Combination Therapy Using Adipose-Derived Stem Cells And Low-Intensity Extracorporeal Shockwave Therapy On Erectile Dysfunction In Streptozotocin-Induced Diabetic Rats

Background(cid:0)Diabetes mellitus (DM) is an important risk factor for erectile dysfunction (ED). Adipose-derived stem cells (ADSCs) and low-intensity extracorporeal shockwave therapy (LI-ESWT) are two emerging therapies for ED. The ecacy of ADSCs combined LI-ESWT in the treatment of diabetes induced erectile dysfunction (DMED) was not completely elucidated. This study aim to investigate combined therapeutic ecacy of ADSCs and LI-ESWT in rat model of DMEDand whether they have interaction. weeks, function was assessed using intracavernous pressure and mean arterial pressure (MAP), after that performed immunohistochemistry (IH).


Background
Erectile dysfunction (ED) is defined as the inability of a male to attain and maintain erection of the penis to permit satisfactory sexual intercourse [1], It is one of the most common symptoms affecting the life quality of middle-aged men [2]. ED is more prevalent in men with diabetes mellitus (DM). A Chinese study showed that the prevalence of ED in diabetics in 2010 was about 75.2% [3]. Diabetes induced erectile dysfunction (DMED), pathogenic manifestations involving endothelial injury, neuropathy, microvascular and fibrousmuscular alterations is usually more serious and difficult to treat than nondiabetic [4,5]. Phosphodiesterase 5 inhibitors (PDE5Is) was considered to be the first-line therapeutic drug for ED however PDE5Is showed lower efficacy in treating DMED [6]. Thus, it becomes a key issue of treat DMED to explore the new treatment.
Adipose-derived stem cells (ADSCs) are stem cells with multiple-differentiation [7] and immunosuppression potential[8] that isolated from adipose tissue. Stem cells have the potential to increase the expression of nerve nitricoxide synthase (nNOS) in cavernous nerves (CN) and are capable of secreting neurotrophic and angiogenic factors [9,10], Which all make ADSCs posses potential therapeutic effect on ED. However there remains a challenge to fully explore clearly the efficacy of ADSCs therapy.
Low-intensity extracorporeal shockwave therapy (LI-ESWT), is an emerging physical therapy for ED currently due to its ability to improve erectile function by stimulating the corpus cavernosum (CC) in patients with ED. LESW has been demonstrated to enhance gene expression related to angiogenesis and proliferation, and improve tissue neovascularization and regeneration [11]. LI-ESWT has been frequently used in clinical study and has achieved excellent therapeutic effects [10,12].
Because stem cells have the potential to restore the function of nerves [9,10] and endothelium, smooth muscle in corpus cavernosum (CC) [13], and LI-ESWT has the potential to improve penile vascular endothelial function [14], we hypothesized that combining these two treatments would improve recovery effect of erectile function than applying the two treatments separately in a rat DMED model. Therefore, in this study, the purpose of the present study was to investigate the efficacy of ADSCs combined with LI-ESWT therapy for DMED and whether have the significant interaction of these two methods in an streptozotocin (STZ)-induced diabetic rat model of ED.

