铝暴露致大鼠骨损伤的分子机制
摘要
为探讨铝暴露致大鼠骨损伤的分子机制,将100只4周龄清洁级Wistar大鼠随机均分成染铝组(430mg·L-1,Al3+)与对照组(蒸馏水),设立5个观测点,每隔30d处死染铝大鼠和对照大鼠各10只。记录大鼠临床症状和体重;用火焰原子吸收分光光度法测定血清、骨与软骨中铝(Al)、钙(Ca)、镁(Mg)、锌(Zn)、铁(Fe)、铜(Cu)、锰(Mn)、硼(B)、锶(Sr)和硒(Se)含量;放射免疫法检测血清中骨钙素(BGP)、甲状旁腺素(PTH)和降钙素(CT)含量;固相夹心ELISA法检测血清中1,25-二羟基维生素D3(1,25-(OH)2-D3)、骨源性碱性磷酸酶(B-ALP)、抗酒石酸酸性磷酸酶(TRACP-5b)、Ⅰ型前胶原羧基末端前肽(PICP)、Ⅰ型胶原C末端肽(CTX-Ⅰ)、Ⅱ型胶原(collagenⅡ)和Ⅱ型胶原C末端肽(CTX-Ⅱ)和蛋白聚糖(Aggrecan)含量;实时荧光定量PCR检测转化生长因子β1(TGF-β1)和骨形态发生蛋白-2(BMP-2)基因mRNA的表达量;双能X线骨密度仪检测骨密度(BMD);并对铝暴露大鼠的骨与软骨组织形态学、骨功能细胞和软骨细胞超微结构进行观察。结果表明:
1大鼠灌胃结晶三氯化铝的LD50为1283.60 mg/kg体重,以430mg/L(Al3+)饮水染铝可成功复制慢性铝中毒大鼠模型。
2光镜观察发现,染铝大鼠骨基质中出现大量软骨样不成熟骨基质,并在染铝120~150d有破骨吸收增强现象;软骨基质结构松散,双细胞“背靠背”分布减少。电镜观察发现,骨细胞细胞核固缩,细胞器数量明显减少,胞质内电子密度较低,线粒体嵴断裂,粗面内质网扩张脱颗粒,在染铝后期线粒体和内质网等膜性结构呈空泡变性;软骨细胞细胞器数量减少,脂滴颗粒减少,基质纤维短小,排列紊乱。
3铝暴露大鼠血清中PTH、CT和1,25-(OH)2-D3含量分别从染铝90(P<0.01)、30(P<0.05)和90d(P<0.01)显著低于对照组,说明铝暴露可通过激素调节途径来间接影响骨代谢。
4铝暴露大鼠骨中矿物质元素Ca、Mg、P及微量元素Zn、Fe、Cu、Mn、B、Sr和Se的含量均不同程度的低于对照组,说明铝暴露可抑制骨矿化元素的沉积。
5血清中BGP,PICP和B-ALP含量分别从染铝60d (P<0.05)、60d(P<0.01)、90d(P<0.01)显著低于对照组;局部调节因子TGF-β1和BMP-2基因mRNA表达量从染铝90d和60d显著低于对照组(P<0.01);说明铝暴露可抑制骨功能细胞活性、代谢酶活性和局部调节因子表达,导致骨生成抑制。
6血清中CTX-Ⅰ从染铝90d极显著高于对照组(P<0.01),TRACP-5b在染铝30d、60d和150d显著低于对照组(P<0.05),但在染铝120d高于对照组。说明铝暴露可诱导代偿性骨吸收增强,导致骨损伤。
7染铝组整体股骨和胫骨BMD在整个实验周期中与对照组相比差异不显著性,但股骨干骺端BMD在染铝120~150d时显著低于对照组(P<0.05),4~6腰椎(L)在染铝150d时显著低于对照组(P<0.05),说明长期铝暴露可诱发骨丢失。
8血清中Ⅱ型胶原含量从染铝30d开始显著低于对照组(P<0.05),CTX-Ⅱ从染铝90d开始极显著高于对照组(P<0.01),Aggrecan在90~120 d显著高于对照组(P<0.05)。说明铝暴露可抑制软骨细胞活性和软骨胶原的合成,并在染铝90d后诱发软骨基质降解增强,导致软骨损伤。
The molecule mechanism of bone injuries induced by aluminum(Al) exposure in rats was studied. Wistar rats (n=100) were divided randomly into two groups. Experimental rats were given drinking water containing aluminum chloride (AlCl3, 430 mg Al3+/L), and control rats were given distilled water for up to 150 days. Ten rats were sacrificed in each group every 30 days. The levels of Al, calcium (Ca), phosphorus (P), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), selenium (Se), boron (B) and strontium (Sr) in serum, bone and cartilage were measured. The serum levels of parathyroid hormone (PTH), calcitonin (CT) and osteocalcin (BGP) were detected using 125I radioimmunoassay (RIA) kits. The serum levels of 1, 25-dihydroxyvitaminD3 (1,25-(OH)2-D3), bone alkaline phosphatase(B-ALP), tartrate-resistantacid phosphatase-5b (TRACP-5b), carboxyterminal propeptide of type I procollagen (PICP), C-telopeptide of type I collagen (CTX-Ⅰ), collagenⅡand C-telopeptide of typeⅡcollagen (CTX-Ⅱ) and aggrecan were detected by solid phase sandwich ELISA. The mRNA expression of TGF-β1 and BMP-2 were detected by real-time Q-PCR. The histomorphology of bone and cartilage and ultrastructure of osteocyte and chondrocyte were observed. The results were shown as follows.
