Effect of diosmetin on ferroptosis of hippocampal cells in rats with bacterial meningitis and the mechanism of SIRT1-Nrf2 signaling pathway / 中华行为医学与脑科学杂志

ABSTRACT

Objective:To explore the effect and mechanism of diosmetin (Dio) on neuronal ferroptosis in rats with bacterial meningitis (BM).Methods:Male SD rats aged 6-7 weeks of SPF grade were selected for the experiment. The BM model was established by injecting group B hemolytic streptococcus into the cisterna magna of cerebellum. Sixty BM model rats were successfully modeled and divided into model group, low-dose Dio group, medium-dose Dio group, high-dose Dio group and inhibitor group according to the random number table method, with 12 rats in each group. Another 12 weight-matched rats were taken as the control group.The rats in the low-dose Dio group, medium-dose Dio group, high-dose Dio group and the inhibitor group were intragastrically administered with Dio at 50 mg/kg, 100 mg/kg, 200 mg/kg and 200 mg/kg, respectively. The rats in the control group were intragastrically administered with an equal volume of 0.9 % sodium chloride solution. On the day of intragastric administration, the rats in the inhibitor group were intraperitoneally injected with SIRT1 pathway inhibitor EX527 (10 mg/kg), and the rats in the other groups were injected with an equal volume of 0.9% sodium chloride solution. The above interventions were performed once a day for 28 consecutive days. Loeffler neurological score was used to evaluate the neurological impairment in rats. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cerebrospinal fluid of rats were detected by ELISA. The number of white blood cells in cerebrospinal fluid was detected by a blood cell analyzer. Glutathione (GSH) was detected by micro-enzyme labeling method, malondialdehyde (MDA) was detected by thiobarbituric acid colorimetric method, reactive oxygen species(ROS) was detected by colorimetry, and Fe 2+ level was detected by ferrozine method. Hematoxylin-eosin staining, Prussian blue staining and TUNEL staining were used to observe the pathological damage, iron accumulation and apoptosis in the hippocampus, respectively.Western blot was applied to measure the expression of transferrin (Tf), proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (Bax), caspase-3 and SIRT1/Nrf2/HO-1/Gpx4 signaling pathway proteins. Graphpad Prism 9.0 was used for data analysis. One-way ANOVA was used for statistical analysis, and SNK- q test was used for further pairwise comparisons. Results:(1) There was a statistically significant difference in neurological function scores among the 6 groups of rats ( F=125.451, P<0.001). The neurological function score of the model group was lower than that of control group, while the neurological function scores of the low-dose Dio group, medium-dose Dio group, and high-dose Dio group were higher than those of the model group (all P<0.05). The neurological function score of the inhibitor group ((2.57±0.26)) was lower than that of high-dose Dio group ((4.34±0.48)) ( P<0.05). (2) There were statistically significant differences in the levels of IL-6, TNF-α and the number of white blood cells in the cerebrospinal fluid of rats among the 6 groups ( F=127.817, 102.413, 180.967, all P<0.001). The levels of IL-6, TNF-α and the number of white blood cells in model group were higher than those of control group(all P<0.05). The levels of IL-6, TNF-α and the number of white blood cells in low-dose Dio group, medium-dose Dio group and high-dose Dio group were lower than those of model group (all P<0.001), and those in inhibitor group were all higher than those in high-dose Dio group(all P<0.001). (3) There were statistically significant differences in iron deposition rate and neuronal apoptosis rate among the 6 groups of rats ( F=90.857, 88.835, both P<0.001). The iron deposition rate ((18.37±3.14)%) and neuronal apoptosis rate ((27.58±2.63)%) in the inhibitor group were higher than those in the high-dose Dio group ((6.35±1.08)%, (14.02±1.87)%) (both P<0.05). (4) The levels of GSH, ROS, MDA, and Fe 2+ in the hippocampus of the 6 groups of rats showed statistically significant differences ( F=54.465, 106.453, 55.969, 105.457, all P<0.001). The GSH content in the inhibitor group ((103.48±8.76) mmol/g) was lower than that in the high-dose Dio group ((133.97±10.54) mmol/g), while the contents of ROS, MDA, Fe 2+ ((225.17±16.32) μmol/mg, (10.73±1.58) μmol/mg, (62.71±5.43) μg/g) were higher than those of the high-dose Dio group ((131.87±11.67) μmol/mg, (4.35±0.87) μmol/mg, (34.86±2.95) μg/g) (all P<0.05). (5)There were statistically significant differences in the protein levels of Tf, PCNA, Bax, caspase-3, SIRT1, Nrf2, HO-1 and Gpx4 in the hippocampus of the 6 groups of rats ( F=120.179, 107.568, 157.265, 98.031, 90.932, 52.283, 59.424, 114.539, all P<0.001). The protein levels of Tf, Bax and caspase-3 in the hippocampus of inhibitor group were higher than those of the high-dose Dio group, while the protein levels of PCNA, SIRT1, Nrf2, HO-1, Gpx4 were lower than those of the high-dose Dio group (all P<0.05). Conclusion:Diosmetin can activate SIRT1/Nrf2/HO-1/Gpx4 signaling pathway, thereby inhibiting neuronal ferroptosis in BM rats.