图1 小鼠束缚后放入自创固定装置
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To observe the effect of electric acupuncture (EA) on promoting the repair of gastric mucosal injury in mice with stress ulcer, and explore the correlation between repair effect of EA and promoting repair factor release and up-regulating hypoxia-inducible factor-1α (HIF-1α) expression.
Male C57BL/6 mice were randomly divided into control group (Sham group), model group (CRS group) and model+EA group (CRS+EA group), with 9 mice in each group. Except for the Sham group, the stress ulcer model (CRS model) was established in the CRS and CRS+EA groups after 3 days of cold-restraint stress, and EA intervention was performed 3 d after modeling. In the CRS+EA group, EA was applied to Zusanli (ST 36) and Liangqiu (ST 34) for 15 min, once in the morning and once in the afternoon, for 2 d. On day 6 of the experiment, the whole stomachs were separated from mice and were sampled to assess the gastric mucosal ulcer index (UI) score. The mRNA expression levels of repair factors [including trefoil factor 2 (TFF2), vascular endothelial growth factor (VEGF) and heat shock protein 70 (HSP70)] were determined by RT-qPCR, and the protein content and mRNA expression level of HIF-1α were determined by Western blot and RT-qPCR.
①The CRS group and the CRS+EA group had significantly higher gastric mucosal UI score than the Sham group (P<0.05); and the CRS+EA group had significantly lower UI score than the CRS group (P<0.05). ②The CRS group had significantly higher levels of VEGF and HSP70 than the Sham group (P<0.05), and the CRS+EA group showed significantly higher levels of TFF2, VEGF and HSP70 than the Sham group (P<0.05). The CRS+EA group had significantly higher levels of TFF2, VEGF and HSP70 than the CRS group (P<0.05). ③There was no statistically significant difference in HIF-1α expression between the CRS group and the Sham group (P>0.05), while the CRS+EA group had significantly higher HIF-1α expression than the Sham group (P<0.05) and the CRS group (P<0.05).