1 The LD50 of AlCl3·6H2O on rats was 1283.60 mg/kg, and the Al exposure model was established successfully in rats on 430mg/L (Al3+).
2 Under optical microscope, much cartilage-like immature matrix was discovered in the bone, and bone resorption was enhanced on days 120~150. The structure of cartilage matrix was loose and the number of chondrocyte was decreased. under electron microscope, the number of organelle and cytoplasmic electronic density decreased, nucleus pyknosed, and mitochondrion mitochondrion crista fractured. Rough endoplasmic reticulum was dilatated and its pellets dropped with the increase of the Al exposure time, and membranaceous structures exhibited vacuolar degeneration. The numbers of organelles and lipid drops decreased in chondrocyte, and matrix fiber became short and disordered with the increase of the Al exposure time.
3 The serum levels of PTH, CT and 1,25-(OH)2-D3 were significantly lower in the Al-treated rats than in the control ones from days 90 (P<0.01), 30 (P<0.05) and 90 (P<0.01), respectively,which show that long-term Al exposure can indirectly inference with bone metabolism acting on hormoral regulation pathway.
4 Al-treated rats showed lower deposition of Ca, Mg, P, Zn, Fe, Cu, Mn, B, Sr and Se compared with control ones. This indicates that Al exposure inhibits the deposition of mineralization elements.
5 The serum levels of BGP, B-ALP and PICP were significantly lower in the Al-treated rats than in the control ones from days 60 (P<0.05), 60 (P<0.01) and 90 (P<0.01), respectively. The mRNA expression of TGF-β1 and BMP-2 were significantly lower in the Al-treated rats than in the control ones from days 90 (P<0.01) and 60 (P<0.01), respectively. These findings suggest that long-term Al exposure inhibits the osteoblasts activity, metabolic enzymes activity and local regulating factor expression. As a result, bone formation was inhibited.
6 Al-treated rats showed significantly higher serum levels of CTX-Ⅰcompared with control rats from day 90 (P<0.01), and lower serum levels of TRACP-5b on days 30, 60 and 150 (P<0.05) while the levels were reversed on day 120. These findings indicate that long-term Al exposure can induce bone injuries by increasing compensatory bone resorption.
