Mechanisms Of Compound Astragalus And Salvia Miltiorrhiza Extract Exerting Anti-keloid Via Tgf-p/smad Signal Transduction Regulated By Mapk Pathway

Background Keloids are dermal fibroproliferative diseases, which are characterized byhyperproliferation of responsive fibroblasts and overabundant deposition ofextracellular matrix (ECM), especially collagen. The underlying mechanisms of keloidpathogenesis remain unclear resulting in no optimal therapeutic approaches available todate. Thus, there is an urgent need to elucidate the molecular mechanism of keloiddevelopment in order to investigate and develop new drugs for keloid treatment thatpoint to the specific targets of keloid pathogenesis. Transforming growth factor-beta1(TGF-β₁) is known to be the critical profibrotic cytokine involved in keloiddevelopment and it transmits a signal mainly through the Smad family. It has beendemonstrated that TGF-β/Smad signal pathway plays an important role in keloidpathogenesis. Our previous in vivo and in vitro experiments demonstrated thatCompound Astragalus and Salvia miltiorrhiza extract (CASE) exerted anti-fibrosiseffects in chronically injured rat liver and inhibited cell invasion in HepG2cells bymediating TGF-β/Smad signal pathway. We have also demonstrated that CASEsignificantly inhibits the proliferation, collagen synthesis and cell invasion in keloidfibroblasts. Therefore, this study proposed to sequentially examine the effects of CASEon cell migration in keloid fibroblasts to further confirm its anti-keloid effects;moreover, we intended to profoundly investigate the effects of CASE on TGF-/Smadsignal transduction pathway in keloid fibroblasts in order to clarify its molecularmechanisms. In addition to the Smad signal, TGF-β1also activates mitogen activated protein kinase (MAPK) pathway, including extracellular signal-regulated kinase (ERK),c-Jun N-terminal kinase (JNK) and p38kinase. Our previous study had discussed themechanisms of TGF-1induced Smad3signal transduction and plasminogen activatorinhibitor-1(PAI-1) transcription activity mediated by MAPK pathway in keloidfibroblasts. Based on these results, the present study was designed to proceed with theresearch on the mechanisms of MAPK pathway regulating other important componentsof the TGF-β/Smad signal transduction pathway in keloid fibroblasts, and further clarifythe action mechanisms of CASE via mediating MAPK pathway.Objectives1. To observe the effects of CASE on cell migration in keloid fibroblasts in order tofurther confirm its anti-keloid effects based on previous studies which haddemonstrated the inhibitory effects of CASE on the proliferation, collagen synthesisand cell invasion in keloid fibroblasts;2. To investigate the effects of CASE on TGF-β/Smad signal transduction pathway inkeloid fibroblasts for the purpose of elucidating its molecular mechanisms;3. To further explore the action mechanisms of CASE via mediating MAPK pathwayfollowing clarification of the mechanisms of TGF-β/Smad signal transductionregulated by MAPK pathway in keloid fibroblasts.Methods1. Cell culture and treatmentKeloid fibroblasts were cultured in serum-free medium for24h with or without CASE(7.5,15,30mg L^-1) treatment, or in the absence or presence of ERK inhibitor (PD98059),JNK inhibitor (SP600125) or p38inhibitor for5h, respectively; subsequently the cellswere treated with TGF-1for the determined concentrations and time depending on thedifferent purposes. The cells of the control group were added to an equal volume of vehicle. Each experiment was replicated at least three times in this study.2. Cell migration in keloid fibroblasts was observed by in vitro scratch assayKeloid fibroblasts were cultured as confluent monolayer followed by scratch-wounding,then pictures were taken to observe cell migration in keloid fibroblasts undermicroscope at0,12,24h time point after