| Background:Diabetic cystopathy (DCP) is a common chronic complication of diabetes, with the incidence of 25%-85%. The clinical manifestations consist of overactive bladder, difficulty urinating and so on, which may further lead to the damage of renal function and urinary tract infections. Urinary tract infections are a common cause of death in patients with diabetes. Due to the diabetic bladder dysfunction, the normal social activities and the basic health of the patients are damaged. What more, the quality of life and even life of the patients are both influenced by diabetic cystopathy. The pathogenesis of DCP is complicated, which mainly consist of diabetic bladder dysfunction and diabetic urethral dysfunction. The mechanisms include the injury of bladder detrusor, the changes of urethral sphincter, the neurological disorders, oxidative stress and so on.DCP is closely related to blood vessels changes and peripheral neuropathy. Nerve growth factor (NGF) is mainly in the sympathetic, which could maintain the normal function of the peripheral nerve. Previous studies suggested that NGF might play an important role in DCP. ProNGF is the precursor of NGF. Whether NGF/ProNGF pathway play a role in urethral dysfunction is still worthy of further study. The previous study indicated that urethral pressure of diabetic rats and bladder relaxation pressure threshold were significantly increased in diabetic rats. The baseline of urethral pressure was also increased, which could be decreased by usingal-adrenoceptor antagonists. However, the expression status of al receptor in diabetic urethra is not clear.DCP is not only related to the urethral dysfunction but also related to the bladder dysfunction. Recent studies suggested that diabetic bladder dysfunction is largely due to the changes of the receptor in detrusor. Cannabinoid receptors (CB receptors) is an important part of the endocannabinoid system, which can affect a variety of intracellular signal transduction pathways, including inhibition of adenylate cyclase, inhibition of calcium channel, activation of potassium channels and activation of MAP kinase corridors. It plays an important role in many physiological activities such as cardiovascular system, immune regulation, the pain signal transduction, neurotransmission and energy metabolism and so on. More and more evidence show that the cannabinoid receptors may exist in the bladder and play an important role in bladder dysfunction. However, the precise mechanism of CB receptor in the pathogenesis of DCP is still unclear.Grape Seed Proanthocyanidin Extracts (GSPE) are a natural polyphenolic compounds extracted from grape seeds. The present research on GSPE is very extensive, involving in the cardiovascular system, the field of diabetic nephropathy, diabetic retinopathy, rheumatoid arthritis, cancer, nervous system and so on. Some studies proved that GSPE played an important role in the protection of diabetic peripheral neuropathy. After the treatment of GSPE, the motor neuron conduction velocity in diabetic rats was significantly improved, accompanied by a reduction of advanced glycation end products, decrease of severe segmental off changes and improvement of Schwann cells. Whether GSPE have a protective effect in DCP is worthy to investigate.Part I The study of the influence of the changes in alphal-Adrenergic Receptor and NGF/ProNGF pathway on the diabetic urethral functionObjective:To investigate the changes of the al-adrenergic receptor and NGF/ProNGF pathway in the ureathra after diabetes induction.Materials and Methods:Female wistar rats were divided into two groups equally at random. The diabetic models were established by injection of STZ. Urethral function was examined by recordings of bladder pressure and urethral perfusion pressure (UPP) after 8 weeks. The expression of al-adrenergic receptor in the urethra was measured via RT-qPCR and ELIS A method. The expression of nerve growth factor (NGF) in the urethra was measured via RT-qPCR and ELISA method. The expression of proNGF, P75NTR and Sortilin in the urethra was measured by western blotting.Results:The average overnight 8-hour fasting serum glucose level of diabetic rats was 445.7 ± 28.58 mg/ml, which was significantly higher than in the control group (96.84 ±18.25 mg/ml) (p< 0.001), which suggested that the diabetic model was established successfully. Diabetic rats showed a gradual weight loss despite taking in more food and water than control rats. The wet bladder and urethra weight in the diabetic group was significantly greater than in control rats.In diabetic rats, the lowest urethral pressure (UPP nadir) during urethral relaxation was significantly higher. Intravenous administration of tamsulosin, an al-adrenoceptor antagonist, significantly decreased the UPP nadir and baseline UPP in diabetic rats.RT-qPCR and western blotting studies showed a statistically significant increase of ala and alb adrenergic receptor in the urethra from the diabetic group. The expression of NGF is significantly decreased in the urethra from the diabetic group while the expression of proNGF was significantly increased (p<0.05). The P75NIR level in the urethras of diabetic rats was decreased compared with control (p<0.05) and there was no significant difference regarding sortilin between two groups.Conclusion:The al-adrenoceptor was increased in the diabetic urethra, which may be the direct reason for high pressure of the diabetic urethra. NGF was decreased in the diabetic urethra while proNGF was increased. The changes of NGF/ProNGF pathway may be another mechanism of diabetic urethral dysfunction. We speculated that the changes of NGF/ProNGF pathway lead to the injury of peripheral nerve injuries of the urethras, further causing the increase