熊果酸对酒精性骨质疏松大鼠骨形成、骨矿化及肠道菌群的影响

发布时间：2018-06-21 14:43

本文选题：熊果酸 + 酒精性骨质疏松　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:通过观察熊果酸对酒精性骨质疏松大鼠股骨骨密度(BMD)、骨钙素(BGP)、骨形成蛋白-2(BMP-2)、血清钙、磷及炎性细胞因子TNF-α和IL-1β等的影响,初步探讨熊果酸对酒精所致骨质疏松大鼠的作用及可能机制,并分析熊果酸对酒精性骨质疏松大鼠肠道菌群多样性的影响。方法:1.动物分组及给药:2月龄SPF级雄性Wistar大鼠90只,大鼠适应性喂养7 d后,按体重随机分为6组(分别为:正常对照组、熊果酸对照组、酒精模型组、熊果酸低、中、高剂量组),每组15只,分笼喂养,每日自由进水,进食。酒精模型组大鼠每日给予50%(V/V)酒精8 m L/kg·bw·d,灌胃2周后,剂量提升至12 m L/kg·bw·d,继续灌胃6周;正常对照组每日灌胃给予生理盐水;熊果酸对照组每日给予150 mg/kg·bw·d熊果酸;熊果酸低、中、高剂量组每日分别灌胃给予50、100、150 mg/kg·bw·d熊果酸,酒精剂量同模型组;实验共持续8周,并记录大鼠每周体重,以调整酒精及熊果酸灌胃量。末次灌胃给药后禁食不禁水12小时,3%戊巴比妥钠麻醉,腹主动脉取血,剥离股骨固定。2.股骨病理学观察:HE染色观察大鼠股骨的病理学变化。3.股骨骨密度(BMD)测定:DEXA骨密度仪检测股骨骨密度。4.采用酶联免疫吸附法(ELISA)检测大鼠血清骨钙素(BGP)、骨形成蛋白-2(BMP-2)浓度的水平。5.采用酶联免疫吸附法(ELISA)检测大鼠血清炎性细胞因子肿瘤坏死因子-α(TNF-α)和白介素-β(IL-1β)含量6.比色法测定血清钙(Ca)浓度;磷钼酸法测定血清磷(P)浓度。7.采用DGGE-PCR技术对粪便肠道菌群进行指纹图谱聚类性分析。结果:1.HE染色观察结果:正常对照组大鼠骨小梁致密、规则且较粗,粗细均匀;而酒精模型组大鼠骨小梁稀松、不规则、粗细不均匀,甚至可见骨小梁断裂;熊果酸中、高剂量组与酒精模型组大鼠相比较,骨小梁致密、规则、较厚、粗细均匀,未见骨小梁断裂;熊果酸低剂量组大鼠改善不明显。2.股骨骨密度:与正常对照组相比较,酒精模型组大鼠的股骨BMD明显降低,且差异具有统计学意义(P0.05)。但经8周熊果酸干预后,可见除熊果酸低剂量组外,熊果酸中、高剂量组大鼠股骨BMD均明显升高,与酒精模型组相比,均具有统计学差异(P0.05)。3.血清BGP、BMP-2结果:酒精模型组与正常对照组相比,BGP含量明显降低,且差异具有统计学意义(P0.05);熊果酸中、高剂量组与酒精模型组相比较,BGP含量均有所升高,且差异有统计学意义(P0.05),但熊果酸低剂量组与酒精模型组相比无统计学差异,仅具有数值上的升高。酒精模型组与正常对照组相比,可见模型组大鼠血清BMP-2含量明显降低,且差异具有统计学意义(P0.05);而熊果酸高剂量组与酒精模型组相比,血清BMP-2含量明显升高,且有统计学差异(P0.05),但熊果酸低、中剂量组血清BMP-2水平变化不大,与酒精模型组相比无统计学差异。4.血清Ca、P结果:酒精模型组大鼠血清Ca、P水平均有明显降低,与正常对照组相比较,差异有统计学意义(P0.05);与酒精模型组相比较,熊果酸中、高剂量组血清Ca和P含量明显回升,且均具有统计学差异(P0.05),但熊果酸低剂量组Ca、P含量虽有数值上的增加,但无统计学差异。5.血清TNF-α和IL-1β结果:相对于正常对照组,酒精模型组TNF-α和IL-1β的水平明显升高,差异具有统计学意义(P0.05);而熊果酸干预后显著抑制了炎性因子的升高,与酒精模型组相比较,TNF-α和IL-1β的水平显著下降,其中熊果酸高剂量组作用明显,且具有统计学差异(P0.05)。6.肠道菌群DGGE-PCR指纹图谱分析:酒精模型组大鼠肠道菌群多样性明显减少且与正常组的相似性较低;而熊果酸高剂量组大鼠肠道菌群结构更加接近于正常组。结论:1.通过对大鼠股骨骨密度测定及股骨病理学观察,初步证实熊果酸对酒精所致的大鼠骨质疏松具有显著的改善作用。2.熊果酸对酒精性骨质疏松的保护机制可能与其能够促进骨形成,降低骨吸收,抑制骨矿物质的流失及炎性细胞因子过表达有关。3.熊果酸能够调节因酒精摄入导致的肠道菌群多样性减少,提示其改善酒精性骨质疏松的作用可能与其对肠道菌群的调节作用有关。
[Abstract]:Objective: To investigate the effect of ursolic acid on femur bone density (BMD), osteocalcin (BGP), bone morphogenetic protein -2 (BMP-2), serum calcium, phosphorus, and inflammatory cytokine TNF- alpha and IL-1 beta in rats with alcoholic osteoporosis, and to investigate the effect and possible mechanism of ursolic acid on alcohol induced osteoporosis rats and to analyze the effect of ursolic acid on alcohol induced osteoporosis. Methods: the effect of the diversity of intestinal microflora in rats. Methods: 1. animal groups and drug delivery: 2 month old SPF male Wistar rats, 90 rats were fed for 7 d. The rats were randomly divided into 6 groups according to their weight (the normal control group, ursolic acid control group, alcohol model group, ursolic acid low, middle, high dose group), 15 rats in each group, fed by cage and free intake of water daily, The rats in the alcohol model group were given 50% (V/V) alcohol 8 m L/kg. BW. D every day. After 2 weeks of gavage, the dosage was increased to 12 m L/kg. BW. D and continued for 6 weeks. The normal control group was given the normal saline daily; the ursolic acid control group was given 150 mg/kg. BW D ursolic acid daily; the ursolic acid was low, middle, and high dose groups were given 50100150 daily to 50100150. Mg/kg. BW. D ursolic acid, alcohol dose and model group; the experiment lasted for 8 weeks, and the rats' weekly weight was recorded in order to adjust alcohol and ursolic acid perfusion. After the last irrigation, the fasting was 12 hours, 3% pentobarbital sodium anaesthesia, the abdominal aorta took blood, and the femoral fixed.2. femur was observed pathological observation: HE staining was used to observe the pathology of the femur of rats. .3. femur bone mineral density (BMD) determination: DEXA bone densitometer detection of femur bone density.4. using enzyme linked immunosorbent assay (ELISA) detection of rat serum osteocalcin (BGP), bone morphogenetic protein -2 (BMP-2) concentration level.5. using enzyme linked immunosorbent assay (ELISA) detection of rat serum inflammatory cytokines tumor necrosis factor alpha (TNF- alpha) and interleukins - beta (IL-1 beta) content 6. colorimetric determination of serum calcium (Ca) concentration; phospho