Effects Of Cyclic Strain On Bioactivities Of Hepatic Stellate Cells

BackgroundOrganisms in various levels of life events are inseparable from a certain degree of mechanical environments. As the basic unit of life, cells are living in a complex mechanical environment. Hepatic stellate cells are the major extracellular matrix cells, which play an important role in the mechanism of liver fibrosis development. It is priority of liver fibrosis research on how to control the activity of hepatic stellate cells and thus reverse the process of hepatic fibrosis. Chronic hepatitis is often accompanied by portal hypertension. High portal pressure occurs usually with sinusoidal expansion, increasing the tension force suffered by hepatic stellate cells. There are few studies about mechanical stretch on the hepatic stellate cells'biological activities. This study try to exert mechanical stretch through the application of programmed cell traction device Flexercell Strain Unit 2000, onto rat cultured hepatic stellate cells to study cyclic strain on the biological activities of the cells. The aim of the present study was to explore the effect and mechanism of mechanical stress on hepatic stellate cell activation, proliferation, ECM production and other functionsMethodsWe used enzymatic digestion-density gradient centrifugation method to isolate rat hepatic stellate cells. The experimental cells consist of 6 groups: Control group (Con), simply Stretch group (MS), stretch + cRGDfV (AM100) group (AM), stretch + SB202190 group (SB), stretch + wortmannin group (wort), stretch + PD98059 group (PD) . Cells were cultured in BioFlex 6-well plates. When cells were grown to an appropriate density, the cultured medium with the DMEM containing 1% FBS culture medium was replaced, and synchronized for 24 hours. The various inhibitors of effective concentration were treated to AM, SB, Wort and PD group, and the control group and simple stretch group only treated with 0.5‰DMSO and 1% BSA in DMEM culture medium. Apart from control group and MS group, other groups were exposed to cyclic tensile strain (stretch conditions: 10% elongation, 0.5 Hz frequency, 24 h time) before one-hour of treatment with inhibitors. When stretch was completed, we detected the cell proliferation, migration and other functions. Total RNA was extracted for real-time quantitative PCR detection of a variety of cell activation associated factor gene expression. Extraction of total protein was for western blotting detection of a variety of signal-line protein expression. The experiment was repeated three times. Data were presented by mean±S.D., and were analysized by SPSS 18.0 software. One-Way ANOVA was used to statistical analysis: data with homogeneity of variance were analysized by LSD-t; Dunnett's T3 was used in data with variance arrhythmia. P <0.05 was considered to be statistically significant.Results1. Cell isolation and identification: Identification of isolated cells by trypan blue staining showed that HSC survival was more than 95%; Desmin,α-SMA immunofluorescence assay and activation proved quiescent HSC cell purity of more than 95%.2. To determine the conditions of stretch on hepatic stellate cells.When the stretch parameter is set to 10% elongation, 0.5 Hz, 24h, the stretch group HSC collagen gene expression generated a marked increase compared with the control group, and it was stable in a variety of conditions of stretch. 10% of elongation and 24hrs duration was sufficient to guarantee enough and stable change in HSC activation. 0.5Hz frequency was much like biological condition in hepatic sinusoidal. Therefore, following experiments were under the stretch condition of 10% elongation, 0.5 Hz frequency, 24-hours duration if there were not any special instructions.3. Mechanical stretch on the biological activity of HSCMechanical stretch can significantly promote the proliferation of HSC, cRGDfV (integrinαvβ3 inhibitor) can inhibit the proliferation of cyclic tensile strain-promoting effects. The experiment also found that cyclic strain significantly promote the alpha-SMA expression of HSC, and the production of TGF-beta 1, CTGF and other factors that promotes fibrosis. The enhancement could be prohibited by cRGDfV, which strongly suggested that cyclical tensile strain promoted the proliferation and activation of HSC, which at least partly activated through the activation of integrinαvβ3. However, we failed to detect the impact of cyclical tensile strain on the production of PDGF-B and its receptor PDGFR-B mRNA expression, indicating the proliferation-promoting and activation- promoting effects of cyclic tensile strain was not related with the PDGF-B pathway. Cyclic tensile strain can significantly promote the mRNA expression of COL1A1 and COL3A1 of HSC, of which COL3A1 is more obvious; the effect can be inhibited by the blockers of integrinαvβ3, cRGDfV.The wound healing assay and Transwell experiments found that cyclic tensile strain alone had no significant effect on the migration of HSC, but it can significantly enhance the PDGF-BB-induced migration of HSC. Blockingαvβ3 integrin activation significantly inhibited PDGF-BB-induced migration of HSC, and inhibited markedly the increased proliferation, activation and migration of HSC. Collective with foregoing findings, the results of this study implied that in the process of development of liver fibrosis, cyclic strain promoted the production of TGF-beta, CTGF and fibrogenetic factors, and it also promoted the proliferation and activation of HSC, which was directly through the integrin pathways; the other hand, by activating integrin-way, collaborative PDGF, to enhance the migration of HSC accelerating the progress of liver fibrosis.4. Cyclical impact of HSC function of tensile strain mechanismResults showed that the various inhibitors used had no significant effects on cell proliferation, and cell apoptosis. Mechanical stretch can significantly enhance the integrinβ3, p38, Akt, ERK1/2 phosphorylation. cRGDfV does not inhibit the phosphorylation of integrinβ3, but it can inhibit FAK phosphorylation level. Akt, p38, ERK1/2 phosphorylation inhibitor, wortmannin, SB202190, PD98059, significantly reduced the level of the corresponding protein phosphorylation in HSC.Cyclic strain can significantly enhance the expression of PCNA protein which resulted in HSC proliferation, and promoteα-SMA, COL1A1 and COL3A1 gene expression. Application cRGDfV or SB202190 can inhibit the proliferation and activation of HSC by tensile strain. Wortmannin (Akt inhibitor) can inhibit tensile strain due to increased expression of COL1A1 and COL3A1 genes, but can not inhibit PCNA orα-SMA. PD98059 (ERK1/2 inhibitors) can not inhibit the proliferation and other biological effects of tensile strain. These results indicated that cyclical effects of tensile strain on the HSC may be via activation of integrinαvβ3 to promote activation of FAK, which can highly activate the MAPK pathway in particular the p38 signal transduction pathway, which resulted in biological effects of HSC.Conclusion1. We successfully isolated static hepatic stellate cells from rats in vitro. The cells can be repeatedly subcultured and automatically activated.2. Cyclic tensile strain enhanced gene expression of collagen Iα1 chain of hepatic stellate cells, especially in the condition of 10% elongation, 0.5Hz frequency and 24 hours for stretch.3. Cyclic tensile strain promoted proliferation, activation of hepatic stellate cells, and its migration under PDGF-BB stimulation. The result tells us cyclic strain can enhance the fibration effect of hepatic stellate cells. therefore we conjecture that cyclic strain may positively feedback on the meintainence and development of portal hypertension.4. Our results showed that Cyclic tensile strain promotes expression and activation of integrinαvβ3 on hepatic stellate cell membrane, this leads to intracellular adhesion kinase FAK phosphorylation, then activates MAPK signal transduction , which promotes the proliferation and activation of hepatic stellate cells through follow-up bioeffects. These tells us integrinαvβ3 plays the key role in the action of cyclic strain on hepatic stellate cells.