Advances in research on mechanism and function of antimicrobial peptides

Abstract

In the dissertation work on the basis of research are substantiated the bactericidal mechanism, barrier protection, anti-inflammatory effect and the relationship between the anti-inflammatory effect and intestinal flora of antimicrobial peptide to develop and applicant drugs for prevention inflammation, maintain intestinal flora balance and improve growth performance. Though antibiotics have good antibacterial and anti-inflammatory effects, they may damage the intestinal barrier function, disrupt the intestinal microbial balance, and disrupt the body's intestinal homeostasis. The problems caused by the long-term large-scale use of antibiotics are becoming more and more serious, and the development of efficient and safe antibiotic substitutes is imminent. Antimicrobial peptides have attracted attention due to their broad-spectrum antimicrobial activity, not have drug resistance, and extensive biological functions. During our resserch we investigated mechanism of the antimicrobial peptide mastoparan X in killing Gram-negative bacteria in vitro and its anti-inflammatory and barrier repair functions in pneumonia and enteritis diseases in vivo, and to further study the relationship between anti-inflammatory and intestinal microbes of MPX. Finally, we explored the anti-inflammatory and barrier repair functions mechanism of MPX in the intestinal epithelial cells, laying a foundation for reducing the use of antibiotics in livestock and poultry breeding, it will help provide certain theoretical and practical value for future applications in livestock and poultry. Our research has found that antimicrobial peptides not only relieve intestinal inflammation, but also have the function of protecting the intestinal barrier. Studies have revealed that MPX had good bactericidal activity in vitro, and the main bactericidal mechanism was to produce bactericidal effect by destroying the integrity of the bacterial cell membrane. MPX had good antibacterial and anti-inflammatory effects in mice. MPX had better effect of killing Actinobacillus pleuropneumoniae and alleviating the symptoms of pneumonia causedby A. pleuropneumoniae which mainly by reducing the level of inflammatory factors to alleviate the symptoms of pneumonia. In addition, the study further found that MPX also could alleviate the intestinal inflammation and barrier function damage infection with E. coli by reducing inflammatory factors and increasing tight junction proteins, and explorethe mechanism of MPX relieve inflammation and maintain intestinal barrier function in intestinal epithelial cells. Finally, the relationship between MPX to relieve intestinal inflammation and intestinal microbes was studied. The results found thatMPX alleviated the intestinal inflammation by changing the diversity of intestinal flora. The results of the studies did not reveal MPX regulate the mechanism of microbial diversity, but intestinal microbial diversity and intestinal flora homeostasis were indeed related to intestinal inflammation, and MPXalleviated the intestinal inflammation was indeed closely related to intestinal flora. A. pleuropneumoniae is the causative agent of highly contagious and fatal respiratory infections, causing substantial economic losses to the global pig industry. Due to increased antibiotic resistance, there is an urgent need to find new antibiotic alternatives for treating A. pleuropneumoniae infections. MPX is obtained from wasp venom and has a killing effect on various bacteria. This study found that MPX had a good killing effect on A.pleuropneumoniae and that the minimum inhibitory concentration (MIC) was 16 µg/mL. The bacterial density of A. pleuropneumoniae decreased 1000 times after MPX (1×MIC) treatment for 1 h, and the antibacterial activity was not affected by pH or temperature. Fluorescence microscopy showed that MPX (1×MIC) destroyed the bacterial cell membrane after treatment for 0.5 h, increasing membrane permeability and releasing bacterial proteins and Ca2+, Na+ and other cations. In addition, MPX (1×MIC) treatment significantly reduced the formation of bacterial biofilms. Quantitative RT-PCR results showed that MPX treatment significantly upregulated the expression of the PurC virulence gene and downregulated that of ApxI, ApxII, and Apa1.In addition, the Sap A gene was found to play an important role in the tolerance of A. pleuropneumoniae to antimicrobial peptides.Therapeutic evaluation in a murine model showed that MPX protects mice from a lethal dose of A. pleuropneumoniae and relieves lung inflammation.This study reports the use of MPX to treat A.pleuropneumonia infections, laying the foundation for the development of new drugs for bacterial infections. Escherichia coli is a facultative anaerobic bacterium that exists in the gastrointestinal tract of humans and animals. It can cause diarrhea, enteritis, destruction of the host's intestinal barrier, and intestinal microecological disturbances. In recent years, due to the abuse of traditional antibiotics, a variety of drug-resistant strains and super bacteria have emerged in an endless stream. Therefore, there is an urgent need to find new alternatives to antibiotics. To explore the effect of MPX against E. coli. The function of MPX against E. coli was detected by MIC, plate count, propidium iodide, NPN and DiSC3(5) permeability testing, immunofluorescence microscope observation, and the impact of MPX stability by temperature, pH, ion. In this study, the results found that MPX has good antibacterial activity against E. coli, and the minimum inhibitory concentration (MIC) was 31.25 