-NR4A1 (1:100, Affinity, China). The following steps were identical to the steps carried out in the ovarian tissues.

Fertility Examination
Ten female rats were randomly selected from each group and lived with the male rats at a ratio of 2:1. After three estrus cycles, the male and female rats were seperated, and the fertility of the female rats was examined. The number of fertile female rats in each group and the number of offspring were counted.

Stromal Cell Culture
The stromal cells were isolated from the ovarian cortex tissue of rats at the age of 3 to 4 weeks using the method published in our previous study [13]. The cells were cultured in McCoy' s 5A medium containing 10% FBS, 1% 100 U/ mL streptomycin sulfate, and 100 U/mL penicillin G. The 1 3 second-generation cells were stained with vimentin, Factor VIII, and Cytokeratin molecules by immunofluorescence for verification.

CCK-8 Cell Viability Assay
CCK8 is a cell proliferation and cytotoxicity assay kit to screen out the appropriate concentration of drugs for experiments. The CCK-8 cytotoxicity kit (Meilong Bio, China) was used to detect the effect of DIM-C-pPhOCH3, a NR4A1 activator, on the viability of stromal cells. The second-generation ovarian stromal cells (5000 cells/well) were seeded in a 96-well plate and incubated overnight. The medium was removed and cultured in a new medium containing different concentrations of DIM-C-pPhOCH3 (0-40 µM) for 24 h. Cell viability was measured according to the instruction of test kit, and read absorbance at 450 nm.

Extraction of Nuclear and Cytoplasmic Proteins
The two cellular components were isolated using a nuclear and cytoplasmic protein extraction kit (Beijing Solarbio Science & Technology Co., Ltd., China) as described in according to the manufacturer's instructions.

Cell Transfection
Rat NR4A1 overexpression adenovirus and control adenovirus were purchased from Hanheng Biological Technology Co., Ltd. (Shanghai, China). The gene information of NR4A1 overexpressed adenovirus was HBAD-r-Nr4a1-3xFlag-EGFP, while that of the control group was HBAD-EGFP. The transfection of the adenovirus was carried out in accordance with the manufacturer's protocol. Fresh medium was replaced 8 h after transfection. And after 48 h, the supernatant of cells was collected and proteins were extracted to detect the transfection efficiency. For siRNA intervention study, the following siRNA and NC-siRNA sequences were used: NR4A1 siRNA, 5'-GCC CUG UAU CCA AGC UCA AdTdT-3', 5'-UUG AGC UUG GAU ACA GGG CdTdT -3'; NC-siRNA, 5'-UUC UCC GAA CGU GUC ACG UdTdT-3', 5'-ACG UGA CAC GUU CGG AGA AdTdT-3'. According to the manufacturer's recommendation, a 3.3 μl aliquot of 20 μmol of siRNA/liter/per well or control siRNA were transfected into stromal cells using a RNAFite transfection reagent. After 48 h transfection, the supernatant of cells was collected for hormone level measurement. And the proteins inside of the cells were isolated to measure the transfection efficiency.

Data Analysis
Data were expressed as mean ± SD. and analyzed by SPSS 22.0 software. Differences between two groups were determined using independent samples t-test or one-way analysis of variance (ANOVA) with LSD test as the post-hoc test.

hUMSCs Identification
To identify the isolated cells as hUMSCs, cells were examined using light microscopy, flow cytometry as well as osteogenic and adipocyte induction. As shown in Supplementary Fig. 1A-a, the morphology of these cells resembled that of fibroblasts. After incubation for 28 days and 14 days, respectively, these cells showed the ability to differentiate into osteoblasts and adipocytes, showing positive staining for Alizarin red S and Oil Red O (Supplementary Fig. 1A-b, A-c). The expression rate of CD44 detected by flow cytometry was 98.7%. And the expression rates of CD90, CD73 and CD105 were all more than 95%, while the expression levels of CD34, CD45 and HLA-DR were all less than 5% (Supplementary Fig. 1B). These results are consistent with previous reports on the phenotypic characteristics of hUMSCs [20].