Electric acupuncture promotes the expression of repair factors (TFF2, VEGF, HSP70) and facilitates the repair of injured gastric mucosa in mice with stress ulcer. The efficacy of EA may be associated with a significant upregulation of HIF-1α expression after treatment.
针刺作为中医学的重要治疗方法,已经普遍被国际主流医学界所认可和接纳,并广泛应用于临床各种疾病的预防和治疗。其中,电针在调理胃肠功能、促进胃动力恢复、治疗胃肠损伤等方面具有显著疗效[
前期研究[
1.1.1 动物
雄性SPF级C57BL/6小鼠27只,体质量22~25 g,购自北京维通利华实验动物技术有限公司。动物生产许可证号:SCXK(京)2016-0006。小鼠饲养于上海中医药大学实验动物中心。动物使用许可证号:SYXK(沪)2020-0009。小鼠自由摄食和饮水,控制饲养温度(21 ℃)和光照时间(上午7:00至晚上21:00)在稳定的条件下。动物实验方案经上海中医药大学实验动物伦理委员会批准(伦理批准号:2019-0014)。
1.1.2 药物与试剂
异氟烷,江苏恒瑞医药有限公司(批号:S190815);磷酸化蛋白酶抑制剂,武汉赛维尔生物科技有限公司(批号:G2007);二喹啉甲酸比色法(BCA)蛋白定量检测试剂盒,武汉赛维尔生物科技有限公司(批号:G2026);抗小鼠甘油醛-3-磷酸脱氢酶(GAPDH)单克隆抗体 ,武汉赛维尔生物科技有限公司(批号:GB12002);抗小鼠HIF-1α抗体,武汉赛维尔生物科技有限公司(批号:GB14165);RNA酶抑制剂,美国Invitrogen公司(批号:c10777000);cDNA反转录试剂盒(First Strand cDNA synthesis Kit),美国Invitrogen公司(批号:K1622);实时荧光定量PCR试剂盒(QuantityNova SYBR Green PCR Kit),德国QIAGEN公司(批号:208052);cDNA反转录随机引物,美国Invitrogen公司(批号:17504044)。
1.1.3 主要仪器
电子天平,德国Sartorius公司(型号:SecuraSQP);电针仪,苏州医疗用品厂有限公司(型号:SDZ-ⅡB);一次性无菌针灸针(规格:0.16 mm×7 mm),苏州天协针灸器械有限公司;动物麻醉机,上海玉研科技仪器有限公司(型号:ABS-100);梯度PCR仪,美国Thermo Fisher Scitific公司(型号:Veriti DX);荧光定量PCR仪,瑞士Roche公司(型号:Roche 480Ⅱ Real Time PCR System);凝胶成像系统,上海天能科技有限公司。
采用冷束缚应激(cold restraint stress,CRS)建立小鼠应激性溃疡模型,参照既往已形成的稳定的CRS小鼠模型建立方法[
图1 小鼠束缚后放入自创固定装置
CRS小鼠应激性溃疡模型稳定性检测:造模3 d后,小鼠呈现安静、萎靡不振、身体蜷缩、寒战等状态,为造模成功的行为学标准;体视镜下可见胃黏膜充血、水肿、全胃散在多发点状/片状糜烂及出血,胃黏膜溃疡指数(UI)评分5分以上,为造模成功的形态学标准。
在实验前16~20 h,小鼠禁止摄入食物,但可以自由饮水。将小鼠按体质量随机分成对照组(Sham组)、模型组(CRS组)及模型+电针组(CRS+EA组),每组9只。
3组小鼠干预方法如下。①Sham组:不进行冷束缚应激的造模和电针干预,其他与③相同;②CRS组:不进行电针干预,其他操作与③相同;③CRS+EA组:建立CRS的小鼠应激性溃疡模型,造模3 d后进行电针干预。
小鼠针灸穴位的定位标准均参考《实验针灸学》[
图2 鼠科动物“足三里”穴(ST36)、“梁丘”穴(ST34)与尾部非经穴图解[
注: ST36为小鼠“足三里”穴,位于膝关节下外侧,腓骨小头下3.5 mm处;ST34为“梁丘”穴,在膝关节上外方2 mm处,股直肌和股外侧肌之间,与外膝眼在一条直线上;Tail non-acupoint为尾巴根部非经非穴。
