| Objective1.To study the effect of T3SS(Type III secretion system)of PA(Pseudomonas Aeruginosa)on the expression of autophagy-related proteins LC3、Beclin-1、p62 in diabetic ulcer wounds and to further investigate its effect on diabetic ulcer wound healing.2.To study the effect of virulence proteins Exo U、Exo S secreted by T3 SS of PA on the expression of autophagy-related proteins LC3、Beclin-1、p62 in diabetic ulcer wounds and to further investigate its effect on diabetic ulcer wound healing.3、To study the effect of rapamycin and gentamicin on the expression of autophagy-related proteins LC3、Beclin-1、p62 in diabetic ulcer wounds and its effect on diabetic ulcer wound healing.Methods Part 1 36 SD rats were randomly divided into six groups:diabetic with PAK infection group(DM+PAK),diabetic with PAK△pcr V-infection group(DM+PAK△pcr V-),diabetic without infection group(DM),non-diabetic with PAK infection group(N+PAK),non-diabetic with PAK△pcr V-infection group(N+PAK△pcr V-),non-diabetic without infection group(NDM).A full-thickness skin ulcer were performed on all rats.Wound healing was observed and PA colonies was counted in ulcer wound at different time points(0,3,7,14,21d)in all groups.We used HE staining to observe morphological changes of wound granulation and detected autophagy-related proteins LC3 、 Beclin-1 、 p62 by immunohistochemistry(IHC)and westernblot(WB)at different time points(0,7,14d).Another,immunofluorescence(IF)was used to detect the expression of LC3 in macrophages of granulation tissue in wound of all rats at different time points(0,7,14d).Part 2 24 SD rats were randomly divided into 4 groups:diabetic with PA103 infection group(DM+PA103),diabetic with PA01 infection group(DM+PA01),non-diabetic with PA103 infection group(N+PA103),non-diabetic with PA01 infection group(N+PA01).Methods were same as Part 1.Part 3 36 SD rats were randomly divided into 4 groups:diabetic with PA103 infection group (DM+PA103),diabetic with PA103 infection and rapamycin intervention group(DM+PA103+Rap),non-diabetic with PA103 infection group(N+PA103),non-diabetic with PA103 infection and rapamycin intervention group(N+PA01+Rap),diabetic with PA103 infection and gentamicin intervention group(DM+PA103+Gen),non-diabetic with PA103 infection and rapamycin intervention group(N+PA103+Gen).Results Part 1 1 Wound healing:Compared with non-diabetic group,there were more black callus on 3th day,more purulent secretions on 7th day,and wound had no shrinkage on 14 th day and was not healed on the 21 th day in diabetic group.Compared with DM and DM + PAK △ pcr V-,there were more black callus,necrosis and swelling around on 3th day,more purulent secretions on 7th day,and more secretions and larger wound area on 21 th day in DM + PAK.2 PA colonies in wound:Compared with the non-diabetic group,the colony growth rate was faster and the rate of descent was slower in diabetic group.Compared with DM + PAK △ pcr V-,PA colonies in DM + PAK began to decrease after 7th day,but the rate was not obvious.3 HE staining showed:Inflammatory cells infiltration gradually increased on 7th and 14 th day in diabetic infection group;Compared with DM+ PAK△pcr V-,DM + PAK respectivelyhad more inflammatory cell infiltration on 7th and 14 th day.In the non-diabetic infection group,the infiltration of inflammatory cells reached the peak on 7th day and decreased on the 14 th day.4 Immunohistochemistry results showed that the expression of LC3 and Beclin-1 increased on 0th,7th and 14 th day,and the expression of p62 protein decreased gradually on 7th and 14 th day in all groups(P <0.05).LC3 and Beclin-1 expression in DM + PAKpcr V-were higher than DM + PAK and p62 expression in DM + PAKpcr V-was lower than DM + PAK on14 th day(P <0.05).Compared with N + PAK,N+PAK△pcr V-had higher LC3 and Beclin-1 expression and lower p62 expression on 7th and 14 th day(P<0.05).Compared with NDM,DM had higher LC3 and Beclin-1 expression and lower p62 expression on 7th and 14 th day(P <0.05).Compared with N + PAK△pcr V-,DM+ PAK had higher LC3 and Beclin-1 expression and lower p62 expression on 14 th day(P <0.05).5 Western blot results were consistent with the results of immunohistochemistry.6 IF results showed: The LC3 expression of macrophages in DM + PAK△pcr V-were higher than DM + PAK group on 14 th day.The LC3 expression