Engineered Adipose-Derived Stem Cells Overexpressing RXFP1 via CRISPR Activation Ameliorate Erectile Dysfunction in Diabetic Rats

Due to the high incidence of diabetes mellitus (DM) and poor response to the first-line treatment of DM-induced erectile dysfunction (DMED), new therapeutic strategies for DMED are needed. Adipose-derived stem cell (ADSC) transplantation is considered a promising treatment modality for DMED but is limited by poor survival and efficacy after transplantation. In this study, we aimed to increase the therapeutic effect of DMED by overexpressing the relaxin family peptide receptor 1 (RXFP1) using a clustered regularly interspaced short palindromic repeats activation (CRISPRa) system in ADSCs. Two lentiviruses carrying the CRISPRa system transfected ADSCs to overexpress RXFP1 (RXFP1-ADSCs). The intracavernous injection of ADSCs was performed in DMED rats induced by the intraperitoneal injection of streptozotocin. Four weeks after transplantation, we measured erectile function and collected specimens of the corpus cavernosum for follow-up detection. The results showed that ADSCs improved erectile function in diabetic rats, and the RXFP1-ADSCs were more significant. We detected reduced levels of oxidative stress, apoptosis and fibrosis together with relative normalization of endothelial and smooth muscle cell function in the penis after ADSC transplantation. RXFP1-ADSCs had more potent efficacy in the above alterations compared to negative control ADSCs due to the high levels of survival and paracrine capacity in RXFP1-ADSCs. The results revealed that RXFP1-ADSC transplantation could partially preserve erectile function in DMED rats associated with the regulation of oxidative stress, apoptosis, fibrosis and endothelial and smooth muscle cell dysfunction. RXFP1 may be the new target for the genetic modification of ADSCs, which benefits the management of DMED.


Introduction
Erectile dysfunction (ED) is one of the common afflictions in male sexual dysfunction, which refers to the inability of the penis to be hard enough for satisfactory sex. The etiology of ED is complex, including aging, nerve damage, drugs, various metabolic abnormalities and other factors [1]. Hypertension, hyperlipidemia, obesity, metabolic syndrome and other metabolic abnormalities can lead to the production of ED or exacerbate the process of ED. Given the large number of patients with diabetes mellitus (DM), DM-induced ED (DMED) has become an area that cannot be ignored in the field of ED. The latest data from the International Diabetes Federation shows that more than 10.5% of adults worldwide suffer from diabetes [2]. In addition, phosphodiesterase type 5 inhibitors (PDE5is) are widely used as a first-line treatment for ED. However, the effective rate of PDE5is is significantly reduced in patients with DMED compared to non-diabetic patients (63% vs. 83%) [3].

Animals
The design of our study was approved by the Committee on Animal Experiments of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (TJH-201910005).
A total of 55 male SD rats (8 weeks old) were included in this study, which were from the Laboratory Animal Center of Tongji Medical College, Wuhan, China. Before the formal experiment, all rats received a one-week adaptation (free access to water and food, suitable temperature). The normal sexual function of all animals was confirmed by paired experiments [15,24,25]. Fasting blood glucose and body weight were recorded throughout the experiment.
Streptozotocin (STZ; Sigma-Aldrich, St Louis, MO, USA; 1%) dissolved in its vehicle (0.1 mol/L citrate phosphate buffer; pH 4.2) was used to model diabetes in rats. Intraperitoneal injection of STZ (60 mg/kg) was performed on 47 rats, while the vehicle was injected intraperitoneally into the remaining 8 rats. Successful diabetes models only referred to rats with fasting blood glucose levels greater than 16.7 mmol/L after 72 h.

Evaluation of Erectile Function
After 4 weeks, we performed the APO experiment again on all rats. Then, the intracavernous pressure (ICP) and arterial pressure were measured under electrical stimulation of the cavernous nerve (15 Hz; 5.0 V; 1 min) to more intuitively assess erectile function. After the evaluation was completed, the corpus cavernosum was divided into sections and stored at −80 • C and in 4% paraformaldehyde, respectively. Specimens were subsequently prepared as frozen sections and paraffin sections.

Western Blot
The protein lysates (RIPA buffer; Boster) of corpus cavernosum and ADSCs were obtained to detect the expressions of related proteins. After the quantification of protein by the BCA assay (Boster), protein samples would be subjected to electrophoresis, transmembrane and incubation with antibodies. The final visualization of the bands was achieved using a ChemiDocTM MP Image System (Bio-Rad Laboratories, Hercules, CA, USA). The details of the primary antibodies used are listed in Table S1.

