Application Of Recombinant Uricase-expressing Genetically Engineered Bacteria In Rat Model Of Hyperuricemia

Normally,the uric acid in the human body is maintained in a balance.When the balance is broken,the uric acid in the body gradually accumulates and eventually develops into hyperuricemia.Currently,the main treatment for hyperuricemia is the use of xanthine oxidase inhibitors to reduce uric acid production.However,with its effective uric acid lowering effect,it will also cause more adverse events.The abnormal accumulation of uric acid in the body can be treated by increasing its excretion in addition to reducing its source.Therefore,one of the keys to successful treatment of hyperuricemia is to increase the excretion of uric acid in the body.Objective:Based on the previous experimental results of our research group,we will apply the constructed recombinant uricase-expressing genetically engineered bacteria and analyze effects of engineered bacteria on lowering uric acid in rat model of hyperuricemia and the practical application value and significance of the recombinant bacteria.Methods:The experimental rats randomly divided into 5 groups were male SPF-grade Sprague-Dawley(SD)rats,with 10 rats in each group,a total of50 rats.Recombinant uricase-expressing genetically engineered E.coli bacteria group(E36eu);empty vector-expressing engineered E.coli bacteria group(E36e);E.coli group(E);carboxymethylcellulose suspension group(NS);and blank control(Con)group.The experiment was carried out for eight weeks,the first four weeks were modeled,and engineered bacteria were applied from the fifth week.Daily measurement of water consumption,food consumption and body weight of each group of rats,and the serum uric acid(SUA)of the rats was measured by enzyme colorimetry every Monday.At the end of the experiment,the blood of each group of rats was collected and counted for eosinophils by Wright staining;the small intestine tissue of each group of rats was analyzed by paraffin section HE staining for histopathological analysis of the small intestine;16S rDNA sequencing technology was used to analyze the intestinal contents;then the fecal matter in the rat's large intestine was taken to detect the allantoin level by an ELISA kit.Results:During the whole experiment,all rats showed a natural growth trend;The serum uric acid levels of rats in the E36 eu,E36e,E,NS groups during the first four weeks were significantly higher than those in the Congroup(P <0.01).The serum uric acid in hyperuricemia rats(E36eu group)administered recombinant uricase-expressing genetically engineered E.coli bacteria from the fifth to eight weeks was significantly lower than the other four groups(P <0.01).This indicated that the application of engineered bacteria effectively reduced serum uric acid in hyperuricemia rats;Compared with the Con group,the eosinophil count in E36 eu,E36e,E,and NS groups increased slightly,but the difference was not significant in statistical analysis;The structure of intestinal mucosa was intact,epithelial cells were closely arranged,and goblet cells were scattered between absorptive cells.There were no pathological changes in cells.The results indicated that the engineered bacteria had no negative effects on intestinal mucosal tissues;Analysis of intestinal contents showed that there was a difference in intestinal species diversity between E36 eu,E36e,E,NS and Con groups,and the composition and abundance of the colonies in each group were different.These results indicated that the engineered bacteria successfully colonized the intestinal tract of the model rats and had an effect on the intestinal flora structure and species diversity of the small intestine;Allantoin in the fecal excrement of hyperuricemia rats(E36eu group)administered recombinant uricase-expressing genetically engineered E.coli bacteria was significantly higher than other groups(p <0.01).Combined the changes of serum uric acid value in hyperuricemia rats and the analysisof intestinal contents,it indicated that the uricase secreted by engineeried bacteria in the small intestine converted uric acid into allantoin and played a role in reducing serum uric acid in hyperuricemia rats.Conclusion:Recombinant uricase-expressing genetically engineered bacteria colonized in the small intestine of hyperuricemia rats.Uricase secreted by engineered bacteria can effectively reduce serum uric acid in hyperuricemia rats through the intestine.The application of engineered bacteria changed the intestinal contents flora composition,in addition,it had no significant adverse effects on rats.