原人参二醇骨骼肌组织内作用靶点肌酸激酶的确证与阐释

发布时间：2018-04-20 00:28

本文选题：人参皂苷 + 原人参二醇　；参考：《南京中医药大学》2017年硕士论文

【摘要】：目的:通过亲和"垂钓"、蛋白质质谱鉴定以及亲和力分析,前期我们发现肌酸激酶(CK-MM)是人参皂苷在骨骼肌组织内的作用靶点,并且该靶点与原形人参皂苷没有相互作用,而与人参次皂苷及苷元有相互作用,其中以与原人参二醇(PPD)的亲和作用最强。在此基础上,本实验进一步采用了多种亲和检测方法对PPD与CK-MM之间的相互作用进行确证,并基于该靶点阐释人参皂苷抗物理疲劳的药效。方法:实验1:原人参二醇与肌酸激酶亲和常数的测定采用生物膜层干涉技术(BLI)、微量热泳动技术(MST)和等温滴定量热技术(ITC)精确测定PPD与CK-MM之间的各类亲和常数,包括亲和力(KD)、结合剂量比(n)、焓变("縃)、熵变("縎),确证两者之间的关系。实验2:原人参二醇与肌酸激酶的分子对接基于分子对接软件,允许PPD和CK构象自由变化,计算对接结果,探讨PPD与CK的结合位点和结合模式,为两者之间的相互作用进一步提供佐证数据。实验3:原人参二醇体外对肌酸激酶活性的影响选择三种不同品系来源的CK-MM:人源CK-MM、兔源CK-MM以及小鼠骨骼肌匀浆液,将其与不同浓度的PPD混合,分析PPD对CK-MM活性的影响。实验4:原人参二醇体内对肌酸激酶活性的影响给予小鼠PPD灌胃后,取双后肢的骨骼肌,采用肌酸激酶试剂盒测定骨骼肌匀浆液中CK-MM的酶活,采用蛋白印迹法(Wester-Blot,WB)和Q-PCR法分析CK-MM的表达,以及采用高效液相色谱法(HPLC)测定组织中磷酸肌酸的含量,探索PPD与CK-MM结合后在体内产生的生物效应。实验5:原人参二醇对小鼠能量代谢的影响正常小鼠给予PPD灌胃,比较小鼠负重游泳前后骨骼肌组织中腺嘌呤核苷三磷酸(ATP)、二磷酸腺苷(ADP)、腺嘌呤核糖核苷酸(AMP)、磷酸肌酸以及乳酸的含量,分析原人参二醇是否有助于延缓组织中乳酸的生成。实验6:原人参二醇对小鼠负重游泳和低张性缺氧的影响通过小鼠的窒息实验以及负重游泳实验,验证PPD是否具有预防涉及能量代谢的病理生理过程的药效。结果:实验1:BLI技术检测表明,PPD与CK-MM之间的亲和力(KD)值为2.53 × l0-5M;ITC技术检测得出两者结合平衡常数(K)为5.38±0.924×105-1,反应结合比(N)为1.05±0.0212 Sites,焓值的变化("縃)为-3494±95.70cal/mol,熵值的变化("縎)为 14.5cal/mol/deg;MST技术测出两者结合的Kd为1.34±0.179× 10-7M。实验2:FTMap分析发现,PPD与肌酸激酶具有三个潜在的结合位点,除ADP位点外(SI、S1'和S1"),尚有邻近位点(S2、S2'和S2")以及二聚体相互作用面;采用Schrodinger软件平台的Glide模块对原人参二醇与这几个潜在结合位点进行了分子对接模拟,发现原人参二醇倾向于结合在S2/S2"位点。原人参二醇可通过羟基、羧基与Hisl91、Glu231、Glu232 形成氢键(S2)或与 Arg320、Gln318、Ser285 形成氢键(S2")。实验3:不管是鼠源性、人源性还是兔源性的CK-MM,PPD体外均可提高其酶活,最大提高幅度约10%左右。不过,PPD的浓度与CK-MM的酶活之间不是线性关系,而是呈倒"U"的关系;随着体外PPD的浓度逐步提高,酶活逐步上升,但是再增加PPD的浓度,酶活开始下降。实验4:PPD灌胃给药对骨骼肌组织中CK-MM的蛋白表达和mRNA表达均没有明显影响,但高剂量PPD可提高肌组织中CK-MM的酶活,提高幅度约5%左右;与此同时,PPD可剂量依赖性地提高骨骼肌组织中磷酸肌酸的含量,最大提高幅度约10%左右。实验5:对于空白组小鼠,与游泳前相比,游泳后骨骼肌组织中乳酸的含量明显升高,约48%;对于给药组小鼠,与游泳前相比,游泳后骨骼肌组织中乳酸的含量也明显升高,约18%。与空白组小鼠相比,给药组小鼠在游泳前骨骼肌组织中磷酸肌酸的含量以及能量储存指数较高;游泳后,空白游泳组的小鼠的能量储存指数以及ATP/ADP的比值均比给药游泳组低。实验6:与空白组相比,PPD可明显延长小鼠负重游泳的时间和窒息致死亡的时间。窒息实验中,低、中以及高剂量组小鼠的生存时间分别延长了 7.6%、12.9%以及15.3%;负重游泳实验中,中以及高剂量组小鼠的游泳时间多了 37.3%以及42.5%。结论:1.CK-MM为人参皂苷在骨骼肌组织中的作用靶点,PPD是与该靶点结合活性较好的人参皂苷在体内的代谢产物之一。2.PPD与CK-MM结合后,可提高CK-MM的酶活。当机体处于相对静止时,CK-MM酶活的提高,可增加组织中磷酸肌酸的含量,提升机体的能量储备;当机体处于耗能状态时,提高的能量储备可给机体提供额外的能量支持,并延缓机体乳酸的生成,拮抗缺氧以及高耗能刺激诱导的病理生理进程。
[Abstract]:Objective: through affinity "fishing", protein mass spectrometry identification and affinity analysis, we found that creatine kinase (CK-MM) is the target of Ginsenoside in skeletal muscle tissue, and that the target has no interaction with the original ginsenoside, but it has interaction with ginsenoside and glycosides, which is related to propanediol (PPD). On this basis, a variety of affinity detection methods have been used to confirm the interaction between PPD and CK-MM. Based on this target, the effect of ginsenoside against physical fatigue is explained. Method: the determination of affinity constant of 1: propanediol and creatine kinase using biofilm interferometry (BLI), micro Calorimetric swimming