Tong Mai Oral Solution, The Chinese Medicine Compound Recipe Of Benefiting Vital Energy And Promoting Blood Flow: Clinical Observation And Experimental Study Of Its Renal Protective Effects On The Patients Suffered From Diabetes Nephropathy

BackgroundRecently, the prevalence rate of diabetes is rising unceasingly in ourcountry. Along with insulin widespread application, the mortality rate ofDiabetes' acute complication,such as ketoacidosis, are obviouslydropped, and the chronic complication such as diabetic nephropathy has becomeone of most common complications with diabetic life extension. In clinicalpractice, diabetes nephrosis is often divided into 3 stages, namely: morningstage, intermediate stage and advanced stage. In different stage, the emphasisof treatment is also different.Professor Yang consider that the Deficiency of Qi and the Blood Stasis isthe mainly etiopathogenesis of diabetic nephropathy, especially when diseaseis morning or intermediate stage, so the therapeutic rule-"benefiting vitalenergy and promoting blood flow" can applied in the treatment of diabeticnephropathy; when their stage are morning or intermediate, the effectivetreatment is the key of pathogenetic condition reverse. So we study themechanism of action of Tongmai Oral Solution to the diabetic nephropathy bythe methods of pharmacodynamics and morphology, in order to provide thesubstantial theory and plenitudinous scientic evidence for Tongmai OralSolution which is composed according to benefiting vital energy and promotingblood flow method cure diabetic nephropathy.Objective.To explore the effect and mechanism of TongMai Oral Solution in the defence and treatment of diabetic nephropathy, and found Tongmai Oral Solution' sfunction superiority stage.Methods:1). Clinical study:This part of the study is controlled. According to degree of proteinuriaand general condition, 63 patients suffered from morning or intermediate stagediabetic nephropathy were divided into treatment and control group. There wasno statistically significant difference between two groups in sex, age,severity of the disease and laboratory items. The patients of the treatmentgroup received management of rongMai Oral Solution while the patients of thecontrol group took the drug of Lotensin. The course of treatment lasted for8 weeks, during which the changes of clinical findings and lab index weremonitored and recorded, including renal function, proteinuria, blood fat,blood pressure, blood glucose, and clinical symptoms and signs. Thedifferences between the symptom index before and after treatment were thencalculated and tested, as well as the quantities of the urine protein of 24hours.2). Experimental Study: (1). PartⅠ. Study of protective effects of TongMai oral solution on thekidney of Diabetes Mellitus rats.a. preparation of animal model. 42 SD male adult rats of specified-pathogenfree were injected with streptozotocin (STZ) intraperitoneally to prepare forDiabetes Mellitus animal model and then randomly divided into 3 groupsincluding model group(M group), TongMai Oral Solution group(T group)andLotensin group(L group) after diabetes was induced successfully. Another groupwas set for experiment control, consisted of normal SD rats.b. Methods of intervention. The rats of T, L, M groups were raised withhigh fat forage every other day while the rats of normal group were raisedwith normal one. Different drugs were perfused into stomach of rats includingTongMai Oral Solution for T group, Lotensin for L group, and sodium chloridefor C and M groups. All the rats were observed closely for 6 weeks. Duringthis period, the rats with values of blood glucose less than 16.8 mmol/L orslight renal pathogenic abnormalities under light microscope were excludedfrom the experimental studies. Several items were recorded to assess renalprotective effects of TongMai oral solution, including general condition, body mass, urine volume, quantities of urinary protein of 24 hours, serum bloodcholesterol total, serum glycerol, blood glucose, blood urea nitrogen, serumcreatinine, and ratio of kidney mass to body mass (K/W).