【作者】 刘燕；

【导师】 姜洁；

【作者基本信息】 山东大学 ， 妇产科学（专业学位）， 2021， 博士

【摘要】 第一部分:坐骨神经子宫内膜异位症模型中PI3K/Akt/mTOR通路促痛机制的研究研究背景:子宫内膜异位症(内异症,Endometriosis,EM)是育龄期常见病、多发病,它影响了约5-10%的育龄妇女[1],主要特征是宫腔外存在子宫内膜间质或腺细胞,其好发部位为卵巢、输卵管以及子宫周围韧带组织,少数的情况也会发生在身体的其他部位[2],如胸膜、心包膜甚至大脑。疼痛是EM的主要症状之一[3],疼痛的表现多种多样,如痛经、慢性盆腔痛、性交痛,甚至出现排尿困难等。内异症的主要治疗方法是激素和手术治疗。然而,术后5年复发率约为50%,其中疼痛的复发早于病灶的发现。而药物对疼痛的疗效并不可靠,随着疼痛的逐渐加重,多数药物会失去作用。因此,急需寻找一种有效的方法治疗子宫内膜异位症。坐骨神经子宫内膜异位症是一种特殊的深部子宫内膜异位症[4],主要表现除疼痛外,还伴随患侧肢体的功能障碍。因发病率低,对其治疗认识不足。目前常用的治疗手段参照普通子宫内膜异位症,可选择口服避孕药、GnRH-a、非甾体抗炎药以及一些中药治疗[5]。一些合适的轻柔运动也可以提供帮助,如散步、瑜伽、游泳和理疗等。当上述治疗方法无效时,可以通过手术将异位的子宫内膜切除,从而达到治疗或缓解坐骨神经子宫内膜异位症疼痛的目的[6]。但是常规的治疗手段对坐骨神经子宫内膜异位症的治疗存在不足。如口服避孕药虽有助于减轻疼痛,但口服避孕药对年龄大于40岁或有高危因素(如糖尿病、高血压、吸烟、血栓史)的患者,有较高的血栓栓塞风险;GnRH-a虽能有效缓解疼痛,长期应用会引起低雌症状及钙质流失等围绝经症状;非甾体抗炎药往往是短期有效,随着时间的延长出现止痛效果下降甚至失去作用;如果选择手术治疗,不仅需要外科医生经验丰富,更需要对于坐骨神经及周围盆骨区域血管及分支较为清楚,才能准确判断异位的子宫内膜,同时常伴随子宫内膜异位组织到达坐骨神经孔或是沿神经和血管至盆骨开口处,此时腹腔镜尚难以到达,需通过臀部到达盆骨背面进行手术,风险都会大大升高。疼痛是中枢神经系统对外周伤害性刺激的反应,内异症疼痛是中枢和外周两种因素相互作用的结果,坐骨神经子宫内膜异位症疼痛机制同样如此,对机制的深入了解,可以帮我们找到有效的治疗内异症疼痛的方法。目前,对内异症疼痛的研究主要集中在外周机制,对中枢机制的研究少之又少。而坐骨神经子宫内膜异位症大鼠模型因其自身的特点较适合研究内异症疼痛的中枢机制。本实验通过建立大鼠坐骨神经内异症模型,模拟临床患者坐骨神经内异症的疼痛症状,从而探查内异症特别是坐骨神经子宫内膜异位症的中枢机制,并以此为基础寻找治疗内异症,特别是坐骨神经子宫内膜异位症疼痛的有效药物,以期在内异症疼痛治疗中带来新的突破。PI3K/Akt/mTOR信号通路参与了神经病理痛、炎性痛的发生。而内异症因其病灶神经末梢形成以及种类的分布被重新定义为神经病理性疼痛。这一通路活化后能够诱导和维持痛敏反应,在中枢参与突触可塑性,在外周参与组织损伤后的痛觉易化过程。PI3K/Akt/mTOR通路在神经性疼痛形成和发展过程中发挥着重要作用,并且这一信号通路直接参与了坐骨神经损伤大鼠模型中对损伤神经元的保护过程[29]。因此,本研究进一步探究PI3K/Akt/mTOR信号通路在坐骨神经子宫内膜异位症疼痛的作用。LY294002是PI3K/Akt/mTOR通路的一种抑制剂,本研究尝试通过LY294002抑制PI3K/Akt/mTOR通路的激活,来探究脊髓背角浅层组织中PI3K/Akt/mTOR信号通路以及其释放的与痛觉相关的神经递质,即P物质(p substance,SP)和降钙素基因相关肽基因(calcitonin gene-related peptide,CGRP),在坐骨神经子宫内膜异位症疼痛中的作用。研究目的:本研究建立大鼠的坐骨神经子宫内膜异位症模型,基于对子宫内膜异位症疼痛机制的认识和理解,在疼痛反应及信号通路蛋白表达两方面研究PI3K/Akt/mTOR信号通路对大鼠子宫内膜异位症疼痛的影响和潜在的促痛机制。研究方法:建立坐骨神经子宫内膜异位症模型,将试验大鼠随机分为假手术对照组(Sham组)、子宫内膜异位症模型对照组(ENDO+DMSO组)和子宫内膜异位症模型给药组PI3K/Akt/mTOR通路抑制剂LY294002治疗组(ENDO+LY294002组),每组8只大鼠。采用蛛网膜下给药方式,通过哈密尔顿微量注射器连接导管,按照适宜计量浓度进行局部微量给药。其后进行如下检测:1.冯·费雷纤维测试和热痛觉敏感度测试,检测三组动物的疼痛水平。2.qPCR 和 Western blot 分别检测 Sham 组、ENDO+DMSO 组和 ENDO+LY294002组病灶侧脊髓背侧角浅层位置组织中PI3K、Akt、mTOR的mRNA水平和蛋白磷酸化水平。3.qPCR 和 Western blot 分别检测 Sham 组、ENDO+DMSO组和 ENDO+LY294002组病灶侧脊髓背侧角浅层位置组织中SP、CGRP和NGF的mRNA及蛋白表达水平。4.使用GraphPad Prism8.0对数据进行统计学分析,统计方法采用包括:ST检验、单变量方差分析、双变量方差分析以及Tukey’s事后检测。研究结果:1.坐骨神经子宫内膜异位症中伴随PI3K/Akt/mTOR通路的激活ENDO组病灶侧脊髓背角浅层位置组织中,PI3K(2.86±0.59vs1,p<0.01)、Akt(3.14±0.69vs1,p<0.01)、mTOR(3.72±0.89vs1,p<0.01)的 mRNA 水平较 Sham 组表达上调,ENDO+LY294002 组 PI3K、Akt、mTOR 的 mRNA 水平明显低于 ENDO 组(PI3K ENDO+LY294002 vs ENDO:1.33±0.47 vs 2.86±0.59,p<0.05;Akt ENDO+LY294002 vs ENDO:2.02±0.52 vs 3.14±0.69,p<0.05;mTOR ENDO+LY294002 vs ENDO:1.64±0.49 vs 3.72±0.89,p<0.01);ENDO+LY294002组PI3K、Akt、mTOR蛋白的磷酸化水平明显低于ENDO组(PI3K ENDO+LY294002 vs ENDO:1.24±0.41 vs 2.72±0.54,p<0.05;Akt ENDO+LY294002 vs ENDO:1.72±0.46 vs 3.41±0.67,p<0.05;mTOR ENDO+LY294002 vs ENDO:2.01±0.43 vs 3.13±0.59,p<0.05),变化同mRNA变化一致。2.PI3K/Akt/mTOR抑制剂改善坐骨神经子宫内膜异位症的痛觉过敏向ENDO+LY294002组动物鞘内注射PI3K/Akt通路抑制剂LY294002后的第2、3、4、5、6小时使ENDO+LY294002组大鼠对于外界机械刺激导致的疼痛耐受性较ENDO组显著增加(2小时ENDO+LY294002 vs ENDO:7.0±1.1 vs 3.9±0.9;3 小时 ENDO+LY294002 vs ENDO:8.2±1.6 vs 3.5±1.0,4 小时ENDO+LY294002 vs ENDO:9.0±1.6 vs 3.7±0.9;5 小时 ENDO+LY294002 vs ENDO:7.9±2.2 vs 4.0±1.0,6小时ENDO+LY294002 vs ENDO:7.0±1.9 vs 3.7±0.8,p<0.05);持续给药后3周内,每3天测量痛域值,自第3天开始,ENDO+LY294002组较ENDO组机械性痛觉过敏明显改善(ENDO+LY294002 vs ENDO:8.4±1.9vs 3.9±1.0,p<0.01),持续至 21 天均明显高于 ENDO 组和ENDO+DSMO 组(ENDO+LY294002 vs ENDO:12.3±2.5 vs 6.9±2.0,p<0.01;ENDO+LY294002 vs ENDO+DSMO:12.3±2.5 vs 6.6±1.9,p<0.01),ENDO 组和ENDO+DSMO组大鼠手术侧后肢的机械性痛觉过敏在任何时间点没有统计学意义,p>0.05。ENDO+LY294002组在用药后3、4、5、6、7小时使模型动物后肢的热刺激耐受性较 ENDO 组显著增加(3 小时 ENDO+LY294002 vs ENDO:8.2±1.6 vs 3.5±1.0;4 小时 ENDO+LY294002 vs ENDO:9.0±1.6 vs 3.7±0.9;5 小时ENDO+LY294002 vs ENDO:7.9±2.2 vs 4.0±1.0;6 小时 ENDO+LY294002 vs ENDO:7.0±1.9 vs 3.7±0.8,7 小时 ENDO+LY294002vs ENDO:5.7±1.4 vs 3.9±1.0,p<0.05)。持续给药3周内,每3天测量痛域值,自第3天开始,ENDO+LY294002组机热痛觉过敏较ENDO组明显改善(ENDO+LY294002 vs ENDO:5.98±1.87 vs 2.75±1.06,p<0.01),持续至 21 天均明显高于 ENDO 组和ENDO+DSMO 组(ENDO+LY294002 vs ENDO:10.37±2.53 vs 5.63±2.01,p<0.01;ENDO+LY294002 vs ENDO+DSMO:10.37±2.53 vs 5.52±1.88,p<0.01),ENDO组和ENDO+DSMO组大鼠手术侧后肢的热痛觉过敏在任何时间点没有统计学意义,p>0.05。3.PI3K/Akt抑制剂改善坐骨神经子宫内膜异位症的痛觉过敏的剂量效应关系模型给药组动物给予不同浓度梯度的LY294002药物干预(5、10、30、50 mg/kg),结果发现当给药浓度为10mg/kg时,相对Sham组,升高了内异症大鼠对机械性痛觉过敏和热痛觉过敏的痛阈值,改善了内异症大鼠的机械性超敏反应和热痛觉过敏,均有显著性差异(P<0.05)。给药浓度在10-30mg/kg范围内,治疗效果与给药浓度呈剂量依赖关系。而给药浓度分别为30mg/kg和5mg/kg的两组动物,其疼痛耐受性并没有显著性差异(p>0.05),因此本项目以下的研究中LY294002药物干预设置为30mg/kg。4.PI3K/Akt/mTOR信号通路影响到SP和CGRP表达检测各组坐骨神经子宫内膜异位症模型动物病灶侧脊髓背角浅层位置组织中的P物质和CGRP的蛋白质和mRNA表达水平。