| Objective: To detect the wound healing effects and further molecular mechanisms of the maggot.Methods:(1)To investigate the effect of living maggot on the infected wound in rats,male SD rats(200g)were selected as the experimental object.First,to make a wound of 2cm × 2cm,the whole skin and subcutaneous superficial fascia were cut on the back of the rats.Besides,the model of the infected wound was established by adding the Staphylococcus aureus into the wounds.Furthermore,rats were randomized into three groups: negative control group(Vaseline gauze covered group),rats in this group were only covered with Vaseline gauze;mupirocin ointment positive control group,rats in this group were smeared with mupirocin ointment and covered with aseptic Vaseline gauze;maggot treatment group,rats in this group were embedded with the living maggot bag in the wound and covered with aseptic gauze.To observe the effect of maggot on the infected wound,the general condition of the wound in the three groups was observed on the 4th,8th and 12 th day,and the index including the change of wound size,infection and new granulation tissue.The modified transparent membrane method was used to record the wound area in rats;the wound tissue was incubated to evaluate the wound infection;the wound tissue was employed for HE staining to observe the histopathological changes of the wound.(2)To detect the healing effect of maggot on the infected wound in rats and explore its further molecular mechanism.The infected wound model was established by dripping Staphylococcus aureus solution into the back wound in rats.After the establishment of the model,rats were treated with three different methods: covered with aseptic Vaseline gauze;smeared with mupirocin and then covered with sterile Vaseline gauze;covered with sterile gauze after the wound was embedded with living maggot bag.The treatment period of the experiment was 12 days.On the 4th,8th and 12 th day of the treatment,the size and infection of the wound were observed to evaluate the effect of the maggot on the wound in rats.At the tissue level,the tissues were selected for HE staining and immunohistochemistry to investigate the histopathological changes and the expression of TGF-β/SMAD pathway.At the molecular level,the tissue proteins were extracted to explore the changes of TGF-β,SMAD2/3 and SMAD4 and the downstream target genes Ras and C-myc.(3)The purpose of this study was to confirm the effect of maggot on angiogenesis in rats wound.Male SD rats(200g)were randomly divided into negative control group,positive control group,and treatment group.The Staphylococcus aureus solution was added to the wound to form the infected wound model in rats.Furthermore,the relevant indexes were evaluated on the 4th,8th and 12 th day after treatment with respective methods.The general situation of wounds was recorded at three-time points;the histopathological changes were detected by HE staining;the effect on neovascularization was further examined;the expression of VEGF-A was directly observed by immunohistochemistry in rat wound.Moreover,at the molecule level,the tissue m RNA and total proteins were extracted to explore the effect of maggot on neovascularization in rat wound.(4)To detect the effect of living maggot on infected wound healing in rats,and further explore the changes of nerve-regeneration.Male SD rats weighing about 200 g were employed to experiment.The whole skin layer and subcutaneous superficial fascia were cut on the back of the rats to make a wound of 2cm × 2cm.Then,the model of the infected wound was established by adding Staphylococcus aureus solution into the rat wound.Rats were randomized into three groups: negative control group(Vaseline gauze covered group);mupirocin ointment positive control group;maggot treatment group.On the 4th,8th and 12 th day,to examine the effect of maggot on the infected wound,the general condition of the wound was recorded;the wound healing was detected by HE staining.Besides,at the histological level,the expression of neuron-specific antigen protein gene product 9.5(PGP9.5)and neuropeptide substance P(SP)were explored by immunohistochemistry to verify the effect of maggot on nerve-regeneration.Results:(1)On the 4th day of treatment,the wound area and the content of bacteria in the mupirocin ointment group were small compared with the other two groups(P< 0.05),and the HE results also showed that the inflammatory cells in the positive control group were significantly less than that in the Vaseline and maggot group.On the 8th and 12 th day,the maggot gradually exhibited the same or even better therapeutic effect as the mupirocin group,which was reflected in the better wound healing,that is,there were fewer bacteria and inflammatory cells in the wound(P< 0.05).(2)On the 4th day of treatment,the expression of TGF-β/SMAD signal pathway relevant protein in the mupirocin group was high than that in the maggot group and the Vaseline group(P< 0.05),which resulted in a better condition at the mupirocin group compared with the other two groups in the wound surface and histopathology.With the prolongation of days,the expression of TGF-β/SMAD pathway relevant protein in the maggot group increased.On the 8 the day,the expression was similar to that in the positive control group,but on the 12 th day,the expression in the maggot group was obviously higher than that in the mupirocin ointment group(P< 0.05).The general condition of the wound and HE staining also confirmed that the wound recovery in the maggot group was better than that in the mupirocin ointment group.Therefore,it is verified that the maggot promotes wound healing by activating TGF-β/SMAD signaling pathway.(3)On the 4th day of treatment,the expression of VEGF-A in the mupirocin group was equal to the maggot group and the Vaseline group,but a better wound surface and histopathology condition occurred in the mupirocin group compared with the other two groups.With the prolongation of days,the expression of VEGF-A in the maggot group increased.On the 8 the day,the expression was similar to that in the positive control group,but on the 12 th day,the expression in the maggot group was obviously higher than that in the mupirocin ointment group(P< 0.01).The general condition of wound and HE staining also confirmed that the wound recovery in the maggot group was better than that in the mupirocin ointment group.Therefore,it is verified that the maggot promotes wound healing by activating VEGF-A-mediated neovascularization.(4)On the 4th day of treatment,the expression of PGP9.5 and SP in the mupirocin group was equal to the maggot group and the Vaseline group.Moreover,a better wound surface and histopathology condition occurred in the mupirocin group compared with the other two groups.With the prolongation of days,the expression of PGP9.5 and SP in the maggot group increased.On the 8 the day,the expression was similar to that in the positive control group,but on the 12 th day,the expression in the maggot group was obviously higher than that in the mupirocin ointment group(P<0.05).The general condition of wound and HE staining also confirmed that the wound recovery in the maggot group was better than that in the mupirocin ointment group.Therefore,it is verified that the maggot promotes wound healing by activating nerve-regeneration.Conclusions: Alive maggot promotes the infected wound healing by activating the TGF-β/SMAD singling pathway in rats.On the one hand,maggot activating the expression of downstream growth-promoting genes.On the other hand,maggot contributes the neovascularization and nerve-regeneration. |
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