of al-adrenoceptor, which may be the reason for high pressure of the diabetic urethra.Part II Cannabinoid receptors 1 and 2 are associated with bladder dysfunction in an experimental diabetic rat modelObjective To investigate diabetes-associated changes in urinary bladder expression of cannabinoid receptors 1 and 2(CB1 and CB2) and the functional role of CB agonists and antagonists in mediating phasic contractions of isolated bladder strips using a streptozotocin-induced diabetic rat model.Materials and Methods The bladder and dorsal root ganglion (DRG) were removed from diabetic rats and age-matched controls 8-10 weeks after diabetes induction. Expression of CB1 and CB2 mRNA was studied using quantitative real-time PCR and protein levels were determined by Western blot analysis. The effect of increasing concentrations (0.1-100mM) of the mixed CB1/CB2 agonist R(+)-WIN 55,212-2 (WIN), selective CB1 antagonist (AM251) and selective CB2 antagonist (AM630) on carbachol-evoked contraction of bladder strips from control and diabetic rats was investigated. WIN-induced alterations of bladder strip contraction were then studied after pre-incubation with AM251 and AM630.Results:The results of Western blot analysis showed that CB1 protein expression was significantly decreased in the diabetic rat bladder and DRG Diabetes was also associated with a significant decrease in the abundance of CB2 protein in the bladder, but not in the DRGThe results of the RT-PCR analysis showed that there was a significant decrease in CB1 mRNA levels in diabetic rat bladder (P<0.001) and DRG (P=0.01) compared with the control rats. A significant decrease in CB2 mRNA was observed in the diabetic rat bladder(P<0.001); however, no significant changes in the abundance of CB2 mRNA were observed in the diabetic rat DRG compared with the control rat(P=0.22).The amplitude and frequency of contractions of control tissues were significantly greater than that of the diabetic tissues (P< 0.05). WIN inhibited the amplitude of carbacholinduced phasic activity in a concentration-dependent manner in both control and diabetic tissues. In contrast to the effects of WIN on the amplitude, the frequency of contraction in bladder tissues was enhanced with increased WIN concentrations. The effect of vehicle (time control) was not significant for any tissue group. The inhibitory effect of WIN on the amplitude of phasic contractions in diabetic tissues was less pronounced than that observed in the age-matched controls (P< 0.05). The effect of WIN on the amplitude of bladder tissue contraction was partially blocked by pre-incubation with AM251 but not with AM630.Conclusion:CB1 and CB2 exist in the bladder tissue and dorsal nerve roots of rats. The expression of CB1 in the bladder of diabetic rats and dorsal root ganglia of expression are significantly decreased compared with the control group. However, there was no significant difference of the expression of CB2 in different group, suggesting that CB1 and CB2 may play a role in diabetic bladder lesions. The changes of CB1 and CB2 may lead to bladder afferent nerve damage, which may be a possible mechanism of DCP.Part III Grape Seed Proanthocyanidin Extracts Attenuates Diabetic Bladder Dysfunction:Effect on Nrf2 PathwayObjective:To investigate whether grape seed proanthocyanidin extracts (GSPE) has a protective effect to the diabetic bladder function and explore the mechanism of the GSPE.Materials and Methods:30 female wistar rats were randomly divided into three groups (Control group, STZ-induced diabetic group eight weeks, GSPE treated diabetic group).Bladder function was determined by simultaneous recordings of intravesical pressure under isovolumetric conditions and urethral perfusion pressure (UPP) in diabetic rats. The activity of SOD and GSH-Px and the quantity of MDA was measured by relevant assay kits. The expression of Nrf2 was studied by immunohistochemistry and westemblot. The cell apoptosis in bladder was determined by TUNEL method. The expression of HO-1, Bax, Bcl2, NGF, proNGF was measured by western blot.Results:Compared with the control group, the bladder contraction pressure in diabetic group decreased and after GSPE administration, the bladder function of the diabetic rats was improved significantly(P<0.05). The bladder structure in diabetic group was damaged and some inflammatory cells infiltration and GSPE could improve it, according to HE staining. Compared with the control group, the quantity of MDA was increased and the activities of SOD and GSH-Px were decreased in diabetic group (P<0.05) while GSPE could reverse the changes of MDA, SOD and GSH-Px. The expression of nuclear erythroid2-related factor2 (Nrf2), which is the key antioxidative transcription factor, is significantly increased by the treatment of GSPE, with the concomitant elevation of downstream hemeoxygenase-1 (HO-1) (P< 0.05). Furthermore, the level of apoptosis in the bladder caused by diabetes was decreased following the administration of GSPE according to the Terminal Deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) assay. Additionally, GSPE affected the expression of apoptosis-related proteins such as Bax and Bcl-2. Furthermore, GSPE showed neuroprotective effects on the bladder and urethral of diabetic rats, as shown by the increased expression of nerve growth factor (NGF) and decreased expression of the precursor of nerve growth factor (proNGF).Conclusion:GSPE improve the state of oxidative stress in diabetic bladder by activating Nrf2. Besides GSPS reduce diabetic bladder cell apoptosis and improve the nerve function So GSPE can protect the diabetic bladder. Besides, GSPE protected the diabetic urethral function by NGF/ProNGF pathway. |
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