| Objective:To explore the therapeutic effect of Cornus officinalis on diabetic nephropathy from in vivo and in vitro aspects and reveal its mechanism in depth while taking the diabetic nephropathy model mice and human mesangial cells (HRMC) as the experimental subjects, and to provide the experimental evidence for the development of the new drugs in diabetic nephropathy.Method:1) in vivo:6-week-old, male,C57BL/6J mice, adaptive feeding for one week and12hours of fasting before modeling, got intraperitoneal injection with0.1ml/10g per body weight for100mg.kg-1dose of streptozotocin at the first day and the fourth day respectively, selected the mice whose blood glucose levels were higher than15mmol.L-1as the diabetic model mice.The model mice were randomly divided into9groups according to the blood glucose levels which were the normal group, model group, habitat group (4g.kg-1), Cornus officinalis group(2g.kg-1), low dose decoction of habitat and Cornus officinalis separately group(3g.kg-1), high dose decoction of habitat and Cornus officinalis separately group(6g.kg-1), low dose decoction of habitat and Cornus officinalis merged group(3g.kg-1), high dose decoction of habitat and Cornus officinalis merged group (6g.kg-1), metformin group(200mg.kg-1),each group had ten mice and all of them were on high AGEs diet.intragastric administration were provided to each group for12consecutive weeks with the medicine of the above doses and the same volume of distilled water were provided to the mice of control group and normal group the same way. Weight. Blood glucose, food intake, water intake and urine output were measured every four weeks and the urine was collected. At the end of the experiment, the mice eyeballs were taken out to get the blood, supernatant was centrifuged and the biochemical markers were measured. The serum insulin levels and serum AGEs content were tested by ELISA. kidneys were taken out quickly, weighed by precise electronic balance after removing the capsule and cleaning, then viscera index was calculated and one kidney was fixed with10%formalin for histopathological examination while the other kidney was preserved into the freezer of-80℃for electron microscopy and immunohistochemistry.2) in vitro, The cultured HRMC was stimulated by30mmol·L-1glucose, on the basis of this model it was intervened by the serum which contains medicine of habitata and Cornus officinalis and it was divided into normal control group (5.5mmol·L-1), high-sugar model group (30mmol·L-1), the habitat group, Cornus officinalis group, decoction of habitat and Cornus officinalis separately group, decoction of habitat and Cornus officinalis merged group. After the culture of48h, the condition of cell proliferation was measured by MTT, cell cycles were measured by flow cytometry, fibronectin protein (FN) and type IV collagen (ColIV) content in the cell culture supernatant were assayed by ELISA and RT-PCR was used to detect expression of transforming growth factor TGF-β.Results:1.Common signs, the average weight gain of model group mice was small and it had significant difference compared with the normal group (P<0.01), the weight of drug-intervention mice group were increased siginificantly, but compared with the normal group there was still a gap and had no statistical significance. food intake, water intake and urine volume of model group mice increased significantly compared with the normal group (P<0.01), with the extension of administration, food intake, water intake,and urine volume significantly reduced in the drug-intervention group,among which high dose decoction of habitat and Cornus officinalis merged group was more obvious.2. Blood biochemical index:Blood glucose:Compared with the normal group, blood glucose was significantly increased with the mice in model group and reached the highest value in the fourth week of the experiment, then decreased gradually, but still much higher than normal (P<0.01). compared with the model group, the effect of lowering the blood glucose in each drug-intervention group was growing gradually and obviously as the duration of the administration increased, especially on the administration of8weeks the blood glucose of habitat group, low dose decoction of habitat and Cornus officinalis separately group, low dose decoction of habitat and Cornus officinalis merged group decreased significantly.On the administration of12weeks, habitat group, high dose decoction of habitat and Cornus officinalis separately group, low dose decoction of habitat and Cornus officinalis merged group, high dose decoction of habitat and Cornus officinalis merged group showed best.Serum insulin:the serum level of insulin in mice of model group was significantly decreased and had significant difference compared with the normal