The Effct Of The Insulin On The Biofilm Formation Of The Pseudomonas Aeroginosa In Wound Infection And On The C-di-GMP Regulation In Diabetic Mice In Vivo And In Vitro

Diabetes-related infectious diseases have become a difficult and important public health problem in China as well as in the Word.Pseudomonas aeruginosa?P.aeruginosa?plays a substantial role in chronic wound infections and cystic fibrosis pneumonia's biofilm infections.The c-di-GMP signal systems employ c-di-GMP as a second messenger which control of many aspects of biological behavior,including the formation and dispersion of P.aeruginosa biofilm,especially regulate the production of exopolysaccharides and surface proteins which functions as extracellular matrix components in the biofilm formed by the bacteria.Previous studies haddemonstrated that biofilm-challenged wounds typically healed longer than non biofilm-challenged of normal wounds in diabetic mouse model.At present,there were more controversy about whether or not insulin treatment improves the P.aeruginosa infections.Chase Watters'groups found such phenomenon that insulin treatment promoted the wild-type P.aeruginosa POA1 biofilm formations.However,how the insulin promoted the biofilm formation in PAO1 had not been explored.And the mechanism which related to the c-di-GMP signaling pathway in P.aeruginosa had not been reported before.To date,there had not been a similar report related to mechanisms on the effects of insulin therapy on the healing of chronic P.aeruginosa infected wounds with different intracellular expression of c-di-GMP.Therefore,the main purpose of this study was to explore the effect of insulin on the healing of P.aeruginosa infection in diabetic mice.And whether or not those effects were related to the regulation of formation and dispersion of bioflim in the intracellular of c-di-GMP signaling pathway.For this reason,three groups of P.aeruginosa strains with different intracellular expression of c-di-GMP as experimental strains were selected in this study.The control strain PAO1,the PAO1/P_lac-yhjH strain with low intracellular expression of c-di-GMP and the PAO1?wspF strain with high intracellular expression of c-di-GMP.In vitro,firstly,the effect of the insulin on the growth of P.aeruginosa with difference intracellular expression of c-di-GMP were detected;Secondly,the effect of insulin on different intracellular expression of c-di-GMP in P.aeruginosa biofilm formation was monitored by 96 well microtiter plates biofilm analysis.Finally,the effect of insulin on the production of c-di-GMP in P.aeruginosa with different intracellular expression of c-di-GMP were investigated.In vivo,firstly,the animal model of P.aeruginosa infection wound in diabetic mice were established,the effects of insulin on the wound healing and the change of body weight in diabetic mice with P.aeruginosa infection wound were observed,and then the effect of insulin on P.aeruginosa bacterial load and the resistance rate to amikacin of P.aeruginosa infection in diabetic mice were observed.HE staining was used to observe the effect of insulin on inflammatory cells in P.aeruginosa infection wound of diabetic mice.RT-qPCR was used to detect the effects of insulin on the expression of RNA for IL-4 and INF-?in wound tissue in diabetic mice infected with P.aeruginosa.PNA-FISH and ConA-FITC staining were used to identify and confirm the existence of P.aeruginosa and it's biofilm in diabetic P.aeruginosa infection wound.We found that the ability of biofilm formation was related to the intracellular expression of c-di-GMP;The insulin could promote biofilm formation and enhance the expression of c-di-GM in the control stain of PAO1 and low intracellular expression of c-di-GMP in PAO1/P_lac-yhjH group in vitro.It indicated that insulin promote biofilm formation by increasing the intracellular expression of c-di-GMP.In the non diabetic group,the fastest would healing was the low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH group,but the slowest would healing was the high intracellular expression of c-di-GMP strain of PAO1?wspF group in the P.aeruginosa infection wound.Which