利拉鲁肽对糖尿病骨质疏松大鼠骨代谢、炎性反应和氧化应激的影响

Effects of liraglutide on the bone metabolism inflammatory response,and oxidative stress in diabetic osteoporosis rats

目的观察利拉鲁肽(liraglutide,LRG)对糖尿病骨质疏松大鼠骨代谢、炎性反应和氧化应激的影响。方法健康雌性SD大鼠,采用数字表法随机分为6组:假手术组(Sham组:分离卵巢但不切除)、糖尿病组(streptozotocin,STZ组:链脲佐菌素诱导制备)、糖尿病+利拉鲁肽组(STZ+LRG组:制备糖尿病模型后采用利拉鲁肽干预)、卵巢去势组(OVX组:切除大鼠双侧卵巢)、糖尿病+卵巢去势组(STZ+OVX组:制备糖尿病模型后切除大鼠双侧卵巢)、糖尿病+卵巢去势+利拉鲁肽组(STZ+OVX+LRG组:制备糖尿病模型后切除大鼠双侧卵巢,并采用利拉鲁肽干预)。药物干预8周后,HE染色观察股骨组织,ELISA法检测炎性因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、白细胞介素6(interleukin-6,IL-6)及氧化应激指标超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malonaldehyde,MDA)、活性氧(reactive oxygen species,ROS)的表达,反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测股骨组织中成骨标志物Nrf2、HO-1、RUNX2、COL1A1 mRNA水平。结果 ELISA结果显示,STZ+LRG组TNF-α(63.142±6.616)ng/L、IL-1β(282.611±47.778)ng/L、IL-6(105.778±14.912)ng/L;STZ+OVX+LRG组TNF-α(97.344±13.622)ng/L、IL-1β(498.537±61.024)ng/L、IL-6(309.778±34.912)ng/L;与其对照组相比,炎性因子的表达均明显降低,差异有统计学意义(P<0.05)。氧化应激指标检测结果显示,STZ+LRG组SOD(12.014±1.499)U/mg、MDA(4.149±0.963)nmol/mg、ROS 28 747.000±3 233.060;STZ+OVX+LRG组SOD(7.430±1.567)U/mg、MDA(5.385±1.164)nmol/mg、ROS 35 803.667±4 729.210;与对照组相比,SOD的活力明显上升,MDA和ROS的含量亦显著下降,差异均有统计学意义(P<0.05)。RT-PCR结果显示,与对照组相比,STZ+LRG组、STZ+OVX+LRG组抗氧化应激指标Nrf2、HO-1及成骨标志物RUNX2、COL1A1 mRNA表达显著升高,差异有统计学意义(P<0.001)。HE染色结果表明,与对照组相比,STZ+LRG组、STZ+OVX+LRG组成骨细胞数量与新生骨小梁数量均明显增加。结论利拉鲁肽对糖尿病并发卵巢切除诱导的骨质疏松大鼠具有显著预防作用,能够改善骨代谢,且该作用部分伴随炎性反应、氧化应激和Nrf2/HO-1信号活性改变。

Objective To observe the effects of liraglutide（ LRG） on bone metabolism,inflammatory response and oxidative stress in diabetic osteoporosis rats. Methods Healthy female SD rats were randomly divided into 6 groups：sham operation group（ Sham group： isolated ovaries but not resected）,diabetes group（ streptozotocin,STZ group： streptozotocin induced preparation）,diabetes ＋ liraglutide treatment group（ STZ ＋ LRG group： treated with liraglutide after establish the diabetic model）,ovarian castration group（ OVX group： resection of rat bilateral ovaries）,diabetes ＋ ovarian castration group（ STZ ＋ OVX group： resection of rat bilateral ovaries after establish the diabetic model）,diabetes ＋ ovarian castration ＋ liraglutide treatment group（ STZ ＋ OVX ＋ LRG group： resection of rat bilateral ovaries after establish the diabetic model,and treatment with liraglutide）. After 8 weeks of drug treatment,HE staining was used to observe the femoral tissue. We detected inflammatory factors such as tumor necrosis factor-α（ TNF-α）,interleukin-1β（ IL-1β）,nterleukin-6（ IL-6） and oxidative stress index of superoxide dismutase（ SOD）,malonaldehyde（ MDA） and reactive oxygen species（ ROS）. RT-PCR was used to detect the mRNA levels of Nrf2,HO-1,RUNX2 and COL1 A1. Results ELISA results suggested that,STZ ＋ LRG group TNF-α（ 63. 142 ± 6. 616） ng/L,IL-1β（ 282. 611 ± 47. 778） ng/L,IL-6（ 105. 778 ±14. 912） ng/L; STZ＋ OVX ＋ LRG group TNF-α（ 97. 344 ± 13. 622） ng/L,IL-1β（ 498. 537 ± 61. 024） ng/L,IL-6（ 309. 778±34. 912） ng/L,compared with its control group,the expression of inflammatory factors was decreased（ P〈0. 05）.The expression of the oxidative stress index demonstrated that STZ ＋ LRG group SOD（ 2. 014± 1. 499） U/mg,MDA（ 4. 149± 0. 963） nmol/mg,ROS 28 747. 000± 3 233. 060; STZ＋OVX＋LRG group SOD（ 7. 430± 1. 567） U/mg）,MDA（ 5. 385±1. 164） nmol/mg,ROS 35 803. 667±4 729. 210,compared with its control group,the SOD activity was increased while the level of MDA and ROS were declined（ P〈0. 001）. The RT-PCR results indicated that,compared with its control group,the mRNA level of anti-oxidative stress index Nrf2,HO-1 and the osteogenic markers RUNX2,COL1 A1 were increased in STZ＋LRG group and STZ＋OVX＋LRG group（ P〈0. 001）. The HE staining implied that,compared with its control group,more osteoblast and newly formed trabeculae was observed in STZ＋LRG group and STZ＋OVX＋LRG group. Conclusion Liraglutide has a significant protective effect on diabetic osteoporosis rats with osteoporosis induced by ovariectomy,which can improve bone metabolism. And the preventine effections at least partially be associated with inflammatory response,oxidative stress and Nrf2/HO-1 signal activity changes.