Korean J Physiol Pharmacol.  2015 Nov;19(6):485-489. 10.4196/kjpp.2015.19.6.485.

Effects of Free Anthraquinones Extract from the Rhubarb on Cell Proliferation and Accumulation of Extracellular Matrix in High Glucose Cultured-Mesangial Cells

• Wang J
• Fang H
• Dong B
• Wang D
• Li Y
• Chen X
• Chen L
• Wei T
• Wei Q

• KMID: 2070787
• DOI: http://doi.org/10.4196/kjpp.2015.19.6.485

Abstract

Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidney, but the efficacy of currently available strategies for the prevention of DN remains unsatisfactory. In this study, we investigated the effects of free anthraquinones (FARs) extract, which was extracted from the rhubarb and purified by macroporous resin DM130 with gradient mixtures of ethanol/water as the lelution solvents, in high glucose-cultured glomerular mesangial cells (MCs). The cell proliferation was determined by CCK-8 assay, the levels of TGF-beta1, CTGF, ColIV and FN proteins in the supernatant of MCs were measured by ELISA assays, and the mRNA levels of these four genes were detected by RT-PCR. The results showed that the increased proliferation of MCs, the mRNA levels and protein expression of TGF-beta1, CTGF, ColIV and FN induced by high glucose were inhibited after the treatment with the FARs extract. This indicated that FARs extract could inhibit cell proliferation and the expression of main extracellular matrix induced by high glucose in MCs. The FARs extract exhibited potential values for prophylaxis and therapy of DN.

Keyword

Cell proliferation; Diabetic nephropathy; Extracellular matrix; Free anthraquinones of rhubarb; Mesangial cells

MeSH Terms

Anthraquinones*
Cell Proliferation*
Diabetic Nephropathies
Enzyme-Linked Immunosorbent Assay
Extracellular Matrix*
Glucose*
Kidney
Mesangial Cells
Rheum*
RNA, Messenger
Sincalide
Solvents
Transforming Growth Factor beta1
Anthraquinones
Glucose
RNA, Messenger
Sincalide
Solvents
Transforming Growth Factor beta1

Figure

• 

  Fig. 1 Typical chromatograms of the three FARs. ##p<0.01, compared with the HG group.

• 

  Fig. 2 The cell proliferation rates in FARs-treated MCs. The cells were exposed to DMEM culture media containing different substances, namely: 5.56 mmol/L glucose in the NG group, 5.56 mmol/L glucose+19.44 mmol/L mannitol in the MA group, 25 mmol/L glucose in the HG group, 25 mmol/L glucose+20 µmol/L DMSO in the DMSO group, 25 mmol/L glucose+5 µg/ml FARs in the FARsL group, 25 mmol/L glucose+10 µg/ml FARs in the FARsM group, 25 mmol/L glucose+20 µg/ml FARs in the FARsH group, respectively. Mean±SD, n=4. **p<0.01, compared with the NG group.

• 

  Fig. 3 The mRNA levels of TGF-β1, CTGF, ColIV and FN in the FARs-treated MCs. (A) Agarose electrophoresis of RT-PCR products amplified from the total RNA extracts of mesangial cells, β-actin was used as the internal standard in each sample. (B~E) RT-PCR data for the mRNA relative quantity of TGF-β1, CTGF, ColIV and FN performed by densitometric analysis. Mean±SD, n=4. **p<0.01, compared with the NG group; ##p<0.01, #p<0.05, compared with the HG group.

• 

  Fig. 4 The relative quantity of TGF-β1, CTGF, ColIV and FN proteins in the supernatant of FARs-treated MCs. (A) The relative quantity of TGF-β1 and CTGF proteins. (B) The relative quantity of Col IV and FN proteins. Mean±SD, n=4. **p<0.01, compared with the NG group; ##p<0.01, #p<0.05, compared with the HG group.

