摘要
目的探讨蒙药巴特日-7通过调控溶质载体家族7成员11/谷胱甘肽过氧化物酶4(SLC7A11/GPX4)信号通路抑制铁死亡缓解脓毒症模型大鼠肠黏膜损伤的作用机制。方法选择SPF级雄性SD大鼠56只,按随机数字表法分为假手术组、模型组、巴特日-7高剂量组(给予630 mg/kg巴特日-7)、巴特日-7中剂量组(给予315 mg/kg巴特日-7)、巴特日-7低剂量组(给予162.5 mg/kg巴特日-7)、美罗培南组(腹腔注射美罗培南105 mg/kg)及核因子E2相关因子2(Nrf2)抑制剂ML385组(腹腔注射ML38530mg/kg),每组8只。采用盲肠结扎穿孔术(CLP)复制脓毒症大鼠模型。光镜及电镜下观察各组大鼠回肠组织的病理损伤情况;采用酶联免疫吸附试验(ELISA)检测各组肠黏膜损伤标志物D-乳酸、二胺氧化酶(DAO)和氧化应激参数超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,以及炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-1β)水平;采用蛋白质免疫印迹试验(Western blotting)检测各组回肠组织铁死亡信号通路关键蛋白Nrf2、血红素加氧酶1(HO-1)、SLC7A11、GPX4和铁蛋白重链1(FTH1)的蛋白质表达水平;采用免疫组化法观察SLC7A11与GPX4在组织微环境中的定位分布规律。结果苏木素-伊红(HE)染色和电镜下观察可见,巴特日-7能减轻回肠黏膜细胞组织炎性浸润水肿和绒毛、腺体、线粒体、内质网等结构损伤。与假手术组比较,模型组肠黏膜损伤和炎症相关因子D-乳酸、DAO、MDA、TNF-α、IL-6、IL-1β均明显升高[D-乳酸(ng/L):1124.01±83.52比523.11±60.55,DAO(ng/L):123.47±12.96比62.24±7.59,MDA(ng/L):5.64±0.39比2.66±0.52,TNF-α(ng/L):83.20±5.39比22.50±3.14,IL-6(ng/L):161.10±8.28比98.81±6.25,IL-1β(ng/L):105.63±8.80比41.39±6.98],SOD活性、氧化应激和铁死亡相关蛋白Nrf2、HO-1、SLC7A11、GPX4、FTH1表达水平均明显降低[SOD(ng/L):72.88±7.14比110.52±7.52,Nrf2蛋白表达(灰度值):0.37±0.07比1.26±0.07,HO-1蛋白表达(灰度值):0.31±0.12比1.01±0.09,SLC7A11蛋白表达(灰度值):0.35±0.11比1.18±0.08,GPX4蛋白表达(灰度值):0.56±0.12比1.63±0.12,FTH1蛋白表达(灰度值):0.45±0.18比1.19±0.11,均P<0.05]。与模型组比较,巴特日-7各剂量组和美罗培南组D-乳酸、DAD、MDA、TNF-α、IL-6、IL-1β均明显降低,SOD活性及氧化应激和铁死亡相关蛋白Nrf2、HO-1、SLC7A11、GPX4、FTH1的蛋白表达水平均明显升高(均P<0.05)。各剂量巴特日-7组的变化以高剂量组最明显[D-乳酸(ng/L):657.41±92.44比1124.01±83.52,DAO(ng/L):82.94±9.56比123.47±12.96,MDA(ng/L):3.54±0.72比5.64±0.39,TNF-α(ng/L):40.66±5.94比83.20±5.39,IL-6(ng/L):120.56±11.21比161.10±8.28,IL-1β(ng/L):54.51±9.66比105.63±8.80,SOD(ng/L):102.43±9.81比72.88±7.14,氧化应激指标:Nrf2蛋白表达(灰度值)为0.86±0.13比0.37±0.07,HO-1蛋白表达(灰度值)为0.82±0.07比0.31±0.12,铁死亡相关蛋白:SLC7A11蛋白表达(灰度值)为0.83±0.12比0.35±0.11,GPX4蛋白表达(灰度值)为1.35±0.17比0.56±0.12,FTH1蛋白表达(灰度值)为0.90±0.17比0.45±0.18,均P<0.05]。Nrf2抑制剂ML385能明显逆转蒙药巴特日-7对脓毒症肠黏膜损伤的保护作用,药量增加,疗效也增加,存在量效关系。结论蒙药巴特日-7可能通过调控SLC7A11/GPX4信号通路上下游因子抑制铁死亡,降低Nrf2/HO-1信号通路氧化应激,缓解脓毒症模型大鼠肠黏膜损伤程度。
Objective To investigate the mechanism by which Mongolian medicine Bateri-7 alleviates intestinal mucosal injury in septic rats by regulating the solute carrier family 7 member 11/glutathione peroxidase 4(SLC7A11/GPX4)signaling pathway to inhibit ferroptosis.Methods A total of 56 male SPF-grade Sprague-Dawley(SD)rats were selected and randomly divided into sham surgery group,model group,high-dose Bateri-7 group(administered Bateri-7 at 630 mg/kg),medium-dose Bateri-7 group(administered Bateri-7 at 315 mg/kg),low-dose Bateri-7 group(administered Bateri-7 at 162.5 mg/kg),Meropenem group(intraperitoneally injected with Meropenem at 105 mg/kg),and nuclear factor-E2-related factor 2(Nrf2)inhibitor ML385 group(intraperitoneally injected 30 mg/kg),with 8 rats in each group.The rat model of sepsis was established by performing a cecal ligation and puncture(CLP).Pathological damage to the ileal tissue of rats was observed using light microscopy staining techniques and electron microscopy;the levels of intestinal mucosal injury markers D-lactic acid,diamine oxidase(DAO),and oxidative stress parameters superoxide dismutase(SOD)activity and malondialdehyde(MDA)content,as well as inflammatory factors tumor necrosis factor-α(TNF-α),interleukins(IL-6,IL-1β),were measured using enzyme-linked immunosorbent assay(ELISA);the expression levels of key proteins in the ferroptosis signaling pathway,including Nrf2,heme oxygenase-1(HO-1),SLC7A11,GPX4,and ferritin heavy chain 1(FTH1),were determined by Western blotting;and the localization distribution of SLC7A11 and GPX4 in the tissue