| Objective:Renal interstitial fibrosis is a common pathological process of the end-stage renal disease.The pathological features are that renal interstitium and renal tubular structures were replaced by a large number of extra cellular matrix,such as collagen?,?,? and laminin.It is a complex process involving inflammatory reaction,oxidative stress,multiple cytokines and signal pathways which can lead to renal interstitial fibrosis,glomerular sclerosis and finally result in the loss of kidney function.Chemerin,a novel hormone made by fat tissue,is described as a inflammatory chemotactic cytokines.This cytokine participates in the establishment of inflammation through NF-?B,JAK/STAT,TGF-?1 signaling pathways and is also correlated with a variety of diseases such as diabetic nephropathy,coronary atherosclerosis,and cancer.Numerous studies have shown that chemerin,as a inflammatory factor,participated in diabetic nephropathy and chronic renal insufficiency.Its expression level was positive correlated with transforming growth factor(TGF-?1),connective tissue growth factor,tumor necrosis factor(TNF-?)and could also predict renal damage.TGF-?1,as a acknowledged and important inflammatory factor,can stimulate the accumulation of extracellular matrix and lead to renal dysfunction.Many studies have found that TGF-?1and collagen ? played an important role in unilateral ureteral obstruction(UUO)model.There were many researches using diabetic nephropathy model to investigate the relationship between Chemerin and renal interstitial fibrosis,rarely about UUO model.Tripterygium wilfordii polyglycoside(TWP)is widely used in the treatment of kidney disease and other autoimmune diseases in the effects of immunomodulatory,anti-inflammatory and anticancer.This is a study to observe the expression level and role of Chemerin,TGF-?1,collagen ? on renal interstitial fibrosis in rats with unilateral ureteral obstruction(UUO)and to evaluate the intervention effect of tripterygium wilfordii polyglycoside.The ultimate objective is to lay an experimental base for the future research and offer new therapeutic targets.Methods: After 1 week of adaptive feeding,the 72 clean grade SD male rats(200±20g)were randomly divided into group A,B,C,D: Sham operation group(separate the left ureter but not ligature),UUO-model group(ligature the left ureter near the renal pelvis section),UUO-model with low dose TWP-treated group and UUO-model with high dose TWP-treated group.Rats in C group and D group received TWP 10mg/kg/d and 20mg/kg/d via gastric irrigation,while the remaining groups were given the same amount of saline.During the experiment,the SD rats were free to eat and drink.We observed the general condition of rats every day in order to ensure their diet and activity were normal,and adjusted the dose according to their weight.Six rats in each group were sacrificed on the 3rd,7th,14 th day for experiments.After washing with 0.9% saline,the kidney tissue was divided into two parts.One part was fixed by 10% neutral formalin and observed the degree of renal histopathological changes through HE staining,Masson staining and immunohistochemical staining.Another part of the kidney tissue needed to freeze quickly in liquid nitrogen and then transferred to-80? ultra low temperature refrigerator for Western Blot.Results:1 Pathological changes of renal tissue1.1 Visible to the naked eye: The color and size of kidney in group A were normal after autopsy.The volume of left kidneys in group B,C,D were significantly larger and their cortex became significantly thinner along with the obstruction time.The volume of kidney in group B was largest,group C and D were all smaller that it.1.2 HE and Masson staining There was no obvious pathological changes in renal tubules and renal interstitium and the kidney structure was normal in group A.Group B: Weobserved that the renal structure was destroyed,renal tubular epithelial cells fall off,part tubules were dilated and infiltrated by slight inflammatory cells and the coloring of collagen fibers were not obvious on the third day.The number of dilated tubules was significantly increased at the seventh day,plenty of infiltrating inflammatory cells,broadening renal interstitial and fibrosis was appeared in renal tissue.We observed an increase of the coloured collagen fibers in the extended renal interstitium.At the fourteenth day,the renal tubule structure was destroyed,the degree of renal interstitial fibrosis worsened and the coloring of collagen fibers were more obvious.In the same time,the most serious was group B,the pathological changes in group C and D were lessened but still serious than group A and the degree of pathological changes in group C was more obviously than group D.2 Immunohistochemical staining2.1 Chemerin: A little chemerin expressed in the renal tubular epithelial cell of group A.The expression of chemerin in the renal tubular epithelial cell of group B,especially in endochylema,was increased significantly.Following time and course progressing,the number of chemerin in the renal tubular epithelial cell were increasing gradually.The differences between group A and group B were statistically significant(P<0.05).Compared with those in group B,the expression of chemerin was decreased significantly in group C and D(P<0.05),and the group D is more obvious,but it is till higher than the same period in group A(P<0.05).2.2 TGF-?1: A little TGF-?1 expressed in the renal tubular epithelial cell of group A.The expression of TGF-?1 in the renal tubular epithelial cell and renal interstitium of group B was increased significantly.Following time and course progressing,the number of TGF-?1 in the renal tubular epithelial cell were increasing gradually.The differences between group A and group B were statistically significant(P<0.05).Compared with those in group B,the expression of TGF-?1 was decreased significantly in group C and D(P<0.05),and the group D is more obvious,but it is till higher than the same period in group A(P<0.05).2.3 Col-?: The renal tubular epithelial cell and renal interstitium of group A expressed a little number of Col-?.The expression of Col-? in the renal tubular epithelial cell of group B was increased significantly.As time goes on,the number of Col-? in renal tubular epithelial cell were increasing gradually.The differences between group A and group B were statistically significant(P<0.05).Compared with group B in the same period,the expression of Col-? was decreased significantly in group C and D(P<0.05),and the group D is more obvious,but it is till higher than the same period in group A(P<0.05).3 Western Blot: Image results show that compared with group A in the same period,the expression of chemerin in group B was decreased significantly on the 3rd,7th,14 th day(P<0.05).Compared with group B in the same period,the expression of chemerin was decreased significantly in group C and D(P<0.05),and the group D is more obvious,but it is till higher than those in group A(P<0.05).Conclusions: Chemerin involved in the developing process of renal interstitial fibrosis,and its expression gradually increased along with the aggravation of renal interstitial injury.This suggests that chemerin may promote renal interstitial fibrosis.TWP maybe play the role of protecting kidneys by down regulating expression of TGF-?1 and collagen ? in renal tubular interstitial via Chemerin,then decrease reduce inflammation and finally reduce and delay renal interstitial fibrosis. |
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