wounded. Normal skin fibroblasts weretreated same as a control.3. Smad2/3phosphorylation and Smad2/3/4complex formation in keloidfibroblasts were detected by immunoprecipitation and western blot methodAfter incubation, keloid fibroblasts were harvested and lysed for total protein extraction.The supernatants of pretreated cell lysates were subjected to immunoprecipitation withanti-Smad2/3antibody. The phosphorylation of Smad2/3at the C-terminal region(pSmad2C, pSmad3C) and at the linker region (pSmad2L, pSmad3L) was thenmonitored by western blot analysis using each domain-specific antibody against thephosphorylation site: anti-pSmad2C (Ser⁴⁶⁵⁴⁶⁷), anti-pSmad3C (Ser⁴²³⁴²⁵),anti-pSmad2L (Ser²⁴⁹²⁵⁴) or anti-pSmad3L (Ser²⁰⁸²¹³). In addition, afterimmunoprecipitation, the Smad2/3/4complex was monitored using anti-Smad4antibody to detect Smad4that bound to Smad2/3. The endogenous Smad2/3expressionand Smad4in cell extracts were monitored as the reference.4. The intracellular localization of phosphorylated Smad2/3and MAPK proteins inkeloid fibroblasts were examined using immunofluorescence assayKeloid fibroblasts were seeded on sterile slides in24-well plates. After incubation, thepretreated cells were fixed and blocked. The cells were then incubated with eachprimary antibody overnight followed by incubation with fluoresceinisothiocyanate-conjugated IgG. After that, the slides were viewed and photographedunder a fluorescence microscope to observe the intracellular localization ofphosphorylated Smad2/3and MAPK proteins in keloid fibroblasts.5. The protein levels of Smad7, PAI-1, and MAPKs in keloid fibroblasts were measured by western blot methodAfter incubation, keloid fibroblasts were harvested and lysed for total protein extraction.The expression of Smad7or PAI-1protein as well as pERK1/2, pJNK1/2, or pp38protein in keloid fibroblasts was measured by western blot method; the expression ofGAPDH and non-phosphorylated ERK1/2, JNK1/2or pp38in keloid fibroblasts wasmeasured as the reference, respectively.6. The PAI-1mRNA level in keloid fibroblasts was detected by real-timequantitative RT-PCRTotal RNA was extracted from keloid fibroblasts after incubation and was reversetranscribed to cDNA followed by SYBR Green I real-time PCR analysis using eachprimer for human PAI-1gene and human-actin gene. Threshold cycles (Ct) value wasutilized to calculate the relative quantization of PAI-1mRNA which normalized to thehousekeeping gene-actin. Melting curves were generated to confirm amplification ofspecific transcripts.Results1. Cell migration in keloid fibroblasts was significantly inhibited by CASEKeloid fibroblasts healed faster than those of normal skin fibroblasts after wounded andwere more sensitive in response to TGF-β₁. Cell migration induced by TGF-β1in keloidfibroblasts was significantly inhibited by CASE in a dose-dependent manner and almostthoroughly inhibited at the concentration of30mg L^-1. In contrast, TGF-β1and CASEhad little effects on cell migration in normal skin fibroblasts.2. TGF-β/Smad signal transduction pathway in keloid fibroblasts was interferedby CASE treatment2.1CASE inhibited the phosphorylation of Smad2/3at C-terminal and linkerregion induced by TGF-β₁in keloid fibroblasts TGF-1markedly induced Smad2/3phosphorylation not only at the C-terminal region(pSmad2C, pSmad3C) but also at the linker region (pSmad2L, pSmad3L) in keloidfibroblasts. CASE significantly inhibited TGF-1-induced pSmad2L and pSmad3Lexpression, whereas exhibited relative weaker effects on pSmad2C and pSmad3C. Inaddition, the expression of pSmad2L and pSmad3C induced by TGF-1was supressedby CASE in a dose-dependent manner.2.2CASE inhibited Smad2/3/4complex formation induced by TGF-β₁in keloidfibroblastsTGF-1treatment obviously induced Smad2/3/4complex formation in keloidfibroblasts, while the Smad2/3/4complex expression induced by