Stromal Cell Identification
To confirm the property of stromal cells isolated from ovarian tissues, the cells were examined by light microscopy and immunofluorescence staining. As shown in Supplementary  Fig. 2B, the primary stromal cells showed fibroblasts-like morphology after adhering to the plate. The characteristics of stromal cells were confirmed by immunofluorescence staining on the specific markers of this type of cells. As shown in Supplementary Fig. 2A, the cell vimentin was shown as positive after staining. However, the staining on the specific epithelial cell marker of cytokeratin and endothelial cell marker of factor VIII was negative. In addition, immunofluorescence assay was performed to detect whether stromal

Effects of hUMSCs Transplantation on Ovarian Tissue Morphology and Function in POI Rats
The ovarian tissues were stained with hematoxylin (H&E) to examine its morphology and follicle number changes in POI rats following hUMSCs transplantation. As shown in Fig. 1A a1-a4 and B, the number of healthy follicles including primitive follicles, primary follicles and secondary follicles in the ovarian tissue at each stage of development were significantly decreased in POI rats. However, more healthy follicles were observed in ovarian tissues of POI rats following hUMSCs transplantation. In contrast, the number of atretic follicles was significantly decreased in ovarian tissues of POI rats after hUMSCs transplantation (P < 0.001, Fig. 1B). There was no significant difference in follicle number between POI + PBS group and POI group (Fig. 1B). To examine the ovarian function of POI rats following hUMSCs transplantation, the hormone levels of E 2 , FSH, LH and progesterone (P) in serum were measured by ELISA kit. As shown in Fig. 1C and D, the expression levels of E 2 and P in the POI group were decreased compared with the control group. After hUMSCs transplantation, the expression of E 2 and P in POI rats was increased. However, the elevated FSH and LH levels in POI rats were decreased following hUMSCs transplantation. In conclusion, these results indicated that hUMSCs transplantation could improve the morphology and endocrine function of ovarian in POI rats.

Effect of hUMSCs Transplantation on Fertility of POI Rats
To explore the effect of hUMSCs transplantation on fertility of POI rats, the number of embryo-implantation and pregnancy rate were examined in the rats of each group. As shown in Fig. 1E, the number of embryo implantation sites in POI group was significantly lower than the control group (P < 0.001). However, the hUMSCs implantation significantly increased the number of embryo implantation in POI rats (P < 0.05). In addition, the results in Table 1 showed that the pregnancy rate of POI rats was significantly lower than that of the control group (P < 0.01), and the situation was significantly improved after hUMSCs transplantation. Taken together, the above data all indicate that hUM-SCs transplantation can help restore the fertility of POI rats.

Effects hUMSCs Transplantation on Ovarian Tissue Fibrosis in POI Rats
To examine the effect of hUMSCs transplantation on the tissue fibrosis in the ovaries of POI rats, the sections were stained with Masson trichrome and Sirus red to visualize the collagens in tissues. The representative images are presented in Fig. 2A, which show collagen stained with blue color ( Fig. 2A a1-a4) or red color ( Fig. 2A b1-b4) in ovary. The experimental results are shown in Fig. 2D, the fibrosis area of the ovarian tissue in the POI group was higher than that in the control group (P < 0.01), while the ovarian fibrosis area in the POI + hUMSCs group was lower than that in the POI + PBS group (P < 0.01). This indicates the increased severity of ovarian tissue fibrosis in POI rats. After the hUMSCs transplantation, the content of collagen is significantly reduced than those shown in POI rats. Furthermore, alpha smooth muscle actin (ɑ-SMA), a specific marker of myofibroblast, and fibrogenesis related molecular markers such as Collagen Type I (Collagen I), Collagen Type III (Collagen III), Fibronection and connective tissue growth factor (CTGF) are examined by immunofluorescence staining. As shown in Fig. 2B and E, the ovarian tissues in POI rats exhibited higher fluorescence signals on each staining than the control group (P < 0.05). Following hUMSCs transplantation, the results showed that the expression of these fibrogenesis related molecular markers in POI rats is significantly decreased with much less red color staining present in the tissues. The quantitation of staining intensity of Fig. 2B was summarized in Fig. 2E. The tissue expression of these fibrosis related molecular markers is further evaluated by ELISA assay. As shown in Fig. 2C, the similar trend as immunofluorescence staining is shown in POI rats following hUMSCs transplantation. Overall, these data indicated that hUMSCs transplantation reduces the number of myofibroblasts and attenuates the degree of ovarian fibrosis in the ovaries of POI rats.

