Study on Serum Markers of Intestinal Barrier Damage in Heroin Addicts at Different Withdrawal Times
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摘要:
目的 分析不同戒断时间内肠道屏障损伤标志物的变化,研究海洛因戒断后肠屏障损伤的机制,为海洛因戒断后精神症状改变和肠屏障之间的关系提供新的方向。 方法 招募14名海洛因戒断1周(HWS1W),24名海洛因戒断3月(HWS3M)的人群和26名健康者为对照组(HCs),用ELISA法测定血清iFABP、D-LDH、DAO、LBP、Zonulin和BDNF。 结果 HWS1W组的血清D-LDH、DAO、LBP、Zonulin和BDNF水平均明显高于HCs组(P < 0.001)。HWS3M组的血清iFABP、D-LDH、DAO和BDNF明显高于HCs组(P < 0.001)。DAO在海洛因戒断1周时明显升高,3个月时明显降低,但仍高于健康对照组(P < 0.001)。iFABP在急性戒断期(1周)没有变化,但在戒断的3个月左右增加。LBP和Zonulin在海洛因戒断组约1周内明显增加,但在HWS3M组和HCs组之间差异无统计学意义(P > 0.05)。HWS1W组和HWS3M组的D-LDH和BDNF的水平明显增加(P < 0.001)。 结论 海洛因戒断1周和3个月均发生肠屏障功能损伤。戒断1周后,肠屏障损伤可能与肠道菌群紊乱、紧密连接蛋白变化、肠道炎症和肠上皮完整性损伤有关。戒断3个月后,肠屏障损伤更可能是肠屏障上皮持续损伤造成的。 Abstract:Objective To investigate the mechanisms of intestinal barrier damage after heroin withdrawal by analyzing changes in markers of intestinal barrier damage at different withdrawal times, providing a new direction for the relationship between altered psychiatric symptoms and the intestinal barrier after heroin withdrawal. Methods 14 1-week heroin withdrawal subjects (HWS1W), 24 3-months heroin withdrawal subjects (HWS3M) and 26 healthy controls (HCs) were recruited, and serum iFABP, D-LDH, iFABP, DAO, LBP, Zonulin, BDNF and D-LDH were determined by ELISA. Results Serum D-LDH, DAO, LBP, Zonulin and BDNF levels in HWS1W group were significantly higher than those in HCs (P < 0.001). Serum iFABP, D-LDH, DAO, and BDNF in HWS3M group were significantly higher than those in HCs group (P < 0.001). DAO increased significantly at 1 week of heroin withdrawal and decreased significantly at 3 months, but was still higher than the healthy control group (P < 0.001). iFABP showed no change in the acute withdrawal period (1 week), but increased at about 3 months of withdrawal. LBP and Zonulin increased significantly in the heroin abstention group for about one week, but there was no significant difference between the HWS3M group and the HCs group (P > 0.05). The levels of D-LDH and BDNF were significantly increased in HWS1W and HWS3M groups (P < 0.001). Conclusions The present study showed that intestinal barrier dysfunction occurred both 1 week and 3 months after heroin withdrawal. After 1 week of abstinence, intestinal barrier damage may be associated with intestinal flora disorder, changes in tight junction proteins, intestinal inflammation and intestinal epithelial integrity damage. After 3 months of abstinence, intestinal barrier damage is more likely to result from persistent damage to the intestinal barrier epithelium. -
