Effect of Melittin on collagen type II expression of rat endplate chondrocytes induced by interleukin 1β_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YE Yuchen ,  ZHANG Changchun , ZHU Kun , XU Gang , HAN Zhongbing , LE Yi

Department of Orthopedics, the First Affiliated Hospital of Bengbu Medical College, Bengbu Anhui, 233030, P.R.China;

Corresponding author：

ZHANG Changchun, Email: zccanhui@sina.com

Keywords：

Melittin; endplate chondrocytes; interleukin 1β; collagen type II; rat

DOI：

10.7507/1002-1892.201609062

Video：

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Objective To observe the effect of Melittin on collagen type II (Col-II) expression of rat endplate chondrocytes (EPCs) induced by interleukin 1β (IL-1β). Methods Primary EPCs from the lumbar vertebra of 4-week-old Sprague Dawley rats were culturedin vitro and identified by morphological observation, toluidine blue staining and Col-II immunofluorescence staining. Then, MTT assay was used to determine the optimal concentration of IL-1 and Melittin. Next, EPCs at passage 3 were randomly divided into 4 groups: no treatment was done in group A as control group; the optimal concentration of IL-1β, Melittin, and both IL-1β and Melittin were used in groups B, C, and D respectively. The expression of Col-II was detected by Western blot after 48 hours intervention. Results Under inverted microscope, the first generation EPCs were polygonal; cell proliferation decreased after fifth generation, and cell morphology changed into fusiform. The acidic mucosubstance in the cytoplasm (such as Aggrecan) was stained dark blue by toluidine blue. After marking Col-II by immunofluorescence, the positive expression of cytoskeleton (green fluorescence) could be observed. MTT assay showed that IL-1β and Melittin could inhibit the EPCs in a dose-dependent manner after intervention of 24 and 48 hours, and the optimal concentrations of IL-1β and Melittin intervention were 10 ng/mL and 1.0 μg/mL respectively. Compared with group A, the expression of Col-II was significantly reduced in group B, and was significantly increased in group C by Western blot assay, but there was no significant difference between group D and group A. The Col-II expression levels of groups A, B, C, and D were 0.991±0.024, 0.474±0.127, 1.913±0.350, and 1.159±0.297 respectively, showing significant difference between the other groups (P<0.05) except between group A and group D (P>0.05). Conclusion Melittin has a protective effect on endplate cartilage, and the research results provide experimental basis for the prevention and treatment of spinal degenerative disease.

Citation： YE Yuchen, ZHANG Changchun, ZHU Kun, XU Gang, HAN Zhongbing, LE Yi. Effect of Melittin on collagen type II expression of rat endplate chondrocytes induced by interleukin 1β. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(3): 345-350. doi: 10.7507/1002-1892.201609062 Copy

