Astragalus and Paeoniae Radix Rubra extract (APE) inhibits hepatic stellate cell activation by modulating transforming growth factor-β/smad pathway

Huang,Weijuan; Li,Lin; Tian,Xiaopeng; Yan,Jinjin; Yang,Xinzheng; Wang,Xinlong; Liao,Guozhen; Qiu,Genquan

doi:10.3892/mmr.2014.3026

Astragalus and Paeoniae Radix Rubra extract (APE) inhibits hepatic stellate cell activation by modulating transforming growth factor-β/smad pathway

Authors:
- Weijuan Huang
- Lin Li
- Xiaopeng Tian
- Jinjin Yan
- Xinzheng Yang
- Xinlong Wang
- Guozhen Liao
- Genquan Qiu
View Affiliations

Affiliations: Department of Scientific Research, Xi'an Medical College, Xi'an, Shaanxi 710061, P.R. China, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat‑sen University, Guangzhou, Guangdong 510060, P.R. China, Department of Pharmacology, Xi'an Medical College, Xi'an, Shaanxi 710061, P.R. China, Department of Traditional Chinese Medicine, First Affiliated Hospital of Xi'an Jiao Tong University, Xi'an, Shaanxi 710061, P.R. China
Published online on: December 1, 2014 https://doi.org/10.3892/mmr.2014.3026
Pages: 2569-2577
Copyright: © Huang et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Previous studies have shown that Astragalus and Paeoniae Radix Rubra extract (APE) is capable of protecting against liver fibrosis in rats. The hypothesis of the present study was that APE exerts its anti‑fibrotic effect by mediating the transforming growth factor β (TGF‑β)/Smad signaling pathway. In order to investigate this hypothesis, a series of assays were designed to detect the effects of APE on cell proliferation, cell invasion and the activation of hepatic stellate cells (HSCs). In addition, the effects of APE on the TGF‑β/Smad signaling pathway were explored, with the aim of elucidating the underlying mechanisms. HSCs were initially isolated from normal rat liver. A number of assays were then employed in order to evaluate the effects of APE on the function of these cells. Cell proliferation was investigated using an MTT assay and cell invasion was observed with the use of transwell invasion chambers. Collagen synthesis was measured with a 3H‑proline incorporation assay and expression of α‑smooth muscle actin was used to determine the extent of HSC activation. Protein expression induced by TGF‑β1 in HSCs was investigated by western blot and immunofluorescence analyses. Plasminogen activator inhibitor type1 (PAI‑1) and urokinase‑type plasminogen activator (uPA) transcriptional activity was measured using reverse transcription polymerase chain reaction. The results demonstrated that APE (5‑80 µg/ml) significantly inhibited fetal bovine serum‑induced cell proliferation in a dose‑dependent manner. Cell invasion and activation of HSCs induced by TGF‑β1 were disrupted by treatment with APE in a dose‑dependent manner. TGF‑β1 was observed to increase the phosphorylation of Smad2/3, while APE administered at higher doses produced inhibitory effects on Smad2/3 phosphorylation. In addition, administration of APE abrogated the TGF‑β1‑induced reduction in Smad‑7 expression in a dose‑dependent manner. The results further indicated that APE treatment not only reduced PAI‑1 expression, but also increased uPA expression in a dose‑dependent manner. In conclusion, APE exerted inhibitory effects on cell proliferation, invasion and activation of HSCs, and the mechanisms underlying these effects may involve the TGF‑β1/Smad pathway.

