Investigation On Its Anti-MRSA Effects Of Emodin In Vitro And In Vivo, And Its Mechanisms

Objective:Methicillin-resistant Staphylococcus aureus (MRSA) is an important Gram-positive bacterium for global hospital infection with high death rate and is difficult to treat. The separation of MRSA increases year by year and its epidemic range continues to expand. Multidrug resistance of MRSA caused by abusing of antibiotics is becoming increasingly serious, and MRSA can produce different degree of resistance to beta lactams, aminoglycosides, macrolides, tetracyclines, fluoroquinolones and sulfonamides and other antibiotics. Glycopeptide antibiotic vancomycin was used as the "last line of defense" in the treatment of MRSA infection. However, vancomycin-intermediary Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. The anti-MRSA new drugs such as linezolid and daptomycin have also been detected. Therefore, investigation of new drugs against MRSA is very urgent and important.The main resistance mechanisms of MRSA against antibiotics include: ① large expression of specific resistance protein penicillin binding protein 2a (PBP2a) encoded by mecA gene which can supplement and compensatory the transpeptidase function of PBP2 inhibited by β-lactam antibiotics to maintain cell wall synthesis,② expression of P-lactamase to destroy β-lactams by hydrolysis, and ③ reducing drug accumulation which mainly related to efflux pump proteins exists in Staphylococcus aureus cell membrane.Research and development of new type of antibiotics is the most effective pathway to fight bacteria. In response to the diffusion of MRSA, people try to obtain drugs with stronger antibacterial activity through bioseparation, chemical synthesis, and structural modification of existing antibiotics. Traditional Chinese herbal medicines have been used for the treatment of infectious diseases for a long time, and the Chinese herbal medicine in China provide rich sourses for the development of natural anti MRSA drugs.With the direct antimicrobial activity as guidance, we previously screened 30 kinds of heat clearing and detoxifying herbs using MRSA standard strains, and we found the crude extract of Polygonum cuspidatum had certain anti-MRSA activity. In this study, Polygonum cuspidatum was chosen for directional separation of the component with direct anti-MRSA effect, then the in vitro and in vivo biological activity and the anti-MRSA mechanism of the active component was studied.Methods:1. The directional isolation of composition and compound from Polygonum cuspidatum based on the combination of pharmaceutical chemistry methods and bioactivity assays1.1. Different extraction parts were obtained by 80% ethanol and organic solvent extract, and the activity of each part was observed by MIC method;1.2 The active extraction part was separated by column chromatography, and the antibacterial activity of each separation composition was observed;1.3 The active composition was purified to obtain the monomeric compound, and the structure of the compound was identified by mass spectrometry and nuclear magnetic resonance.2. In vitro anti-MRSA effect of emodin2.1 Investigation of anti-MRSA activity of emodin through MIC and MBC tests using MRSA252 standard strain and 36 MRSA clinical isolated strains;2.2 Investigation of dynamic sterilization effect of emodin through time kill curve;2.3 Multipassage resistance selection assay was carried out to confirm whether emodin induced MRSA resistance easily.3. In vivo anti-MRSA effect of emodin3.1 The establishment of lethal mouse model and sub lethal mouse model injected with MRSA;3.2 The protective effect of emodin on the survival of mice challenged with a lethal dose of MRSA;3.3 The influence of emodin on the bacterial counts in blood of mice challenged with a sub lethal dose of MRSA.4. Effects of emodin on mammalian cytotoxicity and membrane stability of erythrocyte4.1 The influence of emodin on cell viability of murine peritoneal macrophages RAW264.7 cell lines;4.2 Investigation of the effect of emodin on membrane stability of rabbit erythrocyte using hemolysis test.5. Investigation on anti-MRS A mechanism of emodin5.1 Effects of emodin on major resistance mechanisms of MRSA5.1.1 Biosensor technology was used to observe the Affinity between emodin and PBP2a, and RT-PCR was carried out to observe the effect of emodin on expression of mecA gene;5.1.2 Nitrocefin method was used to observe the effect of emodin on p-lactamase of MRSA252 standard strain;5.1.3 Investigation of the effect of emodin on daunorubicin accumulation in MRSA252 in using fluorescence spectrophotometry.5.2 Effects of emodin on expression of genes related to cell wall synthesis and hydrolysis of MRSA5.2.1 The effect of emodin on MRSA252 morphology observation with scanning electron microscope and transmission electron microscope;5.2.2 Investigation of the effects of emodin on expression of genes related to cell wall synthesis and hydrolysis of MRSA using RT-PCR method.5.3 Effects of emodin on MRSA cell membrane5.3.1 Fluorescence polarization tests were carried out to observe the effect of emodin on MRSA membrane fluidity;5.3.2 Investigation of the effect of emodin on MRSA membrane integrity using nucleic acid probe.Results:1. The anti-MRSA active monomeric compound was separated and purified from Polygonum cuspidatum, and was identified to be Anthraquinone compounds emodin;2. MICs of emodin against MRSA252 standard strain and 36 MRSA clinical strains were between 2 μg/mL and 8 μg/mL, and the MBCs were between 4μg/mL and 32 μg/mL. The MBC/MIC values of emodin were between 1 and 2. Emodin was not easy to cause MRSA resistance;3. Emodin showed significant protective effect on mice challenged with lethal dose of MRSA, and the survival rate of low, medium and high dose of emodin was 12.5%,50% and 87.5%, respectively. Emodin could dose dependently reduced the bacteria counts in blood of mice challenged with sublethal dose of MRSA;4. Emodin (< 32 μg/mL) had no significant effect on RAW264.7 cell line, and emodin (< 64 μg/mL) had no significant effect on membrane stability of rabbit erythrocyte;5. Study on anti-MRSA mechanism of emodin5.1 Emodin had a low affinity with PBP2a and it had no influence on expression of mecA gene. Emodin could not affect the activity of β-lactamase. Emodin could not increase the accumulation of daunorubicin in MRSA;5.2 Emodin could damage the structure of MRSA cell wall and membrane. Emodin had no influence on the expression of genes related to cell wall synthesis and hydrolysis;5.3 Emodin could reduce cell membrane fluidity and disrupt membrane integrity of MRSA.Conclusions:1. Emodin has a significant anti-MRSA activity both in vitro and in vivo with a small possibility to cause resistance.2. Its anti-MRSA effect was tightly related to reducing bacterial membrane fluidity and disrupting membrane integrity, not related to influence on expressions of genes associated with cell wall synthesis and lysis, PBP2a, β-lactamases and drug accumulation.3. Emodin could be further investigated as a leading compound to find new targets and drugs for treatment of MRSA infections.