葡萄糖依赖性肠促胰岛素类似物DAIa2GIP对大鼠膝关节损伤软骨的保护效应
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摘要
背景:已有研究证实,葡萄糖依赖性肠促胰岛素类似物DAla2GIP可直接调节成骨细胞的生长发育,但有关其在软骨细胞上的作用鲜有报道。目的:探讨葡萄糖依赖性肠促胰岛素类似物DAIa2GIP对SD大鼠骨性关节炎软骨损伤的保护效应及潜在机制。方法:将50只雄性SD大鼠(山西医科大学实验动物中心提供)随机分5组,每组10只:正常对照组不进行任何干预;模型对照组、模型治疗组切断双后膝关节前交叉韧带、内侧副韧带并摘除内侧半月板;假手术对照组、假手术治疗组只打开双侧膝关节囊。造模后第8周,模型治疗组、假手术治疗组腹腔注射25 nmol/kg的DAIa2GIP,模型对照组、假手术对照组腹腔注射等量生理盐水,2次/周,注射8周。造模16周后,取各组大鼠膝关节,进行苏木精-伊红染色、番红O-固绿染色、透射电镜观察及免疫组织化学染色。实验方案经山西医科大学伦理委员会批准(2017008)。结果与结论:①苏木精-伊红染色显示,模型对照组软骨4层结构不清;模型治疗组软骨可见4层结构,软骨层较模型对照组增厚;其余3组软骨表面光滑,4层结构清晰;②番红O-固绿染色显示,模型对照组蛋白多糖表达明显不均匀,软骨厚度变薄;模型治疗组蛋白多糖表达基本均匀,软骨厚度较模型对照组增加;其余3组蛋白多糖表达均匀,软骨厚度适中;③透射电镜显示,模型对照组软骨细胞胞质内可见脂滴,线粒体、粗面内质网等细胞器消失;模型治疗组可见数量不等线粒体、粗面内质网等细胞器,并且结构基本恢复正常;其余组细胞结构正常;④免疫组织化学染色显示,与正常对照组比较,模型对照组胶原酶13蛋白表达升高(P <0.05),Ⅱ型胶原蛋白表达降低(P <0.05);与模型对照组比较,模型治疗组胶原酶13蛋白表达降低(P <0.05),Ⅱ型胶原蛋白表达升高(P <0.05);⑤结果表明,DAIa2GIP可能通过抑制关节软骨中胶原酶13表达、减少Ⅱ型胶原的破坏,对SD大鼠骨性关节炎中关节软骨发挥保护作用。
BACKGROUND: Glucose-dependent incretin analogue DAIa2 GIP has been shown to directly regulate the growth and development of osteoblasts, but its effect on chondrocytes is rarely reported.OBJECTIVE: To investigate the protective effect and potential mechanism of glucose-dependent incretin analogue DAIa2 GIP on cartilage injury in Sprague-Dawley rats with osteoarthritis.METHODS: Fifty male Sprague-Dawley rats provided by Laboratory Animal Center of Shanxi Medical University were randomly divided into five groups(n=10/group): normal control group(no intervention), model control group(anterior cruciate ligament and medial collateral ligament resection, and medial meniscus removal at bilateral posterior knees + normal saline), model treatment group(anterior cruciate ligament and medial collateral ligament resection, and medial meniscus removal at bilateral posterior knees + 25 nmol/kg DAIa2 GIP) and sham control group(opening articular capsule of bilateral knees + normal saline), and sham treatment group(opening articular capsule of bilateral knees + 25 nmol/kg DAIa2 GIP). The intraperitoneal administration was given at 8 weeks after modeling, twice weekly for 8 consecutive weeks. The knee joint was removed at 16 weeks after modeling for hematoxylin-eosin staining, safranin O-fast green staining,transmission electron microscope and immunohistochemistry. The study was approved by the Ethics Committee of Shanxi Medical University,approval number: 2017008.RESULTS AND CONCLUSION:(1) Hematoxylin-eosin staining: the four layers of the cartilage structure in the model control group were obscure.The four layers of the cartilage structure were visible in the model treatment group, and the cartilage lay was thicker than that in the model group. In the other three groups, the cartilage surface was smooth with clear four layers.(2) Safranin O-fast green staining: the expression of proteoglycan in the model control group was uneven, and cartilage thickness was thin. The expression of proteoglycan in the model treatment group was even, and the cartilage thickness was increased compared with the model control group. The expression of proteoglycan in the other three groups was even,with moderate cartilage thickness.(3) Transmission electron microscope: there were lipid droplets in the model control group, and mitochondria and rough endoplasmic reticulum disappeared. In the model treatment group, some mitochondria and rough endoplasmic reticulum were observed, and the structure returned to be normal. The other three groups showed the normal structure.(4) Immunohistochemistry: compared with the normal control group, the expression level of collagenase 13 was significantly increased(P < 0.05), and the expression level of collagen type II was significantly decreased in the model control group(P < 0.05). Compared with the model control group, the expression level of collagenase 13 was significantly decreased(P < 0.05), and the expression level of collagen type II was significantly increased in the model treatment group(P < 0.05).(5)To conclude, DAIa2 GIP exerts protective effect on articular cartilage in Sprague-Dawley rats with osteoarthritis possibly by inhibiting the expression of collagenase 13 and reducing collagen type II destruction in articular cartilage.