molybdic acid determination of serum phosphorus (P) concentration.7. clustering analysis of fecal intestinal microflora by DGGE-PCR technology. Results: 1.HE staining results: normal control group of bone trabecula dense, regular and coarse and uniform; and alcohol model group of rat bone trabecula Loose, irregular, uneven, even bone trabecular fracture; ursolic acid, high dose group compared with the alcohol model group, bone trabecula dense, regular, thick, uniform, no bone trabecular fracture; ursolic acid low dose group improved.2. femur bone density: compared with normal control group, alcohol model group rats The BMD of the femur was significantly reduced and the difference was statistically significant (P0.05). But after 8 weeks of ursolic acid dry prognosis, the BMD of the femur in the high dose group of ursolic acid, except the low dose ursolic acid group, was significantly higher than that in the alcohol model group (P0.05).3. serum BGP, BMP-2 results: the alcohol model group was compared with the normal control group. The content of BGP was significantly reduced and the difference was statistically significant (P0.05). In ursolic acid, the content of BGP increased in the high dose group compared with the alcohol model group, and the difference was statistically significant (P0.05), but there was no statistical difference between the low dose ursolic acid group and the alcohol model group, only the increase in the numerical value. The alcohol model group and the normal control group were compared with the alcohol model group. Compared with the group, the serum BMP-2 content of the rats in the model group was significantly lower, and the difference was statistically significant (P0.05), but the serum BMP-2 content in the high dose ursolic acid group was significantly higher than that in the alcohol model group, and there was a statistically significant difference (P0.05), but the ursolic acid was low and the serum BMP-2 level in the middle dose group changed little, and there was no statistical difference compared with the alcohol model group. .4. serum Ca, P results: the serum Ca, P water in the alcohol model group decreased significantly, compared with the normal control group, the difference was statistically significant (P0.05). Compared with the alcohol model group, the serum Ca and P content of the high dose group increased significantly, and all had statistical difference (P0.05), but the low dose ursolic acid group Ca, P content Although there was an increase in numerical value, there was no statistical difference in the results of.5. serum TNF- A and IL-1 beta: compared with the normal control group, the level of TNF- alpha and IL-1 beta in the alcohol model group was significantly higher, the difference was statistically significant (P0.05), while ursolic acid intervention significantly inhibited the increase of inflammatory factors, compared with the alcohol model group, the level of TNF- A and IL-1 beta. The effect of ursolic acid high dose group was obvious, and the effect of high dose ursolic acid group was obvious, and there was statistical difference (P0.05).6. intestinal microflora DGGE-PCR fingerprint analysis: the intestinal microflora diversity of rats in the alcohol model group was significantly reduced and the similarity with the normal group was lower; and the intestinal flora structure of the high dose ursolic acid group was closer to the normal group. The conclusion: 1. links. The measurement of bone mineral density and pathological observation of femur in rats showed that ursolic acid significantly improved osteoporosis in rats induced by alcohol. The protective mechanism of ursolic acid to alcohol induced osteoporosis could promote bone formation, reduce bone absorption, inhibit bone mineral loss and inflammatory cytokine overlay.2.. .3. ursolic acid can regulate the decrease of intestinal microflora caused by alcohol intake, suggesting that its effect on improving alcoholic osteoporosis may be related to the regulation of intestinal microflora.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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