膀胱的主要功能是暫存和協助排尿。糖尿病是一種代謝性疾病，會導致許多並發症，包括糖尿病膀胱功能障礙和下泌尿道症狀。與普通人群相比，糖尿病患者中風的風險是普通人群的兩倍。中風是一種疾病，其病因為流向大腦的血流不暢所導致。尿失禁通常是急性半球中風的後遺症。另一方面，環磷酰胺是臨床上用於治療腫瘤疾病的化療藥物。然而，其有毒代謝物會導致出血性膀胱炎和膀胱過度活動。
Exendin-4 (Ex-4) 是一種腸促胰島素藥物，已獲准用於糖尿病治療和神經元保護。然而，臨床上需要頻繁注射該藥限制了其應用。我們製備了含有Exendin-4的奈米顆粒 (Ex-4-loadedpoly (d,l-lactide-co-glycolide) nanoparticles, PEx-4) 並研究了對糖尿病大鼠的腦缺血/再灌注誘發的腦損傷和排尿功能障礙的影響。使用鏈脲佐菌素 (streptozotocin) 誘導的第一型糖尿病大鼠進行雙側頸動脈閉塞10分鐘的腦缺血模型，我們比較了Ex-4和PEx-4 對前額葉皮層水腫、排尿功能和氧化壓力的影響，並利用免疫組織化學染色來評估對細胞凋亡、細胞焦亡與細胞自噬作用的影響。
電針 (Electroacupuncture, EA) 是一種流行的針灸療法，在傳統經絡理論的基礎上，將針插入體內的穴位並進行最小的電刺激，其中以BL33 (中髎穴，位於人體腰骶部，第三骶後孔中，大鼠則為第二骶後孔中) 為臨床治療中最常用的經絡穴位。我們在環磷酰胺誘導的排尿功能障礙大鼠中使用電針治療評估膀胱中的排尿變化。
內源性大麻素主要通過與外周組織、脊髓和大腦中的 G 蛋白偶聯受體、大麻素受體 1型和 2 型結合來抑制傷害感受。兩種大麻素受體也已在小鼠、大鼠、猴子和人類的膀胱中被發現。我們在環磷酰胺誘導的排尿功能障礙大鼠中探討AM251（一種大麻素受體 1型拮抗劑）評估膀胱中的排尿變化。
結論，具有更強抗氧化活性和持久生物利用度的 PEx-4 通過抑製糖尿病大鼠的氧化壓力、內質網壓力、細胞凋亡、細胞自噬和細胞焦亡信號傳導，有效改善腦缺血/再灌注誘發的腦部和膀胱損傷。另外，電針治療和AM251（一種大麻素受體 1型拮抗劑）可改善 CYP 誘導的大鼠模型中的排尿功能障礙。

The major functions of urinary bladder are temporary storage and assistance in the expulsion of urine. Diabetes mellitus (DM) is a metabolic disorder which leads to many complications including diabetic bladder dysfunction and lower urinary tract symptoms. The risk for stroke is twice as much in DM patients by comparison with the general population. A stroke is a medical condition in which poor blood flow to the brain causes cell death. Urinary incontinence is a sequela of acute hemispheric stroke commonly. In addition, Cyclophosphamide (CYP) is a chemotherapy agents clinically used to treat tumor diseases. However, its toxic metabolite causes hemorrhagic cystitis and bladder hyperactivity.
Exendin-4 (Ex-4) is an incretin mimetic peptide accepted for neuronal protection and diabetes treatment. Nevertheless, the required frequent injections put a limit on its clinical use. Ex-4-loaded poly (d,l-lactide-co-glycolide) nanoparticles (PEx-4) was prepared to investigate the effect on cerebral ischemia/reperfusion (IR) injury associated with cystopathy in diabetic rats. By using ten minutes of bilateral carotid artery occlusion combined with hemorrhage-induced hypotension of IR model in streptozotocin-induced type 1 diabetic (T1DM) Wistar rats, we compared PEx-4 and Ex-4 effect on voiding function, prefrontal cortex edema and oxidative stress including cerebral spinal fluid (CSF) reference HOCl (RHOCl) and H2O2 (RH2O2) levels, endoplasmic reticulum (ER) stress, autophagy, apoptosis and pyroptosis signaling in brain and bladder by immunohistochemistry and western blotting.
