The Antibacterial Effect Of Antibiotics And Photodynamic Therapy On Cutibacterium Acnes Biofilm And Research On Inflammatory Reaction Mechanism Of Cutibacterium Acnes Biofilm-treated Keratinocytes

Cutibacterium acnes(C.acnes)is a key factor in the development acne vulgaris and may lead to the subsequent inflammatory reaction.C.acnes biofilms are widely found in skin lesions at all stages of acne.Our study mainly focuses on the antibacterial effect of antibiotics and 5-aminolevulinic acid photodynamic therapy on C.acnes biofilm and inflammatory reaction mechanism of C.acnes biofilm-treated keratinocytes,and is divided into the following three parts:First,a standard isolate of C.acnes strain ATCC 11827 was used to construct an in vitro biofilm model,and CLSM and XTT reduction methods were used to observe the biofilm formation law.As the results show,the biofilm structure was the most mature on the 8th day.The method of constructing the biofilm model is stable and repeatable,which lays a good experimental foundation for subsequent research.Then we performed five common used antibiotic drug(erythromycin,clindamycin,tetracycline,doxycycline,and minocycline)susceptibility tests on the planktonic and biofilm status of 32 clinical isolates of C.acnes.The drug resistance rates of planktonic were 6.25%,34.38%,3.1 3%,3.1 3%and 0%.The above-mentioned various drug-sensitive of biofilm resistance rates were 18.75%,93.8%,6.25%,31.25%,and 3.13%,respectively.The experimental results revealed that C.acnes biofilm has increased tolerance and resistance ratio planktonic.Then in Chapter 2 we explore the inhibitory effects of ALA-PDT therapy on C.acnes biofilms.We explored the inhibitory effects of ALA-PDT therapy on C.acnes biofilm under different ALA drug concentrations,different ALA incubation times,and different light intensities in three sections,and evaluated by CLSM and XTT reduction methods.The study found that the higher the concentration of ALA drugs(10mM,50mM,100m M),the more severe the destruction of the biofilm structure,and the more significant the inhibition of biofilm activity.The longer the ALA drug incubation time(15min,30min,and 60min),the more severe the destruction of the biofilm structure,and the more obvious the inhibition of biofilm activity.The stronger the light intensity(50J/cm2,100J/cm2,and 200J/cm2),the more severe the destruction of the biofilm structure,and the more significant the inhibition of biofilm activity.The experimental results revealed that high concentration of ALA,long incubation time,and high dose of light intensity can help inhibit C.acnes biofilm.In addition,we also used the new photosensitizer LP4i to study the photodynamic effect of C.acnes biofilm,and found that the concentration of the drug at 10mM and the light intensity of 50J/cm2 can cause significant damage to the structure and vitality of the biofilm.It is suggested that its bactericidal effect is stronger than the classic drug ALA.Further in Chapter 3 we explore how the C.acnes biofilm induces TLR2-mediated inflammatory reaction in human keratinocytes.Firstly,human keratinocytes were induced in vitro using planktonic C.acnes,C.acnes biofilm and blank control.The expression levels of pro-inflammatory factors IL-6,IL-8 and TNF-? in the C.acnes biofilm group were significantly higher than those in the planktonic C.acnes group and the blank control group by q-PCR and ELISA.It shows that the C.acnes biofilm is stronger than planktonic state in inducing the inflammatory response of keratinocytes.Then we used q-PCR,Western blotting,immunofluorescence and ELISA to detect the expression of TLR2 in C.acnes biofilm-induced keratinocytes,and the phosphorylation of its downstream signal molecules MAPK,ERK1/2 and NF-?B.NF-KB-p65 nuclear entry and downstream pro-inflammatory factors IL-6,IL-8 and TNF-? expression.It was found that the C.acnes biofilm can up-regulate the expression of TLR2 receptors in keratinocytes,promote the increase of phosphorylation levels of MAPK,ERK1/2 and NF-?B,promote the nuclear entry of NF-?B-p65 and promote IL-6,IL-8 and TNF-? expression.In addition,we further explored TLR2 and its downstream signaling molecule inhibitors(TLR2 inhibitor(C29),MyD88 inhibitor(ST2825),NF-?B inhibitor(BAY11-7082),p38MAPK inhibitor(SB203580),and ERK1/2inhibitor(U0126))affects the expression of keratinocyte proinflammatory factors under the action of C.acnes biofilm.The results show that inhibitors can down-regulate the inflammatory effects of C.acnes biofilms on keratinocytes.The experimental results revealed that the C.acnes biofilm affected the inflammatory response by inducing keratinocyte TLR2 signaling pathway.