Reference

1. Kanwar YS, Wada J, Sun L, Xie P, Wallner EI, Chen S, Chugh S, Danesh FR. Diabetic nephropathy: mechanisms of renal disease progression. Exp Biol Med (Maywood). 2008; 233:4–11. PMID: 18156300.

2. Gonzalez J, Klein J, Chauhan SD, Neau E, Calise D, Nevoit C, Chaaya R, Miravete M, Delage C, Bascands JL, Schanstra JP, Buffin-Meyer B. Delayed treatment with plasminogen activator inhibitor-1 decoys reduces tubulointerstitial fibrosis. Exp Biol Med (Maywood). 2009; 234:1511–1518. PMID: 19934371.

3. Xu W, Shao X, Tian L, Gu L, Zhang M, Wang Q, Wu B, Wang L, Yao J, Xu X, Mou S, Ni Z. Astragaloside IV ameliorates renal fibrosis via the inhibition of mitogen-activated protein kinases and antiapoptosis in vivo and in vitro. J Pharmacol Exp Ther. 2014; 350:552–562. PMID: 24951279.

4. Wang JY, Yin XX, Wu YM, Tang DQ, Gao YY, Wan MR, Hou XY, Zhang B. Ginkgo biloba extract suppresses hypertrophy and extracellular matrix accumulation in rat mesangial cells. Acta Pharmacol Sin. 2006; 27:1222–1230. PMID: 16923344.

5. Kashihara N, Haruna Y, Kondeti VK, Kanwar YS. Oxidative stress in diabetic nephropathy. Curr Med Chem. 2010; 17:4256–4269. PMID: 20939814.

6. Wang J, Huang H, Liu P, Tang F, Qin J, Huang W, Chen F, Guo F, Liu W, Yang B. Inhibition of phosphorylation of p38 MAPK involved in the protection of nephropathy by emodin in diabetic rats. Eur J Pharmacol. 2006; 553:297–303. PMID: 17074319.

7. Qi W, Chen X, Poronnik P, Pollock CA. Transforming growth factor-beta/connective tissue growth factor axis in the kidney. Int J Biochem Cell Biol. 2008; 40:9–13. PMID: 17300978.

8. Park J, Seo JY, Ha H. Plasminogen activator inhibitor-1 antisense oligodeoxynucleotides abrogate mesangial fibronectin accumulation. Korean J Physiol Pharmacol. 2010; 14:385–390. PMID: 21311679.

9. Sakharova OV, Taal MW, Brenner BM. Pathogenesis of diabetic nephropathy: focus on transforming growth factor-beta and connective tissue growth factor. Curr Opin Nephrol Hypertens. 2001; 10:727–738. PMID: 11706299.

10. Fang F, Wang JB, Zhao YL, Jin C, Kong WJ, Zhao HP, Wang HJ, Xiao XH. A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CCl4-injured rats orally administered rhubarb extract. J Ethnopharmacol. 2011; 137:1492–1497. PMID: 21884773.

11. Gao Q, Qin WS, Jia ZH, Zheng JM, Zeng CH, Li LS, Liu ZH. Rhein improves renal lesion and ameliorates dyslipidemia in db/db mice with diabetic nephropathy. Planta Med. 2010; 76:27–33. PMID: 19639539.

12. Cuéllar MJ, Giner RM, Recio MC, Máñez S, Ríos JL. Topical anti-inflammatory activity of some Asian medicinal plants used in dermatological disorders. Fitoterapia. 2001; 72:221–229. PMID: 11295297.

13. Kuo YC, Meng HC, Tsai WJ. Regulation of cell proliferation, inflammatory cytokine production and calcium mobilization in primary human T lymphocytes by emodin from Polygonum hypoleucum Ohwi. Inflamm Res. 2001; 50:73–82. PMID: 11289657.

14. Yoo MY, Oh KS, Lee JW, Seo HW, Yon GH, Kwon DY, Kim YS, Ryu SY, Lee BH. Vasorelaxant effect of stilbenes from rhizome extract of rhubarb (Rheum undulatum) on the contractility of rat aorta. Phytother Res. 2007; 21:186–189. PMID: 17128434.

