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Experimental Studies of Shenkangwan in Diabetic Nephropathy

Author: LongHaiBo
Tutor: WeiLianBo
School: First Military Medical University
Course: Traditional Chinese Medicine
Keywords: Shenkangwan Diabetic nephropathy Oxidative stress Angiotensin Advanced glycation end products
CLC: R259
Type: PhD thesis
Year: 2007
Downloads: 303
Quote: 1
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Abstract


With the enhance of living standard, the changing of life style and the aging of the population, the incidence of diabetic mellitus (DM) is rising rapidly in world widely, which has become the third non-infectious disease listed behind cardiovascular diseases and tumour in developed country. So, it has become a cosmopolitan public health problem threatened human health. Because of a total incidence of 47.66% in DM, diabetic nephropathy(DN)is one of the primary complications of DM in clinic. And it is a leading cause resulting in death among type 1 DM patients, and only atherosclerosis of coronary and cerebral arteries preponderate over it in severity in type 2 DM patients. According to some surveys, about 17~33.8% renal function failure patients receiving renal replacement treatment(RRT) is resulting from DN, and it is a leading cause resulting in end-stage renal disease(ESRD) in DM patients too. And it was observed that the death rate in DM patients which received RRT, with a survival rate of two years accounting for 50% or so, were far more than that in non-DM patients. So how to treat and prevent the DN effectively is becoming a common concerned problem among the international medical researchers.The pathogenesis of DN is complicated and some of them remain poorly understood. Nowadays, it is widely accepted that oxidative stress(OS) is a primary common mechanism in the occurring of chronic complications of DM. Simultaneously, many researchers have found there was a abnormity of renin-angiotensin system(RAS) in local diabetic kidney tissue, and believed it might play a pivotal-role in the occurrence and progression ofDN. AngiotensinⅡ(AngⅡ) is the principal effector of RAS, and has multiple direct pathopoiesia actions via the combination with type 1 angiotensin receptor(ATIR), including glomerular sclerosis and interstitial fibrosis. Many studies have confirmed that the inhibition of the RAS by the use of angiotensin converting enzyme inhibitors (ACEI) or angiotensinⅡreceptor blockers(ARB) offer renoprotection, reduce the albuminuria excretory rate and delay the progression in DN. In addition to OS and RAS, advanced glycation end products(AGEs), which development its biological effect via combining with receptor for AGEs (RAGEs), also play an important role in the pathogenesis of DN. By interacting with each other AGEs and RAGEs compose a regulation network. Therefore, to illuminate AGEs and RAGEs system possess an important meaning to elucidating the pathogenesis of DN.The use of ACEI and ARB is the most extensive and important therapy in the treatment of the DN in western medicine at present, in addition to the regulation of diets and controlling of the intensive blood glucose. However, the effects of these therapies are proved to be limited, and so far neither one specificness medicine can treat DN effectively. DN belongs to the domain of the "Shenxiao", "Dropsy", "Shennao", "Guange", and so on in traditional chinese medicine(TCM),. "Splenic and renal weaken’is the basal pathogenesis of DN and the"Phlegm and damp", the"Grime and toxin", the"Gore"are the stimulative factors. So the "fundamentality weaken and sign mighty"is the TCM characteristic pathogenesis of DN. Early DN(Ⅰ~Ⅲstages) always appears the symptoms of "Yin weaken"or"both Qi and Yin weaken". Clinic DN(Ⅳstages)always comes forth the symptoms of "splenic and renal weaken", "liquid and humidity logjam" or "gore stagnateing vas". While end DN (Ⅴstages)always emergences the symptoms of"downfall of Yin and Yang in spleen and kidney" and "grime and toxin blocking". So the therapeutic principle of DN inⅢ~Ⅴstages includes "increasing the Qi and toughen the spleen", "nourishing the kidney and maintain Yin" and "impelling blood stream and dredging vas". Although TCM is superior to western medicine in treatment of DN, there are no effective Chinese materia medica to treat the patients with DN, because most of them are still in the period of therapeutic effect observation or experience accumulating.On the basis of