7 There are no statistical significance between Al-treated rats and control ones in the BMD of whole femurs and tibias. However, the BMD of femoral metaphysis and lumber (L4~L6) were significantly lower in the Al-treated rats than in the control ones on days 120~150 and 150 (both P<0.05), respectively. This indicates that long-term Al exposure can induce bone lose.
8 The serum levels of collagenⅡwere significantly lower in the Al-treated rats than in the control ones from day 30 (P<0.05). The serum CTX-Ⅱand aggrecan levels were significantly higher in the Al-treated rats than in the control ones on day 90~150 (P<0.01) and 90~120 (P<0.05), respectively. These findings indicate that Al exposure can inhibit chondrocyte activity and the synthesis of collagenstroma, enhance collagenstroma degradation after 90 days of Al exposure, and then result in cartilage injuries.
1大鼠灌胃结晶三氯化铝的LD50为1283.60 mg/kg体重,以430mg/L(Al3+)饮水染铝可成功复制慢性铝中毒大鼠模型。
2光镜观察发现,染铝大鼠骨基质中出现大量软骨样不成熟骨基质,并在染铝120~150d有破骨吸收增强现象;软骨基质结构松散,双细胞“背靠背”分布减少。电镜观察发现,骨细胞细胞核固缩,细胞器数量明显减少,胞质内电子密度较低,线粒体嵴断裂,粗面内质网扩张脱颗粒,在染铝后期线粒体和内质网等膜性结构呈空泡变性;软骨细胞细胞器数量减少,脂滴颗粒减少,基质纤维短小,排列紊乱。
3铝暴露大鼠血清中PTH、CT和1,25-(OH)2-D3含量分别从染铝90(P<0.01)、30(P<0.05)和90d(P<0.01)显著低于对照组,说明铝暴露可通过激素调节途径来间接影响骨代谢。
4铝暴露大鼠骨中矿物质元素Ca、Mg、P及微量元素Zn、Fe、Cu、Mn、B、Sr和Se的含量均不同程度的低于对照组,说明铝暴露可抑制骨矿化元素的沉积。
5血清中BGP,PICP和B-ALP含量分别从染铝60d (P<0.05)、60d(P<0.01)、90d(P<0.01)显著低于对照组;局部调节因子TGF-β1和BMP-2基因mRNA表达量从染铝90d和60d显著低于对照组(P<0.01);说明铝暴露可抑制骨功能细胞活性、代谢酶活性和局部调节因子表达,导致骨生成抑制。
6血清中CTX-Ⅰ从染铝90d极显著高于对照组(P<0.01),TRACP-5b在染铝30d、60d和150d显著低于对照组(P<0.05),但在染铝120d高于对照组。说明铝暴露可诱导代偿性骨吸收增强,导致骨损伤。
7染铝组整体股骨和胫骨BMD在整个实验周期中与对照组相比差异不显著性,但股骨干骺端BMD在染铝120~150d时显著低于对照组(P<0.05),4~6腰椎(L)在染铝150d时显著低于对照组(P<0.05),说明长期铝暴露可诱发骨丢失。
8血清中Ⅱ型胶原含量从染铝30d开始显著低于对照组(P<0.05),CTX-Ⅱ从染铝90d开始极显著高于对照组(P<0.01),Aggrecan在90~120 d显著高于对照组(P<0.05)。说明铝暴露可抑制软骨细胞活性和软骨胶原的合成,并在染铝90d后诱发软骨基质降解增强,导致软骨损伤。
The molecule mechanism of bone injuries induced by aluminum(Al) exposure in rats was studied. Wistar rats (n=100) were divided randomly into two groups. Experimental rats were given drinking water containing aluminum chloride (AlCl3, 430 mg Al3+/L), and control rats were given distilled water for up to 150 days. Ten rats were sacrificed in each group every 30 days. The levels of Al, calcium (Ca), phosphorus (P), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), selenium (Se), boron (B) and strontium (Sr) in serum, bone and cartilage were measured. The serum levels of parathyroid hormone (PTH), calcitonin (CT) and osteocalcin (BGP) were detected using 125I radioimmunoassay (RIA) kits. The serum levels of 1, 25-dihydroxyvitaminD3 (1,25-(OH)2-D3), bone alkaline phosphatase(B-ALP), tartrate-resistantacid phosphatase-5b (TRACP-5b), carboxyterminal propeptide of type I procollagen (PICP), C-telopeptide of type I collagen (CTX-Ⅰ), collagenⅡand C-telopeptide of typeⅡcollagen (CTX-Ⅱ) and aggrecan were detected by solid phase sandwich ELISA. The mRNA expression of TGF-β1 and BMP-2 were detected by real-time Q-PCR. The histomorphology of bone and cartilage and ultrastructure of osteocyte and chondrocyte were observed. The results were shown as follows.
