Renal Protection Of Astragalus And Its Mechanism In Type 2 Diabetic Rats

In recent years, the incidence of the diabetes mellitus (DM) rises gradually in the world, and the harmfulness makes people pay attention to it more and more. The diabetes nephropathy (DN) is one of the familiar capillaries eomplicationses of the diabetes. The pathogenesis of DN involves many kinds of factors. The relation among Connective tissue growth factor (CTGF), Vascular endothelial growth factor (VEGF) and nephropathy has already become a focus in international research. It has been approved that CTGF plays an important role in the occurrence and development of DN. CTGF is the downstream medium of Transforming growth factor-β₁ (TGF-β₁), but CTGF does not involve in the resisting proliferation and resisting inflammation function of TGF-β₁, and only involve in accelerating cell matrix synthesis function of TGF-β₁.VEGF is the specific mitogen of endothelial cell, and it can promote endothelial cell proliferation and neonatal blood vessel formation, increase vascular permeability. VEGF is the central reason that cause microproteinuria during earlier period of DN, and participates in the renal glomerulus sclerosis formation with other factors.The renal tubule is a vital point that connects the glomerulus with renal stroma. It deserves the further research that the role of renal tubule in the occurrence and development of renal stroma fibrosis.In last Research, it showed that the traditional Chinese medicine, Astragalus, could decrease urinary protein to protect kidney, but the precise mechanism is not yet clear. At present, not yet see the report about the effect of astragalus on CTGF and VEGF in the diabetes mellitus rat, and the effect of astragalus on the renal tubule transformation and differentiation in vitro. So this research is divided into two parts: in vivo and in vitro.Object: In vivo, to observe the effect of astragalus on CTGF and VEGF in type 2 diabetes mellitus rats; and discuss its mechanism; In vitro, to observe the effect of serum containing astragalus on CTGF, α-smooth muscle actin (α-SMA) and otherwise in the renal tubule epithelial cell.Method: (1) in vivo experiment: to establish type 2 diabetes mellitus rat model by unilateral nephrectomy, high-carbonhydrate and high fat diet, STZ injection. The rats were divided into 4 group: â‘ group DM: Establish the model of DM; â‘¡group treated with astragalus; â‘¢group treated with irbesatan(IRB);â‘£group sham-operation. Observe blood-glucose, blood insulin, blood-lipid, kidney weight/body weight, renal glomerulus area and volume, trace albumin in urine etc. Observe the expression of VEGF,CTGF, type IV collagen and Fibronectin (FN) by immunohistochemistry. Measure the expression level of CTGF mRNA in the renal cortex by RT- PCR. (2) in vitro experiment: Incubate the renal tubule epithelial cell(NRK-52E). Divide into 3 group: (Dgroup treated with 10% normal rat serum; (Dgroup treated with 10% normal rat serum and TGF-Pi; ?group treated with 10% containing astragalus rat serum and TGF-pYAfter conincubate 48 hours, observe the morphological change of NRK-52E, observe the expression of CTGF mRNA by the RT-PCR method; the expression of CTGF and a-SMA protein by the Western blot method; the expression of a-SMA by flow cytometry examination.Result: (1) in vivo experiment: the level of blood-glucose, blood insulin, TC, TG, Ccr, trace albumin in urine, kidney weight/body weight, renal glomerulus area and volume in group DM rats, as well as TGF-Pi^ VEGF> CTGF> type IV collagen and FN measured by immunohistochemistry, CTGF measured by Western blot are significantly higher than those in group sham-operation(P<0.05 or PO.01). And those indexes are obviously improved after treated with astragalus (PO.01 or P<0.05). The expression of VEGF in renal glomerulus is positively correlated with renal glomerulus volume, TGF-Pi and urinary albumin (P<0.05). The expression of CTGF is positively correlated with TGF-pi, CO-IV, FN (PO.05 or PO.01). (2) in vitro experiment: There is a little expression of CTGF mRNA in NRK-52E cell in normal control. Then containing TGF-Pi (lOng/ml) serum is added, after stimulating 48hs, the expression of CTGF increases 3.8 times, the significant difference (P<0.01). To add containing astragalus serum at the same time, the stimulation of TGF-|3i to the expression of CTGF can be depressed 43% (P<0.05). The western blot examination shows that the protein level of CTGF in normal NRK-52E cell is low. After stimulated by TGF-pi, the level of CTGF increases 3.6 times (P<0.01). To add containing astragalus serum at the same time, the protein expression of CTGF decreases 39% compared with group treated TGF-pi (PO.05). There is a little protein expression of a-SMA in NRK-52E cell in normal control. Then containing TGF-pi (lOng/ml) serum is added, after stimulating 48hs, the expression of a-SMA significantly increases 4.2 times (PO.01). To add containing astragalus serum at the same time, the expression of a-SMA is significantly depressed 35% (PO.05). The result of flow cytometry examination shows: Stimulated by TGF-Pi (lOng/ml) for 48hs, the a-SMA positive cell expression percentage and average fluorescence intensity both increase (PO.05). In the group treated with containing astragalus serum, the a-SMA positive cell expression percentage and average fluorescence intensity are obviously lower than those in the above group (PO.05).Conclusion: (1) in vivo experiment: The expression of VEGF increases in type 2 diabetes mellitus rats, and the level of VEGF is positively correlated with urinary albumin and kidney weight/body weight. The expression of CTGF increases in type 2 diabetes mellitus rats, and the level is positively correlated with TGF-Pi. Astragalus can depress the expression of VEGF and CTGF. (2) in vitro experiment: There is a little expression of CTGF and a-SMA in normal NRK-52E cell. And TGF-Pi can stimulate CTGF and a-SMA, but containing astragalus rat serum can depress the expression of CTGF and a-SMA.