BACKGROUND: Glucose-dependent incretin analogue DAIa2 GIP has been shown to directly regulate the growth and development of osteoblasts, but its effect on chondrocytes is rarely reported.OBJECTIVE: To investigate the protective effect and potential mechanism of glucose-dependent incretin analogue DAIa2 GIP on cartilage injury in Sprague-Dawley rats with osteoarthritis.METHODS: Fifty male Sprague-Dawley rats provided by Laboratory Animal Center of Shanxi Medical University were randomly divided into five groups(n=10/group): normal control group(no intervention), model control group(anterior cruciate ligament and medial collateral ligament resection, and medial meniscus removal at bilateral posterior knees + normal saline), model treatment group(anterior cruciate ligament and medial collateral ligament resection, and medial meniscus removal at bilateral posterior knees + 25 nmol/kg DAIa2 GIP) and sham control group(opening articular capsule of bilateral knees + normal saline), and sham treatment group(opening articular capsule of bilateral knees + 25 nmol/kg DAIa2 GIP). The intraperitoneal administration was given at 8 weeks after modeling, twice weekly for 8 consecutive weeks. The knee joint was removed at 16 weeks after modeling for hematoxylin-eosin staining, safranin O-fast green staining,transmission electron microscope and immunohistochemistry. The study was approved by the Ethics Committee of Shanxi Medical University,approval number: 2017008.RESULTS AND CONCLUSION:(1) Hematoxylin-eosin staining: the four layers of the cartilage structure in the model control group were obscure.The four layers of the cartilage structure were visible in the model treatment group, and the cartilage lay was thicker than that in the model group. In the other three groups, the cartilage surface was smooth with clear four layers.(2) Safranin O-fast green staining: the expression of proteoglycan in the model control group was uneven, and cartilage thickness was thin. The expression of proteoglycan in the model treatment group was even, and the cartilage thickness was increased compared with the model control group. The expression of proteoglycan in the other three groups was even,with moderate cartilage thickness.(3) Transmission electron microscope: there were lipid droplets in the model control group, and mitochondria and rough endoplasmic reticulum disappeared. In the model treatment group, some mitochondria and rough endoplasmic reticulum were observed, and the structure returned to be normal. The other three groups showed the normal structure.(4) Immunohistochemistry: compared with the normal control group, the expression level of collagenase 13 was significantly increased(P < 0.05), and the expression level of collagen type II was significantly decreased in the model control group(P < 0.05). Compared with the model control group, the expression level of collagenase 13 was significantly decreased(P < 0.05), and the expression level of collagen type II was significantly increased in the model treatment group(P < 0.05).(5)To conclude, DAIa2 GIP exerts protective effect on articular cartilage in Sprague-Dawley rats with osteoarthritis possibly by inhibiting the expression of collagenase 13 and reducing collagen type II destruction in articular cartilage.
引文
[1]Yuan GH,Masukohongo K,Kato T,et al.Immunologic intervention in the pathogenesis of osteoarthritis.Arthritis Rheumatol.2010;48(3):602-611.
[2]韩廷成,周临东,董松林.中医外治法治疗膝骨关节炎的现状与分析[J].现代中西医结合杂志,2012,21(5):566-568.
