| Part I:Research on Literature and TheoryDiabetic bladder dysfunction (DBD) is a complication of diabetes mellitus with chronic hyperglycemic. In course of the development of diabetes mellitus, a variety of patients eventually present symptoms such as prolonged time interval between micturitions, delayed sensation of voiding, micturition weakness, dribbling and incomplete emptying. Urodynamic examination indicates impaired bladder sensation, increased bladder capacity, decreased detrusor contractility and increased residual urine volume. Due to the fact that the physiological activities of bladder are innervated by the peripheral nerves, scientific research on the mechanism of diabetic bladder has been predominantly targeted on neuropathic origin for years. However, it is observable in the clinical setting that lower urinary tract symptoms appear in patients with early diabetes mellitus, yet the symptoms are urinary urgency, frequent urination, nocturia and urge incontinence. Moreover, urodynamic study also suggests detrusor instability of different extents or detrusor hyper-reflexia. These two series of opposite symptoms and signs are deemed to be in correlation with the temporal process of the disease, in which the bladder undergoes two pathological changes in two stages caused by the hyperglycemia. Transition appears from overactive bladder (OAB) in early compensation stage to underactive bladder (UAB) in late decompensation stage. In addition, abnormalities found in the urothelium, neurons, detrusor muscles or urethra can lead to storage or voiding dysfunction of the bladder.Non-adrenergic non-cholinergic (NANC) pathway is found to be significant in bladder physiopathology. Previously, bladder contractility was known to be caused by the binding of muscarinic receptors and acetylcholine released from the parasympathetic nerves. It was discovered later that similar effect was achieved when ATP binded to purinergic receptors P2X. Unusual expression of P2X receptors is always related to cystopathy, in which exploring the role of different subtype of P2X receptors is hence becoming more important. There are literatures showing that P2X1 receptors are distributed along the border of detrusor cells, whereas P2X3 receptors on the nerve bundles beneath the urothelial cells. In pathological conditions, changes in expressions may indicate abnormal activities in bladder sensation and contractility.In Chinese medicine, diabetic cystopathy (DC) or diabetic neurogenic bladder (DNB) was formerly categorized into a disease named "Longbi" (meaning oliguria and/or anuria). The naming is only based on the local lower urinary tract symptoms commonly found in the later stage of diabetes mellitus. It cannot reflect the relativity with diabetes mellitus as a whole. Moreover, it fails to give a transition picture from OAB to UAB. Diabetes mellitus, which is characterized by polydipsia, polyphagia, polyuria and weight loss, is categorized into a disease called "Xiaoke". In this sense, it is suggested that early compensation stage with OAB in DBD remains in the category of "Xiaoke", while late decompensation stage with UAB should be categorized as "Xiaoke with Longbi" for sake of explicitly discriminating "Longbi" alone.Insufficiency of "yang qi" of diabetic patients themselves is a crucial causative factor of diabetes mellitus. The pathological mechanism of the complicated bladder dysfunction, therefore, cannot deny it. In terms of six-meridian differential diagnosis, diabetes mellitus involves six meridians. The fundamental disease location of diabetic bladder dysfunction is mainly in "Taiyang". "Taiyang" refers to the body surface which is characterized as "yang". Weakness in "yang qi" provides chances for pathological qi to invade Taiyang, and eventually intrude from meridian into "fu-organ", causing "abnormality in fu-organ of Taiyang disease". Bladder is indicated as a "fu-organ of Taiyang". With normal function of "qi", the bladder is capable of modulating the transportation and distribution of "Jinye" (body fluid), as well as the excretion of urine and sweat. Abnormal functioning of bladder "qi" leading to impairment in storage and impairment in voiding are two important pathological mechanism of bladder dysfunction."Wuling powder", which is a prescription originated from the Chinese medical classics "Treatise on Exogenous Febrile Diseases", has definite influences in regulating bladder function. In view of the herbal formation,"Fuling" [Poria cocos (Schw.) Wolf], "Zhuling" [Polyporus umbellatus (Pers.) Fries], "Zexie" [Alisma orientale (Sam.) Juzep.] and "Baishu" [Atractylodis macrocephalae Rhizoma] have diuretic effect. Therefore, "Wuling