Study On The Antibacterial And Anti-inflammatory Mechanism Of Berberine Hydrochloride

Objective:The scientific program was directed by the National Natural Science Foundation of China(81603119)and the Beijing Natural Science Foundation(7174316).Berberine([C20H18NO4]+Cl-)belongs to isoquinoline alkaloid,possessing antibacterial ability and having a broad antibacterial spectrum.Despite widespread usage of antibiotics in clinic,the development of drug-resistant bacteria has become a main concern for us,especially methicillin-resistant Staphylococcus(MRSA).MRSA has characteristics of multiple-drug-resistance capacity and high mortality,and it brings threatens to human life to a large extent.Previous studies have found that phenol-soluble modulins(PSMs)in the extracellular matrix of bacteria were the main virulence factors of MRSA,they also found that PSMs accounted for the pathogenicity of MRSA.Accumulation of PSMs polypeptide would help to form bacterial amyloid fibrils,to regulate the formation and the release of bacterial biofilms,and to aggravate inflammatory damage,therefore PSMs can protect the bacteria against the immune system and enhance the pathogenicity of MRSA.In this study,we analysed the interaction between berberine and PSMs peptides to explore the possibility that berberine could inhibit the development of biofilm by inhibiting the formation of amyloid fibrils through interfering or blocking the polymerization of PSMs polypeptide,and this procedure could reduce the possibility of inflammatory damage to the body.Taken together,our data provided new approaches and methods to effective eradiction of MRSA infection in clinic.Materials and Methods1.Materials:Berberine was purchased from Sigma(Sigma Aldrich,St.Louis,MO,USA),all of the procedures were carried out according to the manufacturer's protocol. Methicillin-resistant S.aureus(MRSA-ATCC33591)strain was used in this study,the complete growth medium was Nutritional broth medium(NB,Baltimore,MD,USA),all of the procedures were carried out according to guidelines on ATCC website.2.Methods:(1)In this study,we plotted the growth curve of MRSA-ATCC33591 and we also evaluated the effects of different concentrations of berberine on the growth curve of MRSA-ATCC33591 in nutrition broth utilizing the method of antibacterial rings.We compared and analyzed the experimental group as well as the control group to show significant differences in antibacterial activity.(2)We analyzed the role of berberine on the integrity of biofilm utilizing confocal laser scanning microscopy in this paper.Our data also showed the role of berberine on MRSA biofilm formation and the inhibitory effects of berberine on the biofilm formation of MRSA,as has been described previously.(3)We observed the morphology of extracellular amyloid fibrils in MRSA biofilm at different concentrations of berberine through transmission electron microscopy(TEM).We also reported the effects of berberine on the expression and formation of amyloid fibrils in MRSA biofilm through Congo Red staining in this paper.(4)In this paper we used molecular dynamics simulation to study the interaction between berberine and PSM monomer(Gromacs 4.5 package)and PSM?2(Gromos 96(53A6)force field).(5)Here we established the mouse acute colitis model of DSS-induced acute inflammation and we observed the effects of berberine hydrochloride on inflammatory damage induced by chemoinflammatory agent.(6)Here we observed the role of berberine on inflammatory injury mouse model induced by MRSA or PSMs,and we also studied the anti-inflammatory effect of berberine in MRSA pathogenesis in this paper.Results:(1)Results of the bacteriostatic ring test confirmed that berberine could not directly kill bacteria,but could inhibit the growth characteristics of bacteria.(2)Data from laser scanning confocal microscopy showed that berberine could destroy the continuity of MRSA biofilm.(3)Results of Congo Red staining experiments suggested that berberine could inhibit the formation of extracellular amyloid peptide,and thus could interfere with biofilm formation and stability.(4)Results of Transmission Electron Microscopy(TEM)revealed that berberine inhibited the amyloid formation of MRSA and also suppressed the release of PSMs and the aggregation of PSMs to form amyloid fibrils.(5)The results of the molecular dynamics simulations showed that the binding of the berberine hydrochloride interacting with PSM?2 secreted by MRSA was on the PSM-?2 benzene ring by hydrophobic interaction.(6)Mice colitis model test showed that berberine hydrochloride had a certain effect on the formation of inflammatory bowel disease.(7)Mice's skin abscess model experiment showed that berberine hydrochloride had promising therapeutic effects on the infection caused by drug-resistant bacteria in vivo,and the possible mechanism was expounded.Conclusions:(1)In this study,results of antibacterial ring experiments and confocal laser scanning microscopy analysis proved that the inhibition capacity of berberine on methicillin-resistant Staphylococcus aureus was not directly killing bacteria but destroying the integrity of biofilm to inhibit bacterial growth.(2)Congo Red staining experiments showed that berberine could pass through the drug-resistant biofilm in the inhibition of amyloid fibril formation and then destroyed the stability of biofilm formation.(3)The interaction between berberine and PSM monomer indicates that the binding of berberine to PSM monomer belonged to nonspecific binding,but there was high affinity between berberine and PSM monomer.(4)Results of animal model showed that berberine on MRSA-ATCC33591-induced skin abscess had therapeutic effects,indicating that berberine has anti-inflammatory effects against bacteria in mice.(5)Berberine could interact with the PSMs as secreted by MRSA,and then could affect the formation of PSMs polypeptide biofilm,making berberine a promising target in the inhibition of MRSA infection.