View Figures

View References

1	Ray K: Liver: Hepatic stellate cells hold the key to liver fibrosis. Nat Rev Gastroenterol Hepatol. 11:742014. View Article : Google Scholar
2	Kocabayoglu P and Friedman SL: Cellular basis of hepatic fibrosis and its role in inflammation and cancer. Front Biosci (Schol Ed). 5:217–230. 2013.
3	Mello T, Ceni E, Surrenti C and Galli A: Alcohol induced hepatic fibrosis: role of acetaldehyde. Mol Aspects Med. 29:17–21. 2008. View Article : Google Scholar : PubMed/NCBI
4	Jian YC, Li W, He Y, Jiang M, Liu YB and Xiong WJ: Effect of oxymatrine on hepatic gene expression profile in experimental liver fibrosis of rats. Chin J Integr Med. 18:445–450. 2012. View Article : Google Scholar : PubMed/NCBI
5	Parise ER, de Oliveira AC, Conceicao RD, Amaral AC and Leite K: Response to treatment with interferon-alpha and ribavirin in patients with chronic Hepatitis C virus genotypes 2 and 3 depends on the degree of hepatic fibrosis. Braz J Infect Dis. 10:78–81. 2006. View Article : Google Scholar : PubMed/NCBI
6	Tsolaki E, Athanasiou E, Gounari E, Zogas N, Siotou E, Yiangou M, Anagnostopoulos A and Yannaki E: Hematopoietic stem cells and liver regeneration: differentially acting hematopoietic stem cell mobilization agents reverse induced chronic liver injury. Blood Cells Mol Dis. 53:124–132. 2014. View Article : Google Scholar : PubMed/NCBI
7	Trebicka J, Hennenberg M, Odenthal M, et al: Atorvastatin attenuates hepatic fibrosis in rats after bile duct ligation via decreased turnover of hepatic stellate cells. J Hepatol. 53:702–712. 2010. View Article : Google Scholar : PubMed/NCBI
8	Elsharkawy AM, Oakley F and Mann DA: The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis. Apoptosis. 10:927–939. 2005. View Article : Google Scholar : PubMed/NCBI
9	Moreira RK: Hepatic stellate cells and liver fibrosis. Arch Pathol Lab Med. 131:1728–1734. 2007.PubMed/NCBI
10	Huang Q, Huang R, Zhang S, et al: Protective effect of genistein isolated from Hydrocotyle sibthorpioides on hepatic injury and fibrosis induced by chronic alcohol in rats. Toxicol Lett. 217:102–110. 2013. View Article : Google Scholar : PubMed/NCBI
11	Su LJ, Chang CC, Yang CH, et al: Graptopetalum paraguayense ameliorates chemical-induced rat hepatic fibrosis in vivo and inactivates stellate cells and Kupffer cells in vitro. PLoS One. 8:e539882013. View Article : Google Scholar : PubMed/NCBI
12	Xu MY, Hu JJ, Shen J, Wang ML, Zhang QQ, Qu Y and Lu LG: Stat3 signaling activation crosslinking of TGF-β1 in hepatic stellate cell exacerbates liver injury and fibrosis. Biochim Biophys Acta. 1842.2237–2245. 2014.
13	Lang Q, Liu Q, Xu N, et al: The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats. Biochem Biophys Res Commun. 409:448–453. 2011. View Article : Google Scholar : PubMed/NCBI
14	Chen BL, Peng J, Li QF, Yang M, Wang Y and Chen W: Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis in Schistosoma japonicum-infected mice via transforming growth factor-beta/Smad signaling. World J Gastroenterol. 19:1405–1415. 2013. View Article : Google Scholar : PubMed/NCBI
15	Lee JH, Lee H, Joung YK, et al: The use of low molecular weight heparin-pluronic nanogels to impede liver fibrosis by inhibition the TGF-β/Smad signaling pathway. Biomaterials. 32:1438–1445. 2011. View Article : Google Scholar
16	Hang W, Li L, Tian X, Yan J, Yang X, Wang X, Liao G1 and Qiu G: Astragalus and Paeoniae radix rubra extract inhibits liver fibrosis by modulating the transforming growth factor-β/Smad pathway in rats. Mol Med Rep. Nov 5–2014.(Epub ahead of print). View Article : Google Scholar
