Effect of Pp2cm Gene Silencing on Mouse Macrophage Resistance Against Staphylococcus aureus Infection via TLR Pathway
-
摘要:目的 探究蛋白磷酸酶2Cm(protein phosphatase 2Cm, PP2Cm)的基因Pp2cm沉默对感染金黄色葡萄球菌(Staphylococcus aureus, S. aureus)后巨噬细胞炎症因子表达的影响及作用机制。方法 通过腺病毒(adenovirus, Ad)转染Raw264.7小鼠巨噬细胞系分析了Pp2cm基因敲低对巨噬细胞炎症因子、细胞增殖凋亡和Toll样受体(Toll-like receptor, TLR)信号通路的影响。细胞处理分为4组,包括Ad-Ctrl组、Ad-Pp2cm组、Ad-Ctrl+S. aureus组和Ad-Pp2cm+S. aureus组。分别在细胞中加入对照腺病毒(Ad-Ctrl)或针对Pp2cm基因的腺病毒(Ad-Pp2cm),使用或不使用金黄色葡萄球菌进行炎症诱导。实时荧光定量聚合酶链式反应(real-time fluorescent quantitative polymerase chain reaction, RT-qPCR)检测肿瘤坏死因子α(tumor necrosis factor-alpha, TNF-α)、白细胞介素1β(interleukin 1 beta, IL-1β)、TLR2、TLR4、Toll样受体衔接蛋白(Toll-like receptor adaptor protein, Tirap)和髓样分化因子88(myeloid differentiation factor 88, Myd88)基因表达,蛋白印迹法(Western blot)技术检测PP2Cm蛋白表达,Cell Counting Kit-8(CCK-8)法测定细胞增殖,流式细胞术检测细胞凋亡。结果 Ad-Pp2cm组巨噬细胞Pp2cm mRNA和PP2Cm蛋白表达水平均低于Ad-Ctrl组,差异有统计学意义(P<0.05)。与Ad-Ctrl+S. aureus组相比,Ad-Pp2cm+S. aureus组巨噬细胞中TNF-α与IL-1β基因表达水平升高,差异有统计学意义(P<0.01)。与Ad-Ctrl组巨噬细胞相比,Ad-Pp2cm组巨噬细胞中TLR2、TLR4、Tirap 和Myd88基因表达水平升高,差异有统计学意义(P<0.05)。Ad-Ctrl组与Ad-Pp2cm组巨噬细胞的细胞凋亡和细胞增殖差异无统计学意义。结论 Pp2cm基因沉默促进巨噬细胞对金黄色葡萄球菌感染的炎症响应。TLR通路在巨噬细胞炎症激活过程中发挥重要作用。Abstract:Objective To investigate the effect of silencing protein phosphatase 2cm (Pp2cm) gene on the expression of inflammatory factors in macrophages infected with Staphylococcus aureus (S. aureus) and the mechanisms involved.Methods The effects of Pp2cm knockdown on inflammatory factors, proliferation, apoptosis, and Toll-like receptor (TLR) signaling were analyzed in RAW 264.7 cells, a murine macrophage cell line, transfected with adenovirus (Ad). The cells were divided into four groups, including Ad-Ctrl group, Ad-Pp2cm group, Ad-Ctrl+S. aureus group and Ad-Pp2cm+S. aureus group. Cell transfection was achieved by separately introducing control adenovirus (Ad-Ctrl) or adenovirus targeting the Pp2cm gene (Ad-Pp2cm) and inflammation or the absence of inflammation was induced by applying or not applying S. aureus. The expression of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), TLR2, TLR4, Toll-like receptor adaptor protein (Tirap) and myeloid differentiation factor 88 (Myd88) was determined by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). PP2Cm protein expression was determined by Western blot. Cell proliferation was determined by cell counting kit-8 (CCK-8) assay and cell apoptosis was measured by flow cytometry.Results The expression of Pp2cm gene and PP2Cm protein was downregulated in the Ad-Pp2cm group when compared to the Ad-Ctrl group, with the diference showing statistical significance (P<0.05). When compared to those of the Ad-Ctrl+S. aureus group, macrophages in the Ad-Pp2cm+S. aureus group showed significantly increase in the TNF-α and IL-1β gene levels (P<0.01). Furthermore, the Ad-Pp2cm group demonstrated elevated gene expression levels of TLR2, TLR4, Tirap and Myd88 in macrophages when compared to the Ad-Ctrl group, with the difference showing statistical significance (P<0.05). There were no statistically significant differences in cell apoptosis and proliferation between the Ad-Ctrl and Ad-Pp2cm groups.Conclusions Silencing Pp2cm gene promotes the inflammatory response of macrophages to S. aureus infection. Moreover, the TLR pathway plays an important role in the inflammatory activation of macrophages.
