TSG-6 Inhibits The Growth Of Keloid Fibroblasts By Mediating The TGF-?1/Smad Signaling Pathway

BackgroundPathological scar is a skin fibrosis disease characterized by abnormal proliferation of tissue fibrosis and chronic inflammation.Its pathogenesis is not completely clear,and there is no ideal treatment at present.Therefore,elucidating the pathogenesis of pathological scar and studying the specific target of its pathogenesis and development mechanism become a new direction of developing new drugs for treating pathological scar.Construction of tumor necrosis factor alpha stimulated gene 6(TSG-6)is considered to be a gene involved in a variety of inflammatory reactions.It can inhibit the expression of inflammatory factors such as IL-6 and IL-1beta,inhibit neutrophil infiltration and reduce inflammatory damage.It was found that the expression level of TSG-6 in keloids was significantly lower than that in scarless repaired skin.In our previous experiments in vivo and in vitro,we found that TSG-6 could inhibit the inflammatory reaction of hypertrophic scar in rabbit ears,reduce the degree of inflammation,inhibit the proliferation of keloid fibroblasts,and down-regulate the expression of TGF-beta 1.Therefore,we speculate that the mechanism of its inhibition of pathological scar may be related to the pathway mediated by transforming growth factor beta-1(TGF-beta-1).TGF-beta-1 is considered to be the most critical pathogenic factor of pathological scar.The TGF-beta-1/smad pathway mediated by TGF-beta-1 can be formed by influencing the formation of fibroblasts,inhibiting the degradation of extracellular matrix proteins such as collagen and promoting angiogenesis.Smad2 and Smad3 are phosphorylated by binding to TGF-beta I and II receptors on cell membranes.Activated Smad2/Smad3 complex binds to Smad4 and enters the nucleus,regulating the expression of different genes,thereby affecting the proliferation or apoptosis of fibroblasts and the synthesis or degradation of collagen.This topic intends to further study the effect of TSG-6 on TGF-beta 1/smad signaling pathway in keloid fibroblasts,and further elucidate its anti-keloid effect.Method 1.Culture and passage of human keloid fibroblast(HKF)Collagenase IV was used to digest keloid samples.HKF was isolated and purified.Dulbecco's modified Eagle's medium(DMEM)was used as the medium.Streptomycin,L-glutamine and fetal bovine serum were added to the medium.Generally,5-6 generations of cells were cultured for lentivirus transduction.2.Preliminary determination of the effect of TSG-6 on fibroblast activityCells were divided into two groups,TSG-6 treatment group and control group.Recombinant human TSG-6 was added into TSG-6 group.(1)Cell cycle was determined by flow cytometry: two groups of cells were taken and PI staining was used to observe the number of cells in different time periods per unit area.(2)TUNEL assay was used to detect apoptosis: TUNEL assay was used to observe under optical microscope,and TUNEL positive(brown)cells in cells were counted.(3)Immunohistochemical detection of the effect of TSG-6 on the number of cells expressing proliferative antigen(PCNA)3.Construction and packaging of lentivirus vectorsPrimer synthesis was designed to construct siRNA of TSG-6 with high interference efficiency and stability.The recombinant Lentivirus Expression Vector(Plvx-pour-TSG-6)and recombinant lentivirus interference vector(Plvx-shRNA1-TSG-6)were constructed.Lenti-X p24 Rapid Titer Kit kit was used to detect the titer of lentivirus.When the titer was greater than 106 PFU/mL,the supernatant could be harvested and removed.Cell fragments were preserved at-80 C.4.Recombinant Lentivirus Infection and ScreeningCells were inoculated into 6-well culture dishes after cell concentration was modulated by 5 *107 cells/L.On the second day of culture,viruses were added into 6-well culture dishes,and purinomycin with 8 mg/l concentration was added.The cells were retested after five days.After 48 hours,purinomycin containing 2.5 mg/l was used for cell screening.After one week of screening,TSG-6 overexpression HKF group,TSG-6 interference HKF group and corresponding control group were produced in normal medium.5.Detection of TSG-6 mRNA expression in HKFs transfected by different groupsFive groups of cells were collected,including HKF group,TSG-6 overexpression HKF group,TSG-6 interference HKF group,overexpression control group and interference control group.Total RNA was extracted from cells and synthesized into DNA.Quantitative fluorescence PCR was performed by SYBR Green method.The expression of TSG-6 was detected in all groups of cells.6.MTT assay and flow cytometry to detect cell proliferation and apoptosis at different time after infectionMTT assay was used to detect the proliferation of five groups of cells.Flow cytometry was used to detect the cell apoptosis and cell cycle arrest in the TSG-6 overexpression HKF group,TSG-6 interference HKF group and HKF group.7.Effect of TSG-6 on TGF-beta 1/Smad signaling pathway(1)Immunoprecipitation and Western blot analysisThe cells were divided into six groups: solvent control group,HKF-TGF-beta 1 group,TSG-6 overexpression HKF-TGF-beta 1 group,TSG-6 overexpression HKF-TGF-beta 1 control group,TSG-6 interference HKF-TGF-beta 1 control group,TSG-6 interference HKF-TGF-beta 1 group and TSG-6 interference HKF-TGF-beta 1 control group.In addition to the solvent control group,40 pM TGF-beta 1 was added into each group.After culture,the total cell protein was extracted and immunoprecipitated with mouse anti-smad2/3 antibody.The phosphorylation of Smad2/3 at C-terminal was detected by immunoprecipitation and Western blot analysis.After immunoprecipitation,anti-Smad4 was added to mice and the expression of Smad2/3/4 complex was detected by Western blotting.The same six groups of cells,except the solvent group,were stimulated by TGF-beta 1,cultured and added with corresponding antibodies.The expression of Smad7 and PAI-1 protein was detected by Western blotting.