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OBJECTIVE@#Hypertrophic scars (HS) are a variety of skin tissue fibrosis disease that occurs in human skin, the effective therapeutic method of which is still inaccessible up to now. As a bioactive constituent of a well-known medical plant, Salvia miltiorrhiza (Danshen in Chinese), tanshinone IIA (TSA) is reported to inhibit cell proliferation in HS. Therefore, the aim of this study was to prepare TSA self-soluble microneedles to strengthen its dermal retention and break through the difficulty of significantly thickening epidermal connective tissue and stratum corneum at the HS site. The possible mechanism of action in suppressing HS was studied using human skin fibroblasts (HSF).@*METHODS@#Tanshinone IIA self-dissolving microneedles (TSA-MN) was prepared using a negative mold casting method. The prescription process of microneedle was optimized by Box-Behnken effect surface method. Different media were selected to investigate the ability of transdermal absorption and in vitro release. Furthermore, according to Cell Counting Kit-8 (CCK8) method as well as the Western blot method, the effect of TSA-MN on the biological characteristics of HSF was investigated.@*RESULTS@#With remarkable slow release effect and dermal retention, the release and transdermal properties of TSA-MN in vitro were better than both TSA and ordinary dosage forms. Its effect of HSF confirmed the essential decrease in cell motility during cell proliferation and cell migration in vitro, which plays a significant role in down-regulating the secretion of transforming growth factor-β1 (TGF-β1) in HSF and increasing the expression level of Smad7.@*CONCLUSION@#The prepared TSA self-soluble microneedles is helpful in solving the problem of hypertrophic scars, with a stable dermal retention effect after process optimization.
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Objective: To study the effect and mechanism of tanshinone IIA on human skin fibroblasts cell (HSF). Methods: CCK- 8 method was used to determine the effect of different concentrations of TSA on the proliferation of HSF induced by TGF-β1. The plate cloning ability of HSF treated with different concentrations of TSA (2.5, 5, 10 and 20 μmol/L) for 48 h were analyzed by plate clonogenesis assay. The protein expression of TGF-β1/Smad signaling pathway proteins and α-SMA, VEGFA and COL I were further measured by Western blotting. Results: CCK-8 and plate clonognesis assay results showed that TSA significantly inhibited the proliferation and colony forming efficiency of HSF (P < 0.01). Western blotting results revealed that each concentration group of TSA significantly inhibited the protein levels of p-Smad2 and p-Smad3, and down-regulated the ratio of p-Smad2/Smad2 (P < 0.01). The ratio of p-Smad3/Smad3 was significantly decreased in 5, 10 and 20 μmol/L TSA groups. Additionally, the expression levels of α-SMA, VEGFA and COL I in HSF decreased significantly with the increase of TSA concentration (P < 0.01). Conclusion: TSA exhibits the inhibitory effect on proliferation of HSF, and its mechanism may be related to TGF-β1/Smads signaling pathway.
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Hypertrophic scar( HS) is a very common skin fibrosis disorder after human skin injury and wound healing. The objective of this study was to investigate the efficacy of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B( SAB-TAT-LIP) on proliferation,migration and cell cycle of human skin fibroblasts( HSF),and preliminarily evaluate its effect on prevention and treatment of HS. HSF were cultured in vitro,and MTT assay was used to detect the inhibitory effect of SAB-TAT-LIP on cell proliferation. Cell migration was assessed by Transwell chamber method and scratch method; and cell cycle change was detected by flow cytometry. In vitro cell studies showed that blank liposome basically had no toxic effect on HSF. Different concentrations of SABTAT-LIP inhibited proliferation on HSF in varying degrees after intervention for different periods in a dose and time dependent manner;meanwhile,SAB-TAT-LIP significantly inhibited the migration and invasion of HSF. At the same time,SAB-TAT-LIP could block the cell cycle at G0/G1 phase after intervention for 48 h,P<0.01 as compared with the blank control group. Conclusively,our experimental data quantitatively demonstrate that SAB-TAT-LIP has significant inhibitory effect on cells proliferation,invasion and migration,with blocking effect on G0/G1 phase. This may offer a promising therapeutic strategy for transdermal delivery in prevention and treatment of HS.
Subject(s)
Humans , Benzofurans , Pharmacology , Cell Cycle , Cell Movement , Cell Proliferation , Cell-Penetrating Peptides , Cells, Cultured , Drug Carriers , Fibroblasts , Cell Biology , Liposomes , Skin , Cell BiologyABSTRACT
Objective To prepare the liposomes of salvianolic acid B modified with cell penetrating peptide TAT (SAB-TAT-LIP), of which has effects on preventing and treating hypertrophic scars (HS), and establish the method of quality evaluation, as well as preliminarily investigate the effect on the proliferation and migration of human skin fibroblasts (HSF). Methods Liposomes were prepared by pH gradient reverse-phase evaporation method, and the entrapment efficiency was measured by ultrafiltration. Box-Behnken design was performed to optimize the formulation of liposomes by using encapsulation rate as evaluating index. The physicochemical properties of liposomes including morphology, entrapment efficiency, particle size, zeta potential, in vitro release and transdermal absorption, and stability were studied. In addition, the effect of liposomes on proliferation of HSF was examined by MTT assay, and the effect of liposomes on migration of HSF was investigated by scratching method and Transwell assay. Results Based on the optimal formulation of SAB-TAT-LIP, the entrapment efficiency of salvianolic acid B was (86.70 ± 0.85)%, the average particle size was (219.90 ± 5.09) nm, and the zeta potential was (-9.25 ± 0.92). The in vitro 24 h cumulative release was 62.49% of the total drug with no burst effect. The in vitro 32 h cumulated skin penetration rate was 17.21%, the permeance rate was (28.33 ± 4.9) μg/(cm2∙h), and the retention volume of dermis was (44.39 ± 6.87) μg/cm2. The stability was good when placed at 4 ℃ for 10 d. The in vitro cell studies showed that SAB-TAT-LIP can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts, compared with the control group (P < 0.01). Conclusion The optimized SAB-TAT-LIP have higher encapsulation efficiency, smaller particle size, good sustained release effect, and good dermal retention effect which all satisfy the in vitro release and transdermal regulation of local transdermal preparation, and it can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts in vitro.