Effects of hUMSCs Transplantation on the Expression of NR4A1 Ovarian Tissues of POI Rat
To determine the role of NR4A1 in the ovarian pathological changes of POI rats, the expression of NR4A1 was examined by immunohistochemistry technique in the tissue sections. As shown in Fig. 3A, the expression of NR4A1 in stromal cells of ovarian tissue was significantly decreased in POI rats compared to the control group (P < 0.01). However, the expression of Phospho-NR4A1 was increased than the control group (P < 0.001). Following hUMSCs transplantation, the changes of NR4A1 and Phospho-NR4A1 in POI rats were reversed (P < 0.001). The quantitation of staining intensity of Fig. 3A was summarized in Fig. 3B. These results indicate that the NR4A1 mediates ovarian function changes in POI rats following hUMSCs transplantation.

Effects hUMSC S Supernatant on NR4A1 Expression in Stromal Cells
To investigate the effect of hUMSCs transplantation on NR4A1, the expression of NR4A1 was further examined in vitro. Briefly, the stromal cells were isolated from ovarian tissues of rats. The normal ovarian stromal cells were treated with CDDP and then treated with hUMSCs supernatant. As shown in Figs. 4A and B, the results from immunofluorescence analysis show that the expression of NR4A1 in stromal cells treated with CDDP was significantly decreased compared with the control group (P < 0.001). The quantitation of staining intensity of Fig. 4A and B was summarized in Fig. 4E. Furthermore, the immunoblot analysis was performed on the extraction of nuclear and cytoplasmic proteins.
The results show that the amount of NR4A1 in the cytoplasm in CDDP treated group is much higher than control but lower in the nucleus ( Fig. 4C and F). After treatment with hUMSCs supernatant, the changes of NR4A1 observed in the CDDP treated cells are reversed. Interestingly, the increase of NR4A1 induced by hUMSCs supernatant treatment mainly located in the nucleus. The phosphorylation of NR4A1 in stromal cells was also examined using immunoblot and immunofluorescence techniques. As shown in Fig. 4D, The CDDP treatment increased the expression of Phospho-NR4A1 in stromal cells. However, the expression of Phospho-NR4A1 was decreased after treatment with hUMSCs supernatant (Fig. 4F). These results suggests that the translocation NR4A1 and its phosphoryl change could regulate the pathology changes in ovarian tissues following hUMSCs transplantation.

Effect of NR4A1 Expression on Differentiation of Stromal Cells into Myofibroblasts
To investigate the role of NR4A1 expression in the regulation of stromal cell differentiation, the adenovirus and siRNA transfection was used to affect the expression of NR4A1, respectively. As shown in Fig. 5A, the results through Western Blot analysis showed that the expression of NR4A1 could be significantly reduced by siRNA treatment (P < 0.01) and the expression of NR4A1 in stromal cells was increased after adenovirus transfection (P < 0.05).
To examine the production of Collagen in stromal cells, the supernatants in each group were collected and analyzed by ELISA. The results showed that the content of collagen I and collagen III in the knockdown group achieved by siRNA transfection, was significantly higher than the control group (Fig. 5B) (P < 0.05). However, the content of collagen I and collagen III in the supernatant of NR4A1 overexpression group was lower than the overexpression control group (P < 0.05). The results showed the opposite trend to the production of collagen. Furthermore, Western Blot analysis was performed to examine the expression of α-SMA, a marker of myofibroblasts, in stromal cells treated with siRNA and adenovirus. And the results showed that the expression of α-SMA protein decreased after the overexpression of NR4A1 in stromal cells, compared with the overexpression control group (P < 0.05); on the contrary, when the NR4A1 molecule was low expressed, the expression of α-SMA protein molecule increased compared with the control group (P < 0.05). The expression of fibrosis-related molecules including Fibronection and CTGF in stromal cells after treatment with siRNA and adenovirus was detected by Western Blot. As shown in Fig. 5C-D, compared with the konckdown control group, expressions of fibrosis related proteins such as Fibronection and CTGF increased after transfection of siRNA (P < 0.05). However, compared with the overexpression control group, their expression was decreased after transfection with adenovirus (P < 0.05). These results suggest that NR4A1 reduces stromal differentiation into myofibroblasts and inhibits the expression of fibrogenic factors.