our long-term clinic experience on DN treatment and research on it, we created a new compound preparation with well selected and essential traditional Chinese drug, and named it "Shenkangwan". Long-term use of Shenkangwan in clinic have proved it could treat DN effectively, and has attained national invention patent ("the study on the prescription and craft of Shenkangwan"; patent number: 200610036515.2). Shenkangwan was mainly constituted of huangqi, gordon guryale seed, chrokee rose fruit, leech, herba leonuri, and so on. It has the effect of increasing the Qi and toughen the spleen, nourishing the kidney and constringency, diuresis and detumescence, impeling blood stream and dredging vas. So it accords with the TCM characteristic of pathogenesis ofⅢ~V stages DN fitly and can treat the DN from the fundamentality to the sign. But its eytomolecular mechanism in treating DN is not clear and need farther research. So we carried out the experiment on animal to explore the cytomolecular and genie mechanism of Shenkangwan in treating DN and provide the laboratorial evidence for its clinic application. The details were as follows:ChartⅠRenoprotective effect of Shenkangwan in diabetic nephropathy ratsObjective: To observe the effect of Shenkangwan in treating rats of DN model and protecting their kidneys and provide the laboratorial foundation for further studying its therapeutic mechanism. Methods: Firstly, we established the DM rat models by intraperitoneal injection of streptozotocin(STZ) according to the dose of 55 mg·kg-1body weight. In the following two weeks, we detected the blood glucose, the uric glucose and the urine volume. The rats, which value of blood glucose exceeding 16.7mmol·L-1, uric glucose positive experiment over +++, urinary volume surpassing 150% basal level and observation measurements on urine microalbumin showing positive were taken for the successful DM rat models and calculated the rate of them. The death rate of rats was 17.65% after model created. After 4 weeks, they were randomly divided into 4 groups(n=7): model control group; Shenkangwan group; Irbesartan group and conjunct therapy group. All rats were reared in metabolic cage in the same animal room, fed with standard diet and freely drunk. Other six normal rats were used as normal control group. All rats were treated with corresponding drugs for 8 weeks. During and after the treatment, the general state, blood and uric glucose levels of the rats in every group were observed. After treated in the last time, all rats were put into the metabolizing cage to be measured the volume of their 24 hours urine and drinking water. Then we hocused all the rats with 10% chloral hydrate, collected the blood via ventral artery, gathered the serum by centrifuge and detected the content of the blood glucose, the excretion of the 24 hour urine protein(Upro)and the levels of serum creatinine(SCr), blood urea nitrogen(BUN), triglyceride(TG), total cholesterol(TC). And we took out the kidney of the rats, observed the size and volume of them and measured the kidney weight and relative kidney weight. Then we investigated their renal pathological changes by optical microscope with the coloration method of Hematoxylin and Eosin(HE) and electron microscope.Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results of two groups before and after treatment were analyzed using paired t-test for comparisons. Repeated measurement experiment was used to analyze the statistical significance among several groups. Results after treatment among multi-groups were analyzed using one-way ANOVE, Student-Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P<0.05 was considered to be statistically significant.Results:1.32 rats were used to established the DM models and the rate of successful models was 82.35%(28/34). And the death rate of DM model rats was 14.29%(4/28).2.During the treating eight weeks, The model rats blood glucose were all over 16.7 mmol·L-1, the uric glucose positive experiment were exceeded +++. And they appeared the symptom of the DM such as the quantum of drinking, eating and urinating increased.3. Compare with the normal rats, their following indexes including UN、Cr、TC、TG、24h Upro were increased distinctly(P<0.01). Their kidney weight and relative kidney weight increased distinctly(P<0.01). And kidney tissue came forth about the 3 stages renal pathological lesions according to the stages of Mogensen.4. Compare with the rats in model group, all three treatment