Electroacupuncture (EA) is a popular acupuncture therapy, in which needles are inserted into acupuncture points within the body and a minimal electric stimulation based on traditional meridian theory. We explore Electroacupuncture treatment in CYP-induced bladder dysfunction rats to evaluate the functional alterations of the bladder.
Endocannabinoids inhibit nociception primarily by binding to G-protein coupled receptors, cannabinoid receptors 1 (CB1) and 2 (CB2) , in peripheral tissue, spinal cord and brain. CB1 and CB2 have also been identified in the bladders of mice, rats, monkey and humans. We explore the treatment with AM251 (a CB1 antagonist) in CYP-induced bladder dysfunction rats to evaluate the functional alterations of the bladder.
Subcutaneous administration of PEx-4 displayed long-lasting hypoglycemic effect and higher CSF antioxidant activity than Ex-4 in rats. Our results showed that T1DM enhanced CSF RH2O2, and pIRE-1/pJNK/cleaved caspase-12/CHOP- mediated ER stress, PARP /caspase 3 mediated apoptosis, LC3-II/Beclin-1 mediated autophagy and IL-1β/caspase 1 mediated pyroptosis signaling in the rat bladders and prefrontal cortex. IR caused damage in micturition center, prefrontal cortex edema and further enhanced CSF HOCl and RH2O2 level, ER stress, autophagy, apoptosis and pyroptosis signaling in the T1DM brains and bladders. PEx-4 were more efficient than Ex-4 in attenuating IR-evoked oxidative stress and prefrontal cortex edema in brains and improving voiding dysfunction in bladders of T1DM rats. EA treatment and the treatment with AM251 in CYP-induced rats recovers bladder dysfunctions including intercontraction interval (ICI) , and bladder compliance.
In summary, PEx-4 with long-lasting bioavailability and stronger antioxidant activity confer efficiently therapeutic efficacy to ameliorate IR-evoked brain and bladder injury through inhibiting oxidative stress, ER stress, autophagy, apoptosis and pyroptosis signaling in diabetic rats. Electroacupuncture treatment and the treatment with AM251 also improve bladder dysfunction in CYP-induced rat model.

1.Andersson KE, Arner A (2004) . Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev.;84:935–986.
2.Fowler CJ, Griffiths D, de Groat WC. The neural control of micturition. Nat Rev Neurosci. 2008 Jun;9 (6) :453-66
3.Genuth S, Alberti KG, Bennett P, Buse J, Defronzo R, Kahn R et al. (2003) . Expert Committee on the Diagnosis and Classification of Diabetes Mellitus2, the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Follow-up report on the diagnosis of diabetes mellitus. Diabetes Car. 26: 3160-3167.
4.Daneshgari F, Liu G, Birder L, Hanna-Mitchell AT, Chacko S (2009) . Diabetic bladder dysfunction: current translational knowledge. J Urol 182 (6 Suppl) : S18-26.
5.Song QX, Sun Y, Deng K, Mei JY, Chermansky CJ, Damaser MS. Potential role of oxidative stress in the pathogenesis of diabetic bladder dysfunction. Nat Rev Urol. 2022 Oct;19 (10) :581-596.
6.Wittig L, Carlson KV, Andrews JM, Crump RT, Baverstock RJ. Diabetic Bladder Dysfunction:A Review. Urology. 2019 Jan;123:1-6.
7.Khan Z, Starer P, Yang WC, Bhola A (1990) . Analysis of voiding disorders in patients with cerebrovascular accidents. Urology 35:265-270
8.Sakakibara R. Lower urinary tract dysfunction in patients with brain lesions. Handb Clin Neurol. 2015;130:269-87.
9.Kuriakose D, Xiao Z. Pathophysiology and Treatment of Stroke: Present Status and Future Perspectives. Int J Mol Sci. 2020 Oct 15;21 (20) :7609
10.Han KS, Heo SH, Lee SJ, Jeon SH, Yoo KH (2010) . Comparison of urodynamics between ischemic and hemorrhagic stroke patients; can we suggest the category of urinary dysfunction in patients with cerebrovascular accident according to type of stroke? Neurourol Urodyn 29 (3) :387-390.
11.Kim SE, Shin MS, Kim CJ (2012) . Effects of tamsulosin on urinarybladder function and neuronal activity in the voiding centers of ratswith cyclophosphamide-induced overactive bladder. Int NeurourolJ.;16:13–22.