15. Chen ZQ, Wang JJ. Hypoglycemic and antioxidant effects of Rheum franzenbachii extract in streptozotocin-induced diabetic rats. Pharm Biol. 2010; 48:703–707. PMID: 20645745.

16. Zheng JM, Zhu JM, Li LS, Liu ZH. Rhein reverses the diabetic phenotype of mesangial cells over-expressing the glucose transporter (GLUT1) by inhibiting the hexosamine pathway. Br J Pharmacol. 2008; 153:1456–1464. PMID: 18264122.

17. Wang H, Song H, Yue J, Li J, Hou YB, Deng JL. Rheum officinale (a traditional Chinese medicine) for chronic kidney disease. Cochrane Database Syst Rev. 2012; 7:CD008000.

18. Hyun H, Park H, Jeong J, Kim J, Kim H, Oh HI, Hwang HS, Kim HH. Effects of watercress containing rutin and rutin alone on the proliferation and osteogenic differentiation of human osteoblast-like MG-63 cells. Korean J Physiol Pharmacol. 2014; 18:347–352. PMID: 25177168.

19. Wang X, McLennan SV, Twigg SM. CCN-2 is up-regulated by and mediates effects of matrix bound advanced glycated endproducts in human renal mesangial cells. J Cell Commun Signal. 2011; 5:193–200. PMID: 21630131.

20. Chen S, Hong SW, Iglesias-de la Cruz MC, Isono M, Casaretto A, Ziyadeh FN. The key role of the transforming growth factor-beta system in the pathogenesis of diabetic nephropathy. Ren Fail. 2001; 23:471–481. PMID: 11499562.

21. Guo XH, Liu ZH, Dai CS, Li H, Liu D, Li LS. Rhein inhibits renal tubular epithelial cell hypertrophy and extracellular matrix accumulation induced by transforming growth factor beta1. Acta Pharmacol Sin. 2001; 22:934–938. PMID: 11749778.

22. Li X, Liu W, Wang Q, Liu P, Deng Y, Lan T, Zhang X, Qiu B, Ning H, Huang H. Emodin suppresses cell proliferation and fibronectin expression via p38MAPK pathway in rat mesangial cells cultured under high glucose. Mol Cell Endocrinol. 2009; 307:157–162. PMID: 19524136.

23. Tabolacci C, Oliverio S, Lentini A, Rossi S, Galbiati A, Montesano C, Mattioli P, Provenzano B, Facchiano F, Beninati S. Aloe-emodin as antiproliferative and differentiating agent on human U937 monoblastic leukemia cells. Life Sci. 2011; 89:812–820. PMID: 21978786.

24. Liu K, Park C, Li S, Lee KW, Liu H, He L, Soung NK, Ahn JS, Bode AM, Dong Z, Kim BY, Dong Z. Aloe-emodin suppresses prostate cancer by targeting the mTOR complex 2. Carcinogenesis. 2012; 33:1406–1411. PMID: 22532249.

25. Ibrahim M, Khaja MN, Aara A, Khan AA, Habeeb MA, Devi YP, Narasu ML, Habibullah CM. Hepatoprotective activity of Sapindus mukorossi and Rheum emodi extracts: in vitro and in vivo studies. World J Gastroenterol. 2008; 14:2566–2571. PMID: 18442207.

26. Ibrahim M, Khan AA, Tiwari SK, Habeeb MA, Khaja MN, Habibullah CM. Antimicrobial activity of Sapindus mukorossi and Rheum emodi extracts against H pylori: In vitro and in vivo studies. World J Gastroenterol. 2006; 12:7136–7142. PMID: 17131475.

27. You X, Feng S, Luo S, Cong D, Yu Z, Yang Z, Zhang J. Studies on a rhein-producing endophytic fungus isolated from Rheum palmatum L. Fitoterapia. 2013; 85:161–168. PMID: 23266728.