Histological Alteration
Paraffin sections (4 µm thickness) were prepared for the procedure of immunohistochemistry (IHC) and immunofluorescence (IF). The area and intensity of the positive region reflected the distribution and expression levels of the target molecules. The details of the antibodies used are listed in Table S1. The normal goat IgG (1: 200; GB23303; Servicebio) was used as a negative control in IHC and presented in Figure S2.
Masson trichrome staining was performed using paraffin sections. The red and blue areas represented the component of smooth muscle and collagen, respectively. Resorcinolfuchsin staining was also performed for the detection of elastin levels in the penis. The purple-black part represented elastic fibers, the red part represented collagen fibers, and the yellow part in the background referred to other components. The above indicators could partly reflect the level of fibrosis in the penis.
Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining (Beyotime Biotechnology, Shanghai, China) was performed to detect the apoptotic degree in the penis. When genomic DNA was broken, the exposed 3'-OH could be bound by the probe. It was followed by co-incubation with diaminobenzidine for color development.
Frozen sections (10 µm thickness) were prepared to detect reactive oxygen species (ROS). The probe of Dihydroethidium (DHE; Beyotime Biotechnology) was incubated with tissue slices, and the intensity of red fluorescence could reflect the level of ROS under a fluorescence microscope.

Detection of Special Substances
Tissue homogenate was first prepared from the frozen penis with reference to the respective protocol. The protein concentrations of each sample needed to be determined by the BCA assay (Boster) before the detection of subsequent indicators.
Considering that human RLX-2 is equivalent to rodent RLX-1 [27], the ELISA kits of RLX-1 (BANGYI, Shanghai, China), VEGF, bFGF and cyclic guanosine monophosphate (cGMP; Mbbiology, Jiangsu, China) were used to detect respective target molecules. Different concentrations of standards and 10 uL samples were added to the sample wells coated with relevant antigens. Subsequently, the reaction system containing other reagents was incubated at 37 • C. Absorbance was read at a wavelength of 450 nm using a microplate reader.
A total NO assay kit (Beyotime Biotechnology) was used to detect nitric oxide (NO). The standard was diluted to different gradient concentrations. Then, 60 uL samples, or standards of different concentrations, and other reaction components, were added to each well of 96-well plates. After incubation at 37 • C, color development was recorded with a microplate reader at 540 nm absorbency.
For oxidative activity, malondialdehyde (MDA) and superoxide dismutase (SOD) were chosen to be examined with respective test kits (Beyotime Biotechnology). In addition to the sample's reaction, the standard and the control reaction also needed to be carried out. For the detection of SOD, the absorbance at a wavelength of 450 nm was detected after incubation at 37 • C. For the detection of MDA, the absorbance at a wavelength of 532 nm was caught after a water bath at 100 • C.
In addition, we normalized the above results using the respective protein concentrations. The final results reflected the amount of substance to be measured per unit mass of penile tissue.

Statistical Analyses
The data were expressed as the mean ± standard deviation and analyzed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). The Shapiro-Wilk test was used to determine normal distribution. For normally distributed data, we used one-way ANOVA analysis and Tukey's test for multiple comparisons. For non-normally distributed data, we used the Kruskal-Wallis test and Dunn's test for multiple comparisons. A p-value < 0.05 indicated that the difference was statistically significant.

Preparation and Transfection of ADSCs
As shown in Figure 1A, the surface markers of most ADSCs at passage 4 appeared as follows: CD29 (+), CD31 (−), CD34 (−), CD45 (−), CD90 (+) and CD106 (−). The above indicators showed that the primary cells we isolated from adipose were mesenchymal stem cells. The results of IF were positive for vWF and α-SMA, suggesting that ADSCs could differentiate into endothelial and smooth muscle cells ( Figure 1B). The results of Oil-red-O staining were also positive, meaning that ADSCs could differentiate into adipocytes ( Figure 1C). mally distributed data, we used the Kruskal-Wallis test and Dunn's test for multiple com-parisons. A p-value < 0.05 indicated that the difference was statistically significant.

