| Objective Pressure Injuries are listed as one of the five most common problems in the world that cause serious harm to patients,which brings great pain and financial burden to the families of the patients.And the serious people even endanger lives.The high incidence and difficulty of control have always been a difficult problem in medical care and nursing.The western medical treatment of pressure injury is generally expensive and professional.Although traditional Chinese medicine possesses the advantages of natural economy,it is complex and lacks mechanism research,and external use powder will induce granulomatous lesions.Therefore,it is urgent to develop an external Chinese medicine preparation with simple composition,convenient effect,easy use and clear mechanism.The spraying agent is a new type of traditional Chinese medicine preparation,which can be sprayed into the membrane,which can prolong the time of the drug and improve the efficacy of the medicine.Meanwhile,it isolates the sterilization and is in line with the principle of wet healing of the stress injury,and is convenient to use.The traditional Chinese medicine tortoise shell powder scattered by the turtle shell,Coptis,red powder and borneol,with rot detoxification,blood circulation and pain relief,which is one of the effective prescription for treating skin damage.This study is based on the tortoise shell powder,taken the effective components cholesterol myristate(S8)from the turtle shell,berberine and dextral borneol made into Jinchuang spray film.It has the effect of promoting repair,anti-inflammation,anti-bacteria and promoting absorption.At present,the mechanism of skin injury repair mainly proceeds from the perspective of signal pathways promoting cell proliferation and inhibiting inflammatory response.Studies have shown that the Wnt/?-catenin signaling pathway can promote the proliferation of hair follicle stem cells(HFSCs),while HFSCs play a crucial role in skin repair.Therefore,this study prepared to make a new type of drug,the Jinchuang Spraying Agent,which was used to model the stress injury of early rats,observe its efficacy and explore the mechanism of action from the Wnt/?-catenin signaling pathway and inflammatory mediators of hair follicle stem cells,lay the foundation for the application of traditional Chinese medicine in clinical treatment of Pressure Injuries and the development of new drugs.Methods 1.Establishment early stress injury model of in SD male rats by magnet compression and ischemia-reperfusion cycle.The SD rats after the model were injected into the subcutaneous with Brd U(50mg/kg weight)on the first postoperative day and continuous injection of 7d.2.In the pre-experiment,the concentration screening of Jinchuang spray film was conducted,and the single factor design and orthogonal test were used to screen the film materials and optimize the prescription process.3.To detect the Quality of Jinchuang spray film by correcting tests.4.Experimental grouping and medication: Divide the rats into normal group,model group,positive control group,Jinchuang spraying film(SB)group,cholesterol myristate(S8)group and berberine group(B).The normal group did not do the treatment,the model group was given normal saline,Jinchuang spray group was given Jinchuang spray film,and the positive control group was given 3M spray,1 spray each time;8am,8pm,twice daily(Bid).Observe wound healing in each group of rats,according to the international general PUCH score table,the total effective rate and fee schedule to score of each wound repair,pathological observation skin wound repair,evaluate the efficacy of each group.5.To detect the expression of Wnt/?-catenin signaling pathway in skin wound tissue of rats in each group by Western blot,Quantitative real time polymerase chain reaction(q RT-PCR)and immunofluorescence technology.And the proliferation of cells was detected by Brd U.6.ELISA was used to test Serum levels of CRP,IL-6,and TNF—?;7.Take 1 ~3 d SD rats,according to the methods established in the early stage of the research group to isolate and culture the HFSCs.Use 2-(2-methoxy-4-(phenyl)-3-(4-(phenyl)-5-(2,4-sulpho benzene)-2 h-tetrazolium monosodium salt(CCK 8)to screen Jin Chuang spray film cell drug concentration.Use immunofluorescence,Western blot and RT q PCR technology to detect Wnt3 a,?-catenin,glycogen synthase kinase-3?