Tanod, Wendy Alexander (2019) Potensi Bioaktif Karang Lunak Sarcophyton Sp. Sebagai Anti-Inflamasi Akibat Induksi Lipopolysaccharide – Inhibitor Jalur Myd88 Dependent. Doctor thesis, Universitas Brawijaya.
Abstract
Karang lunak termasuk dalam filum cnidaria, kelas anthozoa, subclass octocorallia dilaporkan menghasilkan senyawa yang berpotensi sebagai senyawa obat. Dilaporkan 11-17 % semua obat yang ditemukan dari laut berasal dari karang lunak. Karang lunak diketahui umumnya memproduksi senyawa turunan terpenoid dan steroid, yang mempunyai efek anti-inflamasi. Proses inflamasi merupakan suatu mekanisme protektif dalam merespons infeksi mikroba atau iritasi atau cedera pada jaringan/organ. Respon inflamasi merupakan respon imunologis kompleks dan diatur ketat terhadap invasi patogen dan cedera jaringan yang sebagian besar dikendalikan oleh sistem imun bawaan (innate) dan adaptif. Sel imun bawaan (innate) seperti makrofag merupakan mediator sentral dari respon inflamasi melalui pengenalan mikroba patogen oleh PRR yang dikenal TLR. Pada penelitian ini, menggunakan Lipopolysaccharide (LPS) dari bakteri Escherichia coli sebagai stimulan respon inflamasi. Reseptor pengenalan LPS, yaitu TLR4. LPS diangkut dalam darah oleh LPS Binding Protein (LBP), ke protein terkait CD14. Interaksi antara CD14 dan LBP, memberikan respons TLR4 terhadap LPS. Interaksi ini menghasilkan stabilisasi, dimerisasi, aktivasi kompleks reseptor dan selanjutnya downstream TLR4 singaling. Signaling TLR4 melalui jalur MyD88-dependent bertanggungjawab terhadap ekspresi sitokin pro-inflamasi. Jalur ini menstimuli aktivasi faktor transkripsi NFκB. Aktivasi NFκB menstimuli produksi sitokin pro-inflamasi (TNF-α, dan IL-6), cell adhesion molecules (ICAM-1 dan VCAM-1) dan iNOS. Untuk mengimbangi produksi sitokin pro-inflamasi, sel T regulator memproduksi sitokin anti-inflamasi IL-10 untuk meredam produksi sitokin pro-inflamasi. Selain memproduksi substansi anti-inflamasi, karang lunak juga dilaporkan memproduksi substansi antioksidan. Respon inflamasi dapat dihambat dengan mencegah stres oksidatif dalam sel. Selain itu, LPS dapat meningkatkan produksi ROS. Produksi ROS memicu pelepasan ikatan Nrf-2/Keap-1, sehingga Nrf-2 dapat teraktivasi dan meregulasi produksi antioksidan endogen (SOD dan HO-1). Tujuan penelitian ini, yaitu 1). Mendapatkan fraksi karang lunak yang menunjukkan aktivitas inhibitori pelepasan NO; 2). Mendapatkan data probabilitas senyawa dan karakter spektrometri fraksi karang lunak yang berpotensi sebagai anti-inflamasi; 3). Mendapatkan fraksi karang lunak yang dapat meningkatkan jumlah relatif sel T naïve, sel T regulator, dan produksi sitokin anti-inflamasi IL-10 setelah induksi LPS secara in vitro dan in vivo; 4). Mendapatkan fraksi karang lunak yang dapat menghambat aktivasi TLR4, ekspresi NFκB, iNOS dan produksi TNF-α dan IL-6 serta menekan ekspresi VCAM-1 dan ICAM-1 setelah diinduksi LPS secara in vitro dan in vivo; dan 5). Mendapatkan fraksi karang lunak yang dapat meningkatkan aktivasi SOD dan HO-1 yang diregulasi oleh Nrf-2, serta dapat menurunkan level MDA pada mencit yang diinduksi LPS. Manfaat penelitian ini, yaitu dapat mengungkap potensi substansi bioaktif karang lunak sebagai anti-inflamasi, dalam menekan respon inflamasi. Hasil penelitian ini juga diharapkan dapat menjadi alternatif bagi dunia farmasi sebagai bahan sediaan agen anti-inflamasi. Metode yang digunakan dalam penelitian ini eksperimen