-
Keywords:
- Staphylococcus aureus /
- Protein phosphatase 2Cm /
- Toll-like receptor
-
多囊卵巢综合征(polycystic ovary syndrome, PCOS)是一种内分泌代谢紊乱性疾病,全世界育龄期女性中的患病率为6%~20%[1]。PCOS以排卵功能障碍、临床或/和生化高雄激素血症以及B超卵巢多囊样改变为特征,常伴有胰岛素抵抗、糖脂代谢紊乱、肥胖、氧化应激增加、长期低级慢性炎症,明显增加抑郁症、2型糖尿病、妊娠期并发症、远期心血管疾病发生的危险性[1-5]。PCOS的病因至今尚不明确,研究表明它可能是一种受表观遗传影响的复杂的多基因疾病,此外可能还与环境因素有关[1, 6]。
血管紧张素Ⅰ转换酶(angiotensin Ⅰ-converting enzyme, ACE)是肾素-血管紧张素系统(renin-angiotensin system, RAS)的关键酶,催化无活性的血管紧张素Ⅰ转化为高活性的血管紧张素Ⅱ(angiotensin Ⅱ, Ang Ⅱ),在调节RAS相关激素水平和多种生理、病理过程中起重要作用[7-8]。ACE基因位于染色体17q23区,由26个外显子和25个内含子构成,定位于第16内含子的一个287 bp 的Alu重复序列的插入(insertion, I)与缺失(deletion, D)多态性(rs4646994)与血清ACE浓度有关,ACE浓度:DD>ID>II基因型[9]。此外,该基因多态性与心脑血管疾病、肾脏疾病、糖尿病并发症等有关[7, 10]。
研究表明RAS和ACE的异常可能促进PCOS发生与发展,在PCOS患者发生高雄激素血症和胰岛素抵抗的过程中起重要作用[8, 11]。迄今已有多篇文章报道了ACE I/D基因多态性与PCOS的关系,但结果并不一致,由于多数研究样本量较小,统计学效率偏低,难以得出明确的结论[8, 12-14]。Ang Ⅱ具有促进氧化应激的作用[15]。PCOS患者常伴有氧化应激的增加,然而ACE I/D变异与PCOS氧化应激的关系仍不清楚。在本研究中,我们采用了一个较大的样本量,分析ACE I/D多态性与PCOS发生的相关性,探讨这个基因变异对临床特征、激素、代谢和氧化应激的影响。
1. 对象与方法
1.1 研究对象
采用回顾性病例-对照研究。选择2006−2019年在四川大学华西第二医院生殖内分泌科门诊就诊的17~44岁PCOS患者1 020例及同期就诊的17~44岁对照妇女825例纳入研究。该研究经四川大学华西第二医院伦理委员会批准(批准号2014-014~PF),所有研究对象均签署了知情同意书。
每个纳入研究的患者均符合2003年在鹿特丹修订的欧洲人类生殖与胚胎学会(European Society of Human Reproduction and Embryology, ESHRE)和美国生殖医学学会(American Society for Reproductive Medicine, ASRM)PCOS诊断标准[16]:在排除其他引起高雄激素血症的疾病后,具有下列条件中的2项,则可确诊为PCOS[17-18]:①稀发排卵和/或不排卵;②临床和/或生化高雄激素血症;③超声检查出多囊卵巢。对于小于20岁的女性而言,雄激素增多症为PCOS确诊的必备条件[19]。对照组为因输卵管梗阻或丈夫不育而不孕的妇女或健康妇女。所有对照女性临床健康,月经周期正常(22~35 d),血液中雄性激素水平正常〔总睾酮(total testosterone, TT)<0.75 ng/mL或游离雄激素指数(free androgen index, FAI)<9.5〕,体格检查无明显痤疮或多毛(F-G评分<6),B超测定卵巢形态正常。
研究对象均排除感染性疾病、自身免疫性/炎症性疾病、肿瘤、心血管疾病、甲状腺功能障碍、肝肾疾病、高泌乳素血症、子宫内膜异位症、促性腺功能低下或早发性卵巢功能不全等。
在进行基因型与生殖激素、糖脂代谢和氧化应激相关指标分析时,为了避免相关干扰因素的影响,要求被纳入的研究对象:在3个月内未服用影响糖脂代谢或激素水平的药物;为非吸烟者;处于月经周期的卵泡早、中期(孕酮水平<9.54 nmol/L);对照组女性非糖尿病患者。在所有研究对象中,符合上述要求的PCOS患者有667例,对照妇女有527例。
1.2 方法
1.2.1 临床资料与标本收集
收集与统计PCOS组和对照组女性的临床资料,包括:年龄、收缩压(systolic blood pressure, SBP)、体质量、腰围、身高、舒张压(diastolic blood pressure, DBP)、臀围、月经情况、多毛F-G评分(Ferriman-Gallwey score, F-G score)、既往史、家族史、痤疮情况、卵巢体积[17](单位为cm3),计算体质量指数(body mass index, BMI)和腰臀比(waist-hip ratio, WHR)。
抽取空腹12 h后的肘静脉血,于4 ℃ 1 500 r/min离心15 min,分别吸取上层血浆和血清,分装后储存于−80 ℃冰箱,用于测定激素、代谢和氧化应激指标。血细胞保存在4 ℃冰箱,用于提取基因组DNA。
1.2.2 基因型分析
ACE基因I/D多态性的PCR扩增引物参照文献[20]:ACE上游引物:5′-CTGGAGACCACTCCCATCCTTTCT-3′,下游引物:5′-GATGTGGCCATCACATTCGTCAGAT-3′,由上海生工生物有限公司合成。PCR反应体系:2 mmol/L MgC12、2.5 μL 10×PCR缓冲液、0.2 mmol/L dNTP、5%二甲基亚砜、各0.2 µmol/L上、下游引物、0.625 U TaqDNA聚合酶(Thermo)、DNA模板2.0 µL(30~80 ng),共25 μL。PCR扩增:95 ℃ 3 min,94 ℃ 1 min,58 ℃ 1 min, 72 ℃ 2 min,32个循环后,在72 ℃条件下延伸7 min。配制2.5%琼脂糖凝胶(含Genecolour荧光试剂)待其凝固后,取10 μL产物进行电泳通过扩增片段确定基因型。其中I等位基因扩增片段为490 bp,D等位基因扩增片段为190 bp。为了控制基因分型质量,随机选择30%以上的DNA样本由不同的操作人员再次进行基因分型。
