鹿茸多肽对IL-1β诱导的大鼠软骨终板细胞增殖和CollagenⅠ、CollagenⅡ、IL-6 mRNA表达的影响
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摘要
目的:观察不同浓度的鹿茸多肽对IL~(-1)β诱导的大鼠椎间盘软骨终板细胞增殖和Ⅰ型胶原(typeⅠcollagen,CollagenⅠ)、CollagenⅡ、白细胞介素(interleukin-6,IL-6) mRNA表达的影响。方法:选取健康SD大鼠(1个月龄),取其脊柱椎间盘软骨终板细胞,并培养至第3代软骨终板细胞进行实验。空白对照组不做任何处理,诱导组只加入10μg·L~(-1)的IL~(-1)β,给药组在培养基中加入10μg·L~(-1)的IL~(-1)β和不同质量浓度的鹿茸多肽(分别为10 mg·L~(-1)、30 mg·L~(-1)、50 mg·L~(-1))。用MTT比色法检测3个时间点(24 h、48 h、72 h)各组椎间盘软骨终板细胞的增殖情况; qP CR法检测CollagenⅠ、CollagenⅡ及IL-6 mRNA的表达。结果:MTT法检测结果:鹿茸多肽10 mg·L~(-1)组的软骨终板细胞增殖与诱导组比较,差异无统计学意义(P> 0. 05);鹿茸多肽30 mg·L~(-1)组和鹿茸多肽50 mg·L~(-1)组能促进软骨终板细胞的增殖,与诱导组比较,差异有统计学意义(P <0. 05,P <0. 01)。qP CR检测结果:IL~(-1)β诱导组的CollagenⅡmRNA的表达降低,CollagenⅠ、CollagenⅡ、IL-6的mRNA的表达升高,与空白对照组比较,差异有统计学意义(P <0. 05);不同质量浓度的鹿茸多肽均能增强CollagenⅡmRNA的表达,降低CollagenⅠ和IL-6 mRNA的表达,差异均有统计学意义(P <0. 05,P <0. 01),30 mg·L~(-1)、50 mg·L~(-1)鹿茸多肽效果更加显著(P <0. 01)。结论:一定浓度的鹿茸多肽能减轻IL~(-1)β对软骨终板细胞增殖的抑制,升高CollagenⅡmRNA的表达,降低CollaoenⅠ、IL-6 mRNA的表达,从而延缓椎间盘的退变。
Objective: To observe the effects of pilose antler polypeptide at different concentrations on the proliferation of rat intervertebral disc chondrocyte endplate cells induced by IL~(-1)β and the expression of type Ⅰ collagen( Collagen Ⅰ),Collagen Ⅱ and interleukin-6( IL-6) mRNA. Methods: Chondrocyte endplate cells of spinal intervertebral disc were harvested from healthy SD rats( 1 month old) and cultured to the third generation of chondrocyte endplate cells. A blank control group was set up without any treatment. The induction group only added 10 μg·L~(-1) of IL~(-1)β. The administration group added 10 μg·L~(-1) of IL~(-1) 1β and different concentrations of velvet polypeptide( 10 mg·L~(-1),30 mg·L~(-1),50 mg·L~(-1),respectively) into the culture medium. The proliferation of intervertebral disc cartilage endplate cells was detected by MTT colorimetry at three time points( 24 h,48 h,72 h). The expression of CollagenI,Collagen Ⅱ and IL-6 mRNA was detected by q PCR. Results: MTT assay showed that there was no significant difference in the proliferation of chondrocyte endplate cells between 10 mg·L~(-1) group and induction group( P >0. 05); 30 mg·L~(-1) group and 50 mg·L~(-1) group could promote the proliferation of chondrocyte endplate cells,which had significant difference compared with induction group( P < 0. 05,P < 0. 01). The results of q PCR showed that the expression of CollagenⅡ mRNA in IL~(-1) β-induced group was decreased,and the mRNA expression of Collagen Ⅰ,Collagen Ⅱ and IL-6 was increased.Compared with the blank control group,the difference was statistically significant( P < 0. 05). The expression of Collagen Ⅱ and the expression of Collagen Ⅰ and IL-6 were enhanced by pilose antler polypeptide at different concentrations,and the difference was statistically significant( P < 0. 05,P < 0. 01). The effect of 30 mg·L~(-1) and 50 mg·L~(-1) pilose antler polypeptide was more significant( P < 0. 01). Conclusion: Certain concentration of pilose antler polypeptide can alleviate the inhibition of IL~(-1)β on the proliferation of cartilage endplate cells,increase the expression of Collagen Ⅱ mRNA and decrease the expression of Collaoen Ⅰand IL-6 mRNA,thus delaying the degeneration of intervertebral disc.Reference citation: LI Wenchao,LIN Yifeng,LIANG Zujian,et al. Effects of Pilose Antler Polypeptide on the Proliferation of Rat Chondrocyte Endplate Cells Induced by IL~(-1) beta and the Expression of Collagen Ⅰ,Collagen Ⅱ and IL-6 mRNA[J]. Acta Chinese Medicine,2019,34( 7) : 1454-1459.