dan metode deskriptif. Penelitian ini dibagi dalam 3 tahap, yaitu 1). Sampling, ekstraksi karang lunak, dan purifikasi; 2). Karakterisasi fraksi karang lunak potensial dan pengujian in silico; 3). Pengujian in vivo dan in vitro. Ekstrak karang lunak potensial (ekstrak kasar dan fraksi) dilakukan pengamatan aktivitas inhibitori pelepasan Nitric Oxide dan penghambatan radikal DPPH. Fraksi karang lunak potensial dianalisis LC-MS serta dikarakterisasi dengan spektrofotometer UV-Vis dan FTIR. Selanjutnya, dipurifikasi dengan kromatografi kolom. Fraksi karang lunak potensial diamati aktivitasnya terhadap parameter inflamasi jalur MyD88 dependent, yaitu TLR 4, NFκB, TNF-α, IL-6, iNOS. Selain itu diamati sel T naïve, xii sel T regulator, IL10, VCAM-1 dan ICAM-1 akibat induksi LPS. Diamati juga potensi substansi antioksidan dari fraksi karang lunak, yaitu SOD, HO-1 dan Nrf-2 dalam mengimbangi stres oksidatif MDA. Pengujian in vivo terdiri dari 6 perlakuan yaitu kontrol normal (tanpa induksi LPS), kontrol negatif (Induksi LPS 4 mg/mL), kontrol positif (LPS + Dexametasone 6 mg/kg), konsentrasi ekstrak 1 (LPS + 50 mg/kg BB), konsentrasi ekstrak 2 (LPS + 125 mg/kg BB), dan konsentrasi ekstrak 3 (LPS + 250 mg/kg BB), dimana masing-masing perlakuan terdiri dari 6 ulangan. Pengujian in vitro memiliki 6 perlakuan yaitu kontrol negatif (tanpa induksi LPS), kontrol negatif (Induksi LPS 0.04 mg/mL), kontrol positif (LPS + Dexametasone 5 mg/mL), konsentrasi ekstrak 1 (LPS + 0.50 mg/mL), konsentrasi ekstrak 2 (LPS + 1.25 mg/mL), dan konsentrasi ekstrak 3 (LPS + 2.50 mg/mL), dimana masing-masing perlakuan terdiri dari 3 ulangan. Pengujian in silico dianalisis dengan Lipinski, SwissADME, PASSonline, pyrex, dan Discovery Studi Visualizer. Sampel karang lunak yang digunakan pada penelitian ini teridentifikasi sebagai Sarcophyton sp. (SCC), dikoleksi dari Teluk Palu, Sulawesi Tengah. Analisis kandungan komponen bioaktif dari ekstrak kasar Sarcophyton sp. (SCC) mengindikasikan kehadiran alkaloid, polifenol dan steroid. Analisis probabilitas senyawa dengan LCMS mendeteksi kehadiran senyawa yang berpotensi kuat sebagai anti-inflamasi, yaitu Coralloidin E; Lobophytol; Sinularolide B; (5Z,9E,11E, 14E) Ethyl 8 hydroxy 13 oxoicosa-5,9,11,14 tetraenoate; Sinulaflexiolide B; Sinulaflexiolide C; Sinulaflexiolide D; Australin A; Australin B; Australin C; dan Australin D. Fraksi DCM Sarcophyton sp. (SCC) menunjukkan aktivitas inhibitori pelepasan NO yang paling baik sebesar 4.85±0.17 μM pada 5 mg/mL sedangkan kontrol LPS 6.00±0.92 μM dan menunjukkan kemampuan dalam menghambat radikal DPPH (IC50=79.36±0.50 μg/mL). Data hasil pengujian menunjukkan bahwa konsentrasi fraksi DCM Sarcophyton sp. (SCC) 125 mg/kg (in vivo) dan 1.25 mg/mL (in vitro) mampu meningkatkan jumlah relatif sel T CD4+ sebesar 69.97±1,44% (in vivo) sedangkan pada kontrol LPS 50.96±1.17% (in vivo); jumlah relatif sel T regulator sebesar 81.83±1.05% (in vivo) dan 44.13±0.81% (in vitro) sedangkan kontrol LPS sebesar 72.97±1.13% (in vivo) dan 25.64±0.93% (in vitro); dan jumlah relatif IL-10 sebesar 8.27±1.08% (in vivo) dan 58.24±0.76% (in vitro) sedangkan jumlah relatif kontrol LPS 7.58±1.01% (in vivo) dan 11.68±0.83% (in vitro). Konsentrasi fraksi DCM Sarcophyton sp. (SCC) 125 mg/kg (in vivo) dan 1.25 mg/mL (in vitro) dapat menurunkan jumlah relatif TLR4 sebesar 15.97±1.37% (in vivo) dan 42.06±0.81% (in vitro) sedangkan pada kontrol LPS sebesar 