| Objective:1.To explore the effect of the virulence factors ExoU,ExoS,ExoT,and ExoY secreted by the T3SS of Pseudomonas aeruginosa on the wound area,colony count,and histomorphology of the wound associated with PA infection in diabetic rats;so as to understand the effects on wound healing.2.To investigate the effect of inducing or inhibiting autophagy on the wound area,colony count and histomorphology of diabetic rats with ulcers and PA infection,so as to understand the effect on wound healing.Methods:168 SD rats were randomly divided into a diabetic group and a normal control group:the two groups were then randomly divided into a rapamycin group,a3-MA group,and a without autophagy intervention group.Each group was infected with six strains and one group without infection.The groups are as follows:DM+3-MA+PA01,DM+3-MA+PA14,DM+3MA+PA01ΔexoTΔexoY,DM+3MA+PA14 ΔexoUΔexoT,DM+3MA+PA14ΔexoUΔexoY,DM+3-MA+PA14ΔexoTΔexoY,DM+3-MA+uninfected.Rats pretreated with rapamycin and without autophagy intervention were divided into 7 similar groups.There were four rats in each group.On the 0th,3rd,7th,14th,and 21st days after infection,we took pictures to calculate the wound area,and took the wound tissue to calculate the number of wound tissue colonies and histomorphological observations.On day 0th,the infection model has not been formed and the wound tissue colony was not counted.Only part of the third day’s samples was observed by transmission electron microscope.Results:1.Basic situation:The rats’blood glucose at the observation end point in the 3-MA treatment group was higher than the baseline value,and the body weight was the heaviest among the three groups.The blood glucose and body weight of the rapamycin treatment group were lower than the baseline value.2.The effects of strains on wound healing:1)Wounds:Diabetes group:On the 3rd and 7th day of infection,the wounds in the ExoU and ExoS groups were significantly worse than those in the other two groups with obvious necrosis and black scabs,and there was purulent exudate in the ExoU group.Shrinkage,necrosis,and exudation were significantly improved by the 14th days.By the 21st day,the wound surface was dry.The wound area of the ExoU group was larger than that of the remaining three groups,but it did not show statistical differences.The wounds in the ExoT and ExoY groups were in good condition.The signs of exudation and slight necrosis were also seen on the 3rd day.The wounds started to improve gradually from the 7th day onwards,and the wounds tended to heal on the 21st day.Compared with the gene knock-out strain,the wild-type strains had worse wound conditions at various time points.The wild-type PA01 and PA14 groups showed blackened wounds on the third day,and there were more wet exudates.On the seventh day,purulent secretions were seen.Wet secretions were also visible in the 21-day wound,bright red in color.The normal control group showed a similar pattern,but the wound condition at each time point was slightly better.ExoS,ExoT and ExoY groups healed completely at the end of observation.2)Colonies:Diabetes group:On the 3rd and 21st days after infection,the wound colony counts in the DM+ExoU group were higher than those in the other three groups;on the 7th day after infection,the colony counts from high to low were:[ExoU(53421.67±7035.31)Vs ExoS(628.23±98.23)vs ExoT(5.31±0.76)vs ExoY(1.92±0.30)CFU×10~6/g].On the 14th day after infection,the differences between the four groups were statistically significant,and the number of colonies from high to low was:[ExoU(4506±854.05)>ExoS(30.31±6.31)>ExoY(0.56±0.12)>ExoT(0.17±0.06)CFU×10~6/g].Over time,in the DM+ExoU and DM+ExoS groups,the number of colonies increased first and then decreased,reaching a peak on the 7th day.The number of colonies in the ExoT and ExoY groups progressed with time,and the number of colonies decreased gradually.On the 14th and 21st days after infection,the colony counts were DM+PA01>DM+ExoS(P=0.003,0.007),and the number of colonies increased first,reached the peak on the 7th day,and then decreased.The DM+ExoS group was more effective than DM The+PA01 group experienced a large decline.At each time point,the number of wound colonies in the DM+PA14 group was higher than that in the DM+ExoU group.The number of wound tissue colonies in the DM+PA14 group was higher than that in the PA01 group at all time points.Normal control group:The number of wound colonies in the ExoU group was higher than that in the ExoS,ExoT and ExoY groups on the 14th and 21st days after