| Background:Wound repair has been a hot topic in the medical domain.With the rapid development of the global economy,these causes such as social conflicts,frequent traffic accidents,animal bites,trauma caused by surgery,and population aging accelerated lead to the high incidence and mortality of trauma.The treatment of trauma costs a lot energy and money,causing a huge burden on individuals and society.With the development of modern wound repair research and the progress of treatment technology,wound healing speed has been significantly accelerated,but a series of problems have also emerged.Scar formation is an important complication of wound healing,which not only seriously affects the beauty of patients,but also has a serious impact on their psychology.Therefore,it is a long-term and necessary measure to find a wound repair drug that attaches equal importance to the healing speed and quality,appropriately regulates the healing speed,and synchronously promotes granulation growth and reepithelialization.Traditional Chinese medicine has a unique understanding and rich clinical experience on how to promote wound repair.Earthworm has been repeatedly reported as a traditional prescription for the treatment of burns and scald wounds.Research found tail-amputated for three-day-old Eisenia foetida prior to naked Eisenia foetida extract contains active material in promoting wound healing.After multi-step active track separations,we obtained active protein bands and got 24 kinds of protein identification,had a preliminary probe on tail-amputated Eisenia foetida promoting cell proliferation.Objective:In the previous study of our research group,it was found that the extracting solution of tail-amputated for three-day-old Eisenia foetida contained active substances to accelerate wound healing.Preliminary studies have been conducted on the signal pathway of tail-amputated Eisenia foetida to promote NIH3T3 proliferation.On the basis of the previous research results,this project intends to continue to explore the relationship between the effect of promoting wound healing of tail-amputated Eisenia foetida and Ras/Raf/MEK/Erk signaling pathway,and explore the effect of tail-amputated Eisenia foetida on wound healing quality,and establish the ICP-MS method to study the correlativity between facilitating wound healing of tail-amputated Eisenia foetida and inorganic elementsMethods:(1)The mRNA expressions of K-Ras,B-Raf,Raf-1 and Erk1/2 genes in Ras/Raf/MEK/Erk signaling pathway of NIH3T3 cells were detected by SYBR real-time fluorescent quantitative PCR.Western blot was used to detect the change of tail-amputated Eisenia foetida(13)on the K-Ras,B-Raf,Raf-1 and Erk1/2 content in the Ras/Raf/MEK/Erk signaling pathway in NIH3T3 cells and the skin wound skins of Wistar rats(2)A rat model of acute skin trauma was established to evaluate the effect of DT on wound healing by observing and measuring the skin wound healing rate.Histological staining methods including HE and Masson and determination of hydroxyprolinic acid were used to evaluate the changes in histopathological morphology,the collagen fibers of skin trauma in Wistar rats at 7,14 and 21 days after surgery.Immunohistochemical method was applied to determine the changes of PDGF and TGF-? in rat skin injury tissues(3)ICP-MS method was established to determine the changes of inorganic elements in rat skin trauma tissues.Results:(1)The results of PCR experiment showed that the mRNA expression of Raf-1,Erk1 and Erk2 genes increased significantly in the treatment group compared with the control group.Western blot results showed that the expression of Erk1/2 in NIH3T3 cells was significantly increased,Erk2 protein was significantly increased in treatment groups.(2)During the experimental period,the activities status of the rats were normal and the skin of the rats in each group basically healed around 21 days.Compared with the blank control group,the wound surface of the DT group was smooth and soft,with smaller scab area.From 0 to 14 days,the skin healing rate of the DT groups was significantly improved compared with that of the blank control group,and the healing rate was up to 98%around 21 days after the modeling.The results of HE staining showed that all rats healed to varing degrees on 7th day to 14th day,the skin structure of the WDT group and DT group was more complete,with more capillaries were found in the corium layer compared with the blank control group.The epidermis of all treatment groups was complete in the 21d,while the epidermis of the DT was thinner,collagen fibers were arranged in a regular manner,and more capillaries were visible.Compared with the blank control group and WDT in the 14th day to 21th day after injury,the area and number of collagen fibers in the injured skin of the DT group were increased with arranged in a parallel and tight manner.The ? collagen in the blank control and WDT group remained basically unchanged for 21 days,while DT group increased significantly.The results of HYP content in skin tissues showed that HYP content in the DT group was significantly higher than that in the blank control group and WDT group(P<0.05)in 7 days and 14 days after surgery.On the 21st day,HYP content in the blank control group was higher than that in the normal rat skin tissue,and that in the skin tissue of the WDT and DT group rats was significantly lower than that in the blank control group(P<0.05).HYP content in the DT group was closer to that in the normal rat skin tissue.Immunohistochemical results showed that the content of PDGF and TGF-? in all groups was the highest on the 7th day after modeling,and then gradually decreased.Compared with the blank control group,PDGF and TGF-(3 were significantly increased in WDT and DT group in 7d(P<0.01).The secretion of PDGF and TGF-? was significantly decreased in WDT group and the DT group(P<0.01)in the 14th to 21st day,and significantly lower than the blank control group.(3)ICP-MS method showed good linearity at 0?250 ?g/L(r>0.999)under optimized experimental conditions.The limit of detection(3?,n=10)is 0.02?13.04 ?g/L.The recoveries(n=6)were 85.4%?119.0%.The content of K,Ca,Mg,Cu,Fe,Zn and Se in the wound tissue was obviously increased by I3 without increasing the content of heavy metal elements.Conclusion:(1)Combined with PCR and Western blot results,it was found that the effect of 13 on promoting wound healing was related to the regulation of Ras/Raf/MEK/Erk signaling pathway.It mainly improves the expression of Erk2.Moreover,it was found that the medium dose had significant promoting effect on the gene expression,but it would not increase with the increase of dose.Therefore,it was speculated that the gene expression of Ras/Raf/MEK/Erk signaling pathway regulated by DT and it was dose-dependent in a certain concentration range.(2)13 could accelerate the growth of granulation tissue on the wound surface,expedite wound healing,and promote the formation of collagen fibers and new blood vessels.In the middle stage of wound healing,the content of PDGF and TGF-? increased to promote cell proliferation and granulation tissue growth.In later healing period,the secretion of PDGF and TGF-? content was decreased to prevent abnormal collagen fiber metabolism resulting in collagen deposition and scar generation.The HYP content will not be increased continuously,and the III collagen was significantly increased.Above all,it indicating that 13 can prevent scar formation and has a better healing quality.(3)ICP-MS method is simple,rapid,sensitive and accurate,and it can be used for simultaneous determination of inorganic elements on wound skin of rats.Tail-amputated Eisenia foetida plays a role in promoting wound healing,which is related to increasing the content of inorganic elements and has a high safety. |
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