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. 2025 Mar 3;73(10):5970–5980. doi: 10.1021/acs.jafc.4c11577

Astragalus Polysaccharides Ameliorate Diabetic Bladder Dysfunction via Normalization of Neuromuscular Conduction

Shaochan Liang †, Siyuan Xu §,∥,†, ShengLian Ye †, Lang Liang †, Hongliang Li †, Jiaye Liu †, Yao Zhang †, Feng Zou †, Xiaodan Liang †, Bo Tan ‡,*, Hongying Cao †,*
PMCID: PMC11908445  PMID: 40032633

Abstract

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Neuromuscular conduction dysfunction-induced underactive bladder (UAB) is a major urological complication associated with diabetes mellitus (DM), and there remain deficiencies in reliable pharmacological treatment options. Astragalus polysaccharides (APS), as an edible active substance in Astragalus membranaceus, have a therapeutic effect on diabetes and its complications. We investigated the effects and mechanism of APS in high-fat-diet-induced (HFD) diabetic UAB mice. APS significantly reduced fasting plasma glucose, insulin, and HOMA-IR index. Furthermore, APS treatment significantly decreased maximum bladder capacity, residual volume, bladder compliance, contraction intervals, empty and full resting pressure, and increased voiding volume and voided efficiency. In addition, APS ameliorated the hyporesponsiveness of purinergic and cholinergic-mediated neuromuscular contraction of the detrusor and improved the dysregulation of inhibitory and excitatory neurotransmission by downregulating the levels of nNOS and VIP, and upregulating ChAT and SP in HFD mice. This study revealed that APS ameliorates diabetic UAB via the normalization of neuromuscular conduction.

Keywords: Astragalus polysaccharides, diabetic bladder dysfunction, neuromuscular conduction, T2DM, inhibitory neurotransmitters, excitatory neurotransmitters

Introduction

Diabetes mellitus (DM), a chronic metabolic disorder, affected approximately 536 million individuals in 2021.1 Type 2 diabetes mellitus (T2DM) accounts for more than 90% of patients with diabetes.2 From a urologic standpoint, diabetic bladder dysfunction (DBD) is one of the diabetic peripheral neuropathies; up to 50% of patients with chronic and poorly controlled DM will develop DBD, in which neuromuscular conduction disorders are the core pathogenesis.3,4

The pathophysiology of DBD involves multiple factors, including detrusor physiology, neuronal injury, and urothelial and urethral dysfunction.5 In general, the pathogenesis of DBD is closely related to abnormal neuromuscular conduction. Contraction and relaxation of the bladder are mediated by complex neural controls, mainly including cholinergic, adrenergic, and nonadrenergic noncholinergic (NANC: including peptidergic and nitrergic neuronal networks) nerve conduction pathways.6

Excitatory transmission of parasympathetic effects in the bladder is mediated by acetylcholine acting on muscarinic receptors to contract the detrusor muscle.7 Choline acetyltransferase (ChAT) is a transferase enzyme responsible for the catalytic biosynthesis of the neurotransmitter acetylcholine.7 Neuronal NO synthase (nNOS) is activated by Ca2+/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiological tone.8 Substance P (SP) produces a rapid contractile response in smooth muscle. Vasoactive intestinal peptide (VIP) inhibits the autonomous contraction of isolated animal bladders.9 In the bladder, the interconnection of excitatory neurotransmission and inhibitory neurotransmission maintains bladder contraction and relaxation. Thus, the neurotransmitters and their transmission pathways in the bladder play a unique role in DBD.

The neuromuscular spectrum of DBD associated with bladder dysfunction is complicated in clinics.10 In addition to overactive bladder (OAB), DBD still leads to flaccid and hypotonic cystopathy characteristics of underactive bladder (UAB) (i.e., poor contractility, increased postvoid residual volume) in female patients.10−12 Clinical research found UAB symptoms in female patients in the early stage of diabetes, which is commonly present in 12 to 45% of women aged over 70 years.11−14 Due to the serious adverse effects of parasympathomimetics and limited evidence regarding the effectiveness of prostaglandins and α-adrenergic blockers, behavioral interventions remain the main therapeutic strategy for UAB.15,16

As the main active ingredient of various antidiabetic herbal medicines, polysaccharides have attracted the attention of numerous researchers.17 Astragalus polysaccharides (APS) are a main active ingredient in Astragalus membranaceus, which is both medicinal and food homologous.18,19 Studies have documented the antidiabetic and insulin-sensitizing effects of APS; the mechanism may involve decreasing the elevated expression and activity of PTP1B in the skeletal muscles of T2DM rats.20,21 In addition, APS can alleviate T2DM by activating the sweet taste receptors pathway, promoting glucose transport, and lipogenesis.22 APS has a promising application in treating diabetes and its chronic complications; particularly in diabetic patients with urinary complications, where the efficacy of pharmacological interventions appears to be limited.

