| Part One:Literature ReviewDiabetic nephropathy (DN) is one of the micro-vascular complication caused by diabetic mellitus (DM) and it has been the leading cause of end-stage renal disease with diabetes-related patients. The concept in understanding the pathogenesis of DN is that the results from a series of specific progressive renal pathological changes that have occurred incidence early in the course of diabetes. The early renal pathological changes is characterized by accumulation of extracellular matrix, leading to expansion of glomerular mesangial regions at the expense of filtration surface area, thickening of the glomerular and tubular basement membranes. Important changes in arterioles and in the renal interstitium also occur as well. These changes cause glomerular hypertension and progressive decline in GFR. Systemic hypertension may accelerate progression. The determinants of these early structural abnormalities are, however, largely remain unknown.Medical research has demonstrated that a number of signal transduction pathways may have played the important roles in onset of DN. Nowadays, the key element of treatments are patient's education, dietary and exercise counseling, monitoring of glucose control, improving of lipid metabolism disorder and taking ACE inhibitors or angiotensin II receptor blockers accordingly. The treatments may in certain extent to improve and control the symptom of DN and prevent ESRD developing. Therefore, it is crucial to better understand and take the necessary aggressive-preventive measures to slow this progressive disease process.Diabetic nephropathy is known as modern medical term and there is no corresponding name has mentioned in ancient Chinese medicine literature. According to pathogenesis and clinical manifestations of Diabetic Nephropathy, it should be categorized in TCM as "wasting-thirsting" or "xiao ke" accompany with its complications, such as "edema", "consumptive disease", "kidney consumption", "dropsy disease", "swollen disease", "turbid urine", "Vomiting and Dysuria Diseases". TCM believes that DN is caused by many factors, its basic pathogenesis is deficiency in origin and excess in superficiality. Deficiency stands for insufficiency in zang-fu organs of qi, blood, yin and yang. Excess represents those pathological products, such as phlegm accumulation, fluid retention, and blood stasis. Yin dysfunction in lung, kidneys, spleen and stomach, caused lung dryness, heat of stomach and deficiency in kidneys Yin. Deficiency in kidney Yin is the main determinants of all. Clinical manifestations shows that intermingled deficiency and excess for many cases. The therapeutic principles are formulated according to the basic theory of TCM and syndrome differentiation. According to the different course of the disease, the conflict between the anti-pathogenic qi and pathogenic depend on strong or weak, advance and retreat, of either side. The principle of treatment is based on syndrome differentiation, development of disease, the strength of human body against the disease, and the function of Zang-fu with its qi and blood, yin and yang to have overall regulation. Treatment is concentrated on replenishing Qi and nourishing Yin as well as removing blood stasis in assistant with tonifying the kidney and liver at early stage. Treatment for invigorate the spleen, warm the kidney supported by benefiting Qi and nourishing blood and removing blood stasis is suggested during metaphase stage; to warm and tonify the spleen and kidney accompany with inducing diuresis and alleviating edema at the advanced stage as a result to reduce proteinuria and improve renal function, controlling the development of the disease. TCM hold unique advantages in treating DN. Pharmacological effects of Chinese medicine and its formulation have multi-level, multi-target and multi-connection features with long term stability of curative effects, which are superior to Western medicine. On clinical practice, it proves that TCM has remarkable curative effects in improving symptoms, protecting renal function, preventing or postpone the progression of renal damage and improving the living quality of patients.The research base on TCM classical prescription "Taohe Chengqi Decoction" has already carried out since 80's regarding to the treatment on type 2 diabetes against the pathogenesis of deficiency in Qi and Yin, blood stasis obstruction of collaterals has gained the notable effects. According to the former studies, Jiangtangsanhuang (JTSH) tablet has been developed for the treatment of diabetic nephropathy under the theoretical basis of stomach and kidney in both physiology and pathology are interaction and interfere. Thus, further study on the mechanism of JTSH tablet for DN in order to be broadly adopted by clinical practice has become profound significance.Part two:Experimental researchObjective:The study had used high cholesterol diet pattern which is closer to the human lifestyle to observe the effects of JTSH tablet on blood glucose, urinary protein in 24hours, renal function, rennin activity, the content of angiotensinⅡ, the expressions of transforming growth factor-β1, the function of PKC and the pathological changes of renal tissue for the early stage of diabetic nephropathy rats, to reveal the mechanism of reno-protective effect of JTSH Tablet against the experimental DN rats. So as to provide experimental basis of clinical service for treating early stage of Diabetic Nephropathy by using purge Heat and blood stasis with nourish qi and yin therapy. Methods:Healthy Wistar rats were randomly divided into normal control group (n=10) with regular feed and diabetic model groups with high cholesterol diet (HCD) throughout the study period. The model groups induced lower-dosage streptozocin (STZ) (30mg/kg) after 28 days of HCD and were randomly divided into three group with fasting blood glucose≥11.1mmol/L:early DN group(n=10), JTSH group(787.5mg·kg-1·d-1,n=12) and Captopril group(4mg·kg-1·d-1,n=12). The course of treatment lasted for 8 weeks. The general status (psychosis, diet, hair color, weight, etc.), blood glucose, glycosuria, renal weight/body weight ratio, urine protein in 24 hours were monitored.