technique (MST) and isothermal titration calorimetry (ITC) accurately determine the affinity constants between PPD and CK-MM, including affinity (KD), combination of dose ratio (n), enthalpy change, entropy change, confirming the relationship between the two. The docking of 2: propanediol and creatine kinase based on molecular docking software allows PPD and CK conformation. The binding sites and binding patterns of PPD and CK were calculated to provide evidence for the interaction between them. The effects of 3: on the activity of creatine kinase in vitro were selected to select the CK-MM: human source CK-MM of three different strains, rabbit source CK-MM and mouse skeletal muscle homogenate. The effect of PPD on the activity of CK-MM was analyzed with the same concentration of PPD. The effect of 4: on the activity of creatine kinase in the experiment of experimental 4: was given to the mice after PPD gavage, the skeletal muscle of the two hind limbs was taken, the enzyme activity of CK-MM in the homogenate of skeletal muscle was measured by Creatine Kinase Kit, and the expression of CK-MM was analyzed by Western blot (Wester-Blot, WB) and Q-PCR method. To determine the content of creatine phosphocreatine in tissue by high performance liquid chromatography (HPLC) and explore the biological effects of PPD and CK-MM in the body. The effect of 5: on energy metabolism in mice was tested by PPD gavage in normal mice, and to compare the adenosine nucleoside three phosphoric acid (ATP) in the skeletal muscle tissue of mice before swimming. Two Adenosine phosphate (ADP), adenine ribonucleotide (AMP), creatine phosphate and lactic acid, analysis of whether propanediol can help delay the formation of lactic acid in tissue. The effect of 6: propanediol on weight swimming and hypoxic anoxia in mice was tested by asphyxia and swimming tests in mice to verify the prevention of PPD. Results of the pathophysiological processes involved in energy metabolism. Results: experimental 1:BLI technique tests showed that the affinity (KD) value between PPD and CK-MM was 2.53 x l0-5M; ITC technology detected the binding equilibrium constant (K) of 5.38 + 0.924 * 105-1, reaction binding ratio (N) of 1.05 + 0.0212 Sites, and the change of enthalpy value ("KD") was -3494 + 95.70cal/mol, entropy value The change ("14.5cal/mol/deg") is 14.5cal/mol/deg; the combination of the MST technique and the 1.34 + 0.179 x 10-7M. experimental 2:FTMap analysis found that PPD and creatine kinase have three potential binding sites, except for ADP loci (SI, S1'and S1 "), and there are adjacent sites (S2, S2', etc.) and two polymer interaction surfaces. The module carried out molecular docking simulation with these potential binding sites. It was found that propanediol was inclined to bind to the S2/S2 "site. Propanediol could be hydroxyl, carboxyl and