22.37±1.48% (in vivo) dan 49.19±0.71% (in vitro); jumlah relatif NFκB sebesar 13.96±0.84% (in vivo) dan 21.99±0.39% (in vitro) sedangkan kontrol LPS 18.38±1.24% (in vivo) dan 22.45±0.19% (in vitro); jumlah relatif TNF-α sebesar 13.95±1.76% (in vivo) dan 31.99±0.70% (in vitro) sedangkan pada kontrol LPS sebesar 17.85±1.32% (in vivo) dan 34.19±0.44% (in vitro); jumlah relatif IL-6 sebesar 21.27±1.10% (in vivo) dan 38.40±0.33% (in vitro) sedangkan jumlah relatif kontrol LPS 25.21±1.29% (in vivo) dan 38.90±0.45% (in vitro); serta menekan ekspresi iNOS pada kategori kuat sebesar 11.11% sedangkan pada kontrol LPS 61.11%. Konsentrasi fraksi DCM Sarcophyton sp. (SCC) 125 mg/kg juga menurunkan level MDA 144.72±18.31 ng/mL sedangkan level MDA pada kontrol LPS 188.06±15.15 ng/mL. Konsentrasi ekstrak DCM Sarcophyton sp. (SCC) 125 mg/kg mampu meningkatkan jumlah relatif SOD (28.58±0.79%); yang menurun akibat induksi LPS (17.04±1.07%); jumlah relatif HO-1 (38.96±1.14%) yang menurun akibat induksi LPS (28.69±1.05%); dan jumlah relatif Nrf-2 (37.34±1.14%) yang menurun karena induksi LPS (27.80±1.15%). Konsentrasi fraksi DCM Sarcophyton sp. (SCC) 125 mg/kg juga mampu menekan ekspresi VCAM-1 pada kategori sedang sebesar 66.65% sedangkan ekspresi VCAM-1 pada kontrol LPS sebesar 83.33%; serta mampu menekan ekspresi ICAM-1 pada kategori sedang sebesar 16.67% sedangkan pada kontrol LPS ekspresi ICAM-1 sebesar 50%. Mekanisme kerja anti-inflamasi fraksi DCM Sarcophyton sp. (SCC), yaitu sebagai inhibitor jalur MyD88 dependent melalui rangkaian MyD88 dependent-IRAK-TRAF-TAK, sehingga menekan fosforilasi dan degradasi IB. Penghambatan fosforilasi dan degradasi IκB mengakibatkan gangguan translokasi dari faktor transkripsi NFκB. Penghambatan aktivasi NFκB ini menyebabkan gangguan produksi sitokin pro-inflamasi TNF-α dan IL-6 serta penekanan ekspresi iNOS. Selain itu, fraksi DCM Sarcophyton sp. (SCC) meningkatkan aktivitas sel T regulator sebagai regulator negatif inflamasi dan mensekresikan sitokin anti-inflamasi IL-10. Produksi IL-10 mengganggu jalur persinyalan xiii TLR4. Fraksi DCM Sarcophyton sp. (SCC) juga mampu menghambat pengenalan LPS dan TLR4 dengan berperan sebagai kompetitif binding dalam pengikatan dengan MD2, sehingga mengganggu interaksi LPS-MD2-TLR4. Substansi antioksidan dalam fraksi DCM Sarcophyton sp. (SCC) mampu menurunkan oxidative stress response element MDA dan meningkatkan jumlah relatif antioxidant response element SOD dan HO-1 yang diregulasi Nrf-2. Hal ini dapat memberikan efek anti-inflamasi dengan memodulasi sinyal inflamasi dan melemahkan respons inflamasi yang diinduksi LPS melalui pengurangan NFκB. Fraksi DCM Sarcophyton sp. (SCC) dapat menghambat ekspresi VCAM-1 dan ICAM-1, diduga sebagai akibat dari deaktivasi faktor transkripsi NFkB. Penghambatan aktivasi NFκB dapat mempengaruhi angiogenesis dan respon inflamasi akibat induksi LPS dan hiperpermeabilitas vaskular. Deaktivasi NFκB, menyebabkan pengurangan ekstravasasi leukosit pada sel endotel mencit yang terinduksi LPS. Penghambatan pelepasan NO meregulasi molekul adhesi sel termasuk ICAM-1 dan VCAM-1. Efek penghambatan ekspresi VCAM-1 dan ICAM-1, sehingga menyebabkan penurunan stres fibrosa, tegangan sentripetal dan retraksi sel. Penurunan ini mengganggu regulasi interaksi leucocyte−endothelial cell adhesion, sehingga mencegah migrasi leukosit keluar dari pembuluh darah antara sel-sel endotel dan ke dalam jaringan secara berlebihan.