12.Wang R, Hong M, Huang J, Zhou N, Zhang Y, Xu S, Liu J, Yuan J, Zhang L, Huang L, Huang P, Tan B, Cao HY. Low-Dose Cyclophosphamide Induces Nerve Injury and Functional Overactivity in the Urinary Bladder of Rats. Front Neurosci. 2021 Oct 1;15:715492.
13.Yuan Z, Tang Z, He C, Tang W (2015) . Diabetic cystopathy: A review. J Diabetes 7 (4) :442-447.
14.Thorens B (1992) . Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci U S A 89 (18) :8641-8645
15.Ahrén B (1998) . Glucagon-like peptide-1 (GLP-1) : a gut hormone of potential interest in the treatment of diabetes. BioEssay 20: 642-651.
16.Eng J, Kleinman WA, Singh L, Singh G, Raufman JP (1992) . Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas. J Biol Chem 267 (11) :7402-7405.
17.Göke R, Fehmann HC, Linn T, Schmidt H, Krause M, Eng J, Göke B (1993) . Exendin-4 is a high potency agonist and truncated exendin- (9-39) -amide an antagonist at the glucagon-like peptide 1- (7-36) -amide receptor of insulin-secreting beta-cells. J Biol Chem 268 (26) :19650-19655.
18.Edwards CM, Stanley SA, Davis R, Brynes AE, Frost GS, Seal LJ et al. (2001) . Exendin-4 reduces fasting and postprandial glucose and decreases energy intake in healthy volunteers. Am J Physiol Endocrinol Metab 281 (1) :E155-161.
19.Nielsen LL, Young AA, Parkes DG (2004) . Pharmacology of exenatide (synthetic exendin-4) : a potential therapeutic for improved glycemic control of type 2 diabetes. Regul Pept 117 (2) :77-88.
20.Iltz JL, Baker DE, Setter SM, Keith Campbell R (2006) . Exenatide: an incretin mimetic for the treatment of type 2 diabetes mellitus. Clin The. 28 (5) :652-665.
21.Chien CT, Fan SC, Lin SC, Kuo CC, Yang CH, Yu TY et al. (2014) . Glucagon-like peptide-1 receptor agonist activation ameliorates venous thrombosis-induced arteriovenous fistula failure in chronic kidney disease. Thromb Haemost 112:1051-1064.
22.Teramoto S, Miyamoto N, Yatomi K, Tanaka Y, Oishi H, Arai H et al. (2011) . Exendin-4, a glucagon-like peptide-1 receptor agonist, provides neuroprotection in mice transient focal cerebral ischemia. J Cereb Blood Flow Metab 31 (8) :1696-1705.
23.Chien CT, Jou MJ, Cheng TY, Yang CH, Yu TY, Li PC (2015) . Exendin-4-loaded PLGA microspheres relieve cerebral ischemia/reperfusion injury and neurologic deficits through long-lasting bioactivity-mediated phosphorylated Akt/eNOS signaling in rats. J Cereb Blood Flow Metab 35 (11) :1790-1803.
24.Kumari A, Yadav SK, Yadav SC (2010) . Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B Biointerfaces 75:1-18.
25.Zhang R, Lao L, Ren K, Berman BM. Mechanisms of acupuncture-electroacupuncture on persistent pain. Anesthesiology. 2014 Feb;120 (2) :482-503.
26.Yang L, WYMQ (2017) . A comparative study of electroacupuncture at Zhongliao (BL33) and other acupoints for overactive bladder symptoms.Front Med;11:129–36
27.Hayn M. H., Ballesteros I., De Miguel F., Coyle C. H., Tyagi S., Yoshimura N., et al. (2008) . Functional and Immunohistochemical Characterization of CB1 and CB2 Receptors in Rat Bladder. Urology 72, 1174–1178
28.Gratzke C., Streng T., Park A., Christ G., Stief C. G., Hedlund P., et al. (2009) . Distribution and Function of Cannabinoid Receptors 1 and 2 in the Rat, Monkey and Human Bladder. J. Urol. 181, 1939–1948.
29.Bakali E., Elliott R. A., Taylor A. H., Willets J., Konje J. C., Tincello D. G. (2013) . Distribution and Function of the Endocannabinoid System in the Rat and Human Bladder. Int. Urogynecol. J. 24, 855–863
30.Boudes M, De Ridder D. Cannabinoid receptor 1 also plays a role in healthy bladder. BJU Int. 2013 Jan;113 (1) :142-3.
