摘要
目的:观察艾灸“天枢”对克罗恩病(CD)小鼠肠道炎症损伤的影响,探讨肿瘤抑制因子p53调控溶质载体家族7成员11(SLC7A11)/谷胱甘肽(GSH)/谷胱甘肽过氧化物酶4(GPX4)轴对CD小鼠肠道铁死亡脂质过氧化的影响。方法:将50只SPF级C57BL/6野生型小鼠随机分为空白组10只、造模组40只。造模组采用蒸馏水配制成浓度为2%的葡聚糖硫酸钠(DSS)溶液制备CD小鼠模型。空白组随机选1只、造模组随机选4只进行模型比较评价,确认模型制备成功后,造模组分为模型组、艾灸组、激艾组、抑制剂组,每组9只,空白组9只。艾灸组小鼠以细艾条灸双侧“天枢”15 min,每天1次,连续干预14 d;激艾组艾灸干预方法同艾灸组,同时于“天枢”穴周围皮下注射激动剂RITA(10 mg/kg),每天1次,连续注射14 d;抑制剂组于“天枢”穴周围皮下注射抑制剂Pifithrin-α(2.2 mg/kg),每天1次,连续注射14 d。干预前与干预后,观察各组小鼠一般情况、体质量和疾病活动指数(DAI)评分。干预后,HE染色法观察小鼠结肠组织形态;ELISA法检测小鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-17和IL-6含量,以及小鼠结肠组织4-羟基壬烯酸(4-HNE)、丙二醛(MDA)、GSH、超氧化物歧化酶(SOD)和亚铁离子(Fe^(2+))含量;Western blot法检测小鼠结肠组织p53、SLC7A11、GPX4蛋白表达。结果:与空白组比较,模型组小鼠毛发干枯,蜷缩聚集,反应迟钝,摄食量下降,可见笼盒内及小鼠肛周出现黏液脓血,大便疏松呈糊状,粪便隐血试验呈阳性,小鼠体质量降低(P<0.05),DAI评分升高(P<0.05);小鼠结肠组织的结构完整性被破坏,腺体肿胀,排列混乱,可见大量炎性细胞浸润及肠上皮断裂缺失;血清TNF-α、IL-1β、IL-17、IL-6含量升高(P<0.05);结肠组织4-HNE、MDA、Fe^(2+)含量与p53蛋白表达升高(P<0.05),SOD、GSH含量与SLC7A11、GPX4蛋白表达降低(P<0.05)。与模型组比较,艾灸组、抑制剂组和激艾组小鼠疾病症状缓解,体质量升高(P<0.05),DAI评分降低(P<0.05);艾灸组和抑制剂组小鼠结肠组织肠上皮连续性均恢复,肠黏膜呈轻度肿胀,腺体排列较整齐,偶见少量炎性细胞散在分布;激艾组小鼠结肠组织肠黏膜肿胀程度较模型组减轻,但减轻程度不及艾灸组和抑制剂组,肠上皮连续性未恢复;艾灸组和抑制剂组血清TNF-α、IL-1β、IL-17、IL-6含量降低(P<0.05);结肠组织4-HNE、MDA、Fe^(2+)含量与p53的蛋白表达降低(P<0.05),GSH、SOD含量与SLC7A11、GPX4蛋白表达升高(P<0.05);激艾组血清TNF-α、IL-1β、IL-17含量降低(P<0.05),结肠组织MDA、Fe^(2+)含量和p53蛋白表达降低(P<0.05),GSH含量和SLC7A11、GPX4蛋白表达升高(P<0.05)。与艾灸组比较,激艾组和抑制剂组血清TNF-α、IL-1β、IL-17、IL-6含量与结肠组织4-HNE含量、p53蛋白表达均升高(P<0.05),体质量与结肠组织SOD、GSH含量以及SLC7A11、GPX4蛋白表达降低(P<0.05);激艾组结肠组织MDA、Fe^(2+)含量升高(P<0.05)。与抑制剂组比较,激艾组血清IL-6含量、结肠组织4-HNE、MDA含量和p53蛋白表达升高(P<0.05),体质量与结肠组织SOD、GSH含量以及GPX4蛋白表达降低(P<0.05)。结论:艾灸CD小鼠“天枢”能够减轻肠道炎症,可能是通过下调p53蛋白表达,增强SLC7A11/GSH/GPX4通路活性,减少脂质过氧化物的产生,从而维持肠道铁稳态,减轻结肠组织铁死亡。
Objective To investigate the effects of moxibustion at"Tianshu"(ST25)on intestinal inflammation in mice with Crohn's disease(CD),and to explore the mechanism of p53 regulating the SLC7A11/GSH/GPX4 axis on intestinal ferroptosis lipid peroxidation in CD mice.Methods Fifty SPF-grade C57BL/6 wild-type mice were randomly divided into a blank group(10 mice)and a modeling group(40 mice).The modeling group was given 2%dextran sulfate sodium(DSS)solution prepared with distilled water to establish a CD mouse model.One mouse was randomly selected from the blank group and four mice were randomly selected from the modeling group for model comparison and evaluation.After confirming successful modeling,the modeling group was divided into a model group,a moxibustion group,an activator group,and an inhibition group,with 9 mice in each group,and 9 mice in the blank group.In the moxibustion group,mice were treated with thin moxa stick at bilateral"Tianshu"(ST25)for 15 min each time,once a day,for 14 consecutive days.The activator group received the same moxibustion intervention as the moxibustion group,and at the same time,RITA(10 mg/kg),an agonist,was injected subcutaneously around"Tianshu"(ST25)once a day for 14 consecutive days.The inhibition group was injected subcutaneously with the inhibitor Pifithrin-α(2.2 mg/kg)around"Tianshu"(ST25)once a day for 14 consecutive days.Before and after intervention,the general condition,body weight,and disease activity index(DAI)of each group were observed.After intervention,HE staining was used to observe the colonic tissue morphology of mice.ELISA was used to detect the serum contents of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-17,and IL-6,as well as the contents of 4-hydroxynonenal(4-HNE),malondialdehyde(MDA),GSH,superoxide dismutase(SOD),and ferrous ion(Fe^(2+))in colonic tissue.Western blot was used to detect the protein expressions of p53,SLC7A11,and GPX4 