NR4A1 Regulates the Stromal Cell Differentiation Following hUMSCs Transplantation in CDDP Treated Stromal Cells
To investigate if the NR4A1 is involved in the differentiation of stromal cells into myofibroblasts following hUMSCs supernatant treatment, the proteins from the cells treated with CDDP and NR4A1 agonist were harvested for Western Blot analysis. As shown in Fig. 6A-B, CDDP decreased the expression of NR4A1 (P < 0.01) and increased the expression of myofibroblast related marker of α-SMA and fibroblast-related factors of Fibronectin and CTGF in stromal cells (P < 0.05). After adding the hUMSCs supernatant, the expression of these molecules was reversed when compared with CDDP treated cells (P < 0.05). Following addition of DIM-C-pPHOCH3, a NR4A1 agonist, the similar trend on these molecules was observed as shown in CDDP cells following hUMSCs supernatant treatment. Moreover, the : Image J software was used to quantify the protein expression in figure C. Data are expressed as the mean ± SD. *: P < 0.05, **: P < 0.01 and ***: P < 0.001 ◂ co-addition of hUMSCs and DIM-C-pPHOCH3 significantly reduced fibrosis markers compared with the hUMSCs or DIM-C-pPHOCH3 group alone. We collected the supernatant of different groups of cells for hormone detection. The results of ELISA assay (Fig. 6C) showed that the secretion of collagen I and collagen III increased after CDDP treatment of stromal cells. In contrast, treatment with hUMSCs supernatant or DIM-C-pPHOCH3 significantly reduced collagen secretion. And the combination of the two is more effective than using them alone. For other molecules associated with fibrosis, we performed immunofluorescence staining experiments. As shown in Fig. 6D-E, the trend of immunofluorescence test results was consistent with that of Western Blot. The data showed that NR4A1 was involved in the reduction of myofibroblast differentiation and the inhibition of fibrosis related markers after hUMSCs treatment. This might explain the mechanism that promotes the ovarian function recovery in POI rats following hUMSCs transplantation in the in vivo study.

The AMPK Signaling Involved in NR4A1 Expression in Ovarian Tissue of POI Rats
To examine if AMPK signal is activated in ovarian tissues of POI rats, the expression of AMPK and Phospho-AMPK was measured in proteins isolated from ovarian tissues using Western Blot. As shown in Fig. 7A and B, the phosphorylation of AMPK was significantly higher in POI rats than the control group (P < 0.01). Following hUMSCs transplantation, the amount of phosphorylated AMPK is decreased (P < 0.05) and the image from immunofluorescence stain in ovarian tissues showed the same results as Western Blot (Fig. 7C-D). Next, we investigated whether AMPK inhibited NR4A1 in the regulation of hUMSCs transplantation treated POI rats. The cultured stromal cells were treated with an AMPK inhibitor of Compound C and the expression of AMPK and NR4A1 was measured by Western Blot. As shown in Fig. 7E and G, CDDP significantly increased AMPK phosphorylation (P < 0.05) and decreased NR4A1 in stromal cells (P < 0.05). After pre-treated with AMPK inhibitor of Compound C before CDDP treatment, the data showed that Compound C significantly inhibited the expression of Phospho-AMPK and increased NR4A1 production in stromal cells (P < 0.05). After adding the hUMSCs supernatant to the cells treated with CDDP, the same trend of Phospho-AMPK decrease and NR4A1 increase was observed as Compound C (P < 0.05). The expression of NR4A1 in different groups of stromal cells was detected by immunofluorescence, and the expression trend was consistent with the detection results of Western Blot (Fig. 7F). The data suggests that the AMPK/NR4A1 signal axis is involved in the ovarian function changes in POI rats following hUMSCs transplantation.

