The Role And Mechanism Of Ac-SDKP In The Process Of Suppressing Epithelial-mesenchymal Transition (EMT) In Silicosis

Objects：Silicosis is an occupational disease. In China, the morbidity ofpneumonoconiosis has still gradually increased year by year due to the rapidgrowth of industry which in turn promoting the industrialization process. Inrecnt years the incidence of pneumonoconiosis has nearly increased by morethan20,000cases every year and by the end of2012, China has recorded807,269occupational diseases totally, among which ninety percent of thecases being pneumonoconiosis (including727,148cases). Furthermore half ofthe pneumonoconiosis cases are silicosis. Therefore in China silicosis is onekind of the most common and serious pneumonoconiosis. At present，it issuggested that bronchial-alveolar epithelial cells to myofibroblasts transitionplays an important role in the formation and development in silicosis. Whilethe silicosis dust is capable of promoting apoptosis of bronchial-alveolarepithelial cells as a potential fibrogenic factor and also mediating the transitionof epithelial-mesenchymal transition (EMT) among which HSP27, one of thetypical proteins in the thirty-three differential proteins be screened out insilicotic rats by proteomics technology, involves in EMT through modulatingthe nuclear transcription factor SNAI1.N-acetyl-seryl-aspartyl-lysylproline (Ac-SDKP) is an anti-fibrotic shortpeptide. Previously, we and other researchers have found that Ac-SDKPinhibits organ fibrosis, such as heart, renal and lung fibrosis. Also our teamhas reported that Ac-SDKP，can regulate induction of TGF-β and its receptorand myofibroblast differentiation and thus suppressing the formation anddevelopment of silicosis. Here we are aimed to explore that if Ac-SDKP cansuppress EMT through modulating the expression of HSP27and further showits anti-fibrotic effects in silicosis. This experiment utilizes animal model invivo and alveolus epithelial cell culture and cell transfection experiment in vitro, comprehensively to explore the regulatory effect of Ac-SDKP on thetarget EMT and key point hsp27, reveal the molecular mechanism of itsanti-fibrotic effects in depth, provide some major theoretical and experimentalfundamentals for developing Ac-SDKP as a new anti-fibrotic drug, meanwhileprovide some valuable information for the prevention and treatment offibrosis of other organs (such as liver、kidney and cardiovascular system.)Methods：SiO2powders were douched as a one-off in the trachea of rat tomake the silicotic model. For in vivo studies, eight groups of animals wereutilized:1) control4w;2) silicotic model4w;3) Single administration ofAc-SDKP4w;4)control8w;5) silicotic model8w;6) Single administration ofAc-SDKP8w;7)Ac-SDKP post-treatment;8) Ac-SDKP pre-treatment. Thehuman type II alveolar epithelial cell line A549was cultured in vitro anddivided into4groups:1) control;2) TGF-1;3) TGF-β1+Ac-SDKP;4)Ac-SDKP. The expression of HSP27、pHSP27、SNAI1、SNAI2、epithelial cellsmarker CK、SP-A、mesenchymal marker α-SMA、vimentin、collagen andcollagen Ⅲ were assessed byImmunohistochemistry、Western blot andRealtime PCR. The colocalization of SP-A and α-SMA、pHSP27and α-SMAwere detected by confocal laser scanning microscopy in rat silicoticmodel.Meanwhile the colocalization of HSP27and SNAI1、pHSP27andSNAI2were also detected by confocal laser scanning microscopy on humantype II alveolar epithelial cell line A549.A549cell line was transiently transfected with the indicated expressionplasmids in vitro and divided into four groups：1）empty vector control;2）empty vector+TGF-β1；3）shRNA plasmid+TGF-β1;4）empty vector+TGF-β1+Ac-SDKP；The expression of HSP27、pHSP27、SNAI1、SNAI2、epithelial cells marker E-cad、mesenchymal marker α-SMA、collagen andcollagen Ⅲwere assessed by Western blot analysis.Results：1Regulatory role of Ac-SDKP in expression of epithelial tissue markersE-cad、SP-A and mesenchymal tissue markers α-SMA、vimentin on EMT insilicotic model of rat Immunohistochemical detection of α-SMA and vimentin showed thatpositive staining for α-SMA and vimentin were only seen in vascular vesselsand trachea smooth muscle cells. While in silicosis model positive expressionsof α-SMA and vimentin were observed in silicotic nodules and interstitialfibrotic area. Of significant interest, Ac-SDKP post-and pre-treatmentmarkedly reduced the appearance area of vimentin and α-SMA in the silicoticnodules and interstitial fibrotic regions. There showed no differences betweencontrol group and single administration of Ac-SDKP group. The results ofconfocal laser scanning microscopy assay indicated that there exsited SP-Apositive cells with red fluorescent marker,which being flat or cuboidal with abigger body sticking in the alveolar wall cavity surface. There also exsitedfewer α-SMA positive cells with green fluorescent marker on alveolar wall.Compared to the control group, there showed much more α-SMA positivecells with green fluorescent marker in silicotic nodules in which existed a fewof SP-A positive cells with red fluorescent marker. Combined with the twofluorescent markers, there