| Objective:With the improvement of people's living standards and the yearning for a better life,the demand for companion animals is increasing day by day,leading to the widespread popularity of various kinds of pets in China.The improved economic also causes the incidence of metabolic syndrome to gradually increase in the population,and this trend is also reflected in pets,such as pet obesity,diabetes,and gout.Hyperuricemia is the basis of the pathogenesis of gout,but there are many studies on gout in animals,while the research on hyperuricemia is relatively insufficient.Therefore,this experiment takes hyperuricemia as the research object and uses bioinformatics to mine the hub genes in the disease.We are also trying to explore whether hydroxychloroquine has a therapeutic effect on hyperuricemia and a potential target of its effect.Methods:We built a rat model of hyperuricemia by taking potassium oxazinate into 6-week-old male SD rats.Hyperuricemia rats were treated with hydroxychloroquine(n=5)and benzbromarone(n=5).The treatment lasted for 4 weeks.During this period,weighing and cage observation were performed weekly.After 4 weeks,the animal's kidney,liver tissue,blood and urine were collected to measure blood uric acid,urine uric acid,blood creatinine,urinary creatinine,urea nitrogen,urine protein,reactive oxygen species,NLRP3,IL-1? and other indicators to assess whether the model was bulit Successfully and the therapeutic effect of hydroxychloroquine.At the same time,the total RNA in the kidney tissue was extracted to construct a transcriptome library,and the next-generation sequencing was performed by Illumina.Bioinformatics analysis mainly uses R packages such as DESeq2 and clusterProfiler for differential expressed gene(DEG)'s calculation and functional enrichment analysis.Additionally,databases such as GEO,GTRD,STRING,PubChem were used for querying,transcription factor target genes prediction,protein-protein interaction analysis,small pharmacal molecule target genes prediction,and molecular docking.Results:The body weight of the rats in the normal group increased steadily,while the weight of the rats in the model group and the two treatment groups increased slowly,and there was no significant difference in the weight of the rats between the model group and the treatment group.Compared with the normal control group,the blood uric acid level in the model group was significantly increased,while the uric acid level was significantly decreased,and there was no statistical difference in urine discharge.At the same time,the serum creatinine,urine creatinine content,renal tissue ROS,liver tissue ROS,IL-1? and NLRP3 of the model group were significantly higher than those of the control group,but the urine protein level only had a tendency to increase and there was also no statistical difference in Serum urea nitrogen and urine urea nitrogen.Compared with the model group,the urine output of rats after treatment has a tendency to increase.The significantly increased urine uric acid and(or)decreased blood uric acid is observed in model group.The levels of blood creatinine and urine creatinine are significantly decreased after treatment.The urinary protein level of rats in the benzbromarone group decreased significantly,while the urea nitrogen and serum urea nitrogen were not statistically different in the two groups compared with the model group.Renal tissue ROS,liver tissue ROS and IL-1? levels were significantly lower than the model group after hydroxychloroquine treatment,and NLRP3 showed a downward trend.Similar results were found in the benzbromarone group.228 up-regulated genes(highly expressed in the model group)and 242 down-regulated genes(lowly expressed in the model group)were calculated between normal and model groups.Functional enrichment analysis showed that the down-regulated gene function of the model group was related to carboxylic acid biosynthesis,carboxylic acid metabolism,cell lipid oxidation metabolism,negative regulation of endoplasmic reticulum stress response,and up-regulated genes were positively correlated with positive regulation of tumor necrosis factor production.By screening the transcription factors related to inflammation in the differential expressed genes,we obtained Nr1d1,Nr1d2,Stat1 and Nfat5.Among them,the expression of Nfat5 was significantly down-regulated after the administration of hydroxychloroquine,and Statl showed a downward trend.In the prediction results of Nfat5 target genes,Spag7 was significantly down-regulated in the model group(P<0.05).209 up-regulated genes and 272 down-regulated genes were calculated between the hydroxychloroquine group and the model group.The enrichment results showed that the hydroxychloroquine group was significantly related to anion transmembrane transport,carboxylic acid metabolism process,body fluid level regulation and renal system.The databases predicted that the number of target genes of hydroxychloroquine and hyperuricemia are 59 and 38,respectively,and the intersection of target genes is PPAR-?.The results of molecular docking showed that PPAR-? residues H266,C285,and S289 might interact with small hydroxychloroquine molecules,and the affinity constant is 10-7.67.The hydroxychloroquine target genes were analyzed for protein-protein interaction,and 11 genes were DEGs between the hydroxychloroquine group and the model group.The functional enrichment showed that the gene set was related to the differentiation of Th17 cells.The results of the weighted gene correlation network analysis showed that the gene set of the yellow module was highly correlated with blood uric acid,and the functional enrichment results of this module showed that it was related to the prolactin signaling pathway.Conclusions:Compared with the control group,the inflammation-related transcription factor Nfat5 was significantly higher in the model group,and decreased significantly after hydroxychloroquine treatment;Spag7 may be one of Nfat5 predicted target genes;The common target gene of hydroxychloroquine and hyperuricemia is Ppar-?;In the results of WGCNA analysis,the blood uric acid-related gene is related to the prolactin signaling pathway. |
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