COXⅦa在中老年男子部分雄激素缺乏综合征中的作用及机理研究
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摘要
中老年男性随着年龄的增长雄激素水平逐渐下降,并出现倦怠、乏力、抑郁、失眠、情绪波动、易怒、性欲减退、勃起功能障碍、体重减轻和骨质疏松等一系列临床症状,这一现象被称为中老年男子部分雄激素缺乏综合征(partial androgen deficiency of the aging male, PADAM)。PADAM和许多疾病有关,如心血管疾病、糖尿病等,老年人群因衰老引发的各种疾病发病率逐年增高,严重影响中老年男性的身体健康和生活质量。关注老年疾病,提高老年人生活质量,延长寿命,成为全球关注的重要医学课题。
COXⅦa(cytochrome c oxidase subunitⅦa)是线粒体呼吸链第4个复合体的核编码亚基,参与该复合体的构成,在线粒体氧化呼吸链的电子传递中发挥重要作用。研究表明,COXⅦa在老年人睾丸组织显著上调,推测其可能与老年雄激素合成功能减退及老年男性雄激素部分缺乏综合征有关,但其具体机制仍不清楚,国内外报道极少见。本研究以D-半乳糖所致亚急性衰老大鼠为PADAM模型,利用基因转染和RNA干扰等现代分子生物学技术,从体内和体外两方面深入研究了COXWa在PADAM中的作用及机制,为研究PADAM的机理和基因靶向治疗提供理论基础。
目的：探讨大鼠出生后不同发育阶段睾丸组织COXⅦa及睾酮合成酶和急性调节蛋白的表达变化并确定其与睾酮浓度的相关性。
1实验动物：选用出生后1周龄、3周龄、5周龄、3月龄和24月龄清洁级雄性SD大鼠,每年龄组6只(1周龄组为12只)；
2睾酮测定：化学发光方法测定血清总睾酮水平,批内和批间变异系数分别为2.2%-2.4%和3.4%-4.7%：
3 RT-PCR方法检测各年龄组睾丸组织COXⅦa、3β-HSD、17β-HSD、P450scc和StARmRNA变化；
4 western blotting、免疫组化法检测各年龄组睾丸组织COXⅦa、3β-HSD、17β-HSD、P450scc和StAR蛋白表达变化和定位。
1大鼠血清睾酮水平：3月龄组、24月龄组、5周龄组、3周龄组和1周龄组依次降低,3月龄组和24月龄组与各组之间差异有统计学意义(p<0.05),其余各组差异无统计学意义(p>0.05)；
2mRNA变化：COXⅦa mRNA水平24月龄组最高、3月龄组最低,与各组之间差异均有统计学意义(p<0.05)；3β-HSD、17β-HSD和P450scc mRNA水平均为3月龄组和5周龄组高于其余各组,差异均有统计学意义(p<0.05)；StARmRNA水平3月龄组最高,5周龄组和24月龄组次之,1周龄组和3周龄组最低,差异有统计学意义(p<0.05)。
3蛋白表达和定位：COXⅦa蛋白表达于间质细胞胞质、胞核及精原细胞胞核,24月龄最高,3月龄组最低；3β-HSD蛋白表达于间质细胞胞质,17β-HSD蛋白表达于间质细胞胞质及精原细胞胞核,P450scc蛋白表达于间质细胞胞质、胞膜及精子细胞胞质,StAR蛋白表达于间质细胞胞质和胞膜以及精原细胞胞质；3β-HSD、17p-HSD、P450scc和StAR蛋白表达均为3月龄组和5周龄组高于其余各组,差异均有统计学意义(p<0.05)。
4相关性分析：COXⅦa蛋白表达与血清睾酮水平、3β-HSD、17β-HSD、P450scc和StAR蛋白表达呈负相关关系,除StAR外,均有统计学意义(p<0.05；p<0.01)。
1不同年龄大鼠睾丸组织COXⅦa表达存在差异,老年鼠表达高于青年鼠,且与血清睾酮浓度呈负相关关系,提示COXⅦa与睾酮合成有关,可能参与了PADAM的发生；
2不同年龄大鼠睾丸组织3β-HSD、17β-HSD、P450scc和StAR表达存在差异,3月龄组和5周龄组高于1周龄组、3周龄组和24月龄组；
3 COXⅦa蛋白表达与3β-HSD、17β-HSD和P450scc蛋白表达呈负相关,提示COXⅦa可能通过上述类固醇合成酶调控睾酮合成,似乎与急性调节蛋白无关。
目的：1构建pEGFP-N1-COXⅦa重组质粒并瞬时转染原代培养的3月龄大鼠Leydig细胞,探讨COXⅦa过表达对睾酮合成能力的影响；
2应用RNA干扰技术抑制COXⅦa在Leydig细胞中的表达,探讨COXWa抑制对睾酮合成能力的影响。
1大鼠睾丸Leydig细胞原代培养和鉴定：根据王晓云等的差速贴壁方法稍加改进,分离纯化Leydig细胞,3β-羟基类固醇脱氢酶化学染色和免疫细胞化学染色鉴定细胞纯度,0.4%台盼蓝溶液检查细胞活力；
2 pEGFP-N1-COXⅦa质粒构建、转染及细胞功能检测
2.1构建pGM-T-COXⅦa克隆质粒：设计扩增COXⅦa的DNA引物,在上、下游引物的5’端分别加入BglⅡ和EcoRⅠ的酶切位点和保护碱基,从24月龄大鼠睾丸组织中RT-PCR扩增COXWa基因，将扩增产物纯化回收、经BglⅡ和EcoRⅠ酶切获得COXⅦa基因片段；用T4连接酶将具有相同BglⅡ和EcoRⅠ粘性末端的pGM-T载体和COXⅦa基因连接,将连接产物转化DH5α感受态细胞,涂板,挑克隆,摇菌抽质粒后酶切鉴定含COXⅦa基因的克隆,大量扩增该克隆后抽质粒,得到pGM-T-COXⅦa克隆质粒；
2.2构建pEGFP-N1-COXⅦa表达质粒：大量扩增含pEGFP-N1空质粒的大肠杆菌,抽提得到pEGFP-N1空质粒；将pGM-T-COXⅦa和pEGFP-N1分别用BglⅡ和EcoRⅠ进行双酶切,酶切片段纯化回收后用T4连接酶进行连接,将连接产物转化DH5a感受态细胞,涂板,挑克隆,摇菌抽提质粒后酶切鉴定含COXⅦa基因的克隆,大量扩增该克隆后抽提质粒,得到pEGFP-Nl-COXⅦa表达质粒；
2.3测序：将RT-PCR扩增的COXⅦa片段和pEGFP-N1-COXⅦa重组质粒送上海生工测序,测序结果与genebank中序列进行比对；
2.4转染细胞：用脂质体介导法将pEGFP-N1-COXⅦa重组质粒转染Leydig细胞,分为空白对照组、空质粒组、转染脂质体组和转染pEGFP-N1-COXⅦa组;
2.5阳性细胞鉴定：在荧光显微镜下观察,有绿色荧光表达者为阳性细胞,并用MitoTracker(?) Red CM-H2XRos孵育,共聚焦激光显微镜观察pEGFP-N1-COXⅦa在细胞内的表达和定位；
2.6细胞功能检测：细胞兰TM检测细胞活力,化学发光法测定培养上清中总睾酮浓度,RT-PCR和western blotting分别检测COXⅦa mRNA和蛋白表达,免疫细胞化学进行蛋白定位。
3.1转染：将筛选出的抑制效应最强的SiRNA转染PADAM模型大鼠的Leydig细胞,分为空白对照组、脂质体组、阴性对照组、RNA干扰组、阳性对照组和荧光标记阴性对照组(观察转染效率)；
3.2转染效率：荧光显微镜下观察荧光标记阴性对照组,判断转染效率；
3.3细胞功能检测：细胞兰TM检测细胞活力,化学发光法测定培养上清中总睾酮浓度,RT-PCR和western blotting分别检测COXⅦa mRNA和蛋白表达。
1分离的Leydig细胞纯度达95%以上,可以满足实验要求；
2pEGFP-N1-COXⅦa质粒经测序证实序列正确；
3过表达COXⅦa的Leydig细胞活力降低,睾酮分泌减少,与空白对照组、转脂质体组和转染空质粒组比较,差异具有统计学意义(p<0.05)；
4 COXⅦa蛋白表达于Leydig细胞胞质和胞核；pEGFP-N1-COXⅦa转染组COXWamRNA和蛋白表达均高于空白对照组、转脂质体组和转染空质粒组,差异具有统计学意义(p<0.05)；
5 RNA干扰抑制了Leydig细胞COXVHamRNA和蛋白表达,均低于空白对照组、脂质体组、阴性对照组,差异具有统计学意义(p<0.05)；