TGF-1was markedlysupressed by CASE in a dose-dependent manner, and almost entirely blocked at theconcentration of15or30mg L^-1.2.3CASE blocked TGF-1-stimulated nuclear translocation of phosphorylatedSmad2/3in keloid fibroblastsThe expression of pSmad2C and pSmad3C was moderately elevated under TGF-1treatment and mostly found in nuclei; while the expression of pSmad2L and pSmad3Lwas markedly enhanced by TGF-1stimulation and localized in both cytoplasm andnuclei. However, both the fluorescence intensity of phosphorylated Smad2/3and theirnuclear translocation induced by TGF-1were significantly inhibited by CASEtreatment.2.4CASE up-regulated TGF-1-decreased Smad7protein level in keloid fibroblastsTGF-1stimulation resulted in decrease of Smad7protein level in keloid fibroblasts,while the decrease of Smad7expression caused by TGF-1was reversed by CASE in adose-dependent manner and back to the level comparable with those of control (withoutTGF-1tratment) at the concentration of30mg L^-1.2.5CASE decreased PAI-1mRNA and protein levels induced by TGF-1in keloidfibroblasts TGF-1induced obvious increase in PAI-1mRNA level in keloid fibroblasts; however,the elevated transcriptional activity of PAI-1stimulated by TGF-1wasdose-dependently suppressed by CASE and the inhibitory effect attained statisticalsignificance at the concentrations of15and30mg L-1. Consistently, the PAI-1proteinlevel increased by TGF-1was almost completely suppressed by CASE atconcentrations of15and30mg L-1.3. TGF-/Smad signal transduction was regulated by MAPK pathway in keloidfibroblastsWe had previously demonstrated that TGF-β₁induced Smad3signal transduction andPAI-1transcription activity was regulated by MAPK pathway in keloid fibroblasts: Thep38pathway participated in the regulation of Smad3phosphorylation at the linkerregion and subsequent Smad2/3/4complex formation; ERK and JNK pathwayscontributed to the regulation of the nuclear translocation of phosphorylated Smad2/3;finally, ERK, JNK and p38pathways all involved in regulating the transcription activityof target gene PAI-1in keloid fibroblasts. The present study continues to explore themechanisms of MAPK pathway regulating other important components in TGF-β/Smadsignal transduction pathway in keloid fibroblasts. The results are presented as follows:3.1Effects of three MAPK inhibitors on Smad2phosphorylation at C-terminal andlinker region induced by TGF-β₁in keloid fibroblastsThe p38inhibitor (SB203580) almost completely inhibited the expression of pSmad2Las well as moderately inhibited pSmad2C expression induced by TGF-1in keloidfibroblasts, whereas ERK inhibitor (PD98059) and JNK inhibitor (SP600125) had littleeffects on Smad2phosphorylation at both C-terminal and linker region.3.2Effects of three MAPK inhibitors on the nuclear translocation ofphosphorylated Smad2stimulated by TGF-1in keloid fibroblastsThe fluorescence intensity of pSmad2C and pSmad2L and their nuclear expression induced by TGF-1were significantly supressed by p38inhibitor (SB203580) in keloidfibroblasts. Although ERK inhibitor (PD98059) and JNK inhibitor (SP600125) had littleeffects on the fluorescence intensity of pSmad2C and pSmad2L, the two inhibitorsblocked the nuclear translocation of pSmad2C and pSmad2L stimulated by TGF-1.3.3Effects of three MAPK inhibitors on TGF-1-decreased Smad7protein level inkeloid fibroblastsTGF-1-decreased Smad7protein level was reversed by p38inhibitor (SB203580) andback to the level compared with those of control (without TGF-1treatment) in keloidfibroblasts. Although ERK inhibitor (PD98059) and JNK inhibitor (SP600125) showeda tendency to increase Smad7protein level, the difference had no statisticalsignificance.3.4Effects of three MAPK inhibitors on TGF-1-induced PAI-1protein level inkeloid fibroblastsAll of the three MAPK inhibitors obviously inhibited the protein expression of