The Effects Of Rhein On The Ratio Of MMP-9/TIMP-1 And Expression Of AP-1 In Kidney Of Diabetic Rats

Background:Diabetic nephropathy(DN) is one of the main reason to result in the end-stage renal disease. The main pathological change of DN is the renal interstitial fibrosis in which extracellular matrix is the main point. The results show that tubular epithelial myofibroblast transdifferentiation (TEMT) plays an important role in renal interstitial fibrosis.Matrix metalloproteinases-9(MMP-9) can specifically degrade the collagen IV in the basement membrane, and collagen IV is the main composition of tubular basement membrane. Tissue inhibitor of metalloproteinase-1 can specificly combine with MMP-9 and keep their activity in a dynamic balance. Extracellular matrix (ECM) rupture status in basement membrane mainly depends on the ratio of MMP-9/TIMP-1.Activator protein-1(AP-1) is a kind of Transcription factor composed of protein Fos and protein Jun. It has multi-functions in cells, participates in gene transcription regulation of a variety of growth factors and cytokines. Some experiments show that the activation of AP-1 is able to enhance the expression of MMP-9, on the invasion and migration of tumor. But the change of AP-1 during tubular interstitial lesions is rarely reported.As the main active ingredients of Rhubarb, Rhein has have protective effects on kidney. Some studies showed that Rhein is able to inhibit the occurrence of TEMT, but whether by adjusting the ratio of MMP-9/TIMP-1 and AP-1 expression and inhibiting the progress of TEMT are unclear. Objective:1. One of the goals of this research is to observe the change of expression of AP-1 and the ratio of MMP-9/TIMP-1 in diabetic rat renal tubular epithelial cells, and the relationship between them.2. This research also aims at observing the effect on TEMT, MMP-9/TIMP-1 ratio and expression of AP-1 after the interfere of rhein using valsartan as a positive control and explore the possible mechanism of rhein on the renal protection.Methods:Healthy male Wistar rats of 8 weeks old were divided into normal group (n=12), diabetic group (n=12), Rhein intervention group (n=12) and Valsartan intervention group (n=12) randomly. Diabetic mellitus rat models were induced using intraperitoneal injection with 55mg/kg STZ. Diabetic rats were treated with rhein 100 mg/kg/d or Valsartan 30 mg/kg /d for 16 weeks after the models were successful established. Normal group and diabetic group were treated with the suspension of sodium cellulose hydroxyl, as well as the solution of intervention group.Six rats of each group were killed at the 8th and 16th weeks. The weight of rat and the Urine protein and Serum creatinine were measured before killing. Kill the rats. Histological changes of the kidney were observed after a Masson stain. The protein expression of E-Cadherin, a-SMA, MMP-9, TIMP-1 and AP-1 in renal tubular epithelial cells were measured using immunohistochemistry. The pearson correlation analysis is used to analyze the correlation of AP-1 and MMP-9/TIMP-1 ratio.Results:1.Comparing to the normal group, the relative area of the renal collagen of diabetic rats increased. The expression of E-cadherin in renal tubular epithelial cells decreased and the expression of a-SMA significantly increase at the level of P=0.05. Comparing to the diabetic group, Rhein and Valsartan intervention groups showed increases in relative area of the renal collagen. The expression of E-cadherin increased, while the expression of a-SMA significantly decreased. But there is no significant differnce between Rhein group and Valsartan group at the P=0.05 level.2.Comparing to the normal group, the expressions of MMP-9 and TIMP-1 in renal tubular epithelial cells of diabetic rats progressively increased with the progression of the disease. The ratio of MMP-9/TIMP-1 reduced gradually. Comparing to the diabetic group, Rhein and Valsartan intervention groups showed decreases in expression of MMP-9 and TIMP-1 of renal tubular epithelial cells. The decrease range of MMP-9/TIMP-1 ratio reduced significantly. But there is no significant differnce between Rhein group and Valsartan group at the P=0.05 level.3.Comparing to the normal group, the expressions of AP-1 in renal tubular epithelial cells of diabetic rats increased significantly along with the progression of the disease (P<0.05). Comparing to the diabetic group, Rhein and Valsartan groups showed significant decreases in expression of AP-1 of renal tubular epithelial cells (P<0.05). But there is no significant differnce between Rhein group and Valsartan group at the P=0.05 level.4.There is a negative correlation between expressions of AP-1 and MMP-9/TIMP-1 ratio for the renal tubular epithelial cells of diabetic rats. The correlation coefficient is-0.693 (P<0.01) at the 8th week and is-0.612 (P<0.01) at the 16th week.Conclusions:1. TEMT exists in the process of DN. 2. During TEMT process of DN rats, the expression of AP-1 increased and the balance of the MMP-9/TIMP-1 ratio was broken in renal tubular epithelial cells. And they are negatively correlated.3. Rhein is able to reduced the expression of AP-1, improve the imbalance of MMP-9/TIMP-1 ratio, and inhibit the process of TEMT in the DN rats.