1.2.3 激素、代谢与氧化应激指标分析
激素和代谢指标送往四川大学华西第二医院检验科测定。使用化学发光法测定血浆胰岛素(insulin, Ins)、TT、血清卵泡刺激素(follicle-stimulating hormone, FSH)、黄体生成素(luteinizing hormone, LH)、性激素结合球蛋白(sex hormone-binding globulin, SHBG)。采用酶法试剂盒测定血糖(glucose, Glu)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol, HDL-C)、三酰甘油(triglycerides, TG)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-C)和总胆固醇(total cholesterol, TC)。
血清丙二醛(malondialdehyde, MDA)浓度和总抗氧化能力(total antioxidant capacity, T-AOC)分别使用相应的试剂盒(南京建成生物工程研究所)测定。血清总氧化状态(total oxidant status, TOS)采用本室建立的微孔板比色方法[5]测定。
上述所有测定的批内变异系数均小于5%,批间变异系数均小于10%。
FAI=(TT×100)/SHBG,稳态模型评估胰岛素抵抗指数(homeostatic model assessment of insulin resistance, HOMA-IR)的计算方法参考以往研究[21]。
1.2.4 统计学方法
PCOS组1 020例和对照组825例是纳入统计分析的总例数,也是进行基因分型的例数;PCOS组667例和对照组527例是进行基因型与生殖激素、糖脂代谢和氧化应激相关指标分析时,排除了部分样本后的例数。采用独立样本t检验和非参数检验(Mann-Whitney U检验),分别用来分析PCOS组与对照组女性之间的连续变量和非正态分布变量。使用卡方分析来评估基因型分布是否符合Hardy-Weinberg平衡以及两组之间的基因型和等位基因频率差异。不同基因型亚组间临床特征、激素、代谢及氧化应激指标的比较采用方差分析。校正年龄、BMI后,不同基因型亚组间相关指标的比较采用协方差分析。P<0.05为差异有统计学意义。
2. 结果
2.1 ACE I/D基因型和等位基因频率的比较
对照组与PCOS组基因型频率分布均符合Hardy-Weinberg平衡(P均>0.05),具有群体代表性。如表1所示,I等位基因频率在PCOS组和对照组中分别为0.644和0.653,D等位基因频率在PCOS组和对照组中分别为0.356和0.347。卡方分析表明ACE基因I/D基因型和等位基因频率在两组间差异无统计学意义(P>0.05)。
表 1 PCOS与对照组之间ACE基因I/D基因型和等位基因频率分布Table 1. Frequencies of ACE gene I/D genotype and allele in PCOS patients compared with the controlsIndex Frequencies (case) Controls
(n=825)PCOS patients
(n=1 020)χ2/P Genotype χ2=0.881,P=0.644 II 0.425 (351) 0.421 (429) ID 0.456 (376) 0.446 (455) DD 0.119 (98) 0.133 (136) Allele χ2=0.377,P=0.539 I 0.653 (1 078) 0.644 (1 313) D 0.347 (572) 0.356 (727) 2.2 ACE I/D多态性不同基因型亚组间临床特征的比较
如表2所示,与对照组相比,PCOS组年龄降低(P<0.05),BMI、WC、WHR、F-G score、痤疮分级评分、SBP、DBP和卵巢体积均升高(P<0.05)。在调整年龄和BMI之后,无论是在PCOS组内,还是在对照组内,不同基因型间临床特征差异均无统计学意义。
表 2 ACE I/D多态性不同基因型亚组间临床特征的比较Table 2. Comparison of clinical features of different genotypes of ACE I/D polymorphism subgroupsIndex Controls PCOS patients Total (n=825) II (n=229) ID (n=241) DD (n=57) Total (n=1 020) II (n=274) ID (n=298) DD (n=95) Age/yr. 28.22±4.10 27.53±3.98 28.05±4.13 29.44±4.32 25.08±4.20# 25.02±4.12 25.02±4.18 25.00±4.74 BMI/(kg/m2) 21.17±2.86 21.06±2.75 21.03±2.59 20.99±3.18 22.90±4.03# 22.82±4.11 23.51±4.30 23.28±4.44 WC/cm 73.79±8.13 73.29±8.34 73.47±7.60 73.68±9.12 79.19±11.05# 78.70±11.22 80.94±11.32 80.43±11.91 WHR 0.82±0.06 0.81±0.06 0.81±0.06 