Experimental Study Of RNAⅢ-inhibiting Peptide For The Prophylaxis Of Staphylococcal Infection On The Orthopedic Implants

ObjectiveThe aim of this study is to synthesize RNAIII-inhibiting peptide (RIP) by the solid-phase synthesis method, then, verify its effects on inhibiting the infection and biofihn formation on the orthopedic implants caused by Staphylococcus in vitro or in vivo. The end-all is to find a new way to prevent Staphylococcus infection of the clinic orthopedic implants.Methods1. The synthesis, purification and verification of RIP: The solid-phase synthesis was used to synthesize the peptide according to the known amino acid sequence of RIP, then, the synthesized peptide was purified by the reverse phase high performance liquid chromatography (RHLC), and detected by mass spectroscopy, amino acid composition analysis, amino acid sequence analysis, Fourier transform infrared spectroscopy ( FTIR) and activity verification.2. The study of RIP inhibiting staphylococcus infection and biofilm formation in vitro and in vivo: RIP's effects on toxins (SEB and hemolysin) production of staphylococcus aureus (S.A) was detected in vitro; The influence of RIP on the adhesion of staphylococcus epidermis (S.E) on the Hela cells was observed with Fluorescence microscope; RIP was cultivated together with S.E ATCC12228 and ATCC35984 respectively, then extra-cellular slime substance(ESS) produced by staphylococcus was detected; The synergistic effect of RIP and antibiotic (levofloxacin ) on the viable count in the biofilm was also included. Based on the above results, rat subcutaneous pocket graft model was established. Before Dacron grafts were implanted into subcutaneous pocket, they were soaked into saline, 10mg/ml RIP, 30mg/ml levofloxacin and admixture, respectively. The survivorship, vital signs and viable cells count in the biofilm of rats of each group were observed,.3. The study of RIP inhibiting staphylococcus infection and biofilm formation on the orthopedic implants in vitro: S.E cells were marked with FITC, then were cultivated together with commonly used biomaterials: Ti alloy, CoCrmo alloy , and UHMWPE,each material has two kinds surface: smooth and rough. Photos were taken with fluorescence microscope and analyzed with Photoshop 9.0 and Image pro plus 6.0 software to study the adhere difference among three materials and RIP's inhibiting Staphylococcus adhesion on the orthopedic biomaterials; SEM and CLSM were also used to study the RIP's effects on the morphology of the biofilm forming on the materials' surface; At last, the living cells and dead cells in the biofilm were marked with SYTO9 and PI respectively, the ratio of living and dead cells among groups was analyzed to study initially RIP's mechanism of action .4. The study of RIP inhibiting staphylococcus infection around the prothesis in rabbit knee arthroplasty: A new rabbits knee arthroplasty model was established, S.E cells were inoculated into operated knees to cause prothesis infection. At the same time RIP and antibiotics were injected into articular cavity. Index including general condition, the change of body temperature and weight, WBC, CRP, ratio of serum copper and zinc, degree of infection and viable cell count in the biofilm on the prothesis were observed to study RIP's prophylaxis effect on the prothesis infection caused by staphylococcus.Results1. The synthesis, purification and verification of RIP: RIP could be synthesized by the solid-phase synthesis method successfully. The purity of RIP was improved to 97.42% by the reverse-phase high performance liquid chromatography (RHLC). The result of mass spectroscopy reveal the polypeptide molecular weight was 914.37, is accordant with theoretic molecular mass; The result of amino acid composition analysis show RIP was composed of Y, S, P, W, T, N, and F, the ratio of amino acids was 0.995:0.950:0.996:0.382:0.987: 1.019:1.00; The amino acid sequence of synthesized RIP was YS-P-W-T-N-F; FTIR detected the stretching and commotion of the keys of NH and CN, according that the same -NH₂ extremity structure with RAP could be concluded; Results of activity verification reveal the peptide had high biological activity.2. RIP could inhibit staphylococcus infection and biofilm formation in vitro and in vivo: RIP could reduce toxins (SEB and hemolysin) production of S.A in vitro; It could also inhibit the adhesion of S.E on the Hela cells according to Fluorescence microscope observing result; moreover, it could also inhibit the secretion of ESS; RIP had synergistic effect with levofloxacin in preventing staphylococcus infection; RIP couldprevent S.A infection around the Dacron grafts in rat subcutaneous pocket graft model ,it could reduced case fatality ratio and cells viable count.3. The results of RIP inhibiting staphylococcus infection and biofilm formation on the orthopedic implants in vitro:The adhesion ability of S.E to Ti alloy, CoCrMo alloy or UHMWPE was different ; Rugosity of materials' surface could also affect staphylococcus' adhesion to the implants. The adhesion to different surface could be ordered as: rough UHWPE > smooth UHWPE > rough CoCrMo > rough Ti > smooth CoCrMo > smooth Ti ; RIP could markedly reduce the adhesion and biofilm formation of S.E on the orthopedic implant materials; Morphology changes of the biofilm from SEM and CLSM observation got the same conclusion; RIP could not kill bacterium directly, however, it could forbid bacterial biofilm formation, according to tow-stain experiment with SYTO9 and PI.4. RIP could prevent staphylococcus infection around the prothesis of rabbits knee arthroplasty: A new rabbits knee arthroplasty model was established successfully, index of RIP + levofloxacin group, including the change of body temperature and weight, WBC, CRP, ratio of serum copper and zinc, degree of infection and viable cell count in the biofilm on the prothesis had an advantage over the controled group, RIP group and levofloxacin group; Associated with antibiotics, RIP could forcefully prevent staphylococcus infection.ConclusionThe solid-phase synthesis method is a good way to synthesize RIP. RIP can prevent infection and biofilm formation of Staphylococcus on the orthopedic implants in vitro and in vivo by interfering in /influencing its "quorum sensing" system and interrupt the communication between cell and cell. This study found a new way to prevent bacterial infection by means of targeting at bacterial QS system, and has very import significance of research and clinic.