Therefore, we induced UAB diabetic mice with a high-fat diet and then investigated the effects of APS on bladder dysfunction in UAB diabetic mice by targeting the cholinergic, nitrergic, and peptidergic neurotransmission pathways.

Materials and Methods

Animal and Diets

A total of 50 specific pathogen-free female C57BL/6J mice were purchased from BesTest BioTech (Zhuhai, China). The rearing environment was 20–24 °C with 50–70% humidity and exposed to a 12/12 h light-dark cycle, with free access to food and water. Fifty mice were adapted for 7 days and then divided randomly into five groups (one cage per five mice) for pharmacodynamic studies of APS: NCD group (normal chow diet, n = 10), HFD group (high-fat diet, 60% calories from fat, n = 10), APS-L group (low-dose Astragalus polysaccharides: 500 mg/kg/d, n = 10), APS-H group (high-dose Astragalus polysaccharides: 1000 mg/kg/d, n = 10), and Tam group (tamsulosin: 0.26 mg/kg/d, n = 10). After feeding HFD for 20 weeks, mice received respective drugs (p.o.) for 3 weeks. HFD was purchased from MediScience (Yangzhou, China). APS with a purity >90% and solubility of 10 mg/mL in distilled water was procured from Solarbio LIFE SCIENCE (Beijing, China). All animal experimental protocols were conducted under the guidelines and supervision of the Institutional Animal Ethics Committee of Guangzhou University of Chinese Medicine (No. ZYD-2021-224).

Glucose Homeostasis and Insulin Sensitivity

Mice fasted for 12 h and underwent fasting glucose measurements. Blood samples were collected from the retinal vein plexus for plasma glucose and plasma insulin testing using commercial kits from Rsbio (Shanghai, China) and ELISA commercial kits from IMD (Hong Kong, China), respectively. Mice had been fasted overnight, and an oral glucose tolerance test was performed by administering glucose (2 g/kg, p.o.). After the mice had been fasted for 6 h, an insulin tolerance test was conducted by injecting insulin (1 U/kg, i.p.). The homeostatic model assessment of insulin resistance (HOMA-IR), [fasting blood glucose (mmol/L) × fasting serum insulin (mIU/L)/22.5], was used to evaluate insulin sensitivity.

Voided Stain on Paper (VSOP) Analysis and Urine Output Testing

Voiding frequency was measured by evaluating the spots revealed by ultraviolet (UV) light exposure of stained VSOP paper for 3 h. The collected paper was imaged in ultraviolet light to visualize the urine. Urine output was collected for 5 h by placing the mice in metabolic cages individually with available food and water.

Cystometry

Cystometry agrees with our laboratory’s previous report.23 After the bladder was emptied, the built-in software was set to zero, and the bladder was filled with sterile saline at a constant rate (1 mL/h). When urine appeared in the external urethra of the mice, the volume of urine excretion was recorded, and the microinjection pump was turned off after three repetitions of the experiment to eradicate any discrepancies.

Histological Test

The bladder was fixed in tissue fixative, sectioned, and embedded in paraffin. The bladder tissues were sectioned at 5 μm. After that, they were stained with H&E and Masson’s trichrome. Image analysis software assessed the parameters mentioned above (Image-Pro Plus 6.0).

