Regulation Of Dracorhodin Perchlorate On Fibroblast Proliferation And Its Mechanism

As the extract of the Dragon's Blood,Dracorhodin always exists in the form of a particular salt,which is called Dracorhodin perchlorate.At the present research,we investigated the effects of Dracorhodin perchlorate on mechanism of promoting fibroblast proliferation and upregulating related proteins phosphorylation levels in wound healing process.It provides a theoretical basis for the treatment of wound repair with Dracorhodin perchlorate.In vitro assay,with the 24 h treatment of different concentrations(0~40?g/mL)of Dracorhodin perchlorate,fibroblast viability was detected by CCK-8 kit.After determining three effective concentrations of Dracorhodin perchlorate,which were used for cell scratch test and flow cytometry to detect the effects of Dracorhodin perchlorate on fibroblast migration and cycle.When the most effective concentration of Dracorhodin perchlorate was determined,we examine the phosphorylation levels of epidermal growth factor receptor(EGFR)?fibroblast growth factor receptors(FGFR)?extracellular regulated protein kinases/cAMP-response element binding protein(ERK/CREB)?c-Jun N-terminal kinase(JNK)and Phosphoinositide 3-kinase/Protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR),moreover,the inhibitors and siRNA of related signaling pathways were used in following experiments to explore mechanism of Dracorhodin perchlorate on promoting fibroblast proliferation.In vivo assays,Dracorhodin perchlorate was made into Ointment through dissolving in Vaseline.After establishing the rat trauma in vivo models,wounds were treated with either DP or Vaseline and DMSO(as control groups),and wound was recorded using a camera at 0,3,7 and 14 d.At 7~thh day,fresh granulation tissue was collected for preparation of pathological sections.In HE staining and SERPINH1antibody immunohistochemical staining assays,the effects of Dracorhodin perchlorate on wound healing and fibroblast proliferation.In vitro assays,the results showed Dracorhodin perchlorate promote fibroblast proliferation at a concentration range of 1.25~5?g/mL,with a peak at 3 mg/mL.Cell scratch test results showed that Dracorhodin perchlorate significantly accelerated cell migration compared to the control group at a concentration of 3?g/mL(p<0.01).The strongest cell migration activity was found within 12-24 h of Dracorhodin perchlorate treatment.Flow cytometry results revealed that Dracorhodin perchlorate improved the cell cycle,compared with the control group,the proportion of G1 phase cells in experimental group significantly reduced(p<0.01),and the proportion of G2and S phase cells increased significantly(p<0.01).In the mechanism exploration experiment,we firstly found that Dracorhodin perchlorate activated ERK signaling pathway.However,ERK inhibitor(PD98059)and ERK siRNA inhibited Dracorhodin perchlorate-induced ERK activation and fibroblast proliferation(p<0.01).In further experiments,we found that Dracorhodin perchlorate significantly upregulated EGFR phosphorylation and activated the downstream ERK/CREB and PI3K/Akt/mTOR signaling pathway(p<0.01).Moreover,the results also showed that EGFR inhibitor(AG1478)abolished Dracorhodin perchlorate-induced relative protein activation and cell proliferation(p<0.01).When ERK inhibitor(U0126)or PI3K inhibitor(LY294002)pretreated cells alone,Dracorhodin perchlorate-induced p-ERK or p-PI3K downstream proteins activation and cell proliferation were suppressed(p<0.01).In addition,CREB inhibitor(ICG001)and mTOR inhibitor(BEZ235)collectively eliminated Dracorhodin perchlorate-induced fibroblast proliferation(p<0.01).The vivo assays results showed that Dracorhodin perchlorate accelerated wound healing significantly(p<0.01),especially at day 7,with the treatment of Dracorhodin perchlorate,new skin tissue stained by HE was found thinner wound area compared to control group(p<0.01),the epidermis was flat and thicker than the control group(p<0.01)and the granulation tissue was densely packed and significantly larger than the control group(p<0.01).Using fibroblast-specific antibody SERPINH1 to stain fibroblasts in tissues at day 7,the results showed that Dracorhodin perchlorate can induced fibroblast proliferation obviously(p<0.01).In conclusion,Dracorhodin perchlorate promoted wound healing in vivo assays and accelerated fibroblast proliferation,which was stimulated by p-EGFR-induced activation of the ERK-CREB and PI3K/Akt/mTOR pathways.