【作者】 孙妲男；

【导师】 唐强；

【作者基本信息】 黑龙江中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 目的观察针康法对糖尿病缺血性脑卒中大鼠运动功能；血清糖脂代谢；血清NF-κKB-p65、 TNF-α、IL-1β；脑缺血皮质区及骨骼肌NF-κB-p65、TNF-α、IL-1β蛋白；骨骼肌IRS-1、 PI3K基因的表达,探讨针康法对于改善糖尿病缺血性脑卒中大鼠神经功能缺损和胰岛素抵抗的相关机制。方法建立糖尿病缺血性脑卒中大鼠模型,将192只大鼠按随机数字法分为6组：空白组、假手术组、模型组、针刺组、康复组、针康组,每组各32只。每组按3、7、14、21天再分为4亚组,每组8只。空白组、假手术组、模型组不进行任何干预,针刺组采用头穴丛刺结合体针治疗,康复组采用跑台训练,针康组采用针刺结合跑台训练治疗。各时间点运用平衡木试验观察大鼠神经功能变化；采用全自动生化分析仪测血TG、TC含量；酶联免疫吸附法测FINS及血清NF-κB-p65、TNF-α、IL-1β含量；免疫组化法检测脑缺血皮质区及骨骼肌NF-KB-p65、TNF-α、IL-1β蛋白表达；Real-Time PCR检测骨骼肌IRS-1mRNA、PI3K mRNA的表达。结果1平衡木试验评分：与假手术组比较,各时间点、各组平衡木试验评分明显增高(P<0.01)。与模型组比较,术后3天、7天、14天康复组评分降低(P<0.05),术后3天、7天、14天、21天针康组评分降低(P<0.05)。与针刺组、康复组比较,术后14天、21天针康组评分降低(P<0.05)。2糖脂代谢：①空腹血糖(FPG)：术后14天、21天,与空自组比较,其余各组大鼠FPG均明显增加(P<0.01)；与假手术组、模型组比较,针康组FPG明显降低(P<0.01)：与针刺组比较,针康组FPG降低(P<0.05)。②血清胰岛素(FINS):与空白组比较,术后14天、21天其余各组大鼠IFINS均明显增加(P<0.01)；与假手术组、模型组比较,术后21天,康复组FINS明显降低(P<0.01)；术后14天、21天,针康组FINS明显降低(P<0.01)。与针刺组比较,术后14天、21天针康组FINS降低(P<0.05)。③胰岛素敏感指数其自然对数(IAI)：与空白组比较,术后14天、21天其余各组大鼠IAI均明显降低(P<0.01)；与假手术组比较,术后21天康复组IAIt增加(P<0.05),术后14天、21天针康组IAI均明显增加(P<0.01)；与模型组比较,术后14天康复组IAI上升(P<0.05),术后14天、21天,针康组IAI均明显上升(P<0.01)；与针刺组比较,术后14天、21天,针康组IAI均上升(P<0.05)。④甘油三酯(TG)：与空白组比较,术后14天、21天其余各组大鼠TG均明显增加(P<0.01)；与假手术组、模型组比较,术后21天针康组TG下降(P<0.05)。⑤胆固醇(TC)：与空白组比较,术后14天、21天其余各组大鼠TC均明显增加(P<0.01)；与假手术组、模型组比较,术后14天、21天针康组TC均下降(P<0.05)；与模型组比较,术后21天康复组TC下降。3血清NF-κB-p65、TNF-α、IL-1β含量：与空白组、假手术组比较,术后各时间点其余各组大鼠血清NF-κB-p65、TNF-α、IL-1β含量均明显增加(P<0.01),假手术组较空白组含量增加(P<0.01)。血清NF-κB-p65与模型组比较,术后各天针康组含量降低(P<0.05)。与针康组比较,术后7天针刺组(P<0.01)、康复组(P<0.05)含量降低不如针康组明显。血清TNF-α与模型组比较,术后7天、14天,针刺组、康复组、针康组含量均明显降低(P<0.01),术后21天康复组含量降低(P<0.05),针康组含量明显降低(P<0.01)。与针康组比较,术后7天、14天针刺组、康复组含量降底不如针康组(P<0.05)。血清IL-1β与模型组比较,针刺组术后3天、7天含量降低(P<0.05)；康复组术后3天、14天含量降低(P<0.05)；针康组术后各时间点含量均明显降低(P<0.01)。与针刺组比较,针康组术后21天含量降低(P<0.05)。4缺血区皮层NF-κB-p65、TNF-α、IL-1β的表达：与空白组、假手术组比较,术后各时间点NF-κB-p65、TNF-α、IL-1β阳性细胞数均明显增加(P<0.01),假手术组较空白组数量增加(P<0.01)。缺血区皮层NF-κB-p65与模型组比较,针康组术后各时间点阳性细胞数降低显著(P<0.01)。与针刺组、康复组比较,针康组术后3天、21天阳性细胞数降低(P<0.05)。缺血区皮层TNF-α与模型组比较,针康组术后各时间点数量明显降低(P<0.01)。与针刺组比较,针康组术后7天数量明显降低(P<0.01),术后3天、21天数量降低(P<0.05)。与康复组比较,针康组术后14天数量降低(P<0.05)。缺血区皮层IL-1β的表达与模型组比较,针康组术后3天、7天、14天数量明显降低(P<0.01),数量降低(P<0.05)。与针刺组、康复组比较,针康组术后3天、7天含量降低(P<0.05)。5骨骼肌NF-κB-p65、TNF-α、IL-1β的表达：术后21天,与空白组比较,各组NF-κB-p65、TNF-α、IL-1β光密度值明显增加(P<0.01)；与假手术组及模型组比较,针刺组、康复组、针康组NF-κB-p65光密度值均明显降低有显著意义(P<0.01)；与针刺组、康复组比较,针康组光密度值明显降低(P<0.01)。与假手术组及模型组比较,针刺组、康复组、针康组TNF-α光密度值均明显降低(P<0.01)；与针刺组、康复组比较,针康组TNF-α光密度值明显降低(P<0.01)。与假手术组及模型组比较,针刺组、康复组、针康组IL-1β光密度值均明显降低(P<0.01)；与针刺组、康复组比较,康复组IL-1β光密度值降低(P<0.05)。6骨骼肌IRS-1mRNA表达：术后21天各组均有IRS-1mRNA的表达,空白组较其他各组表达明显(P<0.01)；与假手术组、模型组比较,针刺组、康复组、针康组IRS-1mRNA的表达水平明显增高(P<0.01)；与针刺组比较,康复组IRS-1mRNA表达增加(P<0.05),针康组IRS-1mRNA表达水平明显增加(P<0.01)；与康复组比较,针康组IRS-1mRNA表达增加(P<0.05)。7骨骼肌PI3KmRNA表达：术后21天各组均有PI3KmRNA的表达,空白组较其他各组表达明显(P<0.01)；与假手术组、模型组比较,针刺组、康复组、针康组PI3KmRNA的表达水平明显增高(P<0.01)；与针刺组、康复组比较,针康组IRS-1mRNA表达增加(P<0.05)。结论：1.针康法能够促进糖尿病缺血性脑卒中大鼠神经功能的康复,针康法优于单纯针刺或康复。2.针康法能够降低糖尿病缺血性脑卒中大鼠糖脂代谢水平,增加胰岛素敏感指数,逆转糖代谢异常,使病态机体得到改善。3.针康法能够降低糖尿病缺血性脑卒中大鼠血清、脑缺血区皮质NF-κB-p65、 TNF-α、IL-1β的含量,进而减少缺血区脑组织炎症的损伤,起到脑保护作用。4.针康法能够减少糖尿病缺血性脑卒中大鼠骨骼肌NF-κB-p65、TNF-α、IL-1β的蛋白表达,缓解微炎状态,使骨骼肌的胰岛素敏感性增加,改善胰岛素抵抗。5.针康法能使糖尿病缺血性脑卒中大鼠骨骼肌IRS-1mRNA、PI3KmRNA的含量增加,针康法可能通过IRS-1/PI3K信号转导通路改善骨骼肌对胰岛素的抵抗。更多还原