6 RNA干扰组细胞活力和睾酮分泌均高于空白对照组、脂质体组和阴性对照组。
COXⅦa具有降低细胞活力、抑制睾酮分泌的作用。
目的：探讨COXⅦa对大鼠睾酮合成的影响及其与类固醇合成酶和急性调节蛋白的关系,利用RNA干扰和中药何首乌饮探索PADAM治疗的新方法。
1 COXⅦa过表达对青年大鼠睾酮合成及类固醇合成酶和蛋白的影响
1.1动物分组：3月龄清洁级SD雄性大鼠随机分为pEGFP-N1-COXⅦa转染组(模型组)、空质粒组、脂质体组、葡萄糖对照组(葡萄糖组)和空白对照组,每组各8只动物；
1.2化学发光法测定各组大鼠血清总睾酮浓度,生物化学方法检测血清SOD、T-AOC活力及MDA含量,RT-PCR和western blotting分别检测COXⅦa、3β-HSD,17β-HSD、P450scc和StAR mRNA和蛋白表达。
2 RNA干扰及何首乌饮对PADAM大鼠睾酮合成及类固醇合成酶和蛋白的影响
2.1建立PADAM大鼠模型：清洁级8周龄雄性SD大鼠,D-半乳糖300mg/kg/d腹腔注射,连续用药50d；
2.2动物分组：正常对照组、PADAM模型组、RNA干扰组、睾酮注射组(阳性对照组)、何首乌饮治疗组和模型对照组,每组各8只动物。
2.3化学发光法测定各组大鼠血清总睾酮浓度,生物化学方法检测血清SOD、T-AOC活力及MDA含量,RT-PCR和western blotting分别检测COXⅦa、3β-HSD、17β-HSD、P450scc和StAR mRNA和蛋白表达。
1青年大鼠转染pEGFP-N1-COXVUa质粒后,与空质粒组、脂质体组、葡萄糖组和空白对照组比较：
1.1血清睾酮浓度降低,差异有统计学意义(p<0.05)；
1.2血清SOD、T-AOC活力下降,MDA含量升高,差异有统计学意义p<0.05)；
1.3 COXⅦa mRNA和蛋白表达增高,差异有统计学意义p<0.05)；
1.43p-HSD、17p-HSD、P450scc和StAR mRNA和蛋白表达下降,差异有统计学意义(p<0.05)；
2 PADAM大鼠RNA干扰组和何首乌饮治疗与模型组、正常对照组比较：
2.1血清睾酮浓度高于模型组,低于正常对照组,差异有统计学意义(p<0.05)；
2.2血清SOD、T-AOC活力高于模型组,低于正常对照组,MDA含量相反,差异有统计学意义(p<0.05)；
2.3 COXⅦa mRNA和蛋白表达较模型组显著下降,仍高于正常对照组,差异有统计学意义(p<0.05)；
2.43β-HSD、17β-HSD、P450scc和StAR mRNA和蛋白表达较模型组升高,但仍低于正常对照组,差异有统计学意义(p<0.05)；
1COXVHa过表达可致青年大鼠睾酮浓度下降,类固醇合成酶和急性调节蛋白表达减少；
2 PADAM模型大鼠经RNA干扰及何首乌饮治疗后,大鼠睾酮合成能力均得到部分恢复,类固醇合成酶和急性调节蛋白表达增加；
3 COXVHa在大鼠衰老过程中发挥作用,可能与类固醇合成酶和急性调节蛋白有关；
4靶向COXⅦa的RNA干扰治疗和何首乌饮治疗可以延缓衰老。
目的：探讨MAPK信号通路是否参与COXWa对Leydig细胞睾酮合成的调节过程。
分别对青年大鼠和PADAM模型大鼠的Leydig细胞转染pEGFP-Nl-COXⅦa重组质粒和RNA Oligo后,进行下列实验：
1流式细胞仪检测细胞凋亡率；
2 RT-PCR检测：3β-HSD、17β-HSD、P450scc和StAR mRNA;
3免疫细胞化学方法检测3β-HSD、17β-HSD、P450scc、StAR、Bax和Bcl-2蛋白表达；
4 western blotting检测3β-HSD、17β-HSD、P450scc、StAR以及Bax、Bcl-2、ERK1/2、p-ERK1/2、JNK、p-JNK、P38、p-P38蛋白表达。
1转染pEGFP-N1-COXⅦa重组质粒后,转染组与对照组、空质粒组和脂质体组比较：
(1)细胞凋亡率增加,差异有统计学意义(p<0.05)；
(2) 3-HSD、17β-HSD. P450sccmRNA减少,差异有统计学意义p<0.05)；StARmRNA差异无统计学意义；
(3)3β-HSD、17β-HSD、P450scc和：StAR蛋白表达减少,差异有统计学意义(p<0.05)；
(4) Bcl-2表达减少,Bax表达增加,Bcl-2/Bax下降,差异有统计学意义(p<0.05)；
(5) ERK1/2、JNK和P38差异无统计学意义；p-ERK1/2和p-JNK显著增加,差异有统计学意义p<0.05)；p-P38未见阳性条带。
2 RNA干扰后,干扰组与阴性对照组、脂质体组和空白对照组比较：
(1)细胞凋亡率减少,差异有统计学意义(p<0.05)；
(2) 3β-HSD、17β-HSD、P450sccmRNA增加,差异有统计学意义(p<0.05)；StARmRNA差异无统计学意义；
(3)3β-HSD、17p-HSD、P450scc和StAR蛋白表达增加,差异有统计学意义(p<0.05)；
(4) Bcl-2表达升高,Bax表达下降,Bcl-2/Bax增大(p<0.05)；
(5) ERK1/2、JNK和P38差异无统计学意义；p-ERK1/2和p-JNK显著减少,差异有统计学意义(p<0.05)；p-P38未见阳性条带。
1在COXⅦa过表达的Leydig细胞,p-ERK1/2和p-JNK高表达,睾酮分泌减少,Bcl-2/Bax下降,细胞凋亡增加,3β-HSD、17β-HSD、P450scc和StAR蛋白表达减少,提示p-ERK1/2和p-JNK抑制类固醇合成酶活性,促进细胞凋亡；
2在COXⅦa干扰的Leydig细胞,p-ERK1/2和p-JNK低表达,睾酮分泌增加,Bcl-2/Bax升高,细胞凋亡减少,3β-HSD、17β-HSD、P450scc和StAR蛋白表达增加,提示抑制ERK1/2和JNK蛋白活化可以上调类固醇合成酶活性,抑制细胞凋亡；
3 COXⅦa可能通过ERK1/2和JNK信号通路发挥作用。
Due to the decrease in androgen levels gradually with their aging, middle-aged males appear fatigue, weakness, depression, insomnia, mood swings, irritability, decreased libido, erectile dysfunction, weight loss and osteoporosis and a series of clinical syndrome, which are known as partial androgen deficiency syndrome of the aging male (PADAM). PADAM is associated with many diseases, such as cardiovascular disease, diabetes and so on. The higher incidence of various age-related diseases seriously affects older men's health and their quality of life. Concerning about the age-related diseases, improving the quality of life of older persons and extending their life span have become the important medical issues of the world's attention.
