| Diabetic foot ulcer(DFU)is a kind of diabetes complication with great harm and high cost of treatment,leading to about 15-25% of patients suffer from amputation,which seriously endangers the survival and quality of life of patients.DFU is susceptible to infection,which is an important factor hindering wound healing.Antibiotics are commonly used to control DFU infection,but the therapeutic effect of antibiotics alone is not ideal due to the bacterial resistance caused by frequent use of antibiotics.Therefore,it is of great significance to explore new interventions to treat DFU infection and promote wound repair.In recent years,platelet-rich gel(PRG)has been used to treat DFU and has shown good anti-infection and promoting healing effects.PRG is produced from platelet-rich plasma(PRP)which is activated by thrombin and/or calcium.Bioactive substances released by activated platelets in PRG are involved in its function.Since PRG does not cause drug resistance and has synergistic effects when used together with antibiotics,this multiple characteristics make it unique and has become a new effective method to treat DFU.Antibacterial peptides,chemokines,complements,peroxides and other substances released by platelets in PRG can directly inhibit or kill pathogens,or play an antibacterial role through chemotaxis and activation of immune cells.Previous studies have confirmed that PRG can inhibit Staphylococcus aureus(S.aureus),staphylococcus epidermidis,escherichia coli,and klebsiella pneumoniae in vitro.S.aureus is a common bacteria living in the skin,which colonizes the wound soon after skin injury occued and is one of the main sources of DFU infection.Keratinocytes are the most important cells that constitute the epidermal structure.During the occurrence of trauma and infection,keratinocytes participate in the skin immune response through phagocytosis of bacteria and secretion a variety of immune mediators and cytokines.In the process of wound healing,keratinocytes proliferate,migrate and differentiate,and complete the process of re-epithelialization to achieve wound healing.Studying the interaction between S.aureus and keratinocytes is helpful to understand the anti-infection and repair mechanism of skin.However,previous studies mainly observed the inhibitory effect of PRG on the growth of S.aureus in vitro from the perspective of antibiotics,but how PRG helps keratinocytes to fight against S.aureus infection is still unclear.In addition to the role of biological antibiotics,PRG can also significantly promote wound healing.Previous studies suggest that a variety of growth factors released by high concentration of platelets in PRG play a coordinating role in accelerating the process of wound healing.Recent studies have also found that platelets can express more than 200 microRNAs,and miR-223,miR-26,miR-23 and miR-21 are highly expressed in platelets,among which miR-21 is directly related to wound healing.It has been reported that miR-21 is a molecule that promotes skin wound healing,so we speculate that miR-21 may play an important role in the treatment of DFU by PRP,but the specific mechanism is still unclear.Programmed cell death factor 4(PDCD4)is a molecule that regulates cell growth and apoptosis and PDCD4 is regulated by miR-21 at the post-transcriptional level.It has been reported that PDCD4 can affect the activity of NF-B signaling pathway,which plays an important regulatory role in inflammation and Immunity.Therefore,we speculated that PRG may affect the activity of NF-κB,regulate wound inflammation,and promote wound healing through regulating PDCD4 mediated by miRNA-21.To explore the above problems,we co-cultured immortalized human keratinocytes(HaCaT cells)and S.aureus under high glucose condition to establish an in vitro model for infected wounds.Then PRP,PRG and extract liquid of platelet-rich gel(EPG)were used in this model to observe their effects on the growth of HaCaT cells and S.aureus,and explore the changes of miR-21 and PDCD4 expression,NF-κB signaling pathway activity and inflammatory cytokine levels in HaCaT cells before and after intervention.The purpose of this study was to evaluate the antibacterial activity and cellular protective effect of different forms of platelet products,and to reveal the possible mechanism of miR-21 in the role of PRG in promoting DFU healing.Part-1 Antibacterial and cell proliferation-promoting role of platelet-rich gel in an in vitro model for infected wounds under high glucose conditionObjective:Building an in vitro model for infected wounds under high glucose condition to simulate keratinocytes infected by S.aureus in DFU,and to study the antibacterial effect of PRP,PRG and EPG on S.aureus and the effect on proliferation of HaCaT cells.Methods:HaCaT cells were co-cultured with S.aureus under high glucose condition to establish an in vitro model for infected wounds,and the effects of different concentrations of S.aureus on cell proliferation were determined.With PPP intervention to the model as control group,the experimental group was divided into PRP intervention group,PRG intervention group and EPG intervention group.The viable bacteria in each group at different time points after the intervention were counted by plate colony counting method,and the effects of different interventions on S.aureus growth were compared.The proliferation of HaCaT cells in single cell culture group,co-culture group of cells and bacteria and EPG intervention group was detected by CCK8 assay.Results:(1)HaCaT cells co-cultured with S.aureus led to a significant decrease to cell proliferation(P<0.05),which was aggravated with the increase of bacterial concentration and the extension of co-culture time,and the cells were completely lost after co-culture with bacteria for 48 hours.