Effection Of Megsin On Matrix Metabolism In Diabetic Mice

Objective: Recently the incidence of diabetes is going up year by year,accompanying with the rising of number of the patients with diabetic nephropathy(DN). DN, as a serious chronic implication of diabetes, has become one of the main etiological factors resulting in chronic renal failure. Glomerular mesangial cell hypertrophy, extracellular matrix accumulation and glomerulosclerosis is the basic pathological feature in the development of DN which is considered to be an irreversible process that eventually leads to end-stage renal failure. Megsin gene expresses superiorly in the mesangial cells. It has shown that the megsin expression strengthens in the IgA nephrosis, is closely related with the matrix proliferation.of the mesangial cell.At present,there is few reports that under diabetes condition the megsin how to express and what is the function.The renal glomerulus principal constituent of extracellular matrix isⅣ-Collagen, LN and textile fiber connection protein. Under the normal conditioon, these ingredients renew unceasingly and in the strict control dynamical equilibrium. The participation kidney extracellular matrix metabolism's enzyme class mainly (plasmin originally stirs up living creature -plasmin system) for the serine proteinase system and the matrix metal proteinase system. The change of matrix synthesis either in the degeneration or the ingredient quantity and the composition , will affect the kidney inherent cell's function, causes the kidney disease's occurrence, Matrix metalloproteinases (matrixmetalloproteinases, MMP) is an important regulation of renal ECM metabolism degradation systems, Including gelatinase, interstitial collagenase, matrix lysin, secretion of elastase and membrane-type MMP and so on, their activeness relies on the zinc ion.The formerly experimental study indicated that in the megsin transgene mouse's anti-renal glomerulus basilar membrane nephritis model, the pathology demonstrated continues extracellular matrix's increasing, Highly prompted that megsin is considered to play the important role in the matrix metabolism regulation as serine proteinase inhibitor (serpin) . Plasminogen is one of megsin biological substrate, it is a common activator of MMPs ,it can directly degrade extracellular matrix, and confirmed its activity as a protease inhibitor, megsin combination with plasminogen, direct inhibition of its degradation of LN, FN and the role of gelatin and can be indirectly block the plasminogen system of matrix metalloproteinases (MMPs) activation, thus inhibiting MMP-9, MMP-2 activity, etc., resulting in its degradation of gelatin and collagen type IV the role of obstruction, but still required depth study to confirm.The experiment detect megsin,IV collagen,MMP-2,TIMP-2 in diabetes mouse with upexpression and inhibit the expression of megsin under the high sugar environment , observeing the megsin expression level and the change tendency and discussing the cell matrix metabolism of the megsin gene in diabetes mouse kidney disease.Method: The clean health 12 weeks male CD1 mouse, altogether 60,after one-sided kidney excision were randomly divides into the surgery group (A group), the diabetes + megsin vecter (B group), diabetes +megsin (C group), diabetes +siRNAmegsin (D group), each 15, the abdominal cavity injection chain urea assists the rhzomorph (Strep tozotocin, STZ) the method preparation diabetes mouse model, at a dose of 150mg/kg ,The diabetic model was considered to be successful when the blood glucose was 16.7mmol/L after 48 hours of STZ injection,.does not meet the above criteria excluding the experimental .Application JAPAN virus hemagglutinin coated plasmid vector via the tail vein injection into megsin cDNA sequence and empty plasmid vector, a weekly injection of 1 times.At the end of 1week,2week,12week , collecting the blood,24-hour urine and renal tissue samples, testing serum creatinine; 24h urinary protein excretion rate, creatinine ; kidney weight / body weight ratio;2μm paraffin section line of HE, 4μm paraffin section immunity group. Application of immunochemistry and Western - blot on kidney tissue mesangial cells type IV-collagen, megsin, MMP-2, TIMP-2expression positioning, qualitative and semi-quantitative analysis. Results:1. 12 weeks, B group,C group,D group,24-hour proteinuria were significantly higher than A group (P <0.01). B group, C group 24-hour urinary protein was higher than the corresponding D group (P <0.01), B group, C group no significant difference between the two groups (P> 0.05).2. Kidney weight / body weight ratio comparison:1week,2 week,the ratio of experimental diabetes B group,C group starts to advance, D group compare with A group is not remarkable (P>0.05), 12 week B group, C group even obviously is higher than A group of (P<0.01), D group to compare the corresponding B group, C group to have reduces (P<0.01).3. Immunohistochemistry and renal tissue protein Westen-bolt: megsin,IV-collagen,TIMP- 2, MMP-2 expression:At the end of 1 week and 2 weeks , the number of glomerular inherent cells in group B increased significantly than in group A (P <0.05), but lower than group C (P <0.01), accompanied with the increase in mesangial matrix; at the end of 2,12 weeks group B,C and group D of mice in the glomerular mesangial cell megsin, IV collagen, TIMP - 2 expression is enhanced showing granular brown coloring, both qualitative and quantitative expression is stronger than A group of mice, there was a significant difference (P <0.01).In group D megsin, IV collagen, TIMP - 2 expression reduce than the same period of group B, group C, there was a significant difference (P <0.01), and at the end of 1,2,12 weeks when the group B, group C, group D of mice in glomerular mesangial cells expressed MMP-2 significantly less than the group A, there was a significant difference (P <0.01), group D with the same period in group B, group C compared to an increase in expression (P <0.01).Conclusion:1.Megsin expressed increasing in diabetic nephropathy,according with the accumulation of extracellular matrix,thedamage of renal function and kidney hypertrophy ,that suggest megsin may play a role in the development of diabetic nephropathy.2.The expression of MMP-2 reduced further, the expression of TIMP-2 enhanced further and IV collagen increased in megsin over-expression diabetic mice, that suggested megsin play a synergistic effect with TIMP-2, inhibitted the activity of MMP and degradation of extracellular matrix, promoted the progress of diabetic nephropathy.3.The imbalance of MMP-2/TIMP-2 system has been alleviated in the renal tissue of lower-expressed megsin diabetic mice, that be confirmed by the transfection of megsin siRNA,and the accumulation of extracellular matrix and the renal dysfunction have been reduced , those suggested that the inhibition of megsin is an effective means of prevention and treatment in early diabetic nephropathy ,and providing a scientific basis for the screening of a new target for diabetic nephropathy...