芍药苷干预对脓毒症大鼠心肌损伤的影响

Paeoniflorin improves myocardial injury via inhibition of Src/VE-cadherin pathway in septic rats

目的研究芍药苷干预对脓毒症大鼠心肌损伤的影响。方法采用随机数字表法将SD大鼠分为4组, 每组10只。(1)对照组:大鼠腹腔注射1.4 ml生理盐水;(2)二甲基亚砜组:大鼠腹腔注射5%二甲基亚砜溶液1.4 ml;(3)脓毒症模型组:大鼠腹腔注射1.4 ml生理盐水, 1 h后腹腔注射0.1 ml(5 mg/kg)脂糖造模;(4)芍药苷干预组:大鼠腹腔注射1.4 ml芍药苷(70 mg/kg), 1 h后腹腔注射0.1 ml(5 mg/kg)脂多糖造模。24 h后处死4组大鼠。酶联免疫吸附测定(ELISA)法测4组大鼠血清心肌肌钙蛋白I(cTnI)及心肌组织中肿瘤坏死因子α(TNFα)、白细胞介素(IL)-6、IL-1β、趋化因子C-X-C基序配体1(CXCL1)、趋化因子C-X-C基序配体2(CXCL2)、血管细胞黏附分子-1(VCAM-1)水平, 伊文思蓝法测4组大鼠心肌组织中伊文思蓝含量;实时荧光定量聚合酶链式反应法测4组大鼠心肌组织中TNFα、IL-6、IL-1β、CXCL1、CXCL2、VCAM-1mRNA表达;Western-Blot法测血管内皮钙黏蛋白、丝裂原活化蛋白激酶p38(p38MAPK)、磷酸化p38MAPK(P-p38MAPK)、磷酸化Src蛋白(P-Src)、Ras相关C3肉毒素底物1(Rac1)蛋白表达。结果与对照组比, 脓毒症模型组cTnI增高[(312.9±17.9)pg/ml比(174.4±17.7)pg/ml, P<0.05], 伊文思蓝含量上升[(23.8±2.9)μg/g 比(5.2±2.0)μg/g, P<0.05], 心肌炎性细胞浸润明显;与脓毒症模型组比, 芍药苷干预组cTnI明显降低[(227.7±15.9)pg/ml, P<0.05], 伊文思蓝含量显著下降[(13.2±2.3)μg/g, P<0.05], 心肌炎性细胞浸润减轻。与对照组比, 脓毒症模型组TNFα、IL-6、IL-1β、CXCL1、CXCL2、VCAM-1增高;与脓毒症模型组比, 芍药苷干预组TNFα[(63.39±9.55)pg/ml 比(126.54±19.17)pg/ml, P<0.05]、IL-6[(64.03±8.82)pg/ml 比(85.60±9.52)pg/ml, P<0.05]、IL-1β[(69.52±9.23)pg/ml比(130.45±15.10)pg/ml, P<0.05]、CXCL1[(2 600.19±379.54)pg/ml比(4 903.89±533.42)pg/ml, P<0.05]、CXCL2[(93.71±10.83)pg/ml比(127.24±13.92)pg/ml, P<0.05]、VCAM-1[(112.22±13.49)pg/ml 比(149.32±15.65 pg/ml), P<0.05]显著下降。与对照组比, 脓毒症模型组TNFα、IL-6、IL-1β、CXCL1、CXCL2、VCAM-1 mRNA表达增高;与脓毒症模型组比, 芍药苷干预组IL-6(相对表达量1.271±0.139 比 1.920±0.191, P<0.05)、IL-1β(相对表达量1.180±0.130 比 1.817±0.191, P<0.05)、VCAM-1(相对表达量1.088±0.144 比 1.460±0.166, P<0.05)mRNA表达降低。与对照组比, 脓毒症模型组P-p38MAPK、P-Src表达增加, 血管内皮钙黏蛋白表达降低。与脓毒症模型组比, 芍药苷干预组p38MAPK(相对表达量1.125±0.078 比 1.520±0.164, P<0.05)、P-p38MAPK(相对表达量 1.639±0.133 比 2.112±0.227, P<0.05)表达均明显下降, 血管内皮钙黏蛋白表达升高。结论芍药苷能改善脓毒症大鼠心脏微血管内皮通透性, 抑制炎症相关蛋白及基因的分泌和表达, 可能与芍药苷抑制Src/血管内皮钙黏蛋白通路有关。