【Abstract】 ObjectiveTo observe the effects of cluster needling of acupuncture combined with treadmill training in Motor function metabolism of serum glucolipid、NF-κB-p65、TNF-α and IL-1β in serum、ischemic region of cerebral cortex and skeletal muscle NF-κB-p65、TNF-α、IL-1β protein and skeletal muscle IRS-1and PI3K expression of gene in rats with Diabetes ischemic stroke. To discuss the probable mechanism applied to improve the neurological function defects and IR in rats.MethodEstablish rat diabetes ischemic stroke model. To randomly divide192rats into six groups, which are Sham operation group, Model group, Acupuncture group, Rehabilitation group and Blank group. And then each group was divided into four subgroups in accordance with the3rd,7th,14th,21st days (n=8). No intervention was given to Sham operation group, Blank group and Model group, while the Acupuncture group was treated with head combined with body acupuncture therapy, the Rehabilitation group was treated with treadmill training, Acupuncture combined with rehabilitation group was treated with acupuncture treatment combined with treadmill training. Each time the changing of rats’ neurological function was observed. TG、TC content were evaluated by net screen test. FINS、NF-KB-p65、TNF-α、IL-1β content were evaluated by ELISA. Ischemic region of cerebral cortex and skeletal muscle NF-κB-p65、TNF-α、IL-1β were evaluated by SABC. And the Real-Time PCR staining was used to observe the expression of skeletal muscle’s IRS-1mRNA、PI3K mRNA.Results1. Scores of Balance Beam test:Compared with Sham operation group, the score of each group was significantly improved at each point (p<0.01). The3rd,7th,14th,21st day after the modeling, the score of Acupuncture combined with rehabilitation group dropped down (p<0.05). And compared with Acupuncture group and Rehabilitation group, the score of Acupuncture combined with rehabilitation group dropped down (p<0.05) on the14th and21st day after the modeling.2.Metabolism of glucolipid:①FPG:Compared with the blank group, the FPG of the remaining groups have been significantly improved on the14th and21st day after the modeling (p<0.01). The FPG of the Acupuncture combined with the rehabilitation group decreased clearly which compared with the Sham operation group and the Model group (p<0.01).And the Acupuncture combined with rehabilitation group’s FPG had been decreased compared with the Acupuncture group (p<0.05).②FINS:Compared with the blank group, the FINS of the remaining groups have been significantly improved on the14th and21st day after the modeling (p<0.01).Compared with the Sham operation group and the Model group, the FINS of the Rehabilitation group decreased clearly on the21st day after the modeling (p<0.01). The acupuncture combined with rehabilitation group’s FINS decreased clearly (p<0.01). And the FINS of acupuncture combined with rehabilitation group’s FINS had a decrease compared with the acupuncture group (p<0.05).③IAI:Compared with the blank group, the IAI of the remaining groups was significantly reduced on the14th and21st day after the modeling (p<0.01). Compared with the sham operation group, the IAI of the rehabilitation group increased clearly on the21st day after the modeling (p<0.05). The acupuncture combined with rehabilitation group’s IAI decreased clearly on the14th and21st day after the modeling (p<0.01). And the IAI of acupuncture group’s IAI was been decreased compared with the model group (p<0.05). The.acupuncture combined with rehabilitation group’s IAI decreased clearly on the14th and21st day after the modeling (p<0.01). Compared with the acupuncture group, the IAI of the acupuncture combined with rehabilitation group had a decrease on the14th and21st days after the modeling (p<0.05).