0.81±0.06 0.85±0.07# 0.85±0.08 0.86±0.07 0.86±0.07 F-G score 0.26±0.76 0.28±0.76 0.26±0.75 0.11±0.36 1.71±2.04# 1.72±2.02 1.71±2.09 1.70±2.08 Acne grade score 0.14±0.35 0.15±0.36 0.14±0.35 0.07±0.26 0.64±0.89# 0.69±0.91 0.59±0.89 0.70±0.94 SBP/mmHg 112.62±11.36 112.14±10.72 112.69±10.82 112.95±13.61 114.25±10.51# 113.97±10.09 115.88±11.10 113.99±10.89 DBP/mmHg 73.56±8.91 73.74±8.45 73.33±8.36 72.33±8.99 75.43±8.72# 75.48±9.51 76.47±8.47 74.34±8.91 Ovarian volume/cm3 7.40±2.75 7.18±2.46 8.15±3.12 7.13±2.07 9.91±4.05# 10.24±4.02 10.04±4.14 10.15±3.90 BMI: Body mass index; WC: Waist circumference; WHR: Waist-hip ratio; F-G score: Ferriman-Gallwey score; SBP: Systolic blood pressure; DBP: Diastolic blood pressure. 1 mmHg=0.133 kPa. # P<0.05, vs. total controls. 2.3 ACE I/D多态性不同基因型亚组间代谢指标分析
如表3所示,调整年龄和BMI之后,与对照组相比,PCOS组血浆胰岛素、HOMA-IR、TG、TC和LDL-C均上升(P<0.05),HDL-C降低(P<0.05)。在进行不同基因型间代谢指标分析时,PCOS组中,与II基因型亚组相比,DD基因型亚组具有更高的HOMA-IR水平(P<0.05);与ID基因型亚组相比,DD基因型亚组具有更高的TC和LDL-C水平(P<0.05)。对照组中代谢指标在不同基因型间无明显差异(P>0.05)。
表 3 ACE I/D多态性不同基因型亚组间代谢水平的比较Table 3. Comparison of metabolic levels of different genotypes of ACE I/D polymorphisms subgroupsIndex Controls PCOS patients Total (n=825) II (n=229) ID (n=241) DD (n=57) Total (n=1 020) II (n=274) ID (n=298) DD (n=95) Fasting Ins/(pmol/L) 62.30±35.71 62.18±36.81 63.42±35.41 58.05±32.72 105.15±71.82# 97.35±58.32 110.28±75.88 112.05±91.48 Fasting Glu/(mmol/L) 5.23±0.47 5.23±0.47 5.26±0.45 5.18±0.59 5.36±0.86 5.32±0.62 5.34±0.85 5.48±1.35 HOMA-IR 2.21±1.29 2.23±1.30 2.24±1.31 2.09±1.12 3.78±3.06# 3.43±2.31 3.93±3.18 4.29±4.31§ TG/(mmol/L) 1.04±0.89 1.00±0.63 1.09±1.15 0.98±0.38 1.44±1.38# 1.43±1.39 1.41±1.03 1.58±2.12 TC/(mmol/L) 4.25±0.72 4.26±0.68 4.25±0.74 4.24±0.78 4.42±0.81# 4.44±0.80 4.35±0.83 4.61±0.75△ HDL-C/(mmol/L) 1.51±0.32 1.51±0.32 1.50±0.32 1.52±0.36 1.38±0.34# 1.40±0.34 1.35±0.32 1.40±0.41 LDL-C/(mmol/L) 2.36±0.63 2.38±0.62 2.35±0.64 2.34±0.67 2.56±0.76# 2.54±0.75 2.54±0.77 2.71±0.73△ Glu: Glucose; HDL-C: High-density lipoprotein cholesterol; HOMA-IR: Homeostatic model assessment of insulin resistance; Ins: Insulin; LDL-C: Low-density lipoprotein cholesterol; TC: Total cholesterol; TG: Triglycerides. Comparisons of all parameters were corrected for differences in age and BMI between the two subgroups, except for the parameters of age and BMI. △P <0.05, vs. the ID genotype subgroup in PCOS patients; §P<0.05, vs. the II genotype subgroup in PCOS patients. #P<0.05, vs. total controls. 2.4 ACE I/D多态性不同基因型亚组间激素水平和氧化应激指标分析