indicated that the would healing of P.a infection was related to the intracellular c-di-GMP signaling pathway.In control strain of PAO1 group and low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH group,insulin-treatment delayed the would healing of db/db diabetic mice with P.aeruginosa would infection probably through promoting more biofilm,more PMNs infiltration and more inflammatory mediators releasing.Which indicated that the effect of insulin on P.aeruginosa infection may also be related to the intracellular c-di-GMP signaling pathway.PART ? THE EFFECT OF INSULIN ON PSEUDOMONAS AEROGINOSA WITH DIFFERENCE INTRACELLAR EXPRESSION OF C-di-GMP IN VITROSection ? THE EFFECT OF INSULIN ON THE GROWTH OF PSEUDOMONAS AEROGINOSA IN EXPERIMENT STRAINSOBJECTIVES:The observation of the effect of insulin on the growth of P.aeruginosa with different expression of c-di-GMP in experiment strains.METHEODS:The experimental strains were resuscitated first,then the concentration of bacteria was amplified by cultured,the concentration of OD₆₀₀was regulated and the concentration of the bacteria were 10 times series diluted to the concentration which we needed.The experimental group was divided into groups according to the insulin concentration,the final concentration of the insulin group was 200nU/ml,200,U/ml,200mU/ml.The bacterial suspension were mixed well and were moved into the 24 hole plates,37?,180 rpm,continuous being cultured,the count of live bacteria was carried out at each observation time point.RESULTS:There was no significant difference among the insulin concentration from 200mU/ml to 200nU/ml,and heat denatured insulin?control group?intervening to P.aeruginosa with different c-di-GMP content including PAO1/P_lac-yhjH,PAO1,PAO1?wspF,in 4hs,8hs,12hs,24hs growth in the colony count.CONCLUSIONS:Different insulin concentrations and heated denatured insulin neither promotes nor inhibits the growth of P.aeruginosa with different expression of c-di-GMP.Section ? THE EFFECT OF INSULIN ON THE ABILITIES OF BIOFILM FORMATION IN PSEUDOMONAS AEROGINOSA EXPERIMENT STRAINSOBJECTIVES:Observation the effect of insulin on the abilities of biofilm formation in P.aeruginosa with different expression of c-di-GMP.METHEODS:The experimental strains(PAO1/P_cdrA-gfp?PAO1/P_lac-yhjH/P_cdrA-gfp?PAO1?wspF/P_cdrA-gfp)were resuscitated first,then the concentration of bacteria was amplified by cultured,the concentration of OD₆₀₀was regulated and the concentration of the bacteria were 10 times series diluted to the concentration which we needed.The experimental group was divided into groups according to the insulin concentration,the final concentration of the insulin group was 200nU/ml,200U/ml,200mU/ml.96 hole plates microtitration biofilm analysis was carried out,37?,110 rpm,continuous cultured for 12 hours,then the semi quantitative method of crystal violet was used for biofilm detection.RESULTS:?1?Insulin can promote biofilm formation in P.aeruginosa with different intracellular expression of c-di-GMP;The largest increase of biofilm formation was the low intracellular expression of c-di-GMP of PAO1/P_lac-yhjH stain,increased from 15.96%to 24.04%,followed by the control strain PAO1,increased from 11.42%to 15.71%,the high intracellular expression of c-di-GMP strain of PAO1?wspF was the least,only increased from 5%to10%;?2?In low intracellular expression of c-di-GMP of PAO1/P_lac-yhjH,200mU/ml and 200nU/ml of insulin concentration increased biofilm formation significantly than the control group?P<0.01?.In control strain of PAO1,200mU/ml and 200?U/ml insulin concentration increased biofilm formation significantly than the control group?P<0.05?.While to the high intracellular expression of c-di-GMP strain of PAO1?wspF,different concentrationofinsulinincreasedbiofilmformationthancontrol group,however,the difference was not statistically significant.CONCLUSIONS:Insulin can promote biofilm formation in low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH and in control strain of PAO1.Insulin had no significant effect on the biofilm formation in high intracellular expression of c-di-GMP strain of PAO1?wspF.Section ? THE