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.

ObjectiveTo investigate the protective effect of folic acid against cholestatic liver injury in mice induced by bis（2-ethylhexyl） phthalate （DEHP） exposure and its mechanism. MethodsICR 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. ResultsCompared 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）. ConclusionFolic 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.