powder" is always regarded as diuretic medication and is often used in ogliuria during bladder voiding dysfunction. Nevertheless, "Guizhi" [Cinnamomi Ramulus] containing "yang-warming and yang-dispersing" effect may give crucial influences to the function of bladder "qi". As a result, some scholars believe that "Wul ing powder" is not only diuretic medication, but also diaphoretic medication. "Yang-warming and qi-converting" effect in this formula helps regulate the fluid metabolism in a normal way. It is perhaps suitable in the stage of bladder storage dysfunction where frequent urination, urinary urgency and increased volume of urine appear.The disease development and symptom transformation usually involve "Taiyin" and "Shaoyin". Warming and tonifying the "yangqi" of Taiyin and Shaoying does not only protect the root of innate and acquired organ functions, but also mean to strengthen the "yang-warming and qi-converting" effect of Wul ing powder on the bladder. As a result, "Huangqi" [Astragali radix], "Fuzi" [Aconiti lateralis radix praeparata] and "Wuyao" [Linderae radix] are added on the basis of "Wuling powder", which is finally known as "Jiawei Wul ing powder". "Huangqi" strengthens Taiyang and Taiyin, "Fuzi" strengthens Shaoyin, and "Wuyao" is used to "warm the kidney and regulate qi".Since the diabetic bladder dysfunction is reported to have a high prevalence and seriously affect patients’quality of life, intervention using Chinese medical prescription may be a way of treatment for them. It is known that the clinical pictures of lower urinary tract are not the same during the compensation stage and decompensation stage, and "Wuling powder" contains dual modulating effects. However, the molecular target and mechanism of "Jiawei Wul ing powder" are still dubious.The investigation proposed that the drug effect of "Jiawei Wuling powder" is modulated through purinergic receptors, so as to achieve the therapeutic purpose in eliminating the symptoms. "Jiawei Wuling powder" was used as the Chinese medical intervention. It was predicted that the expression of P2X1 and P2X3 receptors would decrease, and the relative clinical indexes of bladder storage function would improve. The result of this investigation may help understand the role of purinergic receptors under the dual modulating effect of "Jiawei Wuling powder".Part II:Animal ExperimentObjectiveTo investigate the effect of Chinese medicinal formula "Jiawei Wuling powder" on the expression of purinergic receptors P2X1 and P2X3 on the bladder using experimental Sprage-Dawley rats in compensation stage of diabetic bladder dysfunction as models.Methods110 male SD rats of SPF level were randomly allocated into different groups after adaptive feeding with ordinary feed for 1 week.10 rats were allocated into normal group and the rest were allocated into model groups, including diabetes mellitus group (DM group), tolterodine group (T group), high dose Chinese medicine group (H group), medium dose Chinese medicine group (M group) and low dose Chinese medicine group (L group). Rats in model groups were then fed with high fat feed for 1 month, followed by the intraperitoneal injection of 1% STZ with dosage of 45mg/kg. Venous blood from the tail was obtained after 72 hours. Rats having serum glucose level≥16.67mmol/L, and presenting polydipsia, polyphagia and polyuria were considered as models of diabetes mellitus. Changes in weight and serum glucose were observed every 2 weeks, while amount of water consumed and amount of urine excreted were collected every 4 weeks using metabolic cages. In commencement of the 9th week after STZ induction, rats in T group were intra-gastrically administered with 2ml tolterodine, whereas those in H group, M group and L group with 6ml,3ml and 1.5ml decoction of "Jiawei Wuling powder" respectively. At 12th week, all rats were anaesthetized by intraperitoneal injection of 25% urethane, followed by an abdominal incision to expose the bladders of which a fistula was made on the apex. Normal saline was perfused in a rate of 30ml/h in order to investigate the urodynamic changes in the diabetic rats. Weights of bladder were measured after isolation. With routine dehydration, paraffin embedment and sectioning, the bladder tissues were HE-stained and the pathological conditions were observed under a light microscope. Finally, the expression of purinergic receptors P2X1 and P2X3 was investigated with the use of western blotting and immunohistochemical techniques.ResultsThe percentage of rats in model group presents hyperglycemia was 94.74%. 