海洛因对人的身体和精神健康极为有害[1]。 长期吸食和注射海洛因会导致一些精神问题,缩短寿命,并会损伤整个人体的各个系统,尤其是对神经系统的伤害最为明显[2]。近年来,关于肠道屏障和海洛因滥用之间的相互作用导致精神状态改变的研究也受到关注[3]。
肠道屏障是一个动态的、具有渗透性的物理和免疫保护屏障的复合体。越来越多的证据表明肠道屏障功能的缺陷与胃肠道疾病有关[4]。肠道屏障在免疫调节中也起着很大的作用[5]。有研究表明[3],海洛因成瘾者在戒断期间身体质量指数(body mass index,BMI)下降,血清中的D-乳酸脱氢酶(D-Lactate dehydrogenase,D-LDH)、内毒素和二胺氧化酶(diamine oxidase,DAO)、脂多糖结合蛋白(lipopolysaccharide binding protein,LBP)水平异常,表明有潜在的肠道损害。
肠道屏障变化水平的检测:肠道通透性可以通过测量血清连蛋白Zonulin[6-7]和肠道脂肪酸结合蛋白(intestinal fatty acid binding protein,I-FABP)来测量。Zonulin是1种47 kDa的蛋白,是参与大分子运输的细胞间紧密连接的生理调节器[8]。Zonulin会导致紧密连接的解体,因此被用作肠道屏障损伤的生物标志物。已经观察到Zonulin参与了肠道先天免疫,并在一些自身免疫性疾病中上调[9]。在炎症性肠病 [10]、1型糖尿病[11]和肥胖症患者[12] 中血清Zonulin升高。肠道完整性的另一个潜在标志物是IFABP,也被称为FABP2。这种细胞质蛋白存在于小肠的肠细胞中,水平升高表明肠细胞受损。IFABP调节脂肪酸的代谢,只在肠道上皮细胞中表达。在正常情况下,循环的IFABP浓度处于低水平。当肠道上皮细胞受损时,IFABP被释放到循环中[13]。另外有研究表明[14],脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)在炎症存在的情况下在肠粘膜中显著上调,并通过影响紧密连接蛋白来调节肠道屏障的完整性。 尽管BDNF是一种神经营养因子,但它在一些发育和成熟的外周组织中表达,包括胃肠道[15]。
本研究中,血清中的iFABP、D-LDH、DAO、LBP、Zonulin和BDNF被用来评估海洛因成瘾和健康对照组的肠道屏障功能。海洛因成瘾组分为戒断海洛因1周和3个月。
1. 资料与方法
1.1 受试者资料
所有纳入的受试者都给予书面的知情同意参与,研究得到了昆明医科大学医学伦理委员会的批准((2020)伦审L第34号)。实验组分为海洛因戒断1周组(HWS1W组)和海洛因戒断3个月组(HWS3M组),共2个实验组。实验组入组标准[16]:(1)男性;(2)年龄24~60岁;(3)患者签署知情同意书;(4)有海洛因滥用史,吸毒年限≥5 a。排除标准[16]:(1)既往有颅脑外伤史、脑血管病史、癫痫或严重精神疾病史;(2)最近6个月使用过促进认知的药物;(3)有过其他物质滥用或依赖史(尼古丁除外);(4)有肠道疾病史。正常对照组受试者在昆明地区社会招募,入组标准:(1)男性;(2)年龄24~60岁;(3)患者签署知情同意书。排除标准:(1)既往有颅脑外伤史、脑血管病史、癫痫或严重精神疾病史;(2)最近6个月使用过促进认知的药物;(3)有物质滥用或依赖史(尼古丁除外);(4)有肠道疾病史。HWS1W受试者(n = 14)为2020年12月至2021年4月间从云南省第六强戒毒所招募,戒断海洛因1周。HWS3M组(n = 24)为2020年12月至2021年4月期间从云南省昆明医科大学第一附属医院入院后招募,持续3个月海洛因戒断。HCs组(n = 26)为2020年12月至2021年4月之间招募。从昆明医科大学的体检中心随机选择。经病史、血常规筛查和体检确定,身体健康,无精神障碍史。研究人群的特征,见表1。
表 1 HWS1W、HWS3M、HCs 3组基本情况比较($\bar x \pm s $ )Table 1. Comparison of the three groups of basic conditions ($\bar x \pm s $ )基本情况 HWS1W(n = 14) HWS3M(n = 24) HCs(n = 26) F/t P 年龄(岁) 35.6 ± 6.0 45.7 ± 6.4▲# 36.7 ± 12.3 7.535 0.001* 吸毒时间(a) 14.6 ± 4.3 17.4 ± 5.8 − 1.570 0.125 BMI(kg/m2) 20.3 ± 2.1△☆ 22.6 ± 2.4 23.1 ± 3.2 4.952 0.010* HWS1W:海洛因成瘾戒断1周组;HWS3M:海洛因成瘾戒断3月组;HCs:健康对照组;BMI:身体质量指数。*P < 0.05 ;与HWS1W组比较,▲P < 0.05;与HCS组比较,#P < 0.05;与HWS3M组比较,△P < 0.05;与HWS3M组比较,☆P < 0.05。 1.2 样本处理