Preparation and Transfection of ADSCs
As shown in Figure 1A, the surface markers of most ADSCs at passage 4 appeared as follows: CD29 (+), CD31 (−), CD34 (−), CD45 (−), CD90 (+) and CD106 (−). The above indicators showed that the primary cells we isolated from adipose were mesenchymal stem cells. The results of IF were positive for vWF and α-SMA, suggesting that ADSCs could differentiate into endothelial and smooth muscle cells ( Figure 1B). The results of Oil-red-O staining were also positive, meaning that ADSCs could differentiate into adipocytes ( Figure 1C).
Lentiviruses carrying the CRISPRa system were successfully transfected into ADSCs ( Figure 1D). The results of Western blotting (WB) and qRT-PCR revealed that our genetic modification of ADSCs was effective ( Figure 1E-G). The expression of RXFP1 increased in the two specific RXFP1-targeting sgRNA groups (sgRNA2 and sgRNA3), and the sgRNA2 group had the most noticeable effect. As a result, we applied the ADSCs of the sgRNA2 group to the follow-up animal experiments.  Lentiviruses carrying the CRISPRa system were successfully transfected into ADSCs ( Figure 1D). The results of Western blotting (WB) and qRT-PCR revealed that our genetic modification of ADSCs was effective ( Figure 1E-G). The expression of RXFP1 increased in the two specific RXFP1-targeting sgRNA groups (sgRNA2 and sgRNA3), and the sgRNA2 group had the most noticeable effect. As a result, we applied the ADSCs of the sgRNA2 group to the follow-up animal experiments.

Metabolic and Physiological Parameters
All rats' body weight and fasting blood glucose were similar at the start (all p > 0.05; Figure 2A,B). Eight weeks after the induction of the diabetes model, the body weight decreased, and the blood glucose increased in DM rats compared with the control group (both p < 0.05). Additionally, this trend did not change 4 weeks after ADSC transplantation. These results indicated that the construction of DM rats was successful.

Metabolic and Physiological Parameters
All rats' body weight and fasting blood glucose were similar at the start (all P > 0.05; Figure 2A,B). Eight weeks after the induction of the diabetes model, the body weight decreased, and the blood glucose increased in DM rats compared with the control group (both P < 0.05). Additionally, this trend did not change 4 weeks after ADSC transplantation. These results indicated that the construction of DM rats was successful.
The APO experiment showed that the number of erections was lowest in the DMED group and increased in the treatment groups (all P > 0.05; Figure 2C). In addition, we also measured the arterial pressure and ICP to assess erectile function in each group ( Figure  2D-G). Under the condition that the mean arterial pressure (MAP) was relatively constant, the maximum ICP/MAP and total ICP (AUC, area under the curve) were decreased in the DMED group and increased in the treatment groups (all P < 0.05), and the RXFP1-ADSC group was more significant. The APO experiment showed that the number of erections was lowest in the DMED group and increased in the treatment groups (all p > 0.05; Figure 2C). In addition, we also measured the arterial pressure and ICP to assess erectile function in each group ( Figure 2D-G). Under the condition that the mean arterial pressure (MAP) was relatively constant, the maximum ICP/MAP and total ICP (AUC, area under the curve) were decreased in the DMED group and increased in the treatment groups (all p < 0.05), and the RXFP1-ADSC group was more significant.
The ELISA assays and IHC indicated the presence of RLX-1 in plasma and penile tissue, and its levels in DMED rats were slightly reduced compared to the control group and did not change after ADSC transplantation (p < 0.05; Figure 3A,B,E). The concentrations of VEGF and bFGF declined in the DMED group and recovered to some extent after ADSC transplantation, contrasted with the control group (all p < 0.05; Figure 3C,D). Moreover, RXFP1 was expressed in the corpus cavernosum sinus, and the level in the DMED group was decreased compared with the control group ( Figure S3). The ELISA assays and IHC indicated the presence of RLX-1 in plasma and penile tissue, and its levels in DMED rats were slightly reduced compared to the control group and did not change after ADSC transplantation (P < 0.05; Figure 3A,B,E). The concentrations of VEGF and bFGF declined in the DMED group and recovered to some extent after ADSC transplantation, contrasted with the control group (all P < 0.05; Figure 3C,D). Moreover, RXFP1 was expressed in the corpus cavernosum sinus, and the level in the DMED group was decreased compared with the control group ( Figure S3).

