| Diabetes mellitus (DM) is a progressive chronic endocrinic and metabolic disease with high morbidity and severity, which usually affects eyes, kidneys, heart and brain and other organs. Diabetic nephropathy (DN), caused by diabetic microvascular lesions, is one of the chronic complications of DM. The exact mechanism of DN is not clear yet. Currently, the blood dynamic changes, hyperglydemic biochemic and metabolic abnormity, effect of cell factor and growth factors and other reasons may play a role in this procedure.The main pathological changes of DN are the deposit of glomerular extrcellular matrix (ECM). ECM is composed mainly of type IV collagen and fibronectin,which keep metabolic balance by rapid refresh .When the balance is broken,it will lead to glomerular lesion and even glomerular cirrhosis, and causes DN by the end. Researchers indicated that functional disorders of endothelial cells, secretion of vascular endothelial growth factor (VEGF) by the glomerular podocytic cells and the activativation of mesangial cells, occur during the early period of DN. Expression of VEGF gene elevates under hyperglycemic condition. VEGF's level highly correlates with uremia microprotein and participates in the procedure of nephrosclerosis. Connecture tissue growth factor (CTGF) plays a role in activativation of the fibroblasts and generation and deposit of ECM, and participates in many courses of DN, and mainly induced by transforming growth factorβ(TGF-β) .TGF-βcan upregulates the protein inhibitors such as PAZ and TEMP and also downregulates the matrix degradation enzymes, which suppresses the degradation of newly formed extra cellular matrix, promotes the expression of the matrix surface integrin and strengthen the interaction of matrix. The aggregation of sorbitol as well correlates with DN. Activation of Aldose Reductase (AR),the key rate-limiting enzyme of polyalcohol pathway,plays an important role in the initiation and development of DN. The oncogen c-akt's product PKB/Akt has a crucial effect on cell surviving, glucose metabolism, protein synthesis and so on. AKT, as the downstream molecule of insulin receptor, has an important effect on the insulin metabolism, hormone signal conduction and glucose transshipment. It is the most important thing to establish the ideal DN animal model to illuminate the pathologic mechanisms and choose effective drugs for DN. The heredity DM pure inbred strain animals are the most common choose domesticly or abroadly. But those animals are quite rare and hard to feed. What's more, the pathogenesis of these models is mainly caused by heredity, which does not meet with the clinic characteristic. Feeding with high-calorie food and Streptozotocin, mononephoroctomy, injecting with Doxorubicin hydrochloride and other such kind of methods are used to establish DN model, but the systemic comparison between these methods is not available yet. We compared the models by these methods and find an optimal way to establish DN model.Compound recipes or extracts of traditional Chinese medicines have being used to treat DN extensively and some of them seem to show effects on DN' s prevention and treatment in molecular biochemic and genetic level. Rhizoma Coptidis is exacted from the rhizome of Coptis chinensis Franch , which contains many types of alkaloids , such as Berberine,Palmatine, Coptisine,Worenine,Jatrorrhizine,Magnoflorine etc. Presently, researches on Berberine and Berberastine are more extensive, mainly on the improvement of clinic symptoms, but the molecular mechanisms are still not clear. Few researches are published on other alkaloids. The alkaloid functional targets and the functional mechanisms of Rhizoma Coptidis need to be explored.This subject is the preliminary research on the prevention and treatment and the mechanisms of Total Alkaloids of Rhizoma Coptidis administration on DN rat model in vivo and adopt the method of ethanol circumfluence to extract and decompress to obtain the coptis alkaloid. Establishing DN animal model by cross-combination of feeding with high-calorie food and Streptozotocin, mononephoroctomy, and injecting with Adriamycin and choose the best combination,which would be used to establish DN animal model in the following studies. Grouping with low, medium, high dose of Rhizoma Coptidis and set up the positive control by Metformin Hydrochloride. We preliminary studied on protection and treatment and the mechanisms of Total Alkaloids of Rhizoma Coptidis administration on DN rat model in vivo. And we observe the function of Total Alkaloids of Rhizoma Coptidis on glomerular mesangial cells in vitro. The primary cultured glomerular mesangial cell are used to observe the fuction of Total Alkaloids of Rhizoma Coptidis on cell hyperplasia and the expression of TGF-βand TIMP-1 by hyperglycemic condition. The Metformin Hydrochloride act as control.1. Using ethanol circumfluence 3 times, mixing the filtrate through, decompressing, condensing and vaporing the solvent, obtaining 3.8g crude drugs in per gram of