The Therapeutic Effect of Ginsenoside Rb1 against Mechanical Trauma in a Rat Model of Postpartum Stress Urinary Incontinence

Aims . Te aim of this study was to confrm the repairing efect of ginsenoside Rb1 (GS-Rb1) on mechanical trauma to periurethral tissues caused by childbirth and to explore its potential preventive mechanisms for mechanical trauma-induced stress urinary incontinence. Methods . 48 healthy adult female Sprague–Dawley (SD) rats were randomly divided into four groups: normal control, SUI groups, L-GS-Rb1 groups, and H-GS-Rb1 groups, with 12 rats in each group. Te histopathological examinations of the urethral were performed to detect the morphological changes after repair of periurethral traumatic tissue. Te TGF-β 1/Smad and NRF2/ARE signaling pathways related to periurethral tissue trauma repair were determined by RT-PCR and western blot. Te bladder capacity and LPP were examined in rats. Results . GS-Rb1 signifcantly decreased the number of fragmented and disorganized elastic and muscle fbers in the urethra and anterior vaginal wall of SUI rats. GS-Rb1 also increased the collagen content and reduced damage to the structural integrity of the periurethral myofbers. Furthermore, GS-Rb1 promoted expressions of TGF-β 1, Smad2, Smad3, Smad7, p-Smad3, p-Smad2, and collagens I and III. It also increased the protein levels of Nrf2, GPX1, and MnSOD. Te bladder capacity and LPP of rats in the L-GS-Rb1 group were close to those of rats in the normal groups. Conclusions . Ginsenoside Rb1 promotes the repair of periurethral tissue trauma in the postpartum period and has a preventive efect on the occurrence of stress urinary incontinence.


Introduction
Stress urinary incontinence (SUI) is a highly prevalent condition, particularly among women.Globally, 200 million or more people sufer from urinary incontinence [1,2].Te etiology, like other pelvic foor disorders (PFDs), is related to pelvic foor weakening and/or tears, usually due to obstetric trauma.Te anatomical factors afecting the urethra include changes to the thickness of the urethral mucosa and muscular layer and a decrease in its elasticity that are related to the onset of SUI [3].Histological fndings in periurethral tissues of women with SUI consistently show abnormal collagen remodeling, loss of functional elastic fber networks, and muscle fber rupture [4].At present, treatment for SUI occurs after a diagnosis of SUI is made which results in a high long-term recurrence rate [5].Existing conservative treatments, as well as pelvic foor muscle exercise [6], show that intervention in the early postpartum stage is very benefcial for reducing SUI-related morbidity.However, early postpartum pharmacotherapy for pelvic foor tissue damage caused by mechanical trauma remains limited.
Continued research in this feld has confrmed that the Transforming Growth Factor-β1 (TGF-β1)/Smad pathway mediates ECM remodeling that is associated with SUI pathology.TGF-β1 regulates ECM structure by modulating fbronectin, collagen, and elastin due to the phosphorylation of Smad2 and Smad3 [7].SUI is also associated with oxidative damage resulting from mechanical trauma [8].Many studies have shown that activation of the antioxidant response elements Nuclear factor erythroid2-related factor 2 (Nrf2)/Antioxidant Response Elements (ARE) and the TGF-β1/Smad signaling pathway afect the treatment of SUI [9,10].
Based on these fndings, it is plausible that GS-Rb1 may mitigate oxidative damage and promote matrix and tissue repair associated with mechanical-trauma-induced SUI.Tus, we hypothesized that GS-Rb1 inhibits damage to the urethral sphincter, elastic fbers, and other tissues, and promotes tissue repair.Tis study was conducted to evaluate the efects of GS-Rb1 on the early treatment of mechanical trauma of postpartum periurethral tissue and the prevention of SUI, together with an exploration of the potential underlying mechanisms.