Transplantation of ADSCs Inhibited Oxidative Stress Damage in Penile Tissue
The levels of ROS and MDA were chosen to be examined for detecting the activity of oxidative stress. As shown in Figures 4A,B,E, the levels of ROS and MDA in DMED rats were higher compared with the control group and decreased after treatment of ADSCs, specifically RFXP1-ADSCs (all P < 0.05). Contrarily, SOD, an important antioxidant, exhibited opposite changes in penile tissue (P < 0.05; Figure 4D). The RAGE and NADPH

Transplantation of ADSCs Inhibited Oxidative Stress Damage in Penile Tissue
The levels of ROS and MDA were chosen to be examined for detecting the activity of oxidative stress. As shown in Figure 4A,B,E, the levels of ROS and MDA in DMED rats were higher compared with the control group and decreased after treatment of ADSCs, specifically RFXP1-ADSCs (all p < 0.05). Contrarily, SOD, an important antioxidant, exhibited opposite changes in penile tissue (p < 0.05; Figure 4D). The RAGE and NADPH oxidases, including NOX2 and NOX4, are regarded to be critical in the process of oxidative stress. The results of WB and IHC suggested that the expressions of RAGE, NOX2 and NOX4 were highest in the DMED group and inhibited to a certain extent after ADSC transplantation (all p < 0.05; Figure 4C,F-I).
oxidases, including NOX2 and NOX4, are regarded to be critical in the process of oxidative stress. The results of WB and IHC suggested that the expressions of RAGE, NOX2 and NOX4 were highest in the DMED group and inhibited to a certain extent after ADSC transplantation (all P < 0.05; Figure 4C,F-I).

Transplantation of ADSCs Regulated the NO/cGMP and RhoA/ROCK Pathway in Rats
The results of WB and IF demonstrated that the expressions of eNOS and nNOS were downregulated under long-term diabetes (both p < 0.05; Figure 5A-G). After ADSC transplantation, the expressions of the above two molecules were upregulated but lower than the control group (all p < 0.05). The alteration of the NO/cGMP pathway, as the downstream pathway of eNOS and nNOS, was consistent with the above two molecules ( Figure 5H,I).
The results of WB and IF demonstrated that the expressions of eNOS and nNOS were downregulated under long-term diabetes (both P < 0.05; Figure 5A-G). After ADSC transplantation, the expressions of the above two molecules were upregulated but lower than the control group (all P < 0.05). The alteration of the NO/cGMP pathway, as the downstream pathway of eNOS and nNOS, was consistent with the above two molecules ( Figure  5H,I).  On the contrary, we found the highest expression of RhoA, ROCK1 and ROCK2 in the DMED group (all p < 0.05; Figure 6A-D,F,G). The concentration of Ca 2+ was also significantly reinforced in DMED rats compared with the control rats (p < 0.05; Figure 6E). Moreover, these four indicators were dampened after treatment of ADSCs, in which the more significant effect was found in the RXFP1-ADSCs group. oxide; cGMP = cyclic guanosine monophosphate.
On the contrary, we found the highest expression of RhoA, ROCK1 and ROCK2 in the DMED group (all P < 0.05; Figure 6A-D,F,G). The concentration of Ca 2+ was also significantly reinforced in DMED rats compared with the control rats (P < 0.05; Figure 6E). Moreover, these four indicators were dampened after treatment of ADSCs, in which the more significant effect was found in the RXFP1-ADSCs group.

Transplantation of ADSCs Adjusted Apoptosis in Vivo
The result of TUNEL staining indicated that the apoptosis index in DMED rats was highest among the four groups and declined after treatment of ADSCs but was still lower than the control group (all P < 0.05; Figure 7A,B). The expression of α-SMA and CD31 (the

Transplantation of ADSCs Adjusted Apoptosis In Vivo
The result of TUNEL staining indicated that the apoptosis index in DMED rats was highest among the four groups and declined after treatment of ADSCs but was still lower than the control group (all p < 0.05; Figure 7A,B). The expression of α-SMA and CD31 (the markers of smooth muscle cells and endothelial cells, which are the primary effector cells of the corpus cavernosum) also conformed to the above trend ( Figure 7C-E). Furthermore, we found that the ratio of Bax to Bcl-2, the expression of Caspase 3 and cleaved Caspase markers of smooth muscle cells and endothelial cells, which are the primary effector cells of the corpus cavernosum) also conformed to the above trend ( Figure 7C-E). Furthermore, we found that the ratio of Bax to Bcl-2, the expression of Caspase 3 and cleaved Caspase 3 (C-caspase 3) could be recovered to different degrees under the effects of ADSCs and RXFP1-ADSCs (all P < 0.05; Figure 7F-J). The above results suggested that ADSCs could alleviate apoptosis in vivo, and RXFP1-ADSCs were more significant.