Animals
A total of 150 male's specific pathogen free (SPF) rats aged 8 weeks old (body weight: 300-370g) were purchased from the Animal Center of Henan province, and this was consistent with animal experimental ethics. 130 rats in the interaction analysis group were fasted for 12 hours, and then intraperitoneally injected with streptozotocin (STZ) (Beijing Dingguo Changsheng Biotechnology Co.Ltd, 55 mg/kg). Blood glucose levels were measured at weeks 2, 4, 6 and 8 respectively. Of the 130 rats, 103 rats were diabetic with a randomized blood glucose ≥ 16.7 mmol/L. An intracavernous (IC) injection of phosphate buffer solution (PBS) (Wuhan Seville Biological Technology Co., Ltd) was administered to 20 rats as the normal control group. At 8 weeks after STZ injection apomorphine (APO) test was used to screen the diabetic rats, And the 83 of the 103 (80.58%) rats with negative APO test results were numbered and randomly assigned to four groups: ED control group (ED) was given IC injection of PBS; ADSCs treatment group (ADSCs) was given IC injection of ADSCs suspension; LI-ESWT treatment group (LI-ESWT) was given cavernous shock wave treatment; ADSCs combined with LI-ESWT treatment group (ADSCs + LI-ESWT) was given cavernous shockwave treatment one day after injection of ADSCs. The control group also received an IC injection of PBS.
The Ethics Review Committee of the Life Science of Zhengzhou University approved the study. Animals were euthanized with carbon dioxide at the conclusion of the experiment. This article does not contain any studies with human participants performed by any of the authors ADSC isolation and Intracavernous injection ADSCs were isolated from the inguinal fats of two 4-week-old SPF SD rats 60 80g and cultured as previous standardized method [15].We use MTT assay to detect the proliferation capacity of ADSCs and found the passage 3 ADSCs proliferate fastest, we also performed osteogenic and adipogenic induction and found passage 3 ADSCs could well differentiate into adipocytes and osteocytes after induction. So we use the passage 3 ADSCs as cell suspensions and then intracavernous injection. Under aseptic conditions, the rats in ADSCs, ED and the normal control group were anesthetized. The penis was exposed in each group, and a 24gauge needle was used to inject a total of 1×106 ADSCs (dissolved in 100μl PBS) or only 100μl PBS into the corpus cavernosum (CC). An elastic band was applied to the base of the penis and the pressure was maintained for 2 minutes after the injection.

LI-ESWT treatment
Rats in the LI-ESWT and ADSCs + LI-ESWT groups were treated with shockwaves. After anesthesia, the penis was drawn out of the prepuce in a supine position. Ultrasonic gel was applied on the penis and then the shockwave applicator (HB-ESWT-01 shock wave musculoskeletal pain treatment machine, Seaside Medical Devices Co., Ltd, China) was placed on the penis. A total of 300 (150 on each side of the CC) shocks were delivered at energy level of 0.09 mJ/mm 2 and a frequency of 120/min during each LI-ESWT session [16]. LI-ESWT was repeated 3 times per week with a day's break in between each session, for a total duration of 3 weeks.

Measurement of erectile function
Erectile function was assessed 4 weeks after intervention. The carotid artery and cavernous nerve were exposed to measure the mean arterial pressure (MAP) and the intracavernosal pressure (ICP), respectively. The rats were anesthetized in a supine position.
The major pelvic ganglion (MPG) and CN were exposed through a midline laparotomy. One of the 21-gauge needles that were connected to PE-tubes with heparinized saline (250U/ml, Beijing Solarbio Technology Co., Ltd) was inserted into the carotid artery to measure MAP.
The other one was inserted into CC to measure ICP. PE-tubes were connected to the MD3000 signal acquisition (Anhui Zheng Hua biological instrument and Equipment Co., Ltd.) and processing system (Anhui Zheng Hua biological instrument and Equipment Co., Ltd.). The CNs was stimulated using a stainless steel bipolar hook electrode with the following parameters: 10v, 20Hz, with a pulse width of 0.2 ms for 1 minute. At the same time, the vascular clamp was loosened and the pressure of ICP and MAP were recorded.

Measurement of efficiency and cure rate
No previous study has demonstrated which level of ICP/MAP is the therapeutic efficiency on ED. Our study used the minimum level of ICP/MAP in the normal group as the standard to calculate the effective rate of ADSCs and LI-ESWT for ED, used the lower limit of 95% confidence interval (CI) of ICP/MAP in the normal group as the standard to calculate the cure rate of ADSCs and LI-ESWT for ED. The effective rate = A/B (A=the rats number in treatment group when ICP/MAP level were higher than the minimum of ICP/MAP level in the normal group and B=the total number of samples in the treatment group). The cure rate = C/D (C= the rats number in treatment group when ICP/MAP level were higher than the 95%CI lower limit of ICP/MAP level in the normal group and D refers to the total number of samples in the treatment group).