血液样本收集在EDTA真空管中。采集后1 h内立即置于冰上,在4℃和2000×g下离心10 min,并在-80℃下保存至分析[平均在采集后(4±2月)]。
1.3 检测方法
LBP、DAO、Zonulin、D-LDH、iFABP和BDNF使用市面上的检测试剂盒(酶联免疫吸附法:ELISA)进行检测。所有的分析都是由受过训练的操作人员在同一实验室进行。
1.4 统计学处理
采用SPSS统计软件(IBM,SPSS Statistics 23)进行统计分析。正态分布计量资料以平均值±标准差(
$ \bar x \pm s $ )表示。两独立样本所属总体方差相等采用t检验。3组或更多的连续变量使用单因素方差分析。皮尔逊相关法用于检查2个变量之间的相关性。P < 0.05认为差异具有统计学意义。2. 结果
2.1 研究参与者的特征
该研究招募了38名海洛因成瘾者和26名健康对照者。3组的基本情况,见表1。HWS3M组的年龄高于健康对照组和HWS1W组(P < 0.05)。HWS1W组和HWS3M组在吸毒年限上差异无统计学意义(P > 0.05),HWS1W组的BMI低于其他2组(P < 0.05)。
2.2 海洛因成瘾者在戒断后1周和3个月时肠道屏障功能的血清标志物变化
HWS1W组的血清LBP、Zonulin、D-LDH、DAO和BDNF水平明显高于对照组(P < 0.05),见图1A-1E。HWS3M组的血清D-LDH、DAO、BDNF和iFABP都明显高于对照组(P < 0.05),见图1 C-1F。在HWS1W组,血清Zonulin与LBP呈正相关(r = 0.583,P = 0.029)。所有的血清差异性标志物与年龄、BMI或病程没有相关性,见表2。在HWS3M组,血清DAO与D-LDH呈正相关(r = 0.453,P = 0.03),iFABP与海洛因使用年限呈正相关(r = 0.537,P = 0.008),见表3。
表 2 HWS1W患者肠屏障损伤血清标志物的相关性及其与HWS1W基本情况的相关性[r(P)]Table 2. Correlation of serum markers of intestinal barrier injury in Group HWS1W,Correlation between serum markers of intestinal barrier injury and basic conditions in Group HWS1W [r(P)]项目 D-LDH BDNF DAO LBP Zonulin 年龄 −0.346(0.225) −0.028(0.925) −0.209(0.474) −0.1(0.734) 0.002(0.995) 吸毒时间 −0.332(0.247) −0.339(0.236) −0.17(0.561) −0.014(0.961) −0.297(0.303) BMI 0.101(0.731) 0.275(0.342) 0.208(0.475) 0.096(0.743) 0.123(0.675) D-LDH 1(0.000*) −0.358(0.208) 0.476(0.085) −0.235(0.419) −0.085(0.773) BDNF −0.358(0.208) 1(0.000*) −0.431(0.124) 0.38(0.180) 0.195(0.503) DAO 0.476(0.085) −0.431(0.124) 1(0.000*) −0.356(0.212) 0.279(0.335) LBP −0.235(0.419) 0.38(0.180) −0.356(0.212) 1(0.000*) −0.583(0.029*) Zonulin −0.085(0.773) 0.195(0.503) 0.279(0.335) −0.583(0.029*) 1(0.000*) *P < 0.05。 表 3 HWS3M患者肠屏障损伤血清标志物的相关性及其与HWS3M人体测量数据的相关性[r(P)]Table 3. Correlation of serum markers of intestinal barrier injury in Group HWS3M;Correlation between serum markers of intestinal barrier injury and basic conditions in Group HWS3M [r(P)]项目 iFABP D-LDH DAO BDNF 年龄 −0.007(0.976) −0.179(0.414) −0.174(0.426) −0.384(0.070) 吸毒时间 −0.537(0.008*) 0.236(0.277) 0.065(0.770) −0.009(0.969) BMI −0.191(0.383) 0.039(0.860) −0.209(0.339) −0.016(0.942) iFABP 1(0.000*) −0.151(0.492) −0.029(0.896) 0.012(0.955) D-LDH −0.151(0.492) 1(0.000*) 0.453(0.030*) −0.322(0.134) DAO −0.029(0.896) 0.453(0.030*) 1(0.000*) −0.149(0.497) BDNF 0.012(0.955) −0.322(0.134) −0.149(0.497) 1(0.000*) *P < 0.05。 2.3 海洛因戒断的持续时间和肠道屏障功能
