| Antibiotic resistance is becoming a major crisis endanger public health.Infections diseases caused by drug-resistant bacteria increasingly threaten human lives,such as pneumonia,meningitis,burns and scald infections,diabetic foot ulcer infections,and sepsis.There are limited effective antibiotic agents for treatment of infections caused by vancomycin-resistant Staphylococcus aureus(VRSA),carbapenem-resistant Escherichia coli(CREC),multi-drugresistant Pseudomonas aeruginosa(MRPA)and other drug-resistant bacteria.Once drug resistance occurs,efficacy of drugs from the same class will be greatly reduced.Therefore,the development of new drugs is urgently required.As an important part of host immune defense system,cathelicidin have a great development prospect for its broad-spectrum antibacterial activity,anti-inflammatory activity,immunomodulatory activity,and are not easy to cause bacterial resistance.As a new antiinfective drug candidate,a variety of cathelicidins are currently in clinical research for the treatment of infections.However,as a kind of peptide drug,cathelicidins have some disadvantages such as poor efficacy in vivo and hemolytic toxicity,so it is of great significance to explore new cathelicidin template to solve the problems of stability and toxicity in vivo.There is no cathelicidin drugs in clinical research in China,so the pre-clinical research of cathelicidins will help promote the development of cathelicidin drugs in China.In this thesis,a novel cathelicidin(Hc-CATH)was discovered from sea snake Hydrophis cyanocinctus,and the structure-activity relationship and structure modification were studied systematically,and the modified peptide is intended to be developed into a diabetic foot ulcer gel.The main results of this thesis are as follow:(1)Study on activity screening and structure activity relationship of Hc-CATH.Hc-CATH has broad-spectrum antibacterial activity,and its minimum inhibitory concentration is from 0.16 ?M to 20.67 ?M;Hc-CATH can be directly adsorbed on bacteria,which leads to swelling,rupture and death of bacteria;Hc-CATH can act with LPS and TLR4/MD2 complex respectively,then inhibit MAPK inflammatory pathway and the overexpression of inflammatory factors;the amphipathic alpha helix structure at the N-terminus of Hc-CATH plays an important role in antibacterial and anti-inflammatory activities.In summary,HcCATH is an excellent template for anti-infective peptide drug modification.(2)Structural modification and stability optimization of Hc-CATH:15 modified peptides were designed by N-terminal and C-terminal sequence deletion method,based on the template Hc-CATH.Among modified peptides,HC1,HC2,and HC3 have the strongest antibacterial activity and anti-inflammatory activity.Then stability modification HC1-D2 was designed on the least toxic modification HC1.The results of RP-HPLC show that there is almost no degradation when HC1-D2 incubated with serum for 48 hours,while template HC1 degraded 60%only in 2 minutes.HC1-D2 is effective against drug-resistant bacteria,has the effect of clearing biofilm and rapid sterilization.In vivo experiments,HC1-D2 can reduce the number of infectious bacteria in peritoneal lavage and spleen,increase the survival rate of mice infected by resistant bacteria VRSA,CREC,and MRP A,and significantly reduce the pro-inflammatory cytokines(TNF-?,IL-6)expression.In addition,HC1-D2 can increase the number of peritoneal neutrophils,inflammatory monocytes,and the chemokine MCP-1 in healthy mice,but has no effect on the production of pro-inflammatory cytokine TNF-?,indicating that the regulatory activity of HC1-D2 in organisms is selective.In summary,HC1-D2 can provide reference for the in vivo application of anti-infective peptides.(3)Diabetic foot ulcers are characterized by high amputation rate and high mortality due to drug-resistant bacterial infection,difficult wound healing,and easy recurrence.In this thesis,HC3 with the strongest antibacterial activity and shorter sequence was selected to developed as a topical gel for the treatment of diabetic foot ulcers.In accordance with the pre-clinical chemical drug guidelines,and additional inspection items specific to peptide drugs are added to carry out structural identification,preliminary quality research,stability research,drug efficacy research and preliminary safety evaluation on HC3.The stability inspection results show that HC3 is extremely sensitive to light,humidity,and temperature,so the product needs to be protected from light,sealed and stored at low temperatures.In the rat diabetic ulcer model,compared with sulfadiazine cream,HC3 has a significant effect of clearing wound infection and promoting wound healing.In addition,the preliminary safety investigation has not found any adverse reactions to HC3,which has high safety and is suitable for the development of diabetic foot ulcers drugs.In summary,this study found a novel cathelicidin(Hc-CATH)from the ocean reptiles.We successfully obtained modified HC1 and HC3 with shorter sequence,lower toxicity and retain template activity through structure-activity relationship research and sequence modification.Optimize the stability of HC1 which has lower toxicity,and improved its efficacy in vivo,which can provide data support for the peptide antibiotics application in vivo;the development of gels for HC3 with stronger antibacterial activity,is expected to become a new type of treatment for diabetic ulcers candidate drugs. |
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