ug/mL. MPX bactericidal kinetics study found that MPX had good bactericidal activity within 6 hours. Bacterial permeability studies have shown that MPX could increase the permeability of bacteria, leading to an increase in the protein content of the bacterial supernatant. In addition, NPN, PI and DiSC3(5) results showed that the fluorescence value was positively correlated with MPX. The stability test of MPX found that salt ions, temperature, pH, etc. have a slight influence on its effect. In addition, scanning electron microscopy results showed that the bacteria became smaller and the contents leaked after the action of MPX. The above results showed that MPX has a good bactericidal activity in vitro, laying the foundation for the development of new drugs for the treatment of bacterial infections. To investigate whether theantimicrobial peptide mastoparan X (MPX) was effective against E. coli infection. BALB/c mice infected with E. coli by intraperitoneal injection, which represents a sepsis model. The therapeutic effect of MPX was evaluated in a murine model, revealing that it protected mice from lethal E. coli infection. Furthermore, MPX increased the length of villi and reduced the infiltration of inflammatory cells intothe jejunum. SEM and TEM analyses showed that MPX effectively ameliorated the jejunum damage caused by E. coliand increased the number and length of microvilli. In addition, MPX decreased the expression ofIL-2, IL-6, TNF-α, p-p38 and p-p65in the jejunum and colon. Moreover, MPX increased the expression of ZO-1, occludin and MUC2 in the jejunum and colon, improved the function of the intestinal barrier and promoted the absorption of nutrients. This study suggests that MPX is an effective therapeutic agent for E. coli infection and other intestinal diseases, laying the foundation for the development of new drugs for bacterial infections. We investigated whether the antimicrobial peptide mastoparan X (MPX) was effective against E. coli infection. BALB/c mice infected with E. coli by intraperitoneal injection, which represents a sepsis model. In this study, MPX exhibited no toxicity in IPEC-J2 cells and notably suppressed the levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), myeloperoxidase (MPO) and lactate dehydrogenase (LDH) induced by E. coli. In addition, MPX improved the expression of ZO-1, occludin, and claudin and enhanced the wound healing of IPEC-J2 cells. We have proved that antimicrobial peptides can regulate theintestinal flora and maintain intestinal homeostasis. Previous studies have shown that MPXhadgood therapeutic effect on intestinal inflammation caused by E. coli infection. However, the relationship between the alleviation of intestinal inflammation and the intestinal microbiota by MPX is still unknown. We investigated the correlation between theanti-inflammatory effects of MPX and the regulation of the gut microbiota. In this study, E. coliwas used to induce intestinal inflammation, and the results showed that MPX alleviated weight loss and intestinal pathological changes in necropsy specimens ofE. coli-infected mice. MPX reduced the serum levels of the inflammation-related proteins IL-2, IL-6, TNF-α, MPO and LDH on day 7 and day 28. Furthermore, H&E results showed that MPX increased the length of villi and reduced the infiltration of inflammatory cells intothe jejunum and colon. SEM and TEM resultsshowed that MPX could improve the morphology of jejunum villi and microvilli and increase tight junction protein levels. The16S rRNA sequencing analysis of caecalcontent samplesshowed that species diversity and richnesswere lower in the E. coli-infected group. At the phylum and genus levels, higher abundances of pathogenic Firmicutes, Lachnospiraceae-NK4A-136 and Alistipes bacteria were observed. MPX treatment decreased pathogenic bacterial counts in the E. coli-infected group. Furthermore, MPX increased the abundance of Muribaculaceae. Alpha and Beta analysis showed that there was no significant difference inbacterial community structure between the MPX andcontrol groups. In addition, alterations in theintestinal microbiome of miceaffected physiological functions and metabolic pathways. Overall, this study is the first to investigate the correlation of the effects of MPX on intestinal inflammation and the intestinal microbiota, which alleviate intestinal inflammation by restoring the diversity and functions of the gut microbiota, providing a new perspective regarding thetreatment of enteritis. Thus, the findings of these studies were aimed at clarifying MPX had good antibacterial activity against A. pleuropneumoniae and E. coli infectionsin vitro and in vivo. MPXcould resist the pathological damage to the lung and intestine of mice, and relieve the pneumonia, enteritis and intestinal barrierof mice caused bybacteria and further clarify the mechanism of MPX to relieve intestinal inflammation and enhance the intestinal barrier function atintestinal epithelial cells. Finally, this study found that MPX relieved intestinal inflammation by regulating the diversity of intestinal flora. Based on the materials of the dissertation, methodological recommendations «Prevention of antibiotic resistance through the use of antimicrobial peptides» approved by the Academic Council of SNAU (Protocol № 5, dated 29.12.2021). We recommend using the materials of the dissertation work when studying the courses "Veterinary microbiology", "Veterinary pharmacology" for masters of the Faculty of Veterinary Medicine of Sumy NAU. And for the courses "Veterinary microbiology" and "Veterinary pharmacology" for masters of the Henan Institute of Science and Technology (HIST).