Mechanism Of Autophagy In The Promotion Of Diabetic Wound Healing By PRP

ObjectiveIn vitro experiments were conducted to investigate the relationship between PRP and autophagy and inflammatory factor release in macrophages.The role of autophagy in PPR in promoting the healing of diabetic wounds was investigated in vivo.MethodsThe RAW264.7 macrophage experiment was used as the research object in vitro.Divided into three parts:1.Screening of platelet-rich plasma for macrophage stimulation conditions:Use CCK-8 method and microscope to detect the optimal count concentration and stimulation ratio of PRP.2.The effect of PRP on the autophagy activity of macrophages and the effect of autophagy on the expression of macrophage inflammatory factors:Establiah the model of autophagy by RAPA(the autophagy inducer)and 3-MA(autophagy inhibitor).The expressions of autophagy-related proteins Beclin-1,LC3 Ⅱ(microtubule-associated protein light chain 3 II)and P62 were detected by Western blot.The fluorescence aggregation of LC3 protein was observed by immunofluorescence.The production of TNF-a,IL-10,TGF-β and IL-1β was measured by ELISA.And Griess Reagen method was used to detect the production and release of nitric oxide.3.The effect of PRP on autophagy and inflammatory factors in macrophages:Establiah the macrophage M1/M2 model by LPS/IL-4.Western blot was used to detect the expression levels of autophagy-related proteins Beclin-1,P62 and LC3 Ⅱ.The changes of RAW 264.7 cells were observed under the microscope.The TNF-a,IL-10,TGF-β,IL-1β and Griess Reagen methods were used to detect the production and release of NO.4.Mechanism of platelet-rich plasma to promote wound healing in diabetic mice by regulating autophagy:BKS.Cg-Dock7m+/+Leprdb/Nju TypeⅡ Diabetic Mice as a Research Subject,establishing a Wound Model on the Back.On the 3rd,7th,14th and 21st day,the wound skin tissue was collected,and the wound image was collected to calculate the wound healing rate.Detection of autophagy-related protein expression by Western blot.The wound healing was evaluated by HE staining and Masson staining.The expression of LC3 protein was observed by immunohistochemistry.Results1.Screening of platelet-rich plasma for macrophage stimulation conditions:After RAW 264.7 macrophages were stimulated with different count concentrations and different ratios of PRP,it was found that PRP cells with the highest concentration of 1000×109/L had the highest proliferation rate(1.298±0.528%).The highest proliferation rate of PRP cells with 10%ratio of medium(1.275±0.104%).At the same time,it was found by microscope that 10%of the PRP of 1000×109/L could promote the morphological changes of macrophages,and the cells changed from round to irregular,long-spindle type,and had pseudopods.2.The effect of PRP on the autophagy activity of macrophages and the effect of autophagy on the expression of macrophage inflammatory factors:After autophagy inhibitor 3-MA intervenes in RAW 264.7 macrophages,autophagy is inhibited,autophagy proteins Beclin-1 and LC3 Ⅱ are decreased,and P62 expression is increased.At the same time,the release of pro-inflammatory factors(IL-1β,TNF-α,NO)increased.In contrast,autophagy inducer RAPA up-regulated the level of autophagy,increased expression of Beclin-1 and LC3 Ⅱ,and decreased expression of P62.And up-regulated the levels of anti-inflammatory factors(IL-10,TGF-β).The addition of PRP after 3-MA/RAPA pretreatment can up-regulate the levels of autophagy and anti-inflammatory factors and inhibit the release of pro-inflammatory factors.The same result occurred when PRP was added to macrophages alone.Immunofluorescence showed significant green fluorescence in the RAPA/RAPA+PRP/PPR group.3.The effect of PRP on autophagy and inflammatory factors in macrophages:Microscopic observation revealed that macrophage traits became irregular under LPS/IL-4/PRP intervention,and PRP promoted cell proliferation.Western blot results showed that LPS/IL-4/PRP can promote autophagy.However,LPS,LPS+3-MA group can up-regulate the expression level of inflammatory factors.In contrast,IL-4/RAPA/PRP can increase the expression of anti-inflammatory factors.4.Mechanism of platelet-rich plasma to promote wound healing in diabetic mice by regulating autophagy:After the model of full-thickness skin defect in diabetic mice,the following conclusions can be drawn from the observation of wound healing,HE staining and Masson staining:(1)The normal BSK-M mice group had the fastest healing rate(the healing rate was 92.727±1.278%on the 14th day),and the healing was the best(the inflammation disappeared the fastest,the collagen deposition was the most,the alignment was neat,the epithelialization was complete and the newborn was new blood vessel);(2)Compared with the BSK-M group,the blank group had a slower healing rate(the healing rate was 58.303+4.591%on the 14th day),and the healing was poor(inflammation infiltration was severe,collagen deposition was less,the arrangement was disordered,and the epithelial hyperkeratosis was excessive,basically no new blood vessels).(3)Compared with the blank group,the wound healing rate of the PRP group was faster(the healing rate was 87.456+1.862%on the 14th day),and the healing was improved(inflammation infiltration was alleviated,collagen deposition increased and the arrangement was close to normal tissue,and the epithelialization trend Normal,a small amount of angiogenesis is visible).(4)Compared with the blank group,the 3-MA group slowed the healing rate(the healing rate was 47.233±5.200%on the 14th day),and the wound healing was poor(the inflammatory infiltration was still observed on the 21st day,the tissue was swollen and the epithelium Excessive keratinization,less collagen deposition,no angiogenesis.(5)Compared with the 3-MA group,the 3-MA+PRP group accelerated its healing rate(the healing rate was 66.800±3.995%on the 14th day),and the wound healing was improved compared with the 3-MA group.The results of Western blot and LC3 immunohistochemistry showed that in normal BSK-M mice,the level of autophagy gradually increased after wound formation,and decreased on the 21st day.Compared with the blank group,the level of autophagy is lower,but the overall trend is the same.The level of autophagy was up-regulated in the PRP group compared with the blank group.Autophagy was observed in the 3-MA group,but autophagy was elevated in the 3-MA+PRP group.Conclusion1、For RAW 264.7 macrophages,the optimal count concentration of PRP is 1000×109/L,and the optimal stimulation ratio is 10%.2、(1)PRP can promote autophagy in macrophages;(2)PRR can promote the release of IL-10 and TGF-β from macrophages and inhibit the release of IL-1β,TNF-a and N0.3、(1)PRP can promote the proliferation of macrophages,and the cell morphology becomes irregular long-spindle type,and pseudopods extend.(2)PPR can promote the polarization of macrophages to M2 by regulating autophagy of macrophages.4、Autophagy plays an important role in diabetic wounds.After 3-MA inhibits autophagy,wound healing slows down.While PRP can enhance autophagy in wound tissue and promote wound healing.In summary,PRP can induce the proliferation of RAW 264.7 macrophages by enhancing the level of autophagy.And PRP may induce macrophages polarizate to M2 and release related anti-inflammatory factors.At the same time,PRP can promote the healing of diabetic wounds by inducing autophagy.