二維電泳分析常被用於分析組織或體液中的蛋白質分佈狀況，透過二維電泳分析可比較不同處理的樣品所表現出的蛋白質差異，進而探討相關病理機制。本實驗室體外試驗發現，香椿萃取物TSL-2（5-2）在高濃度時具有過氧化現象，導致精子氧化壓力增加，TSL-6（5-4R）可回復精子所受到之氧化壓力傷害。本實驗利用二維電泳分析法探討在in vivo 的環境中，TSL-6、TSL-2 及TSL-2P 這三種不同成分的香椿萃取物對經由過氧化氫誘發氧化壓力之大鼠睪丸蛋白質表現的影響。大鼠以腹腔注射過氧化氫（1mmol/ kgB.W）誘發氧化壓力，並分別餵食正常飼料（控制組）、Vitamin C、香椿TSL-2（0.053 g/kgB.W/day）、TSL-2P（0.94 g/kgB.W/day）或TSL-6
（0.013 g/kgB.W/day）等五組，八週後犧牲，萃取不同組別各四隻大鼠睪丸蛋白質，以Type 1 及Type 2 兩種方式進行二維電泳分析，Type 1 是將同組別的蛋白質混合進行三重複，以降低大鼠之個體差異對二維電泳再現性的影響，Type 2 是將各隻大鼠蛋白質分別進行二維電泳分析，以減少技術差異。在控制組、Vitamin C 組及香椿TSL-6 這三個組別之間找到13 個表現量差異2 倍以上的蛋白質點。進一步將這些蛋白質點進行質譜分析，一共比對出9 個蛋白質， 分別為tumor rejection antigen gp96 、3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2（HMG-CoA synthase 2）,
Glutathione Transferase Mu 6（GST Mu6）, Cofilin 2, pancreatic trypsin 1, heat shock protein 1-β、peptidylprolyl isomerase A, type II keratin Kb1, heat shock
90kDa protein 1-β。
在控制組、Vitamin C 組及香椿TSL-2 這三個組別之間找到10 個表現量差異1.5 倍以上的蛋白質點。進一步將這些蛋白質點進行質譜分析，一共比對出4 個蛋白質，分別為glutathione S-transferase（GST Mu6）, phospholipid hydroperoxide glutathione peroxidase（PHGPx）, fatty acid binding protein 9, thioredoxin。
在控制組、Vitamin C 組及香椿TSL-2P 這三個組別之間找到3 個表現量差異1.5 倍以上的蛋白質點。進一步將這些蛋白質點進行質譜分析，比對出1 個蛋白質，為phospholipid hydroperoxide glutathione peroxidase（PHGPx）。
利用二維電泳分析的結果發現香椿萃取物TSL-6會影響大鼠睪丸許
多蛋白質的表現，包括與精卵結合相關的gp96、與合成荷爾蒙有關的HMG-CoA合成酶、與蛋白質摺疊有關的Heat shock protein 1 beta、與頂體反應有關的水解酵素Pancreatic trypsin 1以及解毒酵素GST Mu6；香椿萃取物TSL-2及TSL-2P最主要會影響體內之解毒酵素如GST及PHGPx的表現，這些結果顯示，不同香椿萃取物會透過影響不同蛋白質的表現量來調控氧化壓力下睪丸的功能。

Our previous study showed that fractionated Toona sinensis leaf extract TSL-6（5-4R） protected sperm against oxidative damage. In this study, we used 2D-gel electrophoresis to analyze the protein expression of testes in rats under oxidative stress induced by i.p injection of H2O2（1 mmol/ kgB.W.） and
fed with normal diet（Control）, Vitamin C（0.2 g/kg B.W.）, TSL-2（0.053 g/kgB.W/day）, TSL-2P（0.94 g/kgB.W/day） or TSL-6（0.013 g/kgB.W.）for 8 weeks. Two types of 2D-gel electrophoresis were used to minimize
individual variation in Type 1 and to decrease the technique deviation in Type 2. Proteins of four rats in each group were pooled to run triplicate gels in Type 1
while single gel was run for individual rat in Type 2. There are two parameters for selecting spots. First, the spot must express differentially more than 2 or 1.5
folds between each group. Second, any spot must be present in all three triplicates of each composite gel in Type 1, and in at least three of the four individual gels in Type 2. Results of the 2D-gel analysis by ImageMaster 2D
Platinum Software indicated that 13 spots were differentially expressed more than 2 fold among control, Vitamin C and TSL-6 groups. Further identification
of these proteins with MALDI-TOF showed that these proteins were tumor rejection antigen gp96, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 （HMG-CoA synthase 2）, Glutathione Transferase Mu 6（GST Mu 6）, Cofilin
2, pancreatic trypsin 1, heat shock protein 1-β, peptidylprolyl isomerase A, type II keratin Kb1, heat shock 90kDa protein 1-β. These proteins are involved
in detoxification and sperm-oocyte interaction. GST Mu 6 is a member of GST Mu family which is mainly expressed in brain and testis to neutralize many toxic compounds. Pancreatic trypsin 1 is a member of serine protease family
and might be involved in acrosome reaction of mammalian sperm. Tumorrejection antigen gp96 is involved in the formation of functional zona-receptor complex on the surface of mammalian sperm. Heat shock protein 1-β prevents
misfolding of proteins and helps denatured proteins to refold during oxidative stress.
Ten protein spots were differentially expressed more than 1.5 fold among control, Vitamin C and TSL-2 groups. Among them, 4 proteins were identified by MALDI-TOF, including glutathione S-transferase（GST）, phospholipid
hydroperoxide glutathione peroxidase（PHGPx）, fatty acid binding protein 9, thioredoxin。
Among control, Vitamin C and TSL-2P groups, 3 spots were differentially expressed more than 1.5 fold. One protein, phospholipid hydroperoxide glutathione peroxidase（PHGPx）was identified by MALDI-TOF.
In conclusion, Toona Sinensis extracts may regulate the expression of proteins involved in improving sperm-oocyte interaction, detoxification, regulating protein misfolding in rats during oxidative stress.

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