| Objective: Tubulointerstitial fibrosis (TIF) is a common pathological feature of most end-stage kidney diseases. The main cause of tubulointerstitial fibrosis includes the hyperplasia and the phenotype inversion of fibroblasts in renal interstitium and the accumulation of extracellular matrix (ECM). The accumulation of ECM correlates closely to the hyperplasia of fibroblasts and the phenotype inversion of other cells, andα-smooth muscle actin (α-SMA) is the marker of the phenotype inversion of fibroblasts. As a kind of multifunctional cytokine, transforming growth factor-β1 (TGF-β1) can promote the synthesis of ECM and inhibit its degradation, advance the hyperplasia of fibroblasts, participate in the TIF. Adrenomedullin (ADM) is the peptide which consists of 52 amino acids. The study in the near future shows that ADM can inhibit the stimulation of TGF-β1 to synthesis and secretion of collagen. Therefore it can suppress the fibrosis.Safflower in the traditional Chinese medicine has the effects to promoting blood flow, removing blood stasis, and alleviating tumefaction and pain. The redundant accumulation of ECM in kidney corresponds the block of stagnant blood in the theory of the traditional Chinese medicine. So the method of promoting blood flow and removing blood stasis should be used and Safflower was applied. The modern pharmacological study shows that Safflower not only improves the blood circulation, but also relieves the TIF and delays the advancement of chronic renal failure by some mechanisms. The effect of Safflower on TIF correlates closely to inhibiting the expression of TGF-β1. However there is not evidence enough to confirm whether it acts by ADM or not. This experiment had applied Safflower to treating the rats of TIF resulted from unilateral ureteral obstruction (UUO), and the angiotensin receptor antagonist Valsartan acted as the comparison of Safflower. The aim is to approach the antagonism and the mechanism of Safflower on TIF through examining the effect of histomorphology and molecular immunology of Safflower on the obstructed kidneys in rats with UUO.Methods: The 40 female Sprague-Dawley (SD) rats were chosen, and after fed a week, they were put into the metabolic cages to be gotten their urine.After their urine protein and erythrocyte were totally negative, they were randomly divided into four groups: Sham-operation group (SHAM), Model group (UUO), Safflower group (UUOS) and Valsartan group (UUOV). There were 10 rats in every group. Each animal was ligated left ureter except SHAM group. The intragastric administration began since the day before operation, and the same volume isotonic Na chloride was used in SHAM and UUO groups. All rats were sacrificed after 10 days and their left kidneys were taken for pathological study. The methods of immunohistochemistry and in situ hybridization were adopted to detect the expression ofα-SMA, III type collagen (Col III), TGF-β1, ADM and ADM mRNA in renal tissues.Results:1 General state of kidney, kidney weight/body weight (KW/BW) and serum creatinine (Scr), blood urea nitrogen (BUN): The left kidneys in SHAM group had not changed remarkably at the appearance and the size. The left obstructed kidneys in the rest of groups had increased at both volume and weight, and the nephridial tissues were thinner than normal. The figures of KW/BW in UUO (0.0137±0.0045), UUOV (0.0139±0.0048) and UUOS (0.0132±0.0056) groups were higher than that in SHAM (0.0034±0.0002) group remarkably (p<0.001, p<0.001, p<0.001), but there were no obvious differences among the preceding three groups (p>0.05, p>0.05, p>0.05). BUN in UUO (12.07±4.71) group was higher than that in SHAM group (6.92±1.13) (p < 0.01), UUOV(8.18±1.42) and UUOS (8.61±1.61) groups were lower than UUO group remarkably (p<0.01, p<0.01). UUOV group was lower than UUOS group, but there was not statistically significant between them (p>0.05). Scr in UUO group (103.88±28.43) was higher than that in SHAM group (77.22±14.24) (p<0.01). Among the three groups, UUO group was the highest, UUOS group (91.21±15.51) was the next and UUOV group (85.49±20.29) was the lowest. But there was not statistically significant between them (p>0.05, p>0.05, p>0.05).2 The staining of HE and picrosirius of renal tissues: In UUO group, a number of inflammatory cells infiltrated and fibrous tissues were hyperplasia in renal interstitium. Some renal tubules were of atrophy, obliteration or expansion. The glomerular number and area decreased markedly. The renal capsules adhered or expanded and a lot of fibrous tissues proliferated around them. However, the lesions in treatment groups were slighter than that in UUO group.3 The dose of ADM protein in kidney tissue: The dose of ADM protein in SHAM group (9.587±1.83) was the highest in all groups (p<0.001, p<0.001, p<0.001). Except SHAM group, UUOV group (5.81±1.94) was the highest, UUOS group (4.69±0.60) was the next, and UUO group (4.606±0.38) was the lowest. But there