group (P<0.01). after administration of12W, every drug-intervention group had the increasing trendency, among which habitat group, high dose decoction of habitat and Cornus officinalis merged group increased significantly (P<0.01); serum AGEs:the level of serum AGEs in mice of model group was significantly higher than the normal group and it had significant difference (P<0.01). after administration of12W, each drug-intervention group had lower serum AGEs level, among which habitat group, high dose decoction of habitat and Cornus officinalis merged group decreased significantly (P<0.01).3. Related indexes of renal function:24h urinary protein excretion in mice of model group on the administration of4W,8W,12W increased significantly (P<0.01), on the administration of4W,8W,12W,24h urinary protein of drug-intervention group decreased significantly, the administration of12W high dose decoction of habitat and Cornus officinalis merged group decreased most obviously. Scr and BUN of model mice increased significantly (P<0.01), Scr and BUN of each drug-intervention group decreased significantly (P<0.05, P<0.01), among which habitat group, low dose decoction of habitat and Cornus officinalis merged group decreased more significantly at Scr (P<0.01); and as to the reduction of BUN. habitat group, high dose decoction of habitat and Cornus officinalis merged group was the most outstanding (P<0.05). Kidney index:compared with the normal group, model group mice renal index was significantly increased (P<0.01), Compared with model group kidney index of each drug-intervention group had varying degrees of declining (P<0.01).4.Renal pathology:under the light microscope,the glomerular volume of model mice was relatively hypertrophy, mesangial cells were hyperplasia moderately and even severely along with matrix increased.and mesangial area was significantly widened, the pathological changes in each drug-intervention group were mitigate in different degrees.Cells in kidney pathological changes observed under transmission electron microscope: in the model group, mesangial cells proliferated, matrix increased, basement membrane thickening, and compared with model group all the drug-intervention groups had significant improvements, The thickening of the basement membrane was mitigated, but the structure of filtration membrane was basically complete. TGF-β of Immunohistochemistry:a large number of brown deposit was seen in renal tubular epithelial cell cytoplasm of model group and its scope was bigger than that of the normal group. after treatment, the sediments in the renal tubules significantly reduced, in drug-intervention groups. it indicated that the expression of Increasing tendencies of TGF-β in immune group was inhibited after the treatment and the renal fibrosis was mitigated.5. Cell proliferation assay:MTT colorimetry shows that after48h of30mmol· L-1glucose stimulation, the proliferation of HRMC could significantly stimulated and HRMC got different degrees of inhibition under high glucose stimulation in each drug-intervention group.. and the higher concentration the higher inhibitory effect on HRMC.so it showed a certain dose-effect relationship. Flow cytometry showed that high glucose could change the cell cycle of HRMC, make G1phase decrease and S phase increase, after providing habitat and Cornus officinalis, the cell number of G1phase was higher than high sugar group with S phase and G2phase decreased, the inhibitory effect on HRMC’s proliferation by habitat and Cornus officinalis was mainly achieved through the block of cell cycle.6. Influence on the HRMC’s secretion of extracellular matrix (FN, ColIV) and TGF-β:experimental results showed that habitat and Cornus officinalis can inhibit High Glucose-Induced proliferation of the mesangial cell and secretion of extracellular matrix (FN, ColIV), adjust the effect of TGF-β mRNA under high glucose stimulation.Conclusion:the experiment shows that habitat and Cornus officinalis can mitigrate the symptoms of diabetic nephropathy, ameliorate blood glucose,increase insulin, decrease the level of serum AGE, improve renal function and renal pathological changes in renal tissue, inhibit the expression of TGF-β, which contributes to the prevention and treatment of DM nephropathy, retard the development of glomerular sclerosis process. It shows that the habitat and the cornus officinalis can also inhibit the proliferation of human mesangial cells in vitro culture, and block the cell cycle in mesangial cells in G0/G1phase, it also shows a dose-effect relation.The possible mechanism is to improve or delay the sclerosis of DN glomerular through the inhibition of proliferation of the extracellular matrix (ECM) and the expression of TGF-β... |
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