Objective : To investigate the protective effect of dimethyl fumarate(DMF) on maternal intrahepatic cholestasis(ICP) during pregnancy induced by di(2-ethylhexyl) phthalate(DEHP) exposure and its mechanism. Methods : Thirty-two 8-week-old female institute of cancer research(ICR) mice were randomly divided into 4 groups: Ctrl group, DEHP group, DMF group and DEHP+DMF group. DEHP and DEHP+DMF groups were treated with DEHP(200 mg/kg) by gavage every morning at 9:00 a.m. DMF and DEHP+DMF groups were treated with DMF(150 mg/kg) from day 13 to day 16 of gestation by gavage. After completion of gavage on day 16 of pregnancy, maternal blood, maternal liver, placenta, and amniotic fluid were collected from pregnant mice after a six-hour abrosia. The body weight of the mother rats and the body weight of the fetus rats were sorted and analyzed; the levels of total bile acid(TBA), alkaline phosphatase(ALP), aspartate aminotransferase/alanine aminotransferase(AST/ALT) in serum and TBA in liver, amniotic fluid and placenta were detected by biochemical analyzer; HE staining was used to observe the pathological changes of liver tissue; Quantitative reverse transcription PCR(RT-qPCR) was used to detect the expression levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-6, IL-1, IL-18 and NOD-like receptor thermal protein domain associated protein 3(NLRP3) in the liver; Western blot was used to detect the expression of the nuclear factor KappaB(NF-κB) and NLRP3. Results : Compared with the control group, the body weight of the DEHP-treated dams and pups decreased(P<0.05); the levels of TBA, ALP, AST/ALT in the serum of dams and the levels of TBA in the liver, amniotic fluid, and placenta of dams increased(P<0.05); the histopathological results showed that liver tissue was damaged, bile ducts were deformed, and there was inflammatory cell infiltration around them; the levels of inflammation-related factors TNF-α, IL-6, IL-1, IL-18 and NLRP3 transcription in maternal liver increased(P<0.05); the expression of NF-κB and NLRP3 protein in maternal liver significantly increased( P<0. 05). Compared with the DEHP group,the body weight of both dams and fetuses significantly increased in DEHP + DMF group( P<0. 05); the levels of TBA,ALP,AST/ALT in the serum of dams and amniotic fluid of fetuses decreased( P<0. 05); the degree of liver lesions was improved; the transcription levels of inflammation-related factors TNF-α,IL-6,IL-1,IL-18 and NLRP3 in maternal liver decreased( P<0. 05); the expression of NF-κB and NLRP3 protein in maternal liver significantly decreased( P<0. 05). Conclusion DMF can effectively protect the DEHP exposure to lead to female ICP,and its mechanism may be through inhibiting the NF-κB/NLRP3 pathway and reducing liver inflammation.

Objective : To establish a method for measuring major organic acids in the tricarboxylic acid cycle using a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) system, and to investigate the changes in six tricarboxylic acid cycle organic acids(fumaric acid, malic acid, succinic acid, α-ketoglutaric acid, cis-aconitic acid, and citric acid) in the serum, liver, and placenta of mice exposed to di(2-ethylhexyl) phthalate(DEHP) during pregnancy. Methods : The serum, liver and placental samples from pregnant mice were processed and eluted through a Waters ACQUITY UPLC BEH Amide Column(130 Å, 1.7 μm, 2.1 mm × 150 mm) using a gradient elution program. Mobile phase A comprised an aqueous solution of 10 mmol/L ammonium acetate and 5 μmol/L methanephosphonic acid, while mobile phase B consisted of a 90% acetonitrile aqueous solution containing 10 mmol/L ammonium acetate and 5 μmol/L methanephosphonic acid, with a flow rate maintained at 0.35 ml/min. The mass spectrometry detection system utilized an electrospray ionization technique with negative ion mode for multiple reaction monitoring. Results : The correlation coefficients of the standard curves for the six tricarboxylic acid cycle organic acid metabolites were all above 0.996 within the quantitative range. The method's accuracy ranged from 97.14% to 108.26%, with inter-day and intra-day precision relative standard deviation between 1.35% and 6.73%. The matrix effect was between 93.29% and 107.47%, and the extraction recovery rate ranged from 94.82% to 112.57%. Analysis of six tricarboxylic acid cycle organic acids in the liver, serum, and placenta of DEHP-exposed mice during pregnancy showed significant reductions in fumaric acid, malic acid, α-ketoglutaric acid, cis-aconitic acid, and citric acid compared to the control group(P<0.05). Conclusion The HPLC-MS/MS method established in this study for detecting six tricarboxylic acid cycle organic acids in the serum, liver, and placenta of DEHP-exposed pregnant mice is stable, highly sensitive and selective. Prenatal DEHP exposure induced alterations in the levels of tricarboxylic acid(TCA) cycle organic acid metabolites in the liver, serum, and placenta of mice, suggesting that DEHP exposure during pregnancy may interfere with mitochondrial TCA cycle processes. These findings indicate potential value in the diagnosis and treatment of diseases associated with prenatal DEHP exposure.