COXⅦa (Cytochrome c Oxidase SubunitⅦa) is nuclear encoded subunit of complex IV of mitochondrial respiratory chain, involved in the complex composition and plays an important role in electron transfer of mitochondria respiratory chain oxidation. Studies indicate that COXⅦa in the testis of the aging male was significantly increased, suggesting that it may be involved in androgen dysfunction and partial androgen deficiency syndrome of the aging male, but the exact mechanism remains unclear, and there are rare domestic and foreign reports. PADAM model was established by injection of D-galactose. Some techniques of modern molecular biology such as gene transfection and RNA interference were used to study the effects and mechanism of COXⅦa on PADAM rats in vivo and in vitro, aiming to provide the theoretical basis for exploring the mechanism of PADAM and gene targeting therapy.
To study the changes of COXⅦa,3β-HSD,17β-HSD, P450scc and StAR mRNA and acute regulatory protein expression in rat testes of different periodsand the correlation between them and testosterone concentration.
1. Male SD rats aged 1 week,3 weeks,5 weeks,3 months and 24 months were studied, each group 6 animals(1 week group was 12).
2. The serum testosterone was determined by chemiluminescence immunoassay (CLIA), variation coefficient of inter-group and intra-group was 2.2% to 2.4% and 3.4% to 4.7% respectively.
3. RT-PCR was used to examine the changes of COXⅦa,3β-HSD,17β-HSD, P450scc and StAR mRNA of the testicular tissues among the above groups.
4. Western blotting and immunohistochemistry were employed to detect the difference of COXⅦa,3β-HSD,17β-HSD, and P450scc and StAR protein expression of the testicular tissues among the above groups.
1. The testosterone level in the rat serum decreased gradually from 3 months to 24 months,5 weeks,3 weeks and 1 week group. The difference between 3 months,24 months group and the other groups was statistically significant respectively (p<0.05).
2. COXⅦa mRNA level in 24 month group was the highest, and that in 3 month group was the lowest, and the differences between 3 months,24 months group and the other groups were statistically significant(p<0.05); 3β-HSD,17β-HSD and P450scc mRNA levels in 3 months group and 5 weeks group were significantly higher than them in other groups(p<0.05); StARmRNA level in 3 months group was the highest,5 weeks and 24 months group were followed,1 week and 3 weeks group were the lowest, the difference was statistically significant(p<0.05).
3. COXⅦa protein expressed in plasmid and nucleus of Leydig cells and the nucleus of spermatogonia. It was the highest in 24 month group and the lowest in 3 months group.3β-HSD protein was expressed in plasmid of Leydig cells, 17β-HSD protein in plasmid of Leydig cells and nucleus of spermatogonia, P450scc protein in plasmid, nucleus and membrane of Leydig cells, and plasmid of spermatid, StAR protein in the plasmid and membrane of Leydig cells and plasmid of spermatogonia.3β-HSD,17β-HSD, P450scc and StAR protein was significantly higher in 3 months group and 5 weeks group than them in other groups(p<0.05).
4. There was negative correlation between COXⅦa protein and serum testosterone concentration,3p-HSD,17β-HSD, P450scc and StAR protein. The differences were statistically significant except StAR (p<0.05,p<0.01).
1. The expression of COXⅦa had significant change in different periods and it was higher in old rats than in young rats, and was negatively related to serum testosterone concentration, suggesting that COXⅦa may be associated with testosterone synthesis and involved in the occurrence of PAD AM.
2. The expression of 3β-HSD,17β-HSD, P450scc and StAR had significant change in rat testes of different periods, namely,3 months and 5 weeks group was significant higher than 1 week,3 weeks and 24 months group.
3. There was negative correlation between COXⅦa and 3β-HSD,17β-HSD, P450scc, indicating that COXⅦa regulated testosterone synthesis through steroidogenesis enzymes but not StAR.
1. To explore the impact of COXⅦa overexpression to testosterone synthesis by construction pEGFP-N1-COXⅦa recombinant plasmid and transient transfection primary cultured rat Leydig cells extracted from 3 months.
2. To explore the impact of COXⅦa suppression to testosterone synthesis by application of RNA interference inhibition of COXⅦa in Leydig cells.
1. Improved Wang Xiaoyun's differential adhesion speed method was used in the isolation of Leydig cells; 3β-hydroxysteroid dehydrogenase (3β-HsD) staining and immunohistochemical staining were employed in identifying the cell purity,0.4% solution of trypan blue in checking cell viability.
2.pEGPF-N1-COXⅦa plasmid construction, tranfection and function examination
2.1 Construct pEGPF-N1-COXⅦa plasmid:COXⅦa DNA was amplified by RT-PCR from 24 month rat testis and was digested with Bgl II and EcoR I, then was linked to the pGM-T vector by T4 ligase. E. coli including pGM-T-COXⅦa plasmid and E. coli including p-EGPF-N1 plasmid were propagated in a large scale. After extracted, two kinds of plasmid were digested with Bgl II and EcoR I. Then pEGPF-N1 and COXⅦa gene was linked together by T4 linkase.
2.2 Transfection:After the sequence of the recombinant plasmid was proved correct, the recombinant plasmid was transferred into the primary cultured Lydia cells.
2.3 Identification to positive cells:Cells with green fluorescence under the fluorescent microscope were positive. Co focal laser scanning microscopy was used to observe in the expression and localization of pEGFP-Nl-COXⅦa within the cell which were incubated with MitoTracker (?) Red CM-H2Xros together after 20 hours transfected with pEGFP-N1-COXⅦa.
2.4 Function examination:Cell BlueTM Kit was used to determine cell viability, CLIA was used to examine testosterone concentration, RT-PCR and western blotting were used to detect COXⅦa mRNA and protein expression respectively, and immunocytochemistry for protein localization.