English Abstract
Soft corals include in cnidarians phylum, class Anthozoa, subclass Octocorallia are reported to produce compounds that have the potential as drug compounds. Reportedly 11-17% of all drugs found on the sea derived from soft corals. Soft corals are known generally produce terpenoids and steroid derivatives, which have anti-inflammatory effects. The inflammatory process is a protective mechanism in response to microbial infections or irritation or injury to tissues/organs. The inflammatory response is a complex immunological response and strictly regulated against pathogenic invasion and tissue injury that is controlled by innate and adaptive immune systems. Innate immune cells such as macrophage is a central mediator of the inflammatory response through the recognition of microbial pathogens by PRR, which is known TLR. In this research, used Lipopolysaccharide (LPS) from Escherichia coli as a stimulant for the inflammatory response. LPS recognition receptor, namely TLR4. LPS is transported in the blood by LPS Binding Protein (LBP), to CD14-related proteins. The interaction between CD14 and LBP, responding TLR4 to LPS. These interactions result in stabilization, dimerization, activation of the receptor complex, and subsequent downstream TLR4 signaling. TLR4 signaling via the MyD88-dependent pathway is responsible for the expression of pro-inflammatory cytokines. This pathway stimulates the activation of the transcription factor NFκB. Activation of NFκB stimulates the production of pro-inflammatory cytokines (TNF-α, and IL-6), cell adhesion molecules (ICAM-1 and VCAM-1) and iNOS. Regulatory T cells produce anti-inflammatory cytokines IL-10 to reduce the production of pro-inflammatory cytokines to compensate for the production of pro-inflammatory cytokines. Besides producing anti-inflammatory substances, soft corals are also reported to produce antioxidant substances. The inflammatory response can inhibit by preventing oxidative stress in cells. Furthermore, LPS can increase ROS production. ROS triggers the release of Nrf-2/Keap1 bonds so that Nrf-2 can be activated and regulate the production of endogenous antioxidants (SOD and HO-1). The aimed of this study, namely 1). to obtained soft coral species that show NO release-inhibitory activity; 2). to obtained compound probability data and spectrometry characteristics of soft coral fraction that have the potential to be anti-inflammatory; 3). to obtained soft coral fraction which can increase the relative number of naïve T cells, regulatory T cells, and the production of anti-inflammatory cytokines IL-10 after LPS induction in vitro and in vivo; 4). to obtained soft coral fraction that can inhibit the activation of TLR4, NFκB expression and production of TNF-α, IL-6, and suppresses VCAM-1 and ICAM-1 expressions after LPS induction in vitro and in vivo; 5). to got the soft coral fraction that can increase the activation of antioxidant response element SOD and HO-1 which is regulated by Nrf-2 and can reduce MDA levels in LPS-induced mice. The benefits of this research are that it can reveal the potential of soft coral bioactive substances as an anti-inflammatory, in suppressing the inflammatory response. The results of this study are also expected to be an alternative to the pharmaceutical world as an anti-inflammatory agent. The methods used in this study experiments and descriptive. This study divided into two stages: 1). Sampling, soft coral extraction, and purification; 2). Characterization fraction of potential soft corals and in silico assays. 