PAI-1induced by TGF-β₁in keloid fibroblasts among which p38inhibitor (SB203580)exhibited the strongest inhibitory effects.4. The regulatory effects of CASE on MAPK pathway in keloid fibroblastsIn view of the action mechanisms of CASE exerting anti-keloid involved inTGF-β/Smad signal transduction pathway; moreover, TGF-β/Smad signal transductionwas regulated by MAPK pathway in keloid fibroblasts; we therefore further investigatethe regulatory effects of CASE on MAPK pathway in keloid fibroblasts. The results arepresented as follows:4.1Effects of CASE on the phosphorylation of ERK, JNK and p38in keloidfibroblastsThe basic phosphorylation of ERK, JNK and p38were high in keloid fibroblasts andwere enhanced under TGF-β₁stimulation. CASE (7.5,15,30mg L-1) significantly decreased the phosphorylation of ERK, JNK and p38, while the inhibitory effects werecomparable at three concentrations.4.2Effects of CASE on the nuclear expression of phosphorylated ERK, JNK andp38induced by TGF-β₁in keloid fibroblastsThe basic phosphorylated ERK, JNK and p38were mainly localized in the cytoplasm,while the fluorescence intensity and nuclear expression of pERK, pJNK and pp38wereenhanced under TGF-β₁stimulation. However, both the fluorescence intensity and thenuclear translocation of pERK, pJNK and pp38were supreesed by CASE treatment.Conclusions1. CASE significantly inhibited not only the proliferation, collagen synthesis and cellinvasion (previously demonstrated) but also cell migration in keloid fibroblasts,indicating its significant anti-keloid effects.2. Based on the results of2.12.5, CASE markedly inhibited TGF-β₁-inducedphosphorylation of Smad2/3, especially the phosphorylation at linker region, andsubsequently suppressed Smad2/3/4complex formation in keloid fibroblasts. Inaddition, CASE blocked the nuclear translocation of phosphorylated Smad2/3, butup-regulated Smad7expression, and finally decreased the levels of target genePAI-1mRNA and protein. These results suggest that CASE might exert anti-keloideffects via interfering with TGF-β/Smad signal transduction pathway.3. Based on the results of3.13.4, p38inhibitor markedly inhibited TGF-β₁-inducedSmad2phosphorylation at the linker region and up-regulated Smad7expression inkeloid fibroblasts, whereas ERK inhibitor and JNK inhibitor blocked the nucleartranslocation of phosphorylated Smad2. All of the three MAPK inhibitors decreased thelevel of PAI-1protein. These results combined with previous findings indicate that p38pathway mainly regulates the linker phosphorylation of Smad2/3and subsequentSmad2/3/4complex formation as well as Smad7expression; ERK and JNK pathways are mainly responsible for regulation of the nuclear translocation of phosphorylatedSmad2/3; the results further suggest that ERK, JNK and p38pathways all participate inregulating the transcription activity and protein expression of target gene PAI-1.4. Based on the results of4.14.2, CASE not only significantly inhibited thephosphorylation of ERK, JNK and p38, but also blocked the nuclear translocation ofpERK, pJNK and pp38in keloid fibroblasts, suggesting the regulatory effects of CASEon MAPK pathway. Since TGF-β/Smad signal transduction was regulated by MAPKpathway in keloid fibroblasts, thus CASE might also exert anti-keloid effects viaindirectly affecting TGF-β/Smad signal transduction pathway through regulation ofMAPK pathway.In summary, CASE markedly inhibited the proliferation, collagen synthesis as well as cellinvasion and migration in keloid fibroblasts, indicating its significant anti-keloid effects.The mechanisms of CASE might mainly involve in interfering with TGF-β/Smad signaltransduction pathway, and might also implicate in indirectly affecting TGF-β/Smadsignal transduction through regulation of MAPK pathway. Thereby, CASE exertedanti-keloid effects via different ways and acting on the multiple targets.