in colonic tissue.Results Compared with the blank group,the mice in the model group showed dry and dull fur,huddled posture,sluggish responses,reduced food intake,mucus and purulent blood around the anus and in the cages,loose and mushy stools,and positive fecal occult blood tests;the body weight was decreased(P<0.05),and the DAI scores were increased(P<0.05);the structural integrity of the colonic tissue was destroyed,with swollen and disorganized glands,a large number of infiltrating inflammatory cells,and epithelial rupture and loss;the serum levels of TNF-α,IL-1β,IL-17,and IL-6 were elevated(P<0.05);the contents of 4-HNE,MDA,and Fe^(2+)and the protein expression of p53 in colonic tissue were increased(P<0.05),while the contents of SOD and GSH,and the protein expressions of SLC7A11 and GPX4 were decreased(P<0.05).Compared with the model group,the mice in the moxibustion group,inhibitor group,and activator group showed alleviated symptoms;the body weight was increased(P<0.05),and DAI scores were decreased(P<0.05).In the moxibustion and inhibitor groups,the colonic epithelial continuity was restored,with mildly swollen mucosa,orderly arranged glands,and only a few scattered inflammatory cells.In the activator group,the swelling of the colonic mucosa was milder than that in the model group,but the improvement was less than that observed in the moxibustion and inhibitor groups,and epithelial continuity was not restored.The serum levels of TNF-α,IL-1β,IL-17,and IL-6 in the moxibustion group and inhibitor group were decreased(P<0.05);the contents of 4-HNE,MDA,Fe^(2+),and protein expression of p53 in colonic tissue were decreased(P<0.05);the levels of GSH,SOD,and the protein expressions of SLC7A11 and GPX4 were increased(P<0.05).In the activator group,the serum levels of TNF-α,IL-1β,and IL-17 were decreased(P<0.05);the contents of MDA,Fe^(2+),and protein expressions of p53 in the colonic tissue were decreased(P<0.05);and the content of GSH and protein expressions of SLC7A11 and GPX4 were increased(P<0.05).Compared with the moxibustion group,the serum levels of TNF-α,IL-1β,IL-17,and IL-6,as well as the contents of 4-HNE and protein expression of p53,were increased in the activator and inhibitor groups(P<0.05);the body weight,the contents of SOD and GSH,and the protein expressions of SLC7A11 and GPX4 in colonic tissue were decreased(P<0.05).The contents of MDA and Fe^(2+)in colonic tissue in the activator group were increased(P<0.05).Compared with the inhibitor group,the serum level of IL-6,the contents of 4-HNE and MDA,and protein expression of p53 in colonic tissue in the activator group were elevated(P<0.05),while the body weight,the contents of SOD and GSH,and protein expression of GPX4 in colonic tissue were reduced(P<0.05).Conclusion Moxibustion at"Tianshu"(ST25)could alleviate intestinal inflammation in CD mice,possibly by downregulating the protein expression of p53,enhancing the activity of the SLC7A11/GSH/GPX4 pathway,reducing the production of lipid peroxides,maintaining intestinal iron homeostasis,and reducing colonic ferroptosis.
作者
杨翟璨
刘密
陆鹏徽
周竞颖
徐璇
王璐瑶
刘琼
YANG Zhaican;LIU MiA;LU Penghui;ZHOU Jingying;XU Xuan;WANG Luyao;LIU Qiong(School of Acupuncture-Moxibustion,Tuina and Rehabilitation,Hunan University of CM,Changsha 410208,China)
出处
《中国针灸》
北大核心
2026年第1期66-74,共9页
Chinese Acupuncture & Moxibustion
基金
国家自然科学基金资助项目:82474662、81774438
湖南省教育厅科学研究项目:23A0284
湖南省自然科学项目:2023JJ30457
湖南省卫生健康高层次人才重大科研专项:R2023141
长沙市自然科学基金项目:kq2208183
国家中医药管理局2022年青年岐黄学者培养项目:国中医药人教函[2022]256号
湖南省科技创新计划项目:2024JK2132、2024RC1061。
