[雙語翻譯]--外文翻譯--病理周期應(yīng)變通過rhogdiαcaspase-3parp通路引發(fā)人體牙周膜細(xì)胞凋亡（英文）

1、Pathological Cyclic Strain-Induced Apoptosis in Human Periodontal Ligament Cells through the RhoGDIa/ Caspase-3/PARP PathwayLi Wang1., Jinsong Pan1,2., Tingle Wang3, Meng Song1*, Wantao Chen2*1 Department of Stomatology,

2、 First People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 2 Department of Oral and Maxillofacial Surgery,Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shang

3、hai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology,Shanghai, China, 3 Department of Stomatology, Central Hospital of Minhang District, Shanghai, ChinaAbstractAim: Human periodontal ligament

4、(PDL) cells incur changes in morphology and express proteins in response to cyclic strain. However, it is not clear whether cyclic strain, especially excessive cyclic strain, induces PDL cell apoptosis and if so, what me

5、chanism(s) are responsible. The aim of the present study was to elucidate the molecular mechanisms by which pathological levels of cyclic strain induce human PDL cell apoptosis.Materials and Methods: Human PDL cells were

6、 obtained from healthy premolar tissue. After three to five passages in culture, the cells were subjected to 20% cyclic strain at a frequency of 0.1 Hz for 6 or 24 h using an FX-5000T system. Morphological changes of the

7、 cells were assessed by inverted phase-contrast microscopy, and apoptosis was detected by fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide staining followed by flow cytometry. Protein expressio

8、n was evaluated by Western blot analysis.Results: The number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain. The stretched cells were oriented parallel to each

9、 another with their long axes perpendicular to the strain force vector. Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP) protein levels increased in response to pathological cyclic strain over time, while Rho GDP

10、dissociation inhibitor alpha (RhoGDIa) decreased. Furthermore, knock-down of RhoGDIa by targeted siRNA transfection increased stretch-induced apoptosis and upregulated cleaved caspase-3 and PARP protein levels. Inhibitio

11、n of caspase-3 prevented stretch-induced apoptosis, but did not change RhoGDIa protein levels.Conclusion: The overall results suggest that pathological-level cyclic strain not only influenced morphology but also induced

12、apoptosis in human PDL cells through the RhoGDIa/caspase-3/PARP pathway. Our findings provide novel insight into the mechanism of apoptosis induced by pathological cyclic strain in human PDL cells.Citation: Wang L, Pan J

13、, Wang T, Song M, Chen W (2013) Pathological Cyclic Strain-Induced Apoptosis in Human Periodontal Ligament Cells through the RhoGDIa/Caspase-3/PARP Pathway. PLoS ONE 8(10): e75973. doi:10.1371/journal.pone.0075973Editor:

14、 Philip C. Trackman, Boston University Goldman School of Dental Medicine, United States of AmericaReceived May 26, 2013; Accepted August 19, 2013; Published October 10, 2013Copyright: ? 2013 Wang et al. This is an open-a

15、ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Fu

16、nding: This research was supported by grants from the National Natural Science Foundation of China (No. 11172177 and 30973343) and projects of the Shanghai Science and Technology Committee (Grant No. 11DZ2291800). The fu

17、nders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: smgjzh160@

18、sohu.com (MS); chenwantao2002@hotmail.com (WC). These authors contributed equally to this work.IntroductionDuring occlusal load or orthodontic tooth movement, the cells in the periodontal ligament (PDL) are directly subj

19、ected to mechanical stress. The response to mechanical stress is an essential biological reaction [1,2,3,4]. Prediction of tooth mobility under functional loads is a classic issue in dental biomechanics and is especially

20、 important in the development of new solutions for dental restoration, prosthodontics, and orthodontic treatment. The understanding of tooth mobility requires mechanical char- acterisation of the PDL. The PDL is a comple

21、x soft tissue that connects teeth to the surrounding bone, and a common assumption is that it acts as the major element in tooth mobility and stress distribution to supporting tissues [5,6].Apoptosis induced by cyclic st

22、rain is an important determinant of connective tissue destruction in periodontal disease [7]. The application of light orthodontic force causes direct resorption of alveolar bone and tooth mobility, while the application

23、 of excessive orthodontic force results in excessive cyclic strain, which induces local ischaemia, tissue hyalinisation, and cell death in the PDL [8]. Cells undergo death by two major mechanisms: necrosis, in which prim

24、ary damage to the metabolic or membrane integrity of the cell occurs, and apoptosis, which is an internal suicide program contained in all cells [9]. Programmed cell death (apoptosis) [10,11] plays a key role in the regu

25、lation of tissue turnover in long-lived mammals that must integrate multiple physiological as well as pathological death signals.PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e75973RNA Interference an

26、d Inhibitor Treatment The mRNA sequence of human RhoGDIa was acquired from NCBI GenBank. Small interfering RNAs (siRNAs) against human RhoGDIa were designed and synthesised by GenePharma Biological Company (Shanghai, Chi

27、na). The siRNA sequences targeting RhoGDIa were (59R39) GAGAUAGUGUCCGGCAU- GAdTdT and UCAUGCCGGACACUAUCUCdTdT9. After incubation for 24 h, the cells were transfected with siRNA using LipofectamineTM 2000 (Invitrogen, Lif

28、e Technologies) at a final RNA concentration of 100 nM, according to the manufacturer’s instructions. After a 6-h incubation at 37uC in a humidified CO2 incubator, the transfection medium was replaced with DMEM for 18 h

29、prior to induction of cyclic strain. Non-silencing siRNA was used as a negative control (N.C.). For inhibitor studies, human PDL cells were pre-incubated with 10 mM z-DEVD-FMK, a specific caspase-3 inhibitor, for 1 h bef

30、ore cyclic strain was applied. PDL cells under the same conditions except without inhibitor were used as controls.Statistical Analysis Data are presented as means 6 standard deviation (SD) of three separate experiments.