3. RNA Interference
3.1 Transfer:SiRNA Oligo with the most powerful inhibitory effect was screened out to be tranferred into PADAM rat Leydig cells with Lipofectamine 2000. Blank control group, Lipofectamine control, negative control, RNA interference group, positive control and fluorescent labeled negative control group were formed.
3.2 Transfer efficiency:Fluorescent microscope was used to observe fluorescent labeled negative control cells to determine transfer efficiency.
3.3 Function examination:Cell BlueTM Kit was used to determine cell viability, CLIA was used to examine testosterone concentration, RT-PCR and western blotting were used to detect COXⅦa mRNA and protein expression respectively.
1. The purity of isolated Leydig cells was more than 95% in accordance with the test requirements.
2. pEGPF-N1-COXⅦa plasmid sequence was confirmed correct by using the sequencing apparatus.
3. Both the viability and testosterone level of Leydig cells overexpressing COXⅦa was significantly decreased than them of blank control, lipofectamine and empty plasmid group (p<0.05).
4. COXⅦa protein was detected in Leydig cell plasmid and nucleus. COXⅦa mRNA and protein of Leydig cells overexpressing COXⅦa was significantly higher than that of blank control, lipofectamine and empty plasmid group (p<0.05).
5. SiRNA suppressed COXⅦa mRNA and protein expression in the Leydig cells, which were significantly lower than that in blank control, Lipofectamine and negative control group(p<0.05).
6. Both the viability and testosterone level of Leydig cells with SiRNA was significantly higher than that of blank control, Lipofectamine and negative control group (p<0.05).
COXⅦa protein can suppress cell proliferation and testosterone secretion.
To study the impact of COXVIIa to testosterone biosynthesis in rats and the relation between it and steroidogenesis enzymes and acute regulatory protein in vivo and to explore the new therapy by using RNA interference and Polygonum Multiflorum Trunb.
1. The impacts of COXⅦa overexpression to testosterone synthesis, steroidogenesis enzymes and acute regulatory protein in young rats:
1.1 Animal groups:3 months SD male rats were randomly divided into pEGFP-N1-COXⅦa group (M group), empty plasmid group (P group), lipofectamine (L group), glucose control (G group) and blank control group (Bc group), each group 8 animals.
1.2 CLIA was used to determine the concentration of total testosterone in serum. Biochemical methods were used to examine SOD, T-AOC viability and MDA level; RT-PCR and western blotting was used to detect COXⅦa, 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression respectively.
2. The impacts to the Testosterone synthesis and steroidogesis enzyme and protein treated with RNA interference and Polygonum Multiflorum Trunb in PADAM model rats.
2.1 Establishment of PADAM model:clean 8 weeks male SD rats were treated with D-galactose 300mg/kg/d intraperitoneally, for continuous 50d.
2.2 Animal groups:normal control group (Bc group), PADAM model group (M group), RNA interference group (Si group), testosterone injection group (T group), Polygonum Multiflorum Trunb treated group (H group) and model control group (Mc group), each group 8 animals.
2.3 CLIA was used to determine the concentration of total testosterone in serum. Biochemical methods were used to examine SOD, T-AOC viability and MDA level; RT-PCR and western blotting were used to detect COXⅦa, 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression respectively.
1. To compare with P group, L group, G group and Bc group, the expression of the several indicators in M group was as follows:
1.1 Serum testosterone concentration was decreased (p<0.05).
1.2 The SOD and T-AOC activity was decreased, MDA level was increased (p<0.05).
1.3 COXⅦa mRNA and protein expression increased respectively (p<0.05).
1.4 3β-HSD,17p-HSD, P450scc and StAR mRNA and protein expression was decreased respectively (p<0.05).
2. Comparison between Si group, T group and M group and Bc group after RNA interference in PADAM model rats.
2.1 Serum testosterone concentration of Si group and T group was significantly higher than that of M group, while lower than that of Bc group (p<0.05).
2.2 The SOD, T-AOC viability of Si group and T group was higher than that of M group, while lower than that of Bc group, MDA content in the contrary (p<0.05).
2.3 COXⅦa mRNA and protein expression of Si group and T group decreased compared with M group, still higher than that of Bc group (p<0.05).
2.4 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression significantly increased compared with M group, but still lower than that of Bc group (p<0.05).
1. COXⅦa overexpression in young rats could decrease testosterone synthesis and expression of steroidogenesis enzymes and acute regulatory protein.
2. RNA interference in PADAM model rats could lead to partial restoration of testosterone synthesis of rats, increased expression of steroidogenesis enzymes and acute regulatory protein.
3. COXⅦa may be play a role in the aging process and related to steroidogenesis enzymes and the acute-regulated protein.
4. RNA interference targeting COXⅦa gene and Polygonum Multiflorum Trunb can delay senescence of rats.
To explore whether the pathway of ERK and JNK participates in the process that COXⅦa regulates testosterone synthesis of Leydig cells.
Leydig cells extracted from young rats and RADAM model rats were transfected with pEGPF-N1-COXⅦa recombinant plasmid and SiRNA respectively, then the experiments were as followed:
1. Flow Cytometry was used to detect cell apoptosis rate.
2. PCR was used to detect 3β-HSD,17β-HSD, P450scc and StAR mRNA.
3. Immunocytochemistry was used to detect 3β-HSD,17β-HSD, P450scc, StAR, Bax and Bcl-2 protein expression.
4. Western blotting was used to detect 3β-HSD,17β-HSD, P450scc, StAR and Bax, Bcl-2, ERK1/2, p-ERK1/2, JNK, p-JNK, P38, p-P38 protein expression.
1. To compare with control group, empty vector group and Lipofectamine group, the expression of the several indicators in pEGPF-N1-COXⅦa group was as follows: 1.1 The apoptosis rate increased significantly (p<0.05).
1.2 The level of 3β-HSD,17β-HSD, P450scc mRNA decreased significantly (p<0.05), while there was no significant difference of StAR mRNA.
1.3 The expression of 3β-HSD,17β-HSD, P450scc and StAR protein expression decreased significantly (p<0.05).
1.4 Bcl-2 expression decreased, while Bax expression increased, Bcl-2/Bax decreased (p<0.05).
1.5 ERK1/2, JNK and P38 showed no significant difference; p-ERK1/2 and p-JNK increased significantly (p<0.05); p-P38 no positive bands.
2. To compare with the negative control group, Lipofectamine control group and blank control group, the expression of the several indicators in the interference group with was as follows: 1.1 The apoptosis rate decreased significantly (p<0.05). 1.2 The level of 3β-HSD,17P-HSD, P450scc mRNA increased significantly (p<0.05), while there was no significant difference of StAR mRNA. 1.3 The expression of 3β-HSD,17β-HSD, P450scc and StAR protein expression increased significantly (p<0.05). 1.4 Bcl-2 expression increased, while Bax expression decreased, Bcl-2/Bax increased (p<0.05). 1.5 ERK1/2, JNK and P38 showed no significant difference; p-ERK1/2 and p-JNK significantly reduced (p<0.05); p-P38 no positive bands.
