苯乙醇苷通过JNK通路调控脓毒症肠道损伤中铁死亡的机制研究OA

Objective To investigate the protective effect of phenylethanoid glycosides(PhGs),the active constituents of Cistanche tubulosa,on the intestinal barrier in septic rats and to determine whether this effect is mediated through regulation of the c-Jun N-terminal kinase(JNK)signaling pathway to inhibit ferroptosis.Methods Seventy-two specific pathogen-free(SPF)male Sprague-Dawley(SD)rats were randomly assigned to six groups,12 rats in each group,using a random number table:sham operation group,sepsis model group,PhGs group,PhGs+Erastin group,ferroptosis inhibitor deferoxamine(DFO)group,and ferroptosis activator Erastin group.The sham operation group underwent laparotomy with closure only,while sepsis was induced in the remaining groups by cecal ligation and puncture(CLP).The PhGs group received intragastric administration of PhGs at 200 mg/kg after modeling;the PhGs+Erastin group received both intragastric PhGs(200 mg/kg)and intraperitoneal Erastin(20 mg/kg);the DFO group received subcutaneous DFO(20 mg/kg);and the Erastin group received intraperitoneal Erastin(20 mg/kg).Blood and small intestinal tissues were collected 24 hours after modeling.Serum levels of tumor necrosis factor-α(TNF-α)and interleukins(IL-1β,IL-4,IL-10)were measured by enzyme-linked immunosorbent assay(ELISA).Colorimetric assays were performed to determine D-lactate dehydrogenase(D-LDH),malondialdehyde(MDA),and Fe2+levels.Western blotting was used to detect the protein expression levels of phosphorylated JNK(p-JNK)/JNK,glutathione peroxidase 4(GPX4),acyl-CoA synthetase long-chain family member 4(ACSL4),Claudin-1,and diamine oxidase(DAO).Hematoxylin-eosin(HE)staining was performed to evaluate intestinal mucosal pathological damage,and Chiu scoring was applied.Immunohistochemistry was used to assess the percentage of positive expression areas for GPX4,DAO,and JNK.Transmission electron microscopy(TEM)was employed to observe mitochondrial ultrastructure.Results The number of surviving rats at 24 hours in the PhGs group was significantly higher than that in the sepsis model group(8 vs.6,P<0.05).Serum levels of TNF-α(ng/L:30.30±5.06 vs.62.20±3.41),IL-1β(ng/L:273.90±7.99 vs.433.30±8.42),IL-4(ng/L:15.24±1.86 vs.26.40±1.57),IL-10(ng/L:28.07±1.62 vs.53.07±3.36),MDA(μmol/L:4.13±0.41 vs.6.67±0.51),D-LDH(kU/L:6.77±0.55 vs.18.12±1.03),and Fe2+(μmol/L:16.13±0.45 vs.19.20±0.56)were all significantly decreased in the PhGs group compared with the sepsis model group(all P<0.05).Among the above indicators,no statistically significant differences were observed between the PhGs group and the DFO group for all parameters(all P>0.05).Western blotting results showed that the p-JNK/JNK protein expression[(p-JNK/JNK)/β-actin:2.31±0.13 vs.3.41±0.11]and ACSL4 protein expression(0.44±0.02 vs.0.86±0.13)in the PhGs group were significantly lower than those in the sepsis model group,while GPX4 protein expression(GPX4/β-actin:0.48±0.03 vs.0.31±0.01),Claudin-1 protein expression(Claudin-1/β-actin:0.97±0.03 vs.0.69±0.03),and DAO protein expression(DAO/β-actin:0.68±0.04 vs.0.32±0.03)were all significantly restored compared with the sepsis model group(all P<0.05).HE staining demonstrated that the Chiu score in the PhGs group was significantly lower than that in the sepsis model group(1.75±0.71 vs.3.17±0.41,P<0.05).Immunohistochemical results revealed that the percentages of GPX4-positive expression area[(24.29±2.53)%vs.(16.55±2.64)%]and DAO-positive expression area[(16.99±2.34)%vs.(5.64±0.35)%]in the PhGs group were significantly higher than those in the sepsis model group,while the percentage of JNK-positive expression area[(14.32±0.77)%vs.(38.06±1.72)%]was significantly lower(all P<0.05).Transmission electron microscopy observation showed that the mitochondrial structure in the PhGs group was largely intact,with markedly attenuated damage compared with the sepsis model group.Conclusions PhGs exert a protective effect on the intestinal barrier in CLP-induced septic rats.The underlying mechanism may be associated with the inhibition of JNK signaling pathway activation,upregulation of GPX4,and downregulation of ACSL4 expression,thereby suppressing ferroptosis.