和Sham组相比,ENDO+DMSO组P物质和CGRP蛋白表达量较Sham组升高(SP ENDO+DMSO vs Sham:297.2±30 vs 176.7±22.3pg/ml;CGRP NDO+DMSO vs Sham:444.8±36.1 vs 269.4±26.3pg/ml,p<0.01),ENDO+LY294002 组 SP 和 CGRP 蛋白表达量较ENDO+DMSO 组下降(SP ENDO+LY294002 vs ENDO+DMSO:297.2±30 vs 231.1±27.3pg/ml,p<0.01;CGRP ENDO+LY294002 vs ENDO+DMSO:304.8±31.2 vs 444.8±36.1pg/ml,p<0.05)。ENDO+DMSO 组较 Sham 组 SP mRNA 表达升高(2.46±0.49vs1,p<0.01),ENDO+LY294002 组 SP mRNA 表达较 ENDO+DMSO 组下降(ENDO+LY294002 vs ENDO+DMSO:1.74±0.43 vs 2.46±0.49,p<0.05),ENDO+DMSO 组较 Sham组 CGRP mRNA 表达升高(3.05±0.62vs1,p<0.001),ENDO+LY294002 组 CGRP mRNA 表达较 ENDO+DMSO 组下降(ENDO+LY294002 vs ENDO+DMSO:2.11±0.49 vs 3.05±0.62,p<0.05)。通过蛋白质免疫印迹实验检测目标区域SP和CGRP的蛋白水平,发现ENDO组SP和CGRP的蛋白表达量较Sham组显著升高(SP ENDO vs Sham:3.27±0.61vs1;CGRP ENDO vs Sham:3.61±0.72vs1,p<0.001),而予以PI3K/Akt/mTOR信号通路抑制剂LY294002后,ENDO+LY294002组SP和CGRP的蛋白表达量均较ENDO组下调(SP ENDO+DMSO vs ENDO:1.98±0.49 vs 3.27±0.61,p<0.05;CGRP ENDO+DMSO vs ENDO:2.33±0.52 vs 3.61±0.72,p<0.05),但下调后的水平仍然显著高于 Sham组水平。5.抑制PI3K/Akt/mTOR信号通路影响NGF表达检测NGF mRNA表达水平,ENDO+DMSO组NGF的mRNA水平较Sham组显著性升高(2.05±0.33vs1,p<0.05),达到Sham组的2倍,而予以PI3K/Akt/mTOR 信号通路抑制剂 LY294002 处理后,ENDO+LY294002 组 NGF的 mRNA 水平较 ENDO+DMSO 显著性降低(ENDO+LY294002 vs ENDO+DMSO:1.53±0.23 vs 2.05±0.33,p<0.05),但是仍然明显的高于Sham组。检测NGF的蛋白表达水平,ENDO+DMSO组NGF的蛋白水平较Sham组显著性升高(ENDO+DMSO vs Sham:2.95±0.51vs1,p<0.01),达到 Sham 组的近3倍,而予以PI3K/Akt/mTOR信号通路抑制剂LY294002处理后,ENDO+LY294002组NGF的蛋白水平较ENDO+DMSO显著性降低(ENDO+LY294002 vs ENDO+DMSO:1.83±0.36 vs 2.95±0.51,p<0.05),但是仍然明显的高于Sham组。研究结论:1.PI3K/Akt/mTOR信号通路参与坐骨神经内异症疼痛发生发展过程。2.PI3K/Akt/mTOR信号通路通过上调下游的P物质、降钙素基因相关肽和神经生长因子参与调控坐骨神经子宫内膜异位症的痛觉敏感性。3.PI3K/Akt抑制剂LY294002可以改善坐骨神经子宫内膜异位症的痛觉过敏。第二部分:坐骨神经子宫内膜异位症模型中萝卜硫烷镇痛机制的研究研究背景:坐骨神经子宫内膜异位症是一类雌激素依赖的炎性疾病[4]。坐骨神经内异症患者中,发现异位病灶多位于坐骨神经的周围、坐骨神经鞘膜内或者坐骨结节附近。坐骨神经子宫内膜异位症主要的症状是坐骨神经痛和运动障碍。因为坐骨神经子宫内膜异位症的临床表现与腰椎间盘突出症相似,容易误诊为腰椎间盘突出症,导致患者诊断延迟、病情延误[7]。对于坐骨神经子宫内膜异位症,当下的治疗手段非常有限。对坐骨神经子宫内膜异位症而言,原本子宫内膜生长的正常环境位于子宫内,一旦异位内膜生长在坐骨神经周围,则会被机体认定为外源性物质,从而产生免疫炎症和免疫攻击,尝试将其清除。其治疗以手术和药物为主。手术需要外科医生经验丰富,并熟悉坐骨神经周围血管及其分支,手术风险较大。药物治疗可选择口服避孕药、GnRH-a、非甾体抗炎药以及中药[5],但这些治疗都存在不足。寻找一种既能抑制炎症反应减轻疼痛,又能抑制病灶生长的天然药物,可能会对内异症特别是坐骨神经子宫内膜异位症的治疗产生深远的影响。萝卜硫烷(sulforaphane,SFN)是十字花科蔬菜中天然存在的异硫氰酸盐[8],已经被证明了具有抗糖尿病、抗癌和抗菌的功效。最近,还发现萝卜硫烷具有良好的抗炎作用。例如,萝卜硫烷被证明可以抑制细菌脂多糖诱导的炎症,其特征在于降低TNF-α,IL-1β,诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和前列腺素过氧化物合酶2(cyclooxygenase-2,COX-2)的表达,并激活Nrf2,从而发挥神经保护作用。萝卜硫烷还可以通过调节炎症来防止肾脏纤维化和缺血再灌注损伤。此外,萝卜硫烷的另一个重要应用是减轻疼痛。然而,尽管萝卜硫烷在减轻炎症和疼痛方面具有潜力,但至今尚未有萝卜硫烷在坐骨神经子宫内膜异位症治疗中的报道。研究目的:本研究建立大鼠的坐骨神经子宫内膜异位症模型,在病灶体积、疼痛反应及炎症反应三方面观察萝卜硫烷对大鼠坐骨神经子宫内膜异位症疼痛的影响,并进一步探讨萝卜硫烷在坐骨神经子宫内膜异位症模型中镇痛机制。研究方法:建立坐骨神经子宫内膜异位症模型,将实验用大鼠随机分为假手术对照组(Sham组)、子宫内膜异位症模型对照组(ENDO组)、萝卜硫烷给药组(ENDO+SFN组),每组动物各6只。同第一部分研究中构建坐骨神经子宫内膜异位症模型,检测不同浓度萝卜硫烷(5、15、30、60mg/kg)给药对坐骨神经内异症大鼠机械刺激疼痛阈值(PWT)和对热刺激疼痛阈值(PWL)的改变,筛选最佳给药浓度,并进行进一步研究,随后在Sham组、ENDO组、ENDO+SFN组三组中进行如下检测:1.冯·费雷纤维测试和热痛觉敏感度测试,测定PWT和PWL。2.ELISA和Western blot方法分别检测Sham组、ENDO组、ENDO+SFN组大鼠病灶区及血清VEGF表达水平。3.ELISA和qPCR方法分别检测Sham组、ENDO组、ENDO+SFN组大鼠脊髓背角浅层组织炎性细胞因子IL-6、IL-1β,TNF-α蛋白及mRNA表达。4.qPCR和western blot方法分别检测Sham组、ENDO组、ENDO+SFN组大鼠脊髓背角浅层组织iNOS、COX-2mRNA及蛋白表达。5.Western blot检测Sham组、ENDO组、ENDO+SFN组大鼠脊髓背角浅层组织Keap1、Nrf2蛋白表达水平。研究结果:1.萝卜硫烷可以显著改善坐骨神经子宫内膜异位症的痛觉过敏造模前,三组大鼠的疼痛基线水平一致,无显著性差异(P>0.05);ENDO+SFN组在第7天测试机械性刺激引起痛觉过敏和热刺激引起的痛觉过敏较ENDO组改善(机械性痛觉过敏 ENDO+SFN vs ENDO:11.4±2.1 vs 7.2±1.7,p<0.01;热痛觉过敏 ENDO+SFN vs ENDO:6.4±1.1 vs 3.2±1.7,p<0.01),ENDO+SFN 组在第14天测试机械性刺激引起痛觉过敏和热刺激引起的痛觉过敏较ENDO组改善(机械性痛觉过敏 ENDO+SFN vs ENDO:10.8±2.0 vs 5.7±1.9,p<0.01;热痛觉过敏 ENDO+SFN vs ENDO:6.8±1.3 vs 2.7±1.9,p<0.01);ENDO+SFN 组在第21天测试机械性刺激引起痛觉过敏和热刺激引起的痛觉过敏较ENDO组改善(机械性痛觉过敏 ENDO+SFN vs ENDO:14.7±2.1 vs 6.4±1.6,p<0.01;热痛觉过敏ENDO+SFN vs ENDO:9.7±1.7 vs 4.6±1.4,p<0.01);持续至术后28天,随着药物的持续注射,这种效果改善不断增强,与ENDO组比较,有统计学差异(机械性痛觉过敏 ENDO+SFN vs ENDO:16.2±2.3 vs 6.6±1.4,p<0.01;热痛觉过敏 ENDO+SFN vs ENDO:1 1.2±2.0 vs 4.6±1.4,p<0.01)。2.萝卜硫烷改善坐骨神经子宫内膜异位症的痛觉过敏的剂量效应关系模型组动物给予不同浓度梯度的萝卜硫烷干预(5、15、30、60mg/kg),结果发现15mg/kg、30mg/kg、60mg/kg给药,在第7、14、21、28天对模型动物进行机械刺激和热刺激检测,发现与未用药组对机械性超敏反应和热痛觉过敏有显著改善,p<0.05;在15-30mg/kg范围内,治疗效果与给药浓度呈剂量依赖关系;而给药浓度分别为30mg/kg和50mg/kg的两组动物,其疼痛耐受性并没有显著性差异(均p>0.05)。因此本项目以下的研究中萝卜硫烷干预浓度设置为30mg/kg。3.萝卜硫烷通过VEGF抑制坐骨神经子宫内膜异位症模型异位子宫内膜组织的生长坐骨神经子宫内膜异位症造模动物中,ENDO组坐骨神经周围的子宫内膜组织体积增大,ENDO+SFN组大鼠的子宫内膜异位病灶体积明显小于Sham组(ENDO+SFN 组 vs Sham 组:42.6±20 vs 140.2±30 mm3,p<0.001),体积减少超过2/3。同时,ENDO组大鼠血清及病灶区VEGF蛋白水平显著上调,ENDO组较Sham组血清中VEGF蛋白水平提高了 8倍(ENDO vs Sham:185.3±42.0 vs 23.6±12.8 pg/ml,p<0.001),ENDO+SFN 组血清中 VEGF 蛋白水平较 ENDO 组表达下降(ENDO+SFN vs ENDO:185.3±42.0 vs 69.8±33.0 pg/ml,p<0.01),恢复到了 Sham组3倍的水平,相对于ENDO组降低了 2/3。