褪黑素在2型糖尿病骨质疏松大鼠骨组织的药物存留及对骨微结构改善的作用

Drug distribution of melatonin in bone tissue and improvement of bone microstructure in type 2 diabetic osteoporosis rats

目的:探讨不同浓度褪黑素在2型糖尿病骨质疏松(type 2 diabetic osteoporosis,T2DOP)大鼠骨组织中的药物存留情况及对T2DOP大鼠骨微结构的改善作用。方法:选取SD大鼠95只,其中60只采用高糖高脂饲料结合小剂量链脲佐菌素腹腔注射的方法建立T2DOP大鼠模型,2个月后通过检测大鼠血糖及胰岛素敏感指数,确定造模成功45只。随机选取30只造模成功和30只正常SD大鼠行褪黑素分布实验,分别将两组大鼠再根据注射褪黑素的浓度分成造模高浓度(50 mg/kg)组、造模低浓度(10 mg/kg)组、正常高浓度(50 mg/kg)组和正常低浓度(10 mg/kg)组,每组15只;按取材的时间点(5、15、30、60、120 min)再分成5组,每组3只。对每组大鼠骨组织进行预处理,而后采用高效液相色谱法检测骨组织中褪黑素的药物浓度。另取15只造模成功大鼠,根据是否注射褪黑素及其浓度分为单纯造模组、高浓度(50 mg/kg)褪黑素组和低浓度(10 mg/kg)褪黑素组,每组5只,再取5只正常大鼠作为对照(正常大鼠组),处理8周后运用Micro-CT检测骨微结构的改变。结果:药物分布实验结果显示,在腹腔注射褪黑素后各组大鼠骨组织中均有药物存留,均在给药30 min后药物浓度达到最高,120 min后显著降低。其中正常高浓度组与正常低浓度组相比,在5个时间点两组药物浓度无明显差异;造模高浓度组与造模低浓度组相比,在5个时间点两组药物浓度亦无统计学差异。而造模高浓度组在5、15、30、60、120 min 5个时间点的药物浓度分别为(7.613±2.568)ng/ml、(13.983±2.262)ng/ml、(18.816±1.291)ng/ml、(6.172±1.962)ng/ml、(1.112±0.566)ng/ml,均高于正常高浓度组各时间点的药物浓度。Micro-CT结果显示褪黑素治疗8周后,高浓度组的骨密度[(205.72±28.41)g/cm 3]明显低于低浓度组[(223.63±35.41)g/cm 3],但两组均显著高于正常大鼠组[(158.31±31.86)g/cm 3],各组相比差异有统计学意义。结论:外源性褪黑素在骨组织中存在药物分布,且T2DOP大鼠的药物吸收率更高,同时不同浓度的褪黑素在骨组织中的药物分布无差异,均可以改善T2DOP大鼠的骨微结构。

Objective To investigate the medicinal retention of different concentrations of melatonin in the bone tissue of type 2 diabetic osteoporosis(T2DOP)rats and explore to efficacy of improvement of the bone microstructure of T2DOP rats.Methods A total of 95 SD rats were selected,60 of which had intraperitoneal in jection of high-fat diet combined with low-dose streptozotocin establishing a T2DOP rat model.Two months later,45 rats'model was determined to be successful by detecting blood glucose and insulin sensitivity index.30 successful modelling and 30 normal SD rats were randomly selected for melatonin distribution experiment,and were divided into four groups according to the injected melatonin concentration,including modeling rat high concentration group(50 mg/kg),modeling rat low concentration group(10 mg/kg),normal rat high concentration group(50 mg/kg)and normal rat low concentration(10 mg/kg),and there were15 rats in each group.Each group was divided into 5 sub-groups according to the time point of sampling(5,15,30,60,120 min),3 animals per group.The bone tissue of each group was pretreated,and then the melatonin drug distribution in the bone tissue was detected by high performance liquid chromatography(HPLC).Another 15 rats were successfully modeled,and were divided into T2DOP group,high melatonin group(50 mg/kg)and low melatonin group(10 mg/kg),5 rats in each group.5 normal SD rats were taken as controls(control group),and Micro-CT was used to detect changes in bone microstructure after 8 weeks of treatment with melatonin.Results The results of the drug distribution experiment showed that after melatonin was injected intraperitoneally,there were drugs remaining in the bone tissues of the rats in each group.The drug concentration reached the highest after 30 min of administration,and significantly decreased after 120 min.Compared with the normal rat low concentration group,there was no significant difference in the drug concentration between the two groups at 5 time points.However,the drug concentration at the four time points of 5,15,30,and 60 min in the modeling rat high concentration group were 7.613±2.568 ng/ml,13.983±2.262 ng/ml,18.816±1.291 ng/ml,6.172±1.962 ng/ml,1.112±0.566 ng/ml,which were significantly different compared with normal rat high group.Micro-CT results showed that after 8 weeks of melatonin treatment,the bone density of the high concentration group was(205.72±28.41 g/cm3)significantly lower than that in the low concentration group(223.63±35.41 g/cm3),but both groups were significantly higher than the normal rat group(158.31±31.86 g/cm3).Conclusion Exogenous melatonin is distributed in bone tissue,and the drug absorption rate of T2DOP rats is higher.Meanwhile,there is no difference in the distribution of melatonin in bone tissue with different concentrations,and these two concentrations of melatonincan canimprove the bone microstructure of T2DOP rats.