枸杞多糖通过Nrf2/GPX4途径抑制铁死亡改善热射病大鼠结肠损伤

LBP Attenuate Heat Stroke-induced Colonic Injury in Rats by Inhibiting Ferroptosis through Nrf2/GPX4 Pathway

目的探讨枸杞多糖(LBP)对热射病大鼠结肠组织的保护作用及对Nrf2、GPX4、xCT等表达水平的影响。方法将6周龄的40只雄性SPF级大鼠随机分为空白对照(Con)组、热射病(HS)组以及枸杞多糖低、中、高[50、150、450 mg·(kg·d)^(-1)]剂量组。Con组和HS组灌服纯水,剩余各组灌服LBP水溶液,为期4周。灌胃周期结束后,完成热射病大鼠造模并取材。通过HE染色和PAS染色观察结肠组织病理变化;采用TBA法检测血清和组织中丙二醛(MDA)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)水平;采用免疫荧光检测枸杞多糖干预后横结肠组织中Occludin和ZO-1蛋白的表达水平,Western blot检测GPX4、xCT和Nrf2蛋白的表达水平。结果与Con组相比,HS组的结肠黏膜结构被破坏。随着枸杞多糖剂量的增加,热应激所致结肠黏膜损伤程度降低(P<0.05),结肠组织和血清中抗氧化水平逐渐恢复至Con组的水平(P<0.05)。与Con组相比,结肠组织中Occludin、ZO-1、GPX4和xCT蛋白的表达在HS组中均减少(P均<0.05),在LBP-H组中升高(P<0.05)。同时,Nrf2蛋白水平在应激后的HS组中升高(P<0.05),在LBP-H组中继续升高(P<0.05)。结论LBP可激活Nrf2,并通过上调抗氧化因子GPX4和xCT抑制铁死亡过程,进而拮抗热射病模型下因紧密连接蛋白水平降低引起的大鼠结肠损伤。

Objective This study aims to investigate the protective effects of Lycium barbarum polysaccharides(LBP)on colonic tissues of rats with heat stroke and the impact on the expression levels of Nrf2,GPX4,xCT,and other related factors.Methods Forty male of SPF-grade rats at the age of 6 weeks were randomly divided into the following groups:blank control(Con)group,heat stroke(HS)group,and low,medium,and high-dose[50,150,450 mg·(kg·d)^(-1)]LBP groups.The Con and HS groups were administered with pure water,while the remaining groups were administered with LBP aqueous solution for a period of 4 weeks.After the gavage period,heat stroke was induced in rats,and tissue samples were collected.HE and PAS staining were used to evaluate the pathological changes in colonic tissues.TBA method was employed to detect MDA levels CAT levels and SOD levels in serum and tissues.Immunofluorescence was used to validate the expression changes of Occludin and ZO-1 proteins in transverse colonic tissues after LBP intervention.Western blot was utilized to verify the expression changes of GPX4,xCT,and Nrf2 proteins.Results Compared to the Con group,the colonic mucosal structure in the HS group was observed to be damaged.With the increase in LBP dosage,the severity of colonic mucosal damage caused by heat stress significantly decreased(P<0.05),and antioxidant levels in colonic tissues and serum gradually recovered to the levels of the Con group(P<0.05).Compared to the Con group,the expression of Occludin,ZO-1,GPX4,and xCT proteins in colonic tissues decreased in the HS group(P all<0.05)and significantly increased in the high-dose LBP group(P<0.05).Meanwhile,the level of Nrf2 protein significantly increased in the HS group after stress(P<0.05)and continued to rise to a higher level in the high-dose LBP group(P<0.05).Conclusion LBP activates Nrf2,inhibits ferroptosis by upregulating antioxidant factors GPX4 and xCT,and thus antagonizes the colon damage caused by the decreased level of tight junction protein in the rat model of thermal radiation disease.