in macrophages of N+PAK△ pcr V-was higher than that in N+ PAK on7 th and 14 th day.Part 2 1 Wound healing:Compared with DM +PA01,there were more black callus,necrosis and swelling around on 3th day,larger wound area on 14 th day,and more secretions on 21 th day in DM + PA103.2 PA colonies in wound:Compared with DM+ PA01,PA colonies in DM + PA103 increased faster befoer 7th day and decreased slower after 7th day.3 HE staining showed:Compared with DM+ PA01,DM +PA103 respectively had more inflammatory cell infiltration on 7th and 14 th day.Compared with N + PA01,N + PA103 respectively had more inflammatory cell infiltration on 7th and 14 th day.4 Immunohistochemistry results showed that the LC3 and Beclin-1 expression in DM + PA103 were lower and the p62 expression in DM + PA103 was higher than DM + PA01 on 14 th day(P <0.05).Compared with N + PA01,N+PA103 had lower LC3 and Beclin-1 expression and higher p62 expression on 7th day(P <0.05).Compared with N+PA103,DM + PA01 had lower LC3 and Beclin-1 expression and higher p62 expression on 7th and 14 th day(P <0.05).5 Western blot results were consistent with the results of immunohistochemistry.6IF results showed: The LC3 expression of macrophages in DM + PA103 were lower than DM + PA01 on 14 th day.The LC3 expression in macrophages of N+PA01 was higher than that in N+ PA103 on 7th day.Part 3 1 Wound healing:Compared with DM +PA103,there were less purulent secretions on 7th day and the wound area was significantly reduced on 14 th day in DM +PA103+Rap;wound area was significantly reduced on7 th day and tended to heal DM+PA103+Gen..2 PA colonies in wound : Compared with DM + PA103,PA colonies in DM+ PA103+Rap and DM+PA103+Gen increased lower befoer 7th day and decreased faster after 7th day.Compared with N + PA103,the rate of descent in N + PA103+Rap and N+PA103+Gen was faster.3 HE staining showed:Compared with DM+ PA103,DM + PA103 +Rap and DM+ PA103+Gen respectively had less inflammatory cell infiltration on 7th and 14 th day.Compared with N+PA103,N+PA103+Rap and N+PA103+Gen respectively had less inflammatory cell infiltration on 7th and 14 th day.4 Immunohistochemistry results showed that compared with DM + PA103,DM + PA103+Rap had higher LC3 expression on 7th and 14 th day and higher Beclin-1 expression on 14 th day and lower p62 expression on 7th and 14 th day(P <0.05).Compared with N + PA103,the LC3 expression were higher on 7th and 14 th day and the Beclin-1 expression higher on 7th day and the p62 expression were lower on 7th and 14 th day in N + PA103+Rap(P <0.05).Compared with N+PA103+Rap,DM + PA01 +Rap had lower LC3 and Beclin-1 expression and higher p62 expression on 7th and 14 th day(P <0.05).There was no difference in the expression of Beclin-1、LC3and p62 in DM + PA103 and DM + PA103 +Gen at all time points.The expression of Beclin-1、LC3and p62 in N + PA103 +Gen and N +PA103 was no difference at each time point(P> 0.05).5 Western blot results were consistent with the results of immunohistochemistry.6 IF results showed: The LC3 expression of macrophages in DM + PA103+Rap were higher than DM + PA01 on7 th and 14 th day.The LC3 expression in macrophages of N+PA01+Rap were higher than that in N+ PA103 on 7th and 14 th day.Conclusion1 In cutaneous wound healing,autophagy ability is gradually enhanced both in non-diabetic and diabetic wounds.But autophagy in the diabetic group is significantly lower than that in the non-diabetic group,and the wound healing is delayed and impared in diabetic group compared with non-diabetic group.2 T3 SS of PA can inhibit the expression of autophagy-related protein in diabetic wound and the ability of autophagy to clear bacteria was reduced.Besides,Exo U secreted by T3 SS can inhibit autophagy-related protein expression and the ability of autophagy to clear bacteria is significantly reduced,leading more severe necrosis and persistent infection.3 Rapamycin can accelerate wound healing by enhancing autophagy to clear the bacteria both in the state of diabetes or non-diabetes.But its effect on diabetic state is less than non-diabetes.4 Gentamicin can effectively accelerate wound healing by eliminating PA,but it has no significant effect on autophagy-related protein expression both in diabetic wounds or non-diabetic wounds. |
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