不同剂量柴胡加龙骨牡蛎汤对心肌梗死合并焦虑的大鼠心功能、行为学及炎症因子表达的影响

Effects of Different Doses of Chaihu plus Longgu Muli Decoction on Cardiac Function,Behavior,and Expression of Inflammatory Cytokines in Rats with Myocardial Infarction and Anxiety

目的 观察不同剂量柴胡加龙骨牡蛎汤(Chaihu plus longgu muli decoction, CHLM)对心肌梗死(Acute Myocardial Infarction, AMI)合并焦虑的大鼠心功能、行为学、血清内毒素(Lipopolysaccharides, LPS)及炎症因子的影响,筛选最佳干预剂量,为后续研究提供依据。方法 将45只雄性SD大鼠随机分为假手术组,模型组,中药低、中、高剂量组,每组各9只。其中模型组,中药低、中、高剂量组接受为期21 d的空瓶刺激焦虑造模,在第14天时模型组及中药低、中、高剂量组接受冠状动脉前降支结扎术形式的心梗造模。假手术组正常饮水,第14天仅挂线,不结扎。术后第2天,中药低、中、高剂量组分别予柴胡加龙骨牡蛎汤6.8 g/(kg·d)、13.6 g/(kg·d)、27.2 g/(kg·d)灌胃。模型组及假手术组灌胃等体积生理盐水。用旷场实验及高架十字迷宫实验检测大鼠行为学指标(垂直运动分数、水平运动分数、开放臂进入次数占比)(The percentage of open arm entry, OE%)、开放臂时间占比(The percentage of open arm time, OT%)、用心脏超声检测大鼠左室舒张末期容积(Left ventricular end-diastolic volume, LVEDV)、左室收缩末期容积(Left ventricular end-systolic volume, LVESV)、左室射血分数(Left ventricular injection fraction, LVEF)、左室缩短分数(Left ventricular fraction shortening, LVFS)。用心肌组织病理染色客观展示各组心肌细胞炎性浸润及胶原纤维沉积情况。用酶联免疫吸附测定法(Enzyme-linked immunosorbent assay, ELISA)测定血清LPS及白细胞介素-6(Interleukin-6,IL-6)、白细胞介素-1β(Interleukin-1β,IL-1β)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)等炎性因子水平。结果 与假手术组比较,模型组OE%、OT%明显降低,差异有统计学意义(P<0.05,P<0.01)。与模型组比较,中药中剂量组OE%、OT%升高,差异有统计学意义(P<0.05,P<0.01)。与假手术组比较,模型组垂直运动评分、水平运动评分明显降低,差异有统计学意义(P<0.01)。与模型组比较,中药中剂量组垂直运动评分、水平运动评分明显升高,差异有统计学意义(P<0.01)。与假手术组比较,模型组LVESV明显升高(P<0.01),LVEF、LVFS占比明显降低,差异有统计学意义(P<0.01)。与模型组比较,中药中剂量组LVEF、LVFS升高,差异有统计学意义(P<0.05),中药高剂量组LVEF、LVFS升高,差异有统计学意义(P<0.05),中药低剂量组LVEF明显升高,差异有统计学意义(P<0.01)。与假手术组比较,模型组心肌纤维排列不规则、肌纤维不连续、心肌细胞数量减少、核碎裂。中药各组大鼠心肌细胞结构破坏较模型组轻、心肌细胞数量较模型组增多,中药中剂量组细胞结构破坏相对较轻。与假手术组比较,模型组大鼠心肌细胞排列紊乱、间质可见大量胶原纤维及炎性浸润;与模型组比较,中药各组大鼠心肌细胞排列紊乱程度较轻,胶原纤维沉积较少。与假手术组比较,模型组LPS、TNF-α升高(P<0.05),IL-1β、IL-6明显升高(P<0.01)。与模型组比较,中药中剂量组LPS、IL-1β、TNF-α降低(P<0.05),IL-6明显降低(P<0.01);中药低、高剂量组IL-1β降低(P<0.05)、IL-6明显降低(P<0.01)。结论 柴胡加龙骨牡蛎汤中剂量组可以更好地改善心肌梗死合并焦虑大鼠的心功能、焦虑行为及下调炎症因子表达。

Objective To observe the effects of different doses of Chaihu plus Longgu Muli Decoction(CHLM)on cardiac function,behavior,and serum levels of lipopolysaccharide(LPS)and inflammatory cytokines in the rats with acute myocardial infarction(AMI)and anxiety,and to determine the best intervention dose for the follow-up study.Methods Forty-five male SD rats were randomly assigned into a sham operation group,a model group,and low-,medium-,and high-dose CHLM groups(n=9).The other groups except the sham operation group underwent anxiety modeling by empty bottle stimulation for 21 days.On day 14,the modeling of myocardial infarction was carried out by anterior descending coro-nary artery ligation.The sham operation group had free access to water and received only line hanging without ligation on 14th day.On day 2 after operation,CHLM was administrated by gavage at 6.8,13.6,and 27.2 g/(kg·d),respectively,and the model group and sham operation group were administrated with an equal volume of normal saline by gavage.Open field test and elevated plus-maze test were carried out to evaluate the rat behavior based on the crossing and rearing scores,the percentage of open arm entry(OE%),the percentage of open arm time(OT%).Echocardiography was em-ployed to measure the left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV),left ventricular ejection fraction(LVEF),and left ventricular fraction shortening(LVFS).Pathological staining was em-ployed to reveal the inflammatory infiltration and collagen deposition of myocardial cells in the myocardial tissue.Enzyme-linked immunosorbent assay was employed to determine the serum levels of LPS,interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α).Results Compared with the sham operation group,the model group showed reduced OE%and OT%(P<0.05,P<0.01).Compared with those in the model group,the OE%and OT%were increased in the medium-dose CHLM group(P<0.05,P<0.01).Compared with the sham operation group,the model group showed increased LVESV(P<0.01)and reduced LVEF and LVFS(P<0.01).Compared with the model group,the medium-and high-dose CHLM increased the LVEF and LVFS(P<0.05)and the low-dose CHLM in-creased LVEF(P<0.01).Compared with the sham operation group,the model group presented disarrangement of myocar-dial fibers,discontinuous myofibers,decreased myocardial cells,karyorrhexis,and collagen fiber deposition and inflammato-ry infiltration in the interstitial space.The CHLM groups showed slighter structural damage of myocardial cells,more myo-cardial cells,and less deposition of collagen fibers than the model group.The structural damage of cells in the medium-dose CHLM group was mild.Compared with the sham operation group,the model group had elevated levels of LPS(P<0.05),TNF-α(P<0.05),IL-1β(P<0.01),and IL-6(P<0.01).Compared with the model group,the medium-dose CHLM group showed declined levels of LPS(P<0.05),IL-1β(P<0.05),TNF-α(P<0.05),and IL-6(P<0.01);the low-and high-dose groups showed declined levels of IL-1β(P<0.05)and IL-6(P<0.01).Conclusion Medium-dose CHLM can improve the cardiac function,mitigate the anxiety behavior,and down-regulate the expression of inflammatory cytokines in the rats with myocardial infarction and anxiety.