Keratinocyte Electrotaxis And Wound Healing Induced By Physiological Pulse Directed Current Electric Fields

BackgroundWound healing is a dynamic biological process that receiving precise regulation.Re-epithelialization plays a key role in wound healing,whose main feature is epidermal cells migration to the center of the wound.Many factors,such as damage stimuli,chemotactic gradients,contact inhibition,wound space,contact pressure from adjacent cell growth,and mechanical injury are involved in migration inducing of epidermal cells.Previous studies have suggested that electric field is one of the most important factors that triggering epidermal cells migration.Thus,electric field is considered as an effective physical factor improving wound healing.Of which,the constant directed current electric field(CDCEF)is the most widely investigated one so far.However,a long-term exposure to CDCEF always cause an inevitable electrochemical reaction,leading to a harmful side effects on tissue via various manners.Notably,as an intermittent power supply modality,the pulse directed current electric field(PDCEF)not only shortened the contact time with electric field but also spared more breathing space for cells.Therefore,theoretically,all these advantages boost PDCEF to be an ideal candidate for promoting wound recover without significant side effects.Even so,it is still unclear whether PDCEF can induce cell migration,how the related parameters modulation influence cells migration,and whether PDCEF can attenuate the electrochemical side effects when exposure to electric field.The mechanism by which cells sense electric fields is not clear.It may be associated with the relocating of some proteins expressed on cell membrane,including transferrin receptor,low density lipoprotein,acetylcholine,concanavalin A,immunoglobulin E and epidermal growth factor receptor.According to previous literature reports,when receiving the electrical signal,the cells delivered it mainly through the following intracellularly pathways,such as PI3k/AKt pathway,MAPk/ERK pathway,PKC-dependent pathway,ROCK-dependent pathway and differential calcium influx.Among them,the former two have been given the most attention by investigators,but whether cells also share this way to transmit electrical signals when exposure to PDCEF is still unclearIn the past studies,investigators have explored the effect of electricity on regulating skin wound healing by employing alternating current,direct current,and pulsed electricity.Although the results were different,all these results suggest electric field performed the potential ability to promote wound healing.However,due to the side effects of the electric field,the human body is poorly tolerated,which in some extent limited its translation to clinical practice.In view of the high gene similarity between pig and human beings,as well as the high consistency on skin thickness and low shrinkage ratio,the pig is an ideal animal model for skin wound healing researching.Based on the results in vitro,we still need to further examine the efficacy and safety of PDCEF on wound recovering in the pig model.In the meantime,the superiorities of PDCEF should also be expounded when compared with CDCEF.Rapid wound healing requires the following two essential conditions: the first is the cells could effectively migrate toward the center of the wound,the second is to enhance the rate of cell migration.Notably,the electric field markedly triggered the directional migration of cells while improving the motility of the cells.During previous studies,we have observed that constant directed current with physiological power can push cell movement in a short time,in an autophagy activation-dependent manner.Moreover,autophagy suppression dramatically reversed cell motivation under electric field.Thus,we asked that whether the long-term electric field exposure on body has some influencing the level of autophagy in tissue cells.Whether PDCEF promotes wound healing by inducing autophagy,whether PDCEF and CDCEF induce the same level of autophagy is unclear?Thereinto,this study aims at detecting the movement of cells under the action of PDCEF,as well as exploring the effect of its three parameter changes on modulating electrotropism of cells.At the same time,microenvironment changes of cell culture induced by PDCEF and CDCEF were compared.In addition,the potential mechanism of PDCDF-induced cell electrotropism acquired a further exploration.Moreover,to clarify the role of PCEEF in promoting wound healing and its relevant mechanism,we will also establish a skin wound charge model in pig.Methods:1.The motion of the keratinocytes under PDCEF was observed by using the exogenous cell plus model,and the motion direction and speed of the cells were quantitatively analyzed by Image J software.Three parameters of PDCEF were adjusted to observe the effect of each parameter change on its induced cell electrotaxis.The electrochemical changes in the cell culture environment after 6h of PDCEF(150mV/mm,0.1Hz,60%)and CDCEF(150mV/mm)were detected and the proliferation of cells was compared.MAPK/ERK pathway inhibitor U0126(50 M)was used to treat the keratinocytes and detect the effect of this pathway on inducing the keratinocytes electrotaxis in PDCEF(150mV/mm,0.1Hz,60%).The phosphorylation levels of ERK were detected at PDCEF(150mV/mm,0.1Hz,60%)and CDCEF(150mV/mm)with different field strength and time on keratinocytes,to clarify the role of molecular phosphorylation in cell electrotaxis.Furthermore,the direction of cell movement and ERK phosphorylation of keratinocytes were observed in PDCEF(150mV/mm,0.1Hz,60%)for 1.5 hours and then the electric field was turned off for 2 hours,to clarify the relationship between cell movement and ERK phosphorylation level and the continuous stimulation of the electric field.2.The electric application model of skin wound was constructed by using bama pig.Through this model,wound healing progress was observed at 3,6,9 and 12 days under two electric fields of PDCEF(3V,0.1Hz,60%)and CDCEF(3V).HE staining and Masson staining were performed on