Correlation Between TLR4 Inflammatory Pathway And Intestinal Flora In Rats With Gouty Arthritis

Objective: To investigate the relationship between TLR4 inflammatory pathway and intestinal microbe in rats with gouty arthritis.Methods: 5-week-old Wistar male rats were selected to establish models of acute arthritis,hyperuricemia and gouty arthritis,and male rats of the same species were selected to establish blank control group.7 weeks later,the rats were anesthetized by respiratory anesthesia and the abdominal cavity was cut open to collect the blood of the abdominal artery,jejunal colon tissue and feces of the rats.Blood inflammatory indexes of rats were detected by enzyme-linked immunoassay,blood uric acid was detected by uricase,m RNA expression related to TLR4 inflammatory pathway in intestinal tissue of rats was detected by real-time quantitative PCR,expression of related proteins in intestinal tissue of rats was detected by western blotting,and Illumina high-throughput sequencing platform was used to detect 16 S r DNA of intestinal flora in rats.Results: 1)To compare with hyperuricemia group and gouty arthritis group,the serum uric acid value of blank control group is significantly different(P<0.05);the acute arthritis group is statistically significant compared with hyperuricemia group and gouty arthritis group(P<0.05).2)There are significant differences in NF-κB,IL-1β,IL-6 and TNF-α in acute arthritis group,hyperuricemia group and gouty arthritis group compared with blank control group(P<0.05),the levels of NF-κB,IL-1β,IL-6 and TNF-α in gouty arthritis group are significantly higher than those in the other three groups.There are significant differences in NF-κB,IL-1β,IL-6 and TNF-α in acute arthritis group and hyperuricemia group compared with gouty arthritis group(P<0.05).3)The m RNA expression levels of TLR4 and NF-κB in intestinal tissues of blank control group,acute arthritis group,hyperuricemia group and gouty arthritis group are significantly different(P<0.05);TLR4 m RNA of hyperuricemia group and gouty arthritis group is significantly different from blank control group(P<0.05),there is significant difference between acute arthritis group and gouty arthritis group(P<0.05),the m RNA expression of TLR4 in high uric acid group and gouty arthritis group was increased;the m RNA expression of NF-κB in blank control group,hyperuricemia group and gouty arthritis group is significantly different(P<0.05).4)There is significant difference in the expression of NF-κB protein between the blank control group and hyperuricemia group(P<0.05),the TLR4 protein expression level of blank control group and acute arthritis group are significantly different from gouty arthritis group(P<0.05),the expression level of TLR4 protein in gouty arthritis group is higher than that in the other three groups.5)A total of 3275377 ASVs sequences are detected by Illumina high-throughput sequencing platform,including869310 ASVs in blank control group,852301 ASVs in acute arthritis group,697749 ASVs in hyperuricemia group,and 856017 ASVs in gouty arthritis group.Venn diagram can show the number of common and unique ASVs in each group.There are 969 common ASVs in the four groups,5533 unique ASVs in the blank control group,6349 unique ASVs in the acute arthritis group,5106 unique ASVs in the hyperuricemia group,and5041 unique ASVs in the gouty arthritis group.6)The relative abundance of Lactobacillus in blank control group is high(P<0.05),Oscillospira(Spirillaceae)and Clostridiaceae＿Clostridium(Clostridiaceaeaceae)have higher relative abundance in acute arthritis group(P<0.05),the relative abundance of Coprococcus and Prevotella is higher in the hyperuricemia group(P<0.05),the relative abundance of Prevotella and Turicibacter in gouty arthritis group is higher(P<0.05).7)The Alpha diversity index among the four groups shows no statistical significance in the species richness,diversity and evenness of intestinal flora among blank control group,acute arthritis group,high uric acid group and gouty arthritis group(P>0.05).8)Beta diversity analysis among the four groups showed that the species difference between blank control group,hyperuricemia group and gouty arthritis group was statistically significant(P<0.05),the species difference between acute arthritis group,hyperuricemia group and gouty arthritis group was statistically significant(P<0.05),the species difference between hyperuricemia group and gouty arthritis group was statistically significant(P<0.05).9)Metagenome Seq analysis shows there are significant species differences between blank control group and hyperuricemia group(P<0.05),there are significant species differences between blank control group and gouty arthritis group(P<0.05),there are significant species differences between acute arthritis group and hyperuricemia group(P<0.05),there are significant species differences between hyperuricemia group and gouty arthritis group(P<0.05).10)LEf Se analysis was used to analyze each group of marker species.The results show that Gammaproteobacteria,Pseudomonadaceae,Pseudomonadales,Aerococcaceae,Aerococcus,Actinomycetales,Rothia,Streptococcaceae,Methylobacteriaceae,Methylobacterium and Akkermansia are enriched in the blank control group.In acute arthritis group,Clostridia,Oscillospira and Ruminococcaceae are relatively enriched.In hyperuricemia group,Prevotellaceae,Prevotella,Arthrobacter re relatively enriched.In gouty arthritis group,Erysipelotrichi,Erysipelotrichales,Allobaculum,Blautia,Dorea,Acinetobacter,Moraxellaceae,Flavobacteriaceae,Leuconostocaceae,Weissella,Planococcus are relatively enriched.11)Correlation analysis between TLR4 inflammatory pathway and intestinal flora showed that the relative abundance of Clostridium and Pseudomonas bacteria is negatively correlated with TLR4 m RNA(P<0.05),the relative abundance of Prevotella is positively correlated with TLR4 m RNA(P<0.05);the relative abundance of Clostridium bacteria is negatively correlated with NF-κB m RNA(P<0.05),Prevotella is positively correlated with NF-κB m RNA(P<0.05);Clostridium is negatively correlated with TLR4 and NF-κB protein(P<0.05),Lactobacillus and Pseudomonas are negatively correlated with NF-κB protein(P<0.05),Prevotella is positively correlated with NF-κB protein(P<0.05);Lactobacillus,Clostridium and Pseudomonas are negatively correlated with NF-κB,IL-1β,IL-6,TNF-αand TLR4 in serum(P<0.05),Prevotella is positively correlated with NF-κB,IL-1β,IL-6and TNF-α in serum(P<0.05);Clostridium is negatively correlated with blood uric acid(P<0.05),Prevotella is positively correlated with blood uric acid(P<0.05).Conclusions:Intestinal flora diversity decreased in hyperuricemia and gouty arthritis rats,and changes occurred at the level of gate and genus.Intestinal microbial imbalance may participate in the occurrence of gout by affecting the expression of TLR4 inflammatory pathway related proteins.