Objective To investigate the protective effect of folic acid against cholestatic liver injury in mice induced by bis(2-ethylhexyl)phthalate(DEHP)exposure and its mechanism.Methods ICR mice were randomly divided into control group,high-dose folic acid(H-FA)group,DEHP group,DEHP+low-dose folic acid(DEHP+L-FA)group,and DEHP+high-dose folic acid(DEHP+H-FA)group,with 6 mice in each group.The mice in the H-FA group,the DEHP+L-FA group,and the DEHP+H-FA group were given folic acid by gavage at the corresponding dose,and those in the control group and the DEHP group were given an equal volume of PBS solution by gavage.After 2 hours,the mice in the DEHP group,the DEHP+L-FA group,and the DEHP+H-FA group were given corn oil containing 200 mg/kg DEHP,and those in the control group and the H-FA group were given an equal volume of pure corn oil,by gavage for 4 weeks.Body weight and food intake were recorded every day,and blood and liver tissue samples were collected.A biochemical analyzer was used to measure the serum levels of total bile acid(TBA)and alkaline phosphatase(ALP);HE staining was used to observe the histopathological changes of liver tissue;kits were used to measure the content of malondialdehyde(MDA)and superoxide dismutase(SOD)in the liver;LC-MS/MS was used to measure serum bile acid profiles;Western blot was used to measure the expression levels of proteins associated with hepatic bile acid metabolism.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the control group,the daily food intake of the mice in the DEHP group decreased significantly,and the body weight decreased significantly from day 10(P＜0.05),and compared with the DEHP group,the DEHP+L-FA group and the DEHP+H-FA group had basically unchanged body weight and daily food intake(P＞0.05).Compared with the control group,the DEHP group had significant increases in liver weight index and the serum levels of TBA and ALP(all P＜0.05),with enlarged portal area,bile duct deformity and hyperplasia,and a small amount of inflammatory cell infiltration in liver tissue;compared with the DEHP group,the DEHP+L-FA group and the DEHP+H-FA group had a significant reduction in liver weight index(P＜0.01),and the DEHP+H-FA group had significant reductions in the serum levels of TBA and ALP(P＜0.05),with a significant improvement in liver histomorphology and structure after folic acid intervention.Compared with the control group,the DEHP group had a significant reduction in the content of SOD(P＜0.05)and a significant increase in the content of MDA in the liver(P＜0.01),and compared with the DEHP group,the DEHP+H-FA group had significant reductions in the content of MDA and SOD(P＜0.05).Compared with the control group,the DEHP group had significant increases in the serum levels of α-muricholic acid(α-MCA),β-muricholic acid(β-MCA),deoxycholic acid(DCA),lithocholic acid(LCA),taurocholic acid(TCA),taurodeoxycholic acid(TDCA),tauroursodeoxycholic acid(TUDCA),tauro-β-muricholic acid(T-β-MCA),tauro-α-muricholic acid(T-α-MCA),taurohyodeoxycholic acid(THDCA),and taurolithocholic acid(TLCA)(P＜0.05)and a significant reduction in ursodeoxycholic acid(UDCA)(P＜0.05);compared with the DEHP group,the DEHP+H-FA group had significant reductions in the serum levels of DCA,LCA,TCA,TDCA,TUDCA,T-β-MCA,T-α-MCA,THDCA,and TLCA(P＜0.05).Compared with the control group,the DEHP group had significant increases in the protein expression levels of FXR and CYP3A11 in the liver(P＜0.01)and significant reductions in the protein expression levels of CYP7A1 and MRP2(P＜0.01);compared with the DEHP group,the DEHP+L-FA group and the DEHP+H-FA group had significant reductions in the protein expression levels of FXR and CYP3A11 in the liver(P＜0.05)and a significant increase in the protein expression level of MRP2(P＜0.05),and the DEHP+H-FA group had a significant increase in the protein expression level of CYP7A1(P＜0.05).Conclusion Folic acid has a protective effect against cholestatic liver injury in mice induced by DEHP exposure,possibly by regulating bile acid synthesis,catabolism,and transport and maintaining bile acid homeostasis.

Objective:To investigate whether ferroptosis of nerve cells exists in a rat model of neuropathic pain (NP), and to explore the mechanism of O 3 treatment of NP. Methods:Sixty male SD rats were randomly divided into three groups: neuropathic pain model group (NP group), sham operation group (Sham group) and ozone group (O 3 group). The partial sciatic nerve ligation was used in the NP and O 3 groups to construct a neuropathic pain model. The Sham group was subjected to sham surgery. In the O 3 group, 15 μl of O 3 (40 μg/ml) was injected locally at the injury site, and the NP and Sham were injected with the same amount of air, once per day. The mechanical contraction response threshold (MWT) and thermal contraction latency (TWL) of the rats were measured 1 day before the surgery (T0) and 1, 3, 7, 14 days after the surgery (T1 to T4). The spinal cord segments of rats were collected. The expression levels of glutathione peroxidase 4 (GPX4) and long chain fatty acid coenzyme A synthetase 4 (ACSL4) at T1 to T4 was detected using Western Blot. The number of NeuN+ neuron cells in the spinal dorsal horn at T4 was detected using immunofluorescence technology. The specific changes of ferroptosis at T4 was observed by a transmission electron microscopy. Iron deposition in the spinal dorsal horn at T1 to T4 was measured using ferroptosis kits. Results:Compared with the Sham group, rats in the NP group and O 3 group showed decreasing of MWT decreased and shortening of TWL at T2 to T4, decreasing of NeuN+ neurons in spinal dorsal horn at T4, specific changes of ferroptosis in mitochondria at T4, and increasing of iron content in nerve tissue at T2 to T4. Compared with the Sham group, rats in NP group showed decreasing of GPX4 level and increasing of ACSL4 level. Compared with the NP group, rats in the O 3 group showed increasing of MWT and prolonging of TWL at T2 to T4, increasing of the GPX4 level and decreasing of ACSL4 level at T2 to T4, increasing of the number of NeuN+ neuron cells in the spinal dorsal horn, improving of the mitochondrial atrophy of nerve cells, and decreasing of the iron content in nerve tissue at T2 to T4. The above results are statistically significant (all P<0.05). Conclusions:The mechanism of O 3 in treating neuropathic pain may be through inhibition of iron death.