如表4所示,在调整年龄和BMI之后,与对照组相比,PCOS组TT、FAI、LH/FSH等激素指标,TOS、T-AOC、OSI、MDA等氧化应激指标均上升(P<0.05),SHBG水平降低(P<0.05)。
表 4 ACE I/D多态性不同基因型亚组间激素和氧化应激水平的比较Table 4. Comparison of hormone and oxidative stress levels between different genotypes of ACE I/D polymorphism subgroupsIndex Controls PCOS patients Total (n=825) II (n=229) ID (n=241) DD (n=57) Total (n=1 020) II (n=274) ID (n=298) DD (n=95) Hormonal levels TT/(nmol/L) 1.47±0.52 1.49±0.50 1.47±0.53 1.41±0.51 2.34±0.77# 2.34±0.73 2.35±0.82 2.31±0.73 SHBG/(nmol/L) 55.06±27.26 58.62±28.23 51.84±24.89 55.70±31.94 33.49±19.26# 34.97±18.63 33.28±20.37 29.82±16.98△ FAI 3.26±2.07 3.08±1.99 3.42±2.07 3.26±2.36 9.76±7.03# 8.94±6.39 10.20±7.36 10.73±7.56 LH/FSH 1.18±1.30 1.21±0.98 1.15±1.29 1.19±0.94 2.26±1.23# 2.38±1.26 2.12±1.18§ 2.34±1.25 Oxidative stress parameters TOS/(nmol H2O2 Equiv./mL) 11.48±5.38 12.01±5.35 11.26±5.69 10.20±3.83* 15.13±10.58# 15.40±11.67 14.61±8.94 15.95±11.93 T-AOC/(U/mL/min) 14.51±2.67 14.64±2.48 14.48±2.71 14.16±3.18 15.77±3.09# 15.80±3.11 15.78±3.07 15.64±3.10 OSI 0.80±0.41 0.82±0.39 0.79±0.45 0.73±0.29 0.99±0.79# 0.99±0.79 0.97±0.78 1.04±0.81 MDA/(nmol/mL) 3.70±1.09 3.79±1.08 3.66±1.12 3.51±1.02 4.37±1.32# 4.25±1.32 4.42±1.32 4.55±1.29§ FAI: Free androgen index; FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; MDA: Malondialdehyde; OSI: Oxidative stress index; SHBG: Sex hormone-binding globulin; T-AOC: Total antioxidant capacity; TOS: Total oxidant status; TT: Total testosterone. Comparisons of all parameters were corrected for differences in age and BMI between the two subgroups. * P<0.05, vs. the II genotype subgroup in controls; △ P<0.05, vs. the ID genotype subgroup in PCOS patients; § P<0.05, vs. the II genotype subgroup in PCOS patients; # P<0.05, vs. total controls. 在进行不同基因型间激素指标分析时,PCOS组中,DD基因型亚组SHBG水平低于ID基因型亚组(P<0.05);ID基因型亚组LH/FSH比值低于II基因型亚组(P<0.05)。
在进行不同基因型间氧化应激指标分析时,PCOS组中DD基因型亚组MDA水平高于II基因型亚组(P<0.05),对照组中DD基因型亚组TOS水平低于II基因型亚组(P<0.05)。
3. 讨论
ACE基因I/D多态性与PCOS关系的报道并没有得出统一的结论。在白种人与印度人群,DD基因型、ID基因型或(和)D等位基因增加PCOS发生的危险性,是PCOS的遗传危险因素[8, 12-13]。然而,几个较小样本量的研究未能证明ACE基因I/D多态性与中国人PCOS的发生有关[8, 22]。本研究以一个较大的样本量证明ACE I/D变异不是PCOS发生的遗传危险因素,该结果支持以前在中国人群中的研究。在不同种族人群中得出的不同结果,进一步说明了单一基因在PCOS中的低效性,因此,探讨已知基因在PCOS中的作用和地位,有助于对该基因多态性与PCOS的关系提供更全面的认识,对进一步寻找未知基因,阐明其分子生物学机制具有重要意义。
雄激素增多症是PCOS发病机制中的一个关键因素。ACE是调控RAS活性的关键酶,PCOS患者RAS重要成分血清总肾素明显被上调[11],增加RAS活性可能影响下丘脑-垂体-卵巢,提高LH水平,导致卵泡发育与排卵的异常,促进PCOS高雄激素血症的发生[8, 12]。ACE抑制剂(lisinopril)治疗可降低高血压PCOS女性的高雄激素血症[11]。有报道显示在PCOS患者中,ACE基因D等位基因携带者比II基因型携带者有更高的血清LH/FSH比值和睾酮水平[23]。本研究发现在PCOS患者中,DD基因型携带者比ID基因型携带者有更低的SHBG水平,提示ACE基因I/D变异可能通过影响游离睾酮水平,促进PCOS高雄激素血症的发生。