①One and eight weeks after STZ rejection, fasting blood glucose, cholesterol and insulin levels were detected at first and eighth week after STZ-induced;②Renal tissue were detected for the expression of TGF-β1 and activity of PKC by the method of immune histo-chemistry.③Renal Angiotensin converting enzyme(ACE)activity and angiotensinⅡ(AngⅡ) expression were measured by radio immunity.④The renal pathological changes were observed by optics microscope and electronic microscope.Result:1. The various items were observed at the end of 8weeks of experiment. The general conditions and the symptoms of polyphagia, polydipsia, polyuria were improved in JTSH group, the body weight had boosted faster than DN and Captopril groups with obvious significant (P<0.01). JTSH group had significantly reduced FBG, cholesterol, glycerin trimyristate level and increase ISI level after 8 weeks treatment comparing with DN and Captopril groups (P<0.01; P<0.05). It indicated that JTSH tablet have better effect in improving glucose metabolism than Captopril. Besides, decrease obviously in the amount of urine and urine protein of 24 hours of treated groups compared with DN group (P<0.05). Kidney coefficient of JTSH group compared with DN and Captopril groups were decreased (P<0.05 and P<0.01) respectively.2. The radiative immune method and biochemical method were adopt for detecting angiotensin converting enzyme(ACE)activity and the content of angiotensinⅡ(AngⅡ) in renal tissue. The results indicated that ACE activity increased obviously in DN group compared with the others (P<0.01); ACE activity was low in Captopril group compared with the Normal, JTSH and DN groups respectively (P<0.05, P<0.01). It proved that JTSH can suppress ACE activity in certain extend, while its contribution is not as strong as Captopril as angiotensin converting enzyme inhibitor. The content of AngⅡin renal tissue was raised in DN group compared with others (P<0.01). The comparison between two treated groups was no statistical significance. The result showed that both JTSH tablet and Captopril can protect the renal function by reducing and adjusting the AngⅡin renal tissue of the DN rats.3. The rats'renal histopathology was observed by optical microscope and electric microscope at the end of the experiment. It was found that DN group glomerular basement membrane thick, mesangial matrix expansion and part of glomerulor vascular abnormally changed. The treated groups were found the above pathological changes had improved after 8 weeks treatment. It indicates that the damage of renal tissue might have occurred at the very beginning and increased gradually. Glomerular capillaries stromal hyperplasia significantly in DN group and Captopril group compared with JTSH (P<0.05) while index of glomerulosclerosis is increased significantly compared with normal and JTSH group (P<0.01). It looks that JTSH tablets were more effective in improving glomerular sclerosis and renal tubular edema degeneration of DN rats than Captopril, but difference is not outstanding. The result indicates that JTSH can slow down and relieve the kidney damage.4. The immunohisto-chemical Method is used for detecting the expression of TGF-β1 and the activity of PKC in kidney among groups. The expression of TGF-β1 among DN group was significantly higher than normal group (P<0.01), as well as JTSH and Captopril groups (P<0.01). The PKC activity in renal tubular was raised in DN group compared with JTSH and Captopril groups (P<0.01), whereas still higher than the normal group (P<0.01).Conclusion:1. JTSH tablets can significantly reduce FBG, cholesterol and glycerin trimyristate level, as well as ameliorate the insulin resistance. In addition, it can improve "three more and one less" symptoms which embody the holistic concept of TCM.2. JTSH tablets can reduce the excretion of urine protein of the DN rat and suppress the hypertrophy of kidney and reduce renal pathological damage at the same time, so as to relief the renal pathological progression. The mechanism of the improvement might have related to JTSH rectified the disorder of lipid metabolism and abnormal of blood rheology.3. JTSH can inhibit the expression of AngⅡin renal tissue of the DN rats which could be one of the mechanism of improving renal function and relief renal pathological damage.4. JTSH can restrain the PKC and TGF-β1 from over expression which is the important part to reduce the accumulation of extracellular matrix and prevent the renal being damaged.5. The mechanism of reno-protective effect of JTSH tablets may probably worked by inhibiting the Angll activities, controlling and transferring the up raising of PKC activities and reducing the expression of TGF-β1 as a result to reduce the accumulation of glomerular extracellular matrix and improved the renal pathology in order to reach the goal in protecting the renal function.
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