二甲双胍对糖尿病大鼠骨密度及Toll样受体4／核因子kappaB基因表达的影响

Impacts of metformin on bone mineral density and the genetic expression of Toll-like receptor 4/ nuclear factor Kappa B in bone tissue in diabetic rats

目的观察二甲双胍对糖尿病大鼠的骨密度以及骨组织中骨形成蛋白2（BMP-2）和Toll样受体4／核因子KappaB（TLR-4／NF-kB）因子表达的影响。方法将60只5-6周龄健康雄性SD大鼠，分为正常对照组（15只）、实验组（45只）。实验组大鼠给予高糖高脂饲料喂养联合小剂量链脲佐菌素腹腔注射建立糖尿病模型，采用随机数字表法将其分为糖尿病组（16只）和二甲双胍组（15只）。二甲双胍组给予二甲双胍[900mg／（kg·d）]灌胃，正常对照组和糖尿病组大鼠给予等量生理盐水灌胃。16周后用双能X线法检测各组大鼠各部位骨密度及体成分，ELISA法检测各组大鼠血清骨钙素的表达，AMP缓冲液法检测血清碱性磷酸酶（ALP）的表达，Westernblotting法检测各组大鼠BMP-2、TLR-4和NF—KB蛋白的表达，RT-PCR法检测TLR-4和NF-KBmRNA的表达。组间比较采用完全随机设计的单因素方差分析。结果（1）与正常对照组比较，糖尿病组大鼠上肢、大腿和全身骨密度降低[（0．16±0．01）比（0．14±0．02）g／cm2，（0．18±0．02）比（0．14±0．01）g／cm2，（0．19±0．01）比（0．15±0．00）g／cm2；F3．348、12．286、10．616，均P〈0．05[，同时，ALP、骨钙素的表达显著降低[ALP：（130±39）比（62±14）u／L；骨钙素：（2．84±0．52）比（1．94±0．13）μg／g，t=3．273、3．367，均P〈0．05]。TLR．4和NF-kBmRNA及蛋白的表达显著升高，BMP-2表达显著减少（均P〈0．05）。（2）与糖尿病组相比，二甲双胍组大鼠各部位骨密度的变化、骨钙素及ALP表达的升高无统计学意义（均P〉0．05），但TLR-4和NF-kB蛋白及mRNA的表达显著降低（均P〈0．05），BMP-2表达显著增加（0．23±0．05比0．35±0．08，t=4．098，P〈0．05）。结论二甲双胍可抑制TLR-4／NF-kB的表达，促进BMP-2的表达，但在干预时间内（16周）对糖尿病大鼠主要部位如上肢、大腿、全身骨密度无明显影响。

Objective To investigate the effects of metformin on bone mineral density （BMD）, the expression of bone morphogenetic protein 2 （BMP2） and Toll-like receptor 4 （TLR-4）/nuclear factor Kappa B （NF-KB） in diabetic rats. Methods Sixty 4-5 weeks-rid male Sprague-Dawlry （SD） rats were divided into normal control group （NC group, n=15） and experimental group （n=45）. The experimental group was fed with high-sugar high-fat diet. Then a small dose of streptozotocin was injected intraperitoneally. According to random number table, the rats were divided into diabetes group（DM group, n=16） and diabetes ＋metformin group（DM＋MET group, n=15）. The DM＋MET rats were treated with metformin for 16 weeks by garage （900 mg/（kg d））, while rats in other groups were treated with saline by garage. The BMD of different parts in the rats and body composition was obtained with double energy X array（DEXA） scan. The expression of osteocalcin（OCN） in the rats was measured with enzyme linked immunosorbent assay（ELISA）. Protein expression of TLR-4, NF-KB, and BMP-2 was determined with Western blot. mRNA expression of TLR-4 and NF-KB was determined with RT-PCR. Differences among three groups were assessed by one-way analysis of variance. Results Compared with NC group, the BMD of upper limbs and whole body in DM group was decreased（P〈0.05）; The levels of alkaline phosphatase（ALP） and OCN were decreased （（130±39） vs （62± 14） U/L; （2.84±0.52） vs （1.94±0.13） μg/g, t=3.273, 3.367, both P〈0.05）; The levels of TLR-4 and NF-KB were increased （both P〈0.05）; but the values of BMP-2 was decreased （P〈0.05）. There was no significant difference in BMD, OCN and ALP expression between DM group and DM＋MET group （P〉0.05）. Compared to the DM group, the levels of TLR-4 and NF-KB of the DM＋MET group were lower （both P〈 0.05）. And the values of BMP- 2 was higher （0.23 ±0.05 vs 0.35±0.08, t=4.098, P〈0.05）. Conclusions Metformin may inhibit TLR-4/NF-KB expression and increase BMP-2 expression. But it has no significant impact on the BMD of thigh, torso, spine and body in the diabetic rats during the treatment period （16 weeks）.