| ObjectiveBased on the previous studies,this study replicated a model of diabetic neurogenic bladder in rats and used high-throughput,high-density,high-specificity protein chip technology to explore the effect of Modified Wuling Powder on interventional diabetic rat model of neurogenic bladder.The possible mechanism may provide a visual idea for the study of the multi-pathway and multi-target treatment characteristics of Chinese herbal compound.Methods1.Grouping and modelingFifty-five female SD rats weighing 160-180 g passed the quarantine test and were fed adaptively for 1 week.The body weight and blood glucose were measured.There were no significant differences.Fifty-five rats were numbered and divided into blank control groups(referred to as Control,n=8),model group(referred to as Model,n=15),modified wuling powder group(abbreviated as CHM,n=16),clobecholine group(abbreviated as WM,n=16),according to the random table method.And blank group continued normal feeding,the remaining groups were switched to high-fat and high-sugar diets.After 8 weeks,rats in the model group,modified wuling powder group and clobecholine group were injected intraperitoneally with STZ-induced diabetes.The blank control group was injected with the corresponding dose of physiological saline,after 3 days.Measurement of blood glucose ≥16.7 mmol/L was successful in diabetes mellitus.Unsuccessful or dead rats were discarded.The remaining three groups of rats continued to be fed with high-fat and high-sugar diet for 16 weeks to perform urodynamic tests to determine whether the model of diabetic neurogenic bladder rat is successful or not.2.Drug interventionThe doses of modifid wuling powder and clobetastimum were calculated according to the animal kilogram body weight coefficient method.The control and model groups were given the same volume of distilled water.The four groups of administration methods were intragastric administration for 4 weeks,and were performed after completion of urodynamic tests in the week 16.3.Drawing and testing(1)Blood glucose,body weight,and urine volume were measured regularly in experimental rats.Differences between groups and within groups were observed.Animals were observed for their general status to judge whether the models succeeded or not.Urodynamic tests were performed before and after drug intervention to determine whether the model is successful or not and the initial judgment of the effect of drug intervention.(2)After the urodynamic test was completed,the blood was taken and the bladder tissue was fixed in a 4% paraformaldehyde fixative.After alcohol gradient dehydration,transparent xylene,and paraffin embedding,the bladder tissues were cut into approximately 3-4 μ m sections and were observed by HE dye way.(3)After the blood was allowed to stand at room temperature for 4 hours,the serum was collected by centrifugation,detected using RayBiotech kit,and analyzed by GSR-CAA-67 protein chip technology combined with data analysis software.(4)Elisa validation analysis of rat serum protein chip results.Result1.Modeling:(1)General state: Rats in the control group without STZ intraperitoneal injections were full of energy and were responsive.Feed intake,water intake,and body weight all increased steadily.The coat was white and shiny and no hair removal was observed.Bright eyes and gods,hard texture of the stool,less urine,no obvious odor.The rats in the model group,jiawei wuling powder group,and chlorpyrifos group were wilted.After the diabetic model started,they had a violent temperament,with severe bite in the same cage,but they were unresponsive in the later period.They had more drink,more food,body weight loss,and coat colour.Yellowish dull,flake-like hair loss,with more severe neck and back,retinopathy of the eyes of three rats,pale eyes.Loose stools and increased stool volume,urine sour and turbid,urine output.(2)The blood glucose levels in the model group,the Chinese medicine group and the western medicine group increased after the modeling,and the average value was greater than 16.7 mmol/L,suggesting that the diabetes model was successfully established.(3)Compared with the continuous increase in body weight in the blank group,the body weights of the rats in the model group,modified wuling powder group and clobecholine group first increased and then decreased,which was related to the weight loss caused by diabetes after modeling.(4)Urodynamic test: There was no significant difference in the bladder leak point pressure(BLPP)and maximum voiding pressure(MVP)values between the four groups,P>0.05.Compared with the blank group,the increase of residual volume(RV),the decrease of maximal bladder capacity(VE),the urinary rate(MBC)and the increase of bladder compliance of the model group,the Chinese medicine group and the western medicine group were significant in the test indicators,P<0.05.There are no significant difference among the model group and the Chinese medicine group and western medicine group,P>0.05.2.Effects of modified wuling powder on DNB rats:(1)Urodynamic Test: The duration of diabetes was 16 weeks.There was no significant difference between BLPP and MVP in the four groups,P>0.05.Compared with the blank control group,The urinary kinetic parameters of the model group,the modified wuling powder group and the ciclopentine group were increased in RV,VE decreased,and MBC and bladder compliance were increased.The difference was significant(P<0.05 or P<0.01).There was no significant difference among the model group,the modified wuling powder group and the ciclopentine group,P>0.05.It was demonstrated that the detrusor injury and dysuria occurred in the bladder of DNB rats at 16 weeks of diabetes,which may still be in the transitional stage from compensatory to decompensated and bladder function is instable.So there was no significant difference in bladder leak pressure and maximum urination pressure,and the drug had no significant effect on its intervention.(2)Examination of bladder and kidney pathology: Bladder pathology showed that the bladder tissue of the blank group was basically normal,the bladder mucosa epithelial cells proliferated in the model group,the edema of the lamina propria,the infiltration of inflammatory cells,bladder inflammation of the rats of the Chinese medicine group and the western medicine group were all reduced,and the effect of Chinese medicine group was better than that of western medicine group.Kidney histopathology shows that Chinese medicine has a certain protective effect on the kidney,and can improve the inflammatory response of the kidney,relieve renal tubular dilatation,to a certain extent ease the diabetic kidney damage,better than the western medicine group.3.Protein chip detection:(1)Differential protein: There was 1 difference protein between the blank control group and the model group,ICAM-1,and there were 6 differential proteins between the blank group and the Chinese medicine group,namely B7-1,Gas.1.There are 7 differential proteins between IL-2 Ra,IL-22,Nope,Notch-2,and western medicine groups,namely ICAM-1,IL-1b,LIX,TCK-1,TIMP-1,Notch-1.Notch-2,one differential protein between the Chinese medicine group and the model group,namely L-Selectin,there are three differential proteins between the Western medicine group and the model group,namely L-Selectin,PDGF-AA,Notch-2.There are two differential proteins between Chinese medicine group and western medicine group,namely PDGF-AA,RANTES.(2)GO enrichment analysis: There were 414 GO terms that were significantly enriched between the TCM group and the western medicine group.The five biological functions most relevant to the enriched GO term were: a.ERK1/2(extracellular)Positive regulation of kinase.b.Signaling regulation of protein kinase B.c.Inositol lipid-mediated signaling pathways.d.Phosphatidylinositol-mediated signaling pathways.e.Phosphatidylinositol 3-kinase signaling pathway.There was no significant enrichment of GO between blank group and model group,Chinese medicine group and model group.(3)KEGG enrichment analysis: Through analysis,it was found that the most significant between the Chinese medicine group and the western medicine group is the cytokine-cytokine receptor interaction pathway.4.Elisa verification: The verification results are consistent with the results of the protein chip.ConclusionModified Wuling Powder can effectively improve the bladder function of DNB model rats,possibly through the intervention of its inflammatory mechanism. |
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