SUI Model and Experimental Design. Forty-eight
Sprague-Dawley (SD) rats (two-month-old females, weighing 220-250 g) were obtained from the animal experimental center of Shanxi Medical University.Te rats were housed in cages of four at 25 °C ± 2 °C and a 12 h light/ dark cycle, with free access to food and water.All animal procedures were performed per the Guide for the Care and Use of Laboratory Animals and approved by the Animal Experiment Ethics Committee of Shanxi Cancer Hospital (Approval Number 2021001, Shanxi, China).GS-Rb1 doses were obtained from the China Yunnan Teana Pharmaceutical Co. Ltd. (Yunnan, China).Based on the clinical dosage of GS-Rb1 prescribed to Chinese patients (60-180 mg/day), the equivalent dose for rats was calculated using the body surface area conversion method (coefcient 0.018), according to the following formula: Rat dosage � human dosage (mg/day) × 0.018/0.2kg � 5.4/16.2/mg/kg/day.Using this dose as the theoretical reference, the GS-Rb1 concentrations of 4.5 (low dose) and 18 (high dose) mg/day, dissolved in saline, were selected for the low-and high-dose treatment groups, respectively.Te rats were divided into four groups (n � 12 per group): control; SUI; SUI + H-GS-Rb1 (18 mg/kg); SUI + L-GS-Rb1 (4.5 mg/kg).Te animals were anesthetized by intraperitoneal administration of 3% pentobarbital sodium (30 mg/kg) into the cavum abdominis and placed on their backs.A balloon containing 3 mL of saline was inserted into the vagina with a transurethral 12F catheter and left in place for 3 hours.Dysfunctional voiding was apparent in all rats following the procedure.GS-Rb1 (4.5 mg/kg and 18 mg/kg in saline) was administered intragastrically to the SUI + GS-Rb1 group after 24 hours and daily thereafter for one week.In the control and SUI groups, the rats were administered the same volume of saline.After drug withdrawal for 24 hours, the bladder capacity and LPP of the rats were measured, and the rats were euthanized thereafter.Te urethral and anterior vaginal walls of the rats were collected for further analysis.Te urethral and anterior vaginal wall tissues of six rats randomly selected from each group were fxed in 10% bufered formalin (pH 7.4), while the remaining tissue samples were frozen in liquid nitrogen.

Assessment of Urodynamics.
Rats were anesthetized as above and intravesicular catheterization was performed via suprapubic cystostomy using a PE-50 polyethylene tube with a fared end as an anchor.Te bladder was suctioned until it was empty through an inserted epidural catheter, and methylene blue saline at 37 °C was injected at a rate of 10 mL/ h.Te end of the injection coincided with the appearance of the frst drop of saline at the urethral meatus.Te amount of saline injected corresponded to the bladder capacity and was injected using a PE-50 polyethylene tube connected to 50 ml syringe.Te syringe was raised slowly and when the frst drop of saline appeared at the urethral meatus, the height of the syringe was measured and was used to calculate the leak point pressure (LPP).Tis procedure was performed fve times on each rat, and the average LPP was determined.After euthanasia, the urethral and anterior vaginal walls of the rats were harvested.

Histological and Immunohistochemical Analyses.
Te harvested tissues were fxed with 10% formalin, parafnembedded, and sectioned.Masson's trichrome was applied to determine the integrity of the urethral muscle system.Picrosirius red stains for collagen detection and Hart's stain for elastin detection were applied using standard protocols.Immunohistochemistry was performed per protocol (BIOS Biology Co., Ltd., China).Image J software was used to analyze the collagen content, expressed as the collagen volume fraction and calculated by the formula: collagen volume fraction � (collagen-positive blue area/total tissue area) × 100%.

RT-PCR.
Total RNA was extracted using TRIzol (HaDa Biotech, Taiyuan, China) and reverse-transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit (HaDa Biotech).Quantitative PCR was performed on a Real-Time PCR platform (Applied HaDa Biotech) per the provided directions.GADPH was used for normalization, and relative expression was determined by the 2 −∆∆Ct method.Te primer sequences used for the Real-Time PCR are provided in Supplemental Table 1 (Supplementary Material: primer sequence of TGF-β1, Smad3, Smad7, and collagens I and III).

