| Objective: Renal interstitial fibrosis is a common way of kidney disease, induced by various causes, developing into final renal failure. The degree of renal interstitial fibrosis has correlation with the extent of renal function damage. Tubulointerstitial macrophage (MP) accumulation in particular correlates with kidney disease progression, which may directly or indirectly be involved in tubulointerstitial fibrosis[1], and it is the hallmark of irreversible chronic kidney injury. Regulated on activation of normal T cell expressed and secreted (RANTES) is a member belonging to chemotatic factor's family, which can chemotatic and activates MP, leukomonocyte and eosinophils, and can lead to the infiltration of inflammatory cells to the kidney, inducing the substantial expression of various inflammatory factors. Macrophage colony-stimulating factor (M-CSF) is the critical factor that induces strong local MP proliferation[2]. In rat's kidney, M-CSF increased in association with local MP proliferation of unilateral ureteral obstruction (UUO)[3]. Nuclear factor-κB (NF-κB) can regulates expression of a wide range of genes, and participates the process of much inflammatory mediator's signal transducer. Transforming growth factor-β(TGF-β) is an important factor to promote fibrosis, mediating to many processes, such as accumulate cellular damage and extracellular matrix. Ligustrazine is a Chinese herbs with the functions of activating the blood circulation and removing the blood stasis. It's effect of protect kidney include anti-cell proliferation, anti-inflammatory, anti-fibrosis, immuno- regulation and so on. But it's unknown that whether ligustrazine can inhibit the expression of RANTES, M-CSF, thereby reduced the renal infiltrating of MP and interstitial fibrosis. Through UUO, this study is about the effect of Ligustrazine makes on the expressions of RANTES, M-CSF, TGF-β1, NF-κB, and Monocyte/MP infiltration, the degree of the affected renal interstitial fibrosis. We will have a discussion of Ligustrazine on renal interstitial inflammation and fibrosis from the levels of cytokines.Methods: Forty-five male Sprague-Dawly rats weighing 180~220g were randomly divided into sham operated group (group A), unilateral ureteric obstructive operated group (group B) and unilateral ureteric obstructive operation with ligustrazine treatment group (group C), (n=15). After giving intraperitoneal injection of anesthesia with 10% chloral hydrate (3.0ml/kg) to each of the groups of rats, separate the left ureter, ligate at two points with 4.0 silk in the middle and upper 1/3 and cut between the ligatures in order to prevent retrograde urinary tract infection. Rats in group A had their ureters manipulated but not be ligated. Ligustrazine (40mg/Kg/d) began to intraperitoneal injection one day before surgery to the rats in group C. A and B groups received Saline of equal volume by daily peritoneal injection. Rats were killed in the 3, 7, 14 day after surgery. Immunohisto-chemistry was used to measure the level of expression of RANTES, M-CSF, NF-κB, TGF-β1 and CD68-positive cells. The changes of renal tissue were also observed by HE and Masson stain. The results were analyzed semi-quantitatively by the pathological image analysis system. RANTES mRNA was detected by Quantitative real-time PCR (RT-PCR). All the data were analyzed by SPSS 11.5 statistics software, and P value< 0.05 was considered to have statistical significance.Results: 1. Stereology research: Kidneys of the rats in group A had normal appearance. While the kidneys of the obstructed rats became bigger as time went on. Clear water could be found in the bigger kidneys, renal cortex became thinner, renal pelvis expanded and minor calyx oppressed.2. The changes of histopathology: The group A did not found change. The situation among rats in group B that renal interstitial edema was formed, tubular expanded, monocyte came up in tubulointerstitial regions and lymphocytic infiltration existed at the 3th after the survey. That change became obviously at the 7th: Interstitial obviously widened, expansion had various degrees of tubular atrophy and necrosis, epithelial cell degenerated, had oedema and some even died. On the 14th day, substantial inflammatory cell infiltrated and renal interstitial fibrosis aggravated.3. Ligustrazine inhibits the infiltration of monocyte/MP: In group A, mainly no CD68-positive cells expression in nephridial tissue(0.034±0.044~0.042±0.043,P>0.05). In group B, CD68-positive cells were found in renal interstitium at the 3th after the survey. With the time went on, CD68-positive cells were highly expressed (0.268±0.030~0.472±0.048, P<0.05). In group C, the level of CD68-positive cells were lower than that of group B at the same timepoint (0.192±0.068~0.386±0.061, P<0.05), the renal inflammation was released.4. Ligustrazine inhibits the expression of RANTES and RANTES mRNA in nephridial tissue: In group A, the expression of RANTES in renal glomerulus or renal interstitial were trace (0.047±0.020~0.061±0.017, P>0.05). RANTES was mainly expressed in renal glomerulus and renal interstitial, and had a small quantity of expression in blood vesse and proximal convoluted tubule in group B. The area of RANTES increased and reached to the bottom on the 14th day (0.221±0.044~0.453±0.040, P<0.05). The level of RANTES in group C were lower than that of group B at the same timepoint (0.172±0.020~0.374±0.030, P<0.05). The changes of the RANTES mRNA level were consistent with the protein, and closely related to RANTES protein (r=0.520,P<0.05).5. Ligustrazine inhibits the expression of M-CSF: In group A, the expression of M-CSF had a rare quantity in renal glomerulus or renal interstitial (0.039±0.032~0.048±0.034, P>0.05). M-CSF was mainly expressed in renal glomerulus and renal interstitial, and had a small quantity of expression in proximal convoluted tubule in group B and the area of M-CSF increased significantly (0.332±0.051~0.501±0.084, P<0.05); In group C, the expression of M-CSF reduced more significantly than that of group B (0.242±0.044~0.409±0.029,P<0.05). But it was still higher than the level of group A (P<0.05).6. Ligustrazine inhibits the expression of promote fibrosis factors (TGF-β1, NF-κB): From the 3th day, the expressions of TGF-β1 and NF-κB were upregulated in group B, and increased with time went on, reached to the bottom on the 14th day (0.266±0.004~ 0.545±0.070 and 0.270±0.028~0.476±0.054, P<0.05), had statistical significance compared with group A (0.025±0.005~ 0.027±0.001 and 0.015±0.003~0.017±0.007). In group C, both of them were decreased than that in group B at the same timepoint (0.186±0.006~0.424±0.020 and 0.217±0.014~0.409±0.062,P<0.05), but they were still higher than the level of group A (P<0.05).7. RANTES, M-CSF, TGF-β1 were both closely related to CD68 (r=0.857,0.565,0.876,P<0.05), and also positively related to NF-κB (r=0.832,0.550,0.813, P<0.05).Conclusion: Treatment with Ligustrazine in UUO rats markedly limits the renal overexpression of inflammatory mediators like pro-inflammatory factors RANTES, M-CSF and factors of pro-fibrosis TGF-β1, NF-κB. This pathway inhibits the infiltration of monocyte/MP in the earlier period, thereby reduced renal interstitial inflammation and interstitial fibrosis. |
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