Transplantation of ADSCs Reduced Fibrosis in the Corpus Cavernosum
Masson trichrome staining and resorcinol-fuchsin staining are both indicators reflecting the level of fibrosis. The ratio of smooth muscle to collagen significantly de-creased in DMED rats and increased to a certain extent in the treatment group (all p < 0.05; Figure 8A,B). However, the detection of elastic fibers showed the opposite result. The elastin percentage and maximum elastic fiber length in the DMED group were the lowest among the four groups (both p < 0.05; Figure 8C,D,F). Furthermore, WB also verified the above results. The TGFβ1/Smad 2/3/CTGF pathway and the expression of Collagen I, Collagen III and α-SMA of DMED rats all showed a trend opposite to those of control rats and recovered to varying degrees after ADSC transplantation (all p < 0.05; Figure 8E,G-I).

Transplantation of ADSCs Reduced Fibrosis in the Corpus Cavernosum
Masson trichrome staining and resorcinol-fuchsin staining are both indicators reflecting the level of fibrosis. The ratio of smooth muscle to collagen significantly decreased in DMED rats and increased to a certain extent in the treatment group (all P < 0.05; Figure  8A,B). However, the detection of elastic fibers showed the opposite result. The elastin percentage and maximum elastic fiber length in the DMED group were the lowest among the four groups (both P < 0.05; Figure 8C,D,F). Furthermore, WB also verified the above results. The TGFβ1/Smad 2/3/CTGF pathway and the expression of Collagen Ⅰ, Collagen Ⅲ and α-SMA of DMED rats all showed a trend opposite to those of control rats and recovered to varying degrees after ADSC transplantation (all P < 0.05; Figure 8E,G-I).

