| Objective: To investigate the preventive mechanism of Chailing Decoction on chronic cyclosporine A nephropathy by observing the effects of inflammatory reaction on Chronic Cyclosporine A Nephropathy and the infulence of Chailing Decoction on inflammatory cytokines,then to provide the evidence for the clinical application.Methods: Fifty-four Healthy clean Sprague-Dawley(SD)rats were randomly divided into four groups: 24 in CsA group,10 in each of Control group,Valsartan group and TCM group.The rat model with CCN were induced by gavage with CsA 30mg·kg-1·d-1 for 28 days.At the same time,the rats in Valsartan group and TCM group were respectively treated by gavage with valsartan 10mg·kg-1·d-1 and Chailing Decoction 3g·kg-1·d-1 for 28 days,while the rats in the other two groups received physical saline at equal quantity.Every weekend all the rats were weighed and put into the metabolics cages for24-hour urine.6 rats randomly chosen from CsA group in day 8,day 15,day22,day 29,and rest rats in other groups in day 29 were killed to get blood which was used for detecting blood urea nitrogen(BUN)and serum creatinine(Scr),and calculating creatinine clearance rate(Ccr).Kidneys were fixed by10% paraformaldehyde for HE、Masson staining in order to observe the renal pathological changes.Monocyte chemoattractant protein-1(MCP-1),tumor necrosis factor-α(TNF-α),nuclear factor-κB(NF-κB),interleukin-lβ(IL-1β)were detected by immunohistochemistry and Westernblot.The mRNA of MCP-1 、 TNF-α 、 NF-κB 、 IL-1β were detected by reverse transcriptase polymerase chain reaction(RT-PCR).Results:1 The general situation in ratsAll the rats survived and were included in the last statistics.The mentalstate,activity,fur,diet,drinking and urine volume of rats in control group had no obvious changes,while in CsA group,the rats were listless and bovine,the intake of food and water was down-regulated,its fur turned yellow and loose,some even fell off.Compared with CsA group,the general situation of rats in Valsartan group and TCM group were improved.2 The change of pathomorphology in renal tissueHE staining showed that the renal of rats in CsA group,day7,renal interstitial start edema,inflammatory cell infiltration,renal tubular epithelial cells appeared mild degeneration,muddy swollen;day 14,renal interstitial edema,inflammatory cells infiltration,renal tubular epithelial cells degeneration,muddy swollen degree increase day 21 andday 28,a large number of inflammatory cells were infiltrated in renal interstitial,the tubular epithelial cells were denatured,swelling,even necrosis and shed.In control group had no abnormal changes,the tubular epithelial cells arranged regularly without swelling and little inflammatory cells can be seen in renal interstitial.There were still some inflammatory cells infiltrated in renal interstitial with the tubular epithelial cells swelling in Valsartan group and TCM group,but it was significantly lighter than CsA group.Masson staining showed that the renal structure of rats In control group was clear to be seen,the tubular basement membrane was complete,a little collagen deposited in renal interstitial interstitium.In CsA group,day 7,the renal tissue structure was slightly disturbed,the basement membrane of the renal tubule began to thicken,and a small amount of collagen was deposited in the renal interstitial area.day 14,the renal tissue structure was aggravated,the renal tubular basement membrane continued to thicken,Collagen composition increased;day 21,renal tissue structure serious disorder,renal tubular basement membrane thickening,and even twisted,and accompanied by minor rupture,renal interstitial area can be seen a large number of collagen deposition;day 28,renal tissue structure is extremely disorder,Renal tubular basement membrane thickening,accompanied by a large number of twisted,broken,renal interstitial area can be seen a large number of collagencomposition deposition,and was cord-like distribution.There were some degree of collagen deposition in Valsartan group and TCM group,but it was obviously reduced compared with CsA 28 days group.3 The Result of Renal functionCompared with CsA day 7 group,Scr and BUN were increased visibly(P<0.01 or P<0.05)while Ccr was significantly decreased(P<0.01)in CsA day 14,day 21,day 28 group.There’s obvious different in BUN,Scr,Ccr(P<0.01 or P<0.05)Compared with control group,the renal function of rats in CsA 28 group decreased obviously,BUN,Scr increased obviously(P<0.01),Ccr decreased obviously(P<0.01).After medicate,Valsartan group and TCM group improved markedly,Scr and BUN were obviously lower(P<0.01)while Ccr was significantly higher(P<0.01).4 Test the mRNA Expression of MCP-1,TNF-α,NF-κB,IL-1β in Kidney of each group in RT-PCR MethodCompared with CsA day 7 group,the mRNA expression in CsA day 14,day 21,day 28 group were significantly higher(P<0.05 or P<0.01).The mRNA expression in CsA day 14,day 21,day 28 group were significantly different.(P<0.01 or P<0.05)Compared with control group,The mRNA expression in CsA 28 day group significantly higher(P<0.01).Compared with CsA day 28 group,the mRNA expression in Valsartan group and TCM group were significant lower.(P<0.01)5 Test the Protein Expression of MCP-1,TNF-α in Westernblot MethodCompared with CsA day 7 group,the protein expression in CsA day 14,day 21,day 28 group were higher(P<0.01 or P<0.05).The protein expression in CsA day 14,day 21,day 28 group were significantly different.(P<0.01 or P<0.05)Compared with control group,The protein expression in CsA 28 day group significantly higher(P<0.01).Compared with CsA day 28 group,the mRNA expression in Valsartan group and TCM group were significant lower.(P<0.01)6 Test the Protein Expression of NF-κB 、 IL-1β in Kidney in Immunohistochemical MethodCompared with CsA day 7 group,the mRNA expression in CsA day 14,day 21,day 28 group were higher(P<0.01 or P<0.05).The protein expression in CsA day 14,day 21,day 28 group were significantly different.(P<0.01 or P<0.05)Compared with control group,The protein expression in CsA 28 day group significantly higher(P<0.01).Compared with CsA day 28 group,the mRNA expression in Valsartan group and TCM group were significant lower.(P<0.01)Conclusions:1 During the development of CCN,there was a positive correlation between the renal function injury,renal pathological changes,inflammatory reaction and time.2 Chailing Decoction could alleviate the renal pathological damages induced by CsA,and improve renal function by ruducing Scr、BUN and increasing Ccr.3 Chailing Decoction had the effect of down-regulating the over expression of protein and mRNA of MCP-1、TNF-α、NF-κB、IL-1β,thus alleviating renal inflammatory reaction and slowing the process of renal interstitial fibrosis. |
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