根据戒断的时间分析了肠道屏障损伤的标志物。LBP和Zonulin在海洛因戒断1周时与对照组相比明显升高(P < 0.001),3个月后下降到健康对照组的正常水平。D-LDH和BDNF在海洛因戒断1周和3个月时与对照组相比明显增加(P < 0.001)。DAO在海洛因戒断1周时与对照组相比明显升高(P < 0.001),3个月时明显降低,但仍高于健康对照组(P < 0.001)。iFABP在海洛因戒断1周时与对照组相比差异无统计学意义(P > 0.05),但在海洛因戒断3个月时与对照组相比明显升高(P < 0.001),见图1。这里不能排除年龄的因素,因为HWS3M组的年龄与其他2组相比差异有统计学意义(P < 0.05),见表1。
3. 讨论
海洛因成瘾被认为是强迫性药物使用的一种行为状态,在戒毒一段时间后极易复发。海洛因是一种非法的、极易上瘾的毒品。它是阿片类药物中滥用最严重的,是作用最迅速的。反复服用大剂量海洛因会导致身体依赖。 肠屏障和精神疾病之间关联研究具有重要意义。肠屏障是一种动态、可渗透的物理和免疫防御屏障,由肠上皮细胞以及其分泌物和肠上皮下的大量免疫细胞组成。其通过防止抗原分子和微生物随意转移来维持体内平衡。关于胃肠道疾病与精神疾病之间的联系越来越多研究表明胃肠道疾病和精神疾病之间具有共同遗传基础。
前期研究表明[3],海洛因成瘾者的肠道屏障功能存在受损。肠屏障损伤后,肠通透性增加会导致促炎因子的释放[17-19]和慢性全身性炎症,从而可能进一步加重炎症因子对人精神状态和异常行为的调节[20]。例如,焦虑和抑郁患者肠道屏障血清标志物发生改变并与疾病症状呈正相关[21],肠道微生物变化等一些变化也与精神障碍密切相关[22-24]。例如;肠道微生态系统可通过浸润炎性细胞因子影响脑屏障和小胶质细胞神经元状态[25]。浸润性炎症细胞因子、小胶质细胞和星形胶质细胞可能相互激活,驱动慢性炎症并阻止血脑屏障修复。细胞周围损伤和神经元丢失进一步促进认知能力下降[26-27]。因此,深入研究肠道屏障损伤与海洛因滥用者之间的关系,将为今后的海洛因成瘾后精神症状改变研究提供新的方向。对海洛因滥用者肠道屏障功能损害的研究是目前较为有特色的课题。
笔者用血清标记物测量了海洛因滥用者的肠屏障功能。目前认为LBP是由于内毒素进入体内后,LBP作为载体将LPS转运到mCD14或sCD14,进而作用于巨核细胞合成并分泌大量促炎因子和负性炎症因子。高水平的LBP与肠道通透性增加和全身炎症反应的激活有关,这可能是海洛因滥用者在戒断期间更容易感到焦虑的原因[28]。在耐受/免疫反应平衡中,Zonulin是迄今为止唯一参与大分子运输的紧密细胞间连接的生理调节剂。因此,当在遗传易感个体中调节良好的Zonulin蛋白通路失调时,肠道和外部自身免疫、炎症和肿瘤疾病都可能发生[29]。d -乳酸是由胃肠道的原生细菌产生的,人类没有快速代谢它的酶系统。因此,d -乳酸水平升高可能反映了肠黏膜屏障功能异常。血浆中DAO含量的变化是坏死的肠粘膜细胞脱落到肠腔内,DAO进入淋巴血管和肠细胞间隙血流,使DAO含量增加。血DAO含量的升高在一定程度上反映了肠黏膜屏障功能的改善。肠屏障的另一个标记物BDNF被认为是认知能力下降和阿尔茨海默病之间的可能联系,也是海洛因滥用者认知能力下降的可能原因之一[30]。血清iFABP能反映肠上皮细胞的完整性。戒断海洛因约1周的患者iFABP没有变化,这可能是由于研究中的年龄差异。HWS3M组年龄明显高于其他组,iFABP与吸毒持续时间(a)呈正相关。药物滥用时间越长,iFABP水平越高。在本研究中,这些物质被用作与肠道菌群、紧密连接、肠道炎症和肠上皮完整性相关的肠屏障损伤的标志物。
Zonulin和LBP在HWS1W组中均显著升高且呈正相关,这可能是由于海洛因戒断引起的肠道微生物组的变化。然而,HWS3M组的Zonulin和LBP水平与健康对照组一致,这表明在海洛因戒断3个月后,肠道微生物群水平可能会自动调整回正常水平。但肠上皮屏障的损伤仍然存在。
综上所述,本研究表明海洛因戒断1周和3个月均发生了肠屏障功能损伤。戒断1周后,肠屏障损伤可能与肠道菌群、紧密连接、肠道炎症和肠上皮完整性有关。戒断3个月后,肠屏障损伤更可能是肠屏障上皮损伤。这些结果明确了在海洛因戒断短时间内肠道屏障损伤与多种因素有关,这有可能是肠屏障损伤的潜在机制。随着戒断时间增加,肠屏障损伤标志物发生了变化,这表明其肠屏障损伤机制发生了变化。总之,这些结果提示了在海洛因戒断时间不同时,肠屏障损伤机制不同,为海洛因依赖患者肠屏障损伤机制研究提供了新的思路,为海洛因戒断后精神症状改变和肠屏障之间的关系提供了新的方向。
