补中益气汤通过调节Nrf2/PPARγ/GPX4通路抑制铁死亡改善自身免疫性甲状腺炎小鼠甲状腺损伤

Improvement of Thyroid Injury in AIT Mice by Inhibiting Ferroptosis Through Regulation of Nrf2/PPARγ/GPX4 Pathway by Buzhong Yiqitang

目的:基于核因子E2相关性因子2(Nrf2)/过氧化物酶体增殖物激活受体γ(PPARγ)/谷胱甘肽过氧化酶4(GPX4)通路探讨补中益气汤改善自身免疫性甲状腺炎(AIT)小鼠铁死亡的作用机制。方法:将120只SPF级8周龄NOD.H-2h4小鼠按随机数字表法分为空白组、模型组、补中益气汤低、中、高剂量组及西药组,各20只,除空白组外,采用经典的高碘水(0.05%碘化钠)喂养,8周后制成AIT模型。补中益气汤低、中、高剂量组分别予4.78、9.56、19.12 g·kg^(-1)补中益气汤灌胃,西药组给予3.033×10^(-5) g·kg^(-1)硒酵母片混悬液灌胃,空白组及模型组给予等体积蒸馏水灌胃,连续8周后实施取材。苏木素-伊红(HE)染色观察小鼠甲状腺组织病理形态;采用酶联免疫吸附测定法(ELISA)检测血清抗甲状腺过氧化物酶抗体(TPOAb)、抗甲状腺球蛋白抗体(TGAb)含量;试剂盒检测小鼠血清中超氧化物歧化酶(SOD)、丙二醛(MDA)水平;采用免疫荧光检测甲状腺组织中GPX4定位表达;实时荧光定量聚合酶链式反应(Real-timePCR)检测甲状腺组织Nrf2、PPARγ、溶质载体家族7成员11(SLC7A11)、溶质载体家族3成员2(SLC3A2)、溶血卵磷脂酰基转移酶3(LPCAT3)、GPX4 mRNA表达;蛋白免疫印迹法(Western blot)检测甲状腺组织Nrf2、PPARγ、SLC7A11、SLC3A2、LPCAT3、GPX4蛋白表达。结果:与空白组比较,模型组小鼠光镜下可见甲状腺组织内明显的淋巴细胞浸润现象,血清中TGAb、TPOAb含量显著升高(P<0.01),MDA水平显著升高,SOD水平显著降低(P<0.01);甲状腺组织中Nrf2、PPARγ、SLC7A11、SLC3A2、GPX4表达显著降低(P<0.01),LPCAT3的表达显著升高(P<0.01);与模型组比较,补中益气汤各剂量组及西药组小鼠光镜下甲状腺组织内的淋巴细胞浸润程度减轻,血清中TPOAb、TGAb含量明显降低(P<0.05,P<0.01),MDA水平明显降低、SOD水平明显升高(P<0.05,P<0.01);甲状腺组织中Nrf2、PPARγ、SLC7A11、SLC3A2、GPX4表达明显升高、LPCAT3的表达明显降低(P<0.05,P<0.01)。与西药组比较,补中益气汤各剂量组甲状腺组织中Nrf2、PPARγ、SLC7A11、SLC3A2、GPX4、LPCAT3表达总体趋势差异明显(P<0.05,P<0.01)。结论:补中益气汤可有效改善AIT的炎症损伤,其作用机制可能与调控Nrf2/PPARγ/GPX4抑制铁死亡进程有关。

Objective To investigate the mechanism of Buzhong Yiqitang in ameliorating ferroptosis in autoimmune thyroiditis(AIT)mice based on the nuclear factor erythroid 2-related factor 2(Nrf2)/peroxisome proliferator-activated receptorγ(PPARγ)/glutathione peroxidase 4(GPX4)pathway.Method 120 SPF-grade 7-8-week-old NOD.H-2h4 mice were randomly divided into control group,model group,low-,medium-,and high-dose Buzhong Yiqitang groups,and western medicine group,with 20 mice in each group.Except for the control group,all mice were fed with classic high-iodine water(0.05%NaI)to induce AIT models after 8 weeks.The low-,medium-,and high-dose Buzhong Yiqitang groups were administered 4.78,9.56,19.12 g·kg^(-1) of Buzhong Yiqitang,respectively,via gavage.The western medicine group was given 3.033×10^(-5) g·kg^(-1) selenium yeast tablet suspension via gavage,while the control and model groups were given an equal volume of distilled water via gavage.After 8 weeks of continuous treatment,samples were collected.The pathological morphology of mouse thyroid tissue was observed through hematoxylin-eosin(HE)staining,the content of serumantithyroid peroxidase autoantibody(TPOAb)and anti-thyroglobulin antibodies(TGAb)was measured by enzyme-linked immunosorbent assay(ELISA),the kit was used to detect the levels of superoxide dismutase(SOD),and malondialdehyde(MDA)in mouse serum.Immunofluorescence was used to detect the localized expression of GPX4 in thyroid tissue.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the expression of Nrf2,PPARγ,solute carrier family 7 member 11(SLC7A11),solute carrier family 3 member 2(SLC3A2),lysolipid lecithin acyltransferase 3(LPCAT3),and GPX4 mRNA in thyroid tissue.Western blot was used to detect the expression of Nrf2,PPARγ,SLC7A11,SLC3A2,LPCAT3,and GPX4 proteins in thyroid tissue.Result Compared with control group,model group under light microscopy showed significant lymphocyte infiltration in the thyroid tissue,significantly increased levels of TGAb and TPOAb in serum(P<0.01),significantly increased MDA levels and decreased SOD levels in serum(P<0.01),significantly decreased expression of Nrf2,PPARγ,SLC7A11,SLC3A2,and GPX4(P<0.01)in thyroid tissue,while the expression of LPCAT3 was significantly increased(P<0.01).Compared with model group,the Buzhong Yiqitang groups and the western medication group under light microscopy showed lymphocyte infiltration in the thyroid tissue of was decreased,significantly decreased levels of TPOAb and TGAb in serum(P<0.05,P<0.01),decreased MDA levels and increased SOD levels in serum(P<0.05,P<0.01),significantly increased expression of Nrf2,PPARγ,SLC7A11,SLC3A2,and GPX4,while the expression of LPCAT3 was significantly decreased(P<0.05,P<0.01)in the thyroid tissue.Compared with western medication group,Buzhong Yiqitang groups showed significant overall trends in the expression of Nrf2,PPARγ,SLC7A11,SLC3A2,GPX4,and LPCAT3(P<0.05,P<0.01).Conclusion Buzhong Yiqitang can effectively improve the inflammatory injury of AIT,and its mechanism of action may be related to the regulation of Nrf2/PPARγ/GPX4 to inhibit ferroptosis.