(2) PartⅡ. Study of renal protective effects of TongMai oral solutionon the rats of diabetes nephropathy animal model.After diabetes was induced successfully, the rats of diabetes animal modelwere continually raised with high fat food every other day for another 4 weeksin order to induce the diabetes nephropathy in them. The rats, in which diabetesmellitus were induced successfully, were then grouped and treated in a mannerthe same as that of PartⅠ, controlled by normal SD rats. After 6 weeks ofdrugs treatments or placebo managements, all the animals were put to deathand their kidneys were removed for the pathological study including sectionarea of glomerulus (MGA), section area of extracel]ular matrix (MMA),thickness of glemerular basement menbrance (GBM), and the ratio of thesection area of MMA to MGA.PatrⅢ. Study of effects of the TongMai oral solution on the expressionof MMP-9 and its substrates, as well as regulation factor6 renal samples were randomly obtained from the rats of group C, M, andT in PartⅡstudy, respectively. Expression of typeⅣcollagen, MMP-9, TIMP-1,and TGF-β1 were detected by immunohistochemistry. The stained area, areadensity, and photodensity were then observed and recorded. The ratio of stainedarea of MMP-9 to TIMP-1 was also determined.Results:1). Clinical study.(a). there's no statistical significant difference in the generalcondition of the patients, the quantities of urinary protein of 24 hours, andrenal function before treatment. No statistical significance was found in thedifference of blood pressure, values of blood glucose, and values of the bloodfat during the course of treatment.(b).As to the patients of T group, the symptomatic scores and quantity ofurinary protein of 24 hours after treatment decreased obviously compared tothat before the treatment, with statistical significance (P＜0.01).(c).There were no statistical significance found in the difference ofchanges of quantity of urinary protein of 24 hours after treatment betweenthe T group and L group (P＞0.05). However, the changes of symptomatic scores in T group was higher than that of L group after treatment with statisticalsignificance (P＜0.01).2) Experimental Study:(1). PartⅠ. Study of protective effects of TongMai oral solution on thekidney of Diabetes Mellitus rats.a. The changes of body mass and urine volume. The mean values of urine volumein the T, L, and M groups increased concomitant with loss of body mass withstatistical significance (p＜0.01), in contrast to that of C group. Althoughthere' s descent trend in the urine volume and increasing trend in the bodymass of T group compare to that of L and M group, no statistical significancewas demonstrated in the difference of the values of both items among T, L,and M group (P＞0.05).b. Quantity of urinary protein of 24 hours. Quantity of urinary proteinin T, L, and M group increased with statistical significance, compared to thatof C group (P＜0.01). Excretion of urinary protein in 24 hours in T groupdecreased statistically significantly, in contrast to that of M group (P＜0.01).No statistical significance was demonstrated in the difference of dischargeof urinary protein in 24 hours between the L and T group (P＞0.05).c. Changes of blood glucose level. The mean value of blood glucose of T,L, and M group increased with statistical significance, compare to that ofC group (P＜0.01). No statistical significance was demonstrated in thedifferences of values of blood glucose among T, L, and M group (P＞0.05).d. Changes of values of blood urea nitrogen and serum creatinine. There' sno statistical significant difference demonstrated between C, T, L, and M groupe. The cholesterol total(TCh) and glycerol total (TG) fluctuation. The meanvalues of ZCh and TG in T, L, and M group increased statisticallysignificantly, compared that of C group (P＜0.01). In T group, the values ofboth TCh and TG decreased with statistical significance, compared to that ofM and L group (P＜0.01 or P＜0.05).f. Ratio of kidney mass to body mass (K/W). The ratios of K/W of T, L, andM group increased with statistical significance, in contrast to that of C group(P＜0.01). The ratio of K/W of T group was less than that of M group withstatistical significance (P＜0.01). However, no statistical significance wasdemonstrated in the difference of ratio of K/W between the T and L group g. Pathological changes of kidney. Renal pathohistology changes in M group,such as glomerular hypertrophy and widening of mesangial region, were foundworse than that of C group. The similar pathohistology abnormalities were alsodetected in both T and L group but to a less degree. No difference was determinedto be statistically significant as to the severity of renal pathohistoloyabnormality between T and L group.