[3]Gaudin-Audrain C,Irwin N,Mansur S,et al.Glucose-dependent insulinotropic polypeptide receptor deficiency leads to modifications of trabecular bone volume and quality in mice.Bone.2013;53(1):221-230.
[4]Shimazu-Kuwahara S,Kanemaru Y,Harada N,et al.Glucosedependent insulinotropic polypeptide deficiency reduced fat accumulation and insulin resistance,but deteriorated bone loss in ovariectomized mice.J Diabetes Investig.2018.doi:10.1111/jdi.12978.[Epub ahead of print]
[5]Kieffer TJ,McIntosh CH,Pederson RA.Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase iv.Endocrinology.1995;136(8):3585-3596.
[6]Usdin TB,Mezey E,Button DC,et al.Gastric inhibitory polypeptide receptor,a member of the secretin-vasoactive intestinal peptide receptor family,is widely distributed in peripheral organs and the brain.Endocrinology.1993;133(6):2861-2870.
[7]Joo E,Harada N,Yamane S,et al.Inhibition of gastric inhibitory polypeptide receptor signaling in adipose tissue reduces insulin resistance and hepatic steatosis in high-fat diet-fed mice.Diabetes.2017;66(4):868-879.
[8]Nasteska D,Harada N,Suzuki K,et al.Chronic reduction of gip secretion alleviates obesity and insulin resistance under high-fat diet conditions.Diabetes.2014;63(7):2332-2343.
[9]Torekov SS,Harsl?f T,Rejnmark L,et al.A functional amino acid substitution in the glucose-dependent insulinotropic polypeptide receptor(GIPR)gene is associated with lower bone mineral density and increased fracture risk.J Clin Endocrinol Metab.2014;99(4):E729-733.
[10]Holst JJ,Windelov JA,Boer GA,et al.Searching for the physiological role of glucose-dependent insulinotropic polypeptide.J Diabetes Investig.2016;7 Suppl 1:8-12.
[11]Mieczkowska A,Irwin N,Flatt PR,et al.Glucose-dependent insulinotropic polypeptide(gip)receptor deletion leads to reduced bone strength and quality.Bone.2013;56(2):337-342.
[12]Mai X,Lu Z,Lei Z,et al.Phosphorylation of osteopontin in osteoarthritis degenerative cartilage and its effect on matrix metalloprotease 13.Rheumatol Int.2013;33(5):1313-1319.
[13]Wayne JS,Mcdowell CL,Shields KJ,et al.In vivo response of polylactic acid-alginate scaffolds and bone marrow-derived cells for cartilage tissue engineering.Tissue Eng Part A.2005;11(5-6):953-963.
[14]Mankin HJ,Dorfman H,Lippiello L,et al.Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips.Ii.Correlation of morphology with biochemical and metabolic data.B J Bone Joint Surg Am.1971;53(3):523-537.
[15]姜旭,吴成爱,王莹,等.膝关节骨性关节炎中wisp-1调控机制的研究[J].中国骨质疏松杂志,2015,21(5):537-540.
[16]Xu M,Zhang L,Zhao L,et al.Phosphorylation of osteopontin in osteoarthritis degenerative cartilage and its effect on matrix metalloprotease 13.Rheumatol Int.2013;33(5):1313-1319.
[17]Li Y,Lin T,Duan Y,et al.Upregulation of mmp-13 and timp-1expression in response to mechanical strain in mc3t3-e1osteoblastic cells.Bmc Res Notes.2010;3(1):309.
[18]Mieczkowska A,Bouvard B,Chappard D,et al.Glucosedependent insulinotropic polypeptide(gip)directly affects collagen fibril diameter and collagen cross-linking in osteoblast cultures.Bone.2015;74:29-36.
[19]Mabilleau G,Mieczkowska A,Irwin N,et al.Beneficial effects of a n-terminally modified gip agonist on tissue-level bone material properties.Bone.2014;63:61-68.
[20]余洁,许岭翎.肠促胰素与骨代谢[J].中华糖尿病杂志,2017,9(3):200-202.
[21]吴松,石俊俊,卫定禄,等.Dala2gip对淀粉样前体蛋白/早老素1转基因小鼠膝关节软骨损伤保护效应的研究[J].中华实验外科杂志,2017,34(11):1861-1864.