Efect of GS-Rb1 on the Bladder Capacity and LPP of
Rats.SUI was successfully induced in the rat models using the VD method.Te LPP was 35.4 ± 1.4 cm H 2 O in control rats, 30.7 ± 2.4 cm H 2 O in L-GS-Rb1 rats, 27.1 ± 2.8 cm H 2 O in H-GS-Rb1 rats, and 24.5 ± 3.5 cm H 2 O in SUI model rats (Figure 1(a)).Tus, the LPP was lower in SUI rats than in the control or GS-Rb1 rats.Te bladder capacities were abnormally increased in SUI rats (0.95 ± 0.19 ml) relative to the controls (0.53 ± 0.03 mL) and SUI + GS-Rb1 rats (0.56 ± 0.07 ml) but were reduced in the SUI + H-GS-Rb1 rats (0.41 ± 0.07 mL) (Figure 1(b)).

Pathological Efects on Periurethral Tissue in Rats.
Te urethral striated muscles of the control rats were observed to be compact and circumferential on Masson staining.However, the muscle bundles of SUI rats had specifcally changed and showed splitting of the muscle fbers (Figure 2(a)).In addition, the collagen content in tissues obtained from GS-Rb1-treated rats had increased compared with the SUI rats.Picrosirius red staining indicated thinning of the vaginal wall and reduced collagen content of the urethra and vaginal walls in the SUI group compared with the other groups (Figure 2(b)).Te percentage of collagen in the urethral and anterior vaginal walls of the SUI group was signifcantly lower than in other groups (Figure 2(c)).Elastic fbers were tightly connected to the muscle bundles of smooth muscle with organization in control rats that also lined up with the vaginal wall tissue, whereas, in SUI rats, the fbers appeared fragmented and disorganized (Figure 3).

Efect of GS-Rb1 on the Expression of Factors and Proteins
Associated with the TGF-β1/Smad3 Signaling Pathway.Te role of the TGF-β1/Smad3 pathway in the recovery from trauma induced by VD was investigated.Gene expression profles of ECM in the tissue surrounding the urethra tissues were analyzed.Compared with the SUI group, GS-Rb1 treatment resulted in a signifcant increase in the mRNA levels of TGF-β1, Smad3, Smad7, and collagens I and III (Figure 4(a)).Western Blot results further confrmed that the protein levels of the TGF-β1, Smad2, Smad3, Smad7, collagens I and III, and Smad2 and Smad3 phosphorylation were also increased in GS-Rb1-treated groups (Figures 4(b) and 4(c)).

Efects of GS-Rb1 on the Expression of Proteins Associated
with the Nrf2/ARE Axis.Signifcant increases in levels of Nrf2, GPX1, and Mnsod proteins were observed in GS-Rb1treated groups (Figure 5(a)).In the H-GS-Rb1 group, the levels of these proteins were higher than in the L-GS-Rb1 group (Figures 5(b)-5(d)).Te results indicated that GS-Rb1 treatment may be associated with the stimulation of Nrf2/ ARE signaling and inhibition of further tissue damage after mechanical trauma caused by VD.