17	Yu KZ, Liu J, Guo BL, et al: Microscopic research on a multi-source traditional Chinese medicine, Astragali Radix. J Nat Med. 68:340–350. 2013. View Article : Google Scholar : PubMed/NCBI
18	Yusufoglu HS, Alam A, Zaghloul AM, Al-Salkini MA and Alam P: Comparative anti-inflammatory and hepatoprotective activities of astragalus gummifer labill herb and roots in rats. Afr J Tradit Complement Altern Med. 11:268–274. 2014. View Article : Google Scholar : PubMed/NCBI
19	Liang J, Xu F, Zhang YZ, et al: The profiling and identification of the absorbed constituents and metabolites of Paeoniae Radix Rubra decoction in rat plasma and urine by the HPLC-DAD-ESI-IT-TOF-MS(n) technique: a novel strategy for the systematic screening and identification of absorbed constituents and metabolites from traditional Chinese medicines. J Pharm Biomed Anal. 83:108–121. 2013. View Article : Google Scholar : PubMed/NCBI
20	Knook DL, Seffelaar AM and de Leeuw AM: Fat-storing cells of the rat liver. Their isolation and purification. Exp Cell Res. 139:468–471. 1982. View Article : Google Scholar : PubMed/NCBI
21	Majumdar M, Ratho R, Chawla Y and Singh MP: Evaluation of antigenicity and cell mediated immunity of hepatitis E virus patients: using non radioactive MTT assay. Indian J Med Microbiol. 31:64–68. 2013. View Article : Google Scholar : PubMed/NCBI
22	Zhou G, Kandala JC, Tyagi SC, Katwa LC and Weber KT: Effects of angiotensin II and aldosterone on collagen gene expression and protein turnover in cardiac fibroblasts. Mol Cell Biochem. 154:171–178. 1996. View Article : Google Scholar : PubMed/NCBI
23	Hu X, Rui W, Wu C, He S, Jiang J, Zhang X and Yang Y: Compound Astragalus and Salvia miltiorrhiza extracts suppress hepatocarcinogenesis by modulating transforming growth factor-β/Smad sigmaling. J Gastroenterol Hepatol. 29:1284–1291. 2014. View Article : Google Scholar
24	Hashiba M, Ono M, Hyogo H, et al: Glycemic variability is an independent predictive factor for development of hepatic fibrosis in nonalcoholic Fatty liver disease. PLoS One. 8:e761612013. View Article : Google Scholar : PubMed/NCBI
25	Hossain N, Afendy A, Stepanova M, et al: Independent predictors of fibrosis in patients with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 7:1224–1229. 2009. View Article : Google Scholar : PubMed/NCBI
26	Svegliati-Baroni G, Bugianesi E, Bouserhal T, et al: Post-load insulin resistance is an independent predictor of hepatic fibrosis in virus C chronic hepatitis and in non-alcoholic fatty liver disease. Gut. 56:1296–1301. 2007. View Article : Google Scholar : PubMed/NCBI
27	Kowdley KV, Belt P, Wilson LA, et al: Serum ferritin is an independent predictor of histologic severity and advanced fibrosis in patients with nonalcoholic fatty liver disease. Hepatology. 55:77–85. 2012. View Article : Google Scholar
28	Chen HJ, Liang TM, Lee IJ, Huang YT and Lin YL: Scutellariae radix suppresses LPS-induced liver endothelial cell activation and inhibits hepatic stellate cell migration. J Ethnopharmacol. 150:835–842. 2013. View Article : Google Scholar : PubMed/NCBI
29	Katz TM, Goldberg LH and Friedman PM: Nonablative fractional photothermolysis for the treatment of striae rubra. Dermatol Surg. 35:1430–1433. 2009. View Article : Google Scholar : PubMed/NCBI
30	Zhou YX, Chen J, Li JP, Wang YL and Jin XD: Chinese medicinal herbs in treating model rats with hepatic fibrosis. Afr J Tradit Complement Altern Med. 7:104–108. 2010.
31	Vitcheva V, Simeonova R, Krasteva I, Nikolov S and Mitcheva M: Protective effects of a purified saponin mixture from Astragalus corniculatus Bieb., in vivo hepatotoxicity models. Phytother Res. 27:731–736. 2013. View Article : Google Scholar