44 rats finished the experiment, account for 40% of the total rats used. There were 10 rats in normal group and 34 rats in model groups (including 7 in DM group,6 in T group,6 in H group,7 in M group and 8 in L group). During the course of the experiment, the overall situation of rats in normal group was good, but rats in model groups appeared mentally stressed and the hair became brown and sparse. The results of the other indexes are as follows:①Weight: Between-group comparison discovered that rats in L groups are heavier than T groups, but there is no statistical difference between other groups. Within-group comparison found that only rats in DM group decrease in weight, and no statistical difference is found in other groups. ②Serum glucose: Between-group comparison showed that rats in L group have lower serum glucose than that in DM group. However, no significant change is found within groups after medical intervention. ③Amount of water consumed:Between-group comparison discovered that rats in T group, M group and L group have lower amount of water consumed than that in DM group. Within-group comparison found that only rats in T group decrease the water consumed (p=0.018). ④Amount of urine excreted:No difference is found in the amount of urine excreted between rats in different groups, but within-group comparison found the decrement in rats in DM group and T group (p=0.006 and p=0.002). ⑤Urodynamic study:Except H group, the voiding times are increased in rats of model groups than that in normal group. The maximum voiding pressures of rats in DM group and T group are smaller than that in normal group, the difference is more significant in T group (p<0.005). No difference is found between rats in all Chinese medicine groups and rats in normal group and DM group. Except H group, the bladder compliances are higher in model groups than that in normal group, the difference is more significant in T group (p<0.005). No statistical difference is found among model groups. ⑥Bladder weight and bladder weight index:Rats in model groups have heavier bladder weights than that in normal group, the differences are more significant in DM group, H group and M group (p<0.005). The bladder weight is heaviest in M group and is lightest in T group, statistical difference is found. Rats in model groups have higher bladder weight indexes than that in normal group. ⑦Pathological examination: HE-stained slides were observed under light microscope, it was shown that the bladder of rats in model groups have thicker layer of bladder detrusor muscle, cell hyperplasia with varieties of shape, derangement and disruption of muscle bundles, loose structure, significant widened gap between bundles, reduced collagen fibres. ⑧Western Blot:No significant differences of P2X1 and P2X3 expressions are seen amongs all groups. However, P2X3 ratio is down-regulated in model group, that of T group and L group are also down-regulated, while that of M group and H group are up-regulated. ⑨Immunohistochemistry:The expression of P2X1 receptor is mainly on detrusor muscle, urotheliumand vessel wall, that of P2X3 receptor is on detrusor muscle.ConclusionThere are several conclusions:1) The research successfully generated DBD rat models.2) Reduction of weight, blood glucose, water consumption and urine excretion of DBD rats with "Jiawei Wuling powder" are not observed.3) Improvement of voiding time, maximum voiding pressure and bladder compliance in urodynamic study of week 12 DBD rats with "Jiawei Wuling powder" are not discovered.4) Decrement of bladder weight and Repairment of bladder tissue are not found.5) Expression of P2X1 receptors are mainly found in detrusor muscle, uroepitheliem and vessel wall, while that of P2X3 receptors are found in detrusor muscle.6) No influence is found on P2X1 receptor expression by "Jiawei Wuling powder", but mediation is found on P2X3 receptor expression, yet significant difference is not discovered.In addition, several points need to be concerned:1) Despite ineffectiveness in lowering hyperglycemia status by "Jiawei Wuling powder", it perhaps prevents ill-conditioned weight lose. The diuretic effect of the drug may postpone the developmental tread of bladder from OAB phase towards UAB phase. Medium and low dose of drug may help alleviate polydipsia of diabetes mellitus. In terms of symptom treatment, anti-diuretic effect of "Jiawei Wul ing powder" is not found.2) The diuretic effect may help delay the progression of detrusor muscle towards UAB phase.3) Diabetes mellitus down-regulates P2X3 receptor expression. Low dose and medium/high dose of drug show down-regulation and up-regulation of P2X3 receptor expression respectively, indicating that dual modulation of drug may act on P2X3 receptor. |
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