EFFECT OF INSULIN ON THE C-DI-GMP ESPRESSION IN PSEUDOMONAS AEROGINOSA EXPERIMENT STRAINSOBJECTIVES:Observation of the effect of insulin on c-di-GMP expression in P.aeruginosa with different expression of c-di-GMP in experimental strains.METHEODS:The experimental strains(PAO1/P_cdrA-gfp?PAO1/P_lac-yhjH/P_cdrA-gfp?PAO1?wspF/P_cdrA-gfp)were resuscitated first,then the concentration of bacteria was amplified by cultured,the concentration of OD₆₀₀was regulated and the concentration of the bacteria were 10 times series diluted to the concentration we needed.The experimental group was divided into groups according to the insulin concentration,the final concentration of the insulin group was 200nU/ml,200?U/ml,200mU/ml.96 hole plates microtitration biofilm analysis was carried out 37?,185rpm,continuous cultured for 12 hours,then the fluorescence value of GFP and OD₆₀₀00 were detected by enzyme analyzer.The value of GFP was divided by the value of OD₆₀₀,which was the value of relative fluorescence unit?RFU?.RESULTS:?1?Insulin could promote the c-di-GMP expression in control strain PAO1/P_cdrA-gfp and in low intracellular expression of c-di-GMP strains of PAO1/P_lac-yhjH/P_cdrA-gfp group;Between them,compared with the control group,the larger increase of c-di-GMP expression was low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH,increased by 36.27%to47.75%,secondly to control strain of PAO1,increased from 2.99%to43.13%.However,the effect of insulin on the c-di-GMP expression in high intracellular expression of c-di-GMP stain of PAO1?wspF/P_cdrA-gfp was not obvious.?2?Comparing with the control group,200mU/ml,200nU/ml and200?U/ml of insulin concentration increased the c-di-GMP expression significantly in low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH group,?P<0.05?.200mU/ml of insulin concentration was also significantly increased the expression of c-di-GMP in control strain of PAO1group?P<0.01?;But insulin had no significant effect on the c-di-GMP expression in high intracellular expression of c-di-GMP strain of PAO1?wspF.CONCLUSIONS:Insulin promoted the expression of c-di-GMP in low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH/P_cdrA-gfp,and control strain PAO1/P_cdrA-gfp.But it had no significant effect on the c-di-GMP expression in high intracellular expression of c-di-GMP strain of PAO1?wspF/P_cdrA-gfp.PART ? THE EFFECT OF INSULIN ON THE DIABETIC INFECTION WOULD WITH PSEUDOMONAS AEROGINOSA BIOFILM FORMATION AND THE REGULATION OF THE C-DI-GMP IN VIVOSection ? ESTABLISHMET THE ANIMAL MODEL OF PSEUDOMONAS AERUGINOSA WITH DIFFERENCE INTRACELLULAR EXPRESSION OF C-DI-GMP WOUND INFECTIONOBJECTIVE:The P.aeruginosa with difference intracellular expression of c-di-GMP wound infection of animal model was established,which can be prepared for the the following research.METHEODS:1.Preparing for experimental mice:male,8 weeks old db/db mice,blood glucose exceeded 20mmol/L was considered to be the db/db diabetic mice,blood sugar was less than 11.1mmol/L was considered to be db/db non diabetic mice;Mice were adaptive feeded at the SPF level in the animal room of Guangxi Medical University more than one week before experimental.2.Preparation for the bacterial suspension in vitro:the experimental strains were resuscitated first,then the concentration of bacteria was amplified by culturing,the concentration of OD₆₀₀00 was regulated and the concentration of the bacteria were 10 times series diluted to the concentration which we needed.3.On the day of the operation:First,the mice were anesthetized,shaved,and sterilized.Then the whole layer skin of about 1.5cm x1.5cm on back of the mice was removed,then the translucent application was covered,finally,100?L of experimental strains were injected on the wound surface under the surgical membrane.The animal models of chronic P.aeruginosa wound infection in back were established.RESULTS:About three days after the operation,the purulent fluid formation under the surgical membrane was observed indicated that the animal models of P.aeruginosa with difference intracellular expression of c-di-GMP wound infection were successfully established,which