丹参多酚酸对抑郁模型大鼠行为及脑组织炎性因子的影响

Effect of salvianolic acid on behaviors and brain inflammatory factors in depression model rats

目的:探讨丹参多酚酸对慢性温和应激(chronic mild stress,CMS)致抑郁模型大鼠抑郁样行为的影响及其机制。方法:将50只健康雄性清洁级SD大鼠按照随机数字表法分为5组:对照组(nCMS+Nal组)、CMS+生理盐水组(CMS+Nal组)、CMS+氟西汀组(CMS+Flu组)、CMS+丹参多酚酸组(CMS+Sal组)、CMS+氟西汀+丹参多酚酸组(CMS+Flu+Sal组),每组10只。除对照组外,其余4组均接受持续21 d的CMS造模。CMS造模21 d后,按照分组分别给大鼠腹腔注射0.9 %生理盐水(10 mg·kg -1·d -1)、氟西汀(20 mg·kg -1·d -1)、丹参多酚酸(40 mg·kg -1·d -1)、氟西汀(20 mg·kg -1·d -1)+丹参多酚酸(40 mg·kg -1·d -1),连续21 d,给药期间,后4组大鼠继续给予CMS干预。分别于基线水平(第0天)、造模后(第21天)、干预后(第42天)进行强迫游泳和糖水偏爱实验评估抑郁样行为,之后处死大鼠取前额叶皮质和海马,采用RT-qPCR检测Toll样受体4(Toll-like receptor 4,TLR4)和髓样分化因子88(myeloid differentiation primary response 88,MyD88)mRNA的水平。采用Luminex技术检测细胞因子白介素1β(interleukin-1β,IL-1β)、白介素2(interleukin-2,IL-2)、干扰素γ(interferon-γ,IFN-γ)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)水平。采用SPSS 21.0进行数据分析,行为学数据进行重复测量方差分析,分子指标数据用单因素方差分析,Spearman做相关分析。 结果:重复测量方差分析结果显示,在基线水平、造模后和干预后五组大鼠的体质量、糖水偏爱率、强迫游泳静止不动时间的组别与时间的交互作用显著( F=18.238, 6.921,7.591,均 P<0.05)。造模后,与nCMS+Nal组相比,CMS+Flu、CMS+Sal、CMS+Flu+Sal和CMS+Nal组4组大鼠体质量降低、糖水偏爱率降低、强迫游泳静止不动时间增加(均 P<0.05);干预后,与CMS+Nal组相比[体质量(350.15±41.65)g,糖水偏爱率(52.95±11.13)%,静止不动时间(91.40±15.22)s],CMS+Flu、CMS+Sal、CMS+Flu+Sal和nCMS+Nal组大鼠体质量更重[(378.21±30.78)g,(385.12±43.19)g,(391.41±31.21)g,(402.33±18.67)g,均 P<0.05]、糖水偏爱率高[(69.30±15.56)%,(68.12±10.99)%,(71.18±9.51)%,(75.47±11.55)%,均 P<0.05],而强迫游泳静止不动时间降低[(68.81±21.74)s,(66.10±25.51)s,(63.53±22.32)s,(71.21±21.41)s,均 P<0.05]。干预后,CMS+Flu组、CMS+Sal组、CMS+Flu+Sal组三组间体质量、糖水偏爱率和静止不动时间两两比较均差异无统计学意义(均 P>0.05)。干预后,CMS+Flu组、CMS+Sal组、CMS+Flu+Sal组和nCMS+Nal组大鼠前额叶皮质和海马中TLR4 mRNA、MyD88 mRNA的表达水平均低于CMS+Nal组(均 P<0.05)。在前额叶皮质部位,CMS+Flu+Sal组的TLR4 mRNA(0.715±0.358)、MyD88 mRNA(0.739±0.233)表达均低于CMS+Sal组(1.943±0.606,1.815±0.897)(均 P<0.05)。大鼠前额叶皮质与海马部位TLR4 mRNA的表达水平与MyD88 mRNA水平、TNF-α水平、强迫游泳静止不动时间呈正相关,与糖水偏爱率呈负相关(前额叶皮质 r=0.915,0.041,0.027,-0.178;海马 r=0.810,0.070,0.011,-0.153;均 P<0.05)。 结论:丹参多酚酸改善CMS导致的大鼠抑郁样行为,这可能与其抑制脑内TLR4/MyD88信号通路介导的免疫炎性反应有关。

