| Background and purpose:Diabetic foot is one of the main complications of diabetes,and its wounds often remain unhealed.The cost of treatment for diabetic foot care in developed countries accounts for 20% of the total medical resources related to diabetes.In developing countries,this proportion is even as high as 40%,which brings a heavy financial burden to patients and society.Therefore,research on the difficulty of diabetic wounds has been a research hotspot in this field.DMSO is one of the most commonly used solvents and can dissolve many polar or non-polar compounds.Since 1964,Jacob discovered that DMSO can invade the skin to transport small molecules across biofilms,setting off a climax of DMSO.However,by 1965,the FDA banned all clinical studies of DMSO,so that the study of DMSO also entered the freezing point.With the ban on clinical studies of DMSO,the FDA approved a nμMber of clinical studies and drugs related to DMSO.In 2007,DMSO was found to reduce intracranial pressure in closed and craniocerebral injury,and FDA approved DMSO to reduce brain damage in closed brain.A clinical study of tissue swelling,the medicinal value of DMSO was again ignited.At present,the treatment of refractory wounds is more to improve the healing microenvironment of cell growth of wound tissue by physical means,and still lacks a drug that effectively promotes healing of refractory wounds.An article has reported the role of DMSO in promoting wound healing,but the specific mechanism has not yet been elucidated.At the same time,we found that DMSO has the function of promoting the activation of mTOR signaling pathway related to cell proliferation,and whether it affects the fibroblast proliferation cycle through this pathway,and the promotion of refractory wound healing still needs further exploration.In the process of refractory wound healing,fibroblasts and keratinocyte proliferation and migration are important links.Cell proliferation and differentiation are the result of cell cycle,and the cell cycle includes G1 phase,S phase,G2 phase and M phase IV.Stages.The cell cycle is regulated by the cyclin Cyclin,the cyclin-dependent kinase CDK and the cell cycle-dependent kinase inhibitor CKI.CyclinA/B/C/D/E/G/H has been found in the cell cycle.Cyclin D1 and Cyclin E play an important role in promoting the cell cycle from G1 to S phase.The cyclin-dependent kinase CDK is a member of the serine and threonine kinase family.Different cyclin kinases CDK and different cyclin Cyclin bind to different stages of cell cycle regulation,eg CDK2 and cyclin A and E Binding promotes the cell cycle from G1 to S phase.In many difficult-to-heal wounds,fibroblasts often exhibit cell cycle arrest,which hinders cell proliferation and affects wound healing.Translational regulation is an important part of gene expression regulation.Cells can regulate cell life activities by regulating protein translation machines and rapidly synthesizing large amounts of proteins at low mRNA levels.There are two pathways for the translation of eukaryotic mRNA into protein,namely the 5￠-Top pathway and the 5￠-Cap-dependent pathway.Among them,the 5￠-Cap-dependent translation pathway is the main translation pathway of eukaryotic mRNA,and about 80-90% of mRNA,especially most genes involved in cell proliferation and growth are translated into proteins by this pathway.This translational pathway requires the addition of a 7-methylguanine cap structure(m7GpppX)at the 5’ end of the mRNA,which plays an important role in cap-dependent mRNA translation.The eIF4 F complex is a heterotrimeric complex composed of eIF4 E,eIF4G and eIF4 A.The eukaryotic translation initiation factor eIF4 E binds to the 5￠ hat structure of mRNA by specific high affinity,and recruits eIF4 G and eIF4 A to bind to the mRNA 5 end.4E-BP1(a translational negative regulator)binds strongly to eIF4 E and competitively inhibits the interaction of eIF4 E with eIF4G;eIF4G plays a scaffolding role in the complex,which contains eIF4 E and The eIF4 A binding site acts as an adaptor protein for ribosome binding to mRNA;eIF4A has helicase activity,unwinding the double-stranded structure of the 5’ non-coding region of the mRNA,facilitating the ribosome to scan the start codon downstream.The mTOR pathway is a key pathway regulating protein translation.If mTOR molecule is activated by phosphorylation,activated mTOR further induces phosphorylation of 4E-BP1,4E-BP1 is inactivated by phosphorylation,and inactivated 4E-BP1 The 4E-BP1/eIF-4E complex dissociates and releases eIF4 E,which is imprisoned,and eIF4 E,which recruits eIF4 G and eIF4 A to form an eIF4 F initiation factor complex,and binds to the hat structure of the 5￠-end of mRNA.The translation process promotes cell proliferation.This study is based on the previous team’s discovery that DMSO has the ability to activate mTOR signaling,and systematically explore the mechanism of DMSO promoting the healing of refractory wounds in diabetic mice by enhancing fibroblast proliferation.The completion of this project will explain DMSO.The mechanism of promoting and providing theoretical support for the development of new drugs based on DMSO.methodFirst,DMSO treatment of diabetic mouse wound model,observation of wound healing in diabetic mice and detection of the expression level of cell proliferation core protein ki67.Then,diabetic primary mouse fibroblasts were obtained from diabetic mouse model,different concentrations of DMSO were applied to primary mouse fibroblasts,and MTT,RTCA and EDU were used to study the proliferation of primary fibroblasts in diabetic mice.Effects of flow cytometry on cell cycle changes of primary fibroblasts in diabetic mice after DMSO treatment;quantitative RT-PCR and western-blot detection of cell cycle-associated protein and kinase mRNA and protein including Cyclin D1 Expression level;western-blot detects the important signaling pathway of protein translation regulation-mTOR signaling pathway and its downstream key protein expression levels;polysome assay detects the protein translation level of Cyclin D1.result1.Compared with the control group,WB results showed that low concentration of DMSO could increase the expression of core protein Ki67 in wound tissue,and the peak appeared in the 8 days after injury,which accelerated the healing of refractory wounds in diabetic mice.2.MTT,EDU incorporation and RTCA experiments showed that low concentration of low DMSO can promote the proliferation of primary fibroblasts in diabetic mice,5μM concentration showed the strongest results,while DMSO greater than 20μM inhibited cell proliferation.In addition,flow cytometry results showed that DMSO can accelerate the cell cycle of primary fibroblasts from diabetic mice to G phase to S phase,thereby promoting cell proliferation.3.WB results showed that low concentration of DMSO could up-regulate the phosphorylation of mTOR and its downstream protein 4EBP1 to activate the mTOR signaling pathway.Meanwhile,the results of Pull-down assay showed that the eukaryotic protein translation initiation complex regulated by mTOR signal The formation of eIF4 F was increased;WB results confirmed that DMSO significantly up-regulated the expression of Cyclin D1 protein,but RT-PCR results showed that the expression of Cyclin D1 mRNA was not affected by DMSO.The above changes can be inhibited by the mTOR inhibitor rapamycin.Polysome experiments confirmed that DMSO up-regulated the translational level of Cyclin D1 protein.ConclusionDMSO activates this pathway by activating phosphorylation of mTOR,and induces phosphorylation of protein 4EBP1 downstream of this pathway,abolishes the inhibitory effect of 4EBP1 on the binding protein eIF4 E,promotes the formation of the eukaryotic translation initiation complex eIF4 F,and up-regulates Cyclin from the translational level.The expression of D1 promotes the cell cycle of primary fibroblasts in diabetic mice from G1 to S phase,accelerates cell proliferation,and ultimately promotes healing of refractory wounds in diabetic mice. |