31、One-way ANOVA with the Student– Newman–Keuls test was used to compare values and to assess statistical significance (p#0.05).ResultsStretching Force Altered the Morphology of Human PDL Cells The in vitro application of c

32、yclic stretching force altered the morphology of PDL cells. The cells became parallel to one another and were aligned with the long axes perpendicular to the stretching force vector (Fig. 1).Analysis of Apoptosis in Huma

33、n PDL Cells by Annexin V and PI Staining Apoptotic cells were identified by double labelling with annexin V and PI or by labelling with only annexin V. PI labels all dead cells, including necrotic cells and cells in late

34、 stages of apoptosis; cells entering early apoptosis are stained only by annexin V, and viable cells do not stain with annexin V or PI. Figure 2A shows representative dot plots of annexin V and PI staining. The lower lef

35、t quadrant indicates viable cells (V-FITC2/PI2); the lower right quadrant, cells in early apoptosis (V-FITC+/PI2); the upper right quadrant, cells in late apoptosis (V-FITC+/PI+); and theupper left quadrant, necrotic cel

36、ls (FITC2/PI+). In human PDL cells subjected to 20% cyclic strain for 6 h, the rate of apoptosis (cells in early and late stages of apoptosis) was increased slightly, by about 2%, compared with that in control cells. Aft

37、er 24 h of cyclic strain, the apoptosis rate was increased significantly, by about 14%, mainly due to cells entering early apoptosis.Cleavage of Caspase-3 and PARP Increased and RhoGDIa Decreased after Application of Cyc

38、lic Stretching Force As RhoGDIa may be involved in apoptosis of human PDL cells subjected to 20% cyclic strain, the effect of cyclic strain on RhoGDIa expression was examined. Compared with static control cells, PDL cell

39、s subjected to 20% strain at the frequency of 0.1 Hz for 6 or 24 h, respectively, showed lower levels of RhoGDIa protein expression on Western blots (Fig. 2B). In contrast, the expression of cleaved caspase-3 and PARP wa

40、s increased under cyclic strain compared with the expression in static control cells (Fig. 2B).Knock-down of RhoGDIa Sensitises Human PDL Cells to Cyclic Strain-induced Apoptosis and Increases Apoptosis in a Caspase-depe

41、ndent Manner Next, we examined the effect of RhoGDIa knock-down on apoptosis. Cells with RhoGDIa knock-down exhibited an apoptosis rate of approximately 43% before the application of cyclic strain, as measured by flow cy

42、tometry, whereas the apoptosis rate of the negative control cells was approximately 28% (Fig. 3A). In addition, compared with the negative control cells, the RhoGDIa knock-down cells exhibited increased cleavage of caspa

43、se-3 and PARP by Western blot analysis (Fig. 3B).Inhibition of Caspase-3 Confers Resistance to Apoptosis in Human PDL Cells, but does not Decrease Apoptosis Via Other Signalling Pathways Some human PDL cells were incubat

44、ed with z-DEVD-FMK for 1 h to inhibit caspase-3 before applying cyclic strain. The cells that underwent 20% cyclic strain after incubation with z-DEVD-FMK (20% +DEVD group) exhibited about 12% less apoptosis than cells t

45、hat underwent 20% cyclic strain in the absence of z-DEVD- FMK (20% 2DEVD group) and about 2% more apoptosis than the static control group (Fig. 4A). The protein levels of RhoGDIa and cleaved caspase-3 did not differ sign

46、ificantly between the 20% +DEVD and 20% 2DEVD groups, but the 20% +DEVD group exhibited decreased cleavage of PARP. Compared with the static control group, the 20% +DEVD group showed almost no difference in PARP cleavage

47、, but had higher levels of RhoGDIa and cleaved caspase-3 (Fig. 4B).DiscussionMany kinds of cells in the periodontium, including PDL cells (e.g., fibroblasts, cementoblasts, and osteoblasts), vascular cells, and hematopoi

48、etic cells, participate in periodontal remodelling. Human PDL cells play important roles in not only maintenance of the periodontium but also promotion of periodontal regeneration [24,25]. The ability of human PDL cells

49、to sense and respond to physical stresses such as occlusal force is required for periodontal tissue homeostasis and normal development. In the periodontium, applied forces of physiological magnitude regulate cellular pro

50、cesses that are critical for normal tissue maintenance [26]. In contrast, forces of pathological magnitude can induce apoptosis [21,27]. In this study, we used a Flexcell Tension Plus system,Figure 1. Pathological cyclic

51、 strain-induced morphological changes in human PDL cells. A. Cells cultured in the absence of cyclic strain are aligned multi-directionally. B. Cells subjected to 20% strain for 24 h are aligned primarily in one directio

52、n, with the long axis perpendicular to the cyclic strain force vector. The black arrow shows the direction of the stretching force (original magnification, 6 200). doi:10.1371/journal.pone.0075973.g001Rho-GDIa’s Role in