| Objective:Firstly,the effects of autophagy on uric acid-induced oxidative damage of NRK-52 E cells was studied in vitro;Furthermore,the protective effects of Ten Flavor Ru Xiang powder-medicated serum on uric acid-induced oxidative damage of NRK-52 E cells mediated by autophagy mechanism was explored by in vitro experiments;Finally,the protective effects and mechanism of Tibetan medicine Ten Flavor Ru Xiang powder on gouty nephritis rats based on mitophagy pathway were discussed through in vivo experiments,which provided experimental basis for the clinical application and the secondary development of new indications of Ten Flavor Ru Xiang powder.Methods:1.Rat renal tubular epithelial cells(NRK-52E)were cultured in vitro,and the stimulating concentration of uric acid(16 mg/d L)was determined by CCK8 method.The cells were divided into normal group、uric acid group(16 mg/d L),autophagy activation intervention group(uric acid + rapamycin 4 μmol/L)and autophagy inhibition intervention group(uric acid + 3-methyladenine 4 mmol/L).Flow cytometry was used to detect the changes of ROS level and mitochondrial membrane potential in NRK-52 E cells stimulated by uric acid and autophagy intervention agent.Western Blot was used to detect the expression of autophagy-related proteins p62,Beclin-1 and LC3-II in NRK-52 E cells stimulated by uric acid and autophagy intervention.2.SD rats were intragastrically administered with TFRXP(1600 mg/kg)suspension,twice a day.After 7 days,the serum containing TFRXP was taken from the abdominal aorta.NRK-52 E cells were cultured in vitro and treated with uric acid and serum containing TFRXP(2%-10% volume concentration)for 24 hours to determine the best effective final concentration(6%).The cells were divided into normal group、uric acid group(20 mg/d L)、blank serum group(10%)and drug-containing serum group(6%).CCK8 assay was used to detect the viability of NRK-52 E cells in different groups.Flow cytometry was used to detect the changes of ROS level and mitochondrial membrane potential in NRK-52 E cells stimulated by uric acid and autophagy intervention agent.Western Blot was used to detect the expression of autophagy-related proteins p62,Beclin-1,LC3-II and mitophagy pathway proteins PINK1 and Parkin in NRK-52 E cells treated with different groups.3.The gouty nephropathy model of rats was replicated by giving potassium oxonate(750 mg/kg)combined with uric acid(300 mg/kg).SD rats were randomly divided into normal group,model group,TFRXP low,medium and high dose group(200、400、800 mg/kg),allopurinol group(10 mg/kg),with 8 rats in each group for 28 days.The level of blood uric acid(SUA)was detected by automatic biochemical analyzer.The levels of serum creatinine(SCr),blood urea nitrogen(BUN),xanthine oxidase(XOD)in serum and liver tissue,superoxide dismutase(SOD)and malondialdehyde(MDA)in kidney tissue were measured by ELISA.The level of reactive oxygen species(ROS)was detected by chemical fluorescence method.The pathological changes of renal tissue were observed by HE staining.The apoptosis of renal tissue cells were observed by TUNEL staining.The m RNA expression levels of inflammatory factors IL-1β,TNF-α,apoptosis-related factors caspase-3,caspase-9,Bax and Bcl-2 were detected by q RT-PCR.The protein expression levels of inflammatory factors IL-1β,TNF-α,apoptosis-related factors caspase-3,caspase-9,cleaved caspase-3,cleaved caspase-9,Bax and Bcl-2,mitochondrial autophagy pathway PINK1,Parkin and LC3-II were detected by Western Blot.Results:1.Uric acid inhibited the proliferation of NRK-52 E cells,induced the production of ROS,decreased the mitochondrial membrane potential,increased the expression of p62 protein,and decreased the expression of Beclin-1 and LC3-Ⅱ protein(P<0.01).Under the stimulation of uric acid,the autophagy agonist rapamycin intervention inhibited the accumulation of ROS,increased mitochondrial membrane potential,reduced the expression of p62,and promoted the expression of Beclin-1 and LC3-Ⅱ(P<0.01).However,after the intervention of autophagy inhibitor 3-MA,the cells viability was significantly decreased,the level of ROS was significantly increased,the mitochondrial membrane potential was decreased,the expression of p62 was increased,and the expression of Beclin-1 and LC3-Ⅱ were decreased(P<0.05).2.Compared with the uric acid group,the cell viability of the blank serum group had no significant change,while the serum containing TFRXP could significantly improve the cells viability,reduce the increase of ROS content caused by uric acid(P<0.01),restore the decreased mitochondrial membrane potential,and inhibit the expression of p62,and promote the expression of Beclin-1 and LC3-Ⅱ.The expression of PINK1 and Parkin in the mitochondrial autophagy pathway were increased(P<0.01).3.Compared with the normal group,the renal function of the model group was significantly decreased(P<0.05),the uric acid crystal deposition in renal tissue and renal tissue cell apoptosis were obvious,the ROS level and inflammatory factors IL-1βand TNF-α contents were significantly increased(P<0.05),and the expression of mitophagy-related proteins PINK1,Parkin and LC3-Ⅱwere significantly decreased(P<0.01).Compared with the model group,the TFRXP administration group reduced uric acid,ROS,IL-1β and TNF-α levels(P<0.05),reduced urate crystals and cell apoptosis in renal tissues(P<0.01).It down-regulated the expression of pro-apoptotic genes caspase-3,caspase-9,cleaved caspase-3,cleaved caspase-9 and Bax(P<0.05),up-regulated the expression of anti-apoptotic gene Bcl-2,mitophagy-related proteins PINK1,Parkin and LC3-Ⅱ(P<0.01).The effects of the middle and high dose groups were significantly higher than those of the low dose group,and there was no significant difference between the high dose group and the middle dose group.Conclusion:High uric acid can reduce the viability of NRK-52 E cells,induce oxidative damage and inhibit the level of autophagy.Ten Flavor Ru Xiang powder-medicated serum can activate mitophagy pathway,improve cell autophagy level,alleviate oxidative damage caused by uric acid,and improve cell viability.High uric acid can induce renal injury,oxidative stress and inflammatory response in rats.Ten Flavor Ru Xiang powder can improve renal injury in rats with gouty nephropathy by regulating mitophagy through antioxidant,anti-inflammatory and anti-apoptosis effects. |
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