Assessment of Bladder Smooth Muscle Contractility In Vitro

The tension test and formulation of Krebs are the same as the published article in our laboratory.23 In brief, full-thickness longitudinal detrusor strips (0.5 to 1 mm × 5 mm) were prepared and transferred to tissue baths containing 5 mL of Krebs. Each strip was equilibrated for 60 min at 0.5 g. The effects of different concentrations of APS on the contractile responses were measured by electrical-field stimulation (EFS; 32 Hz) to calculate the median effective concentration (EC50) of APS. The contractile responses were measured by α,β-methyleneadenosine 5′-triphosphate trisodium salt (α,β-meATP: 100 μM), EFS (1, 2, 4, 8, 16, 32, 64 Hz; 40 V; duration for 10 s), potassium chloride (KCl: 120 mM), and the dose–response curve to carbachol (CCh) was obtained by adding cumulative concentrations of the agonist (10–8 M to 10–5 M) to the tissue bath. In the end, the weight of each detrusor strip was measured to normalize the force data: [(peak value – base value) (g)]/weight (g).

Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA from the bladder was extracted using the Tissue RNA Purification Kit PLUS (EZBioscience, China), and the cDNA synthesis was obtained and determined from total RNA by using the Color Reverse Transcription Kit (EZBioscience, China). A qPCR assay was performed using an ABI Prism 7500 system (Applied Biosystems, USA) with 2× Color SYBR Green qPCR Master Mix ROX1 plus (EZBioscience, China). Primers were synthesized by Sangon Biotech (Shanghai, China) as follows: ChAT Forward 5′-ACTTCGTCGGAGGCTCTGCTAC-3′ and Reverse 5′-CTGGCTCTTCCTGAACTGCTCTTC-3′; nNOS Forward 5′-AATGGTGGAGGTGCTGGAGGAG-3′ and Reverse 5′-TGTATTCGGTTGAGCCAGGAGGAG-3′; VIP Forward 5′-TGGATGACAGGATGCCGTTTGAAG-3′ and Reverse 5′-TTCCGAGATGCTGCTGCTGATTC-3′; SP Forward 5′-TATTGGTCCGACTGGTCCGACAG-3′ and Reverse 5′-CCGTTCACTGCTCACTGACACAG-3′; GAPDH Forward 5′-CTACCTCATGAAGATCCTGACC-3′ and Reverse 5′-CACAGCTTCTCTTTGATGTCAC-3′.

Western Blot Analysis

Proteins from the bladder were extracted and quantified according to the manufacturer’s protocol (GenScript, Nanjing, China). 20 μg of protein from each bladder sample was fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were hybridized with primary antibodies (ChAT, ABclonal, Wuhan, China; nNOS, BD, New York, USA; GAPDH, VIP, and SP, Affinity, USA), and then incubated with respective secondary antibodies. Clarity Western ECL Substrate (BIO-RAD, California, USA) was used to visualize the protein bands and was captured by using an ePhoto (GenScript, Nanjing, China).

Immunofluorescence

Bladder tissue was collected and sectioned. Sections were incubated with rabbit anti-ChAT primary antibody (1:50; Merck, Canada); antineuronal NOS (nNOS) primary antibody (1:200; Cell Signaling Technology, Danvers, MA); anti-VIP primary antibody (1:100; Affinity, USA); and anti-SP primary antibody (1:50; Santa Cruz Biotechnology, USA). Fluorescence-labeled Alexa Fluor 555, Alexa Fluor 488 (Southern Biotechnology, Birmingham, AL), and Hoechst 33342 (4A Biotech, Beijing, China) were used. Fluorescence was detected as described previously.23 Three visual fields were randomly selected from each bladder tissue to calculate the fluorescence area.

Statistical Analysis

Data were expressed as mean values ± standard error of the mean. Statistical analyses were processed using SPSS 25.0 software. Results were obtained using a t-test or ANOVA. A probability of p < 0.05 was considered statistically significant.

Results

APS Attenuated T2DM Development in HFD-Fed Mice

After being fed a high-fat diet for 20 weeks, mice exhibited obesity; however, a notable reduction in body weight was observed following APS treatment, attributed to a decrease in caloric intake (Figure 1A–C). The HFD-fed mice that developed T2DM showed hyperglycemia, hyperinsulinemia, and a high HOMA-IR index, accompanied by impaired glucose tolerance (Figure 1D–H). In contrast, intragastric administration of APS for 3 weeks significantly ameliorated fasting plasma glucose, plasma insulin levels, and the HOMA-IR index (Figure 1D–H), suggesting that APS is efficacious in preventing the onset of T2DM in HFD-fed mice.

Figure 1.