3). in vivo and in vitro assays. The soft coral extracts (crudes and fractions) observed the inhibitory of Nitric Oxide release activity and inhibition of DPPH radical. The potential soft coral fraction analyzed by LC-MS and characterized by UV-Vis and FTIR spectrophotometers. Furthermore, it purified by column chromatography. The Potential soft coral fraction observed for its activity against the inflammatory parameters MyD88 dependent pathway, namely TLR 4, NFκB, TNF-α, IL-6, iNOS. Additionally observed naïve T cells, regulatory T cells, IL-10, VCAM-1, and ICAM-1 xv expression induced by LPS. The study also observed at the potential antioxidant substances from soft coral fraction, i.e., SOD, HO-1, and Nrf-2 in balancing oxidative stress MDA. In vivo assaying consisted of 6 treatments are normal control (without LPS induction), negative control (LPS induction 4 mg/mL), positive control (Dexamethasone LPS + 6 mg/kg), extract concentration 1 (LPS + 50 mg/kg BW), extract concentration 2 (LPS + 125 mg/kg BB), and extract concentration 3 (LPS + 250 mg/kg BB), where each treatment consisted of 6 replications. In vitro assaying consisted of 6 treatments are normal control (without LPS induction), negative control (LPS induction 0.04 mg/mL), positive control (Dexamethasone LPS + 5 mg/mL), extract concentration 1 (LPS + 0.50 mg/mL), extract concentration 2 (LPS+1.25 mg/mL), and extract concentration 3 (LPS+2.50 mg/mL), where each treatment consisted of 6 replications. The assayed of in silico with Lipinski, SwissADME, PASSonline, pyrex, and Discovery Studio Visualizer. The Soft coral samples used in this study identified as Sarcophyton sp. (SCC) collected from Palu Bay, Central Sulawesi. Analysis of the bioactive component of Sarcophyton sp. (SCC) crude extracts indicates the presence of alkaloids, polyphenols and steroids. The probability analysis of compounds with LC-MS detected the presence of potentially anti-inflammatory compounds, namely Coralloidin E; Lobophytol; Sinularolide B; (5Z,9E,11E, 14E) Ethyl 8 hydroxy 13 oxoicosa-5,9,11,14 tetraenoate; Sinulaflexiolide B; Sinulaflexiolide C; Sinulaflexiolide D; Australin A; Australin B; Australin C; dan Australin D. The DCM fraction of Sarcophyton sp. (SCC) showed the best NO inhibitory release activity of 4.85 ± 0.17 μM at 5 mg/mL while LPS control was 6.00 ± 0.92 μM, and showed the ability to inhibition of DPPH radical (IC50 = 79.36 ± 0.50 μg/mL). The data showed that the concentration 125 mg/kg (in vivo) and 1.25 mg/mL (in vitro) of Sarcophyton sp. (SCC) DCM fraction were able to increase the relative number of CD4+ T cells by 69.97 ± 1.44% (in vivo) while in the LPS control 50.96 ± 1.17% (in vivo); relative number of regulatory T cells was 81.83 ± 1.05% (in vivo) and 44.13 ± 0.81% (in vitro) while the LPS control was 72.97 ± 1.13% (in vivo) and 25.64 ± 0.93% (in vitro); and relative number of IL-10 was 8.27 ± 1.08% (in vivo) and 58.24 ± 0.76% (in vitro) while the relative number of LPS controls was 7.58 ± 1.01% (in vivo) and 11.68 ± 0.83% (in vitro). The concentration 125 mg/kg (in vivo) and 1.25 mg/mL (in vitro) of DCM fraction Sarcophyton sp. (SCC) can reduce the relative number of TLR4 by 15.97 ± 1.37% (in vivo) and 42.06 ± 0.81% (in vitro) while at LPS control it is 22.37 ± 1.48% (in vivo) and 49.19 ± 0.71% (in vitro); relative number of NFκB was 13.96 ± 0.84% (in vivo) and 21.99 ± 0.39% (in vitro) while the LPS control is 18.38 ± 1.24% (in vivo) and 22.45 ± 0.19% (in vitro); relative number of TNF-α was 13.95 ± 1.76% (in vivo) and 31.99 ± 0.70% (in vitro) while the LPS control was 17.85 ± 1.32% (in vivo) and 34.19 ± 0.44% (in vitro); relative number of IL-6 was 21.27 ± 1.10% (in vivo) and 38.40 ± 0.33% (in vitro) while the relative number of LPS controls was 25.21 ± 1.29% (in vivo) and 38.90 ± 0.45% (in vitro); and suppressing iNOS expression in the strong category of 