Objective: To observe the effects of pilose antler polypeptide at different concentrations on the proliferation of rat intervertebral disc chondrocyte endplate cells induced by IL~(-1)β and the expression of type Ⅰ collagen( Collagen Ⅰ),Collagen Ⅱ and interleukin-6( IL-6) mRNA. Methods: Chondrocyte endplate cells of spinal intervertebral disc were harvested from healthy SD rats( 1 month old) and cultured to the third generation of chondrocyte endplate cells. A blank control group was set up without any treatment. The induction group only added 10 μg·L~(-1) of IL~(-1)β. The administration group added 10 μg·L~(-1) of IL~(-1) 1β and different concentrations of velvet polypeptide( 10 mg·L~(-1),30 mg·L~(-1),50 mg·L~(-1),respectively) into the culture medium. The proliferation of intervertebral disc cartilage endplate cells was detected by MTT colorimetry at three time points( 24 h,48 h,72 h). The expression of CollagenI,Collagen Ⅱ and IL-6 mRNA was detected by q PCR. Results: MTT assay showed that there was no significant difference in the proliferation of chondrocyte endplate cells between 10 mg·L~(-1) group and induction group( P >0. 05); 30 mg·L~(-1) group and 50 mg·L~(-1) group could promote the proliferation of chondrocyte endplate cells,which had significant difference compared with induction group( P < 0. 05,P < 0. 01). The results of q PCR showed that the expression of CollagenⅡ mRNA in IL~(-1) β-induced group was decreased,and the mRNA expression of Collagen Ⅰ,Collagen Ⅱ and IL-6 was increased.Compared with the blank control group,the difference was statistically significant( P < 0. 05). The expression of Collagen Ⅱ and the expression of Collagen Ⅰ and IL-6 were enhanced by pilose antler polypeptide at different concentrations,and the difference was statistically significant( P < 0. 05,P < 0. 01). The effect of 30 mg·L~(-1) and 50 mg·L~(-1) pilose antler polypeptide was more significant( P < 0. 01). Conclusion: Certain concentration of pilose antler polypeptide can alleviate the inhibition of IL~(-1)β on the proliferation of cartilage endplate cells,increase the expression of Collagen Ⅱ mRNA and decrease the expression of Collaoen Ⅰand IL-6 mRNA,thus delaying the degeneration of intervertebral disc.Reference citation: LI Wenchao,LIN Yifeng,LIANG Zujian,et al. Effects of Pilose Antler Polypeptide on the Proliferation of Rat Chondrocyte Endplate Cells Induced by IL~(-1) beta and the Expression of Collagen Ⅰ,Collagen Ⅱ and IL-6 mRNA[J]. Acta Chinese Medicine,2019,34( 7) : 1454-1459.
引文
[1]GRUNHAGEN A S,FAIRBANK J.Intervertebral disk nutrition:a review of factors influencing concentrations of nutrients and metabolites[J].Orthop Clin North Am,2011,42(4):465-477.
[2]CHRISTOPHER M.Cytokine Involvement in Biological Inflammation Related to Degenerative Disorders of the Intervertebral Disk:A Narrative Review[J].Chiropr Med,2018,17(1):54-62.
[3]张宇,赵永见,周泉,等.IL-1β诱导小鼠椎间盘软骨终板细胞炎性退变的细胞模型研究[J].免疫学杂志,2012,28(8):665-668.
[4]刘斌,金大地,瞿东滨,等.IL-1β、IL-6、TNF-α在兔腰椎软骨终板退变中的变化及作用[J].中国矫形外科杂志,2007,15(1):64-66.
[5]KARLSEN T A,SHAHDADFAR A,BRINCHMANN J E.Human primary articular chondrocytes,chondroblasts-like cells,and dedifferentiated chondrocytes:differences in gene,micro RNA,and protein expression and phenotype[J].Tissue Eng Part C Methods,2011,17(2):219-227.
[6]林一峰,直彦亮,梁祖建,等.浅谈温养督脉法治疗脊柱退行性疾病的思路与方法[J].新中医,2010,42(8):99-100.
[7]LI Y,YU J M.Research progress in structure-activity relationship of bioactive peptides[J].J Med Food,2015,18(2):147-156.
[8]ZHA E H,LI X X,LI D D,et al.Immunomodulatory effects of a 3.2kDa polypeptide from velvet antler of Cervus nippon Temminck[J].Int Immunopharmacol,2013,16(2):210-213.