1.	Bibby SR, Jones DA, Ripley RM, et al. Metabolism of the intervertebral disc: effects of low levels of oxygen, glucose, and pH on rates of energy metabolism of bovine nucleus pulposus cells. Spine (Phila Pa 1976), 2005, 30(5): 487-496.
2.	Pabst M, Wu SQ, Grass J, et al. IL-1beta and TNF-alpha alter the glycophenotype of primary human chondrocytesin vitro. Carbohydr Res, 2010, 345(10): 1389-1393.
3.	Ahmed S, Wang N, Lalonde M, et al. Green tea polyphenol epigallocatechin-3-gallate (EGCG) differentially inhibits interleukin-1 betainduced expression of matrix metalloproteinase-1 and -13 in human chondrocytes. Pharmacol Exp Ther, 2004, 308(2): 767-773.
4.	Rodriguez AG, Rodriguez-Soto AE, Burghardt AJ, et al. Morphology of the human vertebral endplate. J Orthop Res, 2012, 30(2): 280-287.
5.	Siemionow K, An H, Masuda K, et al. The effects of age, sex, ethnicity, and spinal level on the rate of intervertebral disc degeneration: a review of 1712 intervertebral discs. Spine (Phila Pa 1976), 2011, 36(17): 1333-1339.
6.	徐宏光, 陈小虎, 王弘, 等. 兔椎体软骨终板血管芽增龄性变化. 中华骨科杂志, 2011, 31(4): 351-356.
7.	Liebscher T, Haefeli M, Wuertz K, et al. Age-related variation in cell density of human lumbar intervertebral disc. Spine (Phila Pa 1976), 2011, 36(2): 153-159.
8.	Laffosse JM, Accadbled F, Molinier F, et al. Correlations between effective permeability and marrow contact channels surface of vertebral endplates. J Orthop Res, 2010, 28(9): 1229-1234.
9.	Jeong YJ, Shin JM, Bae YS, et al. Melittin has a chondroprotective effect by inhibiting MMP-1 and MMP-8 expressions via blocking NF-kappaB and AP-1 signaling pathway in chondrocytes. Int Immunopharmacol, 2015, 25(2): 400-405.
10.	Wuertz K, Vo N, Kletsas D, et al. Inflammatory and catabolic signalling in intervertebral discs: the roles of NF-κB and MAP kinases. Eur Cell Mater, 2012, 16(23): 103-119.
11.	Nerlich AG, Bachmeier BE, Schleicher E, et al. Immunomorphological analysis of RAGE receptor expression and NF-kappaB activation in tissue samples from normal and degenerated intervertebral discs of various ages. Ann NY Acad Sci, 2007, 1096: 239-248.
12.	Phillips KL, Cullen K, Chiverton N, et al. Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes. Osteoarthritis Cartilage, 2015, 23(7): 1165-1177.
13.	Kim JS, Ellman MB, Yan D, et al. Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc. Cell Physiol, 2013, 228(9): 1884-1896.
14.	Wuertz K, Quero L, Sekiguchi M, et al. The red wine polyphenol resveratrol shows promising potential for the treatment of nucleus pulposus-mediated painin vitro andin vivo. Spine (Phila Pa 1976), 2011, 36(21): E1373-1384.
15.	Phillips KL, Cullen K, Chiverton N, et al. Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes. Osteoarthritis Cartilage, 2015, 23(7): 1165-1177.
16.	Wang J, Tian Y, Phillips KL, et al. Tumor necrosis factor alpha- and interleukin-1beta-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1. Arthritis Rheum, 2013, 65(3): 832-842.
17.	Phillips KL, Jordan-Mahy N, Nicklin MJ, et al. Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration. Ann Rheum Dis, 2013, 72(11): 1860-1867.
18.	Tian Y, Yuan W, Fujita N, et al. Inflammatory cytokines associated with degenerative disc disease control aggrecanase-1 (ADAMTS-4) expression in nucleus pulposus cells through MAPK and NF-kappaB. Am Pathol, 2013, 182(6): 2310-2321.
19.	Millward-Sadler SJ, Costello PW, Freemont AJ, et al. Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther, 2009, 11(3): R65.
20.	Kim JH, Choi H, Suh MJ, et al. Effect of biphasic electrical current stimulation on IL-1beta-stimulated annulus fibrosus cells usingin vitro microcurrent generating chamber system. Spine (Phila Pa 1976), 2013, 38(22): E1368-1376.

