| Objective:According to statistics,the prevalence rate of chronic kidney disease(CKD)is more than 10% in China,and renal fibrosis is the main pathological basis and common pathway leading to the progression of CKD to end stage renal disease(ESRD)from various causes.For ESRD patients,there is currently no other treatment except renal replacement therapy,but renal replacement therapy also has limitations and is not an ideal treatment plan.Therefore,it is important to find and find ways to delay or even treat renal fibrosis.Flufenidone(AKFPD)is a novel pyridone drug that significantly reduces renal fibrosis by inhibiting activation of NOD-like receptor family Pyrin domain containing 3(NLRP3)inflammasome.Lysosome injury is the main activation pathway of NLRP3 inflammasome.Previous studies have found that several cathepases,especially cathepsin B(CTSB),are involved in the activation of NLRP3 inflammasome based on the cathepsin knockout or knockout model.Lysosome damage is mainly manifested by the change of the stability of the lysosome membrane,which leads to the release of a large amount of lysosomal cathepsin into the cytoplasm,in which CTSB plays a key role in inducing the activation of NLRP3 inflammasome.However,the underlying mechanism of this inhibition is still not completely clear.This study aims to reveal whether AKFPD inhibits renal fibrosis by protecting lysosome damage pathway,explore its molecular mechanism,and identify its new targets,so as to provide a theoretical basis for the clinical application of AKFPD and the development of other new drugs.Methods:A random number table method was used to divide 30 SD male rats into two groups after surgery: postoperative day 3 group and postoperative day 7group.Each group was further divided into normal group,model group and treatment group.Overall,there were six groups: 3d-normal group,3d-model group,3d-treatment group,7dnormal group,7d-model group,7d-treatment group,with 5 rats in each group.The model group and the treatment group underwent left ureter ligation,while the normal group underwent similar operation,but the left ureter was only exposed without ligation.From the first day after surgery to the day of euthanasia,rats in normal group and model group were intragaxed with 0.5% sodium carboxymethylcellulose solution6ml/8kg/d,while rats in treatment group were intragaxed with AKFPD suspension500mg/kg/d.After Anle’s death on day 3 and day 7,left kidney tissue was taken for:(1)HE staining and Masson’s trichrome staining to evaluate renal fibrosis;(2)Western Blotting assay for Fibronectin(FN)and α-smooth muscle actin(α-SMA).(3)Immunofluorescence single stain assay for the expression of Myeloperoxidase(MPO).(4)ELISA method assay Interleukinβ(IL-1β)and Tumor necrosis factor α(TNF-α).(5)Western Blotting assay for NLRP3,Apoptosis-associated speck-like protein(ASC),Cysteinyl aspartate specific proteinase(caspase-1)and its precursors,and IL-1β and its precursors.(6)Western Blotting assay for CTSB and biochemical kit detect the activity of CTSB.(7)Laser confocal immunofluorescence double staining assay for CTSB and NLRP3 co-localization assay.Results:(1)The results of interstitial injury score of HE-stained sections of kidney tissue and collagen fiber positive area score of Masson stained sections with blue staining showed that the scores of UUO rats were significantly higher on the 3rd and 7th postoperative days in the model group compared with the normal group,while the scores were significantly lower on the 3rd and 7th postoperative days in the treatment group compared with the model group,there were significant differences in all comparisons(p <0.01).(2)The results of Western Blotting method showed that the expression of key fibrotic proteins(FN and α-SMA)in UUO rats was significantly increased on the 3rd and 7th postoperative days in the model group compared with the normal group,while that was significantly decreased on the 3rd and 7th postoperative days in the treatment group compared with the model group,there were significant differences in all comparisons(p < 0.01).(3)The results of immunofluorescence single-staining assay showed that the number of MPO-positive cells in UUO rats increased on the 3rd and 7th postoperative days in the model group compared with the normal group,while that decreased on the3 rd and 7th postoperative days in the treatment group compared with the model group,there were significant differences in all comparisons(p < 0.05).(4)The results of ELISA showed that IL-1β and TNF-α were increased in UUO rats on the 3rd and 7th postoperative days in the model group compared with the normal group,while that decreased on the 3rd and 7th postoperative days in the treatment group compared with the model group,there were significant differences in all comparisons(p < 0.05).(5)The results of Western Blotting method showed that NLRP3,ASC,procaspase-1,caspase-1,and pro-IL-1β levels were increased in UUO rats on the 3rd and7 th postoperative days in the model group compared with the normal group,while caspase-1 and IL-1β levels were decreased on the 3rd and 7th postoperative days in the treatment group compared with the model group,there were significant differences in all comparisons(p < 0.05).However,the protein levels of NLRP3,ASC,pro-caspase-1and pro-IL-1β did not change.(6)The results of Western Blotting method showed that the level of CTSB was increased in UUO rats on the 3rd and 7th postoperative days in the model group compared with the normal group,while decreased on the 3rd and 7th postoperative days in the treatment group compared with the model group,there were significant differences in all comparisons(p < 0.05).Meanwhile,the results of the biochemical kit test showed that increased in CTSB activity in UUO rats on the 3rd and 7th postoperative days in the model group compared with the normal group,while decreased on the 3rd and 7th postoperative days in the treatment group compared with the model group,there were significant differences in all comparisons(p < 0.05).(7)The results of laser confocal immunofluorescence double dye experiment showed that the co-localization of CTSB and NLRP3 was significantly increased in UUO rats on the 3rd and 7th postoperative days in the model group compared with the normal group,while that was significantly decreased on the 3rd and 7th postoperative days in the treatment group compared with the model group.Conclusions:(1)Flufenidone can inhibit NLRP3 inflammasome activation-mediated renal inflammatory response and fibrosis in UUO rats.(2)Flufenidone can inhibit the activation of NLRP3 inflammasome by protecting against lysosomal damage in UUO rats. |
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