showed a few of pink cells mostly on the edge ofthe silicotic nodules but less distrubuted in it. This indicated that type IIalveolar epithelial cell existed and proliferated in the silicotic nodules in whichalso exsited α-SMA positive cells.While α-SMA positive cells were muchfewer in Ac-SDKP treatment group（post-treatment group and pre-treatmentgroup）comparing with silicotic model8w group and SP-A positive cells hadthe opposite trend.Western blot analysis further confirmed immunohistochemical results byshowing that silica increases the expressions of α-SMA and vimentin by1.6folds and3.7folds at silicotic4w,1.6folds and2.9folds at silicotic8wcompared with control group respectively, and that Ac-SDKP post-treatmentand pre-treatment significantly ameliorated the expression of α-SMA in ratlung with silicosis to69%and67%， and vimentin to59%and55%respectively. Opposite to the expression of mesenchymal tissue markers, theexpression of epithelial tissue markers E-cad decreased to49%�'�35%andSP-A to50%and21%in silicotic model as compared with control group(4w and8w). After intervention of Ac-SDKP, epithelial tissue markers E-cad in thepost-and pre-treatment group increased to2.3folds and2.6folds, and SP-Aincreased to4.5folds and4.4folds compared with silicotic8w grouprespectively.2Regulatory role of Ac-SDKP in modulating HSP27on EMT in rat withsilicosisImmunohistochemical results showed that there much more positivestaining of HSP27、pHSP27、SNAI1in silicotic nodules and interstitial fibroticregions in silicosis model of rats compared with the control group whichexisted positive expressions only in vascular vessels and trachea smoothmuscle cells. The results of confocal laser scanning microscopy assayindicated that pHSP27（active form of HSP27）with red fluorescent markersshowed less positive in tracheal smooth muscle vessels and alveolar wall butmuch more positive cells with red fluorescent markers in the cytoplasmlocated in silicotic nodules in silicotic model groups. Combined with anothergreen fluorescent marker indicating α-SMA, some cells expressed pink insilicotic nodules.Western blot analysis showed that the expression of HSP27in silicoticmodel was increased to1.5folds and2.1folds, and pHSP27were increased to2.1folds and2.1folds compared to the control group (4w and8w). Whilethere were no differences in statistic analysis between control group and singleadministration of Ac-SDKP group. The most important is that both Ac-SDKPpost-and pre-treatment groups markedly decreased the expression of HSP27to75%and65%，and pHSP27decreased to72%and68%as compared withsilicotic8w group respectively. Similar to the expression of HSP27andpHSP27，the expression of SNAI1in silicosis model also increased to3.1folds and2.7folds，and SNAI2increased to1.8folds and1.5folds ascompared with control group(4w and8w). Also after Ac-SDKP intervention,both Ac-SDKP post-and pre-treatment groups markedly decreased theexpression of SNAI1to62%and54%，and SNAI2decreased to79%and75%as compared with silicotic8w group respectively. The expression of collagen increased to1.7folds and1.6folds，and collagen Ⅲincreased to1.6folds and1.8folds compared with control goup（4w and8w）. In both Ac-SDKP post-and pre-treatment groups the expression of collagen decreased to78%and73%，and collagen Ⅲ decreased to74%and73%comparing with silicoticmodel8w. All the proteins showed no statistic differences between controlgroup and single administration of Ac-SDKP group（4w and8w）.3Regulatory role of Ac-SDKP in expression of epithelial tissue markersE-cad、CK and mesenchymal tissue markers α-SMA、vimentin in culturedhuman A549cell line induced by TGF-β1Immunohistochemical results showed that positive staining of CK locatedmainly in cytoplasm, while the expression of α-SMA and Vimentin showednegative expression with less yellow staining in cytoplasm of human A549cell line in non-induced control group. Following the induction of TGF-β1for72h, the round or polygonal cells become sprindle-shaped with bigger nucleus.The positive staining of α-SMA and vimentin showed filamentary mainlylocated in cytoplasm, while the staining of CK is less obvious in TGF-β1induced group. While after pre-treatment with Ac-SDKP, the intensity of thepositive staining of α-SMA and vimentin showed attenuated and themorphology changes are less sprindle-shaped. On the contrary, the positivestaining of CK showed more intensity. While both morphology changes andthe staining intensity showed no differences between control group and singleAc-SDKP administered group.Western blot analysis indicated that TGF-β1treatment of cultured humanA549cell line resulted in down-regulation of the epithelial tissue markersE-cad by the same50%and CK by the same40%respectively at24h、48h、72h，and up-regulation of the mesenchymal tissue markers α-SMA by1.9folds、2.5folds、1.8folds and vimentin by1.6folds、2.3folds、1.9foldsrespectively compared to non-induced cells at24h、48h、72h. Meanwhile atmRNA level, the expression of E-cad decreased to26%at72h. AfterAc-SDKP intervention, the expression of E-cad increased to1.8folds、1.8folds、2.0folds and CK increased to2.4folds、2.3folds、2.6folds respectively, while the mesenchymal tissue markers α-SMA decreased to63%、72%、66%and vimentin decreased to69%、54%、68%as compared with TGF-β1inducedgroup at24h、48h、72h. Meanwhile the expression of mRNA level of E-cadincreased to3.4folds. All proteins in statistic analysis have no differencesbetween control group and single Ac-SDKP administered group.