1. Bibby SR, Jones DA, Ripley RM, et al. Metabolism of the intervertebral disc: effects of low levels of oxygen, glucose, and pH on rates of energy metabolism of bovine nucleus pulposus cells. Spine (Phila Pa 1976), 2005, 30(5): 487-496.
2. Pabst M, Wu SQ, Grass J, et al. IL-1beta and TNF-alpha alter the glycophenotype of primary human chondrocytesin vitro. Carbohydr Res, 2010, 345(10): 1389-1393.
3. Ahmed S, Wang N, Lalonde M, et al. Green tea polyphenol epigallocatechin-3-gallate (EGCG) differentially inhibits interleukin-1 betainduced expression of matrix metalloproteinase-1 and -13 in human chondrocytes. Pharmacol Exp Ther, 2004, 308(2): 767-773.
4. Rodriguez AG, Rodriguez-Soto AE, Burghardt AJ, et al. Morphology of the human vertebral endplate. J Orthop Res, 2012, 30(2): 280-287.
5. Siemionow K, An H, Masuda K, et al. The effects of age, sex, ethnicity, and spinal level on the rate of intervertebral disc degeneration: a review of 1712 intervertebral discs. Spine (Phila Pa 1976), 2011, 36(17): 1333-1339.
6. 徐宏光, 陈小虎, 王弘, 等. 兔椎体软骨终板血管芽增龄性变化. 中华骨科杂志, 2011, 31(4): 351-356.
7. Liebscher T, Haefeli M, Wuertz K, et al. Age-related variation in cell density of human lumbar intervertebral disc. Spine (Phila Pa 1976), 2011, 36(2): 153-159.
8. Laffosse JM, Accadbled F, Molinier F, et al. Correlations between effective permeability and marrow contact channels surface of vertebral endplates. J Orthop Res, 2010, 28(9): 1229-1234.
9. Jeong YJ, Shin JM, Bae YS, et al. Melittin has a chondroprotective effect by inhibiting MMP-1 and MMP-8 expressions via blocking NF-kappaB and AP-1 signaling pathway in chondrocytes. Int Immunopharmacol, 2015, 25(2): 400-405.
10. Wuertz K, Vo N, Kletsas D, et al. Inflammatory and catabolic signalling in intervertebral discs: the roles of NF-κB and MAP kinases. Eur Cell Mater, 2012, 16(23): 103-119.
11. Nerlich AG, Bachmeier BE, Schleicher E, et al. Immunomorphological analysis of RAGE receptor expression and NF-kappaB activation in tissue samples from normal and degenerated intervertebral discs of various ages. Ann NY Acad Sci, 2007, 1096: 239-248.
12. Phillips KL, Cullen K, Chiverton N, et al. Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes. Osteoarthritis Cartilage, 2015, 23(7): 1165-1177.
13. Kim JS, Ellman MB, Yan D, et al. Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc. Cell Physiol, 2013, 228(9): 1884-1896.
14. Wuertz K, Quero L, Sekiguchi M, et al. The red wine polyphenol resveratrol shows promising potential for the treatment of nucleus pulposus-mediated painin vitro andin vivo. Spine (Phila Pa 1976), 2011, 36(21): E1373-1384.
15. Phillips KL, Cullen K, Chiverton N, et al. Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes. Osteoarthritis Cartilage, 2015, 23(7): 1165-1177.
16. Wang J, Tian Y, Phillips KL, et al. Tumor necrosis factor alpha- and interleukin-1beta-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1. Arthritis Rheum, 2013, 65(3): 832-842.
17. Phillips KL, Jordan-Mahy N, Nicklin MJ, et al. Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration. Ann Rheum Dis, 2013, 72(11): 1860-1867.
18. Tian Y, Yuan W, Fujita N, et al. Inflammatory cytokines associated with degenerative disc disease control aggrecanase-1 (ADAMTS-4) expression in nucleus pulposus cells through MAPK and NF-kappaB. Am Pathol, 2013, 182(6): 2310-2321.
19. Millward-Sadler SJ, Costello PW, Freemont AJ, et al. Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther, 2009, 11(3): R65.
20. Kim JH, Choi H, Suh MJ, et al. Effect of biphasic electrical current stimulation on IL-1beta-stimulated annulus fibrosus cells usingin vitro microcurrent generating chamber system. Spine (Phila Pa 1976), 2013, 38(22): E1368-1376.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of Melittin on collagen type II expression of rat endplate chondrocytes induced by interleukin 1β

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