病灶区域内的VEGF蛋白的表达也有着类似的变化,ENDO组相对于Sham组提高了 5倍(ENDO vs Sham:347.6±67.9vs58.9±27.4pg/ml,p<0.001),ENDO+SFN 组病灶区域内的VEGF 蛋白水平较 ENDO 组降低(ENDO+SFN vs ENDO:156.3±49 vs 347.6±67.9pg/ml,p<0.001),恢复到了 Sham 组 2.5 倍的水平,相对于 ENDO 组降低了超过50%。3.萝卜硫烷抑制坐骨神经子宫内膜异位症中的炎症反应病灶侧坐骨神经附近的脊髓(L4-6)背角浅层组织中,ENDO组脊髓背侧角浅层区域内IL-6蛋白表达量较Sham组升高(ENDO vs Sham:243.7±46.8 vs 74.7± 17.9 pg/mg,P<0.01),ENDO+SFN 组较 ENDO 组 IL-6 蛋白表达量下降(ENDO+SFN vs ENDO:138.3±37vs 243.7±46.8 pg/mg,P<0.05)。ENDO 组脊髓背角浅层区域内IL-1β蛋白表达量较Sham组升高(ENDO vs Sham:154.7±34.1 vs46.3±12.9 pg/mg,P<0.01),ENDO+SFN组较ENDO组IL-1β蛋白表达量下降(ENDO+SFN vs ENDO:71.3±21.2 vs 154.7±34.1 pg/mg,P<0.01)。ENDO 组脊髓背侧角浅层区域内TNF-α蛋白表达量较Sham组升高(ENDO vs Sham:97.4±38.3vs 86.9±19.9 pg/mg,P<0.01),ENDO+SFN 组较 ENDO组 TNF-α 蛋白表达量下降(ENDO+SFN vs ENDO:141.1±28.4 vs 197.4±38.3 pg/mg,P<0.05)。ENDO 组 IL-6、IL-1β、TNF-amRNA 较 Sham 组明显升高,与 ELISA 检测蛋白水平变化相一致。IL-6高了 3.24倍,p<0.001;IL-1β升高了 2.68倍,p<0.01;TNF-α升高了 3.26倍,p<0.001。ENDO+SFN组炎性因子较ENDO组表达下降(IL-6 ENDO+SFN vs ENDO组:1.78±0.5 vs 3.24±0.69,p<0.01;IL-1βENDO+SFN vs ENDO:1.53±0.47 vs 2.68±0.62,p<0.01;TNF-α ENDO+SFN vs ENDO:2.23±0.45 vs 3.26±0.64,p<0.05)。4.萝卜硫烷可以抑制COX2、iNOS的表达病灶侧坐骨神经附近的脊髓(L4-6)背角浅层组织中,ENDO组COX-2的mRNA水平显著性的升高(ENDO vs Sham:3.64±0.76vs1±0.23,p<0.01),达到Sham组的3.6倍,ENDO+SFN组COX-2的mRNA表达水平较ENDO组显著性下调(ENDO+SFN vs ENDO:1.97±0.44 vs 3.64±0.76,p<0.01),下降至 ENDO组约50%;COX-2蛋白水平与mRNA水平变化类似,即ENDO组COX-2蛋白水平较 Sham 组显著性升高超过 3 倍(ENDO vs Sham:3.23±0.63vs1±0.24pg/m,p<0.001),ENDO+SFN 组 COX-2 蛋白量较 ENDO 组下调(ENDO+SFN vs ENDO:1.79±0.4 vs 3.23±0.63pg/ml,p<0.01)。ENDO组iNOS的mRNA和蛋白水平较Sham组升高2.5倍以上,p<0.01,ENDO+SFN组iNOS mRNA和蛋白水平较ENDO组表达下调(mRNA ENDO+SFN vs ENDO:1.84±0.39 vs 2.53±0.54,p<0.05;蛋白 ENDO+SFN vs ENDO:1.98±0.42 vs 2.86±0.57pg/ml,p<0.05),下调幅度约为ENDO组1/3水平。5.Keap1/Nrf2信号通路在坐骨神经子宫内膜异位症中被抑制并可以被萝卜硫烷显著性激活病灶侧坐骨神经附近的脊髓(L4-6)背角浅层组织中,ENDO组Keap1蛋白水平较 Sham 组显著性降低(ENDO vs Sham:0.32±0.16 vs 1±0.18pg/ml,p<0.01),约为对照组的30%水平,ENDO+SFN组Keap1蛋白水平较ENDO组显著升高(ENDO+SFN vs ENDO:0.58±0.19 vs 0.32±0.16pg/ml,p<0.05),表达量比ENDO组增加1倍,达到Sham组的60%水平;ENDO组Nrf2蛋白水平较Sham 组显著性降低(ENDO vs Sham:0.42±0.17 vs1±0.2pg/ml,p<0.01),约为 Sham组的40%水平,ENDO+SFN组Nrf2蛋白水平较ENDO组显著升高(ENDO+SFN vs ENDO:1.67±0.3 vs 0.42±0.17pg/ml,p<0.001),表达量比 ENDO 组增加 3 倍,达到Sham组的1.6倍。研究结论:1.萝卜硫烷可以改善对坐骨神经子宫内膜异位症模型动物痛觉过敏。2.萝卜硫烷可以通过抑制VEGF降低坐骨神经子宫内膜异位症中异位子宫内膜组织的生长速度。3.萝卜硫烷可以有效抑制IL-6、IL-1β、TNF-α、COX2、iNOS表达,减轻坐骨神经子宫内膜异位症疼痛。4.在坐骨神经子宫内膜异位症中Keap1/Nrf2信号通路下调,萝卜硫烷可以上调该信号通路。更多还原

【Abstract】 Part Ⅰ:Study of PI3K/Akt/mTOR pathway pain-promoting mechanism in sciatic nerve endometriosis modelBackground:Endometriosis(EM)has become a common and frequently-occurring disease during childbearing age and about 5-10%of women were influenced by it.The main feature of EM is the endometrial stromal or glandular cells went outside the uterine cavity.Its main pathogenesis is ovary,fallopian tube,and uterine ligament tissue,a few cases also appeared in other parts of the body,such as the pleura,pericardium and even the brain.One of the main symptoms of EM is pain,including dysmenorrhea,chronic pelvic pain,painful intercourse,and even dysuria.Hormone and surgery were the main treatments for EM.However,about 50%patients would feel pain in 5 years after surgery,they could not discover the lesions until the heavy pain.In addition,there is little painkillers for EM.Therefore,it is necessary to find a new and treatment for endometriosisSciatic nerve endometriosis is a rare and special type of deep endometriosis,In addition to pain,sciatic nerve endometriosis is also accompanied by dysfunction of the affected limb.The current treatment are insufficiently understood.At present,the treatments used currently are similar to ordinary endometriosis,including choosing oral contraceptives,GnRH-a,non-steroidal anti-inflammatory drugs and some traditional Chinese medicines.Moreover,some suitable gentle exercises will play a role,such as walking,yoga,swimming,and physical therapy.When the abovementioned treatments do not well work,the ectopic endometrial can be removed by surgery,so as to achieve the purpose of treating or alleviating the pain of sciatic nerve endometriosis.However,the conventional treatments are insufficient to treat the intrauterine ectopic heteropathic disease.For example,although oral contraceptives can help patients relieve pain,it has a higher risk of thromboembolism for patients over 40 years or with have high-risk factors(such as diabetes,hypertension,smoking,history of thrombosis);although GnRH-a can effectively relieve pain,long-term application will cause perimenopausal symptoms such as hypogyny and calcium loss;non-steroidal