11.11% while in the LPS control it was 61.11%. The concentration 125 mg/kg of DCM fraction Sarcophyton sp. (SCC) also decreases MDA level 144.72 ± 18.31 ng/mL while the MDA level in LPS control was 188.06 ± 15.15 ng/mL. The concentration 125 mg/kg of DCM fraction Sarcophyton sp. (SCC) can increase the relative number of SOD (28.58 ± 0.79%); which decreased to LPS-induced (17.04 ± 1.07%); the relative number of HO-1 (38.96 ± 1.14%) decreased to LPS-induced (28.69 ± 1.05%); and the relative number of Nfr-2 (37.34 ± 1.14%) decreased to LPS-induced (27.80 ± 1.15%). The concentration 125 mg/kg of DCM fraction Sarcophyton sp. (SCC) was also able to suppress VCAM-1 expression in the moderate category by 66.65% while VCAM-1 expression in LPS control was 83.33%; and able to suppress ICAM-1 expression in the moderate category by 16.67% while in the LPS control ICAM-1 expression was 50%. It can be concluding from the anti-inflammatory pathway of DCM fractions Sarcophyton sp. (SCC), namely as an inhibitor of the MyD88 dependent pathway through the MyD88 dependent-IRAK-TRAF-TAK interaction, thereby suppressing phosphorylation and degradation of IB. Inhibition of phosphorylation and degradation of IB resulted in impaired translocation of NFκB transcription factors. This inhibition of NFκB activation disrupts the production of pro-inflammatory cytokines TNF-α and IL-6 and suppression of iNOS expression. Also, DCM fraction of Sarcophyton sp. (SCC) increases the activity of regulatory T cells as a negative inflammatory regulator and secures anti-inflammatory xvi cytokines IL-10. IL-10 production interferes with TLR4 signaling pathway. The DCM fraction Sarcophyton sp. (SCC) was also able to inhibit the recognition of LPS and TLR4 by acting as a competitive binding in binding with MD2, thus disrupting the interaction of LPS-MD2-TLR4. The antioxidant substance in DCM fraction of Sarcophyton sp. (SCC) was able to reduce the MDA oxidative stress response element and increase the relative number of antioxidant response elements SOD and HO-1 regulated by Nrf-2. It can provide an anti-inflammatory effect by modulating the inflammatory signal and attenuate the inflammatory response induced by LPS through reducing NFκB. The DCM fraction of Sarcophyton sp. (SCC) can inhibit VCAM-1 and ICAM-1 expression, presumably as a result of deactivation of NFkB transcription factors. Inhibition of NFκB activation can affect angiogenesis and inflammatory response to LPS-induced and vascular hyperpermeability. Deactivation of NFκB causes a reduction in leukocyte extravasation in endothelial cells induced by LPS. Inhibition of NO release regulates cell adhesion molecules including ICAM-1 and VCAM-1. The inhibiting effect of VCAM-1 and ICAM-1 expression causes a decrease in fibrous stress, centripetal stress, and cell retraction. This decrease disrupts the regulation of leucocyte−endothelial cell adhesion interactions, thereby preventing excessive leukocyte migration from the blood vessels between endothelial cells and into the tissues.
Other obstract
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| Item Type: | Thesis (Doctor) |
|---|---|
| Identification Number: | DIS/615.36/TAN/p/2019/061907875 |
| Uncontrolled Keywords: | DRUGS O ANIMAL ORIGINS |
| Subjects: | 600 Technology (Applied sciences) > 615 Pharmacology and therapeutics > 615.3 Organics drugs > 615.36 Drugs of animal origin |
| Divisions: | S2/S3 > Doktor Ilmu Perikanan dan Kelautan, Fakultas Perikanan dan Ilmu Kelautan |
| Depositing User: | Endang Susworini |
| Date Deposited: | 11 Feb 2022 05:09 |
| Last Modified: | 11 Feb 2022 05:09 |
| URI: | http://repository.ub.ac.id/id/eprint/189720 |
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