1. In the Leydig cells overexpressing COXⅦa, p-ERK1/2 and p-JNK expression and the apoptosis rate increased, while testosterone concentration, the ratio of Bcl-2/Bax, and 3β-HSD,17β-HSD, P450scc and StAR protein decreased, suggesting that p-ERK1/2 and p-JNK could suppress steroid synthesis and promote cell apoptosis.
2. The results of RNA interference were in accordance with those of Leydig cells overexpressing COXⅦa.
3. COXⅦa protein exerting its function may be through the route of ERK1/2 and JNK.
COXⅦa(cytochrome c oxidase subunitⅦa)是线粒体呼吸链第4个复合体的核编码亚基,参与该复合体的构成,在线粒体氧化呼吸链的电子传递中发挥重要作用。研究表明,COXⅦa在老年人睾丸组织显著上调,推测其可能与老年雄激素合成功能减退及老年男性雄激素部分缺乏综合征有关,但其具体机制仍不清楚,国内外报道极少见。本研究以D-半乳糖所致亚急性衰老大鼠为PADAM模型,利用基因转染和RNA干扰等现代分子生物学技术,从体内和体外两方面深入研究了COXWa在PADAM中的作用及机制,为研究PADAM的机理和基因靶向治疗提供理论基础。
目的：探讨大鼠出生后不同发育阶段睾丸组织COXⅦa及睾酮合成酶和急性调节蛋白的表达变化并确定其与睾酮浓度的相关性。
1实验动物：选用出生后1周龄、3周龄、5周龄、3月龄和24月龄清洁级雄性SD大鼠,每年龄组6只(1周龄组为12只)；
2睾酮测定：化学发光方法测定血清总睾酮水平,批内和批间变异系数分别为2.2%-2.4%和3.4%-4.7%：
3 RT-PCR方法检测各年龄组睾丸组织COXⅦa、3β-HSD、17β-HSD、P450scc和StARmRNA变化；
4 western blotting、免疫组化法检测各年龄组睾丸组织COXⅦa、3β-HSD、17β-HSD、P450scc和StAR蛋白表达变化和定位。
1大鼠血清睾酮水平：3月龄组、24月龄组、5周龄组、3周龄组和1周龄组依次降低,3月龄组和24月龄组与各组之间差异有统计学意义(p<0.05),其余各组差异无统计学意义(p>0.05)；
2mRNA变化：COXⅦa mRNA水平24月龄组最高、3月龄组最低,与各组之间差异均有统计学意义(p<0.05)；3β-HSD、17β-HSD和P450scc mRNA水平均为3月龄组和5周龄组高于其余各组,差异均有统计学意义(p<0.05)；StARmRNA水平3月龄组最高,5周龄组和24月龄组次之,1周龄组和3周龄组最低,差异有统计学意义(p<0.05)。
3蛋白表达和定位：COXⅦa蛋白表达于间质细胞胞质、胞核及精原细胞胞核,24月龄最高,3月龄组最低；3β-HSD蛋白表达于间质细胞胞质,17β-HSD蛋白表达于间质细胞胞质及精原细胞胞核,P450scc蛋白表达于间质细胞胞质、胞膜及精子细胞胞质,StAR蛋白表达于间质细胞胞质和胞膜以及精原细胞胞质；3β-HSD、17p-HSD、P450scc和StAR蛋白表达均为3月龄组和5周龄组高于其余各组,差异均有统计学意义(p<0.05)。
4相关性分析：COXⅦa蛋白表达与血清睾酮水平、3β-HSD、17β-HSD、P450scc和StAR蛋白表达呈负相关关系,除StAR外,均有统计学意义(p<0.05；p<0.01)。
1不同年龄大鼠睾丸组织COXⅦa表达存在差异,老年鼠表达高于青年鼠,且与血清睾酮浓度呈负相关关系,提示COXⅦa与睾酮合成有关,可能参与了PADAM的发生；
2不同年龄大鼠睾丸组织3β-HSD、17β-HSD、P450scc和StAR表达存在差异,3月龄组和5周龄组高于1周龄组、3周龄组和24月龄组；
3 COXⅦa蛋白表达与3β-HSD、17β-HSD和P450scc蛋白表达呈负相关,提示COXⅦa可能通过上述类固醇合成酶调控睾酮合成,似乎与急性调节蛋白无关。
目的：1构建pEGFP-N1-COXⅦa重组质粒并瞬时转染原代培养的3月龄大鼠Leydig细胞,探讨COXⅦa过表达对睾酮合成能力的影响；
2应用RNA干扰技术抑制COXⅦa在Leydig细胞中的表达,探讨COXWa抑制对睾酮合成能力的影响。
1大鼠睾丸Leydig细胞原代培养和鉴定：根据王晓云等的差速贴壁方法稍加改进,分离纯化Leydig细胞,3β-羟基类固醇脱氢酶化学染色和免疫细胞化学染色鉴定细胞纯度,0.4%台盼蓝溶液检查细胞活力；
2 pEGFP-N1-COXⅦa质粒构建、转染及细胞功能检测
2.1构建pGM-T-COXⅦa克隆质粒：设计扩增COXⅦa的DNA引物,在上、下游引物的5’端分别加入BglⅡ和EcoRⅠ的酶切位点和保护碱基,从24月龄大鼠睾丸组织中RT-PCR扩增COXWa基因，将扩增产物纯化回收、经BglⅡ和EcoRⅠ酶切获得COXⅦa基因片段；用T4连接酶将具有相同BglⅡ和EcoRⅠ粘性末端的pGM-T载体和COXⅦa基因连接,将连接产物转化DH5α感受态细胞,涂板,挑克隆,摇菌抽质粒后酶切鉴定含COXⅦa基因的克隆,大量扩增该克隆后抽质粒,得到pGM-T-COXⅦa克隆质粒；
2.2构建pEGFP-N1-COXⅦa表达质粒：大量扩增含pEGFP-N1空质粒的大肠杆菌,抽提得到pEGFP-N1空质粒；将pGM-T-COXⅦa和pEGFP-N1分别用BglⅡ和EcoRⅠ进行双酶切,酶切片段纯化回收后用T4连接酶进行连接,将连接产物转化DH5a感受态细胞,涂板,挑克隆,摇菌抽提质粒后酶切鉴定含COXⅦa基因的克隆,大量扩增该克隆后抽提质粒,得到pEGFP-Nl-COXⅦa表达质粒；
2.3测序：将RT-PCR扩增的COXⅦa片段和pEGFP-N1-COXⅦa重组质粒送上海生工测序,测序结果与genebank中序列进行比对；
2.4转染细胞：用脂质体介导法将pEGFP-N1-COXⅦa重组质粒转染Leydig细胞,分为空白对照组、空质粒组、转染脂质体组和转染pEGFP-N1-COXⅦa组;
2.5阳性细胞鉴定：在荧光显微镜下观察,有绿色荧光表达者为阳性细胞,并用MitoTracker(?) Red CM-H2XRos孵育,共聚焦激光显微镜观察pEGFP-N1-COXⅦa在细胞内的表达和定位；
2.6细胞功能检测：细胞兰TM检测细胞活力,化学发光法测定培养上清中总睾酮浓度,RT-PCR和western blotting分别检测COXⅦa mRNA和蛋白表达,免疫细胞化学进行蛋白定位。
3.1转染：将筛选出的抑制效应最强的SiRNA转染PADAM模型大鼠的Leydig细胞,分为空白对照组、脂质体组、阴性对照组、RNA干扰组、阳性对照组和荧光标记阴性对照组(观察转染效率)；
3.2转染效率：荧光显微镜下观察荧光标记阴性对照组,判断转染效率；
3.3细胞功能检测：细胞兰TM检测细胞活力,化学发光法测定培养上清中总睾酮浓度,RT-PCR和western blotting分别检测COXⅦa mRNA和蛋白表达。
1分离的Leydig细胞纯度达95%以上,可以满足实验要求；
2pEGFP-N1-COXⅦa质粒经测序证实序列正确；
3过表达COXⅦa的Leydig细胞活力降低,睾酮分泌减少,与空白对照组、转脂质体组和转染空质粒组比较,差异具有统计学意义(p<0.05)；
4 COXⅦa蛋白表达于Leydig细胞胞质和胞核；pEGFP-N1-COXⅦa转染组COXWamRNA和蛋白表达均高于空白对照组、转脂质体组和转染空质粒组,差异具有统计学意义(p<0.05)；