1 The LD50 of AlCl3·6H2O on rats was 1283.60 mg/kg, and the Al exposure model was established successfully in rats on 430mg/L (Al3+).
2 Under optical microscope, much cartilage-like immature matrix was discovered in the bone, and bone resorption was enhanced on days 120~150. The structure of cartilage matrix was loose and the number of chondrocyte was decreased. under electron microscope, the number of organelle and cytoplasmic electronic density decreased, nucleus pyknosed, and mitochondrion mitochondrion crista fractured. Rough endoplasmic reticulum was dilatated and its pellets dropped with the increase of the Al exposure time, and membranaceous structures exhibited vacuolar degeneration. The numbers of organelles and lipid drops decreased in chondrocyte, and matrix fiber became short and disordered with the increase of the Al exposure time.
3 The serum levels of PTH, CT and 1,25-(OH)2-D3 were significantly lower in the Al-treated rats than in the control ones from days 90 (P<0.01), 30 (P<0.05) and 90 (P<0.01), respectively,which show that long-term Al exposure can indirectly inference with bone metabolism acting on hormoral regulation pathway.
4 Al-treated rats showed lower deposition of Ca, Mg, P, Zn, Fe, Cu, Mn, B, Sr and Se compared with control ones. This indicates that Al exposure inhibits the deposition of mineralization elements.
5 The serum levels of BGP, B-ALP and PICP were significantly lower in the Al-treated rats than in the control ones from days 60 (P<0.05), 60 (P<0.01) and 90 (P<0.01), respectively. The mRNA expression of TGF-β1 and BMP-2 were significantly lower in the Al-treated rats than in the control ones from days 90 (P<0.01) and 60 (P<0.01), respectively. These findings suggest that long-term Al exposure inhibits the osteoblasts activity, metabolic enzymes activity and local regulating factor expression. As a result, bone formation was inhibited.
6 Al-treated rats showed significantly higher serum levels of CTX-Ⅰcompared with control rats from day 90 (P<0.01), and lower serum levels of TRACP-5b on days 30, 60 and 150 (P<0.05) while the levels were reversed on day 120. These findings indicate that long-term Al exposure can induce bone injuries by increasing compensatory bone resorption.
7 There are no statistical significance between Al-treated rats and control ones in the BMD of whole femurs and tibias. However, the BMD of femoral metaphysis and lumber (L4~L6) were significantly lower in the Al-treated rats than in the control ones on days 120~150 and 150 (both P<0.05), respectively. This indicates that long-term Al exposure can induce bone lose.
8 The serum levels of collagenⅡwere significantly lower in the Al-treated rats than in the control ones from day 30 (P<0.05). The serum CTX-Ⅱand aggrecan levels were significantly higher in the Al-treated rats than in the control ones on day 90~150 (P<0.01) and 90~120 (P<0.05), respectively. These findings indicate that Al exposure can inhibit chondrocyte activity and the synthesis of collagenstroma, enhance collagenstroma degradation after 90 days of Al exposure, and then result in cartilage injuries.
引文
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