31.Dâmaso AR, Duarte FO, Sene-Fiorese M, et al. (2015) . Experimental diet models in the investigation of obesity In: Andersen ML, Tufik S, eds. Rodent Model as Tools in Ethical Biomedical Research. U.K.: Springer International Publishing, 503–16.
32.Huang KC, Yang CC, Lee KT, Chien CT (2003) . Reduced hemodialysis-induced oxidative stress in end-stage renal disease patients by electrolyzed reduced water. Kidney Int 64: 704-714.
33.Liu WJ, Jin HY, Lee KA, Xie SH, Baek HS, Park TS (2011) . Neuroprotective effect of the glucagon-like peptide-1 receptor agonist, synthetic exendin-4, in streptozotocin-induced diabetic rats. Br J Pharmacol 164 (5) :1410-20.
34.Li F, Liu KF, Silva MD, Meng X, Gerriets T, Helmer KG et al. (2002) . Acute postischemic renormalization of the apparent diffusion coefficient of water is not associated with reversal of astrocytic swelling and neuronal shrinkage in rats. AJNR Am J Neuroradiol 23 (2) :180-188.
35.Nakka VP, Gusain A, Raghubir R (2009) . Endoplasmic reticulum stress plays critical role in brain damage after cerebral ischemia/reperfusion in rats. Neurotox Res 17 (2) :189-202.
36.Fishman RA (1975) . Brain edema. N Engl J Med. 293 (14) :706-711.
37.Perry T, Lahiri DK, Chen D, Zhou J, Shaw KT, Egan JM et al. (2002) . A novel neurotrophic property of glucagon-like peptide 1: a promoter of nerve growth factor-mediated differentiation in PC12 cells. J Pharmacol Exp Ther 300 (3) :958-966.
38.Bertilsson G, Patrone C, Zachrisson O, Andersson A, Dannaeus K, Heidrich J et al. (2008) . Peptide hormone exendin-4 stimulates subventricular zone neurogenesis in the adult rodent brain and induces recovery in an animal model of Parkinson's disease. J Neurosci Res 86 (2) : 326-38.
39.Yao X, Derugin N, Manley GT, Verkman AS (2015) . Reduced brain edema and infarct volume in aquaporin-4 deficient mice after transient focal cerebral ischemia. Neurosci Lett 584:368-372.
40.Andrew J, Nathan PW (1964) . Lesions of the anterior frontal lobes and disturbances of micturition and defaecation. Brain 87:233-262.
41.Terreberry RR, Neafsey EJ (1987) . The rat medial frontal cortex projects directly to autonomic regions of the brainstem. Brain Res 19:639-649.
42.Matsumoto S, Hanai T, Yoshioka N, Shimizu N, Sugiyama T, Uemura H (2006) . Medial prefrontal cortex lesions inhibit reflex micturition in anethetized rats. Neurosci Res 54 (1) :66-70.
43.Ma Y, Hendershot LM (2004) . ER chaperone functions during normal and stress conditions. J Chem Neuroanat 28: 51-65.
44.Oyadomari S, Mori M (2004) . Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11:381-389.
45.Toniolo A, Warden EA, Nassi A, Cignarella A, Bolego C (2013) . Regulation of SIRT1 in vascular smooth muscle cells from streptozotocin-diabetic rats. PLoS One 8 (5) :e65666.
46.Lee J, Hong SW, Park SE, Rhee EJ, Park CY, Oh KW et al. (2014) . Exendin-4 attenuates endoplasmic reticulum stress through a SIRT1-dependent mechanism. Cell Stress Chaperone 19 (5) :649-656.
47.Xu G, Kaneto H, Laybutt DR, Duvivier-Kali VF, Trivedi N, Suzuma K et al. (2007) . Downregulation of GLP-1 and GIP receptor expression by hyperglycemia: possible contribution to impaired incretin effects in diabetes. Diabetes 56 (6) :1551-1558.
48.Tsunekawa S, Yamamoto N, Tsukamoto K, Itoh Y, Kaneko Y, Kimura T, et al. (2007) . Protection of pancreatic beta-cells by exendin-4 may involve the reduction of endoplasmic reticulum stress; in vivo and in vitro studies. J Endocrinol 193 (1) :65-74.
