Abstract
Objective
To investigate the effects of Salvia miltiorrhiza and Ligustrazine Injection (SML) on proliferation and apoptosis of human hepatic stellate cell LX-2 and the expression of N-myc downstreamregulated gene 2 (NDRG2, a tumor suppressor gene).
Methods
HSCs from the LX-2 cell line were cultured in vitro. The proliferative state of different initial LX-2 cell numbers was measured using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. LX-2 cells were plated in 96-well plates at an approximate density of 2.50×104 cells/mL and cultured for 24 h followed by the application of different concentrations of SML (1, 2, 4 and 8 μL/mL). Cell proliferation was measured using the MTT assay at 24 and 48 h. Apoptosis was detected by flow cytometry at 24 h. LX-2 cells were treated with different concentrations of SML and extracted with protein lysis buffer. The levels of NDRG2 and β-catenin were measured by Western blot.
Results
With the exception of the 1 and 2 μL/mL concentrations, 4 and 8 μL/mL SML inhibited cell proliferation in a concentration-dependent manner at 24 and 48 h (P<0.05). With the exception of the 1 and 2 μL/mL concentrations, the NDRG2 expression level was greatly increased in a concentration-dependent manner. However, the level of β-catenin was unaffected.
Conclusion
SML inhibit LX-2 cell proliferation in a concentration-dependent manner, and the mechanism may be associated with NDRG2 over-expression.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Farazi PA, Depinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 2006;6:674–687.
Wu T, Chen JM, Xiao TG, Shu XB, Xu HC, Yang LL, et al. Qinggan Huoxue Recipe suppresses epithelial-tomesenchymal transition in alcoholic liver fibrosis through TGF-beta1/Smad signaling pathway. World J Gastroenterol 2016;22:4695–4706.
Ding LT, Zhu YG, Gu ZQ, Cai LL, Peng JL, Lu GZ. Effect of Salvia miltiorrhiza and Ligustrazine Injection on the early myocardial damage of patients with severe burn. Chin J Burns (Chin) 2012;28:170–172.
Wang XF, Zhao MQ. Ligustrazine and Salvia miltiorrhiza injection solution in complementary therapy of pregnancyinduced hypertension: clinical analysis of 60 cases. J First Mil Med Univ (Chin) 2003;23:969–971.
Zhang L, Wu T, Chen JM, Yang LL, Song HY, Ji G. Tanshinol inhibits acetaldehyde-induced proliferation and activation of hepatic stellate cell-T6. J Integr Med 2012;10:1155–1161.
Li X, Peng XD, Zhang WL, Dai LL. Inhibiting effects of denshensu, baicalin, astragalus and Panax notoginseng saponins on hepatic fibrosis and their possible mechanisms. Chin J Hepatol (Chin) 2008;16:193–197.
Yu BB, Dai LL, Li X, Li D, Jiang ZH. Effect of danshensu on activation of JNK pathway in hepatic stellate cells (HSCs) induced by IL-1 beta. Chin J Hepatol (Chin) 2009;17:451–454.
Xu L, Hui AY, Albanis E, Arthur MJ, O'Byrne SM, Blaner WS, et al. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 2005;54:142–151.
Taimr P, Higuchi H, Kocova E, Rippe RA, Friedman S, Gores GJ. Activated stellate cells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediated apoptosis. Hepatology 2003;37:87–95.
Yang JD. The expression and function study of NDRG2 in liver damage-repair process. Xi'an: Fourth Military Medical University; 2013:64–77.
Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol 2008;214:199–210.
Henderson NC, Mackinnon AC, Farnworth SL, Poirier F, Russo FP, Iredale JP, et al. Galectin-3 regulates myofibroblast activation and hepatic fibrosis. Proc Natl Acad Sci USA 2006;103:5060–5065.
Yoshida K, Matsuzaki K. Differential regulation of TGFbeta/ Smad signaling in hepatic stellate cells between acute and chronic liver injuries. Front Physiol 2012;3:53.
Zheng J, Liu Q, Shi HJ, Ren QY. Progress of liver fibrosis and beta elemene anti liver fibrosis. J Shanxi Med Univ (Chin) 2010;41:935–938.
Shah R, Reyes-Gordillo K, Arellanes-Robledo J, Lechuga CG, Hernandez-Nazara Z, Cotty A, et al. TGF-beta1 upregulates the expression of PDGF-beta receptor mRNA and induces a delayed PI3K-, AKT-, and p70 (S6K)-dependent proliferative response in activated hepatic stellate cells. Alcohol Clin Exp Res 2013;37:1838–1848.
Zheng J. In the interaction of hepatic cacinoma and stellate cells, NDRG2 has effects on metastasis of hepatic cacinoma. Xi'an: The Fourth Military Medical University; 2010:74–87.
Shen L, Qu X, Ma Y, Zheng J, Chu D, Liu B, et al. Tumor suppressor NDRG2 tips the balance of oncogenic TGF-beta via EMT inhibition in colorectal cancer. Oncogenesis 2014;3:1–12.
Cao W, Zhang JL, Feng DY, Liu XW, Li Y, Wang LF, et al. The effect of adenovirus-conjugated NDRG2 on p53- mediated apoptosis of hepatocarcinoma cells through attenuation of nucleotide excision repair capacity. Biomaterials 2014;35:993–1003.
Zhang J, Li S, Yan Q, Chen X, Yang Y, Liu X, et al. Interferon-beta induced microRNA-129-5p down-regulates HPV-18 E6 and E7 viral gene expression by targeting SP1 in cervical cancer cells. PLoS One 2013;8:e84127.
Gao L, Wu GJ, Liu B, Shen MZ, Pan TJ, Yu CG, et al. Upregulation of pVHL along with down-regulation of HIF- 1alpha by NDRG2 expression attenuates proliferation and invasion in renal cancer cells. PLoS One 2013;8:e81366.
Yang J, Zheng J, Wu L, Shi M, Zhang H, Wang X, et al. NDRG2 ameliorates hepatic fibrosis by inhibiting the TGFbeta1/ Smad pathway and altering the MMP2/TIMP2 ratio in rats. PLoS One 2011;6:1–10.
Ji C, Liu J, Li L, Li X, Li S. Effect of tetramethylpyrazine on prohibiting hepatic fibrosis in rats. J Shanxi Med Univ (Chin) 2003;34:293–295.
Han Z. Effect of tiopronin combined with ligustrazine on liver fibrosis. Chin J Integr Tradit West Med Liver Dis (Chin) 2004:14:174-175.
Wu Y, Dai L, Tang J, Guo L. Effect of salavianic acid A (danshensu) on proliferation, activation and TGFβ/BMP receptors in hepatic stellate cells. J Chongqing Med Univ (Chin) 2010:35:1344-1347.
Wang X, Li W. The influence of Salviae miltiorrhizae and Ligustrazine Injection on liver fibrosis indexes when was used in treatment of chronic hepatitis B. Shanxi Med J (Chin) 2012;41:1239–1240.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supported by the National Natural Science Foundation of China (No. 81272490 and 81100764)
Rights and permissions
About this article
Cite this article
Zheng, J., Ma, Lt., Ren, Qy. et al. Anti-fibrotic effects of Salvia miltiorrhiza and Ligustrazine Injection on LX-2 cells involved with increased N-myc downstream-regulated gene 2 expression. Chin. J. Integr. Med. 23, 923–928 (2017). https://doi.org/10.1007/s11655-016-2640-9
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11655-016-2640-9