| Background and purposeBPH (Benign Prostatic Hyperplasia, BPH) is a middle-aged man of common disease. The prevalence of 50-year-old male is 40%, and the prevalence increases with age, the age of 80 to over 80%. With the improvement of living standards of our people and the average life expectancy increases, Chinese men will continue to increase in patients with BPH.Lower urinary tract symptoms (LUTS) in BPH patients are clinically the most important reason for treatment. LUTS include storage symptoms (frequency, urgency, nocturnal, urge incontinence), voiding symptoms and postmicturition symptoms. The storage symptoms of urinary frequency, urgency, urge incontinence and nocturia increased, also known as OAB symptoms. The OAB symptoms have high incidence and serious impact on the quality of life of patients. According to the International Continence Society (ICS) definition of overactive bladder (OAB), it is defined as urgency, with or without urge incontinence, usually with frequency and nocturia, and urinary tract not confirmed infection, metabolic abnormalities or other causes. In Europe, the prevalence of OAB is more than asthma, angina and diabetes and other common chronic diseases. Moreover, OAB have serious negative effects on the quality of life of its people. According to statistics, there is about 1/5 of people with OAB in the United Kingdom. A group of European data shows that male OAB prevalence was 10.8%,12.8% for women. the prevalence of OAB in U.S in men is about 16%, women 16.9%. The prevalence of OAB of adult women over 18 years old in Beijing of China was 4.7%. At present, there is no national epidemiological data on OAB, but according to different regions of China, the incidence of OAB is estimated about 1 million people in China, troubled by the OAB. In LUTS, OAB symptoms have the greatest impact on the daily life of patients. Nearly 50% of the patients reported that OAB symptoms affected their daily lives. Urge incontinence were more likely to yield fracture due to falls, and patients were forced to spend less time in social activities, becoming more withdrawn and more depression. Nocturnal symptoms disturb sleep and affect the physical and mental health of patients. Overall, OAB symptoms seriously affect the quality of life of patients, and it is considered the most troublesome LUTS symptoms.The majority of BPH patients have OAB symptoms. Bates et al reported that 50% to 75% of aged male BPH patients had storage OAB symptoms. BPH is a histological definition. Traditional theory considered the main reason for OAB symptoms as bladder outlet obstruction (BOO) caused by BPH, while ignoring the bladder itself. However, in some BPH patients without BOO or suspected BOO there are varying degrees of OAB, and in part of BPH patients even if prostate BOO had been removed, the improvement of OAB symptoms after surgery is still not obvious. The study of Ameda et al had shown that about 50% to 75% of BPH patients with BOO that was confirmed by urodynamic results had OAB symptoms, while about 46% to 66% of BPH patients with BOO that was confirmed by urodynamic results had DO. After the surgery of removing the obstruction, DO in BPH patients with OAB and BOO would be better. Kageyama et al reported in BPH patients with BOO and DO, after transurethral resection of the prostate to remove the obstruction, DO of 75% of patients disappeared. Many other scholars showed that patients with BPH underwent surgery to remove the obstruction, the patient's voiding symptoms improved, but still about 1/3 of patients with OAB symptoms reservoir can not be improved.The mechanism of storage OAB symptoms of BPH has not yet clarified, is considered relevant detrusor overactivity (DO). According to the International Continence Society (ICS) new definition, DO is defined as'an urodynamic observation characterized by involuntary detrusor contractions during the filling phase which may be spontaneous or provoked'. OAB symptoms (urinary frequency, urgency, nocturia, urge incontinence) of BPH-induced and DO have established contact, although there are differences. From the definition of ICS, we can see that there is a wide discrepancy between the clinical occurrence of OAB and DO. OAB and DO are not directly link since OAB is a symptom related to patients clinical experience during every day life while DO is a pure functional diagnosis in an artificial environment during pressure flow study (PFS). Nevertheless, DO is thought to be the driving force behind OAB symptoms. There are a number of DO and OAB symptoms studies confirmed the significant association between them, such as Coolsaet et al reported detrusor contraction speed of DO was relevant to the opportunity and degree of urgency. Shahab