Discussion
The recent studies have shown that the mesenchymal stem cells (MSCs) transplantation is a promising therapeutic approach due to its lower immunogenicity, higher proliferation capacity and less racially disputed isolation and extraction methods [21][22][23]. Previous studies have shown that transplantation of stem cells is a promising treatment for Alzheimer's disease, cardiovascular disease, and myocardial infarction [24][25][26]. In our previous studies, we have demonstrated that hUMSCs transplantation can improve the ovarian function in POI rats caused by CDDP [13]. However, its exact mechanism is still unclear. In the current study, we have shown that the AMPK/NR4A1 signal axis is involved in the regulation of ovarian function recovery in POI rats following hUMSCs transplantation. The basic pathological changes in POI disease often manifest ovarian tissue fibrosis and follicular development disorders [27,28]. In this report, the same pathological changes were observed in our established animal model of POI rats treated with CDDP. Following hUMSCs transplantation, the extent of ovarian tissue fibrosis was significantly reduced compared to the POI group, providing a good environment for follicular development. Additionally, the ovarian function was recovered with increased sex hormone production such as E 2 and the decrease of FSH, LH secretion. The follicle numbers at each development stage were also significantly increased, which further indicated the recovery of ovarian function following hUMSCs treatment. It has been reported that the number of follicles is limited. Millions of non-growing follicles (NGF) form in the ovaries at about 5 months of gestational age, then gradually decline from puberty until depletion [20]. Our experiments showed that the number of primordial follicles increased in the POI group after stem cell transplantation. It has been reported that ovarian stem cells exist in ovaries and the rearrangement of follicles is related to the microenvironment of ovaries [29,30]. We hypothesized that MSCs may promote primordial follicle rearrangement by regulating [E]: Image J software was used to analyze the average fluorescence intensity of figure D. Data are expressed as the mean ± SD. *: P < 0.05, **: P < 0.01 and ***: P < 0.001 ◂ ovarian fibrosis in POI rat. Finally, we detected the fertility of POI rats and found that the pregnancy was significantly improved after hUMSCs transplantation. These results are consistent with our previously published study [31,32].
To investigate the effects of hUMSCs transplantation on ovarian tissue fibrosis in POI rats, we have examined several fibrosis related biomarkers such as Collagen Type I (Collagen I), Collagen Type III (Collagen III), connective tissue growth factor (CTGF) and fibronectin (FN). The results show that hUM-SCs transplantation is an effective treatment to inhibit ovarian fibrosis and restore ovarian function in POI rats. To investigate the signal pathway that is involved in this pathology change, the study has focuses on the molecular of NR4A1. As reported, the NR4A1 is a class of proteins related to steroid hormone receptors of transcription factors, its biological activities do not require the participation of receptors. Therefore, its activity can be regulated not only at protein level but also at post-transcriptional phosphorylation level [33][34][35]. The study has shown that the NR4A1 expression is involved in the pathogenesis of scleroderma patients [18]. The phosphorylation of NR4A1 has also been implicated in the tissue fibrosis of endometriosis [36]. Therefore, the NR4A1 has been suggested as a possible target to regulate the pathological changes of ovarian tissues in POI disease. In the present study, we found that the total amount of NR4A1 was decreased in ovarian tissues of POI rats. Following transplantation, its expression was significantly increased. For the Phospho-NR4A1, it was increased in POI rats but inhibited following hUMSCs transplantation.
It has been documented that NR4A1 is involved in TGF-β 1 mediated ovarian endometriosis fibrosis [36]. The mechanism of NR4A1 in the regulation of ovarian function recovery in POI rats following hUMSCs transplantation was further investigated in stromal cells isolated from ovarian tissues. The overexpression and knockdown expression of NR4A1 was achieved by transfection of adenovirus and siRNA in the normal stromal cells, and then the molecular expression related to tissue fibrosis in these two situation was detected. We found that the expression of the myofibroblast marker α-SMA was increased, following transfection of siRNA to knock down the expression of NR4A1 in the normal stromal cells. And the expression of fibrosis related molecule Collagen I, Collagen III, CTGF and FN were increased. These findings suggest that NR4A1 plays an important role in the stromal cell transformation into myofibroblasts. The use of NR4A1 agonist has been reported to reduce the expression of fibrosis markers and attenuated fibrogenesis in a nude mouse model of endometriosis [34]. When we used NR4A1 agonist, DIM-C-pPHOCH3 to treat the cells in vitro, and the data showed that NR4A1 agonist could inhibit the differentiation of stromal cells into myofibroblasts induced by CDDP. The results obtained from the in vitro study may help explain the mechanism that is involved in the inhibition of ovarian fibrosis in POI rats following hUMSCs transplantation. And these findings suggest that NR4A1 may be a potential therapeutic target to treat POI disease in the future study.
AMP-activated protein kinase (AMPK) is a heterotrimer αβγAMPK complex composed of three subunits, including α (PRKAA), β (PRKAB) and γ (PRKAG). It has been documented to have an anti-fibrotic effect and its activation can protect tissue fibrosis in a variety of organs, including kidney [37,38], heart [39,40] and liver [41,42]. Studies have shown that the AMPK signal pathway is related to NR4A1 activity [43]. In osteoarthritis, NR4A1 is relatively inactivated by AMPK-dependent phosphorylation under chronic inflammation. And the results from our study has shown that hUMSCs transplantation can reduce AMPK phosphorylation. The expression of NR4A1 was significantly increased following the treatment with AMPK inhibitor. Thus, the AMPK may act as the upstream signal to of NR4A1 which regulates the ovarian function recovery in POI rats following hUMSCs transplantation. Despite promising results from exploring the repair potential of hUMSCs in animal POI models [44], few human clinical trials have been published. There is still a lack of evidence and safety studies on the role of hUMSCs in restoring reproductive aging in published clinical trials, and many specific mechanisms need to be further studied. There is no standard for the number of hUM-SCs transplanted in clinic, the duration of treatment, or the evaluation index. Therefore, hUMSCs transplantation in the clinical treatment of POI is still a great challenge.

