Underactive bladder (UAB) is characterized by a slow urinary stream, hesitancy, and straining to void, with or without a feeling of incomplete bladder emptying, sometimes with storage symptoms. The International Continence Society (ICS) defines DU as “a diagnosis based on urodynamic investigations, generally (but not always) with relevant symptoms and signs, manifest by low detrusor pressure or short detrusor contraction in combination with a low urine flow rate resulting in prolonged bladder emptying and/or a failure to achieve complete bladder emptying within a normal time span (a high post-void residual may be present)” (1).
There is currently no proven treatment for treating UAB. Sericin is a natural hydrophobic polymer produced by silkworms. It has several biological properties including antioxidant, anti-tumor, anti-inflammatory, antibacterial, moisturizing, wound healing, and protective effect on neurons (2). Several studies have reported that sericin prevents lipid peroxidation and eliminates ROS, and enhances the enzymatic antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase, and glutathione (GSH) in the brain and peripheral tissues (3). In addition, several experimental studies have shown that sericin protects dopaminergic and cholinergic neurons primarily through its antioxidant effects. Sericin has also been shown to have an anticholinesterase effect. The enzyme acetylcholinesterase (AChE) is involved in the hydrolysis of the neurotransmitter acetylcholine (ACh) and helps maintain the integrity and permeability of synaptic membranes during neurotransmission. Excessive activity of this enzyme has toxic effects and can lead to the death of neurons. We aimed to investigate the effect of sericin on the expression of RhoA, ROCK-α, ROCK-β, and M2, M3 muscarinic receptor expression, and to measure apoptotic markers in the bladder tissue of UAB following lumbar canal stenosis (LCS) model.

In the present study, a total of 24 female Wistar rats (12-13 weeks and a weight of 220-270 g), were enrolled. Animals were housed in the Neuroscience Research Center's animal shelter at 25 ° C with a 12-hour light / dark cycle and unrestricted access to food and drinking water. 
Rats were randomly divided into 6 groups (6 in each) as follows; 
S200	sericin 200 mg/kg /daily/14 days/P.O + underactive bladder model
S250	sericin 250 mg/kg /daily/14 days/P.O + underactive bladder model
S300	sericin 300 mg/kg /daily/14 days/P.O + underactive bladder model
Model	NS 2 ml/kg/daily/14 days/P.O + underactive bladder model
Sham	NS 2 ml/kg/daily/14 days/P.O + sham surgery
Control	NS 2 ml/kg/daily/14 days/P.O
Due to the solubility of sericin in normal saline, the solutions were prepared immediately before administration.
Model Preparation
To induce UAB model, we used LCS model which previously was established by Sekido et al. Animals were anesthetized with isoflurane. For this purpose, animals were placed in a closed box, gassed with isoflurane 5% in pure oxygen (1 L/min). After the induction of anesthesia, the drug levels were maintained at the appropriate level by a small mask with 0.8–1 L/min oxygen and 1–1.5% isoflurane. Under anesthesia, the L5-L6 vertebral arches were exposed, and a small hole (1.5-2 mm in diameter) were drilled at the fifth vertebral arch, and a rectangular piece (3.5 mm * 5.0 mm * 0.5 mm) of silicone rubber were inserted into the epidural space. The layers of skin, subcutaneous, and muscle were closed using sutures. The urinary bladder was compressed twice daily for emptying in the following days post-procedure, and an antibiotic (ciprofloxacin (1 mg/kg, i.p.) were administered. 
Western blot analysis
Western blot was used to quantitatively assess the expression of RhoA, ROCK-α, ROCK-β, and M2, M3 muscarinic receptor expression, and to measure apoptotic markers, including the levels of caspase 3, 9, Bcl2 and Bax proteins in the bladder tissue.

Western blot results showed that M2 receptor expression decreased significantly following LCS compared to control and sham groups (P <0.01). Expression increased in rats who were treated with sericin. However, the increment of expression in rats with LCS + sericin at doses of 250 and 300 mg was statistically significant compared to the LCS group receiving normal saline (P <0.01). M3 receptor expression decreased in rats with LCS compared to the rats in the control and sham groups (P <0.001). Rats receiving different doses of sericin had higher expression of M3 receptor in the bladder compared to rats in the LCS group receiving normal saline, which was statistically significant (P <0.01).
Our results showed that the expression of Rho A, ROCK-β, and ROCK-α in control and sham groups was higher compared to model groups (P <0.05). Following LCS, they were reduced, and treatment with different doses of sericin increased their expression of them. The results of Tukey post hoc tests showed that the expression of Rho A in the bladder of the groups receiving doses of 250 mg (P <0.05) and 300 mg (P <0.01) compared with the model group of LCS receiving normal saline was significantly higher.
Also, the expression of ROCK-β (ROCK-1) was significantly higher only in the group receiving 300 mg sericin compared to the LCS group receiving normal saline (P <0.05) (Figure 1).
The results of Tukey post hoc tests showed that the expression of ROCK-α (ROCK-2) in the bladder of groups receiving different doses of sericin was not statistically significant compared to the LCS group receiving normal saline.
The analysis of apoptotic factors results showed that the Bax index in LCS groups increased significantly compared to control and sham groups and treated with different doses of sericin reduced this amount in bladder tissue. However, compared to the LCS group receiving normal saline, only the sericin 300 group receiving it was statistically significant (P <0.05). Our results showed that the expression of Bcl-2 in the LCS groups was significantly reduced compared to the control and sham groups (P <0.001). Treatment with different doses of sericin increased the amount that was highest in the group receiving sericin 300 compared to the LCS group receiving normal saline (P <0.01). The ratio of cleaved caspase 3 / Pro-caspase 3, and also cleaved caspase 9 / Pro-caspase 9 increased significantly in the LCS group receiving normal saline compared to the control and sham groups (P <0.001). Treatment with different doses of sericin reduced this amount. However, this decrease in expression was statistically significant in LCS receiving doses of 250 and 300 (P <0.001) (Figure 2).

There is currently no proven treatment for treating UAB. our results revealed that various doses of sericin which has several biological properties may increase the expression of muscarinic receptors, and the expression of Rho A, and ROCK-β (ROCK-1) after decrement of them in LCS rats. In addition, our findings provide evidence for the protective effect of sericin therapy against apoptotic changes in bladder tissue of the underactive bladder model.

Our results revealed the promising role of sericin in the management of the underactive bladder model following the LCS model. It may increase the expression of muscarinic receptors expression after decrement of them in the bladder tissue of LCS rats as well as the expression of Rho A, and ROCK-β (ROCK-1). In addition, our findings provide evidence for the protective effect of sericin therapy against apoptotic changes in bladder tissue of the underactive bladder model.

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Continence 2S2 (2022) 100287
DOI: 10.1016/j.cont.2022.100287