Figure 1

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APS attenuated the T2DM development. (A) 0–20 weeks body weight, (B) body weight changes after APS treatment, (C) daily diet consumption, (D) fasting blood glucose, (E) plasma insulin, (F) homeostatic model assessment of insulin resistance (HOMA-IR), (G) oral glucose tolerance test (OGTT), (H) area under the curve (AUC) of OGTT. Data are expressed as means ± SEM; n = 6–10 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 (APS-L, APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high fat diet); APS-L/H (Astragalus polysaccharides low/high dose: 500/1000 mg/kg); Tam (tamsulosin: 0.26 mg/kg).

APS Ameliorate Abnormal Urine Voiding Patterns

To gain further insight into the effects of APS on HFD-fed mice, urine voiding patterns were evaluated. Mice were placed on filter paper for 3 h and imaged under UV illumination to observe urination frequency (Figure 2A,B). Urine volume was collected using metabolic cages for five h to assess micturition volume. Though the urination frequency did not change significantly, the voiding volume of HFD-fed mice was markedly reduced (Figure 2C,E). APS treatment restored the voiding volume of HFD-fed mice in a time-dependent manner (Figure 2F).

Figure 2.

Figure 2

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APS ameliorate abnormal urine voiding patterns. (A) Schematic of voided stain on paper (VSOP) analysis, (B) representative filter paper imaged with UV illumination in each group, (C) 0–20 weeks VSOP, (D) VSOP after APS treatment, (E) 0–20 weeks voiding volume, (F) voiding volume after APS treatment. Data are expressed as means ± SEM n = 8 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 (APS-L, APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high-fat diet); APS-L/H (Astragalus polysaccharides low/high dose: 500/1000 mg/kg); Tam (tamsulosin: 0.26 mg/kg).

Then, we used cystometry to monitor bladder pressure and contractile activity. Mice initiated bladder contractions at regular intervals with continual filling at 1 mL/h, which were observed as pressure peaks corresponding to micturition events. While observing bladder micturition function, the HFD mice displayed high maximum bladder capacity due to unchanged voiding volume and longer contraction intervals, resulting in bladder compliance damage (Figure 3B,F–H). Moreover, a large increase in the residual volume of HFD mice caused voided efficiency to markedly decline (Figure 3C,E). Meanwhile, the bladder pressure of HFD mice was irregular, with specific performance that increased the empty and full resting pressure (Figure 3J,K). However, APS can markedly decline maximum bladder capacity, residual volume, bladder compliance, contraction intervals, empty and full resting pressure, and increase voided efficiency of HFD-fed mice (Figure 3A–L). This suggests that APS ameliorated the irregular bladder contractile activity of HFD mice and improved the efficiency of micturition, thereby contributing to the bladder urination function.

Figure 3.

Figure 3

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Effect of APS on cystometry in HFD-fed mice. (A) Representative original cystometry recordings in each group, (B) maximum bladder capacity (MBC), (C) residual volume (RV), (D) effective bladder capacity (EBC), (E) voided efficiency (VE), VE = [(MBC – RV)/MBC] × 100%, (F) voiding volume during three repetitions of cystometry, (G) bladder compliance (BC), BC = (MBC/MVP) × 100%, (H) contraction intervals (CI), (I) maximum voiding pressure (MVP), (J) empty resting pressure (ERP), (K) full resting pressure (FRP),(L) FRP subtract FRP. Data are expressed as means ± SEM; n = 6 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 (APS-L, APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high-fat diet); APS-L/H (Astragalus polysaccharides low/high dose: 500/1000 mg/kg); Tam (tamsulosin: 0.26 mg/kg).

The maximum voiding pressure, which reflects initiated bladder contraction, remained steady; thus, urethral dysfunction can be ruled out. Clinically, an increased residual volume and maximum bladder capacity were observed in patients with DBD, attributed to hyporesponsiveness of the bladder detrusor.15 Besides, increased maximum bladder capacity indicated that HFD mice are insensitive to bladder filling; this may be due to abnormalities in bladder neuromuscular conduction. Typically, increased bladder compliance is observed in patients with acute urinary retention or severe detrusor atrophy, accompanied by a large residual volume. Taken together, this result suggests that HFD-fed mice exhibited symptoms analogous to those of clinical UAB diabetic patients, indicating that APS possesses a therapeutic effect on UAB diabetic voiding dysfunction.