(GSK-3?)protein and lymphoid enhancer factor(Lef1),C-myc,G1/S-specific cyclin-D1 gene expression.Results 1.The phase ? stress injury model was successfully established in rats by using 10 h ischemia and 2h reperfusion.2.The effective concentration of each medicine of Jinshen spray coating agent was: S8 1.5 mg/ml,B 2mg/ml,dextral borneol 1 mg/ml,and the best prescription proportion of the film-forming materials was 6% pvp-k30,0.4% HPMC and 30% ethanol.3.The quality test shows that the spray film had a good effect,the liquid and the medicinal film had good properties in all aspects,the stability and applicability were strong and the filming stability was good.It is worth noting that high temperatures should be avoided in storage.4.PUSH scores in different groups were generally lower than those in the model group on 3rd day,7th day ang 14 th day,the difference was statistically significant(P < 0.05).Among them,the PUCH score of SB and S8 group was lower than other groups,the difference was statistically significant(P<0.05)(see figure7).The total efficiency of wound repair in the drug group was higher than that in the model group and the blank control group,and the total effective rate of SB and S8 was the highest.(see table 12)The cost of wound healing due to pressure wounds in different medication groups was different.Compared with group M,the costs of SB and P group were statistically different(P<0.05,P<0.001),and the cost of group P was obviously higher than that of SB group,with statistical difference(P<0.05).5.Western blot results showed that compared with the M group,the expressions of Wnt3 a,?-catenin,Left1,cmyc,and cyclin D1 in SB group were up-regulated,while in the expression of GSK-3? was down-regulated 3 days after surgery(P <0.05);the expressions of Wnt3 a,?-catenin and Cyclin D1 in SB group were up-regulated,while in the expression of GSK-3? was down-regulated 7 days after surgery(P <0.05);the expressions of Wnt3 a,?-catenin and Left1 in SB group were up-regulated,while in the expression of GSK-3? was down-regulated 14 days after surgery(P <0.05).compared with the M group,the expressions of Wnt3 a,?-catenin and Left1 in S8 group were up-regulated,while in the expression of GSK-3? was down-regulated 3 days after surgery(P <0.05);the expressions of Wnt3 a,?-catenin,C-myc and cyclin D1 in S8 group were up-regulated 7days after surgery(P <0.05);the expressions of Left1 in S8 group were up-regulated,while in the expression of GSK-3? 14 days after surgery(P <0.05).The results of RT-q PCR test also indicated that compared with group M,the m RNA expression of Wnt3 a,C-myc in SB groups was enhanced,while the expression of GSK-3? and Cyclin D1 was decreased(P <0.05);the m RNA expression of ?-catenin and C-myc in SB groups was enhanced,while the expression of GSK-3?was decreased 7 days after surgery(P <0.05);the m RNA expression of Cyclin D1 in SB groups was enhanced,while the expression of GSK-3?was decreased 14 days after surgery(P <0.05).the m RNA expression of Wnt3 a and ?-catenin in S8 groups were enhanced,while the expression of GSK-3? was decreased 3 days after surgery(P <0.05);the m RNA expression of ?-catenin and C-myc in S8 groups were enhanced,while the expression of GSK-3? was decreased 7 days after surgery(P <0.05);the m RNA expression of Cyclin D1 in S8 groups were enhanced,while the expression of GSK-3? was decreased14 days after surgery(P <0.05);6.The Brd U and Immunofluorescence results showed that,compared with group M,the expressions of Wnt3 a in SB and S8 groups were up-regulated and mainliy focus on the hair follicle area,while it wasn't clear in B group.The newborn cells in SB and S8 groups increased significantly,mainly in the hair follicle area,and S8 cells increased more significantly..Immunofluorescence showed that compared with the model group,the expression of ?-catenin,Left1,cmyc,and cyclin D1 were enhanced,while the the expression of GSK-3?was decreased 7 days after surgery.7.ELISA detection:compared with the model group,the TNF-?,,IL-6 and CRP levels in SB group and group B were reduced(P <0.01).8.Immunofluorescence showed positive expression of CK15 on cultured cells surface.According to the cck-8 test result,the best drug concentration of SB was 1ug/ml.The expression of ?-catenin,Lef1,C-myc and Cyclin D1 was increased in SB group on HFSCs,while the expression of GSK-3? was decreased.Conclusion 1.It is stable,simple and easy to promote the early pressure injury model of the rats with the magnet oppresses the back with no trauma molding.2.The composition of the Jinchuang spray film(SB)made by this research is more suitable and the film is more stable,which lays a foundation for the development of clinical pressure injury drugs.3.SB may promote the repair of early stress injury in rats by activating the Wnt/ ?-catenin signaling pathway and the inflammatory response.4.SB may activate the Wnt/ ?-catenin signaling pathway to promote the proliferation of HFSCs. |
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