Conclusion
The results of this study suggest that hUMSCs transplantation can repair the ovarian function of POI rats. This process may be realized by inhibiting ovarian fibrosis in POI rats through AMPK/NR4A1 signaling axis. As the number of Fig. 7 Effects of hUMSCs transplantation on AMPK signal in POI rats.
[A]: Western Blot was performed to detect AMPK and Phospho-AMPK expression in ovarian tissues. [B]: The expression of AMPK and Phospho-AMPK protein was quantitated using Image J software.
[C]: Immunofluorescence staining on total AMPK and Phospho-AMPK expression in ovarian tissues. The AMPK and phospho-AMPK signals are stained red. The nucleus was stained by DAPI as blue (Scale bar = 130 µm).
[D]: The mean fluorescence intensity of AMPK and Phospho-AMPK was analyzed by Image J software. [E]: Western blotting analysis of AMPK, Phospho-AMPK and NR4A1 expression in stromal cells.
[F]: Immunofuorescence staining of NR4A1 in stromal cells (Scale bar = 30 µm). The NR4A1 signal was stained as green color. The nucleus was stained by DAPI as blue.
[G]: The expression of NR4A1 and Phospho-AMPK protein was quantitated using Image J software and the mean fluorescence intensity of NR4A1 was analyzed by Image J software. Data are expressed as the mean ± SD. *: P < 0.05, **: P < 0.01 and ***: P < 0.001 ◂ patients receiving chemotherapy increases, so does the number of people who may have POI. The results of this study provide new therapeutic targets and strategies that can be used to promote fertility recovery in these patients.