[9]李振华,赵文海,冷向阳,等.鹿茸多肽对兔骨性关节炎软骨细胞增殖及凋亡调节作用的实验研究[J].世界中西医结合杂志,2009,4(10):701-703.
[10]TOMASZEWSKI K A,WALOCHA J A,MIZIA E,et al.Age-and degeneration-related variations in cell density and glycosaminoglycan content in the human cervical intervertebral disc and its endplates[J].Pol J Pathol,2015,66(3):296-301.
[11]NEWELL N,LITTLE J P,CHRISTOU A,et al.Biomechanics of the human intervertebral disc:A review of testing techniques and results[J].Mech Behav Biomed Mater,2017,69:420-434.
[12]BIBBY S R,JONES D A,RIPLEY R M,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(Phila Pa 1976)[J].Spine,2005,30(5):487-496.
[13]BENNEKER L M,HEINI P F,ALINI M,et al.2004 Young Investigator Award Winner:vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration[J].Spine,2005,30(2):167-173.
[14]BINCH A L A,SHAPIRO I M,RISBUD M.Syndecan-4 in intervertebral disc and cartilage:Saint or Synner[J]?Matrix Biol,2016,52(54):355-362.
[15]SHIRAZIADL A,TAHERI M,URBAN J P,et al.Analysis of cell viability in intervertebral disc:Effect of endplate permeability on cell population[J].Biomech,2010,43(11):1330-1336.
[16]KEPLER C K,MARKOVA D Z,DIBRA F,et al.Expression and relationship of proinflammatory chemokine RANTES/CCL5 and cytokine IL-1βin painful human intervertebral discs[J].Spine,2013,38(11):873-880.
[17]WANG G,HUANG K,DONG Y,et al.Lycori Suppresses EndplateChondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway[J].Cell Physiol Biochem,2018,45(3):1252-1269.
[18]LE MAITRE C L,HOYLAND J A,FREEMONT A J.Catabolic cytokine expression in degenerate and herniated human intervertebral discs:IL-1beta and TNFalpha expression profile[J].Arthritis Res Ther,2007,9(4):R77.
[19]李育刚.腰椎间盘突出患者血清疼痛物质及炎性应激指标的变化研究[J].中国骨与关节外科,2014,7(6):472-474.
[20]KANG J D,GEORGESCU H I,MCINTYRE LARKIN L,et al.Herniated lumbar intervertebra discs spontaneously produce matrix metalloproteinases,nitric oxide,interleukin-6,and prostaglandin[J].Spine,1996,21(3):271-277.
[21]RISBUD M V,SHAPIRO I M.Role of cytokines in intervertebral disc degeneration:pain and disc content[J].Nat Rev Rheumatol,2014,10(1):44-56.
[22]林建华,陈晓东,邓凌霄,等.大鼠软骨细胞复制性老化的体外观察[J].中国修复重建外科杂志,2007,21(11):1228-1232.
[23]蒋萍,蔚芃,赵明才,等.Ⅰ、Ⅱ型胶原蛋白对人软骨细胞生物学特性的影响[J].中国组织工程研究,18(30):4845-4849.
[24]刘兰涛,朱瑜洁,黄博,等.软骨终板干细胞动物体内成骨/软骨的初步研究[J].局解手术学杂志,2016,25(4),235-239.
[25]赵小丹,刘浩,黄富国,等.Ⅰ、Ⅱ型胶原蛋白在不同损伤类型动物模型椎间盘组织内的含量变化规律[J].华西医学,2018,11(13):84-86.
[26]刘宁.温养督脉法治疗腰椎间盘突出症临床疗效与康复观察[J].医学信息,2017,30(12):103.
[27]唐汉武,林一峰,原超,等.温通督脉法治疗退行性腰椎管狭窄症的临床研究[J].时珍国医国药,2016,27(5):1148-1150.
[28]杨光旭,魏雪苗,范红艳,等.鹿茸多肽药理学活性的研究进展[J].吉林医药学院学报,2017,38(2):126-129.
[2]CHRISTOPHER M.Cytokine Involvement in Biological Inflammation Related to Degenerative Disorders of the Intervertebral Disk:A Narrative Review[J].Chiropr Med,2018,17(1):54-62.
[3]张宇,赵永见,周泉,等.IL-1β诱导小鼠椎间盘软骨终板细胞炎性退变的细胞模型研究[J].免疫学杂志,2012,28(8):665-668.
[4]刘斌,金大地,瞿东滨,等.IL-1β、IL-6、TNF-α在兔腰椎软骨终板退变中的变化及作用[J].中国矫形外科杂志,2007,15(1):64-66.