APS Restore the Impaired Bladder Function

We further investigated the bladder function in HFD-fed mice, and different exogenous stimuli—α,β-meATP, CCh, EFS, and KCl—were used in this study to verify the effect of APS on neuromuscular conduction of isolated bladder detrusor strips.

No significant alterations were observed in spontaneous contractions (Figure 4A,F); however, the responsiveness of HFD-fed mice to EFS stimulation significantly declined (Figure 4B,G). The addition of different concentrations of the exogenous M-receptor agonist CCh elicited concentration-dependent contractions in isolated bladder detrusor strips, and HFD-fed mice exhibited significant hyporesponsiveness (Figure 4C,H). The contractile response to the exogenous purinergic receptor agonist α,β-meATP in bladder detrusor strips declined in HFD-fed mice (Figure 4D,I). Nevertheless, no significant differences were observed in response to KCl stimulation (Figure 4E,J). These findings suggest that impaired bladder function is predominantly attributed to neurogenic rather than myogenic abnormalities.

Figure 4.

Figure 4

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APS restore the impaired bladder function. (A) Representative tracing showing spontaneous contraction of bladder smooth muscle, (B) representative tracing showing contraction responses of bladder smooth muscle induced by EFS, (C) representative tracing showing contraction responses of bladder smooth muscle induced by CCh, (D) representative tracing showing contraction responses of bladder smooth muscle induced by α,β-meATP, (E) representative tracing showing contraction responses of bladder smooth muscle induced by KCl, (F) summary of amplitude, (G) summary of bladder smooth muscle contraction induced by electrical field stimulation (EFS, 1–64 Hz), (H) bladder smooth muscle contraction induced by carbachol (CCh) stimulation at 10–8 M to 10–5 M, (I) summary of bladder smooth muscle contraction induced by α,β-meATP (100 μM), (J) summary of bladder smooth muscle contraction induced by potassium chloride (KCl, 120 mM). Data are expressed as means ± SEM n = 6 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 (APS-L, APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high-fat diet); APS (Astragalus polysaccharides: 0.07 mg/mL); Tam (tamsulosin: 0.26 mg/kg).

Preincubation was performed with APS (EC50: 0.07 mg/mL, Supplementary Figure 1) for 10 min. The low contractile response to stimulation of HFD-fed mice bladder strips by EFS, CCh, and α,β-meATP in vitro could be increased (Figure 4G–I). We concluded that APS improved neurogenic abnormalities of HFD mice and finally restored the impaired bladder function, independent of glucose regulation.

Effect of APS on HFD-Fed Mice Bladder Microstructure

To assess the effects of APS on HFD-fed mice bladder microstructure, the bladder wall thickness and smooth muscle/collagen ratio were measured by H&E and Masson staining. The bladder weight of HFD mice remained unchanged (Figure 5B,C). The histomorphological study revealed no significant change in the smooth muscle/collagen ratio (Figure 5D), despite the fact that the bladder wall thickness in HFD-fed mice was significantly increased; APS treatment significantly mitigated this thickening (Figure 5E). In conclusion, although no obvious organic pathology was observed in HFD-fed mice, there was a slight thickening of the bladder wall, and APS ameliorated this condition.

Figure 5.

Figure 5

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Effect of APS on bladder microstructure. (A) Representative H&E and Masson staining of the bladder, (B) bladder weight, (C) bladder weight/body weight (mg/g), (D) ratio of bladder smooth muscle to collagen, (E) bladder wall thickness (μm). Data are expressed as means ± SEM; n = 3–6 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 (APS-L, APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high-fat diet); APS-L/H (Astragalus polysaccharides low/high dose: 500/1000 mg/kg); Tam (tamsulosin: 0.26 mg/kg).

APS Regulate Abnormal Inhibitory Neurotransmission in HFD-Fed Mice

To further investigate the effect of APS on bladder neuromuscular conduction mediated by NANC and cholinergic pathways, we examined the expression levels of nNOS, VIP, and SP in the NANC, and ChAT in the cholinergic.

The data revealed that the mRNA levels of nNOS and VIP, as well as the protein levels of nNOS, were strikingly elevated in HFD-fed mice (Figure 6A,B,E). Upon administration of APS, there was a notable reduction in the mRNA levels of nNOS and VIP, as well as the protein levels of nNOS (Figure 6A,B,E).

Figure 6.