综上所述,本研究表明海洛因戒断1周和3个月均发生了肠屏障功能损伤。戒断1周后,肠屏障损伤可能与肠道菌群、紧密连接、肠道炎症和肠上皮完整性有关。戒断3个月后,肠屏障损伤更可能是肠屏障上皮损伤。这些结果明确了在海洛因戒断短时间内肠道屏障损伤与多种因素有关,这有可能是肠屏障损伤的潜在机制。随着戒断时间增加,肠屏障损伤标志物发生了变化,这表明其肠屏障损伤机制发生了变化。总之,这些结果提示了在海洛因戒断时间不同时,肠屏障损伤机制不同,为海洛因依赖患者肠屏障损伤机制研究提供了新的思路,为海洛因戒断后精神症状改变和肠屏障之间的关系提供了新的方向。
基于海洛因成瘾者此特殊人群受限,本研究采用了横断面数据收集,有一定的局限性。以上局限性将在后续纵向研究中进一步改进。
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表 1 HWS1W、HWS3M、HCs 3组基本情况比较(
$\bar x \pm s $ )Table 1. Comparison of the three groups of basic conditions (
$\bar x \pm s $ )基本情况 HWS1W(n = 14) HWS3M(n = 24) HCs(n = 26) F/t P 年龄(岁) 35.6 ± 6.0 45.7 ± 6.4▲# 36.7 ± 12.3 7.535 0.001* 吸毒时间(a) 14.6 ± 4.3 17.4 ± 5.8 − 1.570 0.125 BMI(kg/m2) 20.3 ± 2.1△☆ 22.6 ± 2.4 23.1 ± 3.2 4.952 0.010* HWS1W:海洛因成瘾戒断1周组;HWS3M:海洛因成瘾戒断3月组;HCs:健康对照组;BMI:身体质量指数。*P < 0.05 ;与HWS1W组比较,▲P < 0.05;与HCS组比较,#P < 0.05;与HWS3M组比较,△P < 0.05;与HWS3M组比较,☆P < 0.05。 表 2 HWS1W患者肠屏障损伤血清标志物的相关性及其与HWS1W基本情况的相关性[r(P)]
Table 2. Correlation of serum markers of intestinal barrier injury in Group HWS1W,Correlation between serum markers of intestinal barrier injury and basic conditions in Group HWS1W [r(P)]
项目 D-LDH BDNF DAO LBP Zonulin 年龄 −0.346(0.225) −0.028(0.925) −0.209(0.474) −0.1(0.734) 0.002(0.995) 吸毒时间 −0.332(0.247) −0.339(0.236) −0.17(0.561) −0.014(0.961) −0.297(0.303) BMI 0.101(0.731) 0.275(0.342) 0.208(0.475) 0.096(0.743) 0.123(0.675) D-LDH 1(0.000*) −0.358(0.208) 0.476(0.085) −0.235(0.419) −0.085(0.773) BDNF −0.358(0.208) 1(0.000*) −0.431(0.124) 0.38(0.180) 0.195(0.503) DAO 0.476(0.085) −0.431(0.124) 1(0.000*) −0.356(0.212) 0.279(0.335) LBP −0.235(0.419) 0.38(0.180) −0.356(0.212) 1(0.000*) −0.583(0.029*) Zonulin −0.085(0.773) 0.195(0.503) 0.279(0.335) −0.583(0.029*) 1(0.000*) *P < 0.05。 表 3 HWS3M患者肠屏障损伤血清标志物的相关性及其与HWS3M人体测量数据的相关性[r(P)]
Table 3. Correlation of serum markers of intestinal barrier injury in Group HWS3M;Correlation between serum markers of intestinal barrier injury and basic conditions in Group HWS3M [r(P)]
项目 iFABP D-LDH DAO BDNF 年龄 −0.007(0.976) −0.179(0.414) −0.174(0.426) −0.384(0.070) 吸毒时间 −0.537(0.008*) 0.236(0.277) 0.065(0.770) −0.009(0.969) BMI −0.191(0.383) 0.039(0.860) −0.209(0.339) −0.016(0.942) iFABP 1(0.000*) −0.151(0.492) −0.029(0.896) 0.012(0.955) D-LDH −0.151(0.492) 1(0.000*) 0.453(0.030*) −0.322(0.134) DAO −0.029(0.896) 0.453(0.030*) 1(0.000*) −0.149(0.497) BDNF 0.012(0.955) −0.322(0.134) −0.149(0.497) 1(0.000*) *P < 0.05。 -
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