[22]邓莉,王海波.膝骨性关节炎动物模型的研究进展[J].广东医学,2018,56(12):1895-1897
[23]Duffy AM,H?lscher C.The incretin analogue d-ala 2 gip reduces plaque load,astrogliosis and oxidative stress in an app/ps1mouse model of alzheimer’s disease.Neuroscience.2013;228:294-300.
[24]李保驰,王维山,董金波,等.MMP3、MMP13在骨性关节炎患者滑膜中的表达及意义[J].中国骨质疏松杂志,2014,20(6):593-596.
[25]包刚,刘永,刘亚丽,等.MMP-13在骨性关节炎滑膜组织中的表达分析[J].中国实验诊断学,2016,20(9):1548-1549.
[26]Hughes A,Oxford AE,Tawara K,et al.Endoplasmic reticulum stress and unfolded protein response in cartilage pathophysiology;contributing factors to apoptosis and osteoarthriti.Int J Mol Sci.2017;18(3).pii:E665.doi:10.3390/ijms18030665.
[27]Li NG,Shi ZH,Tang YP,et al.New hope for the treatment of osteoarthritis through selective inhibition of MMP-13.Curr Med Chem.2011;18(7):977-1001.
[28]Phull AR,Eo SH,Abbas Q,et al.Applications of chondrocytebased cartilage engineering:An overview.BioMed Res Int.2016;2016(2):1879837.
[29]Bollag R,Zhong Q,Ding K,et al.Glucose-dependent insulinotropic peptide is an integrative hormone with osteotropic effects.Mol Cell Endocrinol.2001;177(1-2):35-41.
[30]Zhong Q,Ding KH,Mulloy AL,et al.Glucose-dependent insulinotropic peptide stimulates proliferation and tgf-beta release from mg-63 cells.Peptides.2003;24(4):611-616.
[31]Howard S,Anastassiades T.Differential effects of bone associated factors on newly synthesized anionic glycoconjugates by articular chondrocyte cultures from adult and immature bovines.J Rheumatol.1993;20(12):2083-2094.
[2]韩廷成,周临东,董松林.中医外治法治疗膝骨关节炎的现状与分析[J].现代中西医结合杂志,2012,21(5):566-568.
[3]Gaudin-Audrain C,Irwin N,Mansur S,et al.Glucose-dependent insulinotropic polypeptide receptor deficiency leads to modifications of trabecular bone volume and quality in mice.Bone.2013;53(1):221-230.
[4]Shimazu-Kuwahara S,Kanemaru Y,Harada N,et al.Glucosedependent insulinotropic polypeptide deficiency reduced fat accumulation and insulin resistance,but deteriorated bone loss in ovariectomized mice.J Diabetes Investig.2018.doi:10.1111/jdi.12978.[Epub ahead of print]
[5]Kieffer TJ,McIntosh CH,Pederson RA.Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase iv.Endocrinology.1995;136(8):3585-3596.
[6]Usdin TB,Mezey E,Button DC,et al.Gastric inhibitory polypeptide receptor,a member of the secretin-vasoactive intestinal peptide receptor family,is widely distributed in peripheral organs and the brain.Endocrinology.1993;133(6):2861-2870.
[7]Joo E,Harada N,Yamane S,et al.Inhibition of gastric inhibitory polypeptide receptor signaling in adipose tissue reduces insulin resistance and hepatic steatosis in high-fat diet-fed mice.Diabetes.2017;66(4):868-879.
[8]Nasteska D,Harada N,Suzuki K,et al.Chronic reduction of gip secretion alleviates obesity and insulin resistance under high-fat diet conditions.Diabetes.2014;63(7):2332-2343.
[9]Torekov SS,Harsl?f T,Rejnmark L,et al.A functional amino acid substitution in the glucose-dependent insulinotropic polypeptide receptor(GIPR)gene is associated with lower bone mineral density and increased fracture risk.J Clin Endocrinol Metab.2014;99(4):E729-733.
[10]Holst JJ,Windelov JA,Boer GA,et al.Searching for the physiological role of glucose-dependent insulinotropic polypeptide.J Diabetes Investig.2016;7 Suppl 1:8-12.
