基于Nrf2/SLC7A11/GPX4信号通路介导的铁死亡探讨白头翁汤对溃疡性结肠炎小鼠的作用及机制OA

Objective To investigate the mechanism of Baitouweng Decoction(mainly consist of Pulsatillae Radix 15 g,Phellodendri Chinensis Cortex 12 g,Coptidis Rhizoma 6 g,Fraxini Cortex 12 g)in treating ulcerative colitis(UC)mice,based on ferroptosis mediated by the nuclear factor erythroid 2-related factor 2(Nrf2)/solute carrier family 7 member 11(SLC7A11)/glutathione peroxidase 4(GPX4)signaling pathway.Methods C57BL/6J mice were randomly divided into a normal group,a model group,a Baitouweng Decoction low-dose group(8 g·kg-1),a Baitouweng Decoction high-dose group(16 g·kg-1),a mesalazine group(500 mg·kg-1),and a ferroptosis inhibitor group(Ferrostatin-1,1 mg·kg-1),with 8 mice in each group.A UC mouse model was established by allowing mice free access to 3%dextran sulfate sodium(DSS)solution for 7 consecutive days.Concurrently,each treatment group received the corresponding drug intervention daily.The Baitouwen Decoction low-dose,high-dose groups and the mesalazine group received oral gavage(all at a volume of 20 mL·kg-1),while the ferroptosis inhibitor group received intraperitoneal injection,once daily for 7 consecutive days.The general condition of the mice was observed and recorded,and the disease activity index(DAI)and colonic mucosal damage index(CMDI)were scored.Histopathological changes in colon tissue were observed using hematoxylin and eosin(HE)staining and Alcian blue-periodic acid-Schiff(AB-PAS)staining.The expression levels of Occludin and Claudin-1 in colon tissue were detected by immunohistochemistry.Serum levels of the inflammatory cytokines tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-10(IL-10)were measured by ELISA.Levels of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH),and ferrous ion(Fe²⁺)in colon tissue were detected by micro-methods.Ultrastructural changes in colon tissue were observed by transmission electron microscopy(TEM).The mRNA and protein expression levels of Nrf2,SLC7A11,and GPX4 in colon tissue were detected by qPCR and Western Blot,respectively.Results Compared with the normal group,the model group showed a significant increase in DAI score(P＜0.01);obvious congestion and edema in the colonic mucosa,accompanied by erosion and ulcer formation,and a significant increase in CMDI score(P＜0.01);severely damaged colon tissue structure,with reduced and disordered glands,loss of crypt architecture,accompanied by significant edema,inflammatory cell infiltration,and ulcer formation;a significant decrease in the number of goblet cells with lighter staining,and almost no obvious mucus in gland lumens;significantly decreased positive expression levels of Claudin-1 and Occludin in colon tissue(P＜0.01);significantly elevated serum levels of pro-inflammatory factors TNF-α and IL-6(P＜0.01),and significantly decreased levels of the anti-inflammatory factor IL-10(P＜0.01);significantly increased levels of MDA and Fe2+in colon tissue(P＜0.01),while SOD and GSH levels were significantly decreased(P＜0.01);a marked reduction and disarray in the number of microvilli of colonic epithelial cells,disrupted intercellular junctions,significantly swollen and deformed mitochondria with broken or missing cristae;mRNA and protein expression of Nrf2,GPX4,and SLC7A11 in colon tissue were significantly downregulated(P＜0.01).Compared with the model group,all treatment groups showed a significant decrease in DAI scores(P＜0.05,P＜0.01);the degree of colonic mucosal congestion and edema was significantly alleviated,the scope of erosion was reduced,and CMDI scores were significantly decreased(P＜0.05,P＜0.01);the colonic mucosal structure showed some recovery,with more orderly gland arrangement and reduced degrees of inflammatory cell infiltration and edema;the number of goblet cells significantly increased with deeper staining,and more mucus secretion was visible in gland lumens;positive expression levels of Claudin-1 and Occludin in colon tissue were significantly increased(P＜0.05,P＜0.01);serum levels of TNF-α and IL-6 were significantly decreased(P＜0.05,P＜0.01),while IL-10 levels were significantly increased(P＜0.05,P＜0.01).Compared with the model group,the Baitouwen Decoction low-dose,high-dose groups and the ferroptosis inhibitor group showed significantly decreased levels of MDA and Fe2+in colon tissue(P＜0.05,P＜0.01),and significantly increased levels of SOD and GSH(P＜0.05,P＜0.01);microvilli of colonic epithelial cells were markedly restored,mitochondria showed mild swelling but cristae structures remained clearly visible;mRNA and protein expression of Nrf2,GPX4,and SLC7A11 in colon tissue were significantly upregulated(P＜0.05,P＜0.01).Conclusion Baitouwen Decoction has a significant ameliorative effect on UC mice.Its mechanism may be related to activating the Nrf2/SLC7A11/GPX4 signaling pathway,inhibiting ferroptosis,and thereby alleviating colonic tissue damage.