Figure 6

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Effect of APS on gene and protein expression of inhibitory and excitatory neurotransmitters. (A) nNOS mRNA level, (B) VIP mRNA level, (C) ChAT mRNA level, (D) SP mRNA level, (E) nNOS protein expression, (F) VIP protein expression, (G) ChAT protein expression, (H) SP protein expression, (I) Western blot analysis of nNOS, VIP, ChAT, SP expression in bladder tissue. Data are expressed as means ± SEM; n = 6 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 (APS-L, APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high-fat diet); APS-L/H (Astragalus polysaccharides low/high dose: 500/1000 mg/kg); Tam (tamsulosin: 0.26 mg/kg).

The immunofluorescence staining showed that the expression area of VIP in HFD-fed mice was considerably higher than that in the NCD group and decreased after treatment with APS (Figure 7B,D). However, there is a tendency to raise the area of nNOS, but no statistical discrepancy (Figure 7A,C). These data suggested that APS was effective in regulating the abnormal inhibitory neurotransmission in HFD-fed mice.

Figure 7.

Figure 7

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APS regulate abnormal inhibitory neurotransmission in HFD-fed mice. (A) Distribution of nNOS (green) in bladder tissues, (B) distribution of VIP (green) in bladder tissues, (C) nNOS’s expression area statistics (%) of bladder tissues, (D) VIP’s expression area statistics(%) of bladder tissues. Data are expressed as means ± SEM; n = 6 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 (APS-L, APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high fat diet); APS-L/H (Astragalus polysaccharides low/high dose: 500/1000 mg/kg); Tam (tamsulosin: 0.26 mg/kg).

APS Regulate Abnormal Excitatory Neurotransmission in HFD-Fed Mice

Our subsequent studies demonstrated that the mRNA and protein levels of ChAT and SP, as well as the protein levels of ChAT, pronouncedly decreased in HFD-fed mice (Figure 6C,D,G). After APS administration, the mRNA levels of SP were markedly increased (Figure 6D). Meanwhile, the expression area of ChAT and SP in HFD-fed mice plummeted, and SP expression significantly increased after treatment with APS (Figure 8A–D), which suggested that APS was effective in regulating the abnormal excitatory neurotransmission in HFD-fed mice.

Figure 8.

Figure 8

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APS-regulate abnormal excitatory neurotransmission in HFD-fed mice. (A) Distribution of ChAT (red) in bladder tissues, (B) distribution of SP (red) in bladder tissues, (C) ChAT’s expression area statistics (%) of bladder tissues, (D) SP’s expression area statistics(%) of bladder tissues. Data are expressed as means ± SEM; n = 6 per group. *p < 0.05, **p < 0.01 (HFD vs NCD), #p < 0.05, ##p < 0.01 APS-L (APS-H and Tam vs HFD). NCD (normal chow diet); HFD (high fat diet); APS-L/H (Astragalus polysaccharides low/high dose: 500/1000 mg/kg); Tam (tamsulosin: 0.26 mg/kg).

Taken together, these findings indicate that the normalization of bladder dysfunction in UAB diabetic mice by APS is associated with the downregulation of inhibitory neurotransmitters and the upregulation of excitatory neurotransmitters.

Discussion

DBD is a major urological complication of DM and affects more than half of all diabetic patients.24 Neuromuscular conduction disorders are the core pathogenesis of DBD. Clinical medication is primarily aimed at the treatment of OAB, but some patients exhibit UAB phenotypes; treatment for UAB mainly focuses on behavioral therapy, and further exploration is needed for pharmacotherapy.

Control of glucose levels and energy metabolism is a prerequisite for avoiding diabetic complications. Astragalus membranaceus, a medicinal plant, has the homology of medicine and food. APS is one of the most essential natural active components of it and has a variety of pharmacological activities. APS can regulate blood glucose in conditions such as type I diabetes mellitus, type II diabetes mellitus, and diabetic complications (such as diabetic nephropathy, diabetic cardiomyopathy, and diabetic peripheral neuropathy).17,25 The application of natural polysaccharides in treating dabetes not only alleviates hyperglycemia and regulates insulin resistance but is also effective in preventing complications.25,26

The present study found that HFD mice developed T2DM and insulin resistance accompanied by UAB characteristics of decreased voiding volume, higher maximum bladder capacity and residual volume, hyporesponsiveness of isolated bladder strips, and abnormal urination patterns, which closely resembled those in clinical patients with DBD. We found that APS treatment could attenuate T2DM development in HFD-fed mice. APS ameliorates diabetic bladder dysfunction by regulating cholinergic and NANC-mediated neuromuscular contraction of the bladder in HFD mice.

