Study On Staphylococcus Aureus Skin Infection Model Based On Analytic Hierarchy Process And Intervention Effect Of Pouzolzia Zeylanica Var.microphylla

Objective:In order to establish the technical standard of the dermatosis animal model and the multi-level animal model evaluation system,this experiment simulated clinical skin infections to establish a common animal model of skin infections caused by Staphylococcus aureus,using the analytic hierarchy process?AHP?to establish comprehensive evaluation index weights,screening out high quality animal model,taking Pouzolzia zeylanica extracts as an example of,applying to efficacy evaluation of Traditional Chinese Medicine,which will provide a positive impetus for the prevention and treatment of infectious skin diseases.Method:1.To establish bacterial counting methods:Study on the comparison of counting methods of Staphylococcus aureus based on solid medium:First of all,after comparing the concentration of Staphylococcus aureus in the McFarland's turbidimetry method,plate counting method,and microscopic counting method,then,standard curve of OD value and bacterial concentration was established by the OD value method combined with the plate count method.2.To determine animal skin infection model evaluation system:First of all,the evaluation index of animal skin infection model was investigated,and then preliminary screening comprehensive evaluation index,the hierarchy analysis to determine the hierarchical structure,expert consultation to determine the weight of each evaluation index and to develop the evaluation criteria of the evaluation criteria.3.Study on Animal Model Based on Analytic Hierarchy Process:?1?Study on skin abscess mouse model of Staphylococcus aureus:Eighty-eight?Half male and female?specific pathogen free Kunming mice were randomly divided into 4 groups:the PBS control,low-dose infection,medium-dose infection and high-dose infection,the control group was subcutaneously inoculated with 0.9%sterile saline of 0.1ml/mouse,while the infected groups were subcutaneously inoculated with staphylococcus aureus of10⁸CFU/mouse,10⁹CFU/mouse and 5×10⁹CFU/mouse respectively.?2?Study on skin ulcer mouse model of staphylococcus aureus:One hundred and ninety-eight?Half male and female?specific pathogen free Kunming mice were randomly divided into 9 groups,established skin ulcer by three different methods which were square wound?square wound group?,round wound?round wound group?,scratch wound?scratch wound group?,and then control group was inoculated with 0.9%sterile saline of 0.1ml/mouse,the infected groups were inoculated with staphylococcus aureus of1.36×10⁸CFU/mouse,1.36×10⁹CFU/mouse.?3?Study on skin ulcer rat model of staphylococcus aureus:One hundred and eight?Half male and female?specific pathogen free SD rat were randomly divided into 6 groups,established skin ulcer by two different diameter rounds which were 2cm and 1.5cm,and then control group was inoculated with 0.9%sterile saline of 0.2ml/rat,the infected groups were inoculated with staphylococcus aureus of 3.36×10⁸CFU/rat,3.36×10⁹CFU/rat.The models were evaluated by physical signs,appearance of skin lesions,etiology examination,morphological observation based on AHP.4.Application of Animal Model in Efficacy Evaluation of Pouzolzia zeylanica extracts?PZO?.PZO is a medicinal plant belonging to the genus Polygonaceae.Its main functions are detoxification,swelling,bone formation,and treatment of infectious diseases such as acne edema.We use the above-mentioned animal model of skin micro-organism infection to evaluate the efficacy of its ointment preparation according to the pre-clinical pharmacological research plan for new Chinese medicines.?1?Study on drug treatment of skin abscess mouse induced by staphylococcus aureus:Eighty-four specific pathogen free female Kunming mice were randomly divided into 7 groups:model group,matrix group,low-dose group?0.25g/kg?,medium-dosegroup?0.5g/kg?,high-dose group?1g/kg?,dragon ball ointment group,and mupirocin ointment group,subcutaneously inoculated with staphylococcus aureus of 10⁹CFU/mouse,and then evaluated by body weight change,abscess volume change,reverse culture of skin bacteria,skin pathology.?2?Study on drug treatment of skin ulcer mouse induced by staphylococcus aureus:Seventy-two specific pathogen free male Kunming mice were randomly divided into 6groups:matrixgroup,low-dosegroup?0.25g/kg?,medium-dose group?0.5g/kg?,high-dose group?1g/kg?,dragon ball ointment group,and mupirocin ointment group,established skin ulcer by 1.5cm diameter round,and then inoculated with staphylococcus aureus of 1.54×10⁹CFU/mouse,and then evaluated by body weight change,ulcer area healing rate,reverse culture of skin bacteria,skin pathology,hydroxyproline content.?3?Study on drug treatment