（P>0.05）。4Regulatory role of Ac-SDKP in modulating HSP27in transition fromhuman type II alveolar epithelial cell line A549to myofibroblast induced byTGF-β1Immunohistochemical results indicated that there were no positivestaining of HSP27、pHSP27、SNAI1in human type II alveolar epithelial cellline which showed less yellow staining both in cytoplasm and nucleus.Following the induction of TGF-β1for72h，along with the morphologicalchanges，positive staining of HSP27、 pHSP27located in cytoplasm andSNAI1in nucleus respectively. After intervention of Ac-SDKP，the stainingintensity of HSP27、pHSP27、SNAI1showed obvious attenuated. While bothin morphological and staining intensity there showed no differences betweencontrol group and single Ac-SDKP administered group. Confocal laserscanning microscopy assay indicated that the human type II alveolar epithelialcell changed into sprindle-shaped with obvious green fluorescent intensityexpression of HSP27in the cytoplasm following the induction of TGF-β1,while SNAI1with red fluorescent markers in the nuclear also showed muchmore fluorescent intensity. The expression of SNAI2with red fluorescentmarkers were located both in the cytoplasm and nuclear. After Ac-SDKPintervention，the morphological changes of the cells were not obvious and thefluorescent intensity of HSP27with green fluorescent markers attenuatedalong with SNAI1and SNAI2with red fluorescent markers. There existedmuch more F-actin with obvious green fiber in the cytoplasm along with themorphololgical changes following the induction of TGF-β1. After Ac-SDKPintervention，there were less F-actin with green fiber and the sprindle-shapedchanges were not obvious.Western blot results indicated that comparing to control group, the expression of HSP27significantly increased to1.5folds、1.9folds、1.9foldsand pHSP27increased to1.7folds、2.5folds、3.0folds on human type IIalveolar epithelial cell line A549induced by TGF-β1at24h、48h、72h.Meanwhile at mRNA level, the expression of HSP27also increased to1.9folds as compared with control group at72h. Similar to the trend of HSP27and pHSP27, the transcription nuclear factor SNAI1also showedup-regulating by2.3folds、2.2folds、1.3folds and SNAI2by1.5folds、1.7folds、1.9folds comparing with control group at24h、48h、72h. At mRNAlevel, the trend of SNAI1and SNAI2was similar with protein expression thatincreased by3.1folds and2.3folds as compared with control group.Simultaneously the expression of collagen increased to1.5folds、2.1folds、2.2folds and collagen Ⅲ to1.5folds、1.9folds、2.5folds respectivelycomparing to control group following the induction of TGF-β1at24h、48h、72h. Pre-treatment of Ac-SDKP results in down-regulating of HSP27by71%、62%、74%， pHSP27by56%、44%、44%，SNAI1by50%、71%、75%，SNAI2by65%、64%、66%，collagen by62%、43%、66%and collagenⅢ by66%、52%、44%，comparing to TGF-β1induced group at24h、48h、72h respectively. Meanwhile at mRNA level, the expression of HSP27、SNAI1and SNAI2also decreased to64%、38%、77%respectively as compared withcontrol group at72h. While the above both in protein and mRNA levelshowed no statistic differences significantly at the three time points betweencontrol group and single Ac-SDKP administered group （P>0.05）.5The role and mechanism of Ac-SDKP in the process of suppressingepithelial-mesenchymal transition on transfected human type II alveolarepithelial cell line with plasmid HSP27shRNAWe utilized the mixture of lepo2000and plasmid shRNA to transfecthuman type II alveolar epithelial cell line prior to the induction of TGF-β1for48h. Western blot analysis indicated that the expression of E-cad increasedwhile SNAI1、 α-SMA、 collagen and Ⅲ except SNAI2decreasedsignificantly in statistic analysis in plasmid shRNA group as compared withthe empty vector group following the induction of TGF-β1. After Ac-SDKP intervention, the expression of SNAI1、SNAI2、α-SMA、collagen and Ⅲdecreased sharply while the expression of E-cad increased as compared toTGF-β1induction group（P <0.05）which were similar with plasmid shRNAgroup.Conclusions：1There exited bronchial-alveolar epithelial cells to myofibroblaststransition in the formation of silicotic fibrosis.2The results of the present experiment suggest that Ac-SDKP is capableof suppressing epithelial-mesenchymal transition through regulating theexpression of HSP27which further inhibit collagen synthesis in preventingand treatinging silicosis in vivo and in vitro.3The cell transfection experiment results further verify that Ac-SDKPcan inhibit the expression of HSP27which in turn suppressing theaccumulation of nuclear transcription factor SNAI1and α-SMA， thusincreasing the expression of E-cad and ultimately inhibiting collagen sythesisand deposition in suppressing epithelial-mesenchymal transition in vitro.