| Mesangial proliferative glomerulonephritis is the most common type of primary glomerular disease pathology in our country. The main pathological changes is glomerular mesangial cell proliferation and extracellular matrix deposition. In modern medicine, there is no specific treatment, the disease in clinic is still with glucocorticoid and immunosuppressants and symptomatic treatment is given priority to, it has certain uncertainty and side effects. In recent years, Chinese medicine treatment of the disease has made certain curative effect, and its side effect is small, so the research has the vital significance. Replenishing qi and blood drug most commonly used. Astragalus root and leech are the representative of the above drugs.Its inhibition of proliferation of mesangial cells have different degrees of the literature.Matrix metalloproteinases and its inhibitor is one of the most important enzymes in regulating extracellular matrix, dynamic balance. This research discussed the inhibition mechanism The active ingredients and medicated serum of radix astragali and leeches, and their effective constituents for the proliferation of mesangial cells from the perspective of such factors as MMP-2、TIMP-2、MMP-9、TIMP-1.Objectives:To research the regulating effects of the active ingredients and medicated serum of radix astragali and leeches for such factors as MMP - 2 and TIMP - 2, MMP - 9 and TIMP-1 during the proliferation of mesangial cells induced by lipopolysaccharide (LPS). And to explore the relevant mechanism of the active ingredients and medicated serum of radix astragali and leeches and their effective constituents for treaing mesangial proliferatibe nephritis. Compare yiqi blood medicine in different active ingredients and medicated serum composition formula of strength.Meathods:In vitro culture, the proliferation of mesangial cells is induced by LPS and intervened by the active ingredients and medicated serum of radix astragali and leeches. The experiment were randomly divided into Normal group, Lipopolysaccharide group, Astragalus polysaccharide group, Astragaloside group, Calycosin group, Hirudin group, Astragalus high serum group, Leech high serum group, Astragalus low and leech high serum group, Astragalus high and leech high serum group.The cells were collected after 24 hours.The expression changes in protein of MMP-2 and TIMP-2 were detected by Western Blot, and the expression changes in protein of MMP-9 and TIMP-1 were detected by confocal laser scanning mircroscopy(CLSM).Results:1.Shown by Westing Blot:After the proliferation of HBEH cells induced by LPS, The protein expression of MMP-2 is significantly higher than normal group (P< 0.01). Compared with LPS group, the group of Astragalus polysaccharides, Astragalus glycosides and Calycosin separately group (P< 0.05). They have statistical significance. Hirudin group (P< 0.01),it has a statistically significant. Compared with the Hirudin group, the group of Astragalus polysaccharides, Astragalus glycosides P<0.05, Calycosin separately group (P< 0.01). This shows that Astragalus root, leech monomer of MMP-2 protein expression is falling. The effect of hirudin group is better than that of astragalus membranaceus monomer groups.2. Shown by Westing Blot:After the proliferation of HBEH cells induced by LPS.The protein expression of MMP-2 is significantly higher than normal group (P< 0.01). Compared with LPS group, Astragalus high serum group, Leech high serum group P<0.05. They have statistical significance. Compared with LPS group, Astragalus low and leech high serum group, Astragalus high and leech high serum group P< 0.01. They have statistical significance. Compare each groups in terms of serum. Astragalus high serum group and Astragalus low and leech high serum group, Astragalus high and leech high serum group have statistical significance P< 0.05. This shows that astragalus root and leech serum of MMP - 2 protein expression has reduced effect. Among them. The effect of Astragalus and leeches formula is better than the Astragalus high serum group.3. Shown by Western Blot:After the proliferation of HBEH cells induced by LPS.The protein expression of TIMP-2 is significantly lower than normal group (P< 0.01). Compared with LPS group, the group of Astragalus polysaccharides, Astragalus glycosides and Calycosin separately group (P< 0.05). They have statistical significance. Hirudin group (P≤ 0.01),it has a statistically significant. Hirudin group and astragalus monomer groups have no statistical significance. This shows that Astragalus root, leech monomer of TIMP - 2 protein expression is rising.4. Shown by Westing BlotAfter the proliferation of HBEH cells induced by LPS.The protein expression of TIMP-2 is significantly lower than normal group (P< 0.01). Compared with LPS group, Astragalus high serum, leeches high serum group P> 0.05, they have no obvious effect on TIMP-2 protein expression. Compared with LPS group, Astragalus low and leech high serum group, Astragalus high and leech high