Experimental Study Of The Therapeutic Effect Of Osthole On Hypertrophic Scar And Its Mechanism

BackgroundHypertropihc scar is a common disease secondary to skin injury,often resulting from trauma,burns,etc.Hypertrophic scar often results in unsightly,painful and itchy.If the scar is near a joint,it may also cause functional limitations,causing huge physical and psychological burden to the patient.Hypertrophic scar,a fibrotic disease is caused by excessive wound healing,in which fibroblasts often play a key role,such as secreting extracellular matrix like collagen and contraction to promote wound closure.Understanding the pathogenesis of this disease can be of great help in treatment.Although there are various treatments available,such as surgical excision,steroid hormones and5 fluorouracil,they all have their own side effects or drawbacks,such as causing pain,hyperpigmentation or recurrence.Finding a new treatment,then,remains a challenge.Osthole,a natural coumarin first derived from Cnidium plant has received scholarly attention in recent years because of its wide range of pharmacological effects.Osthole can inhibit the development of many types of cancer,such as lung cancer and cervical cancer.Also,osthole plays an important role in inhibiting fibrosis in a variety of organs,such as the lung,kidney and liver.However,whether it has a therapeutic role in hypertrophic scar,which is a skin fibrotic disease with abnormal proliferative activity and its potential mechanism have not been clearly reported.Epidermal growth factor receptor(EGFR)is recognized as a tyrosine kinase receptor.A number of downstream pathways are activated by the phosphorylation of EGFR,including Phosphatidylinositol-4,5-bisphosphate 3-kinase/ protein kinase B/ mammalian target of rapamycin,(PI3K/AKT/mTOR)signaling pathway,which also has an important place in fibrosis in various organs such as the heart and lung.With the above background knowledge,we hypothesized that hypertrophic scar could be treated by osthole by regulating the phosphorylation of EGFR/PI3K/AKT/mTOR signaling pathway,which was verified in this experiment.AimThe purpose of this study was to investigate whether osthole had a therapeutic effect on hypertrophic scar through the EGFR/PI3K/AKT/mTOR signaling pathway.MethodsIn the first part,we mainly examined the effects of osthole on cellular functions such as proliferation,apoptosis,migration and contraction of hypertrophic scar fibroblasts from in vitro experiments,and then collected treated cells for quantitative polymerase chain reaction(qPCR)and Western blot,to further analyze the possible molecular mechanisms.The proliferative capacity of hypertrophic scar fibroblasts was firstly examined by not only the Cell Counting Kit-8(CCK-8)cell proliferation assay but also the EdU cell proliferation assay.Annexin V/PI staining was conducted to test whether osthole could promote apoptosis in hypertrophic scar fibroblasts.Cell wound healing assay as well as Transwell migration assay were performed and qPCR and Western blot were measured to test whether osthole could inhibit the migration of hypertrophic scar fibroblasts.The expression levels of α-SMA were measured by qPCR and Western blot to determine the contraction ability of hypertrophic scar fibroblasts.Collagen gel contraction assay was used to determine the activation and transformation from hypertrophic scar fibroblasts to myofibroblasts.Finally,the expression levels of collagen type l and Ⅲ,the main components of the extracellular matrix,were measured by qPCR and Western blot to determine the formation of hypertrophic scar.The pathway was predicted through literature review and network pharmacology,and finally,the phosphorylation levels of the proteins involved in the EGFR/PI3K/AKT/mTOR signaling pathway were verified by using Western blot.In the second part,the therapeutic effect of osthole on hypertrophic scarring was validated in vivo by rabbit ear hypertrophic scar models.The rabbit ear hypertrophic scar model was formed by creating an ear wound and leaving it to fully epithelialized,and dosing was initiated in three groups: control group,100 m M osthole group and 200 m M osthole group.One injection was given every 3 days for 8 times in total.Three days after the last osthole administration.The hypertrophic scar tissue was stained with HE to calculate the scar elevation index(SEI)and then with Masson staining to observe the pattern of collagen deposition and to calculate the collagen volume fraction(CVF).ResultsIn the in vitro experiments,we found that osthole inhibited the proliferation of human hypertrophic scar fibroblasts and promoted their apoptosis dose-dependently.It could also inhibit the migration ability of hypertrophic scar fibroblasts by reducing their secretion of MMPs.Osthole inhibited their contractile ability by inhibiting the transformation from fibroblasts to myofibroblasts.Osthole reduced extracellular matrix deposition by inhibiting the expression of collagens type Ⅰ and Ⅲ in hypertrophic scar fibroblasts.The common targets between the target molecules of osthole and molecules related to hypertrophic scar were identified.It was also demonstrated that osthole reduced the phosphorylation levels of EGFR,PI3 K,AKT and mTOR protein molecules by Western blot.Thus,it was confirmed that treatment effect of osthole on hypertrophic scar mainly through the EGFR/P13K/AKT/mTOR signaling pathway.In the in vivo experiment,we first successfully established a rabbit ear scar model and confirmed by HE staining that osthole could significantly reduce the thickness of the dermis of hypertrophic scar tissue and lower the SEI,and the results of Masson staining showed that osthole could make the collagen fibers deposited in the dermis more regular and tightly arranged while reducing the CVF.The ability of osthole to inhibit fibroblast activation,migration and collagen deposition was confirmed by also finding that osthole reduced the m RNA and protein expression of type Ⅰ and Ⅲ collagen,α-SMA and MMP2,MMP9 molecules.ConclusionThis study initially verified the ability of osthole to inhibit hypertrophic scar fibroblasts through the EGFR/PI3K/AKT/mTOR signaling pathway,inhibiting the proliferation,migration,activation,contraction,and extracellular matrix deposition of hypertrophic scar fibroblasts,and thus exert a therapeutic effect on hypertrophic scar,providing a basis for future clinical application.