丹酚酸B对脓毒症小鼠血管平滑肌细胞炎症反应的影响:circACTA2在其中的作用
Effect of salvianolic acid B on inflammatory responses of vascular smooth muscle cells in septic mice:role of circACTA2
张曼莉 1赵景茹 2尹化 1张曼娜 3宋璇 1佟飞1
作者信息
- 1. 河北医科大学第二医院重症医学科,石家庄 050000
- 2. 河北省人民医院神经内科,石家庄 050051
- 3. 河北医科大学第二医院检验科,石家庄 050000
- 折叠
摘要
目的 评价丹酚酸B(Sal B)对脓毒症小鼠血管平滑肌细胞(VSMC)炎症反应的影响及circACTA2在其中的作用.方法 在体实验:健康雄性C57BL/6小鼠81只,6～8周龄,采用随机数字表法分为3组(n=27):假手术组、脓毒症组和Sal B组.采用盲肠结扎穿孔法制作脓毒症模型.模型制备成功后,Sal B组腹腔注射Sal B 7 mg/kg,1次/d,连续2 d.每组随机取20只小鼠用于造模后7 d内测定SBP、DBP、MAP、全血乳酸(Lac)浓度,记录生存情况.造模后48 h时每组随机取7只小鼠,取动脉血管组织,采用免疫荧光染色法测定IL-1β表达,分别采用Western blot法和qRT-PCR法测定IL-1β、TNF-α、IL-6及其mRNA的表达,采用qRT-PCR法测定circACTA2的表达.细胞实验:小鼠动脉VSMC培养后,采用随机数字表法分为6组(n=3):对照组(C组)、LPS组、Sal B组、si-circACTA2+C 组、si-circACTA2+LPS 组和 si-circACTA2+Sal B 组.LPS 组采用 LPS(终浓度1 μg/ml)、Sal B 组采用 LPS(终浓度 1 μg/ml)和 Sal B(终浓度 5 µmol/L)孵育 24 h.si-circACTA2+C组仅用 si-circACTA2 转染 VSMC.si-circACTA2 转染 VSMC 24 h 后,si-circACTA2+LPS 组用 LPS(终浓度 1 µg/ml)、si-circACTA2+Sal B 组采用 LPS(终浓度 1 µg/ml)和 Sal B(终浓度 5 µmol/L)孵育24 h.分别采用Western blot法和qRT-PCR法测定IL-1β、TNF-α、IL-6及其mRNA的表达,采用qRT-PCR法测定circACTA2的表达.结果 在体实验:与假手术组相比,脓毒症组SBP、DBP及MAP降低,全血Lac浓度升高,7 d生存率降低,动脉血管组织IL-1β、TNF-α和IL-6及其mRNA表达上调,circACTA2表达下调(P＜0.05),IL-1β荧光增强.与脓毒症组相比,Sal B组SBP、DBP及MAP升高,全血Lac浓度降低,7 d生存率升高,动脉血管组织IL-1β、TNF-α和IL-6及其mRNA表达下调,circACTA2表达上调(P＜0.05),IL-1β荧光减弱.细胞实验:与C组相比,LPS组IL-1 β、TNF-α和IL-6及其mRNA表达上调,circACTA2表达下调(P＜0.05).与LPS组相比,Sal B组IL-1β、TNF-α和IL-6及其 mRNA 表达下调,circACTA2 表达上调(P＜0.05).与 si-circACTA2+C 组相比,si-circACTA2+LPS组 IL-1β、TNF-α 和 IL-6 及其 mRNA 表达上调(P＜0.05).与 si-circACTA2+LPS 组相比,si-circACTA2+Sal B组IL-1β、TNF-α和IL-6及其mRNA表达差异无统计学意义(P＞0.05).结论 Sal B可减轻脓毒症小鼠VSMC炎症反应,机制可能与促进circACTA2表达有关.