et al reported that DO of BPH patients was associated with the severity of nocturia, and that the magnitude and duration of DO impacted the severity of OAB symptoms. Wadie et al reported that DO affected BPH symptom scores and quality of life score. Hyman et al reported DO was significantly related to urge incontinence. Hashim et al reported that the majority of OAB patients had DO confirmed by urodynamic, while the vast majority of patients with DO showed OAB symptoms. Some other studies reported OAB symptoms had a relationship of 90% of the DO of patients.M receptor antagonist is recognized effective drug for treatment of OAB symptoms. As for the mechanism of M receptor antagonist for the OAB symptoms, the traditional theory presumed that M receptor antagonist blocked postsynaptic (efferent) M receptor on detrusor, thereby inhibiting the parasympathetic nerve endings release the neurotransmitter acetylcholine (Ach)-induced detrusor contraction. However, OAB symptoms occurred on urinary storage and the theory is anticholinergics block the Ach release of the parasympathetic nervous endings, which induced detrusor contraction of voiding phase. But also found that clinical treatment doses of M receptor antagonists improve OAB symptoms patients with urinary urgency and increased bladder capacity, but no evidence that they cause difficulty urinating or urinary retention. Other animal and human studies indicate that intravesical instillation of M receptor antagonist significantly improved storage DO/OAB symptoms. This means, the mechanism of M receptor antagonist for treatment OAB symptoms may be related to inhibition of bladder sensory pathways (afferent nerve), that is to say is related to block M receptors on bladder mucosa.In the past, the bladder mucosa is considered barrier function only, however, there is growing evidence that it also actively involved in bladder sensory function. Recent evidence also shows that M receptor mechanism is related to bladder mucosal sensory function. Bladder mucosa has high-density M receptor, and the stretch of the bladder mucosa would release Ach. Stimulation M receptors in the bladder mucosa can release some substances, such as Ach and ATP, and these substances can regulate the afferent nerve activity. Yoshida et al reported that human bladder tissue in vitro has Ach release, this release of non-neurogenic Ach, at least in part from mucous membranes. Kim et al reported that intravesical instillation of M receptor agonists can produce DO, and DO can also be inhibited by intravesical M-receptor antagonist. They concluded that the reasons of that M receptor antagonists can effectively treat OAB symptoms are not only the inhibition of M receptor-mediated detrusor contraction, but also the blockage of the M receptor on bladder afferent pathway, and they speculated that M receptor on bladder mucosa may play an important role in DO/OAB symptoms. Therefore, the M receptor of bladder mucosa and DO/OAB symptoms closely related, and bladder mucosa is also considered as one target of M receptor antagonist for treatment DO/OAB symptoms.M receptor has five subtypes (M1-M5), and human bladder mucosa express of all M-receptor subtype mRNA. Their expression intensities were:M2R>M3R=M5 >M4R=M1R. MIR was found mainly in the basal cells by immunofluorescence. M2R almost located in Umbrella cells. M3R and M4R were uniformly distributed. M5R gradually reduced from the surface of epithelial to the base.There are still few reports about the M receptor expression in bladder mucosa of human pathological state and the results of the reports are in contradictions. Mukerji et al studied 12 patients with idiopathic detrusor overactivity (IDO) with PUF questionnaire. After bladder biopsies, specimens were stained with M2 and M3 antibody. They found that M2 and M3 receptors were expressed in the epithelium, nerve fibers and detrusor layer. Compared with control group, M2 and M3 receptors on fibroblasts muscle-like cells under urothelium of IDO patients expressed increasingly and were related to urgency and frequency symptom, while M2 and M3 receptors on epithelium and detrusor were unchanged.Mansfield et al observed-the M2 and M3 receptors expression conditions of 20 IDO women patients, and found that M2 receptor expression in bladder mucosa was no change, while M3 receptor expression was decreased. Datta et al found Ml and M3 receptors in submucosal sites downregulationed by immunohistochemistry in patients with DO, and Ml and M3 receptors were negatively correlated to urgency and frequency, and Ml and M3 receptors on bladder epithelial was decreased. There is a clear conflict result of the above M receptor in bladder mucosa, and there is a common characteristic:patients