④TG:Compared with the blank group, the TG of the remaining groups was significantly reduced on the14th and21st day after the modeling (p<0.01). Compared with the sham operation group and the model group, the TG of the acupuncture combined with rehabilitation group dropped down on the21st day after the modeling (p<0.05).⑤TC: Compared with the blank group, the TC of the remaining groups was significantly decreased on the14th and21st day after the modeling (p<0.01). Compared with the sham operation group and the model group, the TC of the acupuncture combined with rehabilitation group dropped down on the14th and21st day after the modeling (p<0.05). Compared with the model group, the TC of the acupuncture group dropped down on the21st days after the modeling.3. The content of NF-κB-p65、TNF-α、IL-1β in serum:Compared with the blank group and the sham operation group, the NF-κB-p65、TNF-α IL-1β content in serum of the remaining groups was significantly decreased on each point after the modeling (p<0.01). Compared with the model group,the content of the NF-KB-p65、TNF-α、IL-1β in serum of the which acupuncture combined with rehabilitation group have been decreased (p<0.01). Compared with the acupuncture combined with rehabilitation group, the NF-KB-p65contents of the acupuncture group decreased (p<0.01) and the rehabilitation group (p<0.05) are not clearer than acupuncture combined with rehabilitation group on the7th day after the modeling. The content’s decrease of the acupuncture group is not clearer than acupuncture combined with rehabilitation group on the14th and21st day after the modeling. Compared with the model group, the TNF-α content of the acupuncture group,the rehabilitation group、 the acupuncture combined with rehabilitation group have been decreased (p<0.01) on the7th and14st day after the modeling, the content of the rehabilitation group(p<0.05) and the acupuncture combined with rehabilitation group(p<0.01) decreased on the21th day after the modeling。Compared with the acupuncture combined with rehabilitation group, The content’s decrease of the acupuncture group and the rehabilitation group is not clearer than it on the7th and14st day after the modeling(p<0.05). Compared with the model group, the IL-1β content of the acupuncture group have been decreased (p<0.05) on the3th and7st day after the modeling; the rehabilitation group decreased (p<0.05) on the3th and14st day; the acupuncture combined with rehabilitation group decreased (p<0.01) on every day. Compared with the acupuncture group, the acupuncture combined with rehabilitation group decreased (p<0.05)on the21st day.4. The expression of NF-κB-p65、TNF-α、IL-1β around ischemic cortex:Compared with the sham operation group and the blank group, there was a significant increase of NF-κB-p65、TNF-α、IL-1β expression in the remaining groups on each point (P<0.01). Compared with blank group, the sham group have been increased (P<0.01). Compared with model group, the NF-KB-p65expression reduced in acupuncture combined with rehabilitation group on every day after modeling (p<0.01). Compared with acupuncture group and rehabilitation group, expression reduced in acupuncture combined with rehabilitation group on3rd and2st day after modeling (p<0.05). Compared with model group, the TNF-α expression reduced in acupuncture combined with rehabilitation group on every day after modeling (p<0.01). Compared with acupuncture group, expression reduced in acupuncture combined with rehabilitation group on7rd (p<0.01) and3rd,21st(p<0.05) day after modeling, Compared with rehabilitation group, expression reduced in acupuncture combined with rehabilitation group on14st (p<0.05) day after modeling。