49.Zhang Y, Wang Q, Zhang J, Lei X, Xu GT, Ye W (2009) . Protection of exendin-4 analogue in early experimental diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol 247 (5) :699-706.
50.Younce CW, Burmeister MA, Ayala JE (2012) . Exendin-4 attenuates high glucose-induced cardiomyocyte apoptosis via inhibition of endoplasmic reticulum stress and activation of SERCA2a. Am J Physiol Cell Physiol 304 (6) :C508-518.
51.Darsalia V, Hua S, Larsson M, Mallard C, Nathanson D, Nyström T et al. (2014) . Exendin-4 reduces ischemic brain injury in normal and aged type 2 diabetic mice and promotes microglial M2 polarization. PLoS One 9 (8) :e103114.
52.Artunc-Ulkumen B, Pala HG, Pala EE, Yavasoglu A, Yigitturk G, Erbas O (2014) . Exenatide improves ovarian and endometrial injury and preserves ovarian reserve in streptozocin induced diabetic rats. Gynecol Endocrinol 4:1-6.
53.Gelber DA, Good DC, Laven LJ, Verhulst SJ (1993) . Causes of urinary incontinence after acute hemispheric stroke. Stroke 24 (3) :378-382.
54.Iltz JL, Baker DE, Setter SM, Keith Campbell R (2006) . Exenatide: an incretin mimetic for the treatment of type 2 diabetes mellitus. Clin The. 28 (5) :652-665.
55.Kastin AJ, Akerstrom V, Pan W (2002) . Interactions of glucagon-like peptide-1 (GLP-1) with the blood–brain barrier. J Mol Neurosci 18 (1–2) :7-14.
56.Kieffer TJ, McIntosh CH, Pederson RA (1995) . Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 136 (8) :3585-3596.
57.Kim S, Jeong J, Jung HS, Kim B, Kim YE, Lim DS et al. (2017) Anti-inflammatory effect of glucagon like peptide-1 receptor agonist, Exendin-4, through modulation of IB1/JIP1 expression and JNK signaling in stroke. Exp Neurobiol 26 (4) :227-239.
58.Lecrux C, McCabe C, Weir CJ, Gallagher L, Mullin J, Touzani O et al. (2008) . Effects of magnesium treatment in a model of internal capsule lesion in spontaneously hypertensive rats. Stroke 39 (2) :448-454.
59.Palamoor M, Jablonski MM (2014) . Comparative study on diffusion and evaporation emulsion methods used to load hydrophilic drugs in poly (ortho ester) nanoparticle emulsions. Powder Technol 253:53-62.
60.Stevens LA, Sellers DJ, McKay NG, Chapple CR, Chess-Williams R (2006) . Muscarinic receptor function, density and G-protein coupling in the overactive diabetic rat bladder. Auton Autacoid Pharmacol 26 (3) :303-309.
61.Yotsuyanagi S, Narimoto K, Namiki M (2006) . Mild brain ischemia produces bladder hyperactivity without brain damage in rats. Urol Int 77 (1) :57-63.
62.Chuang YC, Yoshimura N, Huang CC, Wu M, Tyagi P, Chancellor MB.Expression of E-series prostaglandin (EP) receptors and urodynamiceffects of an EP4 receptor antagonist on cyclophosphamide-inducedoveractive bladder in rats.
63.Pan F, Liu D, Han XM, Li WC, Pang ZL, Li B, Zhang XP, Xiao YJ, Zeng FQ. Urodynamic investigation of cyclophosphamide-induced overactive bladder in conscious rats. Chin Med J (Engl) . 2012 Jan;125 (2) :321-5
64.Wu KC, Chiang BJ, Tsai WH, Chung SD, Chien CT. I-Tiao-Gung extract through its active component daidzin improves cyclophosphamide-induced bladder dysfunction in rat model. Neurourol Urodyn. 2018 Nov;37 (8) :2560-2570
65.Liu X, Liu K, Zhi M (2017) . Effects of electroacupuncture at BL33 on detrusor smooth muscle activity in a rat model of urinary retention. Acupunct Med;35:437–444.
66.Füllhase C, Schreiber A, Giese A, Schmidt M, Montorsi F, Gratzke C, La Croce G, Castiglione F, Stief C, Hedlund P. Spinal neuronal cannabinoid receptors mediate urodynamic effects of systemic fatty acid amide hydrolase (FAAH) inhibition in rats. Neurourol Urodyn. 2016 Apr;35 (4) :464-70.