the tissue sections to compare the conditions of blood vessels,granulation and collagen hyperplasia on the wound under the action of the two electric fields,to clarify the specific healing effects and differences of the two electric fields.Infrared temperature instrument and high-precision pH test paper were used to detect the changes of wound temperature and pH after the application of the two electric fields,and the changes of wound microenvironment caused by the two electric fields were clarified and compared.3.The wound skin tissues were extracted after the application of PDCEF and CDCEF for 3 days,6 days and 9 days.The histoprotein was extracted and the expressions of LC3 and Atg-5 were detected by Western blot to determine the changes in the autophagy level of tissue cells after the two electric fields acted for different periods.Similarly,the expression of LC3 was detected by histological section immunofluorescence assay to determine the changes in autophagy level of tissue cells the two electric fields for different time.4.In vitro,total cell proteins of the keratinocytes were extracted after the two electric fields of PDCEF(150mV/mm,0.1Hz,60%)and CDCEF(150mV/mm)applied for 0h,2h,5h and 8h.And the expressions of LC3 and Atg5 were detected by Western blot to determine the changes of autophagy at the cell level after the two electric fields were applied for different time.The cell motility of the keratinocytes under PDCEF(150mV/mm,0.1Hz,60%)and CDCEF(150mV/mm)after 0h,2h,5h and 8h was observed by using the exogenous cell electric stimulation model,to detect the changes of cell motility after the long time action of two electric fields.Lipofectamine 2000 and Atg5 interfering RNA were used to make keratinocytes that interfered with the expression of Atg5 and LC3.The cells were divided into siRNA group,siRNA with PDCEF group and siAtg5 with PDCEF group.After 5h of in vitro PDCEF(150mV/mm,0.1Hz,60%),total cell protein was collected to detect the autophagy molecule LC3,and the inhibitory effect of siAtg5 on autophagy activation in the electric field was determined.In the same way,the keratinocytes that interfered with the expression of Atg5 were placed in the electric stimulation model,and the cell movement was observed for 5h under the action of PDCEF(150mV/mm,0.1hz,60%),to clarify the effect of autophagy on the electric field induced cell movement.Results:1.PDCEF can obviously induce the electrotaxis of the keratinocytes,and it is field strength-dependent,duty cycle-dependent,and time-dependent.We observed directional electrotaxis with pulsed DCEF,equivalent to that induced by constant DCEF(at the same field strength)when the pulsed DCEF duty cycle was above a threshold.And Application of a higher voltage reduced this duty cycle threshold.The change of pH and temperature in culture medium and electrolyte caused by PDCEF were significantly lower than that of CDCEF,and cell proliferation was significantly reduced after the action of CDCEF,while the level of cell proliferation under the action of PDCEF was not significantly changed.2.When the MAPK/ERK pathway was inhibited by U0126,the effect of PDCEF on cell electrotaxis was significantly reduced,and Western blot indicated that U0126 could significantly inhibit the phosphorylation of ERK under PDCEF.The phosphorylation level of ERK in keratinocytes under PDCEF and CDCEF was field strength-dependent and time-dependent,and the peak time of p-ERK in PDCEF was slightly later than that in CDCEF for 0.5h.When the electric field was removed after the cells were treated with PDCEF for 1.5h,the cell electrotaxis did not disappear immediately,but remained significant for a short period of time.Similar results occurred in the dephosphorylation of p-ERK,which was also a slow process and did not change significantly in a short period of time.3.In pig skin wound model,both PDCEF and CDCEF with physiological strength can promote wound healing,and PDCEF has the most significant effect.PDCEF is superior to CDCEF in promoting wound angiogenesis,granulation hyperplasia and collagen formation.By comparing the changes in wound microenvironment(pH and temperature)in vivo,it was found that the changes in wound pH and temperature caused by PDCEF were significantly lower than those caused by CDCEF,which was more conducive to the stability of wound internal environment.4.At the tissue level,both Western blot results and immunofluorescence results indicated that PDCEF could significantly increase the autophagy level,while the autophagy level tend to decreased under the action of CDCEF.And the cell experiment results suggested that the cell autophagy level under PDCEF remained high,while that increased first and then decreased under CDCEF.Similar results were found in cell motility,which remained high for 8h under PDCEF,but also increased for a short time under CDCEF,and decreased significantly after 5h.After inhibiting the activation of autophagy,the motion of the cells under the PDCEF for 5h was significantly lower than that of the normal cells in the electric field.Conclusions:1.PDCEF can reduce the side effects of electric field and induce the cell electrotaxis equivalent to CDCEF at the same time,and its three parameter changes can affect its electrotaxis induction.2.ERK molecule is one of the important molecules that PDCEF induces the electrotaxis of keratinocytes.PDCEF is able to trigger electrotaxis comparable to a CDCEF by inducing an equivalent level of ERK1/2 phosphorylation to CDCEF.3.PDCEF can significantly induce wound healing in pigs,and is superior to CDCEF in terms of the change of wound granulation and wound microenvironment.4.PDCEF enhances the autophagy level of tissue and cells by activating autophagy,thereby increasing cell motility and promoting wound healing.This study investigated the effect of physiological strength PDCEF on cell electrotaxis and wound healing and its related mechanisms,deepened people's understanding of endogenous wound electric field and the use of exogenous electric field to promote wound healing,and provided new ideas for clinical wound treatment.