could ameliorate the rat’ general state, decrease the above indexes markedly(P<0.01)and mitigate the renal pathological lesion.5. Shenkangwan and Irbesartan have a synergetic renoprotective effect on DN rats.Conclusions:1. Shenkangwan can play a certain renoprotective role in diabetic nephropathy rats.2.Shenkangwan and Irbesartan perform a synergetic renoprotective effect on DN rats. ChartⅡEffect of the Shenkangwan on the renal oxidative stress in diabetic nephropathy ratsObjective: To investigated the effect on the renal oxidative stress in early diabetic nephropathy rats in order to study the renalprotection mechanism of Shenkangwan.Methods: The kidneys used of this study were from the rats in chartⅠ. The renal intrinsic malondialdehyde(MDA) levels and antioxidatant enzyme activities such as glutathione peroxidase(GSH-Px), catalase(CAT) and Cu-Zn superoxide dismutase(SOD) were measured by corresponding methods. Meanwhile, the expression of GSH-Px, CAT, Cu-Zn SOD in the renal tissues of the rats in every group were detected with the method of the reverse transcription-polymerase chain reaction(RT-PCR).Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results after treatment among multi-groups were analyzed using one-way ANOVE、Student- Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups. P<0.05 was considered to be statistically significant.Results:1. Comparing with normal group, the content of MDA was increased significantly and the activity of GSH-Px, CAT and Cu-Zn SOD were down-regulated markedly in the renal tissue of DN model rats(P<0.01). The renal tissue expression of GSH-Px, Cu-Zn SOD mRNA were increased distinctly(P<0.01), but the expression of CAT mRNA was not significantly changed(P>0.05). 2. There was a significant difference in the content of MDA among five groups after treatment(X2=26.753, P<0.001), and the mean rank of DN model was the highest. Compared among three treated groups, that of Irbesartan group was the highest.3.There were significant difference in the activity of GSH-Px, CAT and Cu-Zn SOD(F=100.231、91.88、133.868 respectively; P=0.000). Compare with those in the rats of the DN model group, the activity of GSH-Px, CAT and Cu-Zn SOD in the rats of Shenkangwan, Irbesartan group and conjunct therapy group were up-regulated markedly(P<0.001).Multiple comparison showed no significant difference in these between Shenkangwan and Irbesartan group(P=0.097, 0.452, 0.086 respectively), while compared with conjunct therapy group significant difference were found in these of both two groups(P<0.001).4.Significant difference were observed in the expression level of GSH-Px and Cu-Zn SOD mRNA in the kidney tissue of rats in all groups(X2=23.287, F=29.099, P<0.001), but not in CAT mRNA(P=0.216). The mean rank of the relative expression level of GSH-Px mRNA was the largest in all groups, but that in Skenkangwan was the largest in three treated groups. Compare with that in model group, the relative expression level of Cu-Zn SOD mRNA were markedly up-regulated in all treated groups(P<0.001), while compare with that in conjunct therapy group, significant difference were found in Shenkangwan and Irbesartan groups, but no significant change was found in that between Shenkangwan and Irbesartan groups(P=0.350).Conclusions:1.Shenkangwan can inhibit the renal oxidative stress in early diabetic nephropathy rats.2.Shenkangwan can play a synergistic inhibiting role on the renal oxidative stress with Irbesartan in early diabetic nephropathy rats. ChartⅢEffect of Shenkangwan on the renal angiotensinⅡand its typeⅠreceptor expression in diabetic nephropathy ratsObjective: To explore the effect of Shenkangwan on the expression of AngⅡand AT1R in the kidney tissue of DN model rats.Methods: The blood plasm and kidneys used of this study were from the rats in chartⅠ. Radio-immunity was used to measure the content of AngⅡin the blood plasm and kidneys. The expression of AngⅡand AT1R protein in the renal tissues were analyzed with the method of immunohistochemistry. RT-PCR was used to detect the expression of AT1R mRNA in the tissues.Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results after treatment among multi-groups Were analyzed using one-way ANOVE、Student- Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups. P<0.05 was considered to be statistically significant.Results:1. Comparing with normal group, the content of AngⅡwere increased significantly in all samples of DN model rats(P<0.001). Significant difference were observed in the content of AngⅡboth in the blood plasm and kidney tissues of rats in all five groups(F=50.152, 126.110, respectively, P=0.000). Compare with that in model group, the content of AngⅡboth in the blood plasm and kidney tissues of rats in Shenkangwan group were decreased distinctly(P<0.001). And such did it when compared with Irbesartan and conjunct therapy groups(P<0.001).2. Immunohistochemistry analysis showed significant difference in the renal tissue relative expression of AngⅡin all five groups after treatment(X2=23.837, P=0.000). The mean rank of that in Irbesartan group was the largest in all groups.3.There were significant difference in the relative expression level of AT1R protein and mRNA in the renal tissues in all groups after treatment(X2=24.366, 22.748, respectively; P=0.000). The mean rank of those in model group was the largest in all groups. In three treated groups, that in conjunct therapy group was the smallest, then is that in Irbesartan group.Conclusions:1. Shenkangwan can decrease the expression level of AngⅡboth in the blood plasm and kidney tissue, reduce the content of AT1R in the kidney tissue and inhibit the renal expression of AT1R mRNA in early diabetic nephropathy rats.2. Shenkangwan can antagonize partly the increase of the expression level of AngⅡboth in the blood plasm and kidney tissue induced by Irbesartan.ChartⅣEffect of Shenkangwan on the renal advanced glycation end products and its receptor expression in diabetic nephropathy ratsObjective: To investigated the effect of Shenkangwan on the AGEs and RAGEs in diabetic nephropathy rats.Methods: The blood plasm and kidneys used of this study were from the rats in chartⅠ. Radio-immunity was used to measure the content of AGEs in the blood plasm and kidneys. The expressions of RAGEs protein in the renal tissues were analyzed with the method of immunohistochemistry. RT-PCR was used to detect the expression of RAGEs mRNA in the kidney tissues.Statistical analysis: All values are expressed as the mean±standard deviation((?)±s). Statistical analysis was performed using the statistical package SPSS for Windows Ver. 13.0. Results after treatment among multi-groups were analyzed using one-way ANOVE、Student-Newman-Keuls (SNK) for comparisons and multiple comparisons. If there was heterogeneity of variance, the data should be transformed first, and then analyzed. Provided that variance remained heterogeneity after data transformed, the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups. P<0.05 was considered to be statistically significant.Results:1.Compare with those in normal group, the content of AGEs both in the blood plasm and renal tissue were increased significantly(P<0.01). Compare with those in model group, those in all three treated groups were decreased matrkedly(P<0.01). Significant decrease were found in the content of AGEs both in the blood plasm(P<0.05) and renal tissue(P<0.01) in conjunct therapy group compared with Shenkangwan and Irbesartan groups.2. Compare with those in normal group, the expression level of RAGEs protein and mRNA were up-regulated distinctly(P<0.01) in the renal tissue of rats in model group. Compare with those in model group, those in three treated groups were down-regulated significantly(P<0.01 in Shenkangwan and Irbesartan groups, P<0.05 in conjunct therapy group, respectively). Markedly down-regulations were observed in those in conjunct therapy group compared with those in Shenkangwan and Irbesartan groups(P<0.05). And no significant difference was found in those between Shenkangwan and Irbesartan groups(P>0.05).Conclusions:1. Shenkangwan can decrease the expression level of AGEs both in the blood plasm and kidney tissues, reduce the content of RAGEs in the kidney tissue and inhibit the renal expression of RAGEs mRNA in early DN rats.2. Shenkangwan and Irbesartan have a coordinate repression on AGEs-RAGEs system in DN rats model. Conclusions of the whole paper: We carried out the experiment on animal and cell and proved again that Shenkangwan could treat DN and had a certain protective effect on the kidney of DN. Its therapeutic mechanism was related to retarding OS, reducing the synthesizing AngⅡand down-regulating the expression of AT1R, decreasing the content of AGEs and the expression of RAGEs. And those provided the cytomolecular and genic therapeutic mechanism and laboratorial evidence for its clinic treatment on DN.

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