Compared with model group, the IL-1β expression reduced in acupuncture combined with rehabilitation group on3rd、7th、14th day (p<0.01) and21rd day(p<0.05). Compared with acupuncture group and rehabilitation group, expression reduced in acupuncture combined with rehabilitation group on3rd,7th (p<0.05).5.The expression of skeletal muscle NF-κB-p65/TNF-α IL-1β:On the21st day after modeling, compared with the blank group,there was a significant decrease of NF-κB-p65、 TNF-α、IL-1β expression in the remaining groups (P<0.01), compared with the sham operation group and the blank group,there was a significant increase of NF-κB-p65、TNF-α IL-1β expression in the remaining groups expect of the blank group (P<0.01), compared with acupuncture group, the expression had a significant reduce in rehabilitation group (p<0.05), the expression reduced clearly in acupuncture combined with rehabilitation group (p<0.01), compared with rehabilitation group, the expression reduced in acupuncture combined with rehabilitation group (p<0.05).6. The expression of skeletal muscle IRS-1mRNA:On the21st day after modeling, there was the skeletal muscle IRS-1mRNA expression in every group there was a significant decrease of IRS-1mRNA expression in blank group than any others (P<0.01), compared with the sham operation group and the blank group,there was a significant increase of IRS-1mRNA expression in the remaining groups expect of the blank group (P<0.01), compared with acupuncture group, the expression decreased in rehabilitation group (p<0.05),the expression decreased more clearly in acupuncture combined with rehabilitation group (p<0.01), compared with rehabilitation group, the expression decreased in acupuncture combined with rehabilitation group (p<0.05).7. The expression of skeletal muscle PI3KmRNA:On the21st day after modeling, there was the skeletal muscle PI3KmRNA expression in every group. There was a significant decrease of PI3KmRNA expression in blank group than any others (P<0.01), compared with the sham operation group and the blank group, there was a significant decrease of PI3KmRNA expression in the remaining groups expect of the blank group (P<0.01). Compared with rehabilitation group and acupuncture group, the expression decreased in acupuncture combined with rehabilitation group (p<0.05). Conclusion1. Acupuncture and rehabilitation can promote the recovery of neural function of rats after diabetes ischemic stroke, and its effect is superior to the rehabilitation or acupuncture.2. Acupuncture and rehabilitation can demote the glucolipid metabolism of rats after diabetes ischemic stroke, making insulin more sensitive, and reverse metabolize abnormal, to improve diseased organism status.3. Acupuncture and rehabilitation can demote the NF-κB-p65、TNF-α and IL-1β in brain serum and cerebral ischemic region of rats after diabetes ischemic stroke, and then demote inflammation of ischemic region of cerebral issue and protect the brain.4. Acupuncture and rehabilitation can demote the expression of skeletal muscle NF-κB-p65、TNF-α、IL-1β protein in cerebral ischemic region of rats with diabetes ischemic stroke, to help relieve inflammation, making insulin in skeletal muscle more sensitive and demote insulin resistance.5. Acupuncture and rehabilitation can promote the expression of skeletal muscle IRS-1mRNA、PI3KmRNA protein in cerebral ischemic region of rats with diabetes ischemic stroke. Improvements demote the insulin resistance of skeletal muscle probability by IRS-1/PI3K signal transduction pathway.更多还原