| ObjectiveBased on preliminary study, this research first applied preventive intervention of JiaWei Wuling Powder in STZ-induced diabetic bladder dysfunction model rats. And then assessed preventive effect of herbal formula by animal ethology, histomorphology and urine dynamics. Moreover the study discussed ROCK signaling channel which is considered to be one of potential mechanism of JiaWei Wuling Powder affecting on diabetic bladder dysfunction.Methods1. Animal modeling and grouping70 male SD rats were randomly divided into five groups:10 in normal group and 15 in model group, high-dose group, moderate-dose group and low-dose group. After one-week acclimating breeding and four-week high-fat diet breeding, the diabetic modelling would accept Streptozotocin (STZ) intraperitoneal injection. The DBD model rats would develop naturally after eight weeks.2. InterventionThe high, moderate and low dose group were feed by 1.5ml,3ml and 6ml JiaWei Wuling water decoction. Remaining two groups were feed by 3ml normal saline. The whole course would last for 8 weeks.3. Observations and evaluation① Observed animal behavior and termly measured body weight, glucose and metabolic index. At the 13th week, all of rats would receive urine dynamic tests. Combined with results of all tests mentioned, we would judge whether DBD rats model was successfully made. ② Bladder tissue would fixed by 4% paraformaldehyde, dehydrated by gradient concentration alcohol, transparent by xylene and embedded by paraffin. Then paraffin-embeded samples would sliced into 4μm sections and do hematoxylin-eosin staining. The effect of JiaWei Wuling powder in diabetic bladder dysfunction were evaluated by histomorphology and urine dynamics. ③ assessed effects of JiaWei Wuling powder on protein expression of ROCK signaling channel in diabetic bladder dysfunction model rats bladder tissue.4. Statistical analysisIBM SPSS statistics 22.0 was used for statistical analysis. Measurement data was expressed as mean± standard deviation (±s). If the data follow a normal distribution with or without data processing, ANOVA analysis were used. If not, non-parametric test was used. The test level P<0.05.Results1. Document Research① Diabetic Bladder Dysfunction cutting-edge research.Nowadays, the pathogenesis of diabetic bladder dysfunction is still in great argue. Researchers mainly agreed that diabetic bladder dysfunction is greatly related with endothelium, smooth muscle, blood vessel and nerve damage due to long-term hyperglycemia condition, Time-dependent is one of the most important breakthrough of DBD research. Recently, Cholinergic Receptor, Endothelium NO, Oxidative stress and Nerve Growth Factor are main targets of DBD pathogenesis research. DBD treating strategy is mainly symptomatic therapy without specific medicine and certain efficacy.② The TCM research on DBDThrough ancient literature study, we could learn that diabetic bladder dysfunction could cause by a variety of internal and external pathogenesis, but key pathogenesis is kidney Yang deficiency and inhibited qi transformation, Therefore the treatment when should warm yang and drying dampness.TCM therapies such as herbal formula and acupuncture were widely used in treating DBD. The results indicated TCM therapy was more effective than basic treatment. But documentary evidence evaluation showed low quality of evidence and possibilities of bias. Meanwhile there were few TCM experimental researches studied on DBD and the quality of existing research studies need to be further improved.③ Research on Wuling PowderWuling powder is a popular formula of governing body fluid. Recent study showed the formula could treat more than 100 kinds of disease. The author think that wuling powder could not only induce diuresis, but also has dual-direction regulation of body fluid.④ ROCK Channel diabetes-related researchA number of studies indicated that ROCK signaling pathway had highly relevance with diabetic macroangiopathy and microangiopathy, ROCK has become a potential breakthrough in prevention study of diabetic complication, including neurogenic bladder.2. Experimental Study① Baseline CharacteristicsBefore the intervention of STZ, the body weight of rats had no significant statistical difference (P>0.05), which showed there were no differences in baseline covariates between groups. That is to say, baseline characteristics were comparable between five groups.② Diabetic Bladder Dysfunction rats modelingThe body weight of model group rats was lower than the normal group with a statistically significance (-P<0.05). After STZ intraperitoneal injection, blood glucose of model group rats was significantly increase (P<0.05). The mean is more than 16.9 mmol·L-1, standard of rat diabetic diagnosis. It indicated that this experiments was successfully copy diabetic model. From metabolic index, we could see model group had more food and water consumption and urination (P<0.01). Moreover, model group rats had more urinate time and higher compliance in urine dynamics, which means detrusor damage. Typical DBD histomorphologic changes such as muscles derangement, abnormal muscle cell form and widen muscle bundle were found in bladder tissue HE staining sector. In conclusion, this research successfully made DBD rat model.③ Effect of Jiawei Wuling Powder preventive interventions on DBDThere was a significantly rise in blood glucose in wulingsan groups, with significantly difference compare to normal group (P<0.05) but no difference to model group. The result showed no effect of Jiawei Wuling powder. From metabolic index, only moderate-dose group indicate down-regulate trend with no significantly difference (P>0.05). In urinate dynamic test, all wulingsan groups showed improving trends on urine time and compliance. Among these, moderate-dose groups had significantly amelioration (P<0.05). Under optical microscope, we could find slight histomorphologic damage in wulingsan rat bladder tissue. But compare with model group, there was obvious amelioration in as muscles arrangement and muscle bundle. Above all, Jiawei Wuling powder could improve detrusor function, postpone histomorphologic damage of bladder tissue and somehow prevent the development of DBD.σ Effect of Jiawei Wuling Powder on ROCK channelImmunohistochemistry results showed, ROCK1 and ROCK2 protein expressed in normal rat detrusor cytoplasm. Endoscopic manifestation showed brown section, roughly in line with antibody protocol, However model group showed significantly enhanced expression in ROCK1 and ROCK2 protein. Meanwhile Wulingsan groups down-regulate protein expression.Western Blot results showed that all Herb-formula groups have down-regulate trend of ROCK1 and ROCK2 protein expression, wherein the dose can be significantly reduced expression of ROCK1 (P<0.05).RT-PCR showed that High and moderate dose groups had inhibit ROCK1mRNA and ROCK2mRNA expression with a statistically significant difference, compared with the model group (P<0.05).Conclusion1. Because the pathogenesis is not yet clear, modern medicine treatment of diabetic bladder dysfunction did not reach ideal and specific efficacy, Traditional Chinese medicine has its unique advantage in the prevention and treatment of diabetic bladder dysfunction. Meta analyze shows TCM is more effective than western medicine, but the evidence may recommend in a lower degree. TCM need more large-scale randomized controlled clinical trials and mechanism experimental studies in order to provide a higher quality of evidence based medicine.2. Jiawei Wuling powder could improve detrusor function, postpone histomorphologic damage of bladder tissue and somehow prevent the development of DBD without effect on blood glucose.3. Jiawei Wuling Powder may improve Diabetic Bladder Dysfunction by inhibiting ROCK channel expression. |
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