(2) PartⅡ. Study of renal protective effects of TongMai oral solutionon the rats of diabetes nephropathy animal model.a. changes of body mass and urine volume. The mean value of urine volumein T group decreased while the body mass in T group increased, compared tothat of M and L group. Both the changes of body mass and urine volume werestatistically significantly (P＜0.05 or P＜0.01).b. The blood urea nitrogen. The mean value of blood urea nitrogen of T groupdecreased with statistical significance (P＜0.01 or P＜0.05).c. other items. Outcomes of quantity of urinary protein in 24 hours, TCh,TG, Blood glucose, serum creatinine, and ratio of K/W were referred to thatof PartⅠof the experimental study.d. The renal pathohistology. Microscopic abnormalities of the kidney formrats of M group included widening of mesangial region, thickness of renalbasement membrane, and vacuolar degeneration of renal tubular epithelia. Theserenal changes in M group were worse than that seen in the C group.Although similar renal abnormalities could also be found in T group, it' sobvious that the abnormalities became less serious in T group and L group.No difference was demonstrated between T and L group.e. Ratio of section area of extracellular matrix (MMA) and glomerulus(MGA). it' s higher of ratio of MMA/MGA in M group than that of C group withstatistical significance (P＜0.01). However, in T group the ratio of MMA/MGAwas less than that of both the M and L group (P＜0.01 or P＜0.05).f. The thickness of renal glomerulus basement membrane. The renal basementmembrane got thicker than that of C group with statistical significance(P＜0.01). Compared to that of M group, the GBM of T group was thinner withstatistical significance (P＜0.01).No difference of the same index was foundto be statistical between T and L group (P＞0.05).(3) PartⅢ. Study of effects of the TongMai oral solution on the expression of MMP-9 and its substrates, as well as regulation factor.a. Expression of typeⅣcollagen. The typeⅣcollagen was located in theglomerulus. Strong positive expression was found in group M. According to theoutcomes of immunohistology, the values of area density and photodensity ofpositively stained area were less than that of M group with statisticalsignificance (P＜0.01).b. Expression of MMP-9. The expression of MMP-9 was located mainly inglomerulus and sparse expression could be seen in renal interstitium. It'smore weakly expressed in group M than in C group. The value of area densityand photodensity in MMP-9 positively stained area were higher than that ofM group (P＜0.01).c. Expression of TIMP-1. Expression of TIMP-1 was mainly located inglomerulus with weakly expression located in renal tubule and interstitium.It's stronger positively expressed in M group than in C. The values of areadensity and photodensity of TIMP positively stained area in group T were lowerthan that of group M with statistical significance (P＜0.05).d. Ratio of MMP-9/TIMP-1. It's higher of ratio of MMP-9/TIMP-1 in groupC than that of group M with statistical significance (P＜0.01). However, thisratio in group T was higher than that of group M with statistical significance(P＜0.01). The ratio of MMP-9/TIMP-1 and photodensity of typeⅣcollagenchanged in reverse direction.e. Expression of TGF-β1. Expression of TGF-β1 was located in the renaltubule and interstitium. It's weakly expressed in the glomerulus. It's lowerfor the value of area density and photodensity in group T than that of groupM with statistical significance (P＜0.01).Conclusion(1). Tongmai Oral Solution can improve the patients' clinical symptomsand renal function, decrease the urine protein.(2). The mechanism that Tongmai Oral Solution ameliorated the structureand function damage of DN model rats may be due to enhancement of metabolismof blood fat, up-regulation of the expression of MMP-9, down-regulation ofthe expression of TGF-β1, rebuilding of balance between MMP-9 and TIMP-1,inhibition of thickening of the glomerular basement membrane, increase ofdegeneration of typeⅣcollagen, reduction of extracellular matrixaccumulation, and amelioration of renal pathological lesions. These therapeutic effects may improve renal hypertrophy, regulation of cells, ECM,cytokines, and growth factors resulting in delayed progress of DN.(3). In the course of intervention of DN model rats, excellent renalprotective effects could be obtained not only at the beginning of initiatestage of model induction but at the time when urinary protein appears in theurine of DN model rats. This reveals that the point that the urinary proteinappearances could be regarded as initiate point when Tongmai Oral Solutionis given.