[11]Mieczkowska A,Irwin N,Flatt PR,et al.Glucose-dependent insulinotropic polypeptide(gip)receptor deletion leads to reduced bone strength and quality.Bone.2013;56(2):337-342.
[12]Mai X,Lu Z,Lei Z,et al.Phosphorylation of osteopontin in osteoarthritis degenerative cartilage and its effect on matrix metalloprotease 13.Rheumatol Int.2013;33(5):1313-1319.
[13]Wayne JS,Mcdowell CL,Shields KJ,et al.In vivo response of polylactic acid-alginate scaffolds and bone marrow-derived cells for cartilage tissue engineering.Tissue Eng Part A.2005;11(5-6):953-963.
[14]Mankin HJ,Dorfman H,Lippiello L,et al.Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips.Ii.Correlation of morphology with biochemical and metabolic data.B J Bone Joint Surg Am.1971;53(3):523-537.
[15]姜旭,吴成爱,王莹,等.膝关节骨性关节炎中wisp-1调控机制的研究[J].中国骨质疏松杂志,2015,21(5):537-540.
[16]Xu M,Zhang L,Zhao L,et al.Phosphorylation of osteopontin in osteoarthritis degenerative cartilage and its effect on matrix metalloprotease 13.Rheumatol Int.2013;33(5):1313-1319.
[17]Li Y,Lin T,Duan Y,et al.Upregulation of mmp-13 and timp-1expression in response to mechanical strain in mc3t3-e1osteoblastic cells.Bmc Res Notes.2010;3(1):309.
[18]Mieczkowska A,Bouvard B,Chappard D,et al.Glucosedependent insulinotropic polypeptide(gip)directly affects collagen fibril diameter and collagen cross-linking in osteoblast cultures.Bone.2015;74:29-36.
[19]Mabilleau G,Mieczkowska A,Irwin N,et al.Beneficial effects of a n-terminally modified gip agonist on tissue-level bone material properties.Bone.2014;63:61-68.
[20]余洁,许岭翎.肠促胰素与骨代谢[J].中华糖尿病杂志,2017,9(3):200-202.
[21]吴松,石俊俊,卫定禄,等.Dala2gip对淀粉样前体蛋白/早老素1转基因小鼠膝关节软骨损伤保护效应的研究[J].中华实验外科杂志,2017,34(11):1861-1864.
[22]邓莉,王海波.膝骨性关节炎动物模型的研究进展[J].广东医学,2018,56(12):1895-1897
[23]Duffy AM,H?lscher C.The incretin analogue d-ala 2 gip reduces plaque load,astrogliosis and oxidative stress in an app/ps1mouse model of alzheimer’s disease.Neuroscience.2013;228:294-300.
[24]李保驰,王维山,董金波,等.MMP3、MMP13在骨性关节炎患者滑膜中的表达及意义[J].中国骨质疏松杂志,2014,20(6):593-596.
[25]包刚,刘永,刘亚丽,等.MMP-13在骨性关节炎滑膜组织中的表达分析[J].中国实验诊断学,2016,20(9):1548-1549.
[26]Hughes A,Oxford AE,Tawara K,et al.Endoplasmic reticulum stress and unfolded protein response in cartilage pathophysiology;contributing factors to apoptosis and osteoarthriti.Int J Mol Sci.2017;18(3).pii:E665.doi:10.3390/ijms18030665.
[27]Li NG,Shi ZH,Tang YP,et al.New hope for the treatment of osteoarthritis through selective inhibition of MMP-13.Curr Med Chem.2011;18(7):977-1001.
[28]Phull AR,Eo SH,Abbas Q,et al.Applications of chondrocytebased cartilage engineering:An overview.BioMed Res Int.2016;2016(2):1879837.
[29]Bollag R,Zhong Q,Ding K,et al.Glucose-dependent insulinotropic peptide is an integrative hormone with osteotropic effects.Mol Cell Endocrinol.2001;177(1-2):35-41.
[30]Zhong Q,Ding KH,Mulloy AL,et al.Glucose-dependent insulinotropic peptide stimulates proliferation and tgf-beta release from mg-63 cells.Peptides.2003;24(4):611-616.
[31]Howard S,Anastassiades T.Differential effects of bone associated factors on newly synthesized anionic glycoconjugates by articular chondrocyte cultures from adult and immature bovines.J Rheumatol.1993;20(12):2083-2094.