Discussion
As a metabolic abnormality, DM has a high incidence worldwide and is one of the important causes of ED [1,2]. Due to the limited effects of therapies for DMED, it has troubled a large number of patients and medical workers around the world. Transplantation of ADSCs is a promising treatment for DMED. Given the solid regulatory capacity of RXFP1 in pathological settings, we activated the expression of RXFP1 in ADSCs via CRISPRa to achieve a better therapeutic effect on DMED. In the present study, we successfully expressed high levels of RXFP1 in ADSCs and performed intracavernous injections of them in DMED rats. The results showed that RXFP1-ADSC transplantation partially improved erectile function and had more potent antioxidant, anti-apoptotic and anti-fibrotic abilities than NC ADSCs. In addition, RXFP1-ADSCs could better regulate the content and function of endothelial cells and smooth muscle cells.
Multi-lineage differentiation ability and paracrine factors of mesenchymal stem cells are the two keys to treating ED [9]. The mechanism mainly includes the directed differentiation function of ADSCs into cavernous endothelial cells and smooth muscle cells, as well as a large number of paracrine cytokines, such as VEGF and bFGF. This maintains the number and function of essential cells in the corpus cavernosum and resists pathological changes [28][29][30]. Although the efficiency of differentiation to endothelial or smooth muscle cells is controversial, we cannot yet wholly deny that this mechanism is involved in the treatment of ED with mesenchymal stem cells [30]. In our study, we confirmed in in vitro experiments that ADSCs could differentiate into endothelial and smooth muscle cells, which were the primary effector cells of the corpus cavernosum and participated in the physiological erection. The content and function of endothelial and smooth muscle cells in rats also increased after ADSC transplantation, which may be a combined result of the differentiation of ADSCs and decreased levels of apoptosis. Increased levels of VEGF and bFGF in the penis suggested that paracrine trophic factors of ADSCs promoted the restoration of erectile function. In addition, recent studies have pointed out that exosomes secreted by mesenchymal stem cells also play a protective role in erectile function. Chen et al. and Zhu et al. proposed that ADSC-derived exosomes could independently ameliorate ED in diabetic rats [31,32]. This may be a new option for future cell-free therapy in DMED. PGI2 also plays a protective role in improving penile erection and can be mediated by RLX-1 [33,34]. The activation of RLX-1-RXFP1 signaling in ADSCs may also increase the secretion of PGI2 to upregulate the cAMP pathway in the surrounding corpus cavernous.
Efficacy after transplantation of mesenchymal stem cells is often limited due to various factors, such as (1) shortened cell lifespan resulting from multiple expansion of cells in vitro or (2) a hostile microenvironment at the transplant site. To improve therapeutic efficacy, genetic modification is performed as one of the logical options to enhance cell survival and function [9,35,36]. The activation of RXFP1 may play a protective role in cardiovascular disease [37]. RXFP1 is also involved in the functional exercise of mesenchymal stem cells and increases the therapeutic effect of adult cells [13,14]. Therefore, we reasonably inferred that the upregulation of RXFP1 may also promote the efficacy of ADSCs in lesions. For the purpose of overexpressing RXFP1 in ADSCs, CRISPRa seems to be a good choice. Compared with other existing gene-editing strategies, CRISPRa offers the following advantages: (1) it induces low off-target effects; (2) it is independent of the target gene size and can activate different genes at the same time; (3) it can simultaneously up-or downregulate different genes in target cells; and (4) it is a mutation-independent therapeutic strategy [38]. Moreover, CRISPRa has been successfully used to genetically modify several stem cells, in-cluding ADSCs, BMSCs, induced pluripotent stem cells and other stem cells, to achieve the goal of tissue regeneration or disease treatment [19,39,40]. Given the above theoretical basis, we upregulated RXFP1 expression in ADSCs via CRISPRa, followed by intracavernous injection in DMED rats. Our data showed that ADSCs could increase erectile function through multiple mechanisms, and the effect of RXFP1-ADSC was more potent than that of NC ADSCs. The above results proved that RXFP1 enhanced the therapeutic effect of ADSCs in DMED.
Although DMED is a complex pathological process with multiple factors, oxidative stress plays a critical role. Oxidative stress refers to the imbalance between ROS production and the endogenous antioxidant system. Advanced glycation end-products (AGEs) aggravate ROS production and have an exceptional contribution to oxidative stress, while diabetes is the disease most conducive to the formation of AGEs [41]. Oxidative stress plays a vital role in microvascular injury through various factors [42]. Since the corpus cavernosum happens to be part of the whole body's microvessels, numerous studies have been conducted to ameliorate DMED by reducing oxidative stress [43,44]. The data of our in vivo experiments also revealed the increase of ROS and the decrease of SOD in the penis of diabetic rats, indicating the high level of oxidative stress in DMED. As an essential source of oxidative stress, the activity of NADPH was also confirmed to be enhancive in DMED. The above pathological changes were all reversed to some extent after ADSC transplantation, especially in the case of RXFP1-ADSCs.
Long-term diabetes can also lead to other dysfunctions of the corpus cavernosum, including increased apoptosis and fibrosis, which promote the occurrence or progression of DMED [45,46]. The intracavernous injection of mesenchymal stem cells has been shown to improve erectile function through anti-apoptosis and anti-fibrosis effects [45,47]. The aforementioned protective effects were indeed found in DMED rats after ADSC transplantation in this study, and the upregulation of RXFP1 expression amplified the benefits of ADSCs. The imbalance of smooth muscle relaxation and contraction also participates in the development of DMED. NO is the primary substance that promotes the relaxation of the smooth muscle of the corpus cavernosum and is produced by two enzymes, eNOS and nNOS. The RhoA/ROCK pathway is an important signaling pathway that regulates smooth muscle contraction. Imbalance in the NO/cGMP and RhoA/ROCK pathways also aggravates DMED [44,48,49]. The present study suggested the imbalance of the NO/cGMP and RhoA/ROCK pathways appeared in DMED rats, and ADSC transplantation regulated these two pathways to normal levels. Moreover, oxidative stress can induce or exacerbate the above-mentioned pathological changes, including apoptosis, fibrosis and smooth muscle dysfunction. Reduced levels of oxidative stress contribute to a multifactorial improvement of erectile function in diabetic rats [44,45,48].
Based on the above results, we generalized the possible underlying mechanism of RXFP1-ADSCs in ameliorating DMED (Figure 9). The long-term chronic high-glucose environment in the local tissue of the penis induces an increase in oxidative stress. Histiocytic apoptosis and fibrosis also occur in damaged tissues. At the same time, the content and function of endothelial cells and smooth muscle cells in the penile cavernous sinuses decreased under the harsh local microenvironment. Oxidative stress could, in turn, aggravate levels of apoptosis, fibrosis and endothelial cell and smooth muscle cell dysfunction. The abovementioned multiple complex factors together lead to the decline of erectile function. Transplantation of ADSCs can reverse these pathological changes to a certain extent by differentiating into functional cells and secreting cytokines such as VEGF and bFGF. Moreover, RXFP1-ADSCs can further amplify these beneficial effects and further improve erectile function.
Moreover, the therapeutic effect of mesenchymal stem-cell-derived exosomes on DMED has been confirmed, which was not involved in our experiments. Finally, other subtypes of relaxin and receptors for relaxin also exist besides those explored in this study. Considering there may be cross-effects among them, other subtypes still merit exploration. The above limitations need to be further explored in our future research. Figure 9. Potential mechanism of ADSCs Transplantation in the treatment of DMED. Transplantation of ADSCs improves erectile function in DMED rats through differentiating into functional cells and secreting cytokines such as VEGF and bFGF. Regulation of oxidative stress, apoptosis and fibrosis are involved in this process. The normalization of endothelial and smooth muscle cells' function also follows the above alteration. Moreover, reduced level of oxidative stress greatly benefits the improvement of the pathological environment in the penis. Created with BioRender.com. CRISPR = clustered regularly interspaced short palindromic repeats; sgRNA = single guide RNA; dCas9 = catalytically inactive CRISPR-associated protein 9; RXFP1 = relaxin family peptide receptor 1; ADSCs = adipose-derived stem cells; DMED = diabetes mellitus-induced erectile dysfunction.