Based on cystometry, we verified a significant increase in residual volume, damaged compliance of the bladder, and inability to empty urine efficiently in HFD mice, which are characteristics of the underactive bladder. In homeostasis, low pressures are sustained as bladder filling;27 however, higher empty and full resting pressures were observed in HFD mice, suggesting the urination process and bladder compliance are damaged. The effective bladder capacity represents the ability to store urine during the storage phase; low voided efficiency indicated that it is urinary dysfunction rather than storage dysfunction in HFD mice. The maximum voiding pressure is unchanged, meaning that initiated bladder contraction was constant. Combined with the fact that maximum bladder capacity was enlarged, we deduced it is neurogenic instead of myogenic generating urinary dysfunction. APS treatment significantly ameliorates HFD mice’s abnormal micturition pattern, increases urination efficiency, improves impaired bladder function, and reduces bladder wall thickening. Therefore, APS, which is an extract from the edible medicinal plant Astragalus membranaceus, can provide a safe therapeutic idea for DBD patients who exhibit the UAB phenotype.

The bladder function of micturition is closely related to the regulation of the neuromuscular conduction of the detrusor. Contraction and relaxation of the detrusor are mediated by complex neural controls, mainly including cholinergic, adrenergic, and NANC nerve conduction pathways. Here, we investigated the direct effect of APS on neuron-mediated detrusor contraction through an in vitro tension study. EFS stimulation activates isolated bladder strip motor neurons to release endogenous neurotransmitters, thereby regulating bladder smooth muscle contraction and relaxation. However, isolated bladder detrusor strips of HFD mice showed significant hyporesponsiveness to contractions induced by EFS, but there is no significant difference in contractile responses stimulated by high concentrations of potassium ions, suggesting that HFD mice have abnormal nerve conduction. Meanwhile, isolated bladder detrusor strips of HFD mice showed hyporesponsiveness to contractions induced by CCh and α,β-meATP stimulation, suggesting cholinergic and NANC nerve conduction disorders, respectively. Preincubation with APS can enhance the contractile response from exogenous stimulation of CCh, α,β-meATP, and EFS, which indicates that APS ameliorates underactive bladder by regulating the bladder cholinergic and NANC nerve inactivation of HFD mice. To further investigate the mechanism of APS treatment for DBD, we next study the alteration of bladder neuromuscular conduction mediated by NANC and cholinergic.

DBD is a multifactorial peripheral neuropathy. Dysregulated release of neurotransmitters is the direct reason for bladder neuromuscular conduction failure. The cholinergic neurons of the bladder belong to the excitatory motor neurons (EMNs), and acetylcholine is the primary excitatory neurotransmitter that stimulates the bladder’s smooth muscle contractions.28,29 ChAT is the critical transferase for acetylcholine transport. It has been demonstrated that ChAT inhibitors are effective in voiding function in Zucker diabetic fatty rats.30 We found that both gene and protein expressions of ChAT were reduced in the bladder of DBD mice. The cholinergic neurotransmitter transport was disturbed and led to a detrusor contraction failure during micturition. Apart from cholinergic neurons, SP is the first member of the tachykinin family of peptides, which produces a rapid contractile response in bladder smooth muscle.9 Likewise, the level of gene expression of SP in the bladder was also substantially decreased in the HFD-induced DBD mice. In summary, excitatory neurotransmitter-mediated detrusor contractions were disturbed in HFD-induced DBD mice. Nevertheless, after APS treatment, the gene expression of SP was markedly upregulated.

In addition, NO and VIP act as crucial parts of NANC. The nitrergic neurons of the bladder belong to inhibitory motor neurons (IMNs). NO is a major inhibitory neurotransmitter that mediates NANC signaling, which leads to smooth muscle relaxation and regulates physiological tone.8 VIP is an immunomodulatory neuropeptide widely distributed in neural pathways that regulate micturition, inhibiting the autonomous contraction of isolated animal bladders as well as the human bladder.31 However, the expression of nNOS and VIP both were enhanced in the bladder of mice with HFD feeding, which indicated the inhibitory neurotransmitter was increased to antagonize the excitatory neurotransmitter-mediated detrusor contraction in the current study. Post-APS treatment, the expression of nNOS and VIP waned to normal levels. Taken together, we deduced from the findings that APS alleviates detrusor underactivity and chronic urinary retention by regulating the excitatory and inhibitory neurotransmitters.