5 RNA干扰抑制了Leydig细胞COXVHamRNA和蛋白表达,均低于空白对照组、脂质体组、阴性对照组,差异具有统计学意义(p<0.05)；
6 RNA干扰组细胞活力和睾酮分泌均高于空白对照组、脂质体组和阴性对照组。
COXⅦa具有降低细胞活力、抑制睾酮分泌的作用。
目的：探讨COXⅦa对大鼠睾酮合成的影响及其与类固醇合成酶和急性调节蛋白的关系,利用RNA干扰和中药何首乌饮探索PADAM治疗的新方法。
1 COXⅦa过表达对青年大鼠睾酮合成及类固醇合成酶和蛋白的影响
1.1动物分组：3月龄清洁级SD雄性大鼠随机分为pEGFP-N1-COXⅦa转染组(模型组)、空质粒组、脂质体组、葡萄糖对照组(葡萄糖组)和空白对照组,每组各8只动物；
1.2化学发光法测定各组大鼠血清总睾酮浓度,生物化学方法检测血清SOD、T-AOC活力及MDA含量,RT-PCR和western blotting分别检测COXⅦa、3β-HSD,17β-HSD、P450scc和StAR mRNA和蛋白表达。
2 RNA干扰及何首乌饮对PADAM大鼠睾酮合成及类固醇合成酶和蛋白的影响
2.1建立PADAM大鼠模型：清洁级8周龄雄性SD大鼠,D-半乳糖300mg/kg/d腹腔注射,连续用药50d；
2.2动物分组：正常对照组、PADAM模型组、RNA干扰组、睾酮注射组(阳性对照组)、何首乌饮治疗组和模型对照组,每组各8只动物。
2.3化学发光法测定各组大鼠血清总睾酮浓度,生物化学方法检测血清SOD、T-AOC活力及MDA含量,RT-PCR和western blotting分别检测COXⅦa、3β-HSD、17β-HSD、P450scc和StAR mRNA和蛋白表达。
1青年大鼠转染pEGFP-N1-COXVUa质粒后,与空质粒组、脂质体组、葡萄糖组和空白对照组比较：
1.1血清睾酮浓度降低,差异有统计学意义(p<0.05)；
1.2血清SOD、T-AOC活力下降,MDA含量升高,差异有统计学意义p<0.05)；
1.3 COXⅦa mRNA和蛋白表达增高,差异有统计学意义p<0.05)；
1.43p-HSD、17p-HSD、P450scc和StAR mRNA和蛋白表达下降,差异有统计学意义(p<0.05)；
2 PADAM大鼠RNA干扰组和何首乌饮治疗与模型组、正常对照组比较：
2.1血清睾酮浓度高于模型组,低于正常对照组,差异有统计学意义(p<0.05)；
2.2血清SOD、T-AOC活力高于模型组,低于正常对照组,MDA含量相反,差异有统计学意义(p<0.05)；
2.3 COXⅦa mRNA和蛋白表达较模型组显著下降,仍高于正常对照组,差异有统计学意义(p<0.05)；
2.43β-HSD、17β-HSD、P450scc和StAR mRNA和蛋白表达较模型组升高,但仍低于正常对照组,差异有统计学意义(p<0.05)；
1COXVHa过表达可致青年大鼠睾酮浓度下降,类固醇合成酶和急性调节蛋白表达减少；
2 PADAM模型大鼠经RNA干扰及何首乌饮治疗后,大鼠睾酮合成能力均得到部分恢复,类固醇合成酶和急性调节蛋白表达增加；
3 COXVHa在大鼠衰老过程中发挥作用,可能与类固醇合成酶和急性调节蛋白有关；
4靶向COXⅦa的RNA干扰治疗和何首乌饮治疗可以延缓衰老。
目的：探讨MAPK信号通路是否参与COXWa对Leydig细胞睾酮合成的调节过程。
分别对青年大鼠和PADAM模型大鼠的Leydig细胞转染pEGFP-Nl-COXⅦa重组质粒和RNA Oligo后,进行下列实验：
1流式细胞仪检测细胞凋亡率；
2 RT-PCR检测：3β-HSD、17β-HSD、P450scc和StAR mRNA;
3免疫细胞化学方法检测3β-HSD、17β-HSD、P450scc、StAR、Bax和Bcl-2蛋白表达；
4 western blotting检测3β-HSD、17β-HSD、P450scc、StAR以及Bax、Bcl-2、ERK1/2、p-ERK1/2、JNK、p-JNK、P38、p-P38蛋白表达。
1转染pEGFP-N1-COXⅦa重组质粒后,转染组与对照组、空质粒组和脂质体组比较：
(1)细胞凋亡率增加,差异有统计学意义(p<0.05)；
(2) 3-HSD、17β-HSD. P450sccmRNA减少,差异有统计学意义p<0.05)；StARmRNA差异无统计学意义；
(3)3β-HSD、17β-HSD、P450scc和：StAR蛋白表达减少,差异有统计学意义(p<0.05)；
(4) Bcl-2表达减少,Bax表达增加,Bcl-2/Bax下降,差异有统计学意义(p<0.05)；
(5) ERK1/2、JNK和P38差异无统计学意义；p-ERK1/2和p-JNK显著增加,差异有统计学意义p<0.05)；p-P38未见阳性条带。
2 RNA干扰后,干扰组与阴性对照组、脂质体组和空白对照组比较：
(1)细胞凋亡率减少,差异有统计学意义(p<0.05)；
(2) 3β-HSD、17β-HSD、P450sccmRNA增加,差异有统计学意义(p<0.05)；StARmRNA差异无统计学意义；
(3)3β-HSD、17p-HSD、P450scc和StAR蛋白表达增加,差异有统计学意义(p<0.05)；
(4) Bcl-2表达升高,Bax表达下降,Bcl-2/Bax增大(p<0.05)；
(5) ERK1/2、JNK和P38差异无统计学意义；p-ERK1/2和p-JNK显著减少,差异有统计学意义(p<0.05)；p-P38未见阳性条带。
1在COXⅦa过表达的Leydig细胞,p-ERK1/2和p-JNK高表达,睾酮分泌减少,Bcl-2/Bax下降,细胞凋亡增加,3β-HSD、17β-HSD、P450scc和StAR蛋白表达减少,提示p-ERK1/2和p-JNK抑制类固醇合成酶活性,促进细胞凋亡；
2在COXⅦa干扰的Leydig细胞,p-ERK1/2和p-JNK低表达,睾酮分泌增加,Bcl-2/Bax升高,细胞凋亡减少,3β-HSD、17β-HSD、P450scc和StAR蛋白表达增加,提示抑制ERK1/2和JNK蛋白活化可以上调类固醇合成酶活性,抑制细胞凋亡；
3 COXⅦa可能通过ERK1/2和JNK信号通路发挥作用。
Due to the decrease in androgen levels gradually with their aging, middle-aged males appear fatigue, weakness, depression, insomnia, mood swings, irritability, decreased libido, erectile dysfunction, weight loss and osteoporosis and a series of clinical syndrome, which are known as partial androgen deficiency syndrome of the aging male (PADAM). PADAM is associated with many diseases, such as cardiovascular disease, diabetes and so on. The higher incidence of various age-related diseases seriously affects older men's health and their quality of life. Concerning about the age-related diseases, improving the quality of life of older persons and extending their life span have become the important medical issues of the world's attention.
