The Mechanism Of Diindolemethane Against Staphylococcus Aureus Pneumonia By Targeting Intestinal Flora

Staphylococcus aureus(S.aureus)is an opportunistic pathogen that can cause Staphylococcus aureus pneumonia.The abuse of antibiotics and the tendency of Staphylococcus aureus to develop resistance have led to the widespread prevalence of methicillin-resistant Staphylococcus aureus(MRSA)and other drug-resistant Staphylococcus aureus.Therefore,there is an urgent need for novel anti-infective strategies for Staphylococcus aureus pneumonia.Previous studies have found that intestinal microbiota and its metabolites are involved in the immune regulation of pulmonary bacterial infections and have colonization resistance to drug-resistant bacteria,suggesting that improving intestinal microbiota is a potential treatment strategy for Staphylococcus aureus pneumonia.Phytochemicals have the characteristics of low cost,wide source and low toxicity,and are an important treasure house for the discovery of lead compounds against bacterial infection.In conclusion,it is of great significance to discover lead compounds targeting intestinal microbiota in the treatment of Staphylococcus aureus pneumonia from phytochemicals.In this study,the prophylactic effect of different concentrations of phytochemical diindolemethane(DIM)on Staphylococcus aureus pneumonia was investigated through experimental therapeutics.The results showed that DIM(100 mg/kg,200 mg/kg and400 mg/kg)had good prophylactic effect on Staphylococcus aureus pneumonia,and200 mg/kg DIM could significantly reduce the bacterial load in lung tissue of mice with staphylococcus aureus pneumonia(P < 0.01)and inflammatory response(IL-6,< 0.05;IL-1β,TNF-α,P < 0.01),200 mg/kg can effectively relieve the pathological injury of lung tissue and pulmonary edema.In this study,the antibacterial effect of DIM on S.aureus was further verified by the minimum inhibitory concentration test and growth curve analysis.The results showed that MIC of DIM on S.aureus tested was greater than 1024 μg/m L,and there was no significant inhibitory effect on the growth of MRSA USA300 when the concentration was not higher than 256 μg/m L.The results showed that DIM had no direct inhibitory effect on Staphylococcus aureus.In addition,DIM(no higher than 128 μg/m L)was found to have no significant drug toxicity on host cells(A549 cells)at the cellular level.The content of DIM in stool,blood and lung tissue of mice was determined by HPLC to analyse the absorption and metabolism of the drug in mice.The results showed that DIM was not detected in the above samples of mice,which was lower than the detection limit(0.1 μg/m L).The above studies indicated that DIM played a preventive role in Staphylococcus aureus pneumonia based on nonpathogenic targets.In conclusion,this study further explored the anti-Staphylococcus aureus pneumonia effect of DIM based on intestinal flora and its potential mechanism.For 14 consecutive days,DIM had no significant effects on body mass(diet,water intake and weight gain)and lung organ index of mice,indicating that DIM had no significant toxic effect on mice at effective dose concentration.In this study,an animal model of intestinal flora disorder in mice was established to verify the potential effect of DIM on regulating intestinal flora to prevent Staphylococcus aureus pneumonia.The results showed that DIM could significantly reduce the amount of lung bacteria carried by mice with intestinal flora disorder(P < 0.01)and alleviate the pathological injury of lung tissue.In addition,S.aureus infection caused significant increases in MPO and ROS levels in the lung of the microbiota dysbiosis group(P < 0.01),while DIM treatment significantly reduced MPO and ROS levels(P < 0.01).Inflammatory signaling pathway analysis(protein western blotting test and ELISA test)showed that Staphylococcus aureus infection caused the activation of MAPK inflammatory signaling pathway in mouse lung tissue,and phosphorylated JNK and p38 protein expression levels increased significantly(P < 0.01).DIM treatment significantly decreased the expression of phosphorylated JNK and p38 protein(P < 0.01)and the inflammatory response mediated by Staphylococcus aureus(TNF-α,P <0.05;IL-6、IL-10,P <0.01).Immunofluorescence and PAS staining confirmed that DIM treatment effectively alleviated antibiotic-induced reduction in the expression of tight junction proteins(ZO-1 and Claudin-1)and the number of goblet cells(P < 0.01).16 s RNA high-throughput sequencing results showed that DIM treatment could significantly change the microflora structure of mice(P < 0.01),and increase the proportion of bacteroides,Helicobacter and other microflora.DIM-mediated fecal transplantation significantly reduced lung bacterial load(P < 0.01)and inflammatory response(TNF-α,P<0.05;IL-6、IL-10,P <0.01),alleviated lung pathological injury,and confirmed that DIM played a role in preventing Staphylococcus aureus pneumonia by regulating intestinal flora.In this study,non-targeted metabolomics technology was used to analyze the differential metabolites screening.The results showed that DIM treatment could significantly improve the enrichment of isoferulic acid(IFA),mycophenolic acid(MPA),oxidated glutathione(GSSG)and uridine-5’-disodium diphosphate(UDPD)in the intestine.Metabolite complement-related tests confirmed that IFA supplementation could effectively reduce lung bacterial load(P < 0.01),alleviate pulmonary edema induced by S.aureus(P < 0.05)and pathological damage,inhibit the activation of MAPK inflammatory signaling pathway and the expression of inflammatory factors.In conclusion,this experiment found that DIM treatment(200 mg/kg)regulated the gut microbiota of mice,significantly increased the proportion of Bacteroides and Helicobacter,increased the production of IFA,inhibited the lung MAPK signaling pathway,reduced colony colonization in tissues,alleviated lung pathological injury,and effectively played a role in preventing Staphylococcus aureus pneumonia.This study provides a new idea and lead compound for targeting gut microbiota against Staphylococcus aureus pneumonia,and lays an experimental foundation for further prevention and treatment of drug-resistant Staphylococcus aureus infection.