Ivermectin Reduces Fibrotic Activity Of Scar Fibroblasts By Inhibiting Phosphorylation Of Smad3

Hypertrophic scar(HTS)is a serious dermal disorder symptom,which may be resulted from any injury of deep dermal tissue,especially severe burn,wound and surgery.HTS would lead to hyperpigmentation,pruritus,pain,contraction and would severely impair the quality of life on physiology and psychology by causing cosmetic disturbance and functional deformities.Until now,there are many therapeutic approaches for HTS such as pressure,silicone gel,laser therapy,surgery,corticosteroids and so on.But every treatment has its limitation and side effect and the therapeutic effect is not satisfactory.There is an urgent need to develop more approaches especially pharmacological agents for better preventing and treating hypertrophic scar.HPS formation is an abnormal fibrous wound healing process characterized by over proliferation of local fibroblasts and excessive deposition of collagen and other extracellular matrix(ECM)proteins.In hypertrophic scars,fibroblasts would differentiate into myofibroblasts under the action of TGF-β1 and account for increased ECM synthesis and tissue contraction.Myofibroblast which is a particular type of fibroblast characterized by its abundant expression of α-smooth muscle actin(α-SMA)plays a key role in HTS formation.In the normal healing,MFb is able to promote healing by leading to tissue contraction at the early phase and would disappear through apoptosis at the later phase.The rate of MFbs in HPS can increase to 96%.Comparing with the normal fibroblasts,the proliferation of MFbs is more rapid and the expression of type I collagen and CTGF significantly increase.Due to the alteration of varied regulatory mechanisms,the persistent proliferation of MFbs and increased production of ECM will tremendously accelerate the formation of HTS.Inhibiting the proliferation of scar fibroblasts and decreasing the production of type I collagen,α-SMA and CTGF are the major targets to slow the development of hypertrophic scar.Until now,the underlying mechanisms have not been fully understood,but it has been widely accepted that TGF-β1/Smad3 signaling pathway plays an essential role in HPS formation.It has been certified that TGF-β1 plays an essential role in modulating ECM gene expression and it is a Smad3-dependent process.Smad3 as a major intracellular effector of TGF-β1 is able to translocate into nucleus to regulate the transcription of ECM genes.The activated TGF-β receptorⅠkinase wound activate Smad3 by phosphorylation and p Smad3 will translocate into nucleus and mediate gene transcription.p Smad3 as activated smad3 has been shown to be an important modulator in ECM expression.Multiple studies have showed that Smad3 is over expressed and over phosphorylated in HTS fibroblasts and these lead to improvement of cell proliferation and expression of relevant fibrotic products.They also found that downregulation of Smad3 or p Smad3 could significantly reduce fibrotic reactivity.So,pharmacological agents with inhibition of Smad3 activity might have tremendous clinical potential in the treatment of hypertrophic scars.In this study,researchers in pharmaceutical laboratory at Johns Hopkins University had screened compounds from a library of existing medicines for the inhibitory function of Smad3 protein.they found Ivermectin could inhibit the phosphorylation of Smad3 in HEP3 B cells.Ivermectin is a semi-synthetic macrocyclic lactone derivative of the avermectin family and used to treat parasitic diseases with high security and little side effect.Recently it is found to be an effective and well tolerated agent for the topical treatment of rosacea and displays anti-inflammatory properties.But the effect of ivermectin on HPS fibroblasts has not been reported before.In this vitro study,we tested the effect of ivermectin on fibrotic activity of HTS fibroblasts and found that it could reduce the fibrotic activity by inhibiting Smad3 phosphorylation.Part Ⅰ: Isolating,identifying and culturing the human scar fibroblastsObjective: To isolate and culture the primary human scar fibroblasts.Methods: We collected the HTS tissue that was excised from post burn patients with HTS.Hematoxylin and eosin staining were performed to observe the fibrotic state of the HTS tissue.We isolated the scar fibroblasts from the HTS tissue by enzyme digestion method and cultured conventionally.We observed cell growing status and morphology under the microscope and tested the positive