was not the significant difference between them (p>0.05, p>0.05, p>0.05).4 The expression, distribution and semiquantitative analysis of ADM: The expression of ADM in SHAM group was weakly positive in the renal tubular epithelial cells and the renal glomerulis, UUO group (1364.75±277.43, 1.18±0.13) was weaker than SHAM group (3325.7±377.78, 4.49±0.38) markedly (p<0.001). UUOV (2305.9±281.84, 3.76±0.28) and UUOS group (2330.3±302.86, 3.90±0.39) enhanced markedly compared with UUO group (p<0.001, p<0.001), which means that Safflower and Valsartan can up-regulate the expression of ADM in the protein level. Furthermore, the effect of Safflower was better than that of Valsartan, but there was not the significant difference between them (p>0.05).5 The expression, distribution and semiquantitative analysis of ADM mRNA: The expression of ADM mRNA in SHAM group was strongly positive in the renal tubular epithelial cells, UUO group (294.6±46.39, 1.29±0.89) was markedly weaker than SHAM group (13829.75±1224.49, 10.37±0.49) (p < 0.001). UUOV group (3195.85±621.54, 2.76±0.20) and UUOS group (3311.65±511.12, 2.93±0.16) enhanced markedly compared with UUO group (p<0.001, p<0.001), which means that Safflower and Valsartan can up-regulate the expression of ADM mRNA in the obstructed kidneys. Furthermore, the effect of Safflower was better than that of Valsartan, but there was not the significant difference between them (p>0.05).6 The expression, distribution and semiquantitative analysis of TGF-β1: The weakly positive expression of TGF-β1 in SHAM group was found in the renal tubular epithelial cells and the renal glomerulis. The other groups enhanced markedly in the renal tubular epithelial cells, interstitial cells and the renal glomerulis. UUO group (20104.9±3492.40, 59.61±6.69) was more increased than SHAM group (1962.8±177.07, 7.7±0.49) (p<0.001). UUOV (11367.3±1831.09, 40.67±4.15) and UUOS (9207.9±1559.12, 39.22±3.75) groups descended markedly compared with UUO group (p<0.001, p<0.001), which means that Safflower and Valsartan can inhibit the expression of TGF-β1 in the obstructed kidneys. Furthermore, the effect of Safflower was better than that of Valsartan, and there was the significant difference on positive area (p<0.01), but was not on the integral optical density value (p>0.05).7 The expression, distribution and semiquantitative analysis ofα-SMA: The positive expression ofα-SMA in SHAM group was only found in the vessel wall, and it was not in renal interstitium and intracytoplasm of renal tubular epithelial cells. In UUO group (19745.9±3822.25, 59.07±6.11), it enhanced markedly in renal interstitium compared with SHAM group (1997.6±291.59, 7.58±0.49) (p<0.001). UUOS (9141.5±1526.52, 40.85±5.06) and UUOV (11369.9±1930.25, 41.22±4.09) groups weakened markedly compared with UUO group (p<0.001, p<0.001), which means that Safflower and Valsartan can inhibit the phenotype inversion of interstitial cells effectively. UUOS group was weaker than UUOV group, and there was the significant difference on positive area (p<0.01), but was not on the integral optical density value (p>0.05).8 The expression, distribution and semiquantitative analysis of Col III: Most Col III express in renal interstitium. SHAM group was weakly positive, but the rest of groups enhanced markedly, and they showed the shape of trabs or piece. UUO group (5114.5±315.69, 77.44±8.86) enhanced markedly compared with SHAM group (455.4±34.57, 10.02±0.96) (p < 0.001). UUOV group (3520.5±421.74, 51.14±3.50) and UUOS group (2406.95±375.24, 43.26±3.48) were weaker than UUO group (p <0.001, p<0.001), which means that Safflower and Valsartan can inhibit the expression of Col III in the obstructed kidneys. Furthermore, the effect of Safflower was better than that of Valsartan, and there was the significant difference between them (p<0.001).9 The semiquantitative analysis of the area of TIF: The degree of TIF in UUO group (7448.2±922.92) was more serious than that in SHAM group (854.8±92.26) (p < 0.001). UUOV (5133.4±546.43) and UUOS (4205.8±512.19) groups were smaller markedly than UUO group (p<0.001, p<0.001), which means that Safflower and Valsartan can inhibit TIF effectively. Furthermore, the effect of Safflower was better than that of Valsartan, and there was the significant difference between them (p<0.001).Conclusion:1. Safflower can delay the process of TIF through alleviating the pathological lesion of renal tissue and reducing the deposition of ECM in obstructive kidney.2. Safflower can down-regulate the expression of TGF-β1 in the protein level in the obstructed kidney, inhibit the cell phenotype inversion in the renal tissue and decrease the formation of MyoF in the renal interstitium, thereby it can reduce synthesis of ECM.3. Safflower can up-regulate the expression of ADM and ADM mRNA in the obstructed kidney, so that can take antagonistic effect on TIF by the biological effect of ADM.
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