(2)Immunofluorescence analysisCell grouping was the same as the above experiment.In addition to the solvent control group,after stimulation with TGF-beta 1,the smad2/3 stained cell area was photographed under fluorescence microscopy after immobilization and co-incubation.(3)RT-PCR analysis of PAI-1 gene level in six groups of cellsIn addition to the solvent group,the total RNA was extracted after stimulation by TGF-beta 1,and the expression of PAI-1 was detected by RT-PCR.Result 1.Preliminary determination of morphology of each group and the effect of TSG-6 on HKFs activity 1.1 Cell morphology was initially inoculated with round globular cell clusters.Under inverted microscope,adherent cells changed into long spindle or triangle.1.2 The results of TUNEL assay showed that the cell density of TSG-6 treated group was higher than that of control group.The number of PCNA positive cells in TSG-6 treatment group was lower than that in control group.1.3 TSG-6 affects cell cycle.Flow cytometry PI staining showed that TSG-6 treated cells were blocked in G1 phase.2.The expression of TSG-6 and the expression of TSG-6 in HKFs transfected with lentivirus vector were different in each group.2.1 The target gene fragment of 828 BP was obtained by agarose syrup electrophoresis with the detection of transfection rate.The size of the target gene fragment accorded with TSG-6.Agarose gel electrophoresis showed positive clones after infection.The size of the positive and negative transformed strains was 1179 BP and 198 BP respectively.After BLAST detection,the results of clone sequence identification were 828/828(100%),and the target gene lentiviral vector was successfully constructed.2.2 The expression of TSG-6 after infection by RT-PCR showed that TSG-6 overexpression in HKF group was higher than that in HKF group and Plvx-Puro group,while TSG-6 expression in HKF group was not significantly different from that in Plvx-Puro group.3.Expression of TSG-6 and the effect of TSG-6 on cell proliferation and apoptosis after lentivirus vector transfection into HKFs 3.1 Overexpression of TSG-6 inhibits the proliferation of HKFs.MTT assay results showed that the proliferation of HKFs in PLVX-Puro overexpression group(PLVX-Puro-TSG-6)was significantly lower than that in PLVX-Puro group.At the same time,the proliferation of HKFs in PLVX-shRNA1-TSG-6 transfection group was significantly increased compared with the blank plasmid and PLVX-shRNA1 group.3.2 Overexpression of TSG-6 induced apoptosis of HKFs.The apoptotic rate of TSG-6 overexpression group was 51.92%,significantly higher than that of Plvx-Puro group(19.00%)and HKFs group(3.59%;P < 0.05).3.3 Flow cytometry analysis of TSG-6-regulated cell cycle indicated that cells were inhibited by TSG-6 overexpression in G2/M phase.4.TSG-6 inhibits the TGF-beta 1/Smad signaling pathway of HKFs 4.1 TSG-6 inhibited the phosphorylation of Smad2/3C terminal and the expression of Smad2/3/4 complex.Compared with HKF-TGF-beta group and TSG-6 overexpression HKF-TGF-beta control groups,the expression level of Smad2/3/4 complex in TSG-6 overexpression HKF-TGF-beta 1 group increased significantly.On the contrary,the expression of Smad2/3/4 complex decreased in TSG-6 interference HKF-TGF-beta 1 group.4.2 Compared with HKF-TGF-beta group and TSG-6 overexpression HKF-TGF-beta control groups,the fluorescence intensity of pSmad2 C and pSmad3 C in TSG-6 overexpression HKF-TGF-beta 1 group decreased after the stimulation of TGF-beta 1,suggesting that TSG-6 overexpression HKF-TGF-beta 1 group inhibited intranuclear translocation.4.3 TSG-6 affects the expression of Smad7 and PAI-1 in fibroblasts.After stimulation by TGF-beta 1,the expression of Smad7 in TSG-6 overexpression group is higher than that in other groups,suggesting that TSG-6 can up-regulate the expression of Smad7.RT-PCR results showed that PAI-1 decreased significantly in the TSG-6 overexpression group,but increased significantly in the interference group conclusionTSG-6 inhibits proliferation and apoptosis of keloid fibroblasts and interferes with cell cycle.According to the above studies,the mechanism of TSG-6 inhibiting keloid fibroblasts may be related to the TGF-beta 1/Smad signaling pathway,which can inhibit the phosphorylation of Smad2 and Smad3,the formation of Smad2/3/4 complex and the translocation of Smad2/3 into the nucleus,while up-regulating the expression of negative regulatory protein Smad7 and inhibiting the expression of PAI-1 gene.These results indicate that TSG-6 can prevent pathological scar formation.The potential therapeutic application of Chengzhong provides a basis.