[5]KARLSEN T A,SHAHDADFAR A,BRINCHMANN J E.Human primary articular chondrocytes,chondroblasts-like cells,and dedifferentiated chondrocytes:differences in gene,micro RNA,and protein expression and phenotype[J].Tissue Eng Part C Methods,2011,17(2):219-227.
[6]林一峰,直彦亮,梁祖建,等.浅谈温养督脉法治疗脊柱退行性疾病的思路与方法[J].新中医,2010,42(8):99-100.
[7]LI Y,YU J M.Research progress in structure-activity relationship of bioactive peptides[J].J Med Food,2015,18(2):147-156.
[8]ZHA E H,LI X X,LI D D,et al.Immunomodulatory effects of a 3.2kDa polypeptide from velvet antler of Cervus nippon Temminck[J].Int Immunopharmacol,2013,16(2):210-213.
[9]李振华,赵文海,冷向阳,等.鹿茸多肽对兔骨性关节炎软骨细胞增殖及凋亡调节作用的实验研究[J].世界中西医结合杂志,2009,4(10):701-703.
[10]TOMASZEWSKI K A,WALOCHA J A,MIZIA E,et al.Age-and degeneration-related variations in cell density and glycosaminoglycan content in the human cervical intervertebral disc and its endplates[J].Pol J Pathol,2015,66(3):296-301.
[11]NEWELL N,LITTLE J P,CHRISTOU A,et al.Biomechanics of the human intervertebral disc:A review of testing techniques and results[J].Mech Behav Biomed Mater,2017,69:420-434.
[12]BIBBY S R,JONES D A,RIPLEY R M,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(Phila Pa 1976)[J].Spine,2005,30(5):487-496.
[13]BENNEKER L M,HEINI P F,ALINI M,et al.2004 Young Investigator Award Winner:vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration[J].Spine,2005,30(2):167-173.
[14]BINCH A L A,SHAPIRO I M,RISBUD M.Syndecan-4 in intervertebral disc and cartilage:Saint or Synner[J]?Matrix Biol,2016,52(54):355-362.
[15]SHIRAZIADL A,TAHERI M,URBAN J P,et al.Analysis of cell viability in intervertebral disc:Effect of endplate permeability on cell population[J].Biomech,2010,43(11):1330-1336.
[16]KEPLER C K,MARKOVA D Z,DIBRA F,et al.Expression and relationship of proinflammatory chemokine RANTES/CCL5 and cytokine IL-1βin painful human intervertebral discs[J].Spine,2013,38(11):873-880.
[17]WANG G,HUANG K,DONG Y,et al.Lycori Suppresses EndplateChondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway[J].Cell Physiol Biochem,2018,45(3):1252-1269.
[18]LE MAITRE C L,HOYLAND J A,FREEMONT A J.Catabolic cytokine expression in degenerate and herniated human intervertebral discs:IL-1beta and TNFalpha expression profile[J].Arthritis Res Ther,2007,9(4):R77.
[19]李育刚.腰椎间盘突出患者血清疼痛物质及炎性应激指标的变化研究[J].中国骨与关节外科,2014,7(6):472-474.
[20]KANG J D,GEORGESCU H I,MCINTYRE LARKIN L,et al.Herniated lumbar intervertebra discs spontaneously produce matrix metalloproteinases,nitric oxide,interleukin-6,and prostaglandin[J].Spine,1996,21(3):271-277.
[21]RISBUD M V,SHAPIRO I M.Role of cytokines in intervertebral disc degeneration:pain and disc content[J].Nat Rev Rheumatol,2014,10(1):44-56.
[22]林建华,陈晓东,邓凌霄,等.大鼠软骨细胞复制性老化的体外观察[J].中国修复重建外科杂志,2007,21(11):1228-1232.
[23]蒋萍,蔚芃,赵明才,等.Ⅰ、Ⅱ型胶原蛋白对人软骨细胞生物学特性的影响[J].中国组织工程研究,18(30):4845-4849.
[24]刘兰涛,朱瑜洁,黄博,等.软骨终板干细胞动物体内成骨/软骨的初步研究[J].局解手术学杂志,2016,25(4),235-239.
[25]赵小丹,刘浩,黄富国,等.Ⅰ、Ⅱ型胶原蛋白在不同损伤类型动物模型椎间盘组织内的含量变化规律[J].华西医学,2018,11(13):84-86.
[26]刘宁.温养督脉法治疗腰椎间盘突出症临床疗效与康复观察[J].医学信息,2017,30(12):103.
[27]唐汉武,林一峰,原超,等.温通督脉法治疗退行性腰椎管狭窄症的临床研究[J].时珍国医国药,2016,27(5):1148-1150.
[28]杨光旭,魏雪苗,范红艳,等.鹿茸多肽药理学活性的研究进展[J].吉林医药学院学报,2017,38(2):126-129.