are derived from the DO, while the detection of M receptor subtypes in the bladder mucosa of patients with BPH and OAB symptoms were not reported.In summary, the OAB symptoms of storage phase of BPH have high incidence, seriously affecting the quality of life of patients, but its mechanism is not yet clear, and the current treatment is still not very good. M-receptors in the bladder mucosa may play an important role in the pathogenesis of DO/OAB symptoms. In this thesis, our study detect M receptor subtypes expression and distribution in bladder mucosa of BPH patients with or without DO, and evaluate the relationship between those and the degree of obstruction, clinical symptoms and quality of life, which will help to further explore the pathogenesis of DO/OAB symptoms of storage phase and the mechanism of M receptor antagonist for treatment DO/OAB symptoms, to guide and help clinical treatment of BPH patients with OAB symptoms.Materials and methodsBladder mucosa samples were from the First People's Municipal Hospital, Guangzhou. Collection time was from the June 2010 to the February 2011. Patients were enrolled and screened according to the following selection and exclusion criteria.Inclusion criteria of BPH group:①male patients, aged≥50years;②patients having lower urinary tract symptoms (frequency, urgency, urge incontinence, nocturia);③prostate volume increase (≥20ml) measured by transrectal ultrasound;④free max urinary flow rate<15ml/s;⑤signed the informed consent.Exclusion criteria of BPH group:①patients with urinary tract infection;② patients with bladder stones;③patients with bladder cancer;④Patients had been diagnosed or highly suspected with prostate cancer;⑤patients with diabetes;⑥patients with neurogenic lesions;⑦patients are taking drugs that affect bladder function in 2 months;⑧refused to participate in the study.Inclusion criteria of control group:①male patients, aged≥50 years;②patients with hematuria, bladder cancer, or upper urinary tract tumors need to do cystoscopy or surgery, and without lower urinary tract symptoms (frequency, urgency, urge incontinence, nocturia);③free max urinary flow rate> 15ml/s;④signed the informed consent.Exclusion criteria of control group:①patients with OAB symptoms or obstructive symptoms;②patients with urinary tract infection;③patients with diabetes;④patients with bladder stones;⑤patients with neurogenic lesions;⑥patients with benign prostatic hyperplasia;⑦patients with confirmed urodynamic bladder outlet obstruction;⑧refused to participate in this study.Collect the data of patient demographic, history, physical examination, urinalysis, renal function, international prostate symptom score (IPSS), IPSS questions 2 frequence score, IPSS questions 4 urgency score, IPSS question 7 nocturia score, QOL score, free urinary flow rate, residual urine, prostate volume measured by transrectal B-ultrasound and urodynamics (bladder pressure-volume measurement and pressure-flow study) examination.Patients were strictly selected in accordance with the above criteria, and 26 patients were enrolled.6 patients with bladder cancer, hematuria or ureteral calculi were as a control group.20 cases of BPH were divided into two-groups (BPH with DO group, BPH without DO group) according to whether there were urodynamic DO, and 9 cases in BPH with DO group,11 cases in BPH without DO group.All bladder mucosa specimens were obtained by biopsy through cystoscope or obtained during the operation of bladder open surgery, and those came from a site 2 cm lateral and cephalad from the left ureteric orifice. Once get the mucosa specimens, put into the tissue frozen pipes and immediately put in liquid nitrogen, remove the detrusor on ice under a microscope, then transfer to-80℃refrigerator and stored until use. Before real-time quantitative PCR analysis, all specimens were pathologically confirmed by HE staining diagnosis.M1-M5 receptor expression in the bladder mucosa was analyzed using Real-time quantitative PCR. (1) Preparation of total RNA. (2) Application of Trizol reagent. (3) Use the instrument (ABI 7900, ABI, USA) for the quantitative PCR instrument. (4) Identification of the purity and integrity of RNA. (5) Reverse transcriptase reaction (RT) to obtain cDNA fragments. (6) Quantitative PCR primer design. Before primers designed, login http://www.ncbi.nlm.nih.gov and search CHRM1, CHRM2, CHRM3, CHRM4, CHRM5 and GAPDH gene mRNA sequence in Genbank (accession number followed by:NM000738.2, NM000739.2, NM000740.2, NM000741.2, NM012125.3 and NM002046). On the basis of the primer design sequence, to obtain the sequence by primer design software'DNA Club'through fluorescence quantitative PCR CHRM1, CHRM2, CHRM3, CHRM4, CHRM5 and GAPDH gene primers. Use