胰岛素抵抗在PCOS发病机制中起重要作用。ACE催化产物Ang Ⅱ可以通过改变胰岛素信号通路,减少骨骼肌血流量来降低机体对胰岛素的敏感性[24]。而ACE抑制剂(captopril)能够增加高血压患者对胰岛素的敏感性[11]。对土耳其PCOS患者的研究表明ACE基因DD基因型与增加空腹胰岛素和HOMA-IR有关[25]。本研究表明,在PCOS患者中,携带DD基因型比携带II基因型有更高的HOMA-IR。已有的研究结果支持ACE基因I/D多态性可能通过改变PCOS患者对胰岛素的敏感性来参与PCOS胰岛素抵抗的发生与发展。
在本研究中,我们发现在PCOS患者中,携带DD基因型比携带II基因型有更高的MDA水平,而对照组中,携带II基因型比DD基因型增加TOS水平,倾向于增加MDA水平,提示ACE基因I/D多态性可能影响机体氧化应激状态,在PCOS氧化应激的发生中发挥一定作用。
总之,本研究结果表明ACE基因变异可能与PCOS患者胰岛素抵抗、异常脂血症、高雄激素血症和氧化应激的发生有关。然而,这些假设还需要进一步的研究来证实。此外,我们没有进行血管紧张素Ⅰ转换酶活性和ACE基因表达量的测定。在不同基因型的患者中检测这种酶活性和基因表达量的进一步研究可能为潜在的遗传关联机制提供线索。
* * *
利益冲突 所有作者均声明不存在利益冲突
-
表 1 腺病毒载体序列
Table 1 Adenovirus vector sequence
Gene Vector Target sequence Pp2cm hU6-MCS-CMV-EGFP CCTAGCATCAAGTACGGCAAA Ctrl hU6-MCS-CMV-EGFP TTCTCCGAACGTGTCACGT 表 2 RT-qPCR引物序列
Table 2 RT-qPCR primer sequence
Gene Primer sequence (5′-3′) Product length/bp GAPDH F: AGGTCGGTGTGAACGGATTTG 123 R: TGTAGACCATGTAGTTGAGGTCA Pp2cm F: AAGTACGGCAAACCAATTCCC 132 R: GACTGCGAAGTATAGCACCTC TNF-α F: CCCTCACACTCAGATCATCTTCT 61 R: GCTACGACGTGGGCTACAG IL-1β F: GCAACTGTTCCTGAACTCAACT 89 R: ATCTTTTGGGGTCCGTCAACT TLR2 F: GCAAACGCTGTTCTGCTCAG 231 R: AGGCGTCTCCCTCTATTGTATT TLR4 F: ATGGCATGGCTTACACCACC 129 R: GAGGCCAATTTTGTCTCCACA Tirap F: CCTCCTCCACTCCGTCCAA 100 R: CTTTCCTGGGAGATCGGCAT Myd88 F: TCATGTTCTCCATACCCTTGGT 175 R: AAACTGCGAGTGGGGTCAG GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Pp2cm: protein phosphatase 2cm; TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin 1 beta; TLR: Toll-like receptor; Tirap: Toll-like receptor adaptor protein; Myd88: myeloid differentiation factor 88. -
[1] FARNSWORTH C W, SCHOTT E M, BENVIE A M, et al. Obesity/type 2 diabetes increases inflammation, periosteal reactive bone formation, and osteolysis during Staphylococcus aureus implant-associated bone infection. J Orthop Res,2018,36(6): 1614–1623. DOI: 10.1002/jor.23831
[2] 郭雪雯, 马渝, 肖玲, 等. 糖尿病合并脓毒症研究进展. 重庆医科大学学报,2021,46(9): 1035–1038. DOI: 10.13406/j.cnki.cyxb.002667 [3] BUI T I, GILL A L, MOONEY R A, et al. Modulation of gut microbiota metabolism in obesity-related type 2 diabetes reduces osteomyelitis severity. Microbiol Spectr,2022,10(2): e0017022. DOI: 10.1128/spectrum.00170-22
[4] WHITE P J, MCGARRAH R W, GRIMSRUD P A, et al. The BCKDH kinase and phosphatase integrate BCAA and lipid metabolism via regulation of ATP-citrate lyase. Cell Metab,2018,27(6): 1281–1293.e7. DOI: 10.1016/j.cmet.2018.04.015
[5] BLOOMGARDEN Z. Diabetes and branched-chain amino acids: what is the link? J Diabetes,2018,10(5): 350–352. DOI: 10.1111/1753-0407.12645
