香叶木素对细菌性脑膜炎模型大鼠海马细胞铁死亡的影响及SIRT1-Nrf2信号通路机制

Effect of diosmetin on ferroptosis of hippocampal cells in rats with bacterial meningitis and the mechanism of SIRT1-Nrf2 signaling pathway

目的:探究香叶木素(diosmetin,Dio)对细菌性脑膜炎(bacterial meningitis,BM)大鼠神经元铁死亡的影响及作用机制。方法:选用SPF级6~7周龄雄性SD大鼠,采用大鼠小脑延髓池注射B族溶血性链球菌法建立BM模型,将建模成功的60只BM模型大鼠按照随机数字表法分为模型组、Dio低、中、高剂量组和抑制剂组,每组12只。另取12只体质量相匹配的大鼠作为对照组。Dio低、中、高剂量组和抑制剂组大鼠分别按照50 mg/kg、100 mg/kg、200 mg/kg、200 mg/kg剂量的Dio灌胃,对照组则灌胃等体积0.9%氯化钠溶液,灌胃当天抑制剂组大鼠腹腔注射沉默信息调节因子1(silent mating type information regulation homolog 1,SIRT1)通路抑制剂EX527(10 mg/kg),其余各组大鼠则注射等体积0.9%氯化钠溶液。以上干预1次/d,连续28 d。采用Loeffler神经学评分评估大鼠神经功能损伤情况,ELISA法检测检测大鼠脑脊液中白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),血细胞分析仪检测脑脊液中白细胞数量,采用微量酶标法检测谷胱甘肽(glutathione,GSH)、硫代巴比妥酸显色法检测丙二醛(malondialdehyde,MDA)、比色法测定活性氧(reactive oxygen species,ROS)、亚铁嗪法检测Fe 2+水平,苏木精-伊红染色、普鲁士蓝染色和TUNEL染色分别观察海马组织病理损伤、铁积累及海马区细胞凋亡情况,Western blot检测转铁蛋白(transferrin,Tf)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、半胱氨酸蛋白酶3(caspase-3)及SIRT1/Nrf2/HO-1/Gpx4信号通路蛋白表达。使用Graphpad Prism 9.0进行数据分析,多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果:(1)6组大鼠神经功能评分差异有统计学意义(F=125.451,P<0.001)。模型组大鼠神经功能评分低于对照组,Dio低、中、高剂量组大鼠神经功能评分均高于模型组(均P<0.05),抑制剂组大鼠神经功能评分[(2.57±0.26)分]低于Dio高剂量组[(4.34±0.48)分](P<0.05)。(2)6组大鼠脑脊液中IL-6、TNF-α水平和白细胞数量均差异具有统计学意义(F=127.817,102.413,180.967,均P<0.001),模型组大鼠脑脊液中IL-6、TNF-α水平和白细胞数量均高于对照组(均P<0.05),Dio低、中、高剂量组大鼠脑脊液中IL-6、TNF-α水平和白细胞数量均低于模型组(均P<0.001),抑制剂组大鼠脑脊液中IL-6、TNF-α水平和白细胞数量均高于Dio高剂量组(均P<0.05)。(3)6组大鼠铁沉积比率和细胞凋亡率均差异有统计学意义(F=90.857,88.835,均P<0.001),抑制剂组大鼠铁沉积比率[(18.37±3.14)%]和细胞凋亡率[(27.58±2.63)%]均高于Dio高剂量组[(6.35±1.08)%,(14.02±1.87)%](均P<0.05)。(4)6组大鼠海马组织中GSH、ROS、MDA、Fe 2+含量均差异具有统计学意义(F=54.465,106.453,55.969,105.457,均P<0.001),抑制剂组大鼠GSH含量[(103.48±8.76)mmol/g]低于Dio高剂量组[(133.97±10.54)mmol/g],ROS、MDA、Fe 2+含量[(225.17±16.32)μmol/mg,(10.73±1.58)μmol/mg,(62.71±5.43)μg/g]高于Dio高剂量组[(131.87±11.67)μmol/mg、(4.35±0.87)μmol/mg,(34.86±2.95)μg/g](均P<0.05)。(5)6组大鼠海马组织中Tf、PCNA、Bax、caspase-3、SIRT1、Nrf2、HO-1、Gpx4蛋白表达均差异具有统计学意义(F=120.179,107.568,157.265,98.031,90.932,52.283,59.424,114.539,均P<0.001),抑制剂组大鼠海马组织中Tf、Bax、caspase-3蛋白表达水平高于Dio高剂量组,PCNA、SIRT1、Nrf2、HO-1、Gpx4蛋白表达水平低于Dio高剂量组(均P<0.05)。结论:香叶木素可以启动SIRT1/Nrf2/HO-1/Gpx4信号通路,从而抑制BM大鼠神经元铁死亡。

ObjectiveTo explore the effect and mechanism of diosmetin(Dio)on neuronal ferroptosis in rats with bacterial meningitis(BM).MethodsMale 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 Fe2+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(allP<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-αand 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-αand the number of white blood cells in model group were higher than those of control group(allP<0.05).The levels of IL-6,TNF-αand 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(allP<0.001),and those in inhibitor group were all higher than those in high-dose Dio group(allP<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)%)(bothP<0.05).(4)The levels of GSH,ROS,MDA,and Fe2+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,Fe2+((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(allP<0.05).ConclusionDiosmetin can activate SIRT1/Nrf2/HO-1/Gpx4 signaling pathway,thereby inhibiting neuronal ferroptosis in BM rats.