Effects Of OSM And NR4A1 On Wound Healing Of Donkey Skin By Regulating PI3K/AKT Pathway

Objective:Scar is the final tissue of wound healing in non-fetal mammals.Since wound healing involves three complex and continuous processes,inducing scar free wound healing has become a difficulty in clinical research.Donkey skin is rich in collagen,which is the only raw material of ejiao in traditional Chinese medicine.But the extracellular matrix in donkey wounds does not overdeposit and forms fibrotic keloids.In view of this special phenomenon,our research group used RNA-Seq technology to analyze the differentially expressed genes in the early stage of donkey skin wound healing.Therefore,this study aimed to explore the effects of differential genes OSM and NR4A1 on wound healing of donkey skin through PI3K/AKT pathway.Methods:(1)Primary donkey skin fibroblasts were isolated from Xinjiang donkey back skin by tissue block adhesion method,and separated and purified by pancreatin combined with tissue block differential adhesion method.Cell morphology observation,PCR technique and cell immunofluorescence were used to identify the cell types and purity.The proliferative ability of primary cells was detected by the CCK-8method,and the activity of cells before and after cryopreserved was detected by Trypan blue staining.(2)Construct the overexpression and interference vector of OSM gene,and introduce the foreign gene into donkey skin fibroblasts cultured in vitro by lentivirus and liposome,respectively.The effects of OSM gene differential expression on the biological behavior of donkey skin fibroblasts were detected by CCK-8,cell plate cloning assay and scratch assay.The transcription levels of wound healing related genes(COL1A1,COL3A1,FN,Caspase-3,NR4A1,AKT1)were detected by RT-q PCR.Western blot was used to detect key typeⅠandⅢcollagen in the extracellular matrix.Meanwhile,PI3K inhibitor(PI-103)and activator(740Y-P)were used to explore the relationship between OSM and the PI3K/AKT pathway.(3)Construct the overexpression and interference vector of NR4A1 gene,and introduce the foreign gene into donkey skin fibroblasts cultured in vitro by lentivirus and liposome,respectively.The effects of NR4A1 gene differential expression on the biological behavior of donkey skin fibroblasts were detected by CCK-8,cell plate cloning assay and scratch assay.The transcription levels of wound healing related genes(COL1A1,COL3A1,FN,Caspase-3,OSM,AKT1)were detected by RT-q PCR.Western blot analysis was performed to detect key collagenⅠandⅢand pathway key genes p-AKT and AKT in the extracellular matrix.(4)A circular wound with a diameter of 1.6 cm was established on the donkey’s back skin,and the sh-OSM-2 and sh-NC plasmids were injected into the wound by plasmid injection at five points.The wound healing was observed by photographing,HE and Masson staining.RT-q PCR was used to detect the expression of scar formation related genes at the initial stage of wound proliferation at day 7 and day 17.Results:(1)On the 4th day of adhesion,fibroblasts began to crawl out from the edge of the donkey skin tissue block,and on the 8th day,a large number of fusiform or fusiform cells were observed near the tissue block in the shape of whirlpool.Fibroblasts were identified by cell morphology,PCR and immunofluorescence.The cell growth curve of the third generation was roughly"S"shape,and there was no significant difference in cell viability between pre-cryopreservation and post-resuscitation(P>0.05).(2)The OSM gene differentially expressed cell model of donkey skin fibroblasts was successfully constructed,and the OE-OSM virus concentration titer was 1×10~7 TU/m L,and the best vector(sh-OSM-2)was screened,and the interference efficiency was 65.95%.OSM inhibited proliferation activity,cloning ability and migration ability of donkey skin fibroblasts in vitro.Overexpression of OSM significantly inhibited the transcription levels of COL1A1,COL3A1,AKT1 and FN(P<0.01),and significantly promoted the transcription levels of NR4A1 and Caspase-3(P<0.01).Interference with OSM significantly promoted the transcription levels of COL1A1,COL3A1,AKT1 and FN(P<0.01).Significantly inhibited the transcription levels of NR4A1 and Caspase-3(P<0.01).OSM also significantly inhibited the protein expressions of COL1,COL3,p-AKT and AKT(P<0.01).(3)The NR4A1 gene differentially expressed cell model of donkey skin fibroblasts was successfully constructed,and the OE-NR4A1 virus concentration titer was 2×10~7 TU/m L,and the best vector(sh-NR4A1-1)was screened,with the interference efficiency up to 59.3%.NR4A1 inhibited the fine proliferation activity,cloning ability and migration ability of donkey skin fibroblasts in vitro.Overexpression of NR4A1 significantly inhibited the transcription levels of COL1A1,OSM and FN(P<0.01),and significantly promoted the transcription levels of COL3A1,Caspase-3 and AKT1(P<0.01).Interference with NR4A1 significantly promoted the transcription levels of COL1A1,OSM and FN(P<0.01).The transcription levels of COL3A1 and AKT1 were significantly inhibited(P<0.01).At the protein level,NR4A1 significantly inhibited the expression of COL1,and promoted the expression of COL3,p-AKT and AKT(P<0.01 and P<0.05).(4)Injection of OSM gene sh RNA vector into donkey skin wound accelerated the healing speed.The granulation tissue was slightly higher than the epidermal layer at 17d,and the purulent substance exudated less than the control group.HE staining showed that a large number of cells gathered at the wound site in the control group on the 3rd day and the symptoms were relieved on the 7th day.In the intervention group,the inflammatory reaction was not strong on the 3rd day,but a large number of cells were still visible in the dermis on the 7th day.Masson staining showed that,compared with the control group,collagen deposition was significantly increased in the intervention group,and thick collagen fiber bundles appeared at the same time.RT-q PCR results showed that sh-OSM-2 intervention could significantly reduce the m RNA expressions of OSM,NR4A1and Caspase-3 at the wound site compared with the control group at day 7(P<0.01).The expressions of COL1A1,COL3A1,FN and AKT1 in the wound were significantly higher than those in the control group(P<0.01).At 17 days,the expression levels of OSM and COL3A1 in the intervention group were significantly lower than those in the control group(P<0.05 and P<0.01),and the expression levels of COL1A1,FN,NR4A1,Caspase-3 and AKT1 were significantly higher than those in the control group(P<0.01).Conclusions:(1)The primary donkey skin fibroblasts were successfully isolated by tissue block adhesion method,and the cell viability was determined to meet the requirements of follow-up tests.(1)In donkey skin fibroblasts,OSM can inhibit cell proliferation and migration through the PI3K/AKT signaling pathway and affect the expression of related genes during wound healing,which preliminarily proves that OSM plays an anti-fibrotic role.(3)NR4A1 can inhibit cell proliferation and migration through the PI3K/AKT signaling pathway and affect the expression of related genes in the process of wound healing,which preliminarily proves that part of NR4A1 plays an anti-fibrosis role.(4)In vivo experiments,it was preliminarily proved that OSM played an anti-fibrotic role in wound healing of donkey skin and inhibited scar formation by reducing extracellular matrix deposition.