Hisl91, Glu231, Glu232 to form hydrogen bonds (S2) or Arg320, Gln318, Ser285 to form hydrogen bonds (S2"). Experimental 3: is mouse origin, human origin or Rabbit derived CK-MM, PPD in vitro can improve its enzyme activity, the maximum increase of about 10%. However, the concentration of PPD and the enzyme activity of CK-MM is not linear, but the relationship of inverted "U"; with the increase of PPD concentration in vitro, the enzyme activity gradually rises, but the concentration of PPD increases, and the enzyme activity begins to decline. Experimental 4:PPD gavage to the bone. CK-MM protein expression and mRNA expression were not significantly affected in the iliac muscle tissue, but high dose PPD could increase the enzyme activity of CK-MM in muscle tissue about 5%. At the same time, PPD could increase the content of creatine phosphate in skeletal muscle tissue, and the maximum amplitude was about 10%. Experimental 5: was for blank group mice and swimming. Compared with pre swimming, the content of lactic acid in skeletal muscle after swimming was significantly increased, about 48%. Compared with before swimming, the content of lactic acid in the skeletal muscle of the mice was significantly higher than that before swimming. Compared with the blank group, the content of phosphocreatic acid and the energy storage index of the mice in the skeletal muscle before swimming were higher than that of the blank group, and the content of phosphocreatine and energy storage index were higher in the group of mice after swimming than in the blank group. After swimming, the energy storage index of the mice in the blank group and the ratio of ATP/ADP were lower than those in the swimming group. Compared with the blank group, the experimental 6: significantly prolonged the time of swimming and the time of death in the mice. The survival time of the mice in the low, middle and high dose group was 7.6%, 12.9%, and 15 respectively in the asphyxia experiment. .3%; in the heavy swimming test, the swimming time of mice in the middle and high dose group was 37.3% and 42.5%. conclusion: 1.CK-MM is the target of Ginsenoside in skeletal muscle tissue. PPD is the combination of the target point of ginsenoside, one of the metabolites of the body,.2.PPD and CK-MM, can improve the enzyme activity of CK-MM. The increase of CK-MM enzyme activity can increase the content of creatine phosphocreatine in the tissue and enhance the energy reserve of the body. When the body is in the state of energy consumption, the increased energy reserves can provide additional energy support to the body, delay the formation of the body's lactic acid, antagonize the pathophysiological process induced by oxygen deficiency and high energy consumption.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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