Tortoise Shell Ointment For Acute Skin Wound In Rats Via Regulating Wnt/β-catenin Signal Pathway In HFSCs

ObjectiveThis study extracted the effective monomers for skin healing from the traditional prescription Guiban San,including the effective monomers S8,berberine and nature borneol,to produce Tortoise Shell Ointment(TSO)and administer on acute skin wound.The study observed the effect of TSO on acute skin wound healing on rats,as well as explored whether TSO can accelerate acute wound healing by activating Wnt/(3-catenin signal pathway and promoting HFSCs proliferation.It is to provide ideas for the research of new drugs based on effective monomer components of traditional Chinese medicine for treating skin wounds.Methods1.Extracted the effective monomers S8,berberine and natural borneol and improved the component distribution and dosage form.Low,medium and high doses of TSO were prepared according to different doses.Administered TSO on acute skin wound of rats,to screen for the optimal dose.The quality of TSO was tested by appearance trait observation,stability test and skin irritation test.2.Acute wounds were created on SD rats.Each rat was treated with intraperitoneal injection of 5-bromo-2-deoxyuridineuridine(BrdU)for 3 consecutive days after operation,with a dose of 50 mg/kg according to weight.Rats were randomly divided into five groups:(1)TSO group(applied tortoise shell ointment,with thickness 1-2mm),(2)JWH group(applied Jingwanhong ointment,with thickness 1-2mm),(3)S8 group(applied S8 solution),(4)BBR group(applied Berberine solution),and(5)NS group(applied normal saline).The drug was administered twice a day for 10 days.The wound healing condition was observed,the wound healing rate was calculated,and the hematoxylin-eosin staining and Masson staining were used to evaluate the effect of wound healing effect.3.Immunofluorescence and Western blot were used to detect the expression of P-catenin,LEF1,C-myc in Wnt/β-catenin signal pathway and the expression of CD34 and Integrin β1 in HFSCs.The proliferation of wound cells after BrdU labeling were observed.Results1.TSO was composed of S8,berberine and natural borneol.It has yellow appearance,even and delicate texture,moderate viscosity and good spreading property.There is no obvious irritation to healthy skin and damaged skin,and no abnormal stability test results.The optimal dose screening test showed that the effect of low dose group on promoting wound healing was better,and the time of wound healing was shorter.2.The gross observation of wound repair showed that,the removal of scar in TSO group was more obvious than that in other groups and the healing time of the wound was shorter.On the 5th and 7th day,the wound repair score in TSO group were higher than those in the other 4 groups(P<0.05).On the 5th,7th and 10th day,the wound healing rate in TSO group were higher than that of the other 4 groups(P<0.05).3.The results of hematoxylin-eosin staining showed that the epidermis of TSO group was closely connected with dermis on the 7th day,and the skin appendages increased.The wound healing effect was better than that of other groups.On the 10th day,the repair of the TSO group,S8 group and JWH group was close to the normal epidermis,the stratification was obvious and the structure was dense.The BBR group was covered with thicker epidermal tissue;the NS group still had a little scar coverage.4.The results of Masson staining showed that the time of collagen deposition in TSO group was earlier than that in other groups,and the number of collagen fibers in TSO group was more than that in other groups.On the 10th day,TSO group showed the characteristics of collagen fibers in normal skin tissue compared with other groups..5.The results of immunofluorescence showed that P-catenin,CD34 and Integrin β1 were mainly expressed in the epiphyseal layer and basal layer of the epidermis.LEF1 and C-myc were mainly expressed in the basal layer.Overall,the positive expression intensity of β-catenin,LEF1,C-myc,CD34 and Integrin β1 in TSO group and S8 group were higher than that in the NS group.6.The results of BrdU labeled proliferating cell immunofluorescence showed that the number of proliferatiing cells labeled with BrdU in TSO group and S8 group were higher than that in NS group on the 3rd day after operation.In the proliferative cells,the expression of CD34 and LEF1 was positive at the same time.7.The results of Western bolt showed that the expression level of β-catenin,LEF1,C-myc,CD34,Integrin β1 in TSO group and S8 group were higher than that in NS group at the same time.The expression levels of β-catenin,CD34 and Integrin β1 in TSO group and S8 group were increased in the process of wound repair.The expression level of LEF1 and C-myc were increased in the early and middle stages of repair,but decreased in the later stages of repair.The difference among the three groups was statistically significant(P<0.05).Conclusion1.TSO has clear composition,the preparation process is simple.TSO has stability and safety with uniform and delicate texture,moderate viscosity and good spreading property.2.TSO could accelerate the repair of acute skin wound of SD rats and shorten the time of wound healing.The healing effect was better than that of the single effective monomer of S8 and berberine hydrochloride.3.TSO may promote the repair of skin wounds by regulating the Wnt/β-catenin signaling pathway in HFSCs,thereby promoting the repair of skin wounds.TSO provides an idea for the study of new drugs based on effective monomer components of traditional Chinese medicine for treating skin wounds.