Abstract
Objective To evaluate the effect of Salvianolic acid B(Sal B)on the inflammatory re-sponses of vascular smooth muscle cells(VSMCs)in septic mice and the role of circACTA2.Methods In vivo experiment Eighty-one healthy male C57BL/6 mice,aged 6-8 weeks,were divided into 3 groups(n=27 each)by a random number table method:sham operation group,sepsis group and Sal B group.Sep-sis model was developed by cecal ligation and puncture.After sucessful preparation of the model,Sal B 7 mg/kg/d was intraperitoneally injected once a day for 2 consecutive days in Sal B group.Twenty mice in each group were randomly selected to measure systolic blood pressure(SBP),diastolic blood pressure(DBP),mean arterial pressure(MAP)and whole blood lactic acid(Lac)and to record the survival within 7 days after developing the model.Seven mice in each group were randomly selected at 48 h after developing the model,and the arterial vascular tissues were collected for determination of the expression of interleukin-1beta(IL-1β)(by immunofluorescence staining),expression of IL-1β,tumor necrosis factor-alpha(TNF-α)and IL-6 protein and mRNA(by Western blot and quantitative real-time polymerase chain reaction,re-spectively),and expression of circACTA2(by quantitative real-time polymerase chain reaction).Cell ex-periment Mouse VSMCs were cultured and divided into 6 groups(n=3 each)by a random number table method:control group(C group),lipopolysaccharide(LPS)group,Sal B group,si-circACTA2+C group,si-circACTA2+LPS group,and si-circACTA2+Sal B group.The cells were incubated for 24 h with LPS(fi-nal concentration 1 μg/ml)in LPS group and with LPS(final concentration 1 μg/ml)and Sal B(final con-centration 5 μmol/L)in Sal B group.VSMCs were transfected with si-circACTA2 only in si-circ ACTA2+C group.At 24 h after transfection of si-circACTA2 into VSMCs,the cells were incubated with LPS(final con-centration 1 µg/ml)in si-circACTA2+LPS group and with LPS(final concentration 1 µg/ml)and Sal B(final concentration 5 μmol/L)for 24 h in si-circACTA2+Sal B group.The expression of IL-1β,TNF-α and IL-6 protein and mRNA was detected using Western blot and quantitative real-time polymerase chain reac-tion,and the expression of circACTA2 was determined by the quantitative real-time polymerase chain reac-tion.Results In vivo experiment Compared with sham operation group,SBP,DBP and MAP were signif-icantly decreased,the concentrations of whole blood Lac were increased,7-day survival rate was decreased,the expression of IL-1β,TNF-α and IL-6 protein and mRNA in arterial vascular tissues was up-regulated,circACTA2 expression was down-regulated(P＜0.05),and the fluorescence of IL-1β was enhanced in sep-sis group.Compared with sepsis group,SBP,DBP and MAP were significantly increased,whole blood Lac concentrations were decreased,7-day survival rate was increased,the expression of IL-1β,TNF-α and IL-6 protein and mRNA in arterial vascular tissues was down-regulated,the expression of circACTA2 was up-reg-ulated(P＜0.05),and the fluorescence of IL-1β was weakened in Sal B group.Cell experiment Compared with group C,the expression of IL-1β,TNF-α and IL-6 protein and mRNA was significantly up-regulated,and the expression of circACTA2 was down-regulated in LPS group(P＜0.05).Compared with LPS group,the expression of IL-1β,TNF-α and IL-6 protein and mRNA was significantly down-regulated,and the ex-pression of circACTA2 was up-regulated in Sal B group(P＜0.05).Compared with si-circACTA2+C group,the expression of IL-1β,TNF-α and IL-6 protein and mRNA was significantly up-regulated in si-circACTA2+LPS group(P＜0.05).There were no significant differences in the expression of IL-1β,TNF-α and IL-6 pro-tein and mRNA between si-circACTA2+LPS group and si-circACTA2+Sal B group(P＞0.05).Conclusions Sal B can reduce the inflammatory responses of VSMCs,and the mechanism may be related to promoting the expression of circACTA2 in septic mice.
关键词
丹参酸B/RNA,环状/脓毒症/肌细胞,平滑肌/炎症Key words
Danshensuan B/RNA,circular/Sepsis/Myocytes,smooth muscle/Inflam-mation引用本文复制引用
基金项目
国家自然科学基金青年科学基金(82001229)
河北省自然科学基金-中医药联合基金培育项目(H2023206276)
河北省政府资助临床医学优秀人才培养项目(ZF2023113)
出版年
2024
NETL
NSTL
万方数据