In conclusion, the EMNs and IMNs that regulated the balance of detrusor contraction were disturbed in DBD mice, leading to an underactive bladder. These dysfunctions resulted in poor bladder contractility, increased bladder volume, postvoid residual volume, and damaged bladder compliance. APS modulation of neuromuscular conduction by regulating excitatory and inhibitory neurotransmission ultimately ameliorates abnormalities in diabetic bladder dysfunction.

APS from Astragalus membranaceus, a natural plant polysaccharide, exhibits potential benefits for DM and its complications, as well as the potential for application in functional foods.32 However, clinical pharmacological treatment of DBD does not target its primary cause, as the pathophysiology of DBD has not yet been completely understood.24 There is a lack of a clear molecular mechanism research basis for the study of the pharmacological mechanism in treating DBD. Currently, research on the effects of APS on bladder function primarily focuses on the urothelium.33,34 Based on our cystometry results, APS decreased the maximum bladder volume in HFD-fed mice. This suggests that a potential mechanism by which APS may treat DBD involves its action on the urothelium, potentially modulating the release of factors that influence afferent nerve activity and signaling in the bladder. In general, a more pronounced effect was observed in the APS low-dose group. This phenomenon may be attributed to high doses of APS beyond its therapeutic window. However, the dose-effect relationship of APS remains unclear. It is noteworthy that APS has the effect of immunomodulation. For patients with autoimmune diseases, APS should be used under the supervision of a physician, particularly when immunosuppressive drugs are being administered. Additionally, it is essential to delve into the potential molecular targets, exact mechanism, pharmacokinetics, and pharmacodynamics of APS on DM and its complications through rigorous clinical trials.35

Acknowledgments

This work was funded by the National Natural Science Foundation of China (Grant Nos. 82074107 and 82304842); the Department of Education of Guangdong Province (Grant Nos. 2024KTSCX113 and 2021ZDZX2018); the Basic and Applied Basic Research Foundation of Guangdong Province (Grant No. 2022B1515120073); and the Natural Science Foundation of Guangdong Province (Grant No. 2022A1515220133).

Glossary

Abbreviations

APS

Astragalus polysaccharides

BC

bladder compliance

CCh

carbachol; ChAT, choline acetyltransferase

CI

contraction intervals

DBD

diabetic bladder dysfunction

DM

diabetes mellitus; EBC, effective bladder capacity

EFS

electrical-field stimulation; EMNs, excitatory motor neurons

ERP

empty resting pressure;

FRP

full resting pressure

HFD,

high-fat diet; HOMA-IR, homeostatic model assessment of insulin resistance

IMNs

inhibitory motor neurons

KCl

potassium chloride

MBC

maximum bladder capacity; MVP, maximum voiding pressure

NANC

nonadrenergic noncholinergic

nNOS

neuronal NO synthase

OAB

overactive bladder

OGTT,

oral glucose tolerance test

RV

residual volume

SP

substance P

T2DM

type 2 diabetes mellitus

UAB

underactive bladder

VIP,

vasoactive intestinal peptide

VSOP

voided stain on paper

VE

voided efficiency

α,β-meATP

α,β-methyleneadenosine 5′-triphosphate trisodium salt

Data Availability Statement

The raw data supporting the conclusions of this article will be available from the corresponding author upon reasonable request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c11577.

  • The median effect concentration (EC50) of APS (PDF)

Author Contributions

⊥ S.L. and S.X. contributed equally S.L., S.X., and H.C. designed the research. S.L. and S.X. performed experiments. S.L., S.X., S.Y., L.L., H.L., J.L., Y.Z., F.Z., X.L., and B.T. analyzed data. S.L. wrote the manuscript. S.L., S.X., and H.C. revised the manuscript. All authors read and approved the final study.

The authors declare no competing financial interest.

Supplementary Material

jf4c11577_si_001.pdf (105.5KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jf4c11577_si_001.pdf (105.5KB, pdf)

Data Availability Statement

The raw data supporting the conclusions of this article will be available from the corresponding author upon reasonable request.

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