Objective To study the protective effect and mechanism of paeoniflorin(pae)on myocardial injury in septic rats.Methods Sprague-Dawley(SD)rats were randomly divided into 4 groups with 10 rats in each group.Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml 5%dimethyl sulfoxide(DMSO)solution independently in control group and DMSO group.Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml pae independently,then with 0.1 ml lipopolysaccharide(LPS)1 hour later in sepsis group and pae group.Enzyme linked immunosorbent assay(ELISA)was used to detect serum cardiac troponin I(cTnI)levels and myocardial tissue tumor necrosis factor alpha(TNFα),interleukin(IL)-6,IL-1β,chemokine(C-X-C motif)ligand 1(CXCL1),chemokine(C-X-C motif)ligand 2(CXCL2),vascular cell adhesion molecule 1(VCAM-1)levels.Evans blue(EB)method was used to detect the EB content of myocardial tissue.HE staining method was used to observe the pathological changes,real-time quantitative polymerase chain reaction(RT-qPCR)to detect mRNA expression levels of the above molecules,and Western-blot to detect vascular endothelium-cadherin(VE-cadherin),phosphorylated p38 mitogen-activated protein kinase(P-p38MAPK),phosphorylated Src protein(P-Src),Ras-Related C3 Botulinum Toxin Substrate 1(Rac1)levels.Results Compared with control group,cTnI level and the EB content in sepsis group increased significantly,and the myocardial inflammatory cell infiltration was obvious.The cTnI level and EB content in pae group were significantly reduced,and myocardial inflammatory cell infiltration was reduced[cTnI:(227.7±15.9)pg/ml vs.(312.9±17.9)pg/ml;EB:(13.2±2.3)μg/g vs.(23.8±2.9)μg/g;P<0.05].Compared with control group,the levels of TNFα,IL-6,IL-1β,CXCL1,CXCL2,and VCAM-1 in sepsis group were increased.Compared with sepsis group,the above-mentioned molecular levels of pae group were significantly decreased[TNFα:(63.39±9.55)pg/ml vs.(126.54±19.17)pg/ml;IL-6:(64.03±8.82)pg/ml vs.(85.60±9.52)pg/ml;IL-1β:(69.52±9.23)pg/ml vs.(130.45±15.10)pg/ml;CXCL1:(2600.19±379.54)pg/ml vs.(4903.89±533.42)pg/ml;CXCL2:(93.71±10.83)pg/ml vs.(127.24±13.92)pg/ml;VCAM-1:(112.22±13.49)pg/ml vs.(149.32±15.65)pg/ml,both P<0.05].RT-qPCR results showed that the mRNA expressions of TNFα,IL-6,IL-1β,CXCL1,CXCL2 and VCAM-1 in the sepsis group were increased compared with the control group;Compared with sepsis group,the IL-6 mRNA(1.271±0.139 vs.1.920±0.191,P<0.05),IL-1βmRNA(1.180±0.130 vs.1.817±0.191,P<0.05),VCAM-1 mRNA(1.088±0.144 vs.1.460±0.166,P<0.05)expression decreased significantly in the pae group.Compared with control group,the levels of P-p38MAPK and P-Src in sepsis group increased,and the level of VE-cadherin decreased.Compared with sepsis group,the levels of p38MAPK and P-p38MAPK in pae group were significantly decreased,and the level of VE-cadherin was increased(p38MAPK/β-actin:1.125±0.078 vs.1.520±0.164;P-p38MAPK protein:1.639±0.133 vs.2.112±0.222;both P<0.05).Conclusion Paeoniflorin could improve the permeability of cardiac microvascular endothelium in sepsis rats and inhibit the secretion and expression of inflammation-related proteins and genes,which might be related to the inhibition of Src/VE-cadherin pathway by paeoniflorin.