anti-inflammatory drugs are often effective in the short term.Surgical treatment requires an experienced surgeon with a clearer understanding of the blood vessels,branches of the sciatic nerve and surrounding pelvic region in order to accurately determine the ectopic endometrium.At the same time,it is often accompanied by endometriotic tissue reaching the sciatic nerve foramen or along the nerves and blood vessels to the pelvic opening.Additonally,hysteroscopy is still not easy to be available,and to operate in the back of the pelvis through the buttocks will greatly increase the risk.Pain is the response of the central nervous system to peripheral noxious stimuli.Endometriotic pain is the result of the interaction of central and peripheral factors.The same is true for the pain mechanism of sciatic nerve endometriosis.A deep understanding of the mechanism can help us Find an effective way to treat endometriosis pain.At present,the research on endometriosis pain mainly focuses on the peripheral mechanism,and the research on the central mechanism is rare.The sciatic nerve endometriosis rat model is more suitable for studying the central mechanism of endometriosis pain due to its own characteristics.This experiment established a rat sciatic nerve endometriosis model to simulate the pain symptoms of clinical patients with sciatic nerve endometriosis,so as to explore the central mechanism of endometriosis,especially sciatic nerve endometriosis,and find the treatment of endometriosis based on this.In particular,effective drugs for sciatic nerve endometriosis pain are expected to bring new breakthroughs in the treatment of endometriosis pain.The PI3K/Akt signaling pathway is involved in the occurrence of neuropathic pain and inflammatory pain.Endometriosis is redefined as neuropathic pain due to the formation of nerve endings in the lesion and the distribution of types.The activation of this pathway can induce and maintain hyperalgesia,participate in synaptic plasticity in the center,and participate in the process of pain facilitation after tissue injury in the periphery.PI3K/Akt pathway plays an important role in the formation and development of neuropathic pain,and this signaling pathway directly participates in the protection of ventral motor neurons in a rat model of sciatic nerve injuryLY294002 is an inhibitor of PI3K/Akt pathway.In this study,we tried to explore the PI3K/Akt/mTOR signaling pathway in superficial spinal dorsal horn and the neurotransmitter related to pain released by LY294002 by inhibiting the activation of PI3K/Akt pathway Objective to investigate the role of substance P(SP)and calcitonin gene-related peptide(CGRP)in pain of sciatic nerve endometriosisObjective:In this study,we established a rat model of sciatic nerve endometriosis.Based on the understanding of the pain mechanism of endometriosis,we studied the effect of PI3K/Akt/mTOR signaling pathway on the pain response and the expression of signal pathway proteins,and the potential pain promoting mechanism.Methods:The sciatic nerve endometriosis model(SNEM)was established,and the rats were randomly divided into normal control group(Sham group),endometriosis control group(ENDO+DMSO group),LY294002(inhibitors of PI3K/Akt/mTOR signaling pathway)drug treatment group(ENDO+LY294002 group),with 8 rats in each group.Subarachnoid administration was used.The catheter was connected with Hamilton microinjector,and the local microdose was given according to the appropriate concentration.Later testing1.Von Frey fiber test and thermal pain sensitivity test were used to detect the pain level of sciatic nerve endometriosis model animals2.qPCR and Western blot were used to detect the mRNA and protein phosphorylation levels of PI3K,Akt and mTOR in the superficial layer of spinal dorsal horn around the lesion in Sham group,ENDO+DMSO group and ENDO+LY294002 group.3.qPCR and Western blot were used to detect the mRNA and protein expression levels of SP,CGRP and NGF in the superficial layer of spinal dorsal horn around the lesion in Sham group,ENDO+DMSO group and ENDO+LY294002 group.4.Use graphpad prism 8.0 to analyze the data.The statistical methods include student t-test,univariate analysis of variance,bivariate analysis of variance and Tukey’s post test.Results:1.Accompanying PI3K/Akt pathway activation in sciatic nerve endometriosis Compared with the superficial tissues of dorsal horn of spinal cord in Sham group,the mRNA levels of PI3K,Akt and mTOR were