[6] XUAN L, HOU Y, WANG T, et al. Association of branched chain amino acids related variant rs1440581 with risk of incident diabetes and longitudinal changes in insulin resistance in Chinese. Acta Diabetol,2018,55(9): 901–908. DOI: 10.1007/s00592-018-1165-4
[7] WANG J, LIU Y, LIAN K, et al. BCAA catabolic defect alters glucose metabolism in lean mice. Front Physiol,2019,10: 1140. DOI: 10.3389/fphys.2019.01140
[8] LIU S, LI L, LOU P, et al. Elevated branched-chain alpha-keto acids exacerbate macrophage oxidative stress and chronic inflammatory damage in type 2 diabetes mellitus. Free Radic Biol Med,2021,175: 141–154. DOI: 10.1016/j.freeradbiomed.2021.08.240
[9] PIDWILL G R, GIBSON J F, COLE J, et al. The role of macrophages in Staphylococcus aureus infection. Front Immunol,2020,11: 620339. DOI: 10.3389/fimmu.2020.620339
[10] WANG X, EAGEN W J, LEE J C. Orchestration of human macrophage NLRP3 inflammasome activation by Staphylococcus aureus extracellular vesicles. Proc Natl Acad Sci U S A,2020,117(6): 3174–3184. DOI: 10.1073/pnas.1915829117
[11] GONG Z, ZHANG J, ZHANG S, et al. TLR2, TLR4, and NLRP3 mediated the balance between host immune-driven resistance and tolerance in Staphylococcus aureus-infected mice. Microb Pathog,2022,169: 105671. DOI: 10.1016/j.micpath.2022.105671
[12] WU J, LIU B, MAO W, et al. Prostaglandin E2 regulates activation of mouse peritoneal macrophages by Staphylococcus aureus through Toll-like receptor 2, Toll-like receptor 4, and NLRP3 inflammasome signaling. J Innate Immun,2020,12(2): 154–169. DOI: 10.1159/000499604
[13] LAUTERBACH M A, HANKE J E, SEREFIDOU M, et al. Toll-like receptor signaling rewires macrophage metabolism and promotes histone acetylation via ATP-citrate lyase. Immunity,2019,51(6): 997–1011.e7. DOI: 10.1016/j.immuni.2019.11.009
[14] SRIVASTAVA M, SAQIB U, BANERJEE S, et al. Inhibition of the TIRAP-c-Jun interaction as a therapeutic strategy for AP1-mediated inflammatory responses. Int Immunopharmacol,2019,71: 188–197. DOI: 10.1016/j.intimp.2019.03.031
[15] CRAIG-MUELLER N, HAMMAD R, ELLING R, et al. Modeling MyD88 deficiency in vitro provides new insights in its function. Front Immunol,2020,11: 608802. DOI: 10.3389/fimmu.2020.608802
[16] DOLATABAD M R, GUO L L, XIAO P, et al. Crystal structure and catalytic activity of the PPM1K N94K mutant. J Neurochem,2019,148(4): 550–560. DOI: 10.1111/jnc.14631
[17] WHITE P J, MCGARRAH R W, HERMAN M A, et al. Insulin action, type 2 diabetes, and branched-chain amino acids: a two-way street. Mol Metab,2021,52: 101261. DOI: 10.1016/j.molmet.2021.101261