extract ion. Using ion pair to extract and UV-spectrophotometry method to measure the average content of total Alkaloid of Rhizoma Coptidis were 31.4±1.41%, and HPLC method to determine the average content of berberin of its were 16.42±1.6%.2. Adult male SD rats were divided into five groups: A is normal group: common feed. B is high-lipids and high-sucrose feeding group: high-lipids and high-sucrose feeding. C is high-lipids and high-sucrose feeding+STZ group: high-lipids and high-sucrose feeding for 6w, abdomen administration of STZ. D is high-lipids and high-sucrose feedin g+ Doxorubicin hydrochloride + STZ group: high-lipids and high-sucrose feeding for 4w, abdomen administration of Doxorubicin hydrochloride, abdomen administration of STZ 2 weeks later. E is high-lipids and high-sucrose feeding+mononephroctomy+STZ group: high-lipids and high-sucrose feeding feed for 4w, mononephroctomy, abdomen administration of STZ 2 weeks later. We concluded that E group is the ideal method that the rats appears evidence polydipsia, polyphagia, polyrexia, hypergycemia, hyperglyceremia, microprotein urine, abnormal glomerular filtration ratio and active oxygen, increase of kidney/body weight ratio, marked increase of aldose reductase . The pathology shows the glomerular destroyed, membrane widen, tubular intracellular cell disorders and vacuolization. The super mocropathology showed the glomerular volume increased, base membrane thickened and widened locally, mesangial cell proliferated, extracellular matrix increased, podocytes fused, mitochondria swoll and increased.3.Our initial studies found that Total Alkaloids of Rhizoma Coptidis can decrease the level of blood sugar and lipid of DN rats. This research shows that feeding rats with high-lipids and high-sucrose food for 1 month induced insulin resistance, and administrating 2 weeks after mononephroctomy STZ can induce DN. The model shows hyperglycemia, elevation of body weight slowing down,high kidney/body weight ratio. After 8 weeks microprotein appeared in urine;while after 12 weeks blood insulin and GFR increase,blood urea,crea,TG,glycohemoglobin and urine creatine disordered. MDA of B group decreases significantly, SOD and all types of NOS increase significantly. low, medium, high doses of Total Alkaloids of Rhizoma Coptidis and Metformin Hydrochloride are given respectively to the models, the targets showed different improvements, and the medium dose group improved most significantly. All doses of Total Alkaloids of Rhizoma Coptidis can decrease the AR activity in rat red blood cells. 4.The HE and PAS staining of glomerular showed that the volume of the glomerules increased,membrane widen,tubular epithelial cell disarranged and vacuolized. The super micropathology shows that in the model of rats,the volume of the glomerule increased,basal membrane widened and thickened locally, mesangial cells proliferated, extracellular matrix increased, podocytes fused,mitochondria swoll and increased. After treatment with Total Alkaloids of Rhizoma Coptidis, the pathology of DN improved, but did not converse completely.5.In this DN model,the immunohistochemistry result of the sections of organ shows the OD of VEGF,TGF-β1,CTGF increased and have significant difference. The average OD of those factors got lower than models, but didn't return to a normal level. We adopt RT-PCR to observe the effect of high,medium,and low dose of Total Alkaloid and Metformin feeding on expression of TGF-β1,TIMP-1,MMP-2 mRNA. It showed that expression of TGF-β1,TIMP-1 mRNA elevated, while those of the group treated by Total Alkaloids of Rhizoma Coptidis declined. MMP-2 mRNA of the model declined, the average of the group treatd enhanced. We adopt Western blot to observe the effect of high,medium,and low dose of Total Alkaloid and Metformin feeding on the expression of MMP-9.It showed that level of MMP-9 declined, while after being treated,the level of MMP-9 elevated. It prompted that Total Alkaloid may take effects on TGF-β1,VEGF,CTGF,MMP-9,TIMP-1 and MMP-2 , in order to protect rats from getting Diabetic nephropathy.As the main target cell of DN, glomerular mesangial cells are of high sense in DN pathegenesis by observing its changes under the condition of hyperglycemia. The glomerular mesangial cell cultured in hyperglycemic condition is a good model in vivo. We observed and verified the forms of the primary cultured SD rat renal glomerular mesangial cells. We detected the effect of Total Alkaloids of Rhizoma Coptidis on hyperglycemic GMC and concluded that Total Alkaloids of Rhizoma Coptidis can inhibit the hyperplasia of GMC evidently.We initially observed in a hyperglycemic circumstance, the total Alkaloids of Rhizoma Coptidis system stimulated mesangial cells to express TGF-β1 , activity of TIMP-1 declined, while that of MMP-9 elevated. Those prompt Total Alkaloids of Rhizoma Coptidis possibly upregulates the expression of TGF-β1 , MMP-9 and the TIMP-1 through affecting GMC in vitro.
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