internal reference (glucose-6-phosp.hate dehydrogenase, GAPDH) gene primers, the detection of Ml, M2, M3, M4 and M5 receptor gene expression, while GAPDH gene in each sample were detected between different samples in order to correct for differences in target gene expression because of the differences of total RNA, quality of RNA, and efficiency of reverse transcription reaction. All primers were synthesized by the Invitrogen Corporation. (7) Fluorescence quantitative PCR. (8) To draw quantitative PCR fluorescent labeling curve. (9) Using relative quantitative method for data processing. Statistical analysisThe data was analyzed using SPSS 16.0 software. Age of every group was tested by variance analysis whether there were differences. And according to the literature, the relative mRNA copy number of M1-M5 was shown with median and interquartile, which was the non-parametric test method. M receptor subtypes of BPH with DO group, BPH without DO group and control group was analyzed using the Kruskal-Wallis test. If P<0.05, Mann-Whitney U test was used for multiple comparisons, and data was corrected with Bonferroni. Correlation analysis was tested using Spearman rank correlation between M receptor subtypes expression and storage OAB symptoms (total IPSS scores, IPSS question 2 frequence score, question 4 urgency score and question 7 nocturia score).Results1.26 patients involved this study.6 patients in control group, age (61.5±12.9) years; 9 cases in BPH with DO group, age (73.2±7.7) years; 11 cases in BPH without DO group, age (69.7±10.5) years. There was no statistically significance of age between groups (F=2.412, P=0.112).2. The order of the median positions distribution of standardized M receptor mRNA copy number in the control group, BPH with DO group and BPH without DO group was M3> M5> M4> M1> M2, M4> M3> M5>M1>M2 and M4> M3> M5>M1>M2, respectively.3. The standardization of M1-M5 receptor mRNA copy number among control group, BPH with DO group and BPH without DO group was compared and there was no significant difference. Statistics were M1 (H=1.580, P=0.454), M2 (H= 2.216, P=0.330), M3 (H=0.890, P=0.641), M4 (H=0.449, P=0.799) and M5 (H= 0.022, P=0.989).4. Correlation analysis was tested using Spearman rank correlation between M receptor subtypes expression and storage OAB symptoms (total IPSS scores, IPSS question 2 frequence score, question 4 urgency score and question 7 nocturia score). M5 receptor in the bladder mucosa of BPH with DO group and the IPSS question 4 urgency correlated (rs=-0.688, P=0.040), and the rest data was not correlated. M4 receptor in the bladder mucosa of BPH without DO group and the IPSS question 7 nocturia correlated (rs=-0.608, P=0.047), and there was no correlation between the rest. Conclusion1. The bladder mucosa of BPH patients expresses M1-M5 receptor mRNA.2. The order (proportion) of standardization of M receptor subtypes mRNA copy number in the bladder mucosa of BPH with DO patients and BPH without DO patients is different to that of the control group. M receptor in bladder mucosa may play a role in the pathogenesis of OAB symptoms in patients with BPH.3. The standardization M1-M5 receptor mRNA copy number among control group, BPH with DO group and BPH without DO group was compared and there was no significant difference. DO may have no effect on the expression of M receptor subtypes of the bladder mucosa.4. M5 receptor in the bladder mucosa of BPH with DO group was correlated to the IPSS question 4 urgency. M4 receptor in the bladder mucosa of BPH without DO group was correlated to the IPSS question 7 nocturia.InnovationConventional wisdom holds muscarinic cholinergic receptor antagonist which is the first-line medicine for treatment OAB symptoms works by blocking the M receptor of detrusor efferent pathway, thereby reducing the detrusor contraction. However, therapeutic doses of M receptor antagonists on detrusor contraction had no significant effect, suggesting that M receptor antagonists may inhibit bladder sensory (afferent) to treat OAB symptoms, M receptor expression of bladder mucosa in BPH patients with OAB symptoms may play a key role. So far, M receptor subtypes of bladder mucosa in BPH patients and their clinical significance have not been reported.Features and innovations of this study is the first detection of M receptor subtype distribution and expression of bladder mucosa in BPH patients and to evaluate the relationship between M receptor subtype and clinical symptoms and quality of life, which will help to further explore the DO/OAB symptoms pathogenesis and mechanism of M receptor antagonist, to guide and help the clinical treatment of BPH patients with OAB symptoms.
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