Effects Of Combination Of Thymol And Rosmarinic Acid On Barrier Function,Microflora Structure And Inflammatory Response Of Large Intestine In Rats Challenged By LPS

Studies have shown that Thymol and Rosmarinic Acid as plant extracts have antibacterial,antioxidant and anti-inflammatory effects.In livestock production,Thymol and Rosmarinic Acid have been found to modulate intestinal microflora structure,alleviate inflammatory response and improve intestinal health.The homeostasis of intestinal microbial structure plays an important role in host health,however,intestinal microbiota disorders can lead to intestinal barrier dysfunction,leading to intestinal inflammation and other diseases,which harms host health.However,no studies have been reported on the effects of Thymol and Rosmarinic Acid combination on animal intestinal health.Therefore,this study investigated the effects of the combination of Thymol and Rosmarinic Acid on the barrier function,microflora structure and inflammatory response of the large intestine in rats through the LPS-induced intestinal inflammation model,so as to provide references for its role in alleviating intestinal inflammatory response and improving intestinal health of animals.The study is mainly divided into three parts:1.The antibacterial and antioxidant activity of the combination of Thymol and Rosmarinic Acid in vitroThe purpose of this study was to explore the antibacterial and antioxidant activity of the combination of Thymol and Rosmarinic Acid in vitro.The in vitro bacteriostatic test was to evaluate the inhibitory effect of the combination of Thymol and Rosmarinic Acid on Escherichia coli,Staphylococcus aureus and Salmonella by measuring the minimum inhibitory concentration(MIC)and fractionary inhibitory concentration index(FICI).The antioxidant assay was conducted by constructing free radical system(DPPH and superoxide anion)in vitro to evaluate the free radical scavenging ability of the combination of Thymol and Rosmarinic Acid.The results showed that the FICI of the combination of Thymol and Rosmarinic Acid against Escherichia coli and Salmonella was 0.50,and the FICI of Staphylococcus aureus was 0.75;at the concentration of 1.00 mg/m L,the combination of Thymol and Rosmarinic Acid had significantly higher scavenging ability on DPPH and superoxide anion than that of Thymol and Rosmarinic Acid(P < 0.05).The results showed that the combination of Thymol and Rosmarinic Acid had a strong antibacterial effect and antioxidant capacity,and the effect was better than that of single component Thymol and Rosmarinic Acid.2.Effects of combination of Thymol and Rosmarinic Acid on intestinal morphology,microflora structure and metabolitesThe purpose of this study was to investigate the effects of Thymol and Rosmarinic Acid combination on intestinal morphology,microflora structure and metabolites of rats through LPS-induced intestinal inflammation model.A total of 32 SD rats aged 21 days were randomly divided into 4 groups: challenge control group(LPS),Thymol group(Thy),Rosmarinic Acid group(Ros-A),and Thy × Ros-A group(Thy × Ros-A).Thy group,RosA group and Thy × Ros-A group were treated with 20 mg/kg · BW Thy,20 mg/kg · BW Ros-A,20 mg/kg · BW Thy × Ros-A(1:1)of gavage every day respectively,and the LPS group was gavaged with the same volume of normal saline every day.On the 13 th day of the formal experiment,rats in all treatment groups were intraperitoneally injected with LPS solution of 500 μg/kg · BW to establish the model of acute intestinal inflammation.The results showed that compared with LPS group,the combination of Thymol and Rosmarinic Acid significantly decreased cecal crypt depth,and significantly increased cecal and colonic mucosal thickness(P < 0.05);the composition of intestinal microbial structure and baterial metabolites significantly changed in cecum and colon of Thy × Ros-A group,Compared with the LPS group,the combination of Thymol and Rosmarinic Acid significantly increased the relative abundance of Firmicutes in cecum and colon of rats(P < 0.05),significantly decreased the relative abundance of Bacteroidetes(P < 0.05),and significantly increased the relative abundance of Lactobacillus,Romboutsia,Turicibacter and Clostridium stenosis 1(P < 0.05),significantly decreased the relative abundance of Muribaculaceae and Alistipes in cecum and colon(P < 0.05);compared with LPS group,the combination of Thymol and Rosmarinic Acid significantly increased the concentrations of acetic acid,propionic acid and total SCFAs in cecum and colon.These results indicated that the combination of Thymol and Rosmarinic Acid improved the morphology and structure of cecum and colon,improved the microflora structure of cecum and colon,and increased the concentration of short-chain fatty acids.3.Effect of combination of Thymol and Rosmarinic Acid on barrier function and inflammatory response of large intestineIn this study,LPS-induced rat intestinal inflammation model was used to investigate the effects of the combination of Thymol and Rosmarinic Acid on the barrier function and inflammatory response of the large intestine.The experiment design is the same as in Chapter 2.The results showed that,compared with LPS group,the combination of Thymol and Rosmarinic Acid significantly increased the m RNA relative expression levels of tight junction genes ZO-1,Occludin and Claudin-1 in cecum and colonic mucosa(P < 0.05),and the expression levels were higher or significantly higher than Thymol and Rosmarinic Acid;compared with the LPS group,the combination of Thymol and Rosmarinic Acid significantly decreased the concentrations of IL-6 and MPO in cecum and colon(P < 0.05),significantly decreased the m RNA relative expression levels of TRAF-6,My D88 and IRAK-1 genes(P < 0.05),and the expression levels were lower or significantly lower than Thymol and Rosmarinic Acid;compared with LPS group,the combination of Thymol and Rosmarinic Acid significantly increased the concentrations of GSH-Px and T-AOC in cecal mucosa and T-SOD in colon(P < 0.05),significantly decreased the concentration of MDA in colon(P < 0.05),and the antioxidant activity was better than Thymol and Rosmarinic Acid.These results indicated that the combination of Thymol and Rosmarinic Acid improved intestinal barrier function,alleviated intestinal inflammatory response and improved intestinal health by improving the tight junction function and antioxidant capacity of cecum and colon,and inhibiting the TLR-4/My D88 inflammatory signaling pathway and inflammatory factors,and the effect was better than that of single component Thymol and Rosmarinic Acid.In conclusion,the combination of Thymol and Rosmarinic Acid has strong antibacterial effect and antioxidant capacity.The combination of Thymol and Rosmarinic Acid can improve the morphology and structure of the large intestine,improve the structure of microbiota,and increase the concentration of short-chain fatty acids.The combination of Thymol and Rosmarinic Acid can improve the barrier function and antioxidant capacity of the large intestine,alleviate the inflammatory response of the large intestine,and improve the health status of the large intestine.