Artesunate In Combination With Oxacillin Protect Sepsis Model Mice Challenged With Live Methicillin-Resistant Staphylococcus Aureus (MRSA) And Its Possible Mechanisms

Objective:Gram-positive bacterial sepsis could be triggered by bacteria and bacterial components such as peptidoglycan (PepG) and lipoteichoic acids (LTA), etc. Sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA) has much worse outcome than that induced by methicillin-susceptible Staphylococcus aureus (MSSA) because of multiresistance to a large group of antibiotics, which may ultimately lead to death from septic shock. Therefore, it is very important not only to control bacterial proliferation but also to inhibit inflammation caused by MRSA. However, few anti-sepsis drug is clinical used except activated protein C which is in doubt of its therapeutic effect now. Therefore, it is urgent to investigate new anti-sepsis drugs. Without doubt, it is meaningful if a drug can play roles of both controlling bacterial proliferation and inhibiting inflammation.The exaggerated release of proin?ammatory cytokines are the indicators of sepsis. TNF-αis considered to be an early cytokine while IL-6 is considered to be a later cytokine. Gram-positive bacterial components such as PepG and LTA can trigger proin?ammatory cytokines release via Toll-like receptor 2 (TLR2) - and nucleotide-binding oligomerization domain containing 2 (NOD2) - mediated signal pathways.Flourishing bacterial proliferation will continuously induce proin?ammatory cytokines release, thus leading uncontrolled sepsis even septic shock. MRSA is a superbug due to its multi-resistance to a large group of antibiotics, only sensitive to limited effective antibiotics such as vancomycin, teicoplanin, linezolid and daptomycin. Although excessiveβ-lactamase and increased efflux system also play very important roles for resistance toβ-lactams, the major resistance mechanism of MRSA toβ-lactams is due to the acquisition of additional penicillin-binding protein 2a (PBP2a) encoded by mecA gene. PBP2a can function as a transpeptidase, replacing or compensating the function of the other staphylococcal transpeptidases inhibited byβ-lactams. Therefore, inhibition of PBP2a’s function is presumed to be a good strategy for MRSA control. Unfortunately, there is no successful drug targeting PBP2a used in clinic until now.To date, more than 10 efflux pumps have been described for S. aureus. Most of these pumps belong to the major facilitator superfamily, namely the chromosomally encoded NorA, NorB, NorC, MdeA and SdrM as well as the plasmid-encoded QacA/B pumps. Other types of pumps have also been described for S. aureus such as MepA, a member of the multidrug and toxic compound extrusion family, as well as Smr, which belongs to the small multidrug resistance (SMR) family, and SepA. Although these pumps show different substrate specificity, most of them are capable of extruding compounds of different chemical classes, thus providing the cell with the means to develop a multidrug resistance (MDR) pheno-type or to survive in a hostile environment.Previously, we had demonstrated antimalarial artemisinin and artesunate protected sepsis mice challenged with heat-inactivated Escherichia Coli (E.coli) and MSSA via a mechanism involving a reduction in proinflammatory cytokines release, and artesunate in combination with oxacillin could not only protect mice challenged with heat-inactivated MSSA but also live MSSA via its anti-inflammatory effect. However, there was no report about the effects of artesunate alone or in combination with antibiotics on mice challenged with live MRSA. With these considerations in mind, we undertook the current study to investigate artesunate alone or in combination with antibiotics on mice challenged with live MRSA and the possible molecular mechanisms in vivo and in vitro.Methods:1. The protective effect of AS in combination with OXA on bacterial sepsis model mice challenged with MRSA.1.1. Sepsis mice model challenged with lethal live MRSA was established. The protective effects of AS in combination with OXA were observed.1.2. Inhibition of AS in combination with OXA on blood bacteria number and serum TNF-α, IL-6 releasing of sepsis mice model challenged with sublethal live MRSA was observed.2. Mechanisms of AS as antibacterial potentiator2.1. MICs of AS and different antibacterial agents on WHO-2 and clinical separated strains were observed using micropore dilution, and effects of AS with antibiotics on above strains were observed too. Synergistic effects of AS with antibacterial agents on WHO-2 were observed using dynamic growth curve assay.2.2. Synergistic effect of AS with AMP or AMPS on WHO-2 was compared using dynamic growth curve assay.2.3. Affinity between AS and PBP2a was measured by affinity biosensor. Influences of AS combined with OXA on mecA gene expression on WHO-2 were observed using realtime-PCR.2.4. Accumulation of daunorubicin within bacteria treated with AS was using confocal scanning microscopy and fluorospectrophotometry. Accumulation of oxacillin within bacteria treated with AS was using LC-MS. Effect of AS on bacterial membrance structure was observed using transmission electron microscope. Effect of AS combined with OXA on efflux system of WHO-2 was measured by realtime-PCR.3. Mechanism of AS’anti-inflammatory effect3.1. Inhibition of AS on TNF-α, IL-6 releasing from mice peritoneal macrophages challenged with heat killed WHO-2 were observed using ELISA assay.3.2. Inhibition of AS on TLR2 and NOD2 mRNA and protein expression of mice peritoneal macrophages challenged with heat killed WHO-2 were tested using RT-PCR and western-blot. Inhibition of AS on NF-κB activation of mice peritoneal macrophages challenged with heat killed WHO-2 was tested using ELISA assay.Results:1. The protective effect of AS in combination with OXA on bacterial sepsis model mice challenged with lethal live MRSA was observed.1.1. AS in combination with OXA delayed the death time and decreased the mortality of sepsis mice challenged with lethal live MRSA.1.2. AS in combination with OXA inhibited the blood bacteria number and serum TNF-αand IL-6 of sepsis mice model challenged with sublethal live MRSA.2. Mechanisms of AS as antibacterial potentiator2.1. AS had no antibacterial effect, but AS produced synergistic effect if it combined withβ-lactms to WHO-2.2.2.β-lactmase did not influence the enhance effect of AS on MRSA WHO-2. 2.3. AS combined with OXA inhibited the mecA gene expression especially after addition of OXA or AMP for 6 h. The affinity between AS and PBP2a was higher than affinity between penicillin and PBP2a, teicoplanin and PBP2a.2.4. AS increased accumulation of daunomycin and oxacillin within WHO-2 in dose-dependent and time-dependent manners. AS did not influence the bacterial membrance structure unless in a high concentration. AS combined with OXA inhibited NorA, NorB, NorC mRNA expression of WHO-2, but did not influence the expression of mepA, sepA, medA.3. Mechanism of AS’anti-inflammatory effect3.1. AS inhibited TNF-αand IL-6 release from mice peritoneal macrophages stimulated with heat-killed MRSA WHO-2 in a dose-dependent manner.3.3. AS reduced TLR2 and NOD2 mRNA and protein expressions and NF-κB activation in mice peritoneal macrophages stimulated by heat-killed MRSA WHO-2.Conclusions:1. AS in combination with OXA can significantly protect mice challenged with lethal dose of MRSA WHO-2 than OXA alone and markedly decrease bacterial numbers and TNF-αand IL-6 release in vivo.2. AS has no directly antibacterial effect, but AS increase the sensibility of MRSA WHO-2 and MRSA clinical strain to OXA and AMP. The enhance effect is not related toβ-lactmase, but to the higher affinity between AS and PBP2a and down-regulation of mecA expression. Besides, it also relate to the increased accumulation of antibacterial agents through inhibition of kinds of efflux pumps.3. AS inhibits TNF-αand IL-6 release from peritoneal macrophages through reduction of TLR2 and NOD2 expressions and NF-κB activation.