microenvironment was observed using immunohistochemistry.Results Hematoxylin-eosin(HE)staining and electron microscopy revealed that Bateri-7 alleviated inflammatory infiltration and edema of enterocyte tissues,and reduced damage to structures such as villi,glands,mitochondria,and endoplasmic reticulum.Compared with the sham surgery group,the model group showed significant increases in intestinal mucosal injury and inflammatory factors D-lactic acid,DAO,MDA,TNF-α,IL-6,and IL-1β[D-lactic acid(ng/L):1124.01±83.52 vs.523.11±60.55,DAO(ng/L):123.47±12.96 vs.62.24±7.59,MDA(ng/L):5.64±0.39 vs.2.66±0.52,TNF-α(ng/L):83.20±5.39 vs.22.50±3.14,IL-6(ng/L):161.10±8.28 vs.98.81±6.25,IL-1β(ng/L):105.63±8.80 vs.41.39±6.98],the levels of SOD and the oxidative-stress and ferroptosis-related proteins Nrf2,HO-1,SLC7A11,GPX4 and FTH1 were significantly reduced[SOD(ng/L):72.88±7.14 vs.110.52±7.52,Nrf2 protein expression(gray value):0.37±0.07 vs.1.26±0.07,HO-1 protein expression(gray value):0.31±0.12 vs.1.01±0.09,SLC7A11 protein expression(gray value):0.35±0.11 vs.1.18±0.08,GPX4 protein expression(gray value):0.56±0.12 vs.1.63±0.12,FTH1 protein expression(gray value):0.45±0.18 vs.1.19±0.11,all P<0.05].Compared with the model group,the high-dose Bateri-7 group and meropenem group showed significant decreases in D-lactic acid,DAO,MDA,TNF-α,IL-6,and IL-1β,and significant increases in SOD and oxidative stress and ferroptosis-related proteins Nrf2,HO-1,SLC7A11,GPX4,and FTH1 levels(all P<0.01).The changes in the Baterite-7 groups were most pronounced in the high-dose group[D-lactic acid(ng/L):657.41±92.44 vs.1124.01±83.52,DAO(ng/L):82.94±9.56 vs.123.47±12.96,MDA(ng/L):3.54±0.72 vs.5.64±0.39,TNF-α(ng/L):40.66±5.94 vs.83.20±5.39,IL-6(ng/L):120.56±11.21 vs.161.10±8.28,IL-1β(ng/L):54.51±9.66 vs.105.63±8.80,SOD(ng/L):102.43±9.81 vs.72.88±7.14,oxidative stress indicators:Nrf2 protein expression(gray value):0.86±0.13 vs.0.37±0.07,HO-1 protein expression(gray value):0.82±0.07 vs.0.31±0.12,ferroptosis-related proteins:SLC7A11 protein expression(gray value):0.83±0.12 vs.0.35±0.11,GPX4 protein expression(gray value):1.35±0.17 vs.0.56±0.12,FTH1 protein expression(gray value):0.90±0.17 vs.0.45±0.18,all P<0.05].The Nrf2 inhibitor ML385 significantly reversed the protective effect of Mongolian medicine Bateri-7 on intestinal mucosal injury in sepsis,with increasing dosage correlating with enhanced efficacy,indicating a dose-response relationship.Conclusions Mongolian medicine Bateri-7 may alleviate intestinal mucosal injury in sepsis model rats by regulating upstream and downstream factors of the SLC7A11/GPX4 signaling pathway to inhibit ferroptosis and reduce oxidative stress via the Nrf2/HO-1 signaling pathway.
作者
青格勒
巴特金
乌云必力格
包巴根那
吴超群
特日格乐
关文祥
郝浩
隆云
Qing Gele;Ba Tejin;Wu Yunbilige;Bao Bagenna;Wu Chaoqun;Te Rigele;Guan Wenxiang;Hao Hao;Long Yun(Inner Mongolia Autonomous Region International Mongolian Medicine Hospital,Hohhot O10010,Inner Mongolia Autonomous Region,China;College of Mongolian Medicine,Inner Mongolia Medical University,Hohhot O10065,Inner Mongolia Autonomous Region,China;Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250014,Shandong,China;Peking Union Medical College,Chinese Academy of Medical Sciences,Beijing 100730,China)
出处
《中国中西医结合急救杂志》
2025年第5期561-569,共9页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
内蒙古自治区公立医院高水平临床专科发展示范项目(2023SGGZ137)
内蒙古自治区蒙医药学会科研项目(MXK-162)
内蒙古医学科学院公立医院科研联合基金科技项目(2024GLLH0137)。
关键词
铁死亡
脓毒症
肠黏膜损伤
蒙药巴特日-7
Ferroptosis
Sepsis
Intestinal mucosal injury
Mongolian medicine Bateri-7