significantly increased,and the differences between the two groups were significant(p<0.01).Compared with the ENDO group,the mRNA levels of PI3K,Akt and mTOR were significantly lower in ENDO+LY294002 group(PI3K ENDO+LY294002 vs ENDO:1.33±0.47 vs2.86±0.59,p<0.05;Akt ENDO+LY294002 vs ENDO:2.02±0.52 vs 3.14±0.69,p<0.05;mTOR ENDO+LY294002 vs ENDO:1.64±0.49 vs 3.72±0.89,p<0.01).Compared with the ENDO group,the phosphorylation levels of PI3K,Akt and mTOR were significantly lower in ENDO+LY294002 group(PI3K ENDO+LY294002 vs ENDO:1.24±0.41 vs 2.72±0.54,p<0.05;Akt ENDO+LY294002 vs ENDO:1.72±0.46 vs 3.41±0.67,p<0.05;mTOR ENDO+LY294002 vs ENDO:2.01±0.43 vs 3.13±0.59,p<0.05)2.PI3K/Akt inhibitor improved the dose effect of painful allergies of the intimal endometriosisAt 2,3,4,5 and 6 hours after intrathecal injection of LY294002,the pain tolerance of mechanical stimulation in ENDO+LY294002 group was significantly higher than that of ENDO group(7.0±1.1 vs 3.9±0.9,8.2±.6 vs 3.5±.0,9.0±1.6 vs 3.7±0.9,7.9±2.2 vs 4.0±1.0,7.0±1.9 vs 3.7±0.8,p<0.05);From the third day,the mechanical hyperalgesia of ENDO+LY294002 group was significantly improved than that of ENDO group(8.4±1.9 vs 3.9±1.0,p<0.01).It was significantly higher in ENDO group(12.3±2.5 vs 6.9±2.0,p<0.01)and ENDO+DSMO group(12.3±2.5 vs 6.6±1.9,p<0.01).There was no statistical significance in the mechanical hyperalgesia of hind limb in ENDO group and ENDO+DSMO group at any time point(p>0.05).Compared with ENDO group,ENDO+LY294002 group significantly increased the heat shock tolerance of hind limbs at 3,4,5,6 and 7 hours after treatment(8.2±1.6 vs 3.5±1.0,9.0±1.6 vs 3.7±0.9,7.9±2.2 vs 4.0±1.0,7.0±1.9 vs 3.7±0.8,5.7±1.4 vs 3.9±1.0,p<0.05).From the third day,the hyperalgesia of ENDO+LY294002 group was significantly improved than that of ENDO group(5.98±1.87 vs 2.75±1.06,p<0.01).After 21 days,it was significantly higher than that in ENDO group(10.37±2.53 vs 5.63±2.01,p<0.01)and ENDO+DSMO group(10.37±2.53 vs 5.52±1.88,p<0.01).The thermal hyperalgesia of hind limb in ENDO group and ENDO+DSMO group had no statistical significance at any time point,p>0.05.Injection of the PI3K/Akt inhibitor LY294002 induced by the induction of the apiatic nectar endometriosis,can significantly improve the pain sensitivity of the animal hind limbs;sustained administration can reduce the pain sensitivity of rats to external mechanical stimulation and thermal stimulation.3.Dose effect relationship of PI3K/Akt inhibitor on pain sensitivity in sciatic endometriosisThe model group was given LY294002(5,10,30,50mg/kg)with different concentration gradients.The results showed that when the concentration of the drug was 10mg/kg,the mechanical hypersensitivity and the heat pain allergy were decreased compared with those in the model group due to sciatic neuro endometriosis.The response threshold of the two groups was significantly improved,and there were significant differences(p<0.05).When the concentration of the drug was 10-30mg/kg,the therapeutic effect was dose-dependent.However,there was no significant difference(p>0.05)in pain tolerance between the two groups(30mg/kg and 50mg/kg)Thus,the intervention of LY294002 was set to 30mg/kg in the following study.4.PI3K/Akt/mTOR signaling pathway affects the expression of substance P and calcitonin gene-related peptideThe protein and mRNA expression of substance P and CGRP in the superficial tissue of dorsal horn of spinal cord around the lesions were detected.Compared with sham group,the expressions of substance P and CGRP(444.8±36.1 vs 269.4±26.3pg/ml,p<0.01)in ENDO+DMSO group were significantly higher(SP ENDO+DMSO vs ENDO:297.2±30 vs 176.7±22.3pg/ml,p<0.01;CGRP ENDO+DMSO vs ENDO:444.8±36.1 vs 269.4±26.3pg/ml,p<0.01).The protein expression of substance P and CGRP in ENDO+LY294002 group were lower than those in ENDO+DMSO group(SP ENDO+DMSO vs ENDO:297.2±30 vs 231.1 ±27.3 pg/ml,p<0.01:CGRP ENDO+DMSO vs ENDO:304.8±31.2 vs 444.8±36.1 pg/ml,p<0.05).5.Inhibition of PI3K/Akt/mTOR signaling pathway affects the expression of nerve growth factorLY294002 could inhibit the expression of NGF(1.53±0.23vs1),and the difference was significant(p<0.05).The changes in the protein level of NGF was the same as that of mRNA(2.95±0.51 vs 1.83±0.36),and the difference was significant(p<0.05).The mRNA level of NGF in ENDO+DMSO group was significantly higher than that in sham group(2.05±0.33vs1,p<0.05).The mRNA level of NGF in ENDO+LY294002 group was significantly lower than that in