| Natural products Cichoric acid is a caffeic acid derivative,is found in edible plants and vegetables such as lettuce,echinacea and cabbage It has the effects of enhancing immunity,improving brain function,antioxidant and hypoglycemic effect,and has great potential for development as a food functional factor.Staphylococcus aureus(Staphylococcus aureus,short for "S.aureus")is a widespread foodborne pathogen,can cause a variety of diseases.It is important to note that eukaryotic serine-like Ser/Thr phosphatases(Stp1)and through substrate protein phosphorylation/dephosphorylation play a global regulatory role in S.aureus.Stp1 affects the pathogenicity of S.aureus by regulating virulence factors.Through virtual screening,we found that cichoric acid is an effective Stp1 inhibitor,but its application is greatly limited due to its poor solubility,low stability and low bioavailability.Food grade biopolymer hydrogels are flexible polymer gel materials with three-dimensional network structure between solid and liquid.As drug delivery carriers,they have the advantages of high safety,good biocompatibility,low cost and high commercial availability.Therefore,we prepared food-grade hydrogel with carboxymethyl chitosan and polyvinyl alcohol as raw materials loaded with cichoric acid,which can target Stp1 to reduce the virulence of S.aureus without affecting the growth of S.aureus,avoid the generation of drug resistance and treat S.aureus infection.Specific research results are as follows:(1)Through virtual screening and phosphatase activity experiment,we screened out the natural inhibitor of Stp1 targeting Stp1-cichoric acid.Through molecular dynamics simulation,two binding modes of cichoric acid and Stp1 complex were obtained.The analysis showed that cichoric acid had two inhibition mechanisms of competitive inhibition and non-competitive inhibition on Stp1.The important binding sites were near the active region and the flap region,respectively.Phe150,Leu154,Gln170,Ile173,Thr175 were the key residues for the competitive binding between the cichoric acid and Stp1,and the cichoric acid played a role through competition with the substrate.His153,Val155,Leu156,Pro162,Phe166 are important residues of the non-competitive binding of cichoric acid to Stp1.Cichoric acid binds to Stp1 flap domain and exerts its effect to affect dephosphorylation.The root-mean-square deviation and binding free energy of cichoric acid-Stp1 complex were calculated,which further indicated that the stability and binding affinity of chicori-Stp1 complex were good.(2)According to the phosphatase activity tests of Stp1 and Stp1 mutants,it was determined that the IC50 of cichoric acid to Stp1 was 2.238 μM.After the mutation of Stp1 binding site residues,there was still phosphatase activity,but the inhibitory effect of cichoric acid on them was reduced.Fluorescence quenching experiment and enzyme kinetics experiment further demonstrated two inhibitory mechanisms of cichoric acid on Stp1.In addition,the possible effects of Mn2+ and substrate p NPP were also studied.The experimental results showed that Mn2+ had little effect on the inhibition of Stp1 by cichoric acid,and the increase of the concentration of substrate p NPP caused the decrease of the inhibition of Stp1 by cichoric acid,which confirmed the competitive property of the inhibition of Stp1 by cichoric acid.Fluorescence quantitative PCR showed that cichoric acid down-regulated the key virulence factors.The results of Western blot showed that cichoric acid decreased Hla expression in a dose-dependent manner.Plate test of hemolytic activity found that the presence of cichoric acid can reduce the hemolytic activity of S.aureus.The growth curve and other bacteriostatic experiments showed that cichoric acid had no significant effect on the growth of S.aureus.In conclusion,cichoric acid could treat S.aureus infection mainly by reducing the virulence of S.aureus.(3)A mouse skin trauma model was constructed,and a carboxymethyl chitosan-polyvinyl alcohol-cichoric acid(CMCS-PVA-CA)hydrogel was designed using tetraethyl orthosilicate(TEOS)as a cross-linking agent.Various properties of the hydrogel were characterized and measured.The results showed that CMCS-PVA-CA hydrogel has good stability,water retention property,swelling property and antioxidant capacity.In addition,in the mouse trauma model,the wound surface of the mice was basically healed on the 14 th day after the treatment of the CMCS-PVA-CA hydrogel group,and many new vessels and hair follicles appeared in the pathological sections,indicating that the CMCS-PVA-CA hydrogel had the effect of promoting wound healing.LDH experiment showed that the hydrogel had good biocompatibility and could be used as a wound dressing and popularized in the field of biomedicine. |
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