rate of FSP expression by flow cytometry.Results: Hematoxylin and eosin staining show that the dermal layer was thickening and an increased wavy collagen bundles were deposited.Under the microscope,we found the adherent fibroblast was spindle or star-shaped squamous cell with multi-protrusion.Through flow cytometry,we detected the cells signed by anti-FSP antibody and found that the positive rate was 97.5%.Conclusion: We isolated cells from HTS tissue successfully and testified the cells to be scar fibroblasts.These work would be the base of the following tests.Part II: Testing the effect of ivermectin on production of Smad3 and p Smad3 in normal and HTS fibroblastsObjective: To test the effect of ivermectin on production of Smad3 and p Smad3 in normal and scar fibroblasts.Methods:(1)Western blotting was performed to test the different production of Smad3 and p Smad3 in normal and scar fibroblasts.(2)We designed five groups based on the different concentration of ivermectin: blank control group,0.1μmol/L group,0.3μmol/L group,1.0μmol/L group,3.0μmol/L group.After treated with ivermectin for 48 h,normal and scar fibroblasts were lysed to extract proteins.Western blotting was performed to test the different production of Smad3 and p Smad3 in different groups.Results:(1)Compared with the normal fibroblasts,the production of Smad3 and p Smad3 protein is much higher in scar fibroblasts.(2)For normal fibroblasts,different concentration of ivermectin treatment groups had no significant difference with control group on production of Smad3 and p Smad3(p>0.05).For scar fibroblasts,the production of p Smad3 in different concentration of ivermectin treatment groups was lower than control group(p<0.05)and the effect of 3.0μmol/L group was most obvious.But the production of Smad3 protein in every group had no difference.Conclusion: Ivermectin could inhibit p Smad3 formation in scar fibroblasts,while it has no obvious effect on Smad3 production.Part III: Testing the effect of ivermectin on proliferation and apoptosis of scar fibroblastsObjective: To test the effect of ivermectin on proliferation and apoptosis scar fibroblastsMethods:(1)Proliferation assay.We designed five groups based on the different concentration of ivermectin: blank control group,0.1μmol/L group,0.3μmol/L group,1.0μmol/L group,3.0μmol/L group.CCK-8 assay was used to assess the proliferative activity of HTS fibroblasts for 5 days.(2)Apoptosis assay.We designed three groups based on the different concentration of ivermectin: blank control group,0.3umol/L group,3.0umol/L group.After treatment with ivermectin for 48 h,cell apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit I by flow cytometry.Results:(1)On day 1,no significant change was observed with different concentrations of ivermectin treatment(p>0.05).On day 3,compared with control,ivermectin significantly diminished cell proliferation by 9,14,21 and 28%,at 0.1,0.3,1.0 and 3.0 μmol/L respectively(p<0.05).On day 5,the proliferation rate after ivermectin treatment decreased by 15,31,35 and 38%,at 0.1,0.3,1.0 and 3.0 μmol/L respectively.(2)Compared with control,there was no significant difference of ivermectin treating groups on the rate of apoptosis.Conclusion: Ivermectin is able to inhibit HTS fibroblasts proliferation,while it does not affect cell apoptosis.Part Ⅳ: Testing the effect of ivermectin on the m RNA and protein expression of type I collagen,α-SMA,CTGF in scar fibroblastsObjective: To test the effect of ivermectin on the m RNA and protein expression of type I collagen,α-SMA,CTGF in scar fibroblastsMethods: We designed five groups based on the different concentration of ivermectin: blank control group,0.1μmol/L group,0.3μmol/L group,1.0μmol/L group,3.0μmol/L group.After treated with ivermectin for 48 h,scar fibroblasts were lysed to extract m RNA and proteins.RT-PCR and Western blotting were performed to test the m RNA and protein expression of type I collagen,α-SMA,CTGF in different groups.Results: RT-PCR showed that the m RNA expression of type I collagen,α-SMA,CTGF in ivermectin treating groups was lower than control group(p<0.05).Western blotting indicated that the protein production of type I collagen,α-SMA,CTGF in ivermectin treating groups decreased compared with the control group(p<0.05).The 3.0μmol/L group showed the strongest inhibitory effect on both m RNA and protein levels.Conclusion: Ivermectin is able to inhibit the expression of type I collagen,α-SMA,CTGF in scar fibroblasts on both m RNA and protein levels.