COXⅦa (Cytochrome c Oxidase SubunitⅦa) is nuclear encoded subunit of complex IV of mitochondrial respiratory chain, involved in the complex composition and plays an important role in electron transfer of mitochondria respiratory chain oxidation. Studies indicate that COXⅦa in the testis of the aging male was significantly increased, suggesting that it may be involved in androgen dysfunction and partial androgen deficiency syndrome of the aging male, but the exact mechanism remains unclear, and there are rare domestic and foreign reports. PADAM model was established by injection of D-galactose. Some techniques of modern molecular biology such as gene transfection and RNA interference were used to study the effects and mechanism of COXⅦa on PADAM rats in vivo and in vitro, aiming to provide the theoretical basis for exploring the mechanism of PADAM and gene targeting therapy.
To study the changes of COXⅦa,3β-HSD,17β-HSD, P450scc and StAR mRNA and acute regulatory protein expression in rat testes of different periodsand the correlation between them and testosterone concentration.
1. Male SD rats aged 1 week,3 weeks,5 weeks,3 months and 24 months were studied, each group 6 animals(1 week group was 12).
2. The serum testosterone was determined by chemiluminescence immunoassay (CLIA), variation coefficient of inter-group and intra-group was 2.2% to 2.4% and 3.4% to 4.7% respectively.
3. RT-PCR was used to examine the changes of COXⅦa,3β-HSD,17β-HSD, P450scc and StAR mRNA of the testicular tissues among the above groups.
4. Western blotting and immunohistochemistry were employed to detect the difference of COXⅦa,3β-HSD,17β-HSD, and P450scc and StAR protein expression of the testicular tissues among the above groups.
1. The testosterone level in the rat serum decreased gradually from 3 months to 24 months,5 weeks,3 weeks and 1 week group. The difference between 3 months,24 months group and the other groups was statistically significant respectively (p<0.05).
2. COXⅦa mRNA level in 24 month group was the highest, and that in 3 month group was the lowest, and the differences between 3 months,24 months group and the other groups were statistically significant(p<0.05); 3β-HSD,17β-HSD and P450scc mRNA levels in 3 months group and 5 weeks group were significantly higher than them in other groups(p<0.05); StARmRNA level in 3 months group was the highest,5 weeks and 24 months group were followed,1 week and 3 weeks group were the lowest, the difference was statistically significant(p<0.05).
3. COXⅦa protein expressed in plasmid and nucleus of Leydig cells and the nucleus of spermatogonia. It was the highest in 24 month group and the lowest in 3 months group.3β-HSD protein was expressed in plasmid of Leydig cells, 17β-HSD protein in plasmid of Leydig cells and nucleus of spermatogonia, P450scc protein in plasmid, nucleus and membrane of Leydig cells, and plasmid of spermatid, StAR protein in the plasmid and membrane of Leydig cells and plasmid of spermatogonia.3β-HSD,17β-HSD, P450scc and StAR protein was significantly higher in 3 months group and 5 weeks group than them in other groups(p<0.05).
4. There was negative correlation between COXⅦa protein and serum testosterone concentration,3p-HSD,17β-HSD, P450scc and StAR protein. The differences were statistically significant except StAR (p<0.05,p<0.01).
1. The expression of COXⅦa had significant change in different periods and it was higher in old rats than in young rats, and was negatively related to serum testosterone concentration, suggesting that COXⅦa may be associated with testosterone synthesis and involved in the occurrence of PAD AM.
2. The expression of 3β-HSD,17β-HSD, P450scc and StAR had significant change in rat testes of different periods, namely,3 months and 5 weeks group was significant higher than 1 week,3 weeks and 24 months group.
3. There was negative correlation between COXⅦa and 3β-HSD,17β-HSD, P450scc, indicating that COXⅦa regulated testosterone synthesis through steroidogenesis enzymes but not StAR.
1. To explore the impact of COXⅦa overexpression to testosterone synthesis by construction pEGFP-N1-COXⅦa recombinant plasmid and transient transfection primary cultured rat Leydig cells extracted from 3 months.
2. To explore the impact of COXⅦa suppression to testosterone synthesis by application of RNA interference inhibition of COXⅦa in Leydig cells.
1. Improved Wang Xiaoyun's differential adhesion speed method was used in the isolation of Leydig cells; 3β-hydroxysteroid dehydrogenase (3β-HsD) staining and immunohistochemical staining were employed in identifying the cell purity,0.4% solution of trypan blue in checking cell viability.
2.pEGPF-N1-COXⅦa plasmid construction, tranfection and function examination
2.1 Construct pEGPF-N1-COXⅦa plasmid:COXⅦa DNA was amplified by RT-PCR from 24 month rat testis and was digested with Bgl II and EcoR I, then was linked to the pGM-T vector by T4 ligase. E. coli including pGM-T-COXⅦa plasmid and E. coli including p-EGPF-N1 plasmid were propagated in a large scale. After extracted, two kinds of plasmid were digested with Bgl II and EcoR I. Then pEGPF-N1 and COXⅦa gene was linked together by T4 linkase.
2.2 Transfection:After the sequence of the recombinant plasmid was proved correct, the recombinant plasmid was transferred into the primary cultured Lydia cells.
2.3 Identification to positive cells:Cells with green fluorescence under the fluorescent microscope were positive. Co focal laser scanning microscopy was used to observe in the expression and localization of pEGFP-Nl-COXⅦa within the cell which were incubated with MitoTracker (?) Red CM-H2Xros together after 20 hours transfected with pEGFP-N1-COXⅦa.
2.4 Function examination:Cell BlueTM Kit was used to determine cell viability, CLIA was used to examine testosterone concentration, RT-PCR and western blotting were used to detect COXⅦa mRNA and protein expression respectively, and immunocytochemistry for protein localization.
3. RNA Interference
3.1 Transfer:SiRNA Oligo with the most powerful inhibitory effect was screened out to be tranferred into PADAM rat Leydig cells with Lipofectamine 2000. Blank control group, Lipofectamine control, negative control, RNA interference group, positive control and fluorescent labeled negative control group were formed.
