本文選題：人參皂苷 + 原人參二醇�。� 參考：《南京中醫(yī)藥大學》2017年碩士論文

【摘要】：目的:通過親和"垂釣"、蛋白質(zhì)質(zhì)譜鑒定以及親和力分析,前期我們發(fā)現(xiàn)肌酸激酶(CK-MM)是人參皂苷在骨骼肌組織內(nèi)的作用靶點,并且該靶點與原形人參皂苷沒有相互作用,而與人參次皂苷及苷元有相互作用,其中以與原人參二醇(PPD)的親和作用最強。在此基礎上,本實驗進一步采用了多種親和檢測方法對PPD與CK-MM之間的相互作用進行確證,并基于該靶點闡釋人參皂苷抗物理疲勞的藥效。方法:實驗1:原人參二醇與肌酸激酶親和常數(shù)的測定采用生物膜層干涉技術(BLI)、微量熱泳動技術(MST)和等溫滴定量熱技術(ITC)精確測定PPD與CK-MM之間的各類親和常數(shù),包括親和力(KD)、結合劑量比(n)、焓變("縃)、熵變("縎),確證兩者之間的關系。實驗2:原人參二醇與肌酸激酶的分子對接基于分子對接軟件,允許PPD和CK構象自由變化,計算對接結果,探討PPD與CK的結合位點和結合模式,為兩者之間的相互作用進一步提供佐證數(shù)據(jù)。實驗3:原人參二醇體外對肌酸激酶活性的影響選擇三種不同品系來源的CK-MM:人源CK-MM、兔源CK-MM以及小鼠骨骼肌勻漿液,將其與不同濃度的PPD混合,分析PPD對CK-MM活性的影響。實驗4:原人參二醇體內(nèi)對肌酸激酶活性的影響給予小鼠PPD灌胃后,取雙后肢的骨骼肌,采用肌酸激酶試劑盒測定骨骼肌勻漿液中CK-MM的酶活,采用蛋白印跡法(Wester-Blot,WB)和Q-PCR法分析CK-MM的表達,以及采用高效液相色譜法(HPLC)測定組織中磷酸肌酸的含量,探索PPD與CK-MM結合后在體內(nèi)產(chǎn)生的生物效應。實驗5:原人參二醇對小鼠能量代謝的影響正常小鼠給予PPD灌胃,比較小鼠負重游泳前后骨骼肌組織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,分析原人參二醇是否有助于延緩組織中乳酸的生成。實驗6:原人參二醇對小鼠負重游泳和低張性缺氧的影響通過小鼠的窒息實驗以及負重游泳實驗,驗證PPD是否具有預防涉及能量代謝的病理生理過程的藥效。結果:實驗1:BLI技術檢測表明,PPD與CK-MM之間的親和力(KD)值為2.53 × l0-5M;ITC技術檢測得出兩者結合平衡常數(shù)(K)為5.38±0.924×105-1,反應結合比(N)為1.05±0.0212 Sites,焓值的變化("縃)為-3494±95.70cal/mol,熵值的變化("縎)為 14.5cal/mol/deg;MST技術測出兩者結合的Kd為1.34±0.179× 10-7M。實驗2:FTMap分析發(fā)現(xiàn),PPD與肌酸激酶具有三個潛在的結合位點,除ADP位點外(SI、S1'和S1"),尚有鄰近位點(S2、S2'和S2")以及二聚體相互作用面;采用Schrodinger軟件平臺的Glide模塊對原人參二醇與這幾個潛在結合位點進行了分子對接模擬,發(fā)現(xiàn)原人參二醇傾向于結合在S2/S2"位點。原人參二醇可通過羥基、羧基與Hisl91、Glu231、Glu232 形成氫鍵(S2)或與 Arg320、Gln318、Ser285 形成氫鍵(S2")。實驗3:不管是鼠源性、人源性還是兔源性的CK-MM,PPD體外均可提高其酶活,最大提高幅度約10%左右。不過,PPD的濃度與CK-MM的酶活之間不是線性關系,而是呈倒"U"的關系;隨著體外PPD的濃度逐步提高,酶活逐步上升,但是再增加PPD的濃度,酶活開始下降。實驗4:PPD灌胃給藥對骨骼肌組織中CK-MM的蛋白表達和mRNA表達均沒有明顯影響,但高劑量PPD可提高肌組織中CK-MM的酶活,提高幅度約5%左右;與此同時,PPD可劑量依賴性地提高骨骼肌組織中磷酸肌酸的含量,最大提高幅度約10%左右。實驗5:對于空白組小鼠,與游泳前相比,游泳后骨骼肌組織中乳酸的含量明顯升高,約48%;對于給藥組小鼠,與游泳前相比,游泳后骨骼肌組織中乳酸的含量也明顯升高,約18%。與空白組小鼠相比,給藥組小鼠在游泳前骨骼肌組織中磷酸肌酸的含量以及能量儲存指數(shù)較高;游泳后,空白游泳組的小鼠的能量儲存指數(shù)以及ATP/ADP的比值均比給藥游泳組低。實驗6:與空白組相比,PPD可明顯延長小鼠負重游泳的時間和窒息致死亡的時間。窒息實驗中,低、中以及高劑量組小鼠的生存時間分別延長了 7.6%、12.9%以及15.3%;負重游泳實驗中,中以及高劑量組小鼠的游泳時間多了 37.3%以及42.5%。結論:1.CK-MM為人參皂苷在骨骼肌組織中的作用靶點,PPD是與該靶點結合活性較好的人參皂苷在體內(nèi)的代謝產(chǎn)物之一。2.PPD與CK-MM結合后,可提高CK-MM的酶活。當機體處于相對靜止時,CK-MM酶活的提高,可增加組織中磷酸肌酸的含量,提升機體的能量儲備;當機體處于耗能狀態(tài)時,提高的能量儲備可給機體提供額外的能量支持,并延緩機體乳酸的生成,拮抗缺氧以及高耗能刺激誘導的病理生理進程。
[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.

【學位授予單位】：南京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285

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