炎调方对巨噬细胞中HSP70表达及其效应的影响
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摘要
目的:观察炎调方药物血清对离体巨噬细胞中热休克蛋白70(HSP70)及其他相关细胞因子的调控效应,以验证炎调方治疗脓毒症的作用途径和位点。方法:培养大鼠巨噬细胞RAW 264.7,将培养后的巨噬细胞RAW 264.7分为空白对照组(RAW 264.7+培养液)、正常血清组(RAW 264.7+正常大鼠血清)、炎调方药物血清组(RAW 264.7+炎调方药物血清),每组设4个平行孔。采用MTT比色法测定细胞增殖,确定炎调方药物血清的无毒性剂量范围;采用qRT-PCR方法检测细胞HSP70、核因子-κB(NF-κB)、肿瘤坏死因子(TNF)-α、白介素(IL)-1β、IL-8的mRNA表达;采用Western blot方法检测细胞HSP70、NF-κB蛋白表达,以ELISA方法检测细胞TNF-α、IL-1β、IL-8蛋白表达。结果:炎调方药物血清的无毒性浓度范围包括10%~30%,故后续实验选取20%浓度的炎调方药物血清进行。正常血清组的各项指标与空白对照组比较,其差异均无统计学意义(P>0.05);与空白对照组和正常血清组比较,炎调方药物血清组RAW 264.7细胞的HSP70 mRNA和蛋白表达均明显升高(P<0.01),NF-κB、TNF-α、IL-1β、IL-8的mRNA和蛋白表达均明显降低(P<0.01)。结论:炎调方治疗脓毒症的作用机制可能是上调HSP70的表达,进而抑制NF-κB的活化和相关细胞因子的表达。
Objective: To observe the regulatory effects of drug medicated serum of "Yantiao Prescription"on the heat shock protein 70(HSP70) and other relevant cytokines in macrophages in vitro,and validate the pathway and sites of"Yantiao Prescription"in the treatment of sepsis. Methods: The rat macrophages RAW 264. 7 were cultured and divided into the blank control group treated with culture medium, normal serum group treated with serum from normal rats, and "Yantiao Prescription"drug medicated serum group treated with the drug medicated serum of "Yantiao Prescription",with 4 parallelhole for each group. The cell proliferation was tested by MTT colorimetric method to determine the dose ranges of drug medicated serum of"Yantiao Prescription"with no toxicity. The mRNA expressions of HSP70,NF-κB,TNF-α,IL-1β and IL-8 were detected by qRT-PCR,the protein expressions of HSP70 and NF-κB were detected by Western blot,and the expressions of TNF-α,IL-1β and IL-8 protein were detected by ELISA. Results: The dose ranges of drug medicated serum of"Yantiao Prescription"with no toxicity included 10%-30%,so the 20% concentration of "Yantiao Prescription"drug medicated serum was selected for the further experiment. There were no statistically significant differences on all indexes between the blank control group and normal serum group(P > 0. 05). Compared with the blank control group and normal serum group,the mRNA and protein expressions of HSP70 in the "Yantiao Prescription"drug medicated serum group were significantly increased(P < 0. 01),and the mRNA and protein expressions of NF-κB,TNF-α,IL-1β and IL-8 were obviously decreased(P < 0. 01). Conclusion: The mechanisms of "Yantiao Prescription"on sepsis may be associated with the upregulation of HSP70 expression,which further inhibit the activation of NF-κB and the expressions of relevant cytokines.
Objective: To observe the regulatory effects of drug medicated serum of "Yantiao Prescription"on the heat shock protein 70(HSP70) and other relevant cytokines in macrophages in vitro,and validate the pathway and sites of"Yantiao Prescription"in the treatment of sepsis. Methods: The rat macrophages RAW 264. 7 were cultured and divided into the blank control group treated with culture medium, normal serum group treated with serum from normal rats, and "Yantiao Prescription"drug medicated serum group treated with the drug medicated serum of "Yantiao Prescription",with 4 parallelhole for each group. The cell proliferation was tested by MTT colorimetric method to determine the dose ranges of drug medicated serum of"Yantiao Prescription"with no toxicity. The mRNA expressions of HSP70,NF-κB,TNF-α,IL-1β and IL-8 were detected by qRT-PCR,the protein expressions of HSP70 and NF-κB were detected by Western blot,and the expressions of TNF-α,IL-1β and IL-8 protein were detected by ELISA. Results: The dose ranges of drug medicated serum of"Yantiao Prescription"with no toxicity included 10%-30%,so the 20% concentration of "Yantiao Prescription"drug medicated serum was selected for the further experiment. There were no statistically significant differences on all indexes between the blank control group and normal serum group(P > 0. 05). Compared with the blank control group and normal serum group,the mRNA and protein expressions of HSP70 in the "Yantiao Prescription"drug medicated serum group were significantly increased(P < 0. 01),and the mRNA and protein expressions of NF-κB,TNF-α,IL-1β and IL-8 were obviously decreased(P < 0. 01). Conclusion: The mechanisms of "Yantiao Prescription"on sepsis may be associated with the upregulation of HSP70 expression,which further inhibit the activation of NF-κB and the expressions of relevant cytokines.
引文
[1]施荣,熊旭东,李淑芳,等.炎调方对脓毒症大鼠血清炎症因子的调控作用[J].中国中医急症,2012,21(3):397-398.
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[3]程应东,梁平,张朝军,等.HSP70基因转染对大鼠脓毒症早期外周血细胞因子的影响[J].中国普通外科杂志,2005,14(3):190-193.
[4]Singleton KD,Wischmeyer PE.Effects of HSP70.1/3 gene knockout on acute respiratory distress syndrome and the inflammatory response following sepsis[J].Am J Physiol Lung Cell Mol Physiol,2006,290(5):L956-L961.
[5]何淼,熊旭东,沈晓红.炎调方对脓毒症急性呼吸窘迫综合征(热炽营血证)患者血细胞因子的影响[J].中国中医急症,2013,22(7):1097-1099.
[6]沈晓红,何淼,熊旭东,等.炎调方对脓毒症大鼠肺热休克蛋白70和核因子-κB的调控作用[J].上海中医药大学学报,2013,27(3):69-72.
[2]Schmidt C,Kurt B,H9cherl K,et al.Inhibition of NF-kappa B activity prevents downregulation of alpha1-adrenergic receptors and circulatory failure during CLP-induced sepsis[J].Shock,2009,32(3):239-246.
[3]程应东,梁平,张朝军,等.HSP70基因转染对大鼠脓毒症早期外周血细胞因子的影响[J].中国普通外科杂志,2005,14(3):190-193.
[4]Singleton KD,Wischmeyer PE.Effects of HSP70.1/3 gene knockout on acute respiratory distress syndrome and the inflammatory response following sepsis[J].Am J Physiol Lung Cell Mol Physiol,2006,290(5):L956-L961.
[5]何淼,熊旭东,沈晓红.炎调方对脓毒症急性呼吸窘迫综合征(热炽营血证)患者血细胞因子的影响[J].中国中医急症,2013,22(7):1097-1099.
[6]沈晓红,何淼,熊旭东,等.炎调方对脓毒症大鼠肺热休克蛋白70和核因子-κB的调控作用[J].上海中医药大学学报,2013,27(3):69-72.