were preparing for the following research.CONCLUSIONS:The animal model of P.aeruginosa with difference intracellular expression of c-di-GMP wound infection was established using db/db mice.Section ? DETECTION OF THE OBSERVATION INDEX OF PSEUDOMONAS AERUGINOS WITH DIFFERENCE INTRACELLULAR EXPRESSION OF C-DI-GMP WUOND IN INFECTION MODELOBJECTIVES:To compare the effects of insulin on wound healing,bacteria load of wound tissue and the resistance rate of P.aeruginosa to amikacin in chronic wound infected by P.aeruginosa with different intracellular expression of c-di-GMP in diabetic mice.Monitor the effects of insulin on the invasion of P.aeruginosa biofilm,the infiltration of inflammatory cells and the releasing level of inflammatory mediators in chronic wound infected by P.aeruginosa with different intracellular expression of c-di-GMP content in diabetic mice.METHEODS:Firstly,the animal model of chronic wound infected by P.aeruginosa with different intracellular expression of c-di-GMP in diabetic mice had been established,the square of wound healing were measured and the body weight were weighted before the experiment and at each observation time point in chronic P.aeruginosa infection wound in diabetic mice.The plate colony counting method was used to detect the effect of insulin on P.aeruginosa bacterial load and the resistance rate of P.aeruginosa to amikacin in diabetic mice with chronic P.aeruginosa infection wound.HE staining was used to detect the effect of insulin on inflammatory cells in chronic P.aeruginosa infection wound in diabetic mice.PNA-FISH and ConA-FITC staining were used to identify and confirm the existence of P.aeruginosa and it's biofilm in chronic diabetic P.aeruginosa infection wound.RT-qPCR assay was used to detect the effect of insulin on the inflammatory mediators in chronic P.aeruginosa with different intracellular expression of c-di-GMP infection wound in diabetic mice.RESULTS:?1?The fastest wound healing in P.aeruginosa infection wound was the low intracellular expression of c-di-GMP stain of PAO1/P_lac-yhjH group in non-diabetic mice group,the wound healing of the diabetic with insulin-treated was slower than the the diabetic mice;On day16 post operation in the conrtrol strain of PAO1 group,the wound healing of diabetic with insulin group was slower than the non-insulin treated group in diabetic mice;The slowest wound healing was the high intracellular expression of c-di-GMP strain of PAO1?wspF group.?2?On day4 and day16 post operation,the resistant rate to amikacin of diabetic with insulin group was higher than the non-insulin treatment group in low intracellular expression of c-di-GMP stain of PAO1/P_lac-yhjH in diabetic mice.?3?In the control strain of PAO1 group and the low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH,more PMNs could be seen,more biofilm was formed and more inflammatory mediators were released in the P.aeruginosa wound infection in diabetic with insulin group.However,insulin had no significant effect on wound healing and biofilm formation in high intracellular expression of c-di-GMP strain of PAO1?wspF group.CONCLUSIONS:?1?In non diabetic group,the fastest would healing was the low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH group,but the slowest would healing was the high intracellular expression of c-di-GMP strain of PAO1?wspF group in the P.aeruginosa infection wound.Which indicated that the would healing of P.aeruginosa infection was related to the intracellular c-di-GMP signaling pathway.?2?The increasing of the resistance rate of P.aeruginosa to amikacin caused by insulin in the low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH.?3?In the low intracellular expression of c-di-GMP strain of PAO1/P_lac-yhjH and the control strain PAO1,insulin promoted biofilm formation,insulin-treatment delayed the would healing of db/db diabetic mice with P.aeruginosa would infection probably through promoting more biofilm,more PMNs infiltration and more inflammatory mediators releasing.Which indicated that the effect of insulin on P.aeruginosa infection may also be related to the intracellular c-di-GMP signaling pathway.