ENDO+DMSO group(ENDO+LY294002 vs ENDO+DMSO:1.53±0.23 vs 2.05±0.33,p<0.05).But it was still significantly higher than that in sham group.The protein level of NGF in ENDO+DMSO group was significantly higher than that in sham group(2.95±0.51vs1,p<0.01).After treatment with LY294002,the protein level of NGF in ENDO+LY294002 group was significantly lower than that in ENDO+DMSO group(ENDO+LY294002 vs ENDO+DMSO:1.83±0.36 vs 2.95±0.51,p<0.05),but still significantly higher than that in sham group.Conclusions:1.PI3K/Akt/mTOR signaling pathway participates in the occurrence and development of pain in sciatic neuropathy.2.PI3K/Akt/mTOR signaling pathway is involved in regulating the pain sensitivity of sciatic nerve endometriosis by promoting downstream substance P,calcitonin gene-related peptide and nerve growth factor3.LY294002 can improve the pain sensitivity of sciatic endometriosis by inhibiting the expression of substance P,calcitonin gene-related peptide and nerve growth factor,which is of great significance to develop a new treatment method for sciatic neuropathyPart II:Study on analgesic mechanism of sulforaphane in sciatic nerve endometriosis modelBackground:Sciatic nerve endometriosis is a type of estrogen-dependent inflammatory disease In patients with endometriosis of the sciatic nerve,ectopic lesions are mostly found around the sciatic nerve,within the sciatic nerve sheath,or near the sciatic tuberosityThe main symptoms of sciatic nerve endometriosis are sciatica and dyskinesia.Because the clinical manifestations of sciatic nerve endometriosis are similar to lumbar disc herniation,it is easy to be misdiagnosed as lumbar disc herniation,leading to delays in the diagnosis and condition of patients.For sciatic nerve endometriosis,current treatments are very limited.For sciatic nerve endometriosis,the normal environment for the growth of the endometrium is in the uterus.Once the ectopic endometrium grows around the sciatic nerve,it will be recognized by the body as a foreign substance,resulting in immune inflammation and immunity Attack,try to clear it.Its treatment is based on surgery and drugs.The operation requires the surgeon to be experienced and familiar with the blood vessels around the sciatic nerve and its branches.The operation risk is relatively high.Drug treatment can choose oral contraceptives,GnRH-a,non-steroidal anti-inflammatory drugs and traditional Chinese medicine,but these treatments have shortcomings.Looking for a natural medicine that can not only inhibit inflammation and relieve pain,but also inhibit the growth of lesions,may have a profound impact on the treatment of endometriosis,especially sciatic nerve endometriosis.Sulforaphane(SFN)is a naturally occurring isothiocyanate in cruciferous vegetables and has been proven to have anti-diabetic,anti-cancer and antibacterial effects.Recently,it has been discovered that sulforaphane has a good anti-inflammatory effect.For example,sulforaphane has been shown to inhibit bacterial lipopolysaccharide-induced inflammation,which is characterized by increasing the expression of TNF-α,IL-1β,inducible nitric oxide synthase(iNOS)and prostaglandin peroxidase synthase 2(COX-2),so as to play a neuroprotective effect.Sulforaphane can also prevent renal fibrosis and ischemia-reperfusion injury by regulating inflammation.In addition,another important application of sulforaphane is to relieve pain.However,despite the potential of sulforaphane in reducing inflammation and pain,there have been no reports of sulforaphane in the treatment of sciatic nerve endometriosisPurposes:This study established the rat sciatic nerve endometriosis model,observed the effect of Sulforaphane on the pain of sciatic nerve endometriosis in three aspects:the volume of the lesion,the pain response and the inflammatory response,and further explored the analgesic mechanism of Sulforaphane in the model of sciatic nerve endometriosisMethods:The sciatic nerve endometriosis(SNEM)model was established,and the experimental rats were randomly divided into sham operation group(Sham group),endometriosis control group(ENDO group),and sulforaphane drug treatment group(ENDO+SFN group)with 6 animals in each group.The sciatic nerve endometriosis model was established as in the first part of the study.After the administration of sulforaphane 5,15,30,60 mg/kg,the changes of pain threshold(PWT)caused by mechanical stimulation and pain threshold(PWL)caused by thermal stimulation were detected.And the optimal