Discussion
Vaginal delivery can cause traumatic injury to the pelvic foor tissues, potentially leading to stress urinary incontinence due to damage to the nerves, muscles, and connective tissues responsible for maintaining continence.Tere is evidence that injury to the connective tissue injury is involved in the development of SUI [17].In the present study, we established a mechanical-trauma-induced rat model of postpartum stress urinary incontinence using the VD [18].Te structural changes of urethral and periurethral tissues were investigated.SUI model rats showed visible disruption of the urethral muscle fbers, together with reduced connective tissue and collagen expression.After GS-Rb1 treatment, the urethral microstructure recovered signifcantly relative to rats with untreated SUI.GS-Rb1 mitigated muscle fber damage and increased collagen concentrations.Te urethral wall is rich in loose connective tissue, elastic fber, collagen, and other components, which render the urethral wall with great elasticity and fexibility.Under the action of external forces, the urethral wall can efectively close the urethra and ensure tightness of the urethral closure [19,20].Te histological assessment showed that both low and high doses of GS-Rb1 had excellent therapeutic efcacy on SUI rats.To further evaluate the efect of GS-Rb1 on SUI, bladder capacity and LPP were measured in all rats after 7 days.LPP improved in the GS-Rb1 treatment group.However, the bladder capacity of rats in the SUI group was higher than that of rats in other groups.We speculate that short-term trauma causes edema of urethral tissue, which further causes urinary retention and increases bladder capacity in SUI rats.
TGF-β1 is known to be involved in injury repair processes through its role in cell proliferation, diferentiation,     and survival [20].It plays a crucial role in regulating the ECM, where it phosphorylates Smad2 and Smad3 to stimulate the expression of ECM components such as collagen, fbronectin, and elastin [21].TGF-β1 has also been demonstrated to play a key role in the pathogenesis of SUI, resulting from mechanical trauma [9,21] and has been suggested as a potential target for SUI treatment [22,23].Terefore, we investigated the efects of GS-Rb1 on TGF-β1 and its associated proteins.It was found that the urethral tissue of SUI rats showed reduced levels of TGF-β1, Smad3, p-Smad3, and collagens I and III compared to both the control and GS-Rb1-treated rats.In view of these results, we conclude that TGF-β1/Smad3 signaling may play a critical role in GS-Rb1-mediated repair of tissue subjected to mechanical trauma.
Vaginal distension reduces blood fow to the urogenital organs responsible for continence, resulting in hypoxia and suggesting that ischemia and/or reperfusion injury may be responsible for the resulting damage [24].Nrf2 modulates cellular response to oxidative stress by promoting the transcription of antioxidant genes carrying AREs in their promoter regions [23].In the presence of oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it promotes the transcription of antioxidant genes, such as GPx1, MnSOD, CAT, and HO-1 [25].It has been suggested that mechanical trauma can induce oxidative damage to both the urethral sphincter and the vaginal wall, causing ischemic and hypoxic injury to the pelvic foor tissue and leading to breakages in muscle and elastic fbers and reducing the ECM, resulting in SUI or pelvic foor dysfunction (PFD) [26].Addressing oxidative damage may be critical for the prevention and treatment of SUI [9,10].Tang et al. [7] suggested that mechanical injury-induced ECM remodeling might be associated with the suppression of Nrf2/ARE signaling, leading to inhibition of the TGF-β1/Smad3 signaling pathway.Our study demonstrated that GS-Rb1 treatment reduced tissue injury in SUI rats with increased Nrf2/ARE activation.Te protein levels of Nrf2 were assessed by Western blotting, showing increased expression in SUI + GS-Rb1 rats compared with control rats.Meanwhile, the structure of the damaged tissue was found to be restored in the GS-Rb1-treated groups.Tese fndings suggest that GS-Rb1 may have a protective efect against tissue damage through its antioxidant actions and may promote tissue repair.
Tere are several limitations to this study.First, the adverse efects of GS-Rb1 were not evaluated.Second, pharmacokinetic analysis of ginsenoside Rb1 was not performed in the rats, and thus efective levels of the drug in the blood were not evaluated.In addition, as VD is usually temporary following childbirth, we only undertook a shortterm study, and there are no further data from diferent time points after mechanical trauma.We propose to investigate this topic in depth in our next study to obtain direct evidence of this hypothesis.

Conclusion
In conclusion, GS-Rb1 may mitigate oxidative damage and promote matrix and tissue repair associated with mechanical-trauma-induced SUI.Tus, we speculated that GS-Rb1 attenuates oxidative damage and promotes matrix and tissue repair associated with mechanical trauma-induced SUI.GS-Rb1 may prevent SUI through early treatment of mechanical trauma to postpartum periurethral tissue.

Figure 1 :
Figure 1: Comparison of LPP and bladder capacity in four groups of rats.SUI in rats was induced by VD, then treated with diferent concentrations of GS-Rb1 or saline for one week.Te LPP and bladder capacity in rats was measured.Data are presented as mean ± SD, n � 6. * P < 0.05, * * P < 0.01, * * * * P < 0.0001, ns: P > 0.05.

Figure 2 :Figure 3 :
Figure 2: GS-Rb1 recovered urethral sphincter muscle structure and collagen concentrations.Urethral sphincter muscle structure morphology in rats was detected by Masson's trichrome stain.(b, c).Te collagen content of urethral and anterior vaginal wall tissues in rats was demonstrated by Picrosirius red staining (b), and the percentage of collagen was analyzed (c).Data are presented as mean ± SD, n � 6.* * P < 0.01, * * * P < 0.001.