장기간의 고농도 카페인 노출이 사춘기 골 길이 성장에 미치는 영향에 대한 연구

Abstract

Caffeine has been known to adversely affect fetal skeletal growth and increased incidence of delayed and abnormal fetal skeletal development. Chronic caffeine intake also decreases fetal hepatic insulin-like growth factor 1 production. Thus, it is conceivable that caffeine may disturb long bone growth during pubertal growth spurt. We aimed to investigate the impact of high caffeine exposure on longitudinal bone growth throughout puberty. A total of 51 immature male rats were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 or 180 mg/kg/day for 4 weeks. After sacrifice, final length and weight of long bones were measured, and the tibia processed for histomorphometric analysis. Caffeine caused a significant decrease in body weight gain, accompanied with proportional decreases in lean body mass and body fat. Bone mass and osteogenic activity in vivo assessed using DXA and 18F-NaF PET, respectively were significantly decreased in caffeine-fed groups. Rats fed with caffeine showed a significant shortening and lightening of leg bones and the spinal column compared to the controls. Caffeine feeding delayed changes in the height of growth plates with age along with reduced cell proliferation. In addition, bone formation parameters observed in the secondary spongiosa also significantly reduced in caffeine-fed groups. The direct effect of caffeine on chondrogenic differentiation was evaluated using isolated primary rat chondrocytes. Chondrogenic differentiation markers such as extracellular matrix production, calcification and alkaline phosphatase activity were significantly decreased in caffeine treated cells in vitro. Consistent with these results, expression levels of chondrogenic marker genes such as aggrecan (ACAN), collagen type II (Col2a1) and X (Col10a1) were also reduced in caffeine treated cells in a dose-dependent manner. These results suggest that caffeine is involved in extracellular matrix productions in chondrocytes through the regulation of genes expression level. Our results demonstrated that caffeine altered osteogenic activity, leading to delayed peripubertal longitudinal bone growth and maturation. Given that osteogenic cells undergo dynamic changes in metabolic activity and that the pubertal growth spurt is mainly stimulated by GH/IGF-1 and sex steroids, caffeine could suppress ossification by interfering with both physiologic changes in hormonal secretion and directly inhibiting chondrogenic proliferation and differentiation of the growth plate during this critical period.| 임신 중 카페인 섭취는 태아의 골 성장 지연 및 비정상적 발달을 초래하는 등 태아 골성장에 유해한 것으로 알려져 있다. 또한 지속적인 카페인 노출은 태아의 insulin-like growth factor-1 (IGF-1)합성을 억제하는 것으로 보고된 바 있다. 이는 태아기와 마찬가지로 골성장이 급속히 이루어지는 사춘기 동안에 카페인 노출이 골길이 성장에 영향을 미칠 가능성을 시사하는 소견이다. 그러므로, 본 연구는 사춘기 동안 고농도 카페인에 대한 노출이 골길이 성장에 미치는 영향을 알아보고자, 총 51 마리의 미성숙한 수컷 쥐를 이용하여, 대조군과 120, 180 mg/kg/day 카페인 투여군으로 분류하여 카페인을 4주 동안 위장관 내로 투여하였다. 실험 종료 후, 하지골의 최종 길이와 무게를 측정하고, 경골은 조직계측학적 분석을 위해 사용하였다. 카페인 투여군에서 연령에 따른 체중 증가량은 대조군에 비해 유의한 감소를 보였으며, 이는 근육량 및 체내 지방량 감소와 비례하였다. 골밀도 및 18F-NaF PET으로 측정한 성장판의 골대사 활성이 카페인군에서 현저한 감소를 보였으며, 이러한 생체 내 골활성 및 골밀도의 감소는 하지골의 길이 및 무게 감소를 초래하였다. 또한 카페인 투여군에서 하지골의 성장 지연은 성장판의 형태 조직학적 발달 지연 및 골간단의 골형성 지표 감소를 동반하였다. 부가적으로 성장판 내 연골세포에 대한 카페인의 직접적인 영향을 확인하기 위해 성장판에서 연골세포를 분리, 배양하여 카페인 처치 후 연골세포 분화와 관련된 지표를 분석한 결과, 카페인 처치 용량에 따른 연골질 합성과 석회화 등이 현저히 감소하는 것을 확인하였으며, 마찬가지로 연골분화와 관련된 aggrecan, collagen type II (Col2a1)와 X (Col10a1)의 발현이 감소되는 것을 확인하였다. 이는 카페인이 연골세포 분화 인자의 발현을 조절하므로써 연골세포의 활성과 분화를 억제할 수 있음을 시사하는 소견이다. 결론적으로 사춘기 동안 카페인 노출은 골대사 활성에 영향을 미쳐 골길이 성장 및 성숙에 부정적인 영향을 미칠 수 있음을 알 수 있다. 카페인이 골형성 세포활성을 억제하고 골길이 성장과 관련된 호르몬 (성장호르몬, 성호르몬)의 분비를 억제하는 것으로 보아, 카페인은 골길이 성장이 급속히 이루어지는 중요한 시기에 성장판 내 연골세포에 직접적으로 작용하여 증식과 분화를 억제하고 체내 호르몬 분비에 영향을 미침으로써 골길이 성장을 저하시키는 것으로 보인다.; Caffeine has been known to adversely affect fetal skeletal growth and increased incidence of delayed and abnormal fetal skeletal development. Chronic caffeine intake also decreases fetal hepatic insulin-like growth factor 1 production. Thus, it is conceivable that caffeine may disturb long bone growth during pubertal growth spurt. We aimed to investigate the impact of high caffeine exposure on longitudinal bone growth throughout puberty. A total of 51 immature male rats were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 or 180 mg/kg/day for 4 weeks. After sacrifice, final length and weight of long bones were measured, and the tibia processed for histomorphometric analysis. Caffeine caused a significant decrease in body weight gain, accompanied with proportional decreases in lean body mass and body fat. Bone mass and osteogenic activity in vivo assessed using DXA and 18F-NaF PET, respectively were significantly decreased in caffeine-fed groups. Rats fed with caffeine showed a significant shortening and lightening of leg bones and the spinal column compared to the controls. Caffeine feeding delayed changes in the height of growth plates with age along with reduced cell proliferation. In addition, bone formation parameters observed in the secondary spongiosa also significantly reduced in caffeine-fed groups. The direct effect of caffeine on chondrogenic differentiation was evaluated using isolated primary rat chondrocytes. Chondrogenic differentiation markers such as extracellular matrix production, calcification and alkaline phosphatase activity were significantly decreased in caffeine treated cells in vitro. Consistent with these results, expression levels of chondrogenic marker genes such as aggrecan (ACAN), collagen type II (Col2a1) and X (Col10a1) were also reduced in caffeine treated cells in a dose-dependent manner. These results suggest that caffeine is involved in extracellular matrix productions in chondrocytes through the regulation of genes expression level. Our results demonstrated that caffeine altered osteogenic activity, leading to delayed peripubertal longitudinal bone growth and maturation. Given that osteogenic cells undergo dynamic changes in metabolic activity and that the pubertal growth spurt is mainly stimulated by GH/IGF-1 and sex steroids, caffeine could suppress ossification by interfering with both physiologic changes in hormonal secretion and directly inhibiting chondrogenic proliferation and differentiation of the growth plate during this critical period.