concentration of sulforaphane was selected for further study,and then perform the following tests in the Sham group,ENDO group,and ENDO+SFN group.1.Von Frey Fiber Test and Thermal Pain Sensitivity Test were used to determine the pain threshold of mechanical stimulation(PWT)and the pain threshold of thermal stimulation(PWL)2.ELISA and Western blot were used to detect the VEGF expression in the lesion area of rats in Sham,ENDO,ENDO+SFN groups.3.ELISA and qPCR were used to detect the expression of inflammatory cytokines IL-6,IL-1β,TNF-α protein and mRNA in the superficial spinal dorsal horn of rats in the Sham,ENDO,ENDO+SFN groups4.qPCR and western blot were used to detect the expression of iNOS,COX-2 protein and mRNA in the superficial spinal dorsal horn of rats in the Sham,ENDO,ENDO+SFN groups.5.Western blot were used to detect the expression of Keap1 and Nrf2 in the superficial tissues of the spinal dorsal horn in the Sham,ENDO,ENDO+SFN groupsResults:1.Sulforaphane can significantly improve the hyperalgesia of sciatic nerve endometriosisBefore modeling,the baseline pain levels of the three groups of rats were the same;in the ENDO+SFN group,the test on the 7th day showed that the hyperalgesia caused by mechanical stimulation and the hyperalgesia caused by thermal stimulation were improved compared with the ENDO group(mechanical hyperalgesia ENDO+SFN vs ENDO:1 1.4±2.1 vs 7.2±1.7,p<0.01;thermal hyperalgesia ENDO+SFN vs ENDO:6.4±1.1 vs 3.2±1.7,p<0.01),ENDO+SFN group,the 14th day test showed mechanical stimulation Hyperalgesia caused by hyperalgesia and thermal stimulation was better than ENDO group(mechanical hyperalgesia ENDO+SFN vs ENDO:10.8±2.0 vs 5.7±1.9,p<0.01;thermal hyperalgesia ENDO+SFN vs ENDO:6.8±1.3 vs 2.7±1.9,p<0.01),ENDO+SFN group,the test on the 21st day showed that mechanical irritation caused hyperalgesia and thermal irritation-induced hyperalgesia were improved compared with ENDO group(mechanical hyperalgesia ENDO+SFN vs ENDO:14.7±2.1 vs 6.4±1.6,p<0.01;thermal hyperalgesia ENDO+SFN vs ENDO 9.7±1.7 vs 4.6±1.4,p<0.01),which lasts until 28 days after surgery.With the continuous injection of the drug,this effect is continuously improved,Compared with ENDO group,there are statistical differences(mechanical hyperalgesia ENDO+SFN vs ENDO:16.2±2.3 vs 6.6±1.4,p<0.01;thermal hyperalgesia ENDO+SFN vs ENDO:11.2±2.0 vs 4.6±.4,p<0.01)..2.Dose effect of sulforaphane in improving hyperalgesia of sciatic nerve endometriosisThe model group was given different concentrations of sulforaphane intervention(5,15,30,60mg/kg).The results showed that compared with the model group,the mechanical hypersensitivity and thermal hyperalgesia decreased by sulforaphane with 15mg/kg,30mg/kg,60mg/kg were administered on the 7,14,21,and 28 days,the thresholds of these groups were significantly improved,with significant differences(all p<0.05);in the range of 15-30mg/kg,the therapeutic effect was dose-dependent with the drug concentration.However,the pain tolerance of the two groups with the drug concentration of 30mg/kg and 50mg/kg had no significant difference(p>0.05).Therefore,the intervention concentration of sulforaphane in the following studies of this project was set at 30mg/kg.3.Sulforaphane inhibits the growth of ectopic endometrial tissue in SNEM animals by down-regulating VEGFIn the model animals of sciatic nerve endometriosis,the volume of endometrial tissue around the sciatic nerve increased,and sulforaphane administration had a therapeutic effect(ENDO+SFN group vs Sham Group:140.2±30 vs 42.6±20 mm3,p<0.01),and the volume decreased by more than 2/3.At the same time,the levels of VEGF protein in the serum and the lesion area of the ENDO group were significantly increased,the serum VEGF protein level in ENDO group was 8 times higher than that in Sham group(ENDO vs Sham:185.3±42.0 vs 23.6±12.8 pg/ml,p<0.001);the level of VEGF protein in the serum of ENDO+SFN group was lower than that of ENDO group(ENDO+SFN vs ENDO:185.3±42.0 vs 69.8±33.0 pg/ml,p<0.01),and recovered to 3 times the level of Sham group,compared with ENDO group Reduced by 2/3.The expression of VEGF protein in the lesion area also showed similar changes.Compared with the Sham group,the ENDO group increased by 5 times(ENDO vs 347.6±67.9 vs Sham:58.9±27.4 pg/ml,p<0.001);the VEGF protein level in the lesion area in the ENDO+SFN group was lower than that in the ENDO group(ENDO+SFN vs ENDO:156.3±49 vs 347.6±67.9 pg/ml,p<0.001),and recovered to 2.5 times the level