Immunohistochemistry
For Immunohistochemistry (IH), the penile tissue (midshaft portion) were fixed in fresh 4% paraformaldehyde (Beijing Solarbio Technology Co., Ltd) for 18~24 hours. The fixed tissues were put in paraffin blocks and cut into 5μm sections before mounting on slides. After dewaxing and antigen repair, the slides were incubated with primary antibodies (Table 1)  Statistical analyses were performed with the SPSS 21.0; Measurement data were expressed as mean ± standard deviation (SD). The comparisons of blood glucose and weight were analyzed using one-way ANOVA or LSD; One-way ANOVA was used to test the difference of ICP/MAP between different groups; Univariate ANOVA was used to test the interaction between ADSC and LI-ESWT; We also performed the additive model to analysis interaction of efficacy by calculating the attributable proportion due to interaction (AP), Additionally, relative excess risk due to interaction (RERI) and the synergy index (SI) were also performed to evaluate interactions. All statistical tests were two-tailed and results were considered significant when p<0.05.

Characterization and Construction of animal model
One rat in the ED, one rat in the ADSCs and two rats in the LI-ESWT were dead before the end of experiment ( Figure 1). The final blood glucose levels of STZ-induced diabetic rats were remarkably higher compared to PBS-treated control rats (F=220.51 P<0.05). The final weights of diabetic rats were markedly lower than normal rats (F=45.17, P<0.05). Among ED, ADSCs, LI-ESWT and ADSCs + LI-ESWT groups, blood glucose levels and body weights were not statistically different ( Table 2).         PDE5Is showed less efficacy in treating DMED [17] and the treatment of stem cell therapy and LI-ESWT therapy has been two emerging therapies for ED currently.
Stem cells play an important role in the treatment of ED due to their multipledifferentiation and immunosuppression potential, and have already been explored in different ED models [13,18,19]. As for ED therapy, some study has suggested that ADSCs could improve the therapeutic effect of erectile function in rat models of DMED [20]. In the present study, expression increased of -SMA, vWF and nNOS in CC were observed in ADSCs-injected diabetic rats. These results are consistent with previous findings about diabetic rats [21].
In this study, we explored another treatment to treat the DMED, namely LI-ESWT. Qiu et al utilized LI-ESWT and found that the treatment was able to restore the function of the erectile tissues. However, while our study also produced consistent results that LI-ESWT could improve the therapeutic effect of erectile function in rat models of DMED. LI-ESWT has been known could significantly improve penile vascular endothelial function and penile flow velocity [14,22,23], in addition, DMED involves dysfunction of the nerves, endothelium, and smooth muscle, which may partially explained why the LI-ESWT could improve the therapeutic effect of erectile function in our study.
Although, both ADSCs and LI-ESWT therapies could improve the erectile function, the combined effect of ADSCs and LI-ESWT for DMED are yet to be determined. Thus, our study hypothesized that combining the use of ADSCs and LI-ESWT would significantly improve the treatment of DMED. Our results suggest that combining ADSCs and LI-ESWT could play an important role in the treatment of the DMED. We also explored the interaction between ADSCs and LI-ESWT from a statistical point of view. However, the exact mechanism of erectile function recovery due to dual therapeutics utilizing ADSCs and LI-ESWT still remains unclear.
As we all know, erectile function was closely related to endothelium, smooth muscle and nerves of CC [24], so we selected three relevant markers (  contribute to the recovery of the cavernous nerve or the growth of endothelial cells [25,26].
While the precise molecular mechanism underlying the increased efficacy of the dual treatment is not entirely clear, our results clearly demonstrate that a combination therapy of injecting ADSCs and applying LI-ESWT was more effective than separate treatments at enhancing erectile function.
Although our study demonstrated improved erectile function with a combination therapy of ADSCs and LI-ESWT, investigations should be carried out in more ED animal models and to better evaluate the therapeutic effects and safety for potential clinical application. Our study Page 15/25 used 4 weeks after initial ADSCs and LI-ESWT as the end time point, so it is yet unknown if the reported effects last as long as 6 months or 1 year. Moreover, a limitation of our work is that we use 5-Bromo-2'-deoxyuridine to mark ADSCs, but no labeled cells were detected. The most probable cause is that ADSCs will migrate and play a role in other tissues [27], another cause is that the longevity and specificity of 5-Bromo-2'-deoxyuridine decreases substantially over time [28]. Therefore, the transformation and outcome of the transplanted ADSCs need to be further investigate, more efficient and reliable stem cell labeling techniques are also needed.

Conclusion
We found that ADSCs and LI-ESWT can improve the erectile function of rats, the combination therapy present better effect on ameliorates ED and pathological changes than

Availability of data and material
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests
The authors declare that they have no competing interests.