实验开始后第6天取材。每组中3只用于蛋白质免疫印迹(Western blot)及实时荧光定量逆转录聚合酶链式反应(RT-qPCR)检测,6只用于UI评分。小鼠吸入2%~3%的异氟烷麻醉气体,3~5 min后呈现麻醉状态(镊子夹起胡须无反应),置于操作台,迅速备皮,外科镊夹住皮肤轻提,外科剪沿正中线剪开腹部皮肤,钝性分离皮下、脂肪及肌肉组织,向上翻开大网膜,游离胃组织,沿胃体向上分离胃贲门部,并用缝线结扎,同样沿胃体向下探寻至胃幽门部并结扎。用一次性针筒斜刺入胃体内并注射4%中性甲醛溶液2 mL,两端结扎线向外上提起,顺势摘下小鼠全胃,浸没在20 mL的4%中性甲醛溶液中,固定大约30 min。轻提结扎线,沿着小鼠胃大弯侧全层剖开,暴露胃体内部,在操作板上展开全胃组织,头针作周边固定,用质量分数为0.9%的氯化钠溶液清洗胃内容物。观察胃黏膜的形态学变化,可见胃黏膜呈现多发的点状出血或数个溃疡灶。UI评分5分以上为造模成功的形态学标准。体视镜下可见胃黏膜充血、水肿,全胃散在多发点状/片状糜烂及出血或多个溃疡点,溃疡基底部可见白色或黄白色厚苔,其中个别溃疡点可深达肌层甚至穿孔(见
图3 小鼠造模3 d后胃黏膜形态学变化
电针治疗结束后取小鼠全胃,评估UI评分以及检测修复因子[三叶因子2(TFF2)、血管内皮生长因子(VEGF)、热休克蛋白70(HSP70)]和调控因子HIF-1α表达水平。
1.4.1 胃黏膜损伤修复的检测指标(UI评分)
电针治疗结束后取材小鼠全胃,评估UI评分。UI评分:参照GUTH法[
1.4.2 胃黏膜组织修复因子(TFF2、VEGF、HSP70)和调控因子HIF-1α的mRNA表达
采用RT-qPCR法检测胃组织TFF2、VEGF、HSP70及HIF-1α mRNA表达水平,取-80 ℃冻存的大鼠胃组织,以TRIzol 法提取结肠组织总RNA ,逆转录反应体系为20 μL ,根据试剂说明书操作,按条件进行逆转录。反应条件:40 ℃,30~60 min;95 ℃,5 min。扩增引物由生工生物工程(上海)股份有限公司合成,引物序列见
基因 | 引物序列 | 引物长度/bp |
---|---|---|
VEGF | 上游:5'-ATGAACTTTCTGCTCTCTTGGGTACA-3' | 26 |
下游:5'-GCAGATGTACAAGCCAAGGCGGTG-3' | 24 | |
HSP70 | 上游:5'-GAGGTCACCTTCGACATCGA-3' | 20 |
下游:5'-CTTGCCCGTGCTCTTGTC-3' | 18 | |
TFF2 | 上游:5'-CCCCCATAACAGGACGAAC-3' | 19 |
下游:5'-ATGAAGTTGGAGAAGCAGCAC-3' | 21 | |
HIF⁃1α | 上游:5'-GAAAGCGCAAGTCCTCAAAG-3' | 20 |
下游:5'-TGGGTAGGAGATGGAGATGC-3' | 20 | |
GAPDH | 上游:5'-GGCACAGTCAAGGCTGAGAATG-3' | 22 |
下游:5'-ATGGTGGTGAAGACGCCAGTA-3' | 21 |
注: VEGF为血管内皮生长因子基因,HSP70为热休克蛋白70基因,TFF2为三叶因子2基因,HIF⁃1α为低氧诱导因子⁃1α基因,GAPDH为甘油醛⁃3⁃磷酸脱氢酶。
1.4.3 HIF-1α的蛋白表达水平
采用Western blot法检测胃组织中HIF-1α蛋白表达。取全胃组织,用冷磷酸盐缓冲液(PBS)洗涤去污,切块置于匀浆器;加入组织后在-20 ℃冰上彻底匀浆,将匀浆液振荡;加入RIPA裂解液,冷浴20 min,然后反复吹打,确保细胞完全裂解;12 000 r/min离心10 min,收集上清为总蛋白溶液;再用BCA测蛋白浓度;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)处理45 min。转膜:准备滤纸和聚偏二氟乙烯(PVDF)膜,在垫子上垫海绵、两层滤纸,并除气泡、打湿,25 V恒压转膜过夜。免疫反应:5%的脱脂牛奶封闭1 h,4 ℃孵育一抗3 h,脱色摇床上洗3次,每次5 min;孵育二抗,30 min,脱色摇床上洗3次,每次5 min。化学发光:进行显影和定影,根据不同的光强度调整曝光条件,凝胶图像分析;胶片扫描,存档,整理去色,Alpha软件处理条带光密度值;以β-actin作为内参蛋白,重复3次。