infection.At various time points,the number of wild-type PA colonies was higher than the ExoS and ExoU groups,and the PA14 group was higher than the PA01 group.3)Pathological morphology:Diabetes group:No difference was found between the groups on day 0.Infiltration of inflammatory cells(macrophages,neutrophils,etc.)gradually increased on day 7 after infection,and lymphocytes were in a dense state,and The inflammatory cell density in the ExoU and ExoS groups was always higher than that in the ExoY and ExoT groups.In the ExoU group,endothelial cells and neovascularization increased and fibroblasts increased in the 7th day.Considering the increase in granulation tissue formation,the ExoS,ExoT,and ExoY groups had this phenomenon on the 3rd day.At the 21st day of ExoU,ExoS group,the infiltration was significantly reduced,and there were more fibroblasts,and the wound was in the period of hyperplasia and healing.ExoT and ExoY groups showed normal muscle fibers on the 21st day.Infiltration of inflammatory cells gradually increased to the peak in the DM+PA14 group until the 14th day after infection.Infiltration of inflammatory cells reached the peak on the 7th day after the infection in the DM+PA01 group,and the number of fibroblasts was higher than that in the DM+PA14 group.On the 21st day,the DM+PA01 group had a more regular arrangement of muscle fibers than the DM+PA14 group.At various time points,wild-type PA inflammatory cell infiltration was more significant than ExoU and ExoS groups,the number of fibroblasts was less,and the peak of inflammatory cell infiltration appeared earlier.The normal control group showed a similar pattern.The inflammatory cell infiltration was lower than that of the diabetic group.3.The effect of interventional autophagy on wound healing:1)Wounds:DM+ExoT,DM+ExoU,DM+ExoY,DM+PA01,DM+PA14,N+ExoS,N+ExoY,N+ExoT,N+PA01 group,the wound area were larger after the autophagy induction,and the wound condition was worse than that in the group without autophagy intervention.The DM+Rapamycin+PA01 group showed necrotic-like substances on the wound surface on the third day,and the color was dull.On the 7th and 14th days after infection,there was black scab on the wound surface.The degree of black scab was lighter in the group without autophagy intervention.In the DM+Rapamycin+PA14 group,wound necrosis-like substances were thrown on the 14th and 21st days.The wound margin was irregular,the wet secretions on the wound surface were obvious,and the wound area was still large.In the group without autophagy intervention,the wound surface was dry,small in area,and the granulation tissue was growing well.The wound condition after autophagy inhibition was better than that without autophagy intervention group or induced autophagy group,and the area showed a smaller difference.2)Colonies:DM+ExoY,DM+ExoU,DM+ExoT,DM+PA01,DM+PA14,N+PA01,N+ExoS,N+ExoU groups showed higher colonies after autophagy induced by rapamycin compared with those without autophagy intervention.The colony count of N+ExoT group after 3-MA inhibition of autophagy was lower than that of non-autophagy intervention group.In addition,DM+ExoS,DM+ExoT,DM+ExoU,DM+ExoY,DM+PA01,DM+PA14,N+Both ExoS and N+PA01 groups had lower colony counts after 3-MA inhibition of autophagy than that of induced autophagy.3)Pathology:On the 0th day of infection,no obvious abnormalities were found in each group,regular skin tissues and muscle fibers were arranged regularly,fat cells and a small amount of inflammatory cells were seen,and there were slightly more lymphocytes in the 3-MA treatment group;On the 3rd day,a large number of inflammatory cells were recruited in the rapamycin-treated group,and erythrocyte leakage may be caused by rupture of blood vessels.The number of fibroblasts on the7th day after infection was significantly less than the remaining two groups;By the21st day 3-MA treatment group tended to be normal tissue,with few inflammatory cells.4.DM+PA01ΔexoTΔexoY group’s sample at 3rd showed autophagosomes under electron microscope,and 4 autophagosomes were seen in 8 fields of×2k.Conclusions:1.ExoU-secreting PA strains are less conducive to the healing of infected wounds in diabetic rats than ExoS-secreting PA strains.ExoT and ExoY are less toxic,but they can enhance the adverse effects of ExoU and ExoS on wound healing.2.Induced autophagy is not conducive to the healing of infected wounds in diabetic rats,and inhibition of autophagy can promote wound healing. |
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