[18] LIAN K, GUO X, WANG Q, et al. PP2Cm overexpression alleviates MI/R injury mediated by a BCAA catabolism defect and oxidative stress in diabetic mice. Eur J Pharmacol,2020,866: 172796. DOI: 10.1016/j.ejphar.2019.172796
[19] VORONOVA V, SOKOLOV V, MORIAS Y, et al. Evaluation of therapeutic strategies targeting BCAA catabolism using a systems pharmacology model. Front Pharmacol,2022,13: 993422. DOI: 10.3389/fphar.2022.993422
[20] MU W C, VANHOOSIER E, ELKS C M, et al. Long-term effects of dietary protein and branched-chain amino acids on metabolism and inflammation in mice. Nutrients,2018,10(7): 918. DOI: 10.3390/nu10070918
[21] LIAN N, LUO K, XIE H, et al. Obesity by high-fat diet increases pain sensitivity by reprogramming branched-chain amino acid catabolism in dorsal root ganglia. Front Nutr,2022,9: 902635. DOI: 10.3389/fnut.2022.902635
[22] GART E, Van DUYVENVOORDE W, CASPERS M P M, et al. Intervention with isoleucine or valine corrects hyperinsulinemia and reduces intrahepatic diacylglycerols, liver steatosis, and inflammation in Ldlr-/-.Leiden mice with manifest obesity-associated NASH. FASEB J,2022,36(8): e22435. DOI: 10.1096/fj.202200111R
[23] PAPATHANASSIU A E, KO J H, IMPRIALOU M, et al. BCAT1 controls metabolic reprogramming in activated human macrophages and is associated with inflammatory diseases. Nat Commun,2017,8: 16040. DOI: 10.1038/ncomms16040
[24] ARROYO D S, GAVIGLIO E A, PERALTA RAMOS J M, et al. Phosphatidyl-inositol-3 kinase inhibitors regulate peptidoglycan-induced myeloid leukocyte recruitment, inflammation, and neurotoxicity in mouse brain. Front Immunol,2018,9: 770. DOI: 10.3389/fimmu.2018.00770
[25] JIANG K, GUO S, YANG J, et al. Matrine alleviates Staphylococcus aureus lipoteichoic acid-induced endometritis via suppression of TLR2-mediated NF-kappaB activation. Int Immunopharmacol,2019,70: 201–207. DOI: 10.1016/j.intimp.2019.02.033
-
期刊类型引用(3)
1. 王选艺,孙亚伟,龙雨薇,王俪颖,周渝新,李娜,马雪连,赵红琼,姚刚. 屡配不孕母牛FOXP3、FSHR、FMR1基因多态性与生殖激素相关性分析. 畜牧兽医学报. 2024(06): 2727-2740 . 百度学术
2. 张丽,潘宇,赵阅. 越鞠丸合毓麟珠汤对多囊卵巢综合征不孕症患者脂代谢和HPA轴功能的影响. 中国医院用药评价与分析. 2024(07): 806-809 . 百度学术
3. 梁志超,孙红燕,宋如意,张晴,杨慧,毕亚菊,杨洁. 益肾柔肝法联合屈螺酮炔雌醇用于体重正常型多囊卵巢综合征患者的临床疗效研究. 中国医院用药评价与分析. 2023(07): 784-787 . 百度学术
其他类型引用(3)

开放获取 本文遵循知识共享署名—非商业性使用4.0国际许可协议(CC BY-NC 4.0),允许第三方对本刊发表的论文自由共享(即在任何媒介以任何形式复制、发行原文)、演绎(即修改、转换或以原文为基础进行创作),必须给出适当的署名,提供指向本文许可协议的链接,同时标明是否对原文作了修改;不得将本文用于商业目的。CC BY-NC 4.0许可协议详情请访问 https://creativecommons.org/licenses/by-nc/4.0