3.2 Transfer efficiency:Fluorescent microscope was used to observe fluorescent labeled negative control cells to determine transfer efficiency.
3.3 Function examination:Cell BlueTM Kit was used to determine cell viability, CLIA was used to examine testosterone concentration, RT-PCR and western blotting were used to detect COXⅦa mRNA and protein expression respectively.
1. The purity of isolated Leydig cells was more than 95% in accordance with the test requirements.
2. pEGPF-N1-COXⅦa plasmid sequence was confirmed correct by using the sequencing apparatus.
3. Both the viability and testosterone level of Leydig cells overexpressing COXⅦa was significantly decreased than them of blank control, lipofectamine and empty plasmid group (p<0.05).
4. COXⅦa protein was detected in Leydig cell plasmid and nucleus. COXⅦa mRNA and protein of Leydig cells overexpressing COXⅦa was significantly higher than that of blank control, lipofectamine and empty plasmid group (p<0.05).
5. SiRNA suppressed COXⅦa mRNA and protein expression in the Leydig cells, which were significantly lower than that in blank control, Lipofectamine and negative control group(p<0.05).
6. Both the viability and testosterone level of Leydig cells with SiRNA was significantly higher than that of blank control, Lipofectamine and negative control group (p<0.05).
COXⅦa protein can suppress cell proliferation and testosterone secretion.
To study the impact of COXVIIa to testosterone biosynthesis in rats and the relation between it and steroidogenesis enzymes and acute regulatory protein in vivo and to explore the new therapy by using RNA interference and Polygonum Multiflorum Trunb.
1. The impacts of COXⅦa overexpression to testosterone synthesis, steroidogenesis enzymes and acute regulatory protein in young rats:
1.1 Animal groups:3 months SD male rats were randomly divided into pEGFP-N1-COXⅦa group (M group), empty plasmid group (P group), lipofectamine (L group), glucose control (G group) and blank control group (Bc group), each group 8 animals.
1.2 CLIA was used to determine the concentration of total testosterone in serum. Biochemical methods were used to examine SOD, T-AOC viability and MDA level; RT-PCR and western blotting was used to detect COXⅦa, 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression respectively.
2. The impacts to the Testosterone synthesis and steroidogesis enzyme and protein treated with RNA interference and Polygonum Multiflorum Trunb in PADAM model rats.
2.1 Establishment of PADAM model:clean 8 weeks male SD rats were treated with D-galactose 300mg/kg/d intraperitoneally, for continuous 50d.
2.2 Animal groups:normal control group (Bc group), PADAM model group (M group), RNA interference group (Si group), testosterone injection group (T group), Polygonum Multiflorum Trunb treated group (H group) and model control group (Mc group), each group 8 animals.
2.3 CLIA was used to determine the concentration of total testosterone in serum. Biochemical methods were used to examine SOD, T-AOC viability and MDA level; RT-PCR and western blotting were used to detect COXⅦa, 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression respectively.
1. To compare with P group, L group, G group and Bc group, the expression of the several indicators in M group was as follows:
1.1 Serum testosterone concentration was decreased (p<0.05).
1.2 The SOD and T-AOC activity was decreased, MDA level was increased (p<0.05).
1.3 COXⅦa mRNA and protein expression increased respectively (p<0.05).
1.4 3β-HSD,17p-HSD, P450scc and StAR mRNA and protein expression was decreased respectively (p<0.05).
2. Comparison between Si group, T group and M group and Bc group after RNA interference in PADAM model rats.
2.1 Serum testosterone concentration of Si group and T group was significantly higher than that of M group, while lower than that of Bc group (p<0.05).
2.2 The SOD, T-AOC viability of Si group and T group was higher than that of M group, while lower than that of Bc group, MDA content in the contrary (p<0.05).
2.3 COXⅦa mRNA and protein expression of Si group and T group decreased compared with M group, still higher than that of Bc group (p<0.05).
2.4 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression significantly increased compared with M group, but still lower than that of Bc group (p<0.05).
1. COXⅦa overexpression in young rats could decrease testosterone synthesis and expression of steroidogenesis enzymes and acute regulatory protein.
2. RNA interference in PADAM model rats could lead to partial restoration of testosterone synthesis of rats, increased expression of steroidogenesis enzymes and acute regulatory protein.
3. COXⅦa may be play a role in the aging process and related to steroidogenesis enzymes and the acute-regulated protein.
4. RNA interference targeting COXⅦa gene and Polygonum Multiflorum Trunb can delay senescence of rats.
To explore whether the pathway of ERK and JNK participates in the process that COXⅦa regulates testosterone synthesis of Leydig cells.
Leydig cells extracted from young rats and RADAM model rats were transfected with pEGPF-N1-COXⅦa recombinant plasmid and SiRNA respectively, then the experiments were as followed:
1. Flow Cytometry was used to detect cell apoptosis rate.
2. PCR was used to detect 3β-HSD,17β-HSD, P450scc and StAR mRNA.
3. Immunocytochemistry was used to detect 3β-HSD,17β-HSD, P450scc, StAR, Bax and Bcl-2 protein expression.
4. Western blotting was used to detect 3β-HSD,17β-HSD, P450scc, StAR and Bax, Bcl-2, ERK1/2, p-ERK1/2, JNK, p-JNK, P38, p-P38 protein expression.
1. To compare with control group, empty vector group and Lipofectamine group, the expression of the several indicators in pEGPF-N1-COXⅦa group was as follows: 1.1 The apoptosis rate increased significantly (p<0.05).
1.2 The level of 3β-HSD,17β-HSD, P450scc mRNA decreased significantly (p<0.05), while there was no significant difference of StAR mRNA.
1.3 The expression of 3β-HSD,17β-HSD, P450scc and StAR protein expression decreased significantly (p<0.05).
1.4 Bcl-2 expression decreased, while Bax expression increased, Bcl-2/Bax decreased (p<0.05).
1.5 ERK1/2, JNK and P38 showed no significant difference; p-ERK1/2 and p-JNK increased significantly (p<0.05); p-P38 no positive bands.
2. To compare with the negative control group, Lipofectamine control group and blank control group, the expression of the several indicators in the interference group with was as follows: 1.1 The apoptosis rate decreased significantly (p<0.05). 1.2 The level of 3β-HSD,17P-HSD, P450scc mRNA increased significantly (p<0.05), while there was no significant difference of StAR mRNA. 1.3 The expression of 3β-HSD,17β-HSD, P450scc and StAR protein expression increased significantly (p<0.05). 1.4 Bcl-2 expression increased, while Bax expression decreased, Bcl-2/Bax increased (p<0.05). 1.5 ERK1/2, JNK and P38 showed no significant difference; p-ERK1/2 and p-JNK significantly reduced (p<0.05); p-P38 no positive bands.
1. In the Leydig cells overexpressing COXⅦa, p-ERK1/2 and p-JNK expression and the apoptosis rate increased, while testosterone concentration, the ratio of Bcl-2/Bax, and 3β-HSD,17β-HSD, P450scc and StAR protein decreased, suggesting that p-ERK1/2 and p-JNK could suppress steroid synthesis and promote cell apoptosis.
2. The results of RNA interference were in accordance with those of Leydig cells overexpressing COXⅦa.
3. COXⅦa protein exerting its function may be through the route of ERK1/2 and JNK.
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