Conclusions
RXFP-ADSCs were confirmed to alleviate erectile dysfunction in diabetic rats by increasing the proliferative and paracrine capacity of ADSCs. The possible therapeutic effects of RXFP1-ADSCs were associated with the regulation of oxidative stress, apoptosis and fibrosis. The normalization of endothelial and smooth muscle cell function was also involved in this process. Our research provided new ideas for the application of mesenchymal stem cells and contributed to the development of regenerative and translational medicine.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Flow Diagram of Experimental Design; Figure S2: Negative controls of immunohistochemistry in each group; Figure S3: The expression of RXFP1 in the penis of rats; Table S1: The details of the antibodies used in the study. Figure 9. Potential mechanism of ADSCs Transplantation in the treatment of DMED. Transplantation of ADSCs improves erectile function in DMED rats through differentiating into functional cells and secreting cytokines such as VEGF and bFGF. Regulation of oxidative stress, apoptosis and fibrosis are involved in this process. The normalization of endothelial and smooth muscle cells function also follows the above alteration. Moreover, reduced level of oxidative stress greatly benefits the improvement of the pathological environment in the penis. Created with BioRender.com. CRISPR = clustered regularly interspaced short palindromic repeats; sgRNA = single guide RNA; dCas9 = catalytically inactive CRISPR-associated protein 9; RXFP1 = relaxin family peptide receptor 1; ADSCs = adipose-derived stem cells; DMED = diabetes mellitus-induced erectile dysfunction.
Limitations still existed in our study. Firstly, we did not directly test the survival or resident capacity of ADSCs in high-glucose environments in vitro or in diabetic animals. Moreover, the therapeutic effect of mesenchymal stem-cell-derived exosomes on DMED has been confirmed, which was not involved in our experiments. Finally, other subtypes of relaxin and receptors for relaxin also exist besides those explored in this study. Considering there may be cross-effects among them, other subtypes still merit exploration. The above limitations need to be further explored in our future research.

Conclusions
RXFP-ADSCs were confirmed to alleviate erectile dysfunction in diabetic rats by increasing the proliferative and paracrine capacity of ADSCs. The possible therapeutic effects of RXFP1-ADSCs were associated with the regulation of oxidative stress, apoptosis and fibrosis. The normalization of endothelial and smooth muscle cell function was also involved in this process. Our research provided new ideas for the application of mesenchymal stem cells and contributed to the development of regenerative and translational medicine.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/antiox12010171/s1, Figure S1: Flow Diagram of Experimental Design; Figure S2: Negative controls of immunohistochemistry in each group; Figure S3: The expression of RXFP1 in the penis of rats; Table S1: The details of the antibodies used in the study.