of the Sham group,compared to ENDO group The group has decreased by more than 50%4.Sulforaphane inhibits inflammation in endometriosis of sciatic nerveIn the superficial layer of spinal cord(L4-6)near sciatic nerve,IL-6 protein expression in the superficial area of the dorsal horn of the spinal cord in the ENDO group was higher than that in the Sham group(ENDO vs Sham:243.7±46.8 vs 74.7±17.9 pg/mg,p<0.01),the expression of IL-6 protein in the ENDO+SFN group was lower than that in the ENDO group(ENDO+SFN vs ENDO:138.3±37 vs 243.7±46.8 pg/mg,p<0.05).The expression of IL-1β protein in the superficial region of the spinal dorsal horn in the ENDO group was higher than that in the Sham group(ENDO vs Sham:154.7±34.1 vs 46.3 ±2.9 pg/mg,p<0.01),the expression of IL-1β protein in the ENDO+SFN group was lower than that in the ENDO group(ENDO+SFN vs ENDO:71.3±21.2 vs 154.7±34.1 pg/mg,p<0.01).The expression of TNF-α protein in the superficial area of the spinal dorsal horn in the ENDO group was higher than that in the Sham group(ENDO vs Sham:197.4±38.3 vs 86.9±19.9 pg/mg,p<0.01),the expression of TNF-α in the ENDO+SFN group was lower than that in the ENDO group(ENDO+SFN vs ENDO:141.1±28.4 vs 197.4±38.3 pg/mg,p<0.05).IL-6,IL-1β,and TNF-α mRNA in ENDO group were significantly higher than those in Sham group,which was consistent with the changes in protein levels detected by ELISA.IL-6 was 3.24 times higher,p<0.001;IL-1β was 2.68 times higher,p<0.01;TNF-αwas 3.26 times higher,p<0.001.The expression of inflammatory factors in ENDO+SFN group was lower than that in ENDO group(IL-6 ENDO+SFN vs ENDO group:1.78±0.5 vs 3.24±0.69,p<0.01;IL-1β ENDO+SFN vs ENDO:1.53±0.47 vs 2.68±0.62,p<0.01;TNF-α ENDO+SFN vs ENDO:2.23±0.45 vs 3.26±0.64,p<0.05).5.The COX2 and iNOS expression can be inhibited by sulforaphaneThe mRNA level of COX-2 was significantly increased in ENDO group(3.64 ±0.76),which was 3.6 times higher than that in Sham group(p<0.01),The mRNA expression level of COX-2 in ENDO+SFN group was significantly down regulated(ENDO+SFN vs ENDO:1.97 ±0.44 vs 3.64 ± 0.76,p<0.01)to about 50%of that in ENDO group;the protein level of COX-2 was similar to that in mRNA level,that is,the protein level of COX-2 in ENDO group was more than 3 times higher than that in sham group(3.23±0.63pg/ml),p<0.001;the protein level of COX-2 in ENDO+SFN group was significantly reduced to about half of that in ENDO group(ENDO+SFN vs ENDO:1.79±0.4 vs 3.23±0.63pg/ml,p<0.01).It was found that the mRNA(2.53±0.54)and protein(2.86±0.57 pg/ml)levels of iNOS in ENDO group were 2.5 times higher than those in control group(p<0.01).The mRNA and protein levels of iNOS in ENDO+SFN group were down regulated compared with EDNDO group(mRNA ENDO+SFN vs ENDO:1.84±0.39 vs 2.53±0.54,p<0.05;protein ENDO+SFN vs ENDO 1.98±0.42 vs 2.86±0.57pg/ml,p<0.05),the down-regulation was about 1/3 of the level of ENDO group.6.Keapl/Nrf2 signaling pathway is inhibited in sciatic nerve endometriosis and can be significantly activated by sulforaphaneIn the superficial tissues of the dorsal horn of the spinal cord(L4-6)near the sciatic nerve,it was found that the expression of Keap1 protein was significantly reduced than that in the sham group(ENDO vs Sham:0.32±0.16 vs 1±0.18pg/ml,p<0.01),which was about 30%of the control group.The protein level of Keap1 in ENDO+SFN group was significantly increased than that in ENDO group(ENDO+SFN vs ENDO:0.58±0.19 vs 0.32±0.16pg/ml,p<0.05),which was 1-fold higher than that in ENDO group,reaching 60%of the sham group.The protein level of Nrf2 in ENDO group was significantly lower than that in Sham group(ENDO vs Sham:0.42 ± 0.17 vsl ± 0.2pg/ml,p<0.01),which was about 40%of the level of Sham group.The protein level of Nrf2 in ENDO+SFN group was higher than that in ENDO group(ENDO+SFN vs ENDO:1.67±0.3 vs 0.42±0.17pg/ml,p<0.001),the expression level increased 3 times compared with ENDO group,and reached 1.6 times of Sham group.Conclusions:1.Sulforaphane can treat the pain caused by endometriosis of the sciatic nerve through both anti-inflammatory and analgesic aspects;2.Sulforaphane can inhibit the growth of ectopic endometrial tissue in sciatic nerve endometriosis through VEGF;3.Sulforaphanean can effectively inhibit the expression of IL-6,IL-1β,TNF-α,COX2,iNOS,and relieve the pain of sciatic nerve endometriosis;4.Keap1/Nrf2 signaling pathway is inhibited in sciatic nerve endometriosis,sulforaphane can effectively promote the activation of this signaling pathway.更多还原