| Objective: Diabetic nephropathy (DN), is one of the signs of lesion on systemic microvascular disease and the most common complication of diabetes, the main clinical feature of which is proteinuria, progressive renal dysfunction, edema, hypertension, and renal failure in later stage, it can be considered as one of the crucial causes of death for diabetic patients. The main pathological changes include renal hypertrophy, thickening of glomerular and tubular basement membrane(GBM), mesangial cell proliferation and extracellular matrix (ECM) accumulation induced mesangial expansion which may cause diffuse or nodular glomerulosclerosis and tubulointerstitial fibrosis[1], of which histological basis is the deposition of renal ECM.Studies have found that the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have played an important role in the ECM metabolism and the regulation of matrix formation and degradation in the development of DN. On account of the imbalances of MMPs/TIMPs system leading to inappropriate accumulation of ECM, the proliferation of glomerular mesangial matrix and basement membrane would be thickening, therefore, the regulation of matrix metalloproteinase system had the great significance in reducing the accumulation of ECM for DN, preventing and delaying glomerular sclerosis. At present, There is a lack of highly targeted, effective method to prevent of DN, but traditional Chinese medicine has great potential of clinical prevention and treatment of DN by taking the Multi-target overall regulation and people-oriented individualized treatment plan . In clinical practice, according to DN basic pathogenesis of simultaneous deficiency of both yin and yang, blood stasis obstructing the collaterals and incidental abdominal mass, apply the basic prescription of herbs of supplementing qi and nourisging yin, resolving masses and dredging meridians for curing DN,and pre-studies have confirmed that this prescription has the precise impact of the oxidative stress, podocyte protection, signal transduction pathways, as this study took MMPs/TIMPs system as the cutting point to further explore the development of this prescription effects on DN and its possible mechanism.Methods: 90 healthy male SD rats weighing 200g±10g were carried out left-nephrectomy on the rats'back after being adaptive fed a week, two weeks later, except nephrectomy groups (10), the remaining rats were injected intraperitonally STZ (40mg/kg), 72 hours later blooded from tail vein, which is a successful modeling only if the blood glucos≥16.7mmol/L for three times consecutively, then the model rats were randomly divided into 5 groups: model group, Lrbesartan group, TCM low dose group, TCM middle dose group and TCM high dose group. Irbesartan group were given Irbesartan 15mg/kg/d by gastric perfusion and three TCM groups with 5.29g/kg/d, 10.58g/kg/d, 21.16g/kg/d TCM respectively, and the rest groups received normal saline once a day for 12 weeks, then at the end of the 12th week: collecting 24h urine, blooding from femoral artery, killing the rats, separating cortex and medulla, and detecting the rats 24 h uric albumen ration and renal function, observing each group of pathological changes in rat kidneys by light microscopy and transmission electron microscopy, apply RT-PCR to detecting gene expression of each rat renal cortical MMP-9/TIMP-1Results:1 Weight and hypertrophy index of rats in each group, (kidney weight / body weight)Compared with normal group, the model group and treatment group were underweight (P<0.05); Compared with model group, the treatment group were overweight (P<0.05); body weight among treatment groups was no significant difference (P>0.05). In the normal group, model group and the treatment group, the rats'right kidney weight were no significant difference (P>0.05); with the normal group, model group and each treatment group, the hypertrophy index is higher (P<0.05), but with the model group, each treatment groups, the hypertrophy index is lower (P<0.05), among the each treatment group, the hypertrophy index was no significant difference (P> 0.05).2 24-hour uric albumen ration (UPro) levels in each groupsCompared with the normal group, the UPro levels were increased (P <0.05) in both the model group and each treatment groups; with model group, the UPro level of treatment group were decreased (P<0.05); with the TCM high dose group, The UPro levels of Lrbesartan group and the TCM low dose group were both decreased (P<0.05), in TCM middle dose group there was not statistically significant (P>0.05).And between the Irbesartan and TCM middle dose groups there was not statistically significant (P>0.05) either.3 The renal function in each groupCompared with normal group, the model group and each treatment groups'serum creatinine (Scr) were obviously increased (P<0.05); Compared with model group, the treatment groups were significantly decreased (P<0.05); compared with TCM high dose group, Lrbesartan, TCM low-dose and TCM middle dose group were significantly decreased (P<0.05); Lrbesartan, TCM low and middle dose groups was not statistically significant (P>0.05). About the blood BUN, compared with the normal group, The model group and the treatment groups were significantly increased (P<0.05); with the model group, treatment group were obviously decreased (P<0.05); There was no obvious difference among the treatment groups. (P>0.05).4 Morphological observation of renal pathology4.1 Light microscopy observationNormal group has not appeared renal glomerular hypertrophy, capillary basement membrane didn't become thickening. mesangial cell number and the size of mesangial matrix distribution were normal and no significant tubulointerstitial pathological changes. In model group, there were obvious hypertrophy of renal glomerulus, thickening of GBM, expansion of mesangial matrix and vacuolar/granular degeneration of renal tubular cell. In Irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no significant difference among the treated groups.4.2 Electron microscope observationThe glomerular endothelial cells and Sertoli cell is normal in the normal group, and the basement membrane is integral; In model group, the glomerular basement membrane became thickening, the mesangial matrix is proliferating, accompanied fusion of podocyte. In TCM group and the Lrbesartan group also have been found the pathological changes in varying degrees, but it was less than the model group, which showed that the glomerular basement membrane became thickening, and mesangial matrix is proliferating, accompanied fusion of podocyte. There was no obvious distinguish among the groups.5 The gene expression of renal cortical MMP-9 and TIMP-15.1 Detection of MMP-9 mRNACompared with normal group, the model group and each treatment groups'MMP-9mRNA were significantly decreased (P<0.05); with the model group, the expression of each treatment groups'MMP-9 mRNA were increased (P<0.05), MMP-9 mRNA expression was not statistically significant (P>0.05) among each treatment groups.5.2 Detection of TIMP-1mRNACompared with normal group, the expression of TIMP-1mRNA in model group and each treatment group were obviously decreased (P<0.05); Compared with model group, the expression of TIMP-1mRNA in treatment group were obviously decreased (P<0.05); and with the TCM high dose group, the expression of TIMP-1mRNA in the Irbesartan group and TCM low dose group were obviously decreased (P<0.05), in the TCM middle dose group there was not statistically significant (P>0.05); there was not statistically significant between the Irbesartan and TCM low dose group (P>0.05).Conclusions:1 The herbs of supplementing qi and nourisging yin resolving masses and dredging meridians can significantly reduce the DN rats'urine protein excretion and decrease the blood Scr and serum BUN, in order to protect the renal function, and delay the occurrence and development of DN.2 The herbs of supplementing qi and nourisging yin resolving masses and dredging meridians could effectively inhibit the DN rats glomerular capillary basement membrane to be thickening, mesangial matrix proliferating and fusion of podocytes, thereby delaying the progress of renal pathology.3 The herbs of supplementing qi and nourisging yin resolving masses and dredging meridians could significantly increased expression of MMP-9 mRNA and decreased the TIMP-1mRNA, reduced the accumulation of extracellular matrix ultimately. The prescription can effectively adjust the MMP-9/TIMP-1 system and delay the progress of DN pathologica thereby to achieve the renal protection of DN rats. |
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