实验数据采用SPSS 1.0软件进行统计分析。计量资料以ˉx±s表示。所有数据进行正态性检验,符合正态分布者,多组计量资料之间比较采用单因素方差分析,方差齐者用LSD和SNK法,方差不齐者用Tamhane's T2或Dunnett's T3法;不符合正态分布者采用多组资料的秩和检验。以P<0.05为差异有统计学意义。
与Sham组比较,CRS组、CRS+EA组UI评分显著升高(P<0.05)。与CRS组比较,CRS+EA组UI评分显著下降(P<0.05)。上述说明电针治疗可以促进胃黏膜损伤后的修复,减轻胃黏膜损伤。见
组别 | UI评分 |
---|---|
Sham组 | 0.67±0.33 |
CRS组 | 43.50±18.01* |
CRS+EA组 | 16.33±5.20*# |
注: Sham组为对照组,CRS组为模型组,CRS+EA组为模型+电针组。UI为溃疡指数。与Sham组比较,*P<0.05;与CRS组比较,#P<0.05。
图4 各组小鼠胃黏膜的形态学变化
注: Sham组为对照组,CRS组为模型组,CRS+EA组为模型+电针组。
与Sham组比较,CRS组VEGF、HSP70 mRNA显著升高(P<0.05),CRS组TFF2差异无统计学意义(P>0.05),CRS+EA组TFF2、VEGF、HSP70 mRNA显著升高(P<0.05)。与CRS组比较,CRS+EA组TFF2、VEGF、HSP70 mRNA显著升高(P<0.05)。上述说明电针治疗可以促进修复因子TFF2、VEGF、HSP70 mRNA分泌,可能与电针增强胃黏膜损伤后的修复能力有关。见
组别 | TFF2 | VEGF | HSP70 |
---|---|---|---|
Sham组 | 1.21±0.82 | 1.03±0.27 | 0.97±0.19 |
CRS组 | 2.08±0.74 | 2.33±0.52* | 2.28±0.45* |
CRS+EA组 | 5.23±1.39*# | 3.55±0.68*# | 3.90±0.82*# |
注: TFF2为三叶因子2基因,VEGF为血管内皮生长因子基因,HSP70为热休克蛋白70基因。Sham组为对照组,CRS组为模型组,CRS+EA组为模型+电针组。与Sham组比较,*P<0.05;与CRS组比较,#P<0.05。
与Sham组比较,CRS组HIF⁃1α mRNA及蛋白表达差异无统计学意义(P>0.05),CRS+EA组HIF⁃1α mRNA及蛋白表达显著升高(P<0.05)。与CRS组比较,CRS+EA组HIF⁃1α mRNA及蛋白表达显著升高(P<0.05)。上述说明电针能够上调HIF-1α mRNA及蛋白表达水平,增强胃黏膜损伤的修复。见
组别 | HIF⁃1α mRNA | HIF-1α 蛋白 |
---|---|---|
Sham组 | 1.01±0.11 | 1.00±0.01 |
CRS组 | 1.72±0.86 | 1.52±0.27 |
CRS+EA组 | 5.98±1.28*# | 4.07±0.48*# |
注: HIF⁃1α为低氧诱导因子⁃1α。Sham组为对照组,CRS组为模型组,CRS+EA组为模型+电针组。与Sham组比较,*P<0.05;与CRS组比较,#P<0.05。
图5 各组小鼠胃组织HIF-1a蛋白电泳条带
注: HIF-1α为低氧诱导因子-1α,β-actin为β肌动蛋白。Sham组为对照组,CRS组为模型组,CRS+EA组为模型+电针组。
创伤患者、重症颅脑疾病患者将面临应激性溃疡出血的风险,导致血流动力学剧烈波动,在重症监护室停留时间延长以及死亡风险增加[
足三里穴是足阳明胃经的合穴,它又是胃的下合穴,治疗胃痛、呕吐、腹胀、泄泻、便秘等胃肠系统疾病有明确的疗效。《灵枢·四时气》记载:“胃气逆则呕苦……取三里以下胃气。”明代徐凤所著《针灸大全》记载足三里穴“善治胃中寒”,而高度总结和概括该穴位主治作用的是《四总穴歌》中所述“肚腹三里留”。现代实验研究[
有研究[
研究[
本研究证实,电针促进应激性溃疡胃黏膜损伤的修复能力与HIF-1α的上调、以及修复因子(TFF2、VEGF、HSP70)的表达增强有关,但这种相关性是否具有因果关系,抑或电针是如何通过上游调控机制来上调HIF-1α的表达,促进修复因子的释放,增强应激性溃疡胃黏膜损伤修复,这些尚有待进一步的基础研究来证实。
综上所述,电针治疗可以促进应激性溃疡小鼠修复因子的释放,促进胃黏膜损伤后的修复,显著上调HIF-1α表达水平。本研究结果有助于推动电针应用于应激性溃疡的临床治疗,并为进一步优化针刺对低氧诱导因子为关键调控靶点的电针穴位治疗提供科学依据。
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