(2)Compared with PPP group,PRP,RPG and EPG group can significantly reduce the number of extracellular S.aureus within 24 hours(P<0.05).PRG and EPG inhibited S.aureus strongerly than PRP within 48 hours.After 72 hours of intervention,the number of extracellular S.aureus in the PRG group was significantly lower than that in the EPG group(P<0.05).(3)The antibacterial activity of EPG against S.aureus is positively correlated with its concentration.Compared with the PPP group,the EPG group can significantly reduce the number of intracellular S.aureus(P<0.05).(4)Compared with the single cell culture group,no significant decrease in HaCaT cell proliferation was observed within 24 hours in the EPG intervention group(P>0.05),and significant increase in cell proliferation was observed within the following 24 hours(P<0.05).Furthermore,the proliferation of HaCaT cells was significantly increased at the 36 th and 48 th hours after EPG derectly intervention to uninfected cells(P<0.05).Conclusion:The proliferation of HaCaT cells is impaired by S.aureus,and the degree of damage is positively correlated with the concentration of bacteria and the co-culture time.PRG and EPG have stronger antibacterial activity against S.aureus than PRP,and PRG has longer action time than EPG.However,as liquid,EPG is convenient for precise use and is more suitable for micro experimental operation within 48 hours.As an effective component of PRG,EPG can significantly inhibit the intracellular and extracellular S.aureus,protect HaCaT cells against S.aureus infection and promotes cell proliferation.Part-2 Anti-inflammatory mechanism of platelet-rich gel in an in vitro model for infected wounds under high glucose conditionObjective : DFU is one of the common refractory wounds,and the persistent inflammatory state is one of the obstacles to wound healing.Studies have shown that mi R-21 is a molecule that promotes skin wound healing,and PDCD4,which is regulated by mi R-21,can positively regulate the activity of NF-κB signaling pathway to affect inflammatory response.However,the role of mi R-21 in the treatment of DFU by PRG is still unclear.Therefore,this study used EPG to intervene with the established in vitro model to explore the role of mi R-21 and the possible mechanism of promoting wound healing.Methods:Ha Ca T cells cultured under high glucose condition were divided into four groups: H group,culture of Ha Ca T cells alone;HP group,uninfected Ha Ca T cells interfered with EPG;HS group,co-culture of Ha Ca T cells with S.aureus;HSP group,S.aureus-infected Ha Ca T cells interfered with EPG.Then the expression of mi R-21,PDCD4 and phosphorylated p65(p-p65),the intracellular localization of p65,and the expression and secretion of inflammatory cytokines IL-6,TNF-α and IL-10 were determined in each group at different time points.Results:(1)In the HS group,compared with the H group,the expression of PDCD4 and p-p65 in Ha Ca T cells was significantly up-regulated(P<0.05),p65 was transferred from cytoplasm to nucleus,and the expression and secretion of IL-6 and TNF-α were significantly increased(P<0.05),while IL-10 was significantly decreased(P<0.05).(2)In the HSP group,compared with the HS group,the expressions of PDCD4 and p-p65 were significantly down-regulated(P<0.05),the transfer of p65 to nucleus was reduced,and the expression and secretion of IL-6 and TNF-α were significantly decreased(P<0.05).(3)In the HP group,compared with the H group,the level of mi R-21 in Ha Ca T cells was significantly up-regulated at the 24 th and 48 th hours(P<0.05),accompanied by significantly down-regulated PDCD4 and p-p65 expression(P<0.05),and significantly decreased levels of IL-6 and TNF-α(P<0.05).Conclusion:S.aureus infection leads to the activation of NF-κB signaling pathway in Ha Ca T cells,which induces the expression and secretion of pro-inflammatory cytokines.EPG can play an anti-inflammatory role by reducing the stimulation from bacteria through its antibacterial effect,or it can directly play an anti-inflammatory role by negatively regulating PDCD4 through mi R-21 and then inhibiting the activity of NF-kb signaling pathway.Inhibition of wound inflammation is one of the ways in which PRG promotes DFU healing,and mi R-21,as an intervention target,may provide a new strategy for the prevention and treatment of DFU and other refractory wounds. |
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