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[Objective]To explore the effect of alpha2-macroglobulin(α2M)on superoxide anion(.O2-)content, superoxide dismutase(SOD)activity and the process of cell-to-myofibroblast transformation in human skin fibroblasts (HSF)after X-ray irradiation.[Methods]HSF cells were irradiated with 0,5,10,15 and 20 Gy X-ray.The change of. O2- content and SOD activity in the supernatant of cell culture medium were measured on the first day after irradiation. The protein expression of alpha-smooth muscle actin(α-SMA)was detected by Western blot on the fifth day after irradia-tion.The most sensitive radiation dose is selected.HSF cells were irradiated with the above sensitive dose.Respectively, 1h before irradiation,1 h after irradiation,the experimental group cell culture medium was added to a final concentration of 0.25 mg/mL,0.5 mg/mL of α2M.The change of.O2-content,SOD activity and the protein expression of α-SMA were detected.[Results]HSF cells were irradiated with 5~20 Gy doses of X-ray..O2- content increased,SOD activity de-creased and α-SMA protein expression increased gradually(P<0.05).The addition of α2M at 1 h after 10Gy X-ray irradi-ation reduced the.O2- content,increased the SOD activity and downregulated the protein expression of α-SMA in HSF cells(P<0.05). There was no significant change in the administration at 1 h before irradiation.[Conclusion]HSF cells increased.O2-content significantly,while SOD activity decreased,and the tendency to transform myofibroblasts after X-ray irradiation.α2M can reduce the.O2-content,increase the SOD activity in HSF cells and inhibit the transformation of fibroblasts into myofibroblasts after irradiation.Indicating that α2M can play a role in radiation protection by anti-oxida-tion and anti-fibrosis.
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Objective To investigate the protective effect of lycium barbarum polysaccharide (LBP) on DNA damage of HSF cells induced by UV.Methods We established the model of UV induced photo damage in HSF cells.We detected the viability of HSF cells by using MTT colorimetry.The UV absorption spectrum of LBP was also measured by UV spectrophotometer.The level of ROS was detected by DCFH-DA fluorescent probe method.Comet assay was employed to evaluate the DNA strand breakage damage.Results When the concentration of LBP was less than or equal to 300μg/ml,there was no significant effect on the proliferation of HSF cells (P＞0.05).When the concentration was more than 300 μg/ml,it could inhibit the cell proliferative activities (P＜0.05).Compared to the UV groups,UV+LBP groups can respectively improve the cell proliferation activity (P＜0.05).The absorbance was slight range 280 from 400 nm.Compared with the UV group,the relative fluorescence intensity and the migration distance of UV+ LBP groups were significantly decreased (P＜0.05).Conclusions Lycium barbarum polysaccharide can effectively inhibit the proliferation activity and protect the breakage of DNA strand induced by UV,which is probably due to its action of removing free radicals.
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Objective To determine the inhibitory effect and mechanism of metformin ( MF) on photo-damage of human skin fibroblasts ( HSF) induced by UVA .Methods Human skin fibroblasts were randomly divided into con-trol group, UVA group and UVA+MF group.The proliferation of HSF was detected by CCK-8 assay kit.SA-β-gal staining was performed to evaluate the senescence state .The level of ROS was examined by fluorescence probe DCF-DA staining using flow cytometry .Real-time PCR was used to determine mRNA expression of senescence -asso-ciated signals of MMP 1 and MMP3.The protein expression of MMP 1, MMP3, SOD1 and SOD2 were measured by Western blot .R esults To the proliferation of HSF , 0.01 mmol/L Metformin had no significant effect , but 0.1 and 1 mmol/L Metformin depressed significantly ( P<0.05 ) .Compared with the Control group , it showed that UVA irradiation increased the positive rate of SA-β-gal staining ( P<0.01 ) , the level of ROS ( P<0.05 ) , mRNA and protein expression of MMP1 and MMP3 significantly(P<0.01);Also decreased the expression of SOD1 and SOD2 ( P<0.01) .Compared with the UVA group , it showed that metformin decreased the positive rate of SA-β-gal staining (P<0.05), the level of ROS(P<0.05), mRNA and protein expression of MMP1 and MMP3 significantly(P<0.05);Also increased the expression of SOD 1 ( P<0.01 ) and SOD2.Conclusions Metfomin can inhibit photo-damage of human skin fibroblasts induced by UVA via decreasing ROS and metal matrix protease generation , also the improvement of cellular antioxidant capacity .