of skin ulcer rat induced by staphylococcus aureus:Ninety-six specific pathogen free male Kunming mice were randomly divided into 6 groups:matrix group,low-dose group?0.05g/kg?,medium-dose group?0.1g/kg?,high-dose group?0.2g/kg?,dragon ball ointment group,and mupirocin ointment group,established skin ulcer by1.5cm diameter round,and then inoculated with staphylococcus aureus of2×10⁹CFU/mouse,and then evaluated by body weight change,ulcer area healing rate,reverse culture of skin bacteria,lysozyme content,interleukin-10 and tumor necrosis factor,skin pathology,hydroxyproline content.Results:1.The evaluation index and weight of skin abscess mouse model of staphylococcus aureus were determined by AHP.The evaluation indexes and weights were divided into animal weight change?0.02?,feeding rate?0.01?,sign of fatigue?0.02?,redness and purulent secretion of skin?0.11?,volume changes of abscess?0.13?,skin ulcer rate?0.04?,skin histopathology?0.26?,observation of vital organs?0.04?,blood bacterial culture?0.12?,skin bacterial culture?0.25?;skin ulcer model of evaluation index and weight were divided into weight change?0.02?,feeding rate?0.02?,Sign of fatigue?0.01?,redness and purulent secretion of skin?0.05?,change of areas of ulcer?0.14?,The heal time of ulcer?0.09?,skin histopathology?0.26?,observation of vital organs?0.07?,blood bacterial culture?0.09?and skin bacterial culture?0.25?.2.On solid culture,comparison of traditional bacterial counting methods:microscopic counting method>plate counting method>the McFarland's turbidimetry method;There was a linear correlation between the OD₆₀₀00 value and the plate counting method,linear regression equation was Y=28.98601X-0.17331,OD₆₀₀00 value was X,bacteria concentration was Y×10⁷CFU/ml.3.Study on skin abscess mouse model of staphylococcus aureus:The results showed that 1×10¹⁰CFU/ml>5×10¹⁰CFU/ml>1×10⁹CFU/ml.Study on skin ulcer mouse model of staphylococcus aureus:The results showed that round group>square group>scratch group.Study on skin ulcer rat model of staphylococcus aureus:The results showed that 1.5cm round group>2cm round.4.Study on drug treatment of skin abscess mouse induced by staphylococcus aureus:After drug treatment,compared with the matrix group,the body weight of mice increased significantly and the activity increased.At 6 days after administration,the volume of abscess in mice treated with medium and high dose had significant difference compared with the matrix group.At 3 days after administration,there was a significant difference in reverse culture of skin bacteria with high-dose,and at 8 days after the administration,there was a significant difference in reverse culture of skin bacteria with low,medium and high-dose.Study on drug treatment of skin ulcer mouse induced by staphylococcus aureus:After drug treatment,compared with the matrix group,the body weight of mice increased significantly and the activity increased.At 3 days after administration,compared with the matrix group,there was a significant difference in healing rate of the ulcer area between the medium and high doses.At 8 days after administration,there was a significant difference in the reverse culture of skin bacteria between the medium and high doses,and high dose group significantly increased the content of hydroxyproline in the skin.Study on drug treatment of skin ulcer rat induced by staphylococcus aureus:After drug treatment,compared with the matrix group,the body weight of rat increased significantly and the activity increased,at 5 days after the administration,there was a significant difference in healing rate of low,medium and high dose.At 6 days after administration,there was a significant difference in the reverse culture of skin bacteria between the low,medium and high doses.Compared with the matrix group,medium and high doses groups were significantly decreased the content of Interleukin-10 and tumor necrosis factor.and significantly increased the content of hydroxyproline in the skin.Conclusion:1.AHP is a scientific and effective evaluation method.The skin abscess mouse model can be injected subcutaneously with 1×10⁷CFU¹×10⁹CFU.The skin ulcer mouse model of staphylococcus aureus can be established by round wound with 1.0×10⁸CFU¹×10⁹CFU.The skin ulcer rat model of staphylococcus aureus can be established by 1.5cm round wound with 2.0×10⁸CFU².0×10⁹CFU.2.Pouzolzia Zeylanica?L.?Benn.Var.Microphylla?Wedd.?W.T.Wang has a significant effect on skin abscesses caused by Staphylococcus aureus infection.It has significant anti infection and skin healing effects on skin ulcers caused by skin infection in mice and rats,and there is a dose-effect dependence relationship.The anti-infective effect is related to the inhibition of bacterial growth and the synthesis or release of inflammatory factors.