serum group P< 0.05. Compare each groups in terms of serum, they have no statistical significance P> 0.05. This shows that Astragalus and leeches formula have a rising effect on TIMP - 2 protein expression.5. Shown by immunofluorescence method:There had a small amount of MMP-9 HBEH protein expression in normal group.The red fluorescence intensity is high in the LPS group, indicating LPS can induce the proliferation of HBEH cells and then the high expressiong of MMP-9 protein in the cell. Compared with LPS group, the group of Normal, Astragalus polysaccharide, Astragaloside, Calycosin, and Hirudin had statistical significance(P< 0.01). Compared with the Hirudin group, the group of Astragalus polysaccharides, Astragalus glycosides and Calycosin separately group (P< 0.05).These instructioned Astragalus and Leech monomer of MMP-9 protein expression showed a marked decline of function. The effect of hirudin group is better than that of astragalus membranaceus monomer groups. Compared with LPS group, the group of Normal, Astragalus high serum, Leech high serum, Astragalus low and leech high serum, Astragalus high and leech high serum all have statistical significance.(P< 0.01). Compare each groups in terms of serum. Astragalus high serum group and Astragalus high and leech high serum group have statistical significance P< 0.05. These instructioned Astragalus and Leech drug serum of MMP-9 protein expression showed a marked decline of function..6. Shown by immunofluorescence method:There had a large amount of TIMP-1 HBEH protein expression in normal group.The red fluorescence intensity is low in the LPS group, indicating LPS can induce the proliferation of HBEH cells and then the low expressiong of TIMP-1 protein in the cell. Compared with LPS group, the normal group have a remarkable statistical significance. (P< 0.01). The group of Astragalus polysaccharide Calycosin had statistical significance. (P< 0.05). The group of Astragaloside and Hirudin have a remarkable statistical significance(P< 0.01). Compared with the Hirudin group, the group of Astragalus polysaccharides P< 0.05. These instructioned Astragalus and Leech monomer of TIMP-1 protein expression showed a marked increase of function, The effect of hirudin group is better than that of astragalus membranaceus monomer groups. Compared with LPS group, Astragalus high serum group had no statistical significance(P> 0.05),the group of Leech high serum had no statistical significance (P< 0.05),the group of Astragalus low and leech high serum, Astragalus high and leech high serum all have statistical significance.(P≤ 0.01). Compare each groups in terms of serum. Astragalus high and leech high serum group and Astragalus high serum, Leech high serum, Astragalus low and leech high serum had a remarkable statistical significance. (P< 0.01). Other serum groups show no statistical difference. These instructioned Astragalus and Leech drug serum of TIMP-1 protein expression showed a marked increase of function. As a whole, the effect of Astragalus high and leech high serum group is better than other serum groups.Conclusion:1.Each monomer group conspicuously decrease the high expression of MMP-2 protein in the cell after the proliferation of HBEH induced by LPS. The effect of hirudin group is better than that of astragalus membranaceus monomer groups.2.Each serum group conspicuously decrease the high expression of MMP-2 protein in the cell after the proliferation of HBEH induced by LPS. Among them, The effect of Astragalus and leeches formula is better than the Astragalus high serum group.3.Each monomer group conspicuously increase the low expression of TIMP-2 protein in the cell after the proliferation of HBEH induced by LPS. Compare each group, they have no statistical significance.4.The group of Astragalus high serum and Leech high serum had no obvious influence on expression of TIMP-2 protein. The group of Astragalus low and leech high serum, Astragalus high and leech high serum had a rising effect on TIMP-2 protein expression. Compare each groups in terms of serum, they have no statistical significance.5. Each monomer group conspicuously decrease the high expression of MMP-9 protein in the cell after the proliferation of HBEH induced by LPS. The effect of hirudin group is better than that of astragalus membranaceus monomer groups.6. Each serum group conspicuously decrease the high expression of MMP-9 protein in the cell after the proliferation of HBEH induced by LPS.7. The group of Astragalus polysaccharides and Hirudin conspicuously increase the low expression of TIMP-1 protein in the cell after the proliferation of HBEH induced by LPS.8. The group of Leech high serum, Astragalus low and leech high serum, Astragalus high and leech high serum conspicuously increase the low expression of TIMP-1 protein in the cell after the proliferation of HBEH induced by LPS. The effects of Astragalus high and leech high serum group is better than other serum groups. |
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