Objective To investigate the effect of salvianolic acid on depressive behavior in depression model rats induced by chronic mild stress(CMS)and its mechanism.Methods Fifty healthy male clean grade Sprague-Dawley(SD)rats were divided into five groups according to a random number table with 10 in each group:control group(nCMS+Nal group),CMS+normal saline group(CMS+Nal group),CMS+fluoxetine group(CMS+Flu group),CMS+salvia acid group(CMS+Sal group),CMS+fluoxetine+Salvia acid group(CMS+Flu+Sal group).Except the control group,the rats in the other four groups were all received CMS modeling for 21 days.Twenty-one days after CMS modeling,rats were intraperitoneally injected with 0.9%normal saline(10 mg·kg-1·d-1),fluoxetine(20 mg·kg-1·d-1),salvia acid(40 mg·kg-1·d-1),fluoxetine(20 mg·kg-1·d-1)+salvia acid(40 mg·kg-1·d-1)for 21 days.During the administration period,rats in the other four groups continued to receive CMS intervention for 21 days.Forced swimming test and sucrose preference test were conducted at baseline(day 0),after modeling(day 21)and after intervention(day 42)so as to evaluate depression like behavior.Then the rats were sacrificed and the hippocampus and prefrontal cortex were taken.The mRNA levels of Toll like receptor 4(TLR4)and myeloid differentiation primary response 88(MyD88)were detected by RT-qPCR.The cytokines including interleukin-1β(IL-1β),interleukin-2(IL-2),interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)were detected by Luminex technique.SPSS 21.0 was used for statistical analysis.Repeated measurement ANOVA was used for behavioral data analysis,one-way ANOVA was used for molecular index data analysis,and Spearman was used for correlation analysis.Results The results of repeated measurement ANOVA showed that the interaction effects between group and time of body mass,sucrose preference,forced swimming immobility time were significant at baseline,after modeling and after intervention(F=18.238,6.921,7.591,all P<0.05).After modeling,compared with nCMS+Nal group,the rats in CMS+Flu group,CMS+Sal group,CMS+Flu+Sal group and CMS+Nal group had lower body weight,lower sucrose preference rate and longer forced swimming immobility time(all P<0.05).After intervention,compared with CMS+Nal group(body weight(350.15±41.65)g,sucrose preference(52.95±11.13)%,static time(91.40±15.22)s),the body weight((378.21±30.78)g,(385.12±43.19)g,(391.41±31.21)g,(402.33±18.67)g,all P<0.05)and sucrose preference((69.30±15.56)%,(68.12±10.99)%,(71.18±9.51)%,(75.47±11.55)%,all P<0.05)of CMS+Flu group,CMS+Sal group,CMS+Flu+Sal group and nCMS+Nal group were all increased,while the forced swimming immobility time((68.81±21.74)s,(66.10±25.51)s,(63.53±22.32)s,(71.21±21.41)s,all P<0.05)were shorter(all P<0.05).After intervention,among the body weight,sucrose preference and the immobility time of CMS+Flu group、CMS+Sal group and CMS+Flu+Sal group,there were no differences between each two groups(all P>0.05).After intervention,the levels of TLR4 mRNA and MyD88 mRNA in prefrontal cortex and hippocampus of CMS+Flu group,CMS+Sal group,CMS+Flu+Sal group and nCMS+Nal group were all lower than those in CMS+Nal group(all P<0.05).In prefrontal cortex,the levels of TLR4 mRNA(0.715±0.358)and MyD88 mRNA(0.739±0.233)in CMS+Flu+Sal group were lower than those in CMS+Sal group(1.943±0.606,1.815±0.897)(both P<0.05).The level of TLR4 mRNA in prefrontal cortex and hippocampus of rats were positively correlated with the level of MyD88 mRNA and TNF-αlevel and forced swimming immobility time and negatively correlated with sucrose preference rate(prefrontal cortex r=0.915,0.041,0.027,-0.178,all P<0.05;hippocampus r=0.810,0.070,0.011,-0.153,all P<0.05).Conclusion The antidepressant effect of salvianolic acid is presumedly achieved by inhibiting the immunoinflammatory response mediated by the TLR4/Myd88 signaling pathway in CMS rats.