32	D’Argenio G, Mazzone G, Ribecco MT, et al: Garlic extract attenuating rat liver fibrosis by inhibiting TGF-β1. Clin Nutr. 32:252–258. 2013. View Article : Google Scholar
33	Li J, Li J, Li S, et al: Ameliorative effect of grAPE seed proanthocyanidin extract on thioacetamide-induced mouse hepatic fibrosis. Toxicol Lett. 213:353–360. 2012. View Article : Google Scholar : PubMed/NCBI
34	Lang Q, Liu Q, Xu N, et al: The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats. Biochem Biophys Res Commun. 409:448–453. 2011. View Article : Google Scholar : PubMed/NCBI
35	Liu X, Yang Y, Zhang X, et al: Compound Astragalus and Salvia miltiorrhiza extract inhibits cell invasion by modulating transforming growth factor-beta/Smad in HepG2 cell. J Gastroenterol Hepatol. 25:420–426. 2010. View Article : Google Scholar
36	Tian XP, Yin YY and Li X: Effects and mechanisms of Acremoniumterricola milleretal mycelium on liver fibrosis induced by carbon tetrachloride in rats. Am J Chin Med. 39:537–550. 2011. View Article : Google Scholar : PubMed/NCBI
37	Cai Y, Zhou CH, Fu D and Shen XZ: Overexpression of Smad ubiquitin regulatory factor 2 suppresses transforming growth factor-beta mediated liver fibrosis. J Dig Dis. 13:327–334. 2012. View Article : Google Scholar : PubMed/NCBI
38	Sferra R, Vetuschi A, Catitti V, et al: Boswellia serrata and Salvia miltiorrhiza extracts reduce DMN-induced hepatic fibrosis in mice by TGF-beta1 downregulation. Eur Rev Med Pharmacol Sci. 16:1484–1498. 2012.PubMed/NCBI
39	Freudlsperger C, Bian Y, Contag WS, et al: TGF-β and NF-κB signal pathway cross-talk is mediated through TAK1 and SMAD7 in a subset of head and neck cancers. Oncogene. 32:1549–1559. 2013. View Article : Google Scholar
40	Bian EB, Huang C, Wang H, et al: Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol Lett. 224:175–185. 2014. View Article : Google Scholar
41	Hamzavi J, Ehnert S, Godoy P, et al: Disruption of the Smad7 gene enhances CCI4-dependent liver damage and fibrogenesis in mice. J Cell Mol Med. 12:2130–2144. 2008. View Article : Google Scholar : PubMed/NCBI
42	Dooley S, Hamzavi J, Breitkopf K, et al: Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology. 125:178–191. 2003. View Article : Google Scholar : PubMed/NCBI
43	Ji H, Tang H, Lin H, Mao J, Gao L, Liu J and Wu T: Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation. Biomed Rep. 2:787–792. 2014.PubMed/NCBI
44	Lin Z, Jiang L, Yuan C, et al: Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1. J Biol Chem. 286:7027–7032. 2011. View Article : Google Scholar : PubMed/NCBI
45	Wang B, Li W, Chen Y, et al: Coexpression of Smad7 and UPA attenuates carbon tetrachloride-induced rat liver fibrosis. Med Sci Monit. 18:R394–R401. 2012. View Article : Google Scholar
46	Tang LX, He RH, Yang G, Tan JJ, Zhou L, Meng XM, Huang XR and Lan HY: Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro. PLoS One. 7:e313502012. View Article : Google Scholar : PubMed/NCBI
47	Salas AL, Montezuma TD, Farina GG, Reyes-Esparza J and Rodriguez-Fragoso L: Genistein modifies liver fibrosis and improves liver function by inducing uPA expression and proteolytic activity in CCl4-treated rats. Pharmacology. 81:41–49. 2008. View Article : Google Scholar
48	Ghosh AK and Vaughan DE: PAI-1 in tissue fibrosis. J Cell Physiol. 227:493–507. 2012. View Article : Google Scholar
49